EA040014B1 - METHODS FOR THE TREATMENT OF PATIENTS WITH MALIGNANT TUMORS USING FARNESIL TRANSFERASE INHIBITORS - Google Patents

Description

Technical field

The present invention relates to the field of molecular biology and biology of malignant tumors. Methods are provided for using specific immune-related genes as biomarkers to predict clinical sensitivity and therapeutic response to treatment with a farnesyl transferase inhibitor in a subject having cancer. Also provided herein is a kit for implementing these methods.

State of the art

Stratification of patient populations to improve therapeutic response rates is becoming increasingly valuable in managing the clinical course of disease in cancer patients. Farnesyl transferase inhibitors (FTIs) are drugs useful in the treatment of cancers such as leukemia, lymphoma, and certain solid tumors. However, patients respond differently to FTI treatment. Thus, methods for predicting the ability of a cancer patient to respond to FTI treatment or methods for selecting patients for FTI treatment represent an unmet need. The methods and compositions of the present invention meet this need and provide other associated benefits.

The essence of the invention

This document provides methods for selecting populations of patients with malignant tumors for treatment using FTI. The methods presented herein are based in part on the discovery that the genotype and expression level of specific immunological genes can be used to predict the ability of a cancer patient to respond to FTI treatment.

In some embodiments, the methods provided herein for treating cancer in a subject comprise (a) typing a KIR in a subject, where the subject is a carrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeutically effective amount of FTI to the subject. In some embodiments, the subject is a carrier of KIR2DS2. In some embodiments, the subject is a carrier of KIR2DS5. In some embodiments, the subject is a carrier of KIR2DS2 and KIR2DS5.

In some embodiments, the methods provided herein further comprise HLA typing in a subject prior to subjecting the subject to FTI treatment, wherein the subject is an HLA-C2 carrier. In some embodiments, the subject is a carrier of both KIR2DS2 and HLA-C2.

In some embodiments, typing a KIR in a subject includes determining the presence of a KIR gene in a sample from the subject. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a bone marrow sample. In some embodiments, the sample is peripheral blood mononuclear cells (PBMC). In some embodiments, the sample is enriched in natural killer (NK) cells.

In some embodiments, KIR typing is performed by sequencing, polymerase chain reaction (PCR), microarray DNA analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, immunoblot analysis, or enzyme-linked immunosorbent assay (ELISA). In one embodiment, KIR typing is performed by PCR. In one embodiment, KIR typing is performed through DNA microarray analysis. In one embodiment, KIR typing is performed by immunoblot or ELISA assay.

In some embodiments, the methods provided herein for treating cancer in a subject comprise (a) determining the expression level of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM in a sample from the subject, where (i) the expression level KIR2DS2 in the sample is higher than the reference level of KIR2DS2 expression; (ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression; (iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression; (iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression; or any combination of these is present; and (b) administering a therapeutically effective amount of the FTI to the subject.

In some embodiments, the methods provided herein for treating cancer in a subject comprise (a) determining expression levels of KIR2DS2 and KIR2DL2 or KIR2DS5 and KIR2DL5 in a sample from a subject, wherein (i) the ratio of KIR2DS2 expression level to KIR2DL2 expression level in the sample is greater than reference ratio; or (ii) the ratio of the level of expression of KIR2DS5 to the level of expression of KIR2DL5 in the sample is higher than the reference ratio; (b) administering a therapeutically effective amount of the FTI to the subject.

In some embodiments, the sample is a blood sample. In some embodiments, the sample is a bone marrow sample. In some variants

- 1 040014 implementation of the sample is a mononuclear cells of peripheral blood (PBMC). In some embodiments, the sample is enriched in NK cells. In some embodiments, the NK cells are further propagated in vitro.

In some embodiments, determining the level of expression of the biomarker includes determining the level of the biomarker protein. Methods for determining the level of a biomarker protein can be an immunohistochemistry (IHC) method, immunoblot analysis, flow cytometry (FACS), or ELISA. In some embodiments, the biomarker protein level is measured by ELISA.

In some embodiments, determining the level of expression of a biomarker includes determining the level of mRNA of the biomarker. Methods for determining the mRNA level of a biomarker can be q-PCR, RT-PCR, RNA sequencing, microarray analysis, SAGE, MassARRAY or FISH method. In some embodiments, the mRNA level of the biomarker is measured by q-PCR or RT-PCR.

In some embodiments, the subject is a cancer patient. In some embodiments, the subject has a hematologic malignancy. In some embodiments, the subject has a solid tumor. A solid tumor may be a benign tumor or a malignant tumor. In some other embodiments, the subject has a precancerous condition. The hematologic malignancy may be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), natural killer cell lymphoma (NK lymphoma), natural killer cell leukemia (NK leukemia), skin T-cell lymphoma ( CTCL), peripheral T-cell lymphoma (PTCL), or chronic myeloid leukemia (CML). In some embodiments, the patient is an MDS patient. A patient with MDS may have very low risk MDS, low risk MDS, intermediate risk MDS, or high risk MDS. In some embodiments, the patient is a lower risk MDS patient who may have very low risk MDS, low risk MDS, intermediate risk MDS. In some embodiments, the patient is an AML patient. In some embodiments, the implementation of the patient with AML is a patient after induction of remission or after transplantation. In some embodiments, the AML patient is over 60 years of age or otherwise ineligible for remission induction.

In some embodiments, the methods provided herein for selecting a cancer patient for FTI treatment comprise (a) typing a KIR in a subject, where the subject is a carrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeutically effective amount of FTI to the subject. In some embodiments, the methods are a) KIR typing in a subject, where the subject is a carrier of KIR2DS2 or KIR2DS5, b) HLA typing in a subject, where the subject is a carrier of HLA-C2, and (c) administering a therapeutically effective amount of FTI to the subject. In some embodiments, the subject is a carrier of both KIR2DS2 and HLA-C2.

In some embodiments, methods for selecting a cancer patient for FTI treatment comprise (a) determining the expression level of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM in a sample from a subject, wherein (i) the expression level of KIR2DS2 in the sample is higher than the reference level of expression of KIR2DS2; (ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression; (iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression; (iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression; or any combination of these is present; and (b) administering a therapeutically effective amount of the FTI to the subject.

In some embodiments, the methods provided herein for selecting a cancer patient for FTI treatment comprise (a) determining the expression levels of KIR2DS2 and KIR2DL2 or KIR2DS5 and KIR2DL5 in a sample from a subject, where (i) the ratio of the expression level of KIR2DS2 to the expression level of KIR2DL2 in the sample higher than the reference ratio; or (ii) the ratio of the level of expression of KIR2DS5 to the level of expression of KIR2DL5 in the sample is higher than the reference ratio; (b) administering a therapeutically effective amount of the FTI to the subject.

In one embodiment, the methods provided herein for treating MDS in a subject comprise (a) typing a KIR in a subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeutically effective amount of tipifarnib to the subject. The methods may further include HLA typing in a subject, where the subject is an HLA-C2 carrier. In some embodiments, the subject is a carrier of both KIR2DS2 and HLA-C2. In some embodiments, the MDS is a lower risk MDS.

Provided herein are methods for selecting cancer patient populations for FTI treatment based on the mutation status of Ras. In some embodiments, provided herein are methods for predicting the ability of a cancer patient to respond to FTI treatment based on the status of Ras mutations in a sample from the patient. In some embodiments, provided herein are methods of treating

- 2 040014 malignant tumor in a subject using a therapeutically effective amount of FTI.

In some embodiments, provided herein are methods of treating cancer in a subject, comprising (a) determining the presence or absence of a Ras mutation in a sample from the subject, wherein the Ras mutation includes a K-Ras mutation or an N-Ras mutation, and then (b) administering a therapeutically effective amount of FTI to the subject, if the sample is determined to be free of a K-Ras mutation or an N-Ras mutation.

In some embodiments, the methods include determining the presence or absence of an amino acid substitution at a codon selected from the group consisting of G12, G13, and Q61 from K-Ras. In some embodiments, the methods include determining the presence or absence of an amino acid substitution at a codon selected from the group consisting of G12 G13 and Q61 from N-Ras.

In some embodiments, the patient is treated with FTI if the sample is determined not to have an amino acid substitution in G12, G13, and Q61 from K-Ras, and also not to have an amino acid substitution in G12, G13, and Q61 from N-Ras. In some embodiments, the patient is treated with FTI if the sample is determined not to have any K-Ras mutation or any NRas mutation. In some embodiments, the patient is treated with FTI if the sample is determined to have wild-type K-Ras and wild-type N-Ras.

In some embodiments, the subject is a cancer patient. In some embodiments, the subject has a hematologic malignancy. In some embodiments, the subject has a solid tumor. A solid tumor may be a benign tumor or a malignant tumor. In some other embodiments, the subject has a precancerous condition. The hematologic malignancy may be chronic myelomonocytic leukemia (CMML), myeloproliferative neoplasia (MPN), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), juvenile myelomonocytic leukemia (JMML), chronic myeloid leukemia (CML), natural killer cell lymphoma (NK lymphoma), natural killer cell leukemia (NK leukemia), cutaneous T-cell lymphoma (CTCL), or peripheral T-cell lymphoma (PTCL). In some embodiments, the patient is a CMML patient. In some embodiments, the patient is an MDS patient. In some embodiments, the patient is an AML patient.

In some embodiments, provided herein are methods of treating CMML in a subject: (a) determining the presence or absence of a K-Ras mutation in a sample from the subject, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if wild K-Ras is detected in the sample. type.

In some embodiments, provided herein are methods of treating CMML in a subject: (a) determining the presence or absence of an N-Ras mutation in a sample from the subject, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if wild N-Ras is detected in the sample. type.

Provided herein are methods for selecting cancer patient populations for FTI treatment based on the presence of an H-Ras mutation. In some embodiments, provided herein are methods for predicting the ability of a cancer patient to respond to FTI treatment, methods for selecting a population of cancer patients for FTI treatment, and methods for treating cancer in a subject using a therapeutically effective amount of FTI based on the presence of an H-mutation. Ras in a sample from a patient.

In some embodiments, provided herein are methods of treating cancer in a subject, comprising (a) determining the presence or absence of an HRas mutation in a sample from the subject, then (b) administering a therapeutically effective amount of FTI to the subject if the sample is determined to have an H-Ras mutation. . In some embodiments, the HRas mutation may be an amino acid substitution at G12, G13, and Q61 of the H-Ras.

In some embodiments, provided herein are methods of treating cancer in a patient, comprising (a) determining the presence or absence of an HRas mutation, a K-Ras mutation, and an N-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to the subject if the sample has an H-Ras mutation but not a KRas mutation or an N-Ras mutation. In some embodiments, the methods comprise (a) determining whether a cancer patient has an H-Ras and wild-type K-Ras and wild-type N-Ras mutation, then (b) administering a therapeutically effective amount of FTI to the subject. In some embodiments, the FTI is tipifarnib.

In some embodiments, the subject has a hematologic malignancy. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is HPV negative. In some embodiments, the cancer is hepatocellular carcinoma, head and neck cancer, salivary gland tumor, thyroid tumor, urothelial cancer, breast cancer, melanoma,

- 3 040014 gastric cancer, pancreatic cancer or lung cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the cancer is a salivary gland tumor. In some embodiments, the cancer is a thyroid tumor.

In some embodiments, provided herein is a method for treating HNSCC in a subject based on the presence of an H-Ras mutation. In some embodiments, the HNSCC may be HPV negative HNSCC. In some embodiments, the HNSCC may be recurrent/repeated HNSCC. In some embodiments, the HNSCC may be a metastatic HNSCC. The methods provided herein include (a) determining the presence or absence of an H-Ras mutation in a sample from a subject having HNSCC, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if the sample is determined to have an H-Ras mutation.

In some embodiments, provided herein is a method of treating a salivary gland tumor in a subject based on the presence of an H-Ras mutation. In some embodiments, the salivary gland tumor may be HPV negative. In some embodiments, the salivary gland tumor may be an advanced salivary gland tumor. In some embodiments, the salivary gland tumor may be a metastatic salivary gland tumor. The methods provided herein include (a) determining the presence or absence of an H-Ras mutation in a sample from a subject having a salivary gland tumor, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if the sample is determined to have an H-Ras mutation.

In some embodiments, provided herein is a method for treating thyroid cancer in a subject based on the presence of an H-Ras mutation. In some embodiments, the thyroid cancer may be HPV negative. In some embodiments, the thyroid cancer may be advanced thyroid cancer. In some embodiments, the thyroid cancer may be a metastatic thyroid cancer. The methods provided herein include (a) determining the presence or absence of an H-Ras mutation in a sample from a subject having thyroid cancer, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if the sample is determined to have an H-Ras mutation.

In some embodiments, the sample is a tumor biopsy. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a peripheral blood sample. In some embodiments, the sample is a serum sample. In some embodiments, the sample is a bone marrow sample. In some embodiments, the sample is peripheral blood mononuclear cells (PBMC).

In some embodiments, the Ras mutation status is determined by analysis of nucleic acids obtained from a sample. In some embodiments, the Ras mutation status is determined by analysis of proteins obtained from a sample. In some embodiments, Ras mutation status is determined by sequencing, polymerase chain reaction (PCR), DNA microarray analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, denaturing high performance liquid chromatography (DHPLC), or restriction fragment length polymorphism analysis ( RFLP). In some embodiments, the Ras mutation status is determined by multiplex PCR. In some embodiments, the mutation status of Ras is determined by next generation sequencing.

In some embodiments, the FTI is selected from the group consisting of tipifarnib, lonafarnib (SCH-66336), CP-609,754, BMS-214662, L778123, L744823, L739749, R208176, AZD3409, and FTI277. In some embodiments, FTI is administered at a dose of 1-1000 mg/kg body weight. In one embodiment, the FTI is tipifarnib. In some embodiments, tipifarnib is administered at a dose of 200-1200 mg twice daily (b.i.d.). In some embodiments, tipifarnib is administered at a dose of 600 mg daily orally. In some embodiments, tipifarnib is administered at a dose of 300 mg b.i.d. orally for 3 of 4 weeks in repeating 4-week cycles. In some embodiments, tipifarnib is administered at a dose of 600 mg b.i.d. orally for 3 of 4 weeks in repeating 4-week cycles. In some embodiments, tipifarnib is administered at a dose of 900 mg b.i.d. orally in alternating weeks (one week of administration, one week of rest) in repeating 4-week cycles (days 1-7 and 15-21 of repeating 28-day cycles). In some embodiments, tipifarnib is administered at a dose of 1200 mg b.i.d. orally on alternating weeks (days 1-7 and 15-21 of recurring 28-day cycles). In some embodiments, tipifarnib is administered at a dose of 1200 mg b.i.d. orally on days 1-5 and 15-19 of repeating 28-day cycles. In some embodiments, patients are subjected to at least three cycles

- 4 040014 treatment. In some embodiments, patients are subjected to at least six treatment cycles.

In some embodiments, the methods provided herein also include exposing the subject to a second therapy. The second therapy may be radiotherapy. In some embodiments, the methods provided herein also include administering to a subject a second therapeutically effective amount of a second active ingredient, or subjecting the subject to maintenance therapy. In some embodiments, the second active ingredient is a DNA hypomethylating agent, a therapeutic antibody that specifically binds a cancer antigen, a hematopoietic growth factor, a cytokine, an anticancer agent, an antibiotic, a cox-2 inhibitor, an immunomodulatory agent, an antithymocyte globulin, an immunosuppressive agent, a corticosteroid, or their pharmacological derivative. In some embodiments, the second active ingredient is a DNA hypomethylating agent such as azacitidine or decitabine. In some embodiments, the second active ingredient is an anti-PD1 antibody or an anti-PDL1 antibody.

In some embodiments, the kits provided herein for predicting the ability of a cancer patient to respond to FTI treatment comprise at least one agent for typing a KIR in a cancer patient and an adjuvant where the cancer patient is predicted to be is capable of responding to FTI treatment if the cancer patient is a carrier of KIR2DS2 or KIR2DS5. The kits may further comprise an HLA typing agent where the cancer patient is predicted to be able to respond to FTI treatment if the cancer patient is an HLA-C2 carrier.

In some embodiments, the kits provided herein for predicting the ability of a cancer patient to respond to FTI treatment comprise at least one agent for determining the expression of at least one biomarker in a sample from a cancer patient, and an auxiliary agent, wherein the biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5 and GZMM; and wherein a cancer patient is predicted to be responsive to FTI treatment if (i) the expression level of KIR2DS2 in the sample is higher than the reference level of KIR2DS2 expression;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression;

(v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression;

(vi) the ratio of the level of expression of KIR2DS2 to the level of expression of KIR2DL2 in the sample is higher than the reference ratio; or (vii) the ratio of the level of expression of KIR2DS5 to the level of expression of KIR2DL5 in the sample is higher than the reference ratio;

or any combination of these is present.

Brief description of the figures

In FIG. 1A shows the correlation of KIR2DS5 expression levels with clinical outcome for AML patients treated with tipifarnib. SD stands for stable disease; PD stands for progressive disease; CR stands for complete response; HI stands for improvement in hematological parameters. In FIG. 1B shows the correlation of KIR2DS5 expression levels with progression-free survival (PFS) of AML patients treated with tipifarnib.

In FIG. 2A shows the correlation between the ratio of KIR2DS2 expression to KIR2DL2 and PFS expression in AML patients treated with tipifarnib. In FIG. 2B shows the correlation between the ratio of KIR2DS2 expression level to KIR2DL2 expression level and overall survival (OS) of AML patients treated with tipifarnib.

Fig. 2A: Cox proportional hazards regression analysis.

Parameter b SE Wald statistics R Exp.(b) 95% CI expected(b) 2DS/2DL -7.4132 2.0012 13.7227 0.0002 0.0006 0.0000-0.0299

Fig. 2B: Cox proportional hazards regression analysis.

b SE Wald statistics R Exp. (b) 95% CI expected (b) 2DS2/2DL2 ratio -5.3430 1.6871 10.0296 0.0015 0.0048 0.0002-0.1283

On both Figs. 3A and 3B show no correlation between the ratio of KIR2DS2 expression to KIR2DL2 and OS expression in AML patients treated with non-FTI chemotherapeutic agents. In FIG. 3A: Patients were treated with high doses of cytarabine and mitoxantrone. In FIG. 3B: Patients were treated with high doses of cytarabine and mitoxantrone/intense chemotherapy.

In FIG. 4A shows the correlation between the ratio of KIR2DS5 expression level to KIR2DL5A expression level and both PFS and OS of AML patients treated with tipifarnib. In FIG. 4B shows no correlation between the ratio of KIR2DS5 expression to KIR2DL5A and OS expression in AML patients treated with non-FTI chemotherapeutic agents (cytarabine and mitoxantrone).

Fig. 4A: Cox proportional hazards regression analysis.

Tipifarnib/PFS b SE Wald statistics R Exp. (b) 95% CI expected(b) Ratio 2DS5/2DL5A -3.5254 1.2351 8.1480 0.0043 0.0294 0.0026-0.3272

In FIG. 5 shows the correlation of GZMM expression levels with clinical outcome for AML patients treated with tipifarnib.

In FIG. 6A shows the correlation between GZMM expression level and survival in AML patients treated with tipifarnib. In FIG. 6B shows no correlation between GZMM expression level and survival in AML patients treated with non-FTI chemotherapeutic agents (cytarabine and mitoxantrone).

Fig. 6A: Cox proportional hazards regression analysis.

(Tipifarnib) GZMM/OS b -0.5642 SE 0.1652 Wald statistics 11.6675 P 0.0006 Exp. (b) 0.5688 95% CI expected(b) 0.4122-0.7850 GZMM/PFS -0.5856 0.1809 10.4780 0.0012 0.5568 0.3913-0.7923

In FIG. 7A shows the correlation of KIR2DS2 expression levels with clinical outcome for AML patients treated with tipifarnib. In FIG. 7B shows the specific correlation of KIR2DS2 expression levels with clinical outcome for AML patients treated with tipifarnib (left panel) but not with non-FTI chemotherapeutic agents (right panel).

In FIG. 8 shows the correlation between wild-type N-RAS status and prolonged progression-free survival (PFS) in AML patients treated with tipifarnib.

In FIG. 9 shows a higher response rate to tipifarnib treatment in AML patients with wild-type N-RAS compared to patients with mutant N-RAS.

Detailed description

1. Definitions.

As used herein, the singular refers to one or more than one grammatical entity. By way of example, a biomarker refers to one biomarker or more than one biomarker.

As used herein, the term NK cell, or natural killer cell, refers to a type of bone marrow-derived large granular lymphocyte that shares a common progenitor with T cells but lacks B cell or T cell surface markers. NK cells typically make up 10-15% of all circulating lymphocytes. NK cells are protective cells of the innate immune system, recognizing structures on the surface of virus-infected cells or tumor cells and destroying these cells by releasing cytotoxins. NK cells can be activated without prior exposure to an antigen.

To selectively kill infected or tumor cells, NK cells must distinguish healthy cells from diseased cells. The cytolytic activity of human NK cells is modulated by the interaction of inhibitory and activating membrane receptors, which are expressed on the surface of NK cells, with MHC (HLA) class I molecules, which are expressed by non-NK cells, including tumor cells or bone marrow transplant recipient cells. Killer cell immunoglobulin-like receptors (KIR; or CD158) mapped to chromosome 19q13.4.3-5 constitute a family of MHC-I (HLA-A, -B, -C) binding receptors that regulate the activation threshold of NK cells (Valiante el at., Immunity 7:739-751(1997)).

In humans, the HLA class I complex is 2000 bp long. and contains approximately 20 genes. Within the class I region, there are genes encoding well-characterized class I MHC molecules designated HLA-A, HLA-B, and HLA-C. In addition, there are unclassified class I genes that include HLA-E, HLA-F, HLA-G, HLA-H, HLA-J and HLA-X, as well as a new family known as MIC. While HLA-A and -B play a role, interactions between KIR and HLA-C molecules predominate in preventing NK cells from attacking healthy autologous cells.

- 6 040014 (Colonna et al., PNAS, 90:1200-12004 (1993); Moesta AK et al., Front Immunol. 3:336 (2012)).

The HLA-C gene has many alleles, including HLA-C1 and HLA-C2, based on the presence of asparagine or lysine at amino acid position 80 in the mature protein (Mandelboim et al., 1996). Moreover, HLA-C1 contains a conserved serine residue at amino acid position 77, while asparagine is present in HLA-C2 at the same position. Thus, at least three genotypes can be distinguished with respect to HLA-C: a genotype having both HLA-C1 and HLA-C2 (heterozygous HLA-C1/HLA-C2), a genotype having either HLA-C1 (homozygous HLA-C1/HLA-C1) or HLA-C2 (homozygous HLA-C2/HLA-C2) and a genotype lacking both HLA-C1 and HLA-C2.

As used herein, the term KIR genes refers to genes encoding KIR receptors on NK cells. The KIR genes cluster in one of the most variable regions in the human genome in terms of both gene content and sequence polymorphism. This extensive variability forms the NK cell repertoire, in which KIR is expressed on the cell surface in a combinatorial manner. Interactions between KIRs and their respective ligands on target cells result in either positive or negative signals that regulate NK cell function.

The KIR genes are inherited in two main haplotypes: A and B. Haplotype A has only one activating receptor, KIR2DS4, which is inactivated in most of the US population due to a 22 bp deletion. The KIR haplotype B includes 22 KIR2DS2 and 16 KIR2DS5 alleles present in ~45% and ~25% of American Caucasians, respectively. There is a strong linkage disequilibrium between KIR2DS2 (activating) and KIR2DL2 (inhibiting). DNA methylation maintains allele-specific expression of the KIR gene (eg, the CpG island in the KIR2DS2 promoter extends from -160 to +26 and has 6 cytosine-rich regions) (Moesta AK et al., Front Immunol. 3:336 (2012)).

So far, at least 14 distinct KIR genes have been identified, which are KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DS1. These genes share extensive sequence homology. Each gene is 9-16 kb long, divided into 8-9 exons, which encode a signal peptide, two or three extracellular domains, a stem, a transmembrane region, and a cytoplasmic tail. These genes vary with respect to their presence or absence on different KIR haplotypes, creating considerable diversity in the number of KIR genotypes observed in a population. For example, some individuals may only carry seven of the 14 KIR genes, while other individuals may carry 12 of the 14 KIR genes. Each KIR gene encodes either an inhibitory or an activating KIR. For example, KIR2DS2 and KIR2DS5 are both activating KIRs, and KIR2DL2 and KIR2DL5 are both inhibitory KIRs. One particular KIR gene can have multiple alleles. For example, KIR2DL5 includes two alleles, KIR2DL5A and KIR2DL5B. Thus, four genotypes can be distinguished with respect to KIR2DL5: genotypes having both KIR2DL5A and KIR2DL5B, genotypes having either KIR2DL5A or KIR2DL5B, and genotypes lacking KIR2DL5.

An exemplary amino acid sequence and corresponding nucleic acid coding sequence for human KIR2DS2 (GENBANK: GQ921920.1; GI: 261362473) are shown below.

- 7 040014

MSLMVVSMVCVGFFLLQGAWPHEGVHRKPSLLAHPGPLVKSEETVILQCWSDVRFEHFL LHREGKYKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVIT GLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRFSAGPKVNGTFQADFPL GPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHVLIGTS WKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA (SEQ ID NO: 1)

ATGTCGCTCATGGTCGTCAGCATGGTGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTG

GCCACATGAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGTCCCCTGGTGAAATCA GAAGAGACAG T CAT С С T G СAAT G Τ T G G T CAGAT G T GAG G Τ Τ T GAG GAG TTCCTTCTG CACAGAG AGGGGAAGTATAAGGACACTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAGGCCAA CTTCTCCATCGGTCCCATGATGCAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACT CACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATG AGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTTTGGCAGGAGAGAGCGTGACCTTGTC CTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAACGT AGGTTCTCTGCAGGGCCCAAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCA CCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCCTATGAGTGGTCAAACTC GAGTGACCCACTGCTTGTTTCTGTCACAGGAAACCCTTCAAATAGTTGGCCTTCACCCACTGAA CCAAGCTCCAAAACCGGTAACCCCAGACACCTGCATGTTCTGATTGGGACCTCAGTGGTCAAAA TCCCTTTCACCATCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCTGC TGTAATGGACCAAGAGCCTGCAGGGAACAGAACAGTGAACAGCGAGGATTCTGATGAACAAGAC CATCAGGAGGTGTCATACGCATAA (SEQ ID NO: 2)

An exemplary amino acid sequence and corresponding nucleic acid coding sequence for human KIR2DL2 (GENBANK: EU791546.1; GI: 209512828) are shown below.

MSLMWSMACVGFFLLQGAWPHEGVHRKPSLLAHPGRLVKSEETVILQCWSDVRFEHFL LHREGKFKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVIT GLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHECRFSAGPKVNGTFQADFPL GPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVIGNPSNSWPSPTEPSSKTGNPRHLHILIGTS

- 8 040014

WIILFILLFFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKI

TRPSQRPKTPPTDIIVYTELPNAESRSKWSCP (SEQ ID NO : 3 )

ATGTCGCTCATGGTCGTCAGCATGGCGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTG

GCCACATGAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGTCGCCTGGTGAAATCA

GAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCAGGTTTGAGCACTTCCTTCTGCACAGAG

AAGGGAAGTTTAAGGACACTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAAGCCAA

CTTCTCCATCGGTCCCATGATGCAAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACT

CACTCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATG

AGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTC

CTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAATGT

AGGTTCTCTGCAGGGCCCAAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCA

CCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAACTC

GAGTGACCCACTGCTTGTTTCTGTCATAGGAAACCCTTCAAATAGTTGGCCTTCACCCACTGAA

CCAAGCTCTAAAACCGGTAACCCCCGACACCTGCACATTCTGATTGGGACCTCAGTGGTCATCA

TCCTCTTCATCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCTGCGGT

AATGGACCAAGAGTCTGCAGGGAACAGAACAGCGAATAGCGAGGACTCTGATGAACAAGACCCT

GAG GAG G T GAG AT AC AC AC AG T T GAAT C AC TGCGTTTT C AC AC AGAGAAAAAT C AC TCGCCCTT

C T CAGAG G C C CAAGACAC C C C CAACAGATATCAT CGT GTAGACGGAAC T T CCAAAT GC T GAGT C

CAGATCCAAAGTTGTCTCCTGCCCATGA (SEQ ID NO :4)

An exemplary amino acid sequence and corresponding nucleic acid coding sequence for human KIR2DS5 (GENBANK: AJI81015.1; GI: 754367842) are shown below.

MSLMVISMACVAFFLLQGAWPHEGFRRKPSLLAHPGPLVKSEETVILQCWSDVMFEHFL

LHREGTFNHTLRLIGEHIDGVSKGNFSIGRMTQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVIT

GLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRLPAGPKVNRTFQADFPL

DPATHGGTYRCFGSFRDSPYEWSKSSDPLLVSVTGNSSNSWPSPTEPSSETGNPRHLHVLIGTS

WKLPFTILLFFLLHRWCSNKKNASVMDQGPAGNRTVNREDSDEQDHQEVSYA (SEQ ID

NO: 5)

ATGTCGCTCATGGTCATCAGCATGGCGTGTGTTGCGTTCTTCTTGCTGCAGGGGGCCTG

GCCACATGAGGGATTCCGCAGAAAACCTTCCCTCCTGGCCCACCCAGGTCCCCTGGTGAAATCA

GAAGAGACAGTCATCCTGCAATGTTGGTCAGATGTCATGTTTGAGCACTTCCTTCTGCACAGAG

AGGGGACGTTTAACCACACTTTGCGCCTCATTGGAGAGCACATTGATGGGGTCTCCAAGGGCAA

CTTCTCCATCGGTCGCATGACACAAGACCTGGCAGGGACCTACAGATGCTACGGTTCTGTTACT

CACTCCCCCTATCAGTTGTCAGCGCCCAGTGACCCTCTGGACATCGTGATCACAGGTCTATATG

AGAAACCTTCTCTCTCAGCCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTC

- 9 040014

CTGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGGGAAGGGGAGGCCCATGAACGT

AGGCTCCCCTGCAGGGCCCCAAGGTCAACAGAACATTCCAGGCCGACTTTCCTCTGGACCCTGCCA

CCCACGGAGGGACCTACAGATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAAGTC

AAGTGACCCACTGCTTGTTTCTGTCACAGGAAACTCTTCAAATAGTTGGCCTTCACCCACTGAA

CCAAGCTCCGAAACCGGTAACCCCAGACACCTACACGTTCTGATTGGGACCTCAGTGGTCAAAC

TCCCTTTCACCATCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCATC

TGTAATGGACCAAGGGCCTGCGGGGAACAGAACAGTGAACAGGGAGGATTCTGATGAACAGGAC

CATCAGGAGGTGTCATACGCATAA (SEQ ID NO:6)

An exemplary amino acid sequence and corresponding coding sequence for the human KIR2DL5A nucleic acid (GENBANK: ABM92655.1 GI: 124245538) are shown below.

MSLMVISMACVGFFLLQGAWTHEGGQDKPLLSAWPSAVVPRGGHVTLLCRSRLGFTIFS

LYKEDGVPVPELYNKIFWKSILMGPVTPAHAGTYRCRGSHPRSPIEWSAPSNPLVIWTGLFGK

PSLSAQPGPTVRTGENVTLSCSSRSSFDMYHLSREGRAHEPRLPAVPSVDGTFQADFPLGPATH

GGTYTCFSSLHDSPYEWSDPSDPLLVSVTGNSSSSSSSPTEPSSKTGIRRHLHILIGTSVAIIL

FIILFFFLLHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTYAQLDHCVFTQTKITSPS

QRPKTPPTDTTMYMELPNAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI (SEQ ID NO:7)

ATGTCGCTCATGGTCATCAGCATGGCGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTG

GACACATGAGGGTGGACAGGACAAGCCCTTGCTGTCTGCCTGGCCCAGCGCTGTGGTGCCTCGA

GGAGGACATGTGACTCTTCTGTGTCGCTCTCGTCTTGGGTTTACCATCTTCAGTCTGTACAAAG

AAGATGGGGTGCCTGTCCCTGAGCTCTACAACAAAATATTCTGGAAGAGCATCCTCATGGGCCC

TGTGACCCCTGCACACGCAGGGACCTACAGATGTCGGGGTTCACACCCGCGCTCCCCCATTGAG

TGGTCGGCACCCAGCAACCCCCTGGTGATCGTGGTCACAGGTCTATTTGGGAAACCTTCACTCT

CAGCCCAGCCGGGCCCCACGGTTCGCACAGGAGAGAACGTGACCTTGTCCTGCAGCTCCAGGAG

CTCATTTGACATGTACCATCTATCCAGGGAGGGGAGGGCCCATGAACCTAGGCTCCCTGCAGTG

CCCAGCGTCGATGGAACATTCCAGGCTGACTTTCCTCTGGGCCCTGCCACCCACGGAGGGACCCT

ACACATGCTTCAGCTCTCTCCATGACTCACCCTATGAGTGGTCAGACCCGAGTGACCCACTGCT

TGTTTCTGTCACAGGAAACTCTTTCAAGTAGTTCATCTTCACCCACTGAACCAAGCTCAAAAAACT

GGTATCCGCAGACACCTGCACATTCTGATTGGGACCTCAGTGGCTATCATCCTCTTCATCATCC

TCTTCTTCTTTCTCCTTCATTGCTGCTGCTCCAACAAAAAGAATGCCTGCTGTAATGGACCAAGA

GCCTGCCGGGGACAGAACAGTGAACAGGGAGGACTCTGATGATCAAGACCCTCAGGAGGTGACA

TAT G CACAG T T G GAT CAC TGCGTTTT CACAOAGACAAAAAT CAC TTCCCCTTCT CAGAG G C C CA

AGACAC C T C CAACAGATAC CAC CAT G TACAT G GAAC T T C CAAAT G C TAAG C CAAGAT CAT T G T C

TCCTGCCCATAAGCACCACAGTCAGGCCTTGAGGGGATCTTCTAGGGAGACAACAGCCCTGTCT

CAAAACCGGGTTGCTAGCTCCCATGTACCAGCAGCTGGAATCTGA (SEQ ID NO :8)

An exemplary amino acid sequence and corresponding nucleic acid coding sequence for human KIR2DL5B (GENBANK: ABM92657.1 GI: 124245542) are shown below.

- 10 040014

MSLMVVSMACVGFFLLQGAWTHEGGQDKPLLSAWPSAVVPRGGHVTLLCRSRLGFTIFS LYKEDGVPVPELYNKIFWKSILMGPVTPAHAGTYRCRGSHPRSPIEWSAPSNPLVIWTGLFGK PSLSAQPGPTVRTGENVTLSCSSRSSFDMYHLSREGRAHEPRLPAVPSVDGTFQADFPLGPATH GGTYTCFSSLHDSPYEWSDPSDPLLVSVTGNSSSSSSSPTEPSSKTGILRHLHILIGTSVAIIL FIILFFFLLHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTYAQLDHCVFTQTKITSPS QRPKTPPTDTTMYMELPNAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI (SEQ ID NO:9)

ATGTCGCTCATGGTCGTCAGCATGGCGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTG

GACACATGAGGGTGGACAGGACAAGCCCTTGCTGTCTGCCTGGCCCAGCGCTGTGGTGCCTCGA

GGAGGACATGTGACTCTTCTGTGTCGCTCTCGTCTTGGGTTTACCATCTTCAGTCTGTACAAAG

AAGATGGGGTGCCTGTCCCTGAGCTCTACAACAAAATATTCTGGAAGAGCATCCTCATGGGCCC TGTGACCCCTGCACACGCAGGGACCTACAGATGTCGGGGTTCACACCCGCGCTCCCCCATTGAG TGGTCGGCACCCAGCAACCCCCTGGTGATCGTGGTCACAGGTCTATTTGGGAAACCTTCACTCT CAGCCCAGCCGGGCCCCACGGTTCGCACAGGAGAGAACGTGACCTTGTCCTGCAGCTCCAGGAG CTCATTTGACATGTACCATCTATCCAGGGAGGGGAGGGCCCATGAACCTAGGCTCCCTGCAGTG CCCAGCGTCGATGGAACATTCCAGGCTGACTTTCCTCTGGGCCCTGCCACCCACGGAGGGACCT ACACATGCTTCAGCTCTCTCCATGACTCACCCTATGAGTGGTCAGACCCGAGTGACCCACTGCT TGTTTСTGTCACAGGAAACTСTTCAAGTAGTTCATCTTСACССACTGAACCAAGСTСCAAAACT GGTATCCTCAGACACCTGCACATTCTGATTGGGACCTCAGTGGCTATCATCCTCTTCATCATCC TCTTCTTCTTTCTCCTTCATTGCTGCTGCTCCAACAAAAAGAATGCTGCTGTAATGGACCAAGA GCCTGCCGGGGACAGAACAGTGAACAGGGAGGACTCTGATGATCAAGACCCTCAGGAGGTGACA TATGCACAGTTGGATCACTGCGTTTTCACACAGACAAAAATCACTTCCCCTTCTCAGAGGCCCA AGACAC С T С СAACAGATAC СAC CAT G TAGAT G GAAC T T С СAAAT G С TAAG С CAAGAT CAT T G T C TCCTGCCCATAAGCACCACAGTCAGGCCTTGAGGGGATCTTCTAGGGAGACAACAGCCCTGTCT CAAAACCGGGTTGCTAGCTCCCATGTACCAGCAGCTGGAATCTGA (SEQ ID NO:10)

As used herein, the term KIR typing refers to a method for determining the genotype of KIR genes in a subject, comprising determining the presence or absence of one or more specific KIR genes or alleles in the subject's genome. KIR typing may also include determining the copy number of one or more specific KIR genes or alleles in the subject's genome.

As used herein, the term HLA typing refers to a method for determining the genotype of HLA genes in a subject, comprising determining the presence or absence of one or more specific HLA genes or alleles in the subject's genome. HLA typing also includes determining the copy number of one or more specific HLA genes or alleles in a subject's genome.

Granzyme M (GZMM) is a serine protease expressed in a variety of subgroups of cytotoxic lymphocytes. The granulation-exocytosis pathway is the main mechanism by which cytotoxic lymphocytes destroy virus-infected cells and tumor cells. In this pathway, cytotoxic lymphocytes release granules containing the pore-forming protein perforin and a family of serine proteases known as granzymes (GZM) into the immune synapse. The formation of pores by perforin facilitates the penetration of granzymes into the target cell, where they can activate various pathways of cell death. There are five human granzymes: GZMA, GZMB, GZMH, GZMK, and GZMM. Of the five GZMs, GZMM is a marker for NK cells or NKT cells, and GZMH is a marker for cytotoxic T cells (Poot, Cell Death and Differentiation 21:359-368 (2014)).

An exemplary amino acid sequence and corresponding coding sequence for the human GZMM nucleic acid (NCBI Ref: NM_020535.3 GI:65508540) are provided below.

- 11 040014

MEACVSSLLVLALGALSVGSSFGTQIIGGREVIPHSRPYMASLQRNGSHLCGGVLVHPK

WVLTAAHCLAQRMAQLRLVLGLHTLDSPGLTFHIKAAIQHPRYKPVPALENDLALLQLDGKVKP

SRTIRPLALPSKRQVVAAGTRCSMAGWGLTHQGGRLSRVLRELDLQVLDTRMCNNSRFWNGSLS PSMVCLAADSKDQAPCKGDSGGPLVCGKGRVLAGVLSFSSRVCTDIFKPPVATAVAPYVSWIRK

IVTGRSA (SEQ ID NO: 11)

ATGGAGGCCTGCGTGTCTTCACTGCTGGTGCTGGCCCTGGGGGCCCTGTCAGTAGGCAG CTCCTTTGGGACCCAGATCATCGGGGGCCGGGAGGTGATCCCCCACTCGCGCCCGTACATGGCC TCACTGCAGAGAAATGGCTCCCACCTGTGCGGGGGTGTCCTGGTGCACCCAAAGTGGGTGCTGA CGGCTGCCCACTGCCTGGCCCAGCGGATGGCCCAGCTGAGGCTGGTGCTGGGGCTCCACACCCT GGACAGCCCCGGTCTCACCTTCCACATCAAGGCAGCCATCCAGCACCCTCGCTACAAGCCCGTC CCTGCCCTGGAGAACGACCTCGCGCTGCTTCAGCTGGACGGGAAAGTGAAGCCCAGCCGGACCA TCCGGCCGTTGGCCCTGCCCAGTAAGCGCCAGGTGGTGGCAGCAGGGACTCGGTGCAGCATGGC CGGCTGGGGGCTGACCCACCAGGGCGGGCGCCTGTCCCGGGTGCTGCGGGAGCTGGACCTCCAA GTGCTGGACACCCGCATGTGTAACAACAGCCGCTTCTGGAACGGCAGCCTCTCCCCCAGCATGG TCTGCCTGGCGGCCGACTCCAAGGACCAGGCTCCCTGCAAGGGTGACTCGGGCGGGCCCCTGGT GTGTGGCAAAGGCCGGGTGTTGGCCGGAGTCCTGTCCTTCAGCTCCAGGGTCTGCACTGACATC TTCAAGCCTCCCGTGGCCACCGCTGTGGCGCCTTACGTGTCCTGGATCAGGAAGGTCACCGGCC GATCGGCCTGA (SEQ ID NO:12)

As used herein, the term subject refers to a human. The subject may be a human or a non-human mammal such as a dog, cat, bovid, equine, mouse, rat, rabbit, or transgenic species thereof. The subject may be a patient or a patient with a cancer.

As used herein, the term malignant tumor or carcinogenic refers to a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of malignant tumors include, but are not limited to, hematologic malignancies (eg, multiple myeloma, lymphoma, and leukemia) and solid tumors. As used herein, the term premalignant condition refers to a condition associated with an increased risk of cancer that, if left untreated, can lead to cancer. A premalignant condition can also refer to a non-invasive malignancy that has not progressed to an aggressive, invasive stage.

As used herein, the term treat, treatment and treatment, when applied to a patient with a cancer, refers to an action that reduces the severity of the cancer or delays or retards the progression of the cancer, including (a) slowing the growth of the cancer or stopping development of the cancer; and (b) causing regression of the cancer, or delaying or minimizing one or more of the symptoms associated with the presence of the cancer.

As used herein, the term definition refers to the use of any form of measurement to assess the presence of a substance, either quantitatively or qualitatively. The measurement can be relative or absolute. Measuring the presence of a substance may include determining whether a substance is present or absent, or determining the amount of a substance.

As used herein, the term carrier, when used in connection with a gene, refers to a subject whose genome contains at least one copy of a gene, and when used in connection with an allele of a gene, refers to a subject whose genome contains at least one copy of a specific allele. . For example, a carrier of KIR2DS2 refers to a subject whose genome contains at least one copy of KIR2DS2. If a gene has more than one allele, a gene carrier refers to a subject whose genome contains at least one copy of at least one allele of the gene. For example, the KIR2DL5 gene has two known alleles, KIR2DL5A and KIR2DL5B. A KIR2DL5A carrier refers to a subject whose genome contains at least one copy of the KIR2DL5A allele; a KIR2DL5B carrier refers to a subject whose genome contains at least one copy of the KIR2DL5B allele. A KIR2DL5 carrier refers to a subject whose genome contains at least one copy of KIR2DL5A, KIR2DL5B, or both. As another example, an HLA-C2 carrier refers to

- 12 040014 to a subject whose genome contains at least one copy of the HLA-C2 allele. The subject may be homozygous for HLA-C2/HLA-C2 or heterozygous for HLA-C1/HLA-C2.

As used herein, the term administer, administer, or administer refers to the act of delivering or causing a compound or pharmaceutical composition to be delivered to a subject in the manner described herein or otherwise known in the art. Administration of a compound or pharmaceutical composition includes directing that the compound or pharmaceutical composition is to be delivered to a patient. Exemplary administration forms include oral dosage forms such as tablets, capsules, syrups, suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM) or intraperitoneal (IP); transdermal dosage forms, including creams, jellies, powders or patches; buccal dosage forms; powders for inhalation, sprays, suspensions and rectal suppositories.

As used herein, the term therapeutically effective amount of a compound, when used in connection with a disease or disorder, refers to an amount sufficient to provide a therapeutic benefit in the treatment of the disease or disorder, or in the management of the disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a compound means an amount of a compound, alone or in combination with other drugs, that provides a therapeutic benefit in the treatment of a disease or disorder or in the management of the disease or disorder. The term includes an amount that improves overall treatment, reduces or stops symptoms, or improves the therapeutic efficacy of another drug. The term also refers to the amount of a compound that elicits a significant biological or medical response in a biological molecule (e.g., protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human being sought by a researcher, veterinarian, physician, or clinical staff.

As used herein, the term sample refers to a material or mixture of materials containing one or more components of interest. A sample from a subject refers to a sample obtained from a subject, including samples derived from biological tissue or fluid obtained, accessed or collected in vivo or in situ. The sample can be obtained from an area of the subject's body containing precancerous or malignant cells or tissues. Such samples may be, but are not limited to, organs, tissues, fractions, and cells isolated from a mammal. Illustrative samples include bone marrow, whole blood, partially purified blood, peripheral blood mononuclear cells (PBMCs), and tissue biopsies. Illustrative samples also include a cell lysate, cell culture, cell line, tissue, oral tissue, gastrointestinal tissue, organ, organelle, body fluid, blood sample, urine sample, skin sample, and the like.

As used herein, the term biomarker refers to a gene that may either be present or absent in individual subjects, or may be present but differentially expressed in individual subjects. The presence of a biomarker, including the level of expression of the biomarker, in a sample from a subject may indicate the subject's ability to respond to a particular treatment, such as FTI treatment.

As used herein, the term expresses, or expression when used in connection with a gene, refers to the process by which the information carried by a gene is manifested as a phenotype, including transcription of the gene into messenger RNA (mRNA), subsequent translation of the mRNA molecule into a polypeptide chain. and its assembly into the final protein.

As used herein, the term biomarker RNA product refers to the RNA transcript transcribed from the biomarker, and the term biomarker protein product refers to the protein or polypeptide translated from the biomarker RNA product.

As used herein, the term biomarker expression level refers to the amount or accumulation of a biomarker expression product, such as, for example, the amount of biomarker RNA product (biomarker RNA level) or the amount of biomarker protein product (biomarker protein level). When a biomarker is a gene with more than one allele, the expression level of the biomarker refers to the total accumulation of expression products of all existing alleles for that gene, unless otherwise indicated. For example, the expression level of KIR2DL5 refers to the total expression level of both KIR2DL5A and KIR2DL5B, unless otherwise indicated.

As used herein, the term reference expression level refers to a predetermined expression level of a biomarker that can be used to determine the significance of the expression level of a biomarker in a sample from a subject. The reference level of biomarker expression may be the level of expression of the biomarker in a sample from a healthy individual. The reference level of biomarker expression may also be a cut-off value determined by one of skill in the art through statistical analysis of the expression levels of the biomarker in a sample and the ability to respond to treatment of individuals in the sample. For example, through

- 13 040014 analysis of GZMM expression levels in individuals from the sample and the ability of these individuals to respond to FTI treatment, a person skilled in the art can determine the cut-off value as a reference level of GZMM expression, where the subject is likely to be able to respond to FTI treatment if the level of GZMM expression subject is higher than the reference expression level.

As used herein, the term responsive or responsive, when used in connection with treatment, refers to the effectiveness of a treatment to reduce or lessen the symptoms of the disease being treated. For example, a patient with a cancer is capable of responding to FTI treatment if the FTI treatment effectively inhibits the growth of the cancer or causes arrest of the development of the cancer, causes regression of the cancer, or delays or minimizes one or more of the symptoms associated with the presence of the cancer in that patient. patient.

The ability of a cancer patient to respond to a particular treatment can be characterized as a complete or partial response. A complete response, or CR, refers to the absence of clinically detectable disease with normalization of previously abnormal radiographic findings, bone marrow and cerebrospinal fluid (CSF) analysis, or abnormal amounts of monoclonal proteins. Partial response, or PR, refers to at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in all measurable tumor burden (i.e., the number of malignant cells present in a subject , or the measured volume of tumor masses, or the amounts of abnormal monoclonal proteins) in the absence of new lesions.

One of skill in the art will appreciate that the clinical standards used to determine CR, PR, or other level of a patient's ability to respond to treatment may vary for different types of cancers. For example, for hematopoietic malignancies, patients capable of responding to a particular treatment can be defined as patients having a complete response (CR), partial response (PR), or improvement in hematological parameters (HI) (Lancet et al., Blood 2:2 ( 2006)). HI can be defined as any number of blast cells in the bone marrow less than 5%, or a decrease in the number of blast cells in the bone marrow by at least half. On the other hand, patients unable to respond to a particular treatment can be defined as patients having either progressive disease (PD) or stable disease (SD). Progressive disease (PD) can be defined as either a >50% increase in % of blasts in the bone marrow or circulating blasts compared to baseline, or the appearance of new circulating blasts (at least 2 consecutive cases). Stable disease (SD) can be defined as any response that does not meet the criteria for CR, PR, HI, or PD.

As used herein, the term probability refers to the possibility of an event. A subject likely to be able to respond to a particular treatment when a condition is met means that the likelihood for a subject to be able to respond to a particular treatment is higher when the condition is met than when the condition is not met. The likelihood of being able to respond to a particular treatment may be higher, for example, by 5%, 10%, 25%, 50%, 100%, 200% or more in a subject when a particular condition is met, compared to a subject when the condition is not met. For example, a cancer patient likely to be able to respond to FTI treatment when the subject is a carrier of KIR2DS2 means that the probability for the subject to be able to respond to FTI treatment is 5%, 10%, 25%, 50%, 100%, 200% or more higher in a subject that is a carrier of KIR2DS2 versus a subject that is not a carrier of KIR2DS2. As another example, a subject likely to be able to respond to tipifarnib treatment when the level of GZMM expression in a sample from the subject is higher than the reference level of GZMM expression means that the probability for the subject to be able to respond to tipifarnib treatment is 5, 10, 25, 50 , 100, 200% or more higher in a subject whose GZMM expression level is higher than the reference GZMM expression level compared to a subject whose GZMM expression level is lower than the reference expression level.

Ras proteins are GTPases that regulate proliferation and the transfer of biological information from extracellular signals to the nucleus. Mammalian cells express three ras genes encoding four Ras proteins, which are H-Ras, N-Ras, KA-Ras and K _B -Ras. KA-Ras and K _B -Ras are also generically referred to as K-Ras. Ras proteins exist either in an active state associated with GTP or an inactive state associated with GDP. Mutant RAS proteins accumulate in a GTP-bound conformation due to defective intrinsic GTPase activity and/or resistance to inactivation by GTPase-activating proteins (GAPs). Mutations that lock Ras proteins in their GTP-bound, activated state result in uncontrolled growth and malignant transformation. K-Ras mutations resulting in glycine to valine substitution at K-Ras catalytic sites, resulting in loss of GTPase activity and subsequent permanent binding of GTP to RAS (Yokota, Anti-Cancer Agents in Medicinal Chemistry, 12:163-171(2012)) . Substitution of other amino acids, such as aspartate and valine at codon 12 and aspartate at codon 13, can result in the larger amino acid side chains protruding into the protein's GDP/GTP binding pocket, interfering with GTP hydrolysis. As a result of these conformational and structural changes, EGFR signaling becomes deregulated in response to constitutive activation of the K-Ras protein (Herreros-Villanueva et al., Clinica Chimica Acta 431 (2014) 21:1-220).

An exemplary amino acid sequence and corresponding nucleic acid coding sequence of the human K-Ras isoform A (K _A -Ras) (GENBANK: NM_033360.3 GI: 575403058) are shown below.

MTEYKLVWG AGGVGKSALI ¹ IQLIQNHFVD EYDPTIEDSY RKQWIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVPMVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLV REIRQYRLKK ISKEEKTPGC VKIKKCIIM (SEQ ID NO:13) ATGACTGAAT ATAAACTTGI ¹ GGTAGTTGGA GCTGGTGGCG TAGGCAAGAG TGCCTTGACG ATACAGCTAA TTCAGAATCA TTTTGTGGAC GAATATGATC CAACAATAGA GGATTCCTAC AGGAAGCAAG TAGTAATTGA TGGAGAAACC TGTCTCTTGG ATATTTCTCGA CACAGCAGGT CAAGAGGAGT ACAGTGCAAT GAGGGACCAG TACATGAGGA CTGGGGAGGG CTTTCTTTGT GTATTTCCA TAAATAATAC TAAATCATTT GAAGATATTC ACCATTATAG AGAACAAATT AAAAGAGTTA AGGACTCTGA AGATTGTACCT ATGGTCCTAG TAGGAAATAA ATGTGATTTG CTTCTAGAA CAGTAGACAC AAAACAGGCT CAGGACTTAG CAAGAAGTTA TGGAATTCCT TTTATTGAAA CATCAGCAAA GACAAGACAG AGAGTGGAGG ATGCTTTTTA TACATTGGTG AGGGAGATCC GACAATACAG ATTGAAAAAAATCAGCAAAG AAGAAAAGAC TCCTGGCTGT GTGAAAATTA

AAAATGCAT TATAATGTAA (SEQ ID NO:14)

An exemplary amino acid sequence and corresponding nucleic acid coding sequence for human K-Ras (K _B -Ras) isoform B (GENBANK: NM_033360.3 GI: 575403058) is shown below.

MTEYKLVWG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQWIDGET

CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI

- 15 040014

KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ

GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM (SEQ ID NO: 15)

ATGACTGAAT ATAAACTTGT GGTAGTTGGA GCTGGTGGCG TAGGCAAGAG

TGCCTTGACG ATACAGCTAA TTCAGAATCA TTTTGTGGAC GAATATGATC

CAACAATAGA GGATTCCTAC AGGAAGCAAG TAGTAATTGA TGGAGAAACC

TGTCTCTTGG ATATTTCTCGA CACAGCAGGT CAAGAGGAGT ACAGTGCAAT

GAGGGACCAG TACATGAGGA CTGGGGAGGG CTTTCTTTGT GTATTTGCCA

TAAATAATAC TAAATCATTT GAAGATATTC ACCATTATAG AGAACAAATT

AAAAGAGTTA AGGACTCTGA AGATTGTACCT ATGGTCCTAG TAGGAAATAA

ATGTGATTTG CCTTCTAGAA CAGTAGACAC AAAACAGGCT CAGGACTTAG

CAAGAAGTTA TGGAATTCCT TTTATTGAAA CATCAGCAAA GACAAGACAG

GGTGTTGATG ATGCCTTCTA TACATTAGTT CGAGAAATTC GAAAACATAA

AGAAAAGATG AGCAAAGATG GTAAAAAGAA GAAAAAGAAG TCAAAGACAA

AGTGTGTAATTATGTAA (SEQ ID NO: 16)

An exemplary amino acid sequence and corresponding nucleic acid coding sequence for human N-Ras (GENBANK: NM_002524.4 GI: 334688826) are shown below.

MTEYKLVWG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQWIDGET

CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNSKSF ADINLYREQI

KRVKDSDDVP MVLVGNKCDL PTRTVDTKQA HELAKSYGIP FIETSAKTRQ

GVEDAFYTLV REIRQYRMKK LNSSDDGTQG CMGLPCWM (SEQ ID NO: 17)

ATGACTGAGT ACAAACTGGI ¹ GGTGGTTGGA GCAGGTGGTG TTGGGAAAAG CGCACTGACA ATCCAGCTAA TCCAGAACCA CTTTGTAGAT GAATATGATC CCACCATAGA GGATTTCTTAC AGAAAACAAG TGGTTATAGA TGGTGAAACC TGTTTGTTGG ACATACTGGA TACAGCTGGA CAAGAAGAGT ACAGTGCCAT GAGAGACCAA TACATGAGGA CAGGCGAAGG CTTCCTCTGT GTATTTCCA TCAATAATAG CAAGTCATTT GCGGATATTA ACCTCTAGAG GGAGCAGATT AAGCGAGTAA AAGACTCGGA TGATGTACCT ATGGTGCTAG TGGGAAAACAA GTGTGATTTG CCAACAAGGA CAGTTGATAC AAAACAAGCC CACGAACTGG CCAAGAGTTA CGGGATTCCA TTCATTGAAA CCTCAGCCAA GACCAGACAG GGTGTTGAAG ATGCTTTTTA CACACTGGTA AGAGAAATAC GCCAGTACCG AATGAAAAAA CTCAACAGCA GTGATGATGG GACTCAGGGT TGTATGGGAT

TGCCATGTGT GGTGATGTAA (SEQ ID NO: 18)

An exemplary amino acid sequence and corresponding coding sequence for a human H-Ras nucleic acid (GENBANK: CR536579.1 GI: 49168641) are shown below.

- 16 040014

MTEYKLVWG AGGVGKSALI ¹ IQLIQNHFVD EYDPTIEDSY RKQWIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHQYREQI KRVKDSDDVP MVLVGNKCDL AARTVESRQA QDLARSYGIP YIETSAKTRQ GVEDAFYTLV REIRQHKLRK LNPPDESGPG CMSCKCVLS (SEQ ID NO:19) ATGACGGAAT ATAAGCTGGI ¹ GGTGGTGGGC GCCGGCGGTG TGGGCAAGAG TGCGCTGACC ATCCAGCTGA TCCAGAACCA CTTTGTGGAC GAATACGACC CGAGTATAGA GGATTCCTAC CGGAAGCAGG TGGTCATTG TGGGGAGACG TGCCTGTTGG ACATCCTGGA TACCGCCGGC CAGGAGGAGT ACAGCGCCAT GCGGGACCAG TACATGCGCA CCGGGGAGGG CTTCCTGTGT GTGTTTCCA TCAACAACAC CAAGTCTTTT GAGGACATCC ACGAGTAGAG GGAGCAGATC AAACGGGTGA AGGACTCGGA TGACGTGCCCC ATGGTGCTGG TGGGGAACAA GTGTGACTG GCTGCACGCA CTGTGGAATC TCGGCAGGCT CAGGACCTCG CCCGAAGCTA CGGCATCCCC TACATCGAGA CCTCGGCCAA GACCCGGCAG GGAGTGGAGG ATGCCTTCTA CACGTTGGTG CGTGAGATCC GGCAGCACAAA GCTGCGGAAG CTGAACCCTC CTGATGAGAG TGGCCCCGGC TGCATGAGCT

GCAAGTGTGT GCTCTCCTGA (SEQ ID NO:20)

Ras isoforms are farnesylated.

Farnesyl transferase (FTase) plays critical roles in post-translational modifications of Ras proteins. A way to interfere with Ras function is to inhibit FTase, an enzyme that attaches a 15-carbon isoprenyl group to Ras proteins, through farnesyl transferase inhibitors (FTIs). FTIs are a class of biologically active anticancer drugs that inhibit farnesylation of a wide range of target proteins, including Ras. FTIs block Ras activation through FTase inhibition, ultimately leading to cell growth arrest. Thus, FTIs were predicted to be effective drugs for the treatment of malignant tumors.

Thirty percent of all cancers express oncogenically activated Ras. The high prevalence of mutant Ras, found in 30% of all human cancers, makes this pathway an attractive target for anticancer drug development. Ras mutation(s) leading to a constitutively active RAS pathway was originally predicted to serve as a biomarker for patient response to FTI, based on preclinical evidence that FTIs can block RAS-transformed cells. (Raponi et al., Blood 111:2589-96 (2008)). Contrary to the common understanding described herein, there are unexpected findings that cancer patients having wild-type K-Ras and N-Ras are more responsive to FTI treatment compared to patients having mutant K-Ras or N -Ras, and that selection of cancer patients based on Ras mutation status may improve the overall response rate to FTI treatments such as tipifarnib treatment.

As used herein, the term Ras mutation refers to an activating mutation in the ras gene or Ras protein. A Ras mutation can refer to either a genetic change in the DNA sequence of one of the ras genes resulting in the activation of the corresponding Ras protein, or a change in the amino acid sequence of the Ras protein resulting in its activation. Thus, the term Ras mutation, as used herein, does not include a change in the ras gene that does not result in the activation of the Ras protein, or a change in the Ras protein sequence that does not result in its activation. Accordingly, a sample or subject that does not have any Ras mutation as used herein can still have a mutation in the ras gene that does not affect the activity of the Ras protein, or a mutation that disrupts the activity of the Ras protein, or have a mutation in the Ras protein that does not affect its activity, or a mutation that disrupts its activity. A sample or subject may have multiple copies of the ras gene. A sample or subject may also have both a wild-type Ras protein and a mutant Ras protein. As used herein, a sample or subject having a Ras mutation may also have a copy of the wild-type ras gene and/or wild-type Ras protein. sample or

- 17 040014 a subject defined as having wild-type Ras, as used herein, refers to a specimen or subject that has only the wild-type ras gene and wild-type Ras protein, and does not have Ras mutations. Accordingly, a sample or subject defined as having wild-type K-Ras, as used herein, refers to a sample or subject that has only the wild-type kras gene and wild-type K-Ras protein, and does not have K-Ras mutations. A sample or subject defined as having wild-type N-Ras, as used herein, refers to a sample or subject that has only the wild-type nras gene and wild-type N-Ras protein, and does not have N-Ras mutations.

The Ras protein may be K-Ras, N-Ras, H-Ras, or any combination thereof. K-Ras may be K _A -Ras, KB-Ras, or both. In some embodiments, the mutation is a missense mutation that locks the Ras protein in its GTP-bound activated state. In some embodiments, the mutation results in an amino acid substitution at one or more of codons 12, 13, 61 of the Ras protein.

In some embodiments, the Ras mutation is a K-Ras mutation. In some embodiments, the K-Ras mutation is a mutation in K _A -Ras, K _B -Ras, or both. The K-Ras mutation may include at least one mutation in a codon selected from the group consisting of G12, G13 and Q61 of K _A -Ras, KB-Ras, or both. In some embodiments, the KA-Ras mutation may include at least one mutation selected from the group consisting of amino acid substitutions

G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C, G13D,

G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V.

In some embodiments, the K _B -Ras mutation may include at least one mutation selected from the group consisting of amino acid substitutions

G12C, G12D, G12A, G12V, G12S, G12F, G12R,

G12N, G13C, G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V.

In some embodiments, the Ras mutation is an N-Ras mutation. In some embodiments, the N-Ras mutation may include at least one mutation in a codon selected from the group consisting of G12, G13, G15, G60, and Q61. In some embodiments, the N-Ras mutation may include at least one mutation in a codon selected from the group consisting of G12, G13, and Q61. In some embodiments, the N-Ras mutation may include at least one mutation selected from the group consisting of amino acid substitutions

G12C, G12D, G12F, G12S, G12A, G12V, G12R,

G13C, G13R, G13A, G13D, G13V, G15W, G60E, Q61P, Q61L, Q61R,

Q61K, Q61H and Q61E.

In some embodiments, the Ras mutation is an H-Ras mutation. In some embodiments, the H-Ras mutation may include at least one mutation in a codon selected from the group consisting of G12, G13, and Q61. In some embodiments, the NRas mutation may include at least one mutation selected from the group consisting of G12R, G12V, G13C, G13R, Q61L, and Q61R amino acid substitutions.

2. Farnesyl transferase inhibitors for the treatment of malignant tumors.

2.1. farnesyl transferase inhibitors.

Provided herein are methods for treating cancer using FTI in a selected cancer patient or in a selected population of cancer patients. Representative FTIs roughly belong to two classes (Shen et al., Drug Disc. Today 20:2 (2015)). FTIs in the first class have a farnesyl diphosphate (FPP) backbone. For example, FPP analogues with a malonic acid (Ta) group have been reported to be FPP competitors (Duez, S. et al., Bioorg. Med. Chem. 18:543-556(2010)). In addition, imidazole-containing derivatives linked via an acidic substituent and a peptidyl chain have also been synthesized as bisubstrate FTIs, and the developed bisubstrate inhibitors have better affinity than FPP. FTIs in the second class are peptidomimetic molecules that can be divided into two groups, namely thiol and nonthiol FTIs. Against thiol FTIs, for example L-739749, a selective FTI peptidomimetic, has shown potent antitumor activity in nude mice without systemic toxicity (Kohl, N. E. et al., PNAS 91:9141-9145 (1994)). In addition, a variety of thiol inhibitors have also been developed, such as tripeptidyl FTIs (Lee, H-Y. et al., Bioorg. Med. Chem. Lett. 12:1599-1602 (2002)).

For nonthiol FTIs, heterocycles have thus been widely used to replace thiol groups for contact with the zinc ion at the binding site. According to the structures of the pharmacophore groups, non-thiol FTIs can be divided into three classes. The first class is characterized by various mo

- 18 040014 nocyclic rings such as L-778123, FTI in phase I clinical trials for solid tumors and lymphoma. L-778123 binds at the CAAX peptide site and competes with the CAAX substrate farnesyl transferase. The second class is represented by tipifarnib in phase III studies and BMS214662 in phase III studies consisting of a variety of monocyclic rings and bicyclic rings (Harousseau et al., Blood 114:1166-1173 (2009)). A representative class 3 inhibitor is lonafarnib, which is active in both Ras-dependent and non-Ras-dependent malignancies and is in phase III clinical trials to combat carcinoma, leukemia, and myelodysplastic syndrome. Lonafarnib is a tricyclic core FTI containing a central seven-membered ring fused to two six-membered aromatic rings.

Thus, FTIs as described herein can take many forms but share the necessary inhibitory function of interfering with or reducing farnesylation of proteins involved in cancer and proliferative diseases.

Numerous FTIs are within the scope of the invention and include those described in US Patent Nos.

5976851; 5972984; 5972966;

5968965; 5968952; 6187786; 6169096; 6037350; 6177432; 5965578; 5965539; 5958939; 5939557; 5936097; 5891889; 5889053; 5880140; 5872135; 5869682; 5861529; 5859015; 5856439; 5856326; 5852010; 5843941; 5807852; 5780492; 5773455; 5767274; 5756528; 5750567; 5721236; 5700806; 5661161; 5602098; 5585359; 5578629; 5534537; 5532359; 5523430; 5504212; 5491164; 5420245; and 5238922 G

The entire contents of which are therefore provided by reference.

FTIs within the scope of the invention also include those described in Thomas et al., Biologies 1: 415-424 (2007); Shen et al., Drug Disc. Today 20:2 (2015); Appels et al., The Oncologist 10:565-578(2005), the entire contents of which are hereby incorporated by reference.

In some embodiments, the FTIs include arglabin (i.e., 1(R)-10-epoxy-5(S),7(S)-guai3(4),11(13)-diene-b,12-olide described in WO 98/28303 (NuOncology Labs)); peryl alcohol as described in WO 99/45912 (Wisconsin Genetics); SCH-66336 (lonafarnib) i.e. (+)-(R)-4-[2-[4-(3,10-dibromo-8chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridine-11 -yl)piperidin-1-yl]-oxoethyl]piperidine1-carboxamide described in U.S. Patent No. 5874442 (Schering); L778123 i.e. 1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinon described in WO 00/01691 (Merck); L739749, i.e. the 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl]-pentyloxy-3-phenylpropionylmethionine sulfone compound described in WO 94/10138 (Merck); FTI-277 i.e. methyl {N-[2-phenyl-4-N[2(R)-amino3-mercaptopropylamino]benzoyl]}methionate (Calbiochem); L744832, i.e. 1-methylethyl ester (2S)-2-[[(2S)-2-[(2S, 3S)-2-[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy] -1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)butanoic acid (Biomol International L.P.); CP-609,754 (Pfizer), i.e., (R)-6-[(4-chlorophenyl)-hydroxyl-(1-methyl-1-H-imidazol-5-yl)methyl]-4-(3- ethynylphenyl)-1-methyl-2(1 H)-quinolinone and (R)-6-[(4-chlorophenyl)-hydroxyl-(3-methyl-3-H-imidazol-4-yl)methyl]-4- (3-ethynylphenyl)-1-methyl-2-(1H)-quinolinone; R208176 (Johnson & Johnson), i.e. JNJ-17305457, or (R)-1-(4-chlorophenyl)-1-[5-(3-chlorophenyl)tetrazolo[1,5-a]quinazolin-7-yl]-1-(1-methyl-1H- imidazol-5-yl)methanamine; AZD3409 (AstraZeneca), i.e. (S)-isopropyl 2-(2-(4-fluorophenethyl)-5-((((2S,4S)-4-(nicotinoylthio)pyrrolidin-2-yl)methyl)amino)benzamido)-4-(methylthio) butanoate; BMS 214662 (Bristol-Myers Squibb), i.e. (R)-2,3,4,5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulfonyl)1H-1,4-benzodiazepine-7-carbonitrile described in WO 97/30992 (Bristol Myers Squibb) and the Pfizer compounds (A) and (B) described in WO 00/12498 and WO 00/12499.

In some embodiments, FTIs are non-peptide, so-called small molecule drugs, such as quinolines or quinoline derivatives, including 7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-2, 3-dihydro-o-1H,5H-benzo[y]quinolizin-5-one, 7-(3-chlorophenyl)-9-[(4-chlorophenyl)-1H-imidazol-1-ylmethyl]-1,2 -dihydro-o-4H-pyrrolo[3,2,1-y]quinolin-4-one, 8[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl),methyl]-6- (3-chlorophenyl)-1,2-dihydro-4H-pyrrolo[3,2,1p]quinolin-4-one and 8-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl) methyl]-6-(3-chlorophenyl)-2,3-dihydro1H,5H-benzo[p]quinolizin-5-one.

Tipifarnib is a non-peptidomimetic FTI (Thomas et al., Biologies 1: 415-424 (2007)). It is a derivative of 4,6-disubstituted-1-methylquinolin-2-one ((B)-6[amino(4-chlorophenyl)(1-methyl-1 H-imidazol-5-yl)methyl]-4-( 3-chlorophenyl)-1-methyl-2(1 H)-quinolinone)) obtained by optimizing the quinolone leader compound identified in the compound library screening. Tipifarnib competitively inhibits the CAAX FTase peptide binding site and is an unusually potent and highly selective inhibitor of farnesylation. Tipifarnib is not a geranylgeranyltransferase I inhibitor. Tipifarnib has a controlled safety profile as monotherapy, is acceptably well tolerated in humans, and requires twice-daily dosing to achieve effective plasma concentrations.

Tipifarnib is synthesized by condensation of the 1-methylimidazole anion with a 6-(4chlorobenzoyl)quinolone derivative, followed by dehydration. The quinolone intermediate is prepared in four steps by N-phenyl-3-(3-chlorophenyl)-2-propenamide cyclization, acylation, oxidation and N-methylation. Tipifarnib has been identified from the Janssen ketoconazole and retinoic acid catabolism programs as a key structural feature in Ras prenylation. Tipifarnib is a potent in vitro FTase inhibitor and is active when administered orally in a variety of animal models. Tipifarnib activity as a monotherapy was observed in a population of unselected tumors (AML, MDS/CMML, urothelial cancer, breast cancer, PTCL/CTCL), although the phase III clinical trial failed to show an improvement in overall survival.

In some embodiments, provided herein is a method for treating cancer in a subject using an FTI or a pharmaceutical composition comprising an FTI, or selecting a patient with a cancer for treatment with an FTI. Pharmaceutical compositions provided herein contain therapeutically effective amounts of FTI and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the FTI is tipifarnib; arglabin; peryl alcohol; lonafarnib (SCH-66336); L778123; L739749; FTI-277; L744832; R208176; BMS 214662; AZD3409; or SR-609,754. In some embodiments, the FTI is tipifarnib.

2.2. Compositions.

FTI formulations may be prepared in the form of suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration, or in the form of sterile solutions or suspensions for ophthalmic or parenteral administration. , as well as preparations for transdermal patches and dry powder inhalers. Typically, FTIs are formulated into pharmaceutical compositions using technologies and methods well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, Seventh Edition 1999).

In the compositions, effective concentrations of FTI and pharmaceutically acceptable salts are mixed with a suitable pharmaceutical carrier or vehicle. In specific embodiments, the FTI concentrations in the compositions are effective to deliver an amount that, upon administration, treats, prevents, or alleviates one or more of the symptoms and/or progression of cancers, including hematologic cancers and solid tumors.

The compositions can be formulated for administration in a single dose. To formulate the composition, a weighed fraction of the FTI is dissolved, suspended, dispersed, or otherwise mixed with the selected vehicle at an effective concentration such that the condition being treated is alleviated or alleviated. Pharmaceutical carriers or bases suitable for administering the FTIs provided herein include any such carriers known to those skilled in the art to be suitable for a particular route of administration.

In addition, FTI may be formulated as the sole pharmaceutically active ingredient in a composition or may be combined with other active ingredients. Liposomal suspensions, including tissue-targeted liposomes, such as tumor-targeted liposomes, may also be useful as pharmaceutically acceptable carriers. They can be obtained in accordance with methods known to specialists in this field. For example, liposome formulations can be prepared as is known in the art. Briefly, liposomes such as multilamellar vesicles (MLVs) can be prepared by drying egg phosphatidylcholine and brain phosphatidylserine (7:3 molar ratio) on the inside of the vial. A solution of the FTI provided herein in divalent cation-free phosphate buffered saline (PBS) is added and the vial is shaken until the lipid film is dispersed. The resulting vesicles are washed to remove all non-encapsulated compounds, pelleted by centrifugation and then resuspended in PBS.

The FTI is included in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically beneficial effect in the absence of undesirable side effects in the patient being treated. A therapeutically effective concentration can be determined empirically by testing compounds in the in vitro and in vivo systems described herein and then extrapolating human doses from them.

The concentration of FTI in a pharmaceutical composition may depend on the rate of absorption, tissue distribution, inactivation and clearance of FTI, physicochemical characteristics of FTI, dosing schedule and amount administered, as well as other factors known to those skilled in the art. For example, the amount delivered is sufficient to alleviate one or more

- 20 040014 symptoms of malignant tumors, including hematopoietic malignant tumors and solid tumors.

In specific embodiments, a therapeutically effective dose should result in a serum concentration of the active ingredient from about 0.1 ng/mL to about 50,100 μg/mL. In one embodiment, the pharmaceutical compositions provide a dose of from about 0.001 mg to about 2000 mg of the compound per kilogram of body weight per day. Pharmaceutical unit dosage forms are prepared to provide from about 1 mg to about 1000 mg, and in specific embodiments, from about 10 to about 500 mg of the desired active ingredient or combination of desired ingredients per unit dosage form.

The FTI may be administered all at once or may be divided into a number of smaller doses for administration at some time intervals. It is understood that the exact dose and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro testing data. It should be noted that the values of concentrations and doses can also be changed depending on the severity of the condition to be alleviated. In addition, it should be understood that for any particular subject, particular dosage regimens will need to be adjusted over time according to the needs of the individual and the professional judgment of the person administering or directing the administration of the compositions, and that the concentration ranges provided herein are illustrative only and are not intended to limit the scope or practice of the claimed compositions.

Thus, effective concentrations or amounts of one or more of the compounds described herein, or their pharmaceutically acceptable salts, are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical, or topical administration to prepare pharmaceutical compositions. The compounds are included in an amount effective to alleviate one or more symptoms or to treat, delay progression or prevent. The concentration of the active compound in the composition may depend on the rate of absorption, tissue distribution, inactivation and excretion of the active compound, dosing schedule, amount administered, particular formulation, as well as other factors known to those skilled in the art.

The compositions are intended to be administered by a suitable route, including, but not limited to, oral, parenteral, rectal, topical, and topical. For oral administration, capsule and tablet formulations can be prepared. The compositions are in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical administration may include any of the following: a sterile diluent such as water for injection, saline, fatty oil, polyethylene glycol, glycerin, propylene glycol, dimethylacetamide, or other synthetic solvent; antimicrobials such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates and phosphates; and tonicity agents such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, pens, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.

In cases where the FTI has insufficient solubility, methods can be used to solubilize the compounds. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as TWEEN®, or dissolution in aqueous sodium bicarbonate.

After mixing or adding the compound(s), the resulting mixture may be a solution, suspension, emulsion, or the like. The form of the resulting mixture depends on a number of factors, including the intended route of administration and the solubility of the compound in the chosen carrier or base. An effective concentration is sufficient to alleviate the symptoms of the disease, disorder, or condition being treated, and may be empirically determined.

Pharmaceutical compositions are presented for administration to humans and animals in unit dosage forms such as tablets, capsules, pills, powders, granules, sterile solutions or suspensions for parenteral administration, and solutions or suspensions for oral administration, and oil-in-water emulsions containing suitable amounts compounds or their pharmaceutically acceptable salts. The pharmaceutical therapeutically active compounds and their salts are formulated and administered in unit dosage forms or multiple dosage forms. Unit dosage forms, as used herein, refer to physically discrete units suitable for human and animal subjects, and individually packaged as is known in the art. Each unit dose contains a predetermined amount of a therapeutically active compound sufficient to provide the desired therapeutic effect, in association with the necessary pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. Unit dosage forms may be administered in fractions or pluralities. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container for administration in the form of divided unit doses. Examples of multiple dosage forms include vials, tablet or capsule bottles, or pint or gallon bottles. Thus, a multiple dosage form is a plurality of unit doses not separated by packaging.

Sustained release formulations may also be obtained. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the compound provided herein, where the matrices are in the form of molded articles such as films or microcapsules. Examples of sustained release matrices include iontophoresis patches, polyesters, hydrogels (e.g., poly(2hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-O-(-)-3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow release of molecules for over 100 days, specific hydrogels release proteins for shorter periods of time. When encapsulated compounds remain in the body for a long time, they can denature or aggregate as a result of exposure to humidity at 37° C., resulting in a loss of biological activity and possible changes in their structure. It is possible to develop rational ways of stabilization, depending on the mechanism of action involved. For example, if the mechanism of aggregation is found to be the formation of intermolecular S--S bonds through thio-disulfide exchange, stabilization can be accomplished through modification of sulfhydryl residues, lyophilization from acidic solutions, control of moisture content, use of suitable additives, and development of specific polymer matrix formulations.

It is possible to prepare dosage forms or compositions containing the active ingredient in the range of 0.005-100%, with the addition of the remainder using a non-toxic carrier. For oral administration, a pharmaceutically acceptable non-toxic composition is prepared by incorporating any of the commonly used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talc, cellulose derivatives, croscarmellose sodium, glucose, sucrose, magnesium carbonate, or sodium saccharin. Such formulations include solutions, suspensions, tablets, capsules, powders, and sustained release formulations such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters. , polylactic acid and others. Methods for preparing these compositions are known to those skilled in the art. Provided compositions may contain about 0.001-100% of the active ingredient, in specific embodiments, about 0.1-85% or about 75-95%.

FTI or pharmaceutically acceptable salts thereof can be prepared with carriers that protect the compound from rapid elimination from the body, such as sustained release formulations or coatings.

The composition may include other active compounds to obtain the desired combination of properties. The compounds provided herein, or their pharmaceutically acceptable salts as described herein, may also be administered with another pharmacological agent generally known in the art to be of value in the treatment of one or more of the diseases or medical conditions. referred to herein above, such as diseases associated with oxidative stress.

The lactose-free compositions provided herein may contain excipients well known in the art and listed, for example, in United States Pharmacopeia (USP) SP (XXI)/NF (XVI). Typically, lactose-free compositions contain the active ingredient, binder/filler and lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Exemplary lactose-free dosage forms contain the active ingredient, microcrystalline cellulose, pregelatinized starch and magnesium stearate.

Also included are anhydrous pharmaceutical compositions and dosage forms containing the compound provided herein. For example, the addition of water (eg 5%) is widely accepted in the pharmaceutical field as a simulation of long term storage to determine characteristics such as shelf life or stability of formulations over time. See, for example, Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pp. 379-80.

- 22 040014

In fact, water and heat increase the decomposition of some compounds. Thus, the effect of water on the composition can be of great importance, since moisture and/or moisture is usually taken into account during the manufacture, handling, packaging, storage, transport and use of compositions.

The anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture ingredients and under low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms containing lactose and at least one active ingredient containing a primary or secondary amine are anhydrous if significant contact with moisture and/or moisture is expected during manufacture, packaging and/or storage.

The anhydrous pharmaceutical composition should be prepared and stored in a manner that maintains its anhydrous nature. Accordingly, anhydrous compositions are packaged using materials known to prevent exposure to water so that they can be included in appropriate formulary sets. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (eg, vials), blister packs, and strip packs.

Oral pharmaceutical dosage forms are solid, gel or liquid. Solid dosage forms are tablets, capsules, granules and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets that can be enteric-coated, sugar-coated, or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

In specific embodiments, the formulations are solid dosage forms such as capsules or tablets. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of a similar nature: a binder; diluent; a disintegrant; lubricant; glidant; sweetener; flavoring agent.

Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia gum, gelatin solution, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium, and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include sodium croscarmellose, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar, and carboxymethylcellulose. Coloring agents include, for example, any of the approved certified water-soluble dyes FD and C, mixtures thereof; and water-insoluble FD and C dyes suspended in alumina hydrate. Sweeteners include sucrose, lactose, mannitol and artificial sweeteners such as saccharin and any amount of spray-dried flavors. Flavoring agents include natural flavors extracted from plants, such as fruits, and synthetic blends of compounds that provide a pleasant sensation, such as, but not limited to, peppermint and methyl salicylate. Humectants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Antiemetic coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

When the unit dosage form is a capsule, it may contain, in addition to the material of the above type, a liquid carrier such as a fixed oil. In addition, the unit dosage forms may contain various other materials that modify the physical form of the unit dosage form, such as sugar coatings and other enteric agents. The compounds may also be administered in the form of an elixir component, suspension, syrup, wafer, openable capsule, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetener and specific preservatives, colors, colors and flavors.

Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents and wetting agents. Enteric-coated tablets, due to the enteric coating, withstand the action of gastric acid and dissolve or disintegrate in the neutral or alkaline environment of the intestine. Sugar-coated tablets are compressed tablets coated with various layers of pharmaceutically acceptable substances. Film-coated tablets are compressed tablets coated with a polymer or other suitable coating. Multi-compressed tablets are compressed tablets made by more than one compression cycle using the aforementioned pharmaceutically acceptable substances. Coloring agents can also be used in the above dosage forms. Flavorings and sweeteners are used in compressed tablets, sugar-coated, multi-compressed and chewable tablets. Flavorings and sweeteners are particularly useful in making chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water emulsions or water-in-oil emulsions.

Elixirs are transparent sweetened water-alcohol preparations. Pharmaceutically acceptable carriers for use in elixirs include solvents. Syrups are concentrated aqueous solutions of sugar, such as sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small beads in another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers and preservatives. Suspensions employ pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules for reconstitution into liquid oral dosage forms include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used in effervescent granules for reconstitution into liquid oral dosage forms include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propyl paraben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include gelatin, gum arabic, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, veegum, and gum arabic. Diluents include lactose and sucrose. Sweeteners include sucrose, syrups, glycerin, and artificial sweeteners such as saccharin. Humectants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified FD and C water soluble dyes and mixtures thereof. Flavoring agents include natural flavors extracted from plants, such as fruits, and synthetic mixtures of compounds that impart a pleasant taste sensation.

For a solid dosage form, a solution or suspension, for example in propylene carbonate, vegetable oils or triglycerides, is encapsulated in a gelatin capsule. Such solutions and their preparation and encapsulation are described in US Pat. Nos. 4,328,245; 4409239; and 4,410,545. For a liquid dosage form, a solution, such as polyethylene glycol, can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, such as water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate), and other similar vehicles, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other formulations that may be used include, but are not limited to, formulations containing a compound provided herein, dialkylated mono- or polyalkylene glycol, including but not limited to 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol dimethyl ether -350, polyethylene glycol-550 dimethyl ether, polyethylene glycol-750 dimethyl ether, where 350, 550 and 750 refer to the approximate average molecular weight of polyethylene glycol, and one or more antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate , vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholic solutions, including a pharmaceutically acceptable acetal. The alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents containing one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, lower alkyl aldehyde di(lower alkyl) acetals such as acetaldehyde diethyl acetal.

In all embodiments, tablet and capsule formulations can be coated, as is known to those skilled in the art, to modify or delay the release of the active ingredient. Thus, for example, they can be coated using conventional enteric coatings such as phenyl salicylate, waxes and cellulose acetate phthalate.

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Parenteral administration, usually characterized by injection, subcutaneous, intramuscular, or intravenous, is also provided herein. Formulations suitable for injection may be prepared in conventional forms, in the form of liquid solutions or suspensions, in solid forms suitable for dissolution or suspension in a liquid prior to injection, or in the form of emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic excipients such as wetting agents or emulsifiers, pH buffering agents, stabilizers, solubility enhancers and other such agents such as sodium acetate, sorbitan monolaurate. , triethanolamine oleate and cyclodextrins. Implantation of a sustained release or delayed release system so as to maintain a constant dosage level is also contemplated herein. Briefly, the compound provided herein is dispersed in a solid internal matrix, e.g. polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of acrylic and methacrylic acid esters, collagen, cross-linked polyvinyl alcohol and cross-linked partially hydrolysed polyvinyl acetate, which is surrounded by an outer polymeric membrane, such as polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl copolymers l chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, ionomeric polyethylene terephthalate, butyl rubber, epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, which is insoluble in body fluids. The compound diffuses through the outer polymeric membrane during the release rate control step. The percentage of active compound contained in such parenteral compositions depends to a large extent on its particular nature, as well as on the activity of the compound and the needs of the subject.

Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administration. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately before use, including tablets for subcutaneous preparations, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with the base immediately before use, and sterile emulsions. Solutions may be either aqueous or non-aqueous.

For intravenous administration, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickeners and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous bases, non-aqueous bases, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, sequestering or chelating agents, and other pharmaceutically acceptable agents.

Examples of aqueous bases include sodium chloride injection, Ringer's solution for injection, isotonic dextrose solution for injection, sterile water for injection, Ringer's solution with dextrose and lactate for injection. Non-aqueous parenteral bases include vegetable fatty oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobials at bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multi-dose containers, including phenols or cresols, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl esters of p-hydroxybenzoic acid, thimerosal, benzalkonium chloride and benzethonium chloride . Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifiers include Polysorbate 80 (TWEEN® 80). Means that sequester or chelate metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol as water-miscible bases and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The FTI concentration is adjusted so that the injection provides an effective amount of the compound to produce the desired pharmacological effect. The exact dose depends on the age, body weight and condition of the patient or animal, as is known in the art. A single dose of parenteral preparations is packaged in an ampoule, vial or syringe with a needle. All preparations for parenteral administration must be sterile as known and practiced in the art.

By way of illustration, intravenous or intra-arterial infusion of a sterile aqueous solution containing FTI is an effective route of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing the active material, injected as needed to produce the desired pharmacological effect.

Injectable preparations are designed for local and systemic administration. Typically, a therapeutically effective dose is to contain a concentration of at least about 0.1% wt./wt. up to about 90% wt./wt. or more, for example more than 1% wt./wt. the active compound in the tissue(s) being treated. The active ingredient may be administered all at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the exact dose and duration of treatment is a function of the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro testing data. It should be noted that the values of concentrations and doses can also be changed depending on the age of the individual being treated. In addition, it should be understood that for any particular subject, particular dosing regimens will need to be adjusted over time according to the needs of the individual and the professional judgment of the person administering or directing the administration of the formulations, and that the concentration ranges provided herein are illustrative only. and are not intended to limit the scope or practice of the claimed formulations.

The FTI may be suspended in finely divided or other suitable form, or may be derivatized to provide a more soluble active product or to form a prodrug. The form of the resulting mixture depends on a number of factors, including the intended route of administration and the solubility of the compound in the chosen carrier or base. The effective concentration is sufficient to relieve the symptoms of the condition and can be determined empirically.

Also of interest herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be diluted and formulated as solids or gels.

A sterile lyophilized powder is prepared by dissolving the FTI provided herein, or a pharmaceutically acceptable salt thereof, in a suitable solvent. The solvent may contain a stability enhancing excipient or other pharmacological component of the powder or diluted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate, or other similar buffer known to those skilled in the art, in one embodiment, at approximately neutral pH. By subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, the desired formulation is obtained. As a rule, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single dose (including but not limited to 10-1000 mg or 100-500 mg) or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions such as from about 4° C. to room temperature.

By diluting this lyophilized powder with water for injection, a formulation for parenteral use is obtained. For dilution add about 1-50 mg, about 5-35 mg or about 9-30 mg of lyophilized powder per ml of sterile water or other suitable vehicle. The exact amount depends on the selected compound. This amount can be determined empirically.

Mixtures for topical administration are prepared as described for topical or systemic administration. The resulting mixture may be a solution, suspension, emulsion, or the like, and are in the form of creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, sprinklers, sprays, suppositories. , dressings, skin patches, or any other formulation suitable for topical administration.

The FTI or a pharmaceutical composition containing the FTI can be formulated as an aerosol for topical administration, such as inhalation (see, for example, US Pat. especially asthma). These formulations for administration to the respiratory tract may be in the form of an aerosol or inhaler solution, or in the form of a fine powder for inhalation, alone or in combination with an inert carrier such as lactose. In such a case, the composition particles may have a diameter of less than 50 or less than 10 microns.

FTI or a pharmaceutical composition containing FTI can be formulated for local or

- 26 040014 local administration, such as topical administration to the skin and mucous membranes, such as in the eye, in the form of gels, creams and lotions and for administration to the eye or for intracisternal or intraspinal administration. Topical administration is envisaged for transdermal delivery, as well as for administration to the eyes or mucous membranes, or for inhalation therapies. Nasal solutions of the active compound may also be administered, alone or in combination with other pharmaceutically acceptable excipients. These solutions, particularly those intended for ophthalmic use, can be formulated in the form of 0.01-10% isotonic solutions, pH about 5-7, using suitable salts.

Other routes of administration, such as the use of transdermal patches and rectal administration, are also contemplated herein. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories as used herein refer to solid bodies for insertion into the rectum that melt or soften at body temperature to release one or more pharmaceutically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are bases or carriers and agents for increasing the melting point. Examples of bases include cocoa butter (theobromine oil), glycerol-gelatin, carbovax (polyoxyethylene glycol), and suitable mixtures of mono-, di- and triglycerides of fatty acids. Combinations of various bases can be used. Means for increasing the melting point of suppositories include spermaceti and wax. Rectal suppositories can be obtained by pressing or by molding. An exemplary weight of a rectal suppository is about 2-3 g. Tablets and capsules for rectal administration are made using the same pharmaceutically acceptable materials and by the same methods as for oral formulations.

The FTI or pharmaceutical composition comprising the FTIs provided herein can be administered via controlled release methods or via delivery devices well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in US Patent Nos.

3916899; 3536809; 3598123; and 4008719, 5674533, 5059595, 5591767,

5120548, 5073543, 5639476, 5354556, 5639480, 5733566, 5739108,

5891474, 5922356, 5972891, 5980945, 5993855, 6045830, 6087324,

6113943, 6197350, 6248363, 6264970, 6267981, 6376461,6419961,

6589548, 6613358, 6699500 and 6740634, the contents of each of which are given herein by reference. Such dosage forms can be used to provide sustained or controlled release of FTI using, for example, hydropropyl methylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof to provide the desired release profile in varying proportions. . Suitable controlled release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein.

All controlled release pharmaceutical products have the common goal of improving drug therapy over that achieved by their non-controlled equivalents. In one embodiment, the use of optimally designed controlled release formulations in medical treatment is characterized by the minimum amount of drug used to cure or control a condition in a minimum amount of time. In specific embodiments, the benefits of controlled release formulations include extended drug potency, reduced dosing frequency, and improved patient compliance. In addition, controlled release formulations can be used to influence the time of onset of action or other characteristics, such as blood levels of the drug, and may thus influence the occurrence of side (eg, adverse) effects.

Most controlled release formulations are designed to initially release an amount of drug (active ingredient) that quickly produces the desired therapeutic effect, and gradually and continuously release other amounts of drug to maintain that level of therapeutic effect over an extended period of time. To maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

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Controlled release of the active ingredient can be stimulated through a variety of conditions, including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

In specific embodiments, FTI can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other routes of administration. In one embodiment, a pump may be used (see, Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl J. Med 321:574 (1989) In another embodiment, polymeric materials can be used In another embodiment, the controlled release system can be placed in the vicinity of the therapeutic target, i.e., thus only a fraction of the systemic dose is needed (See, for example, Goodson, Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984).

In some embodiments, the controlled release device is administered to a subject in the vicinity of a site of unacceptable immune activation or tumor. Other controlled release systems are discussed in Langer's review (Science 249:1527-1533 (1990). F can be dispersed in a solid internal matrix, e.g. , polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone-carbonate copolymers, hydrophilic polymers such as acrylic and methacrylic acid ester hydrogels, collagen, cross-linked polyvinyl alcohol and cross-linked partially hydrolyzed polyvinyl acetate, which is surrounded by an outer polymer membrane, e.g. polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorine polyvinyl chloride, vinyl chloride-vinyl acetate copolymers, vinylidene chloride, ethylene-propylene, ionomeric polyethylene terephthalate, butyl rubber, epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, which is insoluble in body fluids. The active ingredient then diffuses through the outer polymeric membrane in a release rate control step. The percentage of the active ingredient contained in such parenteral compositions depends to a large extent on its specific nature, as well as on the needs of the subject.

An FTI or an FTI pharmaceutical composition may be packaged in the form of articles containing a packaging material, a compound, or a pharmaceutically acceptable salt thereof provided herein, which is used to treat, prevent, or alleviate one or more symptoms or progression of cancers, including hematologic malignancies and solid tumors, and a label indicating that the compound, or a pharmaceutically acceptable salt thereof, is used to treat, prevent, or alleviate one or more symptoms or progression of a malignant tumor, including hematologic malignancies and solid tumors.

The products shown in this document contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those skilled in the art. See, for example, US Pat. packaging materials suitable for the selected formulation and intended route of administration and treatment. Provide a wide range of compositions of the compounds and compositions presented in this document.

2.3. Doses.

In some embodiments, a therapeutically effective amount of the FTI-containing pharmaceutical composition is administered orally or parenterally. In some embodiments, the pharmaceutical composition contains tipifarnib as an active ingredient and is administered orally in an amount of 1 to 1500 mg/kg daily, either in the form of a single dose or divided into more than one dose, or more specifically in an amount from 10 to 1200 mg/kg daily. In some embodiments, the pharmaceutical composition contains tipifarnib as an active ingredient and is administered orally at 100 mg/kg daily, 200 mg/kg daily, 300 mg/kg daily, 400 mg/kg daily, 500 mg/kg daily, 600 mg/kg daily, 700 mg/kg daily, 800 mg/kg daily, 900 mg/kg daily, 1000 mg/kg daily, 1100 mg/kg daily, or 1200 mg/kg daily. In some embodiments, the FTI is tipifarnib.

In some embodiments, FTI is administered at a dose of 200-1500 mg daily. In some embodiments, FTI is administered at a dose of 200-1200 mg daily. In some embodiments, FTI is administered at a dose of 200 mg daily. In some embodiments, FTI is administered at a dose of 300 mg daily. In some embodiments, FTI is administered at a dose of 400 mg daily. In some embodiments, FTI is administered at a dose of 500 mg daily. In some embodiments, FTI is administered at a dose of 600 mg daily. In some embodiments, FTI is administered at a dose of 700 mg daily. In some embodiments, FTI is administered at a dose of 800 mg daily. In some embodiments, FTI is administered at a dose of 900 mg daily. In some embodiments, FTI is administered at a dose of 1000 mg daily. In some embodiments, FTI is administered at a dose of 1100 mg daily. In some embodiments, FTI is administered at a dose of 1200 mg daily. In some embodiments, FTI is administered at a dose of 1300 mg daily. In some embodiments, FTI is administered at a dose of 1400 mg daily. In some embodiments, the FTI is tipifarnib.

In some embodiments, FTI is administered at a dose of 200-1400 mg b.i.d. (i.e. twice a day). In some embodiments, FTI is administered at a dose of 300-1200 mg b.i.d. In some embodiments, FTI is administered at a dose of 300-900 mg b.i.d. In some embodiments, FTI is administered at a dose of 600 mg b.i.d. In some embodiments, FTI is administered at a dose of 700 mg b.i.d. In some embodiments, FTI is administered at a dose of 800 mg b.i.d. In some embodiments, FTI is administered at a dose of 900 mg b.i.d. In some embodiments, FTI is administered at a dose of 1000 mg b.i.d. In some embodiments, FTI is administered at a dose of 1100 mg b.i.d. In some embodiments, FTI is administered at a dose of 1200 mg b.i.d. In some embodiments, the FTI is tipifarnib.

As one of skill in the art would understand, the dosage will vary depending on the dosage form used, the condition and sensitivity of the patient, the route of administration, and other factors. The exact dosage will be determined by the practitioner in light of factors related to the subject in need of treatment. The dosage and administration is adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the subject's age, weight and sex, diet, time and frequency of administration, drug combination(s), sensitivity reactions, and resistance/response to treatment. During the cycle of treatment, the daily dose can be changed. In some embodiments, the initial dose may be titrated down during the treatment cycle. In some embodiments, the initial dose may be titrated upward during the treatment cycle. The final dose may depend on the occurrence of dose-dependent toxicity and other factors. In some embodiments, FTI is administered at an initial dose of 300 mg daily and increased to a maximum dose of 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 mg daily. In some embodiments, FTI is administered at an initial dose of 400 mg daily and increased to a maximum dose of 500, 600, 700, 800, 900, 1000, 1100, or 1200 mg daily. In some embodiments, FTI is administered at an initial dose of 500 mg daily and increased to a maximum dose of 600, 700, 800, 900, 1000, 1100, or 1200 mg daily. In some embodiments, FTI is administered at an initial dose of 600 mg daily and increased to a maximum dose of 700, 800, 900, 1000, 1100, or 1200 mg daily. In some embodiments, FTI is administered at an initial dose of 700 mg daily and increased to a maximum dose of 800, 900, 1000, 1100, or 1200 mg daily. In some embodiments, FTI is administered at an initial dose of 800 mg daily and increased to a maximum dose of 900, 1000, 1100, or 1200 mg daily. In some embodiments, FTI is administered at an initial dose of 900 mg daily and increased to a maximum dose of 1000, 1100, or 1200 mg daily. Dose increases can be done all at once or in steps. For example, an initial dose of 600 mg daily can be increased to a final dose of 1000 mg daily by increasing by 100 mg per day for 4 days, or by increasing by 200 mg per day for 2 days, or by increasing by 400 mg all at once. In some embodiments, the FTI is tipifarnib.

In some embodiments, FTI is administered at a relatively high starting dose and titrated down to a lower dose, depending on the patient's response and other factors. In some embodiments, FTI is administered at an initial dose of 1200 mg daily and reduced to a final dose of 1100 mg, 1000 mg, 900 mg, 800 mg, 700 mg, 600 mg, 500 mg, 400 mg, or 300 mg daily. In some embodiments, FTI is administered at an initial dose of 1100 mg daily and reduced to a final dose of 1000, 900, 800, 700, 600, 500, 400, or 300 mg daily. In some embodiments, FTI is administered at an initial dose of 1000 mg daily and reduced to a final dose of 900, 800, 700, 600, 500, 400, or 300 mg daily. In some embodiments, FTI is administered at an initial dose of 900 mg daily and reduced to a final dose of 800, 700, 600, 500, 400, or 300 mg daily. In some embodiments, FTI is administered at an initial dose of 800 mg daily and reduced to a final dose of 700, 600, 500, 400, or 300 mg daily. In some embodiments, FTI is administered at an initial dose of 600 mg daily and reduced to a final dose of 500, 400, or 300 mg daily. Dose reductions can be done all at once or in steps. In some embodiments, the FTI is tipifarnib. For example, an initial dose of 900 mg daily can be reduced to a final dose of 600 mg daily by decreasing by 100 mg per day for 3 days or by reducing by 300 mg all at once.

The treatment cycle can have a different length. In some embodiments, the treatment cycle may be 1, 2, 3, 4, 5, 6, 7, 8 weeks, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some variants

- 29 040014 max implementation, the treatment cycle is 4 weeks. The treatment cycle may have an intermittent schedule. In some embodiments, a 2 week treatment cycle may comprise a 5 day dosing followed by a 9 day rest. In some embodiments, a 2 week treatment cycle may comprise a 6 day dosing followed by an 8 day rest. In some embodiments, a 2 week treatment cycle may comprise a 7 day dosing followed by a 7 day rest. In some embodiments, a 2 week treatment cycle may comprise an 8 day dosing followed by a 6 day rest. In some embodiments, a 2 week treatment cycle may comprise a 9 day dosing followed by a 5 day rest.

In some embodiments, FTI is administered daily for 3 out of 4 weeks in repeated 4 week cycles. In some embodiments, the FTI is administered daily in alternating weeks (one week of administration, one week of rest) in repeating 4-week cycles. In some embodiments, FTI is administered at a dose of 300 mg b.i.d. orally for 3 of 4 weeks in repeating 4-week cycles. In some embodiments, FTI is administered at a dose of 600 mg b.i.d. orally for 3 of 4 weeks in repeating 4-week cycles. In some embodiments, FTI is administered at a dose of 900 mg b.i.d. orally in alternating weeks (one week of administration, one week of rest) in repeating 4-week cycles. In some embodiments, FTI is administered at a dose of 1200 mg b.i.d. orally on alternating weeks (days 1-7 and 15-21 of repeating 28-day cycles). In some embodiments, FTI is administered at a dose of 1200 mg b.i.d. orally on days 1-5 and 15-19 of repeating 28-day cycles.

In some embodiments, use of the 900 mg tipifarnib b.i.d. regimen may be taken. in alternate weeks. In this regimen, patients are given an initial dose of 900 mg, po, b.i.d. on days 1-7 and 15-21 of the 28-day treatment cycles. In some embodiments, patients are subjected to two treatment cycles. In some embodiments, patients are subjected to three treatment cycles. In some embodiments, patients are subjected to four treatment cycles. In some embodiments, patients are subjected to five treatment cycles. In some embodiments, patients are subjected to six cycles of patient treatment. In some embodiments, the implementation of patients is subjected to seven cycles of treatment patients. In some embodiments, patients are subjected to eight treatment cycles. In some embodiments, patients are subjected to nine cycles of patient treatment. In some embodiments, patients are subjected to ten treatment cycles. In some embodiments, patients are subjected to eleven treatment cycles. In some embodiments, patients are subjected to twelve treatment cycles. In some embodiments, patients are subjected to more than twelve treatment cycles.

In the absence of uncontrolled toxicity, subjects may continue to be treated with tipifarnib for up to 12 months. The dose may also be increased to 1200 mg b.i.d. if the subject tolerates the treatment well. A 300 mg dose step reduction may also be included to control for treatment-related toxicity that occurs during treatment.

In some other embodiments, tipifarnib is administered orally at a dose of 300 mg b.i.d. daily for 21 days, followed by 1 week off, in 28-day treatment cycles (21-day schedule; Cheng DT, et al., J Mol Diagn. (2015) 17(3):251-64). In some embodiments, the 5-day dosage is in the range of 25 to 1300 mg b.i.d. followed by a 9-day rest (5-day schedule; Zujewski J., J Clin Oncol., (2000) Feb;18(4):927-41). In some embodiments, a 7-day b.i.d. is taken. dosing followed by a 7-day rest (7-day schedule; Lara PN Jr., Anticancer Drugs., (2005) 16(3):317-21; Kirschbaum MH, Leukemia., (2011) Oct;25(10): 1543-7). In a 7-day schedule, patients may be given an initial dose of 300 mg b.i.d. with a dose increase of 300 mg to a maximum planned dose of 1800 mg b.i.d.. In a study with a 7-day schedule, patients can also receive tipifarnib b.i.d. on days 1-7 and days 15-21 of 28-day cycles at doses up to 1600 mg b.i.d..

FTI can inhibit the growth of mammalian tumors when administered in a twice daily dosing schedule. The introduction of FTI in a single dose daily for one to five days can cause a marked suppression of tumor growth, lasting for at least 21 days. In some embodiments, FTI is administered in a dosage range of 50-400 mg/kg. In some embodiments, FTI is administered at 200 mg/kg. Dosing regimens for specific FTIs are also well known in the art (eg US Pat. No. 6,838,467, the entire contents of which are incorporated herein by reference). For example, suitable doses for the compounds arglabin (WO 98/28303), peryl alcohol (WO 99/45712), SCH-66336 (US patent No. 5874442), L778123 (WO 00/01691), 2(S)-[2(S )-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl]pentyloxy-3phenylpropionylmethionine sulfone (WO 94/10138), BMS 214662 (WO 97/30992), AZD3409; compounds Pfizer A and B (WO 00/12499 and WO 00/12498) are described in the descriptions of the aforementioned patents, the contents of which are given herein by reference, or known to a person skilled in the art, or can be easily determined by a person skilled in the art.

- 30 040014

For peryl alcohol, the drug may be administered at 1-4 grams per day for a 150 lb (68 kg) human patient. Preferably at 1-2 grams per day for a 150 lb (68 kg) human patient. SCH-66336 can generally be administered in a single dose of about 0.1 to 100 mg, more preferably about 1 to 300 mg, according to the particular application. Compounds L778123 and 1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2piperazinone can be administered to a human patient in an amount between about 0.1 mg/kg body weight to about 20 mg/ kg of body weight per day, preferably between 0.5 mg/kg of body weight to about 10 mg/kg of body weight per day.

Pfizer Compounds A and B can be administered at doses ranging from about 1.0 mg up to about 500 mg per day, preferably from about 1 to about 100 mg per day in single or divided (ie, multiple) doses. Therapeutic compounds are typically administered in daily doses ranging from about 0.01 to about 10 mg per kg of body weight per day, in single or divided doses. BMS 214662 can be administered in a dosage range of about 0.05 to 200 mg/kg/day, preferably at less than 100 mg/kg/day in a single dose or in 2-4 divided doses.

2.4. Methods of combined therapy.

In some embodiments, FTI treatment is carried out in combination with radiotherapy or radiation therapy. Radiotherapy includes the use of γ-radiation, x-rays and/or targeted delivery of radioactive isotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton irradiation (US Pat. Nos. 5,760,395 and 4,870,287; the entire contents of all of which are hereby incorporated by reference), and UV irradiation. It is most likely that all these factors cause a wide range of DNA damage, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance.

In some embodiments, a therapeutically effective amount of a pharmaceutical composition containing FTI is administered that effectively sensitizes the tumor in the host to radiation (US Pat. No. 6,545,020, the entire contents of which are hereby incorporated by reference). The radiation may be ionizing radiation, and in particular gamma radiation. In some embodiments, gamma rays are emitted by linacs or by radioactive isotopes. Irradiation of the tumor with radioactive isotopes can be external or internal.

The irradiation may also be X-ray irradiation. Dosing ranges for x-rays range from daily doses of 50 to 200 x-rays for long periods of time (3-4 weeks) to single doses of 2000-6000 x-rays. Dosing ranges for radioactive isotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation, and uptake by neoplastic cells.

In some embodiments, administration of the pharmaceutical composition is started up to one month, in particular up to 10 days or weeks, prior to tumor irradiation. In addition, if the irradiation of the tumor is fractional, the administration of the pharmaceutical composition is maintained in the interval between the first and last irradiation session.

The amount of FTI, radiation dose, and intervals between radiation doses depend on a series of parameters, such as the type of tumor, its location, the response of patients to chemotherapy or radiotherapy, and ultimately the physician and radiologist determine them on a case-by-case basis.

In some embodiments, the methods provided herein further comprise administering a therapeutically effective amount of a second active agent or maintenance therapy. The second active ingredient may be a chemotherapeutic agent. A chemotherapeutic agent or drug can be classified by their mechanism of activity within the cell, such as whether they can affect the cell cycle and at what stage. Alternatively, an agent can be classified based on its ability to directly cross-link DNA, intercalate into DNA, or induce chromosomal and mitotic aberrations through its effect on nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetoginins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pankriastatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterin, prednemustine, trofosfamide and uramustine; nitrosureas such as carmustine, chlorzotocin, fotemu

- 31 040014 stin, lomustine, nimustine and ranimnustine; antibiotics such as endiine antibiotics (eg calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dinemycin, including dinemycin A; bisphosphonates such as clodronate; esperamycin; as well as the chromophore neocarcinostatin and related chromophores of chromoproteins - enediin antibiotics, aclacinomysins, actinomycin, autramycin, azaserin, bleomycins, sactinomycin, carabicin, carminomycin, carcinophyllin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-oxo-norubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marsellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, rhodubocine, quelamycin , streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin and trimetrexate; purine analogs such as fludarabine, β-mercaptopurine, thiamiprin and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; adrenal suppressants such as mitotane and trilostane; folic acid compensator such as frolinic acid; aceglatone; aldofosfamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfornitin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pyrarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK; razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anvidin); urethane; vindesine; decarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); cyclophosphamide; taxoids, eg paclitaxel and docetaxel, gemcitabine; b-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantron; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (eg CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, trans-platinum, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

The second active ingredients may be large molecules (eg proteins) or small molecules (eg synthetic inorganic, organometallic or organic molecules). In some embodiments, the second active ingredient is a DNA hypomethylating agent, a therapeutic antibody that specifically binds a cancer antigen, a hematopoietic growth factor, a cytokine, an anticancer agent, an antibiotic, a cox-2 inhibitor, an immunomodulatory agent, an antithymocyte globulin, an immunosuppressive agent, a corticosteroid, or their pharmacologically active mutant or derivative.

In some embodiments, the second active ingredient is a DNA hypomethylating agent, such as a cytidine analog (eg, azacitidine) or 5-azadeoxycytidine (eg, decitabine). In some embodiments, the second active agent is a cytoreductive agent, including, but not limited to, induction therapy, topotecan, hydrea, PO etoposide, lenalidomide, LDAC, and thioguanine. In some embodiments, the second active ingredient is mitoxantrone, etoposide, cytarabine, or valspodar. In some embodiments, the second active ingredient is mitoxantrone plus valspodar, etoposide plus valspodar, or cytarabine plus valspodar. In some embodiments, the second active ingredient is idarubicin, fludarabine, topotecan, or ara-C. In some other embodiments, the second active ingredient is idarubicin plus ara-C, fludarabine plus ara-C, mitoxantrone plus ara-C, or topotecan plus ara-C. In some embodiments, the implementation of the second active substance is quinine. Other combinations of the agents mentioned above may be used and doses may be determined by the physician.

For any particular type of cancer described herein, treatments as described herein or otherwise available in the art may be used in combination with FTI treatment. For example, drugs that can be used in combination with FTI include belinostat (Beleodac®) and pralatrexate (Folotin®) marketed by Spectrum Pharmaceuticals, romidepsin (Istodax®) marketed by Celgene, and brentuximab vedotin (Adcetris®) (against ALCL) marketed by Seattle Genetics; drugs that can be used in combination with FTI include azacitidine (Vidaza®) and lenalidomide (Revlimid®) marketed by Celgene and decitabine (Dacogen®) marketed by Otsuka and Johnson &Johnson; medicines that can be used in combination with FTI

- 32 040014 against thyroid cancer include vandetanib (Caprelsa®) from AstraZeneca, sorafenib (Nexavar®) from Bayer, cabozantinib (Kometrik®) from Exelixis, and lenvatinib (Lenvima®) from Eisai.

Non-cytotoxic drugs such as pralatrexate (Folotin®), romidepsin (Istodax®), and belinostat (Beleodak®) can also be used in combination with FTI treatment.

In some embodiments, the implementation of the second active substance is an immunotherapeutic agent. In some embodiments, the second active ingredient is an anti-PD1 antibody or an anti-PDL1 antibody.

In some embodiments, it is contemplated that the second active ingredient or second drug used in combination with FTI may be administered before, during, or after FTI treatment. In some embodiments, a second active ingredient or second drug used in combination with FTI may be administered prior to treatment for FTI. In some embodiments, the second active ingredient or second drug used in combination with the FTI may be administered at the same time as the FTI treatment. In some embodiments, the implementation of the second active substance or the second drug used in combination with FTI, you can enter after treatment with FTI.

FTI treatment can also be done in combination with bone marrow transplantation. In some embodiments, FTI is administered prior to bone marrow transplantation. In other embodiments, FTI is administered after bone marrow transplantation.

3. Immunological genes as biomarkers for the treatment of FTI.

Provided herein are methods for selecting cancer patients for treatment with a farnesyl transferase inhibitor (FTI). The methods presented herein are based in part on the discovery that genotypes and expression levels of specific genes that are associated with natural killer cell (NK cell) activity correlate with the clinical benefit of FTI treatment. Specifically, genotyping of the KIR genes and HLA genes and expression levels of biomarkers including KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5 and GZMM can be used to predict the ability of a cancer patient to respond to FTI treatment. As presented herein, in addition to expression levels of individual biomarkers, the relative ratio of expression levels between specific biomarkers, such as the ratio of KIR2DS2 expression level to KIR2DL2 expression level, or the ratio of KIR2DS5 expression level to KIR2DL5 expression level, can also be used to predict a patient's ability to with cancer respond to FTI treatment. Accordingly, provided herein are methods for predicting the ability of a cancer patient to respond to FTI treatment, methods for selecting a population of cancer patients for FTI treatment, and methods for treating cancer in a subject using a therapeutically effective amount of FTI based on the genotype or expression levels of these biomarkers in sample from the patient. Also provided herein are compositions and kits for predicting the ability of a cancer patient to respond to FTI treatment.

Farnesyl transferase (FTase) plays critical roles in post-translational modifications of Ras proteins. FTIs are a class of biologically active anticancer drugs that inhibit farnesylation of a wide range of target proteins, including Ras. Ras proteins play a key role in cell growth stimulation signaling, and mutation of the ras gene results in permanent activation of the protein, ultimately leading to uncontrolled cell proliferation. The high prevalence of mutant ras genes found in 30% of all human malignant tumors makes this pathway an attractive target for the development of anticancer drugs. A way to interfere with Ras function is to inhibit FTase, an enzyme that attaches a 15-carbon isoprenyl group to Ras proteins, by FTI. FTIs block Ras activation through FTase inhibition, ultimately leading to cell growth arrest. Thus, FTIs were predicted to be effective drugs for the treatment of malignant tumors.

However, a correlation between ras mutations and FTI response has not been shown in past clinical studies (Karp et al., Blood 97:3361-3369 (2001); and US Patent Publication 20070048782)). While several early clinical trials focused on cancers with high ras mutation rates, response rates were disappointingly low in these trials. (Mesa Lancet Oncol 6:279-286 (2006); Rao et al., J Clin Oncol 22:3950-3957 (2004))

Early studies of tipifarnib, an FTI, were performed in AML patients with poor risk and no prior treatment (CTER-20, phase II), and AML patients with relapsed/refractory AML (INT-17, phase II). In a phase III study of tipifarnib, compared with best supportive care (BSC), failed to show an improvement in overall survival. Numerous genes/proteins have been associated in the literature with FTI activity (AKAP13, mDIA, etc.) (Raponi et al., Clin Cancer Res. 13:2254-60 (2007); Kamasani et al., Cancer Biology & Therapy, 6 :1418-1423 (2007)), and gene expression profiling assays in bone marrow samples from 2 AML studies

- 33 040014 (CTER-20, INT-17) identified the expression ratio of 2 genes: RASGRP1 (T-cell signaling) and ARTX (DNA repair protein) as a potential biomarker of tipifarnib activity in AML (Raponi et al., Blood. 111 :2589-96(2008)). However, a subsequent prospective study using the ratio of 2 genes in bone marrow blasts as inclusion criterion failed to show a significant clinical benefit of tipifarnib in AML (Lancet et al., Blood (ASH) 120: Abstract 1508(2012)).

The present invention has identified a variety of immunological genes as biomarkers associated with better prognosis for FTI treatment, and novel methods are presented herein for selecting patients for FTI treatment. In contrast to previously identified markers such as RASGRP1, which have been found to be associated with good prognosis not only for FTI treatment but also for other standard chemotherapy, the immune-related biomarkers identified in the present invention are specifically associated with the clinical benefit of FTI treatment. but not by means of other chemotherapeutic agents.

Biomarkers as identified herein include KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, GZMM, as well as the specific ligand for KIR2DS2, HLA-C2. Carriers of KIR2DS2 and HLA-C2 have been shown to be predisposed to MDS. (Serio et al., Blood (ASH Annual Meeting Abstracts) 2006 108: Abstract 2670; Cook et al., Blood 2004; 103: 1521-152) The immunological biomarkers identified in the present invention are all genes associated with NK cells . The discovery that an FTI, such as tipifarnib, selectively targets cancers with specific KIR genotypes or expression patterns described herein may be based at least in part on the mechanism that specific NK cells with specific genotypes or expression patterns KIRs can induce autoimmunity. Such NK cells can also down-regulate antigen presentation, kill or down-regulate specific T cell subtypes. By inhibiting KIR-RAS signaling, FTI can modulate or inhibit NK cell activity, promote cytotoxicity against cancer cells by modulating the patient's own immune system. As well as by inhibiting KIR-RAS signaling, FTI can modulate or inhibit the activity of NK cells and other immunocytes against normal hematological cells and their progenitors, reducing or eliminating the need for erythrocyte or platelet transfusion or hematological growth factor administration.

3.1. KIR typing and HLA typing.

As presented herein, the genotype of the KIR genes and HLA genes of a subject may be indicative of the subject's likelihood of responding to FTI treatment. A cancer patient who is a carrier of KIR2DS2, KIR2DS5, or HLA-C2 is likely to be able to respond to FTI treatment. Accordingly, KIR typing in cancer patients and selective treatment of those carrying KIR2DS2 or KIR2DS5 may increase the overall response rate of cancer patients to FTI treatment. In addition, HLA typing in cancer patients and selective treatment of those who carry HLA-C2 may further increase the overall response rate of cancer patients to FTI treatment.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a therapeutically effective amount of FTI to a subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5. In some embodiments, provided herein is a method of treating cancer in a subject by typing a KIR in the subject and administering a therapeutically effective amount of FTI to the subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5. In some embodiments, the subject is a carrier of KIR2DS2. In some embodiments, the subject is a carrier of KIR2DS5. In some embodiments, the subject is a carrier of both KIR2DS2 and KIR2DS5. In some embodiments, the subject is also not a carrier of KIR2DL2. In some embodiments, the subject is also not a carrier of KIR2DL5. In some embodiments, the subject is a KIR2DS2 carrier but not a KIR2DL2 carrier. In some other embodiments, the subject is a KIR2DS5 carrier but not a KIR2DL5 carrier.

In some embodiments, methods for treating cancer in a subject as provided herein further comprise HLA typing in the subject and administering a therapeutically effective amount of FTI to a subject that is an HLA-C2 carrier. In some embodiments, the subject is a carrier of both KIR2DS2 and HLA-C2. In some embodiments, the subject is a carrier of both KIR2DS5 and HLA-C2. In some embodiments, the subject is a carrier of KIR2DS2, KIR2DS5, and HLA-C2. In some embodiments, the implementation of the subject, which is a carrier of HLA-C2, is homozygous HLAC2/HLA-C2. In some embodiments, the subject is heterozygous for HLA-C1/HLA-C2.

In some embodiments, provided herein is a method for selecting a cancer patient for FTI treatment by KIR typing, where the cancer patient

- 34 040014 Cancer is selected for FTI treatment if the cancer patient is a carrier of KIR2DS2 or KIR2DS5. In some embodiments, provided herein is a method for predicting the likelihood for a cancer patient to be responsive to FTI treatment by KIR typing, and determining that the cancer patient is likely to be able to respond to FTI treatment if the cancer patient is a carrier. KIR2DS2 or KIR2DS5. In some embodiments, the method further comprises administering a therapeutically effective amount of FTI to a cancer patient. In some embodiments, the subject is a carrier of KIR2DS2. In some embodiments, the subject is a carrier of KIR2DS5. In some embodiments, the subject is a carrier of both KIR2DS2 and KIR2DS5. In some embodiments, the subject is also not a carrier of KIR2DL2. In some embodiments, the subject is also not a carrier of KIR2DL5. In some embodiments, the subject is a KIR2DS2 carrier but not a KIR2DL2 carrier. In some other embodiments, the subject is a KIR2DS5 carrier but not a KIR2DL5 carrier.

In some embodiments, methods for selecting a cancer patient for FTI treatment or predicting the likelihood for a cancer patient to be responsive to FTI treatment, as provided herein, further comprise HLA typing in the subject and administering a therapeutically effective amount of FTI to the subject who is a carrier. HLA-C2. In some embodiments, the subject is a carrier of both KIR2DS2 and HLA-C2. In some embodiments, the subject is a carrier of both KIR2DS5 and HLA-C2. In some embodiments, the subject is a carrier of KIR2DS2, KIR2DS5, and HLAC2. In some embodiments, the implementation of the subject, which is a carrier of HLA-C2, is homozygous for HLA-C2/HLA-C2. In some embodiments, the subject is heterozygous for HLA-C1/HLA-C2.

KIR typing methods are well known in the art. Exemplary KIR typing methods are described in WO 2012047985; Lebedeva et al., Hum Immun., 68(9):789-96 (2007); Gonzalez et al., Hum Immun., 70 (10) : 858-63 (2009); Yun et al., Blood (ASH Annual Meeting Abstracts) 106: Abstract 1407 (2005) (see also Yun et al., Clin Immunol. 123 (3) : 272-280 (2007).); Leung et al., J Immun. 174:6540-6545 (2005); Dinauer et al., US 2008/0280289 (see also WO 2005/046459, selected sections; and KIR Genotyping Product Brochure 2004.); Chen et al., WO 2009/051672. see also PCT/US2008/011671; Trachtenberg et al, Patent Application Publication No. US 2008/0213787 (see also WO 2007/041067.); Houtchens et al., Immunogenetics. 59(7):525-37 (2007).; Gomez-Lozano et al., Tissue Antigens 59(3):184-193 (2002); and Shilling et al., J Immunol. 168:2307-2315 (2002); US Pat. Uhrberg et al., Immunity 7:753-763 (1997); Gomez-Lozano et al., Tissue Antigens 59:184-193 (2002); Cook et al., Hum. Immunology 64:567-571 (2003); Crum et al., Tissue Antigens 56:313-326 (2000); Middleton et al., Transplant immunology 10:147-164 (2002); Ross et al., Nature Biotech., 16:1347-1351 (1998); Fei et al., Rapid Comm. Mass. Spec, 14:950-959 (2000); Fei et al., NAR 26 (11) :2827-2828 (1998); Amexis et al., PNAS 98(21) 12097-12102 (2001); Li et al., Electrophoresis 20:1258-1265 (1999); Buetow et al., PNAS 98(2) 581-584 (2001); Storm et al., Methods in Mol. Biol., 212:241-262 (2003); Parham, Immunology Lett. 92:11-13 (2004); and MassARRAY™ Homogenous Mass EXTEND™ (hME) Assay, Sequenom®, Application Notes, Bulletin #1021; the full contents of each of which are hereby incorporated by reference.

In addition, several KIR genotyping kits are available including Inno-Train, KIRReady Gene Product Brochure 9/2005; Miltenyi Biotec, KIR Typing Kit Product Brochure 2009; Invitrogen, KIR Genotyping SSP Kit Product Brochure 11/2006; and Tepnel Lifecodes, KIR Genotyping Product Brochure 6/2005, the entire contents of each of which are hereby incorporated by reference.

The methods provided herein can be carried out by any method described herein or otherwise known in the art. In some embodiments, provided herein is a method of treating cancer in a subject using FTI by KIR typing, or selecting a patient with cancer for FTI treatment by KIR typing, where KIR typing is by sequencing, polymerase chain reaction (PCR), microarray analysis DNA, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, immunoblot analysis, or enzyme-linked immunosorbent assay (ELISA). In some embodiments, KIR typing is performed through DNA microarray analysis. In some embodiments, KIR typing is performed by ELISA. In some embodiments, KIR typing is performed via sequencing. In some embodiments, KIR typing is performed by next generation sequencing (NGS).

In some embodiments, KIR typing can be performed by PCR. In some embodiments, KIR typing can be performed by PCR using a sequence specific primer (SSP). In some embodiments,

- 35 040014 SSPs include SSPs that are specific for the amplification of KIR2DL2, KIR2DL5, KIR2DS2, KIR2DS5, or any combination thereof. In some embodiments, KIR typing can be performed by PCR using a sequence-specific oligonucleotide probe (SSOP). In some embodiments, KIR typing can be performed by PCR using sequence-based typing (SBT). In some embodiments, KIR typing can be performed through DNA microarray analysis. In some embodiments, KIR typing may be performed by MS. In some embodiments, KIR typing may be performed by array-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF). As one of skill in the art would understand, KIR typing can be accomplished by any of the methods described herein or otherwise known in the art.

HLA typing methods are well known in the art. Initially, the most widely used DNA typing method to identify these alleles was restriction fragment length polymorphism (RFLP) analysis. In addition to restriction fragment length polymorphism (PCR-RFLP), another method has been hybridization of amplified PCR products with sequence-specific oligonucleotide probes (PCR-SSO) to separate HLA alleles (see, Tiercy et al. (1990) Blood Review 4: 9 -15, the contents of which are hereby incorporated by reference). For this method, it is necessary to obtain a PCR product of the HLA locus of interest and then dot it onto nitrocellulose membranes or strips. Each membrane is then hybridized with a sequence-specific probe, washed and then analyzed by X-ray film exposure or by colorimetric analysis depending on the detection method. Similar to the PCR-SSP method, probes are obtained for the region of allelic polymorphism responsible for different HLA alleles. Each sample must be hybridized and analyzed with different probes at least 100-200 times for complete class I and II typing. Hybridization and detection methods for PCR-SSO typing include the use of non-radioactively labeled probes, microplate formats, and the like. (See, for example, Saiki et al. (1989) Proc. Natl. Acad. Sci., U.S.A. 86: 6230-6234; Erlich et al. (1991) Eur. J. Immunogenet. 18(1-2): 33 -55, Kawasaki et al (1993) Methods Enzymol 218:369-381, all of which are hereby incorporated by reference in their entirety).

A molecular typing method using amplification with sequence specific primers (PCR-SSP) has been described (see Olerup and Zetterquist (1992) Tissue Antigens 39: 225-235). This PCR-SSP method is simple, useful and fast since the detection step is much simpler. In SSP-PCR, allelic sequence-specific primers amplify only the complementary template allele, allowing high-resolution detection of genetic diversity. This method allows determination of HLA type simply by whether amplification products (collectively referred to as amplicons) are present or absent after PCR. In PCR-SSP, detection of amplification products is typically performed by agarose gel electrophoresis followed by staining of the gel with ethidium bromide (EtBr).

Another way of typing HLA is SSCP single-strand conformational polymorphism. Briefly, single strand PCR products of different HLA loci are separated by non-denaturing polyacrylamide gel electrophoresis (PAGE). Single strands migrate to a unique location based on the composition of their base pairs. By comparison with known standards, typing can be deduced. It can be used to determine true homozygosity.

Other HLA typing methods, including, but not limited to, sequencing, polymerase chain reaction (PCR), DNA microarray analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, immunoblotting analysis, or enzyme-linked immunosorbent assay (ELISA), also can be used in the methods presented herein. In some embodiments, the sequencing may be NGS. Other methods are described in US patent No. 6670124, US 5468611, US 8435740, US 8771951 and US 20130267613; the entire contents of which are hereby incorporated by reference. Various other HLA typing methods known in the art can also be used in the methods presented herein.

For example, single nucleotide polymorphism (SNP) analysis can be used for HLA typing. In SNP analysis, different HLAs can be typed based on polymorphisms at position 77 in HLA-C and at position 83 in HLA-B and -A. SNP analysis can be performed in the HT7900 from Applied Biosystems following the allelic discriminant analysis protocol provided by the manufacturer. The assay primers are designed to amplify all alleles of a particular type of HLA (such as HLA-C) as well as the amplicon containing the polymorphic region of interest. Two probes are developed with one mismatch between them. Each probe bound only one group of alleles and was fluorescently labeled with 6FAM or VIC at the 5' end. The probes also contained Taqman® minor groove binding protein (MGB) with a non-fluorescent quencher (NFQ) (Applied Biosystems). For HLA-C, the forward primer can be

5'-TTGGGACCGGGAGACACAG-3' (SEQ ID NO: 46),

- 36 040014 and the reverse primer may be

5'-CGATGTAATCCTTGCCGTC-3' (SEQ ID NO: 47).

The probes used for HLA-C1 and HLA-C2 may be 6FAM-CCGAGTGAG CCTGC-MGBNFQ (SEQ ID NO: 48) and VIC-CCGAGTGAA CCTGC-MGBNFQ (SEQ ID NO: 49), respectively. Each assay reaction mix can contain a 250 nM probe and 20 ng of genomic DNA in 1x Taqman genotyping premix from Applied Biosystems (USA).

In some embodiments, KIR typing or HLA typing is performed as an adjunct diagnosis in the treatment of FTI. Concomitant diagnosis can be performed at the clinical center where the subject is being treated. Concomitant diagnosis may also be performed at a center other than the clinical center where the subject is being treated.

In some embodiments, the KIR typing or HLA typing methods include obtaining a sample from a subject. The subject may be a patient with a malignant tumor. The sample may be a whole blood sample, a bone marrow sample, a partially purified blood sample, a PBMC, or a tissue biopsy sample. In some embodiments, the sample is a bone marrow sample from a cancer patient. In some embodiments, the sample is a PBMC from a cancer patient. In some embodiments, the sample is enriched in NK cells. NK cells can be enriched from bone marrow, whole blood, or partially purified blood from a cancer patient. In some embodiments, the NK cells are further expanded in vitro prior to KIR typing.

3.2. KIR expression and GZMM expression.

As presented herein, the level of expression of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM in a sample from a subject may be indicative of the subject's likelihood of being responsive to FTI treatment. A cancer patient whose KIR2DS2 expression level is higher than the reference KIR2DS2 expression level is likely to be able to respond to FTI treatment. A cancer patient whose KIR2DS5 expression level is higher than the reference KIR2DS5 expression level is likely to be able to respond to FTI treatment. A cancer patient whose GZMM expression level is higher than the reference GZMM expression level is likely to be able to respond to FTI treatment. A cancer patient whose KIR2DL2 expression level is lower than the reference KIR2DL2 expression level is likely to be able to respond to FTI treatment. A cancer patient whose KIR2DL5 expression level is lower than the reference KIR2DL5 expression level is likely to be able to respond to FTI treatment. Accordingly, detecting the expression level of one or more of these biomarkers in cancer patients and selectively treating cancer patients meeting one or more of the conditions described above can increase the overall response rate of cancer patients to FTI treatment.

In some embodiments, the expression level of KIR2DL5 is the sum of the expression levels of KIR2DL5A and KIR2DL5B. In some embodiments, the expression level of KIR2DL5 is the expression level of KIR2DL5A. In some embodiments, the expression level of KIR2DL5 is the expression level of KIR2DL5B.

Also provided herein are methods of using the ratio of expression levels of specific biomarkers to predict the likelihood of a subject being responsive to FTI treatment. For example, a high ratio of KIR2DS2 expression level to KIR2DL2 expression level (2DS2/2DL2 ratio) may indicate that the subject is likely to be able to respond to FTI treatment. Similarly, a high ratio of KIR2DS5 expression level to KIR2DL5 expression level (2DS5/2DL5 ratio) may indicate that the subject is likely to be able to respond to FTI treatment. Accordingly, detection of the expression level of these biomarkers in cancer patients and selective treatment of cancer patients whose 2DS2/2DL2 ratio is higher than the reference ratio, or whose 2DS5/2DL5 ratio is higher than the reference ratio, or both, can increase the overall response rate of cancer patients to FTI treatment.

In some embodiments, provided herein is a method of treating cancer in a subject by administering a therapeutically effective amount of FTI to the subject, wherein (i) the expression level of KIR2DS2 in a sample from the subject is higher than a reference level of KIR2DS2 expression;

(ii) the level of expression of KIR2DL2 in the sample from the subject is lower than the reference level of expression of KIR2DL2;

(iii) the level of expression of KIR2DS5 in the sample from the subject is higher than the reference level of expression of KIR2DS5;

- 37 040014 (iv) the level of expression of KIR2DL5 in the sample from the subject is lower than the reference level of expression

KIR2DL5; or (v) the level of expression of GZMM in the sample from the subject is higher than the reference level of expression

GZMM;

or any combination of these is present.

In some embodiments, provided herein is a method of treating cancer in a subject by (a) determining the expression level of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM in a sample from the subject, where (i) the expression level KIR2DS2 in the sample is higher than the reference level of KIR2DS2 expression;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression; and (b) administering a therapeutically effective amount of the FTI to the subject.

In some embodiments, provided herein is a method for selecting a cancer patient for FTI treatment by determining the expression level of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM in a sample from a cancer patient, where the patient with a cancer is selected for FTI treatment if (i) the expression level of KIR2DS2 in the sample from the subject is higher than the reference level of KIR2DS2 expression;

(ii) the level of expression of KIR2DL2 in the sample from the subject is lower than the reference level of expression of KIR2DL2;

(iii) the level of expression of KIR2DS5 in the sample from the subject is higher than the reference level of expression of KIR2DS5;

(iv) the level of expression of KIR2DL5 in the sample from the subject is lower than the reference level of expression of KIR2DL5; or (v) the level of GZMM expression in the sample from the subject is higher than the reference level of GZMM expression;

or any combination of these is present.

In some embodiments, the methods provided herein include treating a subject with a therapeutically effective amount of FTI, or selecting a patient with cancer for treatment with FTI, where one of five conditions is met:

(i) the level of expression of KIR2DS2 in a sample from a subject or patient with a malignant tumor is higher than the reference level of expression of KIR2DS2 (condition 1);

(ii) the level of expression of KIR2DS5 in the sample from the subject or patient with a malignant tumor is higher than the reference level of expression of KIR2DS5 (condition 2);

(iii) the level of expression of KIR2DL2 in a sample from a subject or patient with a malignant tumor is lower than the reference level of expression of KIR2DL2 (condition 3);

(iv) the level of expression of KIR2DL5 in a sample from a subject or patient with a malignant tumor is lower than the reference level of expression of KIR2DL5 (condition 4); and (v) the level of GZMM expression in the sample from the subject or cancer patient is higher than the reference level of GZMM expression (condition 5).

One of skill in the art will appreciate that satisfaction of any one of the five conditions described above may indicate that the subject is likely to be able to respond to FTI treatment. Accordingly, each condition can independently serve as a criterion for selecting a patient for FTI treatment in order to increase the overall response rate. One of skill in the art will also appreciate that combinations of two or more conditions can also serve as patient selection criteria for FTI treatment that are more selective than a single condition and can potentially provide a higher overall response rate. Accordingly, this document also provides a method of using any combination or permutation of the above conditions to select a patient for FTI treatment.

In some embodiments, the method provided herein comprises treating a subject with a therapeutically effective amount of FTI or selecting a cancer patient for FTI treatment, wherein the subject or cancer patient satisfies two of the five conditions described above, such as conditions 1 and 2, 1 and 3, 1 and 4, 1 and 5, 2 and 3, 2 and 4, 2 and 5, 3 and 4, 3 and 5, or 4 and 5. In some embodiments, the subject or patient with cancer satisfies the conditions 1 and 2. In some embodiments, the subject or patient with cancer satisfies conditions 1 and 3. In some embodiments, the subject or patient with cancer satisfies conditions 1 and 4. In some embodiments, the subject or patient with cancer satisfies conditions 1 and 5. In some embodiments, the subject or patient with cancer satisfies conditions 2 and 3. In some embodiments, the subject or patient with cancer satisfies conditions 2 and 4. In some embodiments, the subject or patient with cancer satisfies conditions 2 and 5. In some embodiments, the subject or patient with cancer satisfies conditions 3 and 4. In some embodiments, the subject or patient with cancer satisfies conditions 3 and 4. embodiments, the subject or patient with cancer satisfies conditions 3 and 5. In some embodiments, the subject or patient with cancer satisfies conditions 4 and 5. For example, in some embodiments, the method comprises treating the subject with a therapeutically effective amount of FTI or selecting a patient with cancer for the treatment of FTI, wherein the expression level of KIR2DS2 in the subject sample is higher than the reference KIR2DS2 expression level, and where the GZMM expression level in the subject sample is higher than the reference GZMM expression level. As another example, in some embodiments, the method comprises treating a subject with a therapeutically effective amount of FTI, or selecting a cancer patient for FTI treatment, where the expression level of KIR2DS5 in the subject's sample is higher than a reference level of KIR2DS5 expression and the expression level of KIR2DL2 in the sample is subject is lower than the reference level of KIR2DL2 expression.

In some embodiments, the method provided herein comprises treating a subject with a therapeutically effective amount of FTI or selecting a patient with cancer for FTI treatment, wherein the subject or patient with cancer satisfies three of the five conditions described above, such as conditions 1, 2 and 3; 1, 2 and 4; 1, 2 and 5; 1, 3 and 4; 1, 3 and 5; 1, 4 and 5; 2, 3 and 4; 2, 3 and 5; 2, 4 and 5; or 3, 4, and 5. In some embodiments, the subject or patient with cancer satisfies conditions 1, 2, and 3. In some embodiments, the subject or patient with cancer satisfies conditions 1, 2, and 4. In some embodiments, the subject or patient with cancer satisfies conditions 1, 2, and 5. In some embodiments, the subject or patient with cancer satisfies conditions 1, 3, and 4. In some embodiments, the subject or patient with cancer satisfies conditions 1, 3, and 5. In some embodiments, the subject or patient with cancer satisfies conditions 1, 4, and 5. In some embodiments, the subject or patient with cancer satisfies conditions 2, 3, and 4. In some embodiments, the subject or patient with cancer satisfies conditions 2, 3, and 5. In some embodiments, a subject or patient with cancer satisfies conditions 2, 4, and 5. In some embodiments, a subject or patient with cancer satisfies conditions 3, 4, and 5. For example, in some other embodiments, the method comprises treating a subject with a therapeutically effective amount of FTI or selecting a patient with cancer. a tumor for treating FTI, wherein the expression level of KIR2DS2 in the subject sample is higher than the reference KIR2DS2 expression level, the expression level of KIR2DL2 in the subject sample is lower than the reference KIR2DL2 expression level, and the GZMM expression level in the subject sample is higher than the reference GZMM expression level.

In some embodiments, the method provided herein comprises treating a subject with a therapeutically effective amount of FTI or selecting a patient with cancer for FTI treatment, wherein the subject or patient with cancer satisfies four of the five conditions described above, such as conditions 1, 2 , 3 and 4; 1, 2, 3 and 5; 1, 2, 4 and 5; 1, 3, 4 and 5; or 2, 3, 4, and 5. In some embodiments, the subject or patient with cancer satisfies conditions 1, 2, 3, and 4. In some embodiments, the subject or patient with cancer satisfies conditions 1, 2, 3, and 5. B in some embodiments, the subject or patient with cancer satisfies conditions 1, 2, 4, and 5. In some embodiments, the subject or patient with cancer satisfies conditions 1, 3, 4, and 5. In some embodiments, the subject or patient with cancer satisfies conditions 2, 3, 4 and 5.

In some embodiments, the method provided herein comprises treating a subject with a therapeutically effective amount of FTI or selecting a cancer patient for FTI treatment, wherein the subject or cancer patient satisfies all five of the above conditions, namely where (i) the level expression of KIR2DS2 in the sample from the subject is higher than the reference level of expression of KIR2DS2;

(ii) the level of expression of KIR2DL2 in the sample from the subject is lower than the reference level of expression of KIR2DL2;

(iii) the level of expression of KIR2DS5 in the sample from the subject is higher than the reference level of expression of KIR2DS5;

(iv) the level of expression of KIR2DL5 in the sample from the subject is lower than the reference level of expression of KIR2DL5; and (v) the level of expression of GZMM in the sample from the subject is higher than the reference level of expression

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GZMM.

In addition to the level of expression of individual biomarkers, the ratio of expression levels between two biomarkers can also serve as a criterion for selecting a patient for FTI treatment in order to increase the response rate. In some embodiments, provided herein is a method of treating cancer in a subject by administering a therapeutically effective amount of FTI to the subject, wherein (i) the ratio of KIR2DS2 expression level to KIR2DL2 expression level (2DS2/2DL2 ratio) in a sample from the subject is higher than the reference 2DS2 ratio. /2DL2; or (ii) the ratio of KIR2DS5 expression level to KIR2DL5 expression level (2DS5/2DL5 ratio) in the sample from the subject is higher than the reference 2DS5/2DL5 ratio.

In some embodiments, provided herein is a method of treating cancer in a subject by determining expression levels of KIR2DS2 and KIR2DL2, or expression levels of KIR2DS5 and KIR2DL5 in a sample from a subject, where (i) the 2DS2/2DL2 ratio in the sample is higher than the reference 2DS2/ 2DL2; or (ii) the 2DS5/2DL5 ratio in the sample is higher than the reference 2DS5/2DL5 ratio;

and administering a therapeutically effective amount of the FTI to the subject.

In some embodiments, provided herein is a method for selecting a cancer patient for FTI treatment by determining expression levels of KIR2DS2 and KIR2DL2, or KIR2DS5 and KIR2DL5 expression levels in a sample from a cancer patient, wherein the cancer patient is selected for FTI treatment if (i) the 2DS2/2DL2 ratio in the sample is higher than the reference 2DS2/2DL2 ratio; or (ii) the 2DS5/2DL5 ratio in the sample is higher than the reference 2DS5/2DL5 ratio;

and administering a therapeutically effective amount of the FTI to the subject.

In some embodiments, the method provided herein comprises treating a subject with a therapeutically effective amount of FTI, or selecting a cancer patient for FTI treatment, where the 2DS2/2DL2 ratio in a sample from a subject or cancer patient is higher than a reference 2DS2 ratio. /2DL2. In some embodiments, the method of the present invention comprises treating a subject with a therapeutically effective amount of FTI, or selecting a cancer patient for FTI treatment, where the 2DS5/2DL5 ratio in a sample from a subject or cancer patient is higher than a reference 2DS5/2DL5 ratio. In some embodiments, the method of the present invention comprises treating a subject with a therapeutically effective amount of FTI or selecting a cancer patient for FTI treatment, where the 2DS2/2DL2 ratio in a sample from a subject or cancer patient is higher than a reference 2DS2/2DL2 ratio, and the 2DS5/2DL5 ratio in a sample from a subject or cancer patient is higher than the reference 2DS5/2DL5 ratio.

In some embodiments, the methods provided herein for selecting a cancer patient for FTI treatment may also be based on the level of expression of individual biomarkers, as well as the ratio of expression levels between biomarkers. In some embodiments, the method comprises treating a subject with a therapeutically effective amount of FTI, or selecting a cancer patient for FTI treatment, where the 2DS2/2DL2 ratio in a sample from a subject or cancer patient is higher than a reference 2DS2/2DL2 ratio, and the subject or a patient with a malignant tumor satisfies at least one of the five conditions described above regarding the individual level of expression of biomarkers. In some embodiments, the method comprises treating a subject with a therapeutically effective amount of FTI, or selecting a cancer patient for FTI treatment, where the 2DS5/2DL5 ratio in a sample from a subject or cancer patient is higher than a reference 2DS5/2DL5 ratio, and the subject or a patient with a malignant tumor satisfies at least one of the five conditions described above regarding the individual level of expression of biomarkers. In some embodiments, the method comprises treating a subject with a therapeutically effective amount of FTI, or selecting a cancer patient for FTI treatment, where the 2DS2/2DL2 ratio in a sample from the subject or cancer patient is higher than a reference 2DS2/2DL2 ratio and the ratio 2DS5 /2DL5 in a sample from a subject or patient with cancer is higher than the reference ratio 2DS5/2DL5, and the subject or patient with cancer satisfies at least one of the five conditions described above regarding the individual level of biomarker expression.

In some embodiments, the expression level of KIR2DL5 is the sum of the expression levels of KIR2DL5A and KIR2DL5B. In some embodiments, the expression level of KIR2DL5 is the expression level of KIR2DL5A. In some embodiments, the expression level of KIR2DL5 is the expression level of KIR2DL5B. Thus, in some embodiments, the 2DS5/2DL5 ratio is the ratio of the expression level of KIR2DS5 to the total expression levels of KIR2DL5A and KIR2DL5B. In some embodiments, the 2DS5/2DL5 ratio is the ratio of KIR2DS5 expression level to KIR2DL5A expression level. In some embodiments, the 2DS5/2DL5 ratio is the ratio of KIR2DS5 expression level to KIR2DL5B expression level.

To reiterate, five conditions for selecting a subject or patient with cancer for FTI treatment based on the level of expression of a single biomarker include: condition 1: the level of expression of KIR2DS2 in a sample from a subject or patient with cancer is higher than the reference level of KIR2DS2 expression; condition 2: the level of expression of KIR2DS5 in a sample from a subject or patient with a malignant tumor is higher than the reference level of expression of KIR2DS5; condition 3: the level of expression of KIR2DL2 in a sample from a subject or patient with a malignant tumor is lower than the reference level of expression of KIR2DL2; condition 4: the level of expression of KIR2DL5 in a sample from a subject or patient with a malignant tumor is lower than the reference level of expression of KIR2DL5; Condition 5: The expression level of GZMM in a sample from a subject or cancer patient is higher than the reference level of GZMM expression. Any combination or permutation of these conditions can be further combined with any one or both of the criteria based on expression ratio (2DS2/2DL2 ratio and 2DS2/2DL2 ratio) as criteria for selecting a patient for FTI treatment.

For example, in some embodiments, the method provided herein comprises treating a subject with a therapeutically effective amount of FTI, or selecting a cancer patient for FTI treatment, where the 2DS2/2DL2 ratio in a sample from a subject or cancer patient is greater than the reference ratio is 2DS2/2DL2, and the level of GZMM expression in the sample from the subject is higher than the reference level of GZMM expression. In some embodiments, the method comprises treating a subject with a therapeutically effective amount of FTI, or selecting a cancer patient for FTI treatment, where the 2DS5/2DL5 ratio in a sample from the subject or cancer patient is higher than a reference 2DS5/2DL5 ratio, and the level expression of KIR2DS2 in the sample from the subject is higher than the reference level of KIR2DS2 expression. One skilled in the art will appreciate that the scope of the present invention is not limited to these exemplary combinations and includes any combinations or permutations of individual patient selection criteria for FTI treatment as described herein.

As described herein, the KIR genotype, HLA genotype, and expression profile of certain NK cell-associated genes can be used to predict the ability of a cancer patient to respond to FTI treatment. Accordingly, provided herein are methods for selecting cancer patients for FTI treatment, or methods for treating patients using FTI, comprising first typing the KIR in the patient or determining expression profiles of the biomarkers identified herein to assess whether the patient is likely to respond to treatment. The methods may further include HLA typing in a patient. One of skill in the art will appreciate that these methods can be used independently as criteria for selecting patients for FTI treatment. In addition, the methods can also be used in conjunction with other patient stratification methods to further increase the response rate of a patient population to FTI treatment. For example, in some embodiments, the methods provided herein further include determining the status of ras gene mutations and selecting a patient for FTI treatment when the patient has a particular ras mutation, such as a K-ras mutation, an N-ras mutation, or an H-ras mutation, such as described in more detail below. In some embodiments, the methods provided herein further comprise determining ras gene mutation status and selecting a patient for FTI treatment when the patient has wild-type K-ras and wild-type N-ras. In some embodiments, the methods provided herein further include determining the status of the ras gene mutations and selecting a patient for FTI treatment when the patient has an H-ras mutation. In other embodiments, the methods provided herein may further include using the ratio of 2 genes between RASGRP1 and ARTX as an additional criterion for selecting patients for FTI treatment (US Patent No. 7932036, the entire contents of which are hereby incorporated by reference). The methods described herein or otherwise known in the art can be used to determine the status of mutations in the ras gene or the expression of other biomarkers such as RASGRP1 or ARTX. In some embodiments, the mutation status of a ras gene, such as H-ras, can be determined by NGS.

In some embodiments, the methods provided herein include determining the level of expression of a biomarker. In some embodiments, the expression level of the biomarker may be the level of the biomarker protein. In some embodiments, the level of expression of a biomarker may be the level of an RNA biomarker. Any method as described herein or otherwise known in the art for determining the level of a protein or RNA level of a gene can be used to determine the level of expression of a biomarker of the present invention.

Exemplary methods for detecting or quantifying mRNA levels include, but are not limited to, PCR-based methods, Northern blotting, ribonuclease protection assays, and the like. The sequence of an mRNA (eg, mRNA of a biomarker such as CRBN or CAP, or a fragment thereof) can be used to generate a probe that is at least partially complementary.

The probe can then be used to detect the mRNA sequence in the sample using any suitable assay such as PCR based methods, Northern blotting, dip probe assays, and the like.

Conventional methods known in the art for quantifying mRNA expression in a sample include Northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNase protection assays (Hod, Biotechniques 13:852-854 (1992)); and polymerase chain reaction (PCR) (Weis et ah, Trends in Genetics 8:263-264 (1992)). Alternatively, antibodies can be used that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid DNA-RNA duplexes or DNA-protein duplexes. Representative sequencing-based gene expression analysis methods include Serial Gene Expression Analysis (SAGE) and Gene Expression Analysis by Massive Parallel Characteristic Sequence Sequencing (MPSS).

A sensitive and flexible quantitative method is PCR. Examples of PCR methods can be found in the literature. Examples of PCR assays can be found in US Pat. No. 6,927,024, the entire contents of which are incorporated herein by reference. Examples of RT-PCR methods can be found in US Pat. No. 7,122,799, the entire contents of which are incorporated herein by reference. The fluorescent in situ PCR method is described in US Pat. No. 7,186,507, the entire content of which is incorporated herein by reference.

It should be noted, however, that other nucleic acid amplification methods (ie, other than PCR) can also be used in the nucleic acid analysis methods described herein. For example, suitable amplification methods include ligase chain reaction (see, for example, Wu & Wallace, Genomics 4:560-569, 1988); strand displacement analysis (see, for example, Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992; US Pat. No. 5,455,166); and several transcription-based amplification systems, including the methods described in US Pat. Nos. 5,437,990; 5409818; and 5399491; transcription-based amplification system (TAS) (Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173-1177, 1989); and self-sustaining sequence replication (3SR) (Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874-1878, 1990; WO 92/08800). Alternatively, methods to amplify the probe to detectable levels can be used, such as amplification using Q replicase (Kramer & Lizardi, Nature 339:401-402, 1989; Lomeli et al., Clin. Chem. 35: 1826-1831, 1989) . A review of known amplification methods is provided, for example, in Abramson and Myers in Current Opinion in Biotechnology 4:41-47 (1993).

mRNA can be isolated from the original tissue sample. General methods for isolating mRNA are well known in the art and are described in standard molecular biology textbooks including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). In particular, RNA isolation can be performed using a purification kit, buffer kit and protease from commercial manufacturers such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy minicolumns. Other commercially available RNA isolation kits include MASTERPURE® Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.) and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). Tumor-derived RNA can be isolated, for example, by cesium chloride density gradient centrifugation.

In some embodiments, the first step in obtaining a gene expression profile by PCR is reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. In other embodiments, a combined reverse transcription-polymerase chain reaction (RT-PCR) reaction can be used, for example, as described in US Pat. Nos. 5,310,652; 5322770; 5561058; 5641864; No. 5,693,517. Two common reverse transcriptases are avian myeloblastosis virus (AMV-RT) reverse transcriptase and Moloney murine leukemia virus (MMLV-RT) reverse transcriptase. The reverse transcription step is typically started with primers, using specific primers, random hexamers, or oligo-dT primers, depending on the conditions and purpose of obtaining an expression profile. For example, isolated RNA can be reverse transcribed using the GENEAMP™ RNA PCR Kit (Perkin Elmer, Calif USA) according to the manufacturer's instructions. The resulting cDNA can then be used as a template in a subsequent PCR reaction.

In some embodiments, reverse transcription-real-time detection PCR (qRT-PCR) can be used to both detect and quantify RNA targets (Bustin, et al., 2005, Clin. Sci., 109:365-379) . Examples of methods based on qRT-PCR can be found, for example, in US patent No. 7101663, the entire content of which is given herein by reference. PCR devices with real-time detection such as Applied

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Biosystems 7500 are commercially available, as are reagents such as TaqMan sequence detection chemicals.

For example, TaqMan® gene expression assays may be used, following the manufacturer's instructions. These kits are pre-designed gene expression assays for rapid, reliable detection and quantification of human, mouse and rat mRNA transcripts. You can use TaqMan® or analysis using 5'-nuclease, as described in US patent No. 5210015; 5487972; and 5804375; and in Holland et al., 1988, Proc. Natl. Acad. sci. USA 88:7276-7280. TAQMAN® PCR typically uses the 5' nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect the nucleotide sequence located between the two PCR primers. The probe is not elongated by the Taq DNA polymerase enzyme and is labeled with a fluorescent reporter dye and a fluorescence quenching dye. Any laser-induced radiation from the reporter dye is quenched by the quenching dye when the two dyes are localized in close proximity, as they are localized on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-independent manner. The resulting probe fragments are dissociated in solution, and the signal from the released reporter dye is released from the quenching effect of the second fluorophore. One reporter dye molecule is released for each new molecule synthesized, and the detection of an unquenched reporter dye provides a basis for quantitative interpretation of the data.

Any method suitable for detecting a degradation product can be used in a 5'-nuclease assay. Often the detection probe is labeled with two fluorescent dyes, one of which is capable of quenching the fluorescence of the other dye. Dyes are attached to the probe, preferably one is attached to the 5' end and the other is attached to the interior so that quenching occurs when the probe is in the unhybridized state and so that cleavage of the probe by the 5'-3' exonuclease activity of the DNA polymerase occurs between the two dyes.

Amplification results in splitting of the probe between dyes with a concomitant cessation of quenching and an increase in fluorescence observed from the initially quenched dye. The degradation product accumulation is monitored by measuring the increase in fluorescence of the reaction. US Pat. Nos. 5,491,063 and 5,571,673, both incorporated herein by reference, describe alternative methods for detecting probe degradation that occurs in parallel with amplification. 5'-nuclease assay data can initially be expressed as Ct, or cycle threshold. As discussed above, fluorescence values are recorded during each cycle and represent the amount of product amplified up to that point in the amplification reaction. The point at which the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).

To minimize errors and the effect of variability between samples, PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues and is not affected by experimental processing. The RNAs most commonly used to normalize gene expression patterns are mRNAs for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin housekeeping genes.

Primers and probes for PCR are designed based on the intron sequences present in the gene to be amplified. In this embodiment, the first step in primer/probe design is to refine the boundaries of intron sequences within genes. This can be done through publicly available software such as the DNA BLAT software developed by Kent, W., Genome Res. 12(4):656-64 (2002), or by means of the BLAST software, including variants thereof. The subsequent steps follow well established PCR primer and probe design techniques.

To exclude non-specific signals, it may be important to mask repetitive sequences within introns when designing primers and probes. This can easily be done using the Repeat Masker program, available online from Baylor College of Medicine, which screens DNA sequences against a library of repeat elements and returns a query sequence in which the repeat elements are masked. The masked intron sequences can then be used to design primer and probe sequences using any of the commercial or otherwise publicly available primer/probe design software packages such as Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Rozen and Skaletsky (2000) Primer3 on the WWW for general users and biological programmers. In: Krawetz

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S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386).

Factors to consider when designing PCR primers include primer length, melting point (T _m ) and G/C content, specificity, primer complementary sequences, and 3' end sequence. Typically, optimal PCR primers are typically 17-30 bases long and contain about 20-80%, eg about 50-60% G+C bases. T _m between 50 and 80°C, for example, from about 50 to 70°C, as a rule, are preferred. For additional guidance on the design of PCR primers and probes, see, for example, Dieffenbach et ah, General Concepts for PCR Primer Design in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133-155; Innis and Gelfand, Optimization of PCRs in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, TN Primerselect: Primer and probe design. Methods Mol. Biol. 70:520-527 (1997), the entire contents of which are hereby expressly incorporated by reference.

An exemplary PCR program, for example, is 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, then 60°C for 1 minute. To determine the number of cycles at which the fluorescence signal associated with the accumulation of a particular amplicon crosses a threshold (denoted as CT), the data can be analyzed, for example, using the 7500 Real-Time PCR System Sequence Detection software v.1.3 using the relative quantitative assessment with comparison of ST. Using this method, yield is expressed as a fold change in expression levels. In some embodiments, the implementation of the threshold level can be selected by automatic determination by software. In some embodiments, the cut-off level is set above background, but low enough to be within the exponential growth region of the amplification curve.

RNA sequencing, also referred to as shotgun whole transcriptome sequencing (WTSS), refers to the use of high-throughput sequencing techniques for cDNA sequencing to obtain information regarding the RNA content of a sample. Publications describing RNA sequencing include: Wang et al., Nature Reviews Genetics 10 (1): 57-63 (January 2009); Ryan et al. BioTechniques 45 (1): 81-94 (2008); and Maher et al., Nature 458 (7234): 97-101 (January 2009); the entire contents of which are hereby incorporated by reference.

Differential gene expression can also be identified or confirmed using the microarray method. In this method, polynucleotide sequences of interest (including cDNA and oligonucleotides) are printed or arrayed onto a microarray substrate. The array-form sequences are then hybridized to specific DNA probes from cells or tissues of interest.

In one embodiment of the microarray method, PCR-amplified inserts of cDNA clones are applied to a substrate in a dense array. Preferably, at least 10,000 nucleotide sequences are applied to the substrate. Genes in microarray form, immobilized on a microarray of 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated by incorporating fluorescent nucleotides through reverse transcription of RNA isolated from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity for each DNA spot on the array. After rigorous washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method such as a CCD camera. Quantification of the hybridization of each element included in the array allows an assessment of the occurrence of the corresponding mRNA. Using two-color fluorescence, separately labeled cDNA probes derived from two RNA sources are pairwise hybridized to the array. The relative occurrence of transcripts from two sources corresponding to each indicated gene is thus determined simultaneously. The miniaturized hybridization scale allows a convenient and fast assessment of the expression pattern for a large number of genes. Such methods have been shown to have the sensitivity needed to detect rare transcripts expressed at low copy numbers per cell and to reproducibly detect at least approximately two-fold differences in expression levels (Schena et al., Proc. Natl. Acad. Sci USA 93(2): 106-149 (1996)). Microarray analysis can be performed with commercially available equipment following manufacturer's protocols, such as using Affymetrix GENCHIP™ technology or Incyte microarray technology.

Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need to obtain an individual hybridization probe for each transcript. First, a sequence tag (approximately 10-14 bp) is obtained that contains enough information to uniquely identify the transcript, provided that the tag is derived from a unique position within each transcript. A plurality of transcripts are then joined together to form serial molecules that can be sequenced, revealing the identity of multiple labels at the same time. The expression pattern of any population of transcripts can be quantified by determining the occurrence of individual marks and identifying the gene corresponding to each mark. For more details, see, for example, Velculescu et ah, Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).

MassARRAY technology (Sequenom, San Diego, Calif.) is an automated, high-throughput method for analyzing gene expression using mass spectrometry (MS) for detection. According to this method, after RNA isolation, reverse transcription and PCR amplification, the cDNA is subjected to primer extension. The cDNA-derived primer extension products are purified and arrayed on a chip that is preloaded with the components needed to prepare samples for MALTI-TOF MS. The various cDNAs present in the reaction are quantified by analyzing the peak areas in the resulting mass spectra.

The mRNA level can also be measured by a hybridization based assay. A typical mRNA analysis method may comprise the steps of 1) obtaining surface-bound probes of interest; 2) hybridizing the mRNA population with surface-bound probes under conditions suitable for specific binding; (3) post-hybridization washes to remove nucleic acids not bound by hybridization; and (4) detecting hybridized mRNAs. The reagents used in each of these steps and the conditions for their use may vary depending on the particular application.

Any suitable assay platform can be used to determine the level of mRNA in a sample. For example, the assay can be in the form of dipping probes, a membrane, a chip, a disk, a test strip, a filter, a microsphere, a glass slide, a multiwell plate, or an optical fiber. The assay system may have a solid support to which a nucleic acid corresponding to the mRNA is attached. The solid support may comprise, for example, a plastic, silicone, metal, resin, glass, membrane, particle, precipitate, gel, polymer, shell, sphere, polysaccharide, capillary, film, plate, or glass slide. The assay components can be obtained and packaged together in the form of an mRNA detection kit.

The nucleic acid can be labeled, if desired, to obtain a population of labeled mRNAs. Typically, the sample can be labeled using methods well known in the art (e.g., using DNA ligase, terminal transferase, or RNA backbone labeling, etc.; see, e.g., Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y.). In some embodiments, the sample is labeled using a fluorescent label. Illustrative fluorescent dyes include, but are not limited to, xanthene dyes, fluorescein dyes, rhodamine dyes, fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX ), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), N,N,N',N'-tetramethyl-6-carboxyrodamine (TAMRA or T), 6- carboxy-X-rhodamine (ROX or R), 5-carboxyrodamine 6G (R6G5 or G5), 6-carboxyrodamine 6G (R6G6 or G6) and rhodamine 110; cyanine dyes, for example Cy3, Cy5 and Cy7 dyes; Alexa dyes, such as Alexa-fluoro-555; coumarin, diethylaminocoumarin, umbelliferone; benzimide dyes, eg Hoechst 33258; phenanthridine dyes, eg Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, BODIPY dyes, quinoline dyes, pyrene, fluorescein, chlorotriazinyl, R110, eosin, JOE, R6G, tetramethylrhodamine, lyssamine, ROX, naphthofluorescein, and the like.

Hybridization can be carried out under suitable hybridization conditions, the stringency of which can be varied as desired. Typical conditions are suitable for producing solid surface probe/target complexes between complementary binding participants, i.e. between the considered surface-bound probes and complementary mRNAs in the sample. In specific embodiments, stringent hybridization conditions can be applied.

Hybridization is generally carried out under stringent hybridization conditions. Standard hybridization techniques (eg, under conditions suitable to ensure specific binding of target mRNAs in a sample to probes) are described in Kallioniemi et al., Science 258:818-821 (1992) and WO 93/18186. Several general method manuals are available, such as Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993). For a description of methods suitable for in situ hybridization, see Gall et al., Meth. Enzymol. 21:470-480 (1981); and Angerer et al., in Genetic Engineering: Principles and Methods (Setlow and Hollaender, Eds.) Vol. 7, pgs 43-65 (Plenum Press, New York 1985). The choice of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, stringency of wash conditions, etc., may depend on the design of the experiment, including the source of the sample, the nature of the binding agents, the degree of complementarity expected, etc., and they can be determined as the subject of experiments generally accepted by experts in this field. One skilled in the art will clearly appreciate that alternative but compatible hybridization and wash conditions can be used to provide conditions of similar stringency.

After the mRNA hybridization procedure, surface-bound polynucleotides, as a rule,

- 45 040014 is washed to remove unbound nucleic acids. Washing can be carried out using any convenient washing method, where the washing conditions are generally stringent, as described above. Hybridization of the target mRNAs with the probes is then detected using standard methods.

In some embodiments, the methods provided herein include determining the mRNA level of one or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM using one of the methods described herein or otherwise known herein. areas. In one embodiment, the methods include determining the level of KIR2DS2 mRNA in a sample from a subject. In one embodiment, the methods include determining the level of KIR2DL2 mRNA in a sample from a subject. In one embodiment, the methods include determining the level of KIR2DS5 mRNA in a sample from a subject. In one embodiment, the methods include determining the level of KIR2DL5 mRNA in a sample from a subject. In one embodiment, the methods include determining the level of GZMM mRNA in a sample from a subject.

In some embodiments, the KIR2DL5 mRNA level is the total mRNA level of KIR2DL5A and KIR2DL5B. In some embodiments, the KIR2DL5 mRNA level is the KIR2DL5A mRNA level. In some embodiments, the KIR2DL5 mRNA level is the KIR2DL5B mRNA level.

In some embodiments, the methods provided herein include determining mRNA levels of two or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM. In some embodiments, the methods include determining mRNA levels of three or more of these biomarkers. In some embodiments, the methods include determining mRNA levels of four or more of these biomarkers. In some embodiments, the methods include determining mRNA levels for all five of these biomarkers.

In some embodiments, the methods provided herein include determining the levels of KIR2DS2 and KIR2DL2 mRNA in a sample from a subject or cancer patient, and calculating the ratio of KIR2DS2 mRNA level to KIR2DL2 mRNA level (2DS2/2DL2 mRNA ratio). In some embodiments, the methods further comprise determining levels of KIR2DS5 and KIR2DL5 mRNA in a sample from a subject or cancer patient and calculating the ratio of KIR2DS5 mRNA level to KIR2DL5 mRNA level (2DS5/2DL5 mRNA ratios). In some embodiments, the methods further comprise determining levels of KIR2DS2 and KIR2DL2 mRNA in a sample from a subject or patient with cancer, and levels of mRNA of KIR2DS5 and KIR2DL5 in a sample from a subject or patient with cancer, and calculating both the 2DS2/2DL2 mRNA ratio and mRNA ratios 2DS5/2DL5.

In some embodiments, the mRNA level of one or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM is determined by qPCR, RT-PCR, RNA sequencing, microarray analysis, SAGE, MassARRAY, or FISH. In some embodiments, the mRNA level of one or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM is determined by qPCR or RT-PCR.

In some embodiments, the methods provided herein include determining the protein level of one or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM. The biomarker protein level can be detected by a variety of immunohistochemistry (IHC) methods or other immunoassay methods.

IHC staining of tissue sections has been shown to be a reliable way to assess or detect the presence of proteins in a sample. Immunohistochemistry methods use an antibody as a probe and to visualize cellular antigens in situ, typically by chromogenic or fluorescent methods. Thus, antibodies or antisera, preferably polyclonal antisera and most preferably monoclonal antibodies specific for each marker, are used to detect expression. As discussed in more detail below, antibodies can be detected by direct labeling of the antibodies themselves, for example using radioactive labels, fluorescent labels, hapten labels such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, an unlabeled primary antibody is used in combination with a labeled secondary antibody containing antisera, polyclonal antisera, or a monoclonal antibody specific for the primary antibody. Protocols and kits for immunohistochemistry are well known in the art and are commercially available. Automated systems for slide preparation and IHC are commercially available. The Ventana® BenchMark XT system is an example of such an automated system.

Standard immunological and immunoassay methods can be found in Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991). In addition, immunoassays can be performed in any of several configurations, which are extensively reviewed in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, above. For a general overview of immunoassays see also Methods

- 46 040014 in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Ten, eds., ^7th ed. 1991).

Common assays for detecting the level of a biomarker protein include non-competitive assays such as sandwich assays and competitive assays. Typically, an assay such as an ELISA assay can be used. ELISA assays are known in the art, for example for analyzing a wide variety of tissues and samples, including blood, plasma, serum, or bone marrow.

A wide variety of immunoassay methods are available using this assay format, see, for example, US Pat. These include single site assays and dual site assays, or sandwich assays of non-competitive types, as well as traditional competitive binding assays. These assays also include direct binding of the labeled antibody to the target biomarker. Sandwich assays are conventional assays. There are a number of variations on the sandwich analysis method. For example, in a typical direct assay, an unlabeled antibody is immobilized on a solid substrate and the sample to be tested is brought into contact with the bound molecule. After a suitable incubation period for a period of time sufficient to allow the formation of an antibody-antigen complex, then a second antibody specific for the antigen labeled with a reporter molecule capable of producing a detectable signal is added and incubated, allowing time sufficient for the formation of another antibody complex. -antigen-labeled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observing the signal produced by the reporter molecule. The results can either be qualitative, by simple observation of a visible signal, or they can be quantified by comparison with a control sample containing known amounts of the biomarker.

Direct assay options include simultaneous assays, in which both the sample and the labeled antibody are added simultaneously to the bound antibody. These methods are well known to those skilled in the art, including any minor modifications as will be readily apparent. In a typical direct sandwich assay, the first antibody with specificity for a biomarker is either covalently or passively bound to a solid surface. The solid surface may be glass or polymer, where the most common polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. Solid supports may be in the form of tubes, beads, microplate discs, or any other surface suitable for immunoassay. Coupling methods are well known in the art and typically consist of cross-linking covalent bonding or physical adsorption, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a sufficient period of time (e.g. 2-40 min or overnight if more convenient) and under suitable conditions (e.g. room temperature to 40°C, for example, between 25 and 32°C inclusive) to allow binding of any subunit present in the antibody. After an incubation period, the solid phase with the antibody subunit is washed, dried and incubated with a second antibody specific for the biomarker portion. The second antibody is linked to a reporter molecule, which is used to show the binding of the second antibody to a molecular marker.

In some embodiments, flow cytometry (FACS) can be used to detect the level of a biomarker protein. Surface proteins (such as KIR) can be detected using antibodies against specific biomarkers. The flow cytometer detects and records the intensity of the fluorochrome labeled antibody, which indicates the level of expression of the biomarker. Non-fluorescent cytoplasmic proteins can also be observed by staining permeabilized cells. The dye can be either a fluorescent compound capable of binding to specific molecules, or a fluorochrome labeled antibody to bind to a selected molecule.

An alternative method involves immobilizing the target biomarkers in a sample and then exposing the immobilized target to a specific antibody, which may or may not be labeled with a reporter molecule. Depending on the amount of target and the signal strength of the reporter molecule, the bound target can be detected by direct labeling using an antibody. Alternatively, a second labeled antibody specific for the first antibody is subjected to a target-first antibody complex to form a tertiary target-first antibody-second antibody complex. The complex is detected by a signal emitted from a labeled reporter molecule.

In the case of an enzyme immunoassay, the enzyme is conjugated to a second antibody, usually via glutaraldehyde or periodate. As is clearly understood, however, there are a wide variety of different conjugation methods readily available to those skilled in the art. Common enzymes, including horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, and others, are discussed in this document. The substrates to be used with specific enzymes are generally chosen to produce, after hydrolysis with the appropriate enzyme, a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to use fluorogenic substrates that form a fluorescent product, in contrast to the chromogenic substrates mentioned above. In all cases, the enzyme labeled antibody is added to the antibody-molecular marker complex, allowed to bind, and then the excess reagent is washed off. A solution containing the appropriate substrate is then added to the antibody-antigen-antibody complex. The substrate can react with the enzyme associated with the second antibody to form a qualitative visual signal that can be further quantified, usually spectrophotometrically, to provide an indication of the amount of biomarker that was present in the sample. Alternatively, fluorescent compounds such as fluorescein and rhodamine can be chemically attached to antibodies without changing their binding capacity. When activated by illumination with light of a specific wavelength, the fluorochrome labeled antibody absorbs light energy, inducing a state of excitation of the molecule, followed by emission of light of a characteristic color that can be visually detected using a light microscope. As with EIA, the fluorescently labeled antibody is allowed to bind to the first antibody-molecular marker complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of a suitable wavelength, the observed fluorescence indicates the presence of a molecular marker of interest. Immunofluorescence and EIA methods are both very well developed in the art and are discussed in this document.

In some embodiments, the methods provided herein include determining the protein level of one or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM in a sample from a subject or patient with cancer using one of the methods described herein or otherwise known in the art. In one embodiment, the method includes determining the level of the KIR2DS2 protein in a sample from a subject or patient with a cancer. In one embodiment, the methods of the present invention include determining the level of the KIR2DL2 protein in a sample from a subject or patient with a cancer. In one embodiment, the method includes determining the level of the KIR2DS5 protein in a sample from a subject or patient with cancer. In one embodiment, the method includes determining the level of the KIR2DL5 protein in a sample from a subject or patient with cancer. In one embodiment, the method includes determining the level of GZMM protein in a sample from a subject or patient with cancer.

In some embodiments, the KIR2DL5 protein level is the sum of the KIR2DL5A and KIR2DL5B protein levels. In some embodiments, the KIR2DL5 protein level is the KIR2DL5A protein level. In some embodiments, the KIR2DL5 protein level is the KIR2DL5B protein level.

In some embodiments, the methods provided herein include determining a protein level for two or more of these biomarkers. In some embodiments, the methods include determining protein levels for three or more of these biomarkers. In some embodiments, the methods include determining protein levels for four or more of these biomarkers. In some embodiments, the methods include determining protein levels for five of these biomarkers.

In some embodiments, the methods provided herein further comprise determining levels of KIR2DS2 and KIR2DL2 proteins in a sample from a subject or patient with cancer and calculating the ratio of KIR2DS2 protein level to KIR2DL2 protein level (2DS2/2DL2 protein ratios). In some embodiments, the methods further comprise determining the levels of the KIR2DS5 and KIR2DL5 proteins in a sample from a subject or cancer patient and calculating the ratio of KIR2DS5 protein level to KIR2DL5 protein level (2DS5/2DL5 protein ratios). In some embodiments, the methods further comprise determining the levels of the KIR2DS2 and KIR2DL2 proteins in a sample from a cancer subject or patient, and the levels of KIR2DS5 and KIR2DL5 proteins in a sample from a cancer subject or patient, and calculating both the 2DS2/2DL2 protein ratio and protein ratios 2DS5/2DL5.

In some embodiments, the protein level of one or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM is determined by Western blot, ELISA, immunohistochemistry, flow cytometry, bead cytometry, or mass analysis. spectroscopy. In some embodiments, the protein level of one or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM is determined by ELISA.

3.3. Samples.

In some embodiments, methods for KIR typing, HLA typing, or methods for determining either an mRNA level or a protein level of one or more biomarkers further comprise obtaining a sample from a subject. The subject may be a mammal, such as a human. The subject may be male or female, and may be an adult, child, or neonate. The subject may be a patient. The patient may be a cancer patient. Samples can be analyzed during the time period during the active phase of the cancer (eg, lymphoma, MDS, or leukemia) or when the cancer is inactive. In specific embodiments, more than one sample may be obtained from a subject.

In some embodiments, KIR typing methods, HLA typing methods, or methods for determining either the mRNA level or the protein level of one or more biomarkers are performed as an adjunct diagnosis for the treatment of FTI. Concomitant diagnosis can be performed at the clinical center where the subject is being treated. Concomitant diagnosis may also be performed at a center other than the clinical center where the subject is being treated.

In specific embodiments, the sample used in the methods provided herein includes body fluids of the subject. Non-limiting examples of body fluids include blood (e.g. peripheral whole blood, peripheral blood), blood plasma, bone marrow, amniotic fluid, intraocular fluid, bile, lymph, menses, serum, urine, cerebrospinal fluid surrounding the brain and spinal cord, synovial fluid surrounding the joints of bones.

In one embodiment, the sample is a bone marrow sample. Methods for obtaining bone marrow samples are well known in the art, including, but not limited to, bone marrow biopsy and bone marrow aspiration. Bone marrow has a liquid part and a harder part. During a bone marrow biopsy, a sample of the hard part is taken. During aspiration of the bone marrow, a sample of the liquid part is taken. A bone marrow biopsy and a bone marrow aspiration may be performed at the same time and referred to as a bone marrow examination.

In some embodiments, the sample is a blood sample. The blood sample can be obtained using conventional methods as described, for example, in Innis et al., editors, PCR Protocols (Academic Press, 1990). Leukocytes can be isolated from blood samples using conventional methods or commercially available kits such as the RosetteSep kit (Stein Cell Technologies, Vancouver, Canada). Leukocyte subpopulations, such as mononuclear cells, NK cells, B cells, T cells, monocytes, granulocytes, or lymphocytes, can be further isolated using conventional methods, such as magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, Calif.) or fluorescence-activated cell sorting (FACS) (Becton Dickinson, San Jose, California).

In one embodiment, the blood sample is from about 0.1 ml to about 10.0 ml, from about 0.2 ml to about 7 ml, from about 0.3 ml to about 5 ml, from about 0.4 ml to about 3.5 ml or from about 0.5 ml to about 3 ml. In another embodiment, the blood sample is approximately 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2, 5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 ml.

In some embodiments, the sample used in the present methods includes a biopsy (eg, a tumor biopsy). The biopsy may be from any organ or tissue, such as skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph nodes, hair, spleen, brain, breast, or other organs. Any biopsy technique known to those skilled in the art can be used to isolate a sample from a subject, such as open biopsy, closed biopsy, core biopsy, incisional biopsy, excisional biopsy, or fine needle aspiration biopsy.

In one embodiment, the sample used in the methods provided herein is obtained from a subject prior to subjecting the subject to treatment for a disease or disorder. In another embodiment, the sample is obtained from the subject during the subject's exposure to treatment for a disease or disorder. In another embodiment, the sample is obtained from the subject after subjecting the subject to treatment for a disease or disorder. In various embodiments, the implementation of the treatment includes the introduction of FTI to the subject.

In specific embodiments, the sample used in the methods presented herein includes a plurality of cells. Such cells may include any cell type, such as stem cells, blood cells (eg, PBMC), lymphocytes, NK cells, B cells, T cells, monocytes, granulocytes, immunocytes, or tumor cells or cancer cells. In specific embodiments, the sample used in the methods provided herein comprises a plurality of enriched NK cells from a blood sample or a bone marrow sample from a subject or patient with cancer. Specific cell populations can be generated using a combination of commercially available antibodies (eg, Quest Diagnostic (San Juan Capistrano, Calif.); Dako (Denmark)).

In specific embodiments, the implementation of the sample used in the methods presented herein, comes from diseased tissue, such as from an individual with a malignant tumor (eg, lymphoma, MDS or leukemia). In some embodiments, cells can be obtained from tumor cells, or cancer cells, or tumor tissues, such as tumor biopsies or tumor explants. In particular embodiments

- 49 040014 the number of cells used in the methods presented herein can range from a single cell to about 10 ⁹ cells. In some embodiments, the number of cells used in the methods provided herein is approximately 1x10 ⁴ , 5x10 ⁴ , 1x105, 5x105, 1x10 ⁶ , 5x10 ⁶ , 1x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , or 5x10 ⁸ .

The number and type of cells collected from a subject can be monitored, for example, by measuring changes in cell morphology and surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining using tissue-specific or marker-specific cells antibodies), fluorescence activated cell sorting (FACS), magnetic field activated cell sorting (MACS), by checking cell morphology using light or confocal microscopy, and/or by measuring changes in gene expression using methods well known in the art, such as PCR and gene expression profiling. These methods can also be used to identify cells that are positive for one or more specific markers. Fluorescence activated cell sorting (FACS) is a well known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laser excitation of the fluorescent groups in the individual particles results in a small electrical charge allowing the electromagnetic separation of positive and negative particles from the mixture. In one embodiment, specific antibodies or ligands for cell surface markers are labeled with different fluorescent labels. The cells are passed through a cell sorter, allowing cells to be separated based on their ability to bind the antibodies used. FACS-sorted particles can be deposited directly into individual wells of 96-well or 384-well plates for ease of separation and cloning.

In specific embodiments, subsets of cells are used in the methods provided herein. Methods for sorting and isolating specific cell populations are well known in the art and may be based on cell size, morphology, or intracellular or extracellular markers. Such methods include, but are not limited to, flow cytometry, flow sorting, FACS, bead-based separation such as magnetic cell sorting, size-based separation (e.g., using a sieve, array of obstacles, or filter), microfluidic sorting, separation based on antibodies, precipitation, affinity adsorption, affinity extraction, density gradient centrifugation, laser capture microdissection, etc. In some embodiments, the sample includes enriched NK cells sorted by one or more of the methods described herein or otherwise known in the art. In one embodiment, enriched NK cells are further expanded in vitro prior to being subjected to KIR typing, HLA typing, or expression level analysis of one or more biomarkers selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM.

The sample may be a whole blood sample, a bone marrow sample, a partially purified blood sample, a PBMC, or a biopsy sample. In some embodiments, the sample is a bone marrow sample from a cancer patient. In some embodiments, the sample is a PBMC from a cancer patient. In some embodiments, the sample is enriched in NK cells from bone marrow, whole blood, or partially purified blood. In some embodiments, the NK cells are further propagated in vitro. Methods for obtaining a sample from a subject and methods for preparing a sample to determine either the mRNA level or the protein level of one or more biomarkers are well known in the art.

3.4. Reference levels and reference ratios.

Provided herein are methods of treating a subject using a therapeutically effective amount of FTI or selecting a cancer patient for FTI treatment based on KIR typing, expression level (either mRNA level or protein level) of one or more individual biomarkers selected from the group, consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5 and GZMM, or any or both of the ratio of expression levels between biomarkers (2DS2/2DL2 ratios; 2DS5/2DL5 ratios). In some embodiments, a cancer patient is selected for FTI treatment if the cancer patient is a carrier of KIR2DS2 or KIR2DS5 or both. In some embodiments, a cancer patient is selected for FTI treatment if (i) the expression level of KIR2DS2 in a sample from the subject is higher than a reference level of KIR2DS2 expression;

(ii) the level of expression of KIR2DL2 in the sample from the subject is lower than the reference level of expression of KIR2DL2;

(iii) the level of expression of KIR2DS5 in the sample from the subject is higher than the reference level of expression of KIR2DS5;

- 50 040014 (iv) the level of expression of KIR2DL5 in the sample from the subject is lower than the reference level of expression

KIR2DL5;

(v) the level of GZMM expression in the sample from the subject is higher than the reference level of GZMM expression;

(vi) the ratio of KIR2DS2 expression level to KIR2DL2 expression level in the sample from the subject is higher than the reference ratio of KIR2DS2 expression level to KIR2DL2 expression level; or (vii) the ratio of KIR2DS5 expression level to KIR2DL5 expression level in the sample from the subject is higher than the reference ratio of KIR2DS5 expression level to KIR2DL5 expression level;

or any combination of (i)-(vii) is present.

The methods provided herein may further include determining a reference expression level for each individual biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, or a reference ratio between biomarker expression levels, including a 2DS2/2DL2 reference ratio and a 2DS5 reference ratio. /2DL5. In some embodiments, the reference level of biomarker expression is the level of expression of the biomarker in a sample from a healthy individual, or the average or median level of expression of the biomarker in multiple samples from one or multiple healthy individuals. In some embodiments, the reference biomarker expression level is the average expression level of the biomarker in samples from 2, 3, 5, 10, 15, 20, 30, 40, 50 or more healthy individuals. In some embodiments, the reference level of biomarker expression is the median level of biomarker expression in samples from 2, 3, 5, 10, 15, 20, 30, 40, 50 or more healthy individuals.

In some embodiments, the reference level of KIR2DS2 expression is the level of KIR2DS2 expression in a sample from a healthy individual, or the average or median level of KIR2DS2 expression in multiple samples from one or multiple healthy individuals. In some embodiments, the reference level of KIR2DL2 expression is the level of KIR2DL2 expression in a sample from a healthy individual, or the average or median level of KIR2DL2 expression in multiple samples from one or multiple healthy individuals. In some embodiments, the reference level of KIR2DS5 expression is the level of KIR2DS5 expression in a sample from a healthy individual, or the average or median level of KIR2DS5 expression in multiple samples from one or multiple healthy individuals. In some embodiments, the reference level of KIR2DL5 expression is the level of KIR2DL5 expression in a sample from a healthy individual, or the average or median level of KIR2DL5 expression in multiple samples from one or multiple healthy individuals. In some embodiments, the reference level of GZMM expression is the level of GZMM expression in a sample from a healthy individual, or the average or median level of GZMM expression in multiple samples from one or multiple healthy individuals.

In some embodiments, the methods provided herein further comprise determining a reference ratio between expression levels of two biomarkers, such as a reference 2DS2/2DL2 expression ratio or a reference 2DS5/2DL5 expression ratio. In some embodiments, the reference biomarker expression ratio is the ratio of biomarker expression in a sample from a healthy individual, or the average or median ratio of biomarker expression in multiple samples from one or multiple healthy individuals. In some embodiments, the implementation of the reference ratio of expression of two biomarkers is the average ratio of expression of biomarkers in samples from 2, 3, 5, 10, 15, 20, 30, 40, 50 or more healthy individuals. In some embodiments, the reference ratio of expression of two biomarkers is the median ratio of expression of biomarkers in samples from 2, 3, 5, 10, 15, 20, 30, 40, 50 or more healthy individuals.

In some embodiments, the 2DS2/2DL2 reference ratio is the 2DS2/2DL2 ratio in a sample from a healthy individual, or the mean or median 2DS2/2DL2 ratio across multiple samples from one or multiple healthy individuals. In some embodiments, the 2DS5/2DL5 reference ratio is 2DS5/2DL5 in a sample from a healthy individual, or the mean or median of 2DS5/2DL5 across multiple samples from one or multiple healthy individuals.

In some embodiments, a reference biomarker expression level, or a reference ratio between expression levels of two biomarkers, may be determined based on historical data from prior clinical studies, including outcome for a patient group, namely the ability of patients to respond to FTI treatment, as well as biomarker expression levels or ratio expression levels between biomarkers for a group of patients. A number of statistical methods are well known in the art for determining a reference level (or referred to as a cutoff value) of one or more biomarkers when used to predict a patient's ability to respond to a particular treatment or to stratify patients for a particular treatment.

One method of the invention includes analyzing gene expression profiles for the biomarkers identified herein that distinguish responders from non-responders to determine a reference level of expression for one or more biomarkers. Comparison between responders and non-responders can be made using the Mann-Whitney U-test, chi-square test, or Fisher's exact test. Descriptive statistics analyzes and comparisons can be made using SigmaStat software (Systat Software, Inc., San Jose, CA, USA).

In some embodiments, a classification and regression tree analysis (CART) may be taken to determine a benchmark. CART analysis is based on a binary recursive splitting algorithm and allows the identification of complex interactions of prognostic variables that may not be obvious from more traditional methods such as multiple linear regression. Binary recursive splitting refers to an analysis that is: 1) binary, meaning that there are two possible outcome variables, namely responders and non-responders, with the effect of dividing patients into 2 groups; 2) recursive, which means that the analysis can be carried out many times; and 3) splitting, which means that the entire data set can be divided into parts. This analysis also has the ability to exclude predictive variables with poor performance. The classification tree can be built using Salford Predictive Modeler v6.6 (Salford Systems, San Diego, CA, USA).

The articles of the invention are representations of gene expression profiles that can be used to predict the ability of a cancer patient to respond to FTI treatment, translated into a medium that can be automatically read, such as computer-readable media (magnetic, optical, etc.). The products may also include instructions for evaluating gene expression profiles on such media. For example, the products may include a CD-ROM containing computer instructions for comparing gene expression profiles for the biomarkers described above. The articles may also contain gene expression profiles digitally recorded on them so that they can be compared with gene expression data from patient samples. Alternatively, the profiles may be recorded in various presentation formats. Clustering algorithms, such as those built into the OMNIVIZ and TREE VIEW computer programs mentioned above, can aid visualization.

Receiver performance analysis (ROC) can be used to determine a reference expression level or a reference expression ratio, or to test the overall predictive value of individual genes and/or multigene classifiers. An overview of ROC analysis can be found in Soreide, J Clin Pathol 10,1136 (2008), the entire contents of which are hereby incorporated by reference.

The reference level can be determined from the ROC curve for the training set to provide both high sensitivity and high specificity. Exclusion cross-validation (LOOCV) can be used to determine how many biomarkers to include in the prediction. Record the response rates for the excluded samples based on different numbers of genes. The performance of predictions with different numbers of genes can be assessed based on misclassification error rate, sensitivity, specificity, p-values to measure the separation of the Kaplan-Meier curves for the two predicted groups.

You can use the highest scoring pair (TSP) algorithm pioneered by Geman et al. (2004). Essentially, the algorithm ranks all pairs of genes (genes i and j) based on the absolute difference (Dij) in the frequency of the event when gene i is more expressed than gene j in samples among classes C1-C2. In cases where there are multiple high-scoring pairs (where everyone shares the same Dij), the top pair is selected based on the second rank score, which measures the amplitude at which expression levels shift from one class to another within a gene pair. The top pair with the highest absolute Dij>2-fold frequency in all samples can be selected as a candidate pair. The candidate pair can then be evaluated on an independent test set of data. Exclusion cross-validation (LOOCV) can be performed on the training data set to evaluate the performance of the algorithm. The performance of forecasts can be evaluated based on the maximum error rate due to misclassification. All statistical analyzes can be performed using R (R Development Core Team, 2006).

An overview of methods and statistical tools that can be used to determine a benchmark can be found in James Westgard, Ph.D., Basic Methods Validation, 3 ^d edition (2008), the entire contents of which are hereby incorporated by reference. Specific references are to Section 9 (How is a reportable range of a method determined) and Section 15 (How is a reference interval verified).

The clinically detectable range (CRR) is the range of analyte levels that can be measured by a method that allows dilution, concentration, or other sample pretreatment used to extend the range of direct analytical measurement. As presented in Basic Methods Validation by Dr. Westgard, the experiment to be performed is often referred to as a linearity experiment, although there is no technical requirement that the method provide a linear response unless a calibration is used.

- 52 040014 two points. This range may also be referred to as the linear range, analytical range, or process operating range.

The recorded range is estimated by checking the linearity of the graph. This check may involve manually drawing the best straight line over the linear portion of the points, drawing a line connecting the points through all points, then comparing with the best straight line, or fitting a regression line through the points in the linear range. There are more complex statistical calculations recommended in some guidelines, such as the Clinical and Laboratory Standards Institute (CLSI) protocol EP-6, to assess the linearity of analytical methods. However, it is generally accepted that the detectable range can be adequately determined by visual assessment, ie. by manually constructing the best straight line corresponding to the lowest points in the series. The Clinical and Laboratory Standards Institute (CLSI) recommends at least a minimum of 4 to preferably 5 different concentration levels. More than 5 may be used, particularly if it is necessary to maximize the upper limit of the recording range, but 5 levels are convenient and almost always sufficient.

The reference interval is typically established by analyzing samples obtained from individuals meeting carefully defined criteria (reference sample group). Protocols such as those of the International Federation of Clinical Chemistry (IFCC) Expert Group on the Theory of Reference Values and CLSI describe exhaustive systematic processes that use carefully selected groups of samples to establish reference intervals. These protocols typically require a minimum of 120 reference individuals for each group (or subgroup) to be characterized.

The CLSI-approved guide C28-A2 describes various methods for laboratories to verify the transfer of established reference intervals to an individual laboratory, including

1) an intuitive solution, where the laboratory staff simply reviews the information received and subjectively confirms that the reference intervals are acceptable for the patient population and testing methods accepted by the laboratory;

2) confirmation for 20 samples, where experimental verification is carried out by collecting and analyzing samples from 20 individuals representing a reference population of samples;

3) evaluation using 60 samples, where experimental verification is carried out by collecting and analyzing samples from 60 individuals representing a reference population of samples, and the actual reference interval is determined and compared with the declared or published interval using a statistical formula comparing the means and standard deviations of the two populations ; And

4) calculation by comparative method, where it is possible to adjust or correct the declared or published reference intervals based on the observed methodological bias and the mathematical relationship between the methods used.

One skilled in the art will appreciate that the reference levels of expression of the biomarkers described herein, as well as the reference ratios between two biomarkers, can be determined by one or more of the methods as presented herein or by other methods known in the art.

Accordingly, in some embodiments, the methods provided herein include

a) determining a reference level of expression of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, GZMM; And

b) administering a therapeutically effective amount of FTI to a cancer patient if (i) the expression level of KIR2DS2 in a sample from the cancer patient is higher than a reference KIR2DS2 expression level;

(ii) the level of expression of KIR2DL2 in a sample from a patient with a malignant tumor is lower than the reference level of expression of KIR2DL2;

(iii) the level of expression of KIR2DS5 in a sample from a patient with a malignant tumor is higher than the reference level of expression of KIR2DS5;

(iv) the level of expression of KIR2DL5 in a sample from a patient with a malignant tumor is lower than the reference level of expression of KIR2DL5; or (v) the level of GZMM expression in the sample from the cancer patient is higher than the reference level of GZMM expression;

or any combination of (i)-(v) is present.

In some embodiments, the expression level of KIR2DL5 is the sum of the expression levels of KIR2DL5A and KIR2DL5B. In some embodiments, the expression level of KIR2DL5 is the expression level of KIR2DL5A. In some embodiments, the expression level of KIR2DL5 is the expression level of KIR2DL5B.

Accordingly, in some embodiments, the implementations presented herein

- 53 040014 methods include

a) determination of a reference mRNA level of a biomarker selected from the group consisting of

KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, GZMM; And

b) administering a therapeutically effective amount of FTI to a cancer patient if (i) the level of KIR2DS2 mRNA in a sample from the cancer patient is higher than a reference level of KIR2DS2 mRNA;

(ii) the level of KIR2DL2 mRNA in a sample from a cancer patient is lower than the reference level of KIR2DL2 mRNA;

(iii) the level of KIR2DS5 mRNA in a sample from a cancer patient is higher than the reference level of KIR2DS5 mRNA;

(iv) the level of KIR2DL5 mRNA in a sample from a cancer patient is lower than the reference level of KIR2DL5 mRNA; or (v) the level of GZMM mRNA in the sample from the cancer patient is higher than the reference level of GZMM mRNA;

or any combination of (i)-(v) is present.

In some embodiments, the KIR2DL5 mRNA level is the sum of the KIR2DL5A and KIR2DL5B mRNA levels. In some embodiments, the KIR2DL5 mRNA level is the KIR2DL5A mRNA level. In some embodiments, the KIR2DL5 mRNA level is the KIR2DL5B mRNA level.

Accordingly, in some embodiments, the methods provided herein include

a) determining a reference level of a biomarker protein selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, GZMM; And

b) administering a therapeutically effective amount of FTI to a cancer patient if (i) the level of KIR2DS2 protein in a sample from the cancer patient is higher than a reference level of KIR2DS2 protein;

(ii) the level of KIR2DL2 protein in a sample from a cancer patient is lower than the reference level of KIR2DL2 protein;

(iii) the level of KIR2DS5 protein in the sample from the cancer patient is higher than the reference level of KIR2DS5 protein;

(iv) the level of KIR2DL5 protein in a sample from a cancer patient is lower than the reference level of KIR2DL5 protein; or (v) the GZMM protein level in the sample from the cancer patient is higher than the reference GZMM protein level;

or any combination of (i)-(v) is present.

In some embodiments, the KIR2DL5 protein level is the sum of the KIR2DL5A and KIR2DL5B protein levels. In some embodiments, the KIR2DL5 protein level is the KIR2DL5A protein level. In some embodiments, the KIR2DL5 protein level is the KIR2DL5B protein level.

In some embodiments, the methods provided herein include

a) determination of the 2DS2/2DL2 reference ratio or the 2DS5/2DL5 reference ratio; And

b) administering a therapeutically effective amount of FTI to a cancer patient if (i) the 2DS2/2DL2 ratio in the sample from the cancer patient is higher than the reference 2DS2/2DL2 ratio; or (ii) the 2DS5/2DL5 ratio in a sample from a cancer patient is higher than the reference 2DS5/2DL5 ratio;

or both (i) and (ii) are present.

In some embodiments, the 2DS2/2DL2 ratio is the ratio of the level of KIR2DS2 mRNA to the level of KIR2DL2 mRNA. In some embodiments, the 2DS2/2DL2 ratio is the ratio of the KIR2DS2 protein level to the KIR2DL2 protein level. In some embodiments, the 2DS5/2DL5 ratio is the ratio of the level of KIR2DS5 mRNA to the level of KIR2DL5 mRNA. In some embodiments, the 2DS5/2DL5 ratio is the ratio of the KIR2DS5 protein level to the KIR2DL5 protein level.

3.5. Malignant tumors.

Provided herein are methods for treating cancer in a subject with FTI, and methods for selecting patients with cancer for FTI treatment. The cancer may be a hematopoietic cancer or a solid tumor. Also provided herein are methods for treating a pre-malignant condition in a subject using FTI and methods for selecting patients with a pre-malignant condition for treatment with FTI. IN

- 54 040014 In some embodiments, the FTI is tipifarnib.

In some embodiments, provided herein are methods for treating hematopoietic cancer in a subject using FTI or selecting patients with cancers for FTI treatment. Hematologic malignancies are malignant tumors of the blood or bone marrow. Examples of hematologic (or hematogenous) malignancies include leukemias, lymphoma, and myelodysplastic syndrome (MDS).

Leukemia refers to malignant neoplasia of hematopoietic tissues. Various forms of leukemia are described, for example, in US patent No. 7393862 and provisional patent application US No. 60/380842, filed May 17, 2002, the entire contents of which are incorporated herein by reference. Although viruses have been reported to cause several forms of leukemia in animals, the causes of leukemia in humans remain largely unknown. The Merck Manual, 944-952 ( ^17th ed. 1999). Transformation to malignancy typically occurs in a single cell in two or more stages, followed by proliferation and clonal expansion. In some leukemias, specific chromosomal translocations have been identified with corresponding leukemia cell morphology and specific clinical features (eg, translocations 9 and 22 in chronic myelocytic leukemia, and 15 and 17 in acute promyelocytic leukemia). Acute leukemias are predominantly undifferentiated cell populations, while chronic leukemias are more mature cell forms.

Acute leukemias are divided into lymphoblastic (ALL) and non-lymphoblastic (ANLL) types. The Merck Manual, 946-949 ( ^17th ed. 1999). They can be further subdivided according to their morphological and cytochemical manifestations according to the Franco-American-British (FAB) classification or according to their type and degree of differentiation. The use of B- and T-cell-specific and myeloid antigen-specific monoclonal antibodies best aids classification. ALL is predominantly a disease of childhood, which is determined by the results of laboratory tests and examination of the bone marrow. ANLL, also known as acute myelogenous leukemia or AML, occurs at any age and is the more common acute leukemia in adults; it is the form usually associated with radiation as an etiological factor. In some embodiments, provided herein are methods for treating patients with AML using FTI or methods for selecting patients for treatment with FTI.

Standard treatments for patients with AML usually include 2 phases of chemotherapy (chemo): remission induction (or induction) and consolidation (post-remission therapy). The goal of the first part of treatment (induction of remission) is to get rid of as many leukemic cells as possible. The intensity of treatment may depend on the age and health of the individual. Intensive chemotherapy is often given to people under the age of 60. Slightly older patients in good general health may benefit from similar or slightly less intense treatment. People who are much older or have poor general health are not suitable for intensive chemotherapeutic agents.

In younger patients, such as those under 60 years of age, induction often involves treatment with 2 chemotherapeutic agents, cytarabine (ara-C) and an anthracycline drug such as daunorubicin (daunomycin) or idarubicin. Sometimes a third drug, cladribine (leustatin, 2-CdA), is also administered. Chemotherapy is usually given in a hospital and continues for about a week. In rare cases where the leukemia has spread to the brain or spinal cord, cerebrospinal fluid (CSF) chemotherapy may also be given. Radiotherapy can also be used.

Induction is considered successful if remission is achieved. However, AML in some patients may be resistant to induction. Patients who respond to induction are then subjected to further treatment to destroy the remaining leukemic cells and help prevent recurrence, which is called consolidation. For younger patients, the main options for consolidation therapy are: multiple cycles of high-dose cytarabine (ara-C) chemotherapy (known in some cases as HiDAC); transplantation of allogeneic (donor) stem cells; and transplantation of autologous stem cells.

Chronic leukemias are described as being lymphocytic (CLL) or myelocytic (CML). The Merck Manual, 949-952 ( ^17th ed. 1999). CLL is characterized by the appearance of mature lymphocytes in the blood, bone marrow, and lymphoid organs. The hallmark of CLL is persistent absolute lymphocytosis (>5000/μl) and an increase in the number of lymphocytes in the bone marrow. In most patients with CLL, there is also a clonal expansion of lymphocytes with B-cell characteristics. CLL is a disease of middle or older age. In CML, a characteristic feature is the predominance of granulocytes at all stages of differentiation in the blood, bone marrow, liver, spleen, and other organs. In a symptomatic patient at diagnosis, the total leukocyte count (WBC) is usually around 200,000/mcL, but can be as high as 1,000,000/mcL. CML is relatively easy to diagnose due to the presence of the Philadelphia chromosome. Fine

- 55 040014 Bone marrow stromal cells are known to support the progression of CLL disease and resistance to chemotherapy. Disruption of interactions between CLL cells and stromal cells is an additional target of CLL chemotherapy.

In addition, other forms of CLL include prolymphocytic leukemia (PLL), large granular lymphocyte leukemia (LGL), hairy cell leukemia (HCL). Cancer cells in PLL are similar to normal cells called prolymphocytes, immature forms of B-lymphocytes (B-PLL) or T-lymphocytes (T-PLL). Both B-PLL and T-PLL tend to be more aggressive than the normal type of CLL. Malignant cells in LGL are large and have features of either T cells or NK cells. Most LGL leukemias are slow growing, however a small number are more aggressive. HCL is another lymphocyte malignancy that tends to progress slowly and accounts for approximately 2% of all leukemias.

Malignant cells are of the B-lymphocyte type, but differ from the malignant cells seen in CLL.

Chronic myelomonocytic leukemia (CMML) is classified as myelodysplastic/myeloproliferative neoplasia in the 2008 World Health Organization hematopoietic tumor classification. Patients with CMML have high blood monocyte counts (at least 1000 per mm ³ ). Two classes - myelodysplastic and myeloproliferative - are distinguished on the basis of the level of the number of leukocytes (threshold 13 g/l). Often, the number of monocytes is much higher, which causes the total white blood cell count to become very high as well. Normally, abnormal cells are present in the bone marrow, but the number of blast cells is less than 20%. In approximately 15-30% of patients with CMML, the process continues until the development of acute myeloid leukemia. The diagnosis of CMML is based on a combination of morphological, histopathological, and chromosomal abnormalities in the bone marrow. The Mayo predictive model classified patients with CMML into three risk groups based on: increased absolute monocyte count, presence of circulating blasts, hemoglobin <10 g/dl, and platelets <100 x 10 ⁹ /l. Median survival was 32 months, 18.5 months, and 10 months in the low, intermediate, and high risk groups, respectively. The Groupe Francophone (GFM) index divided patients with CMML into three risk groups based on: age >65 years, WBC>15χ10 ⁹ /L, anemia, platelets <100x10 ⁹ /L, and ASXL1 mutation status. After a median follow-up of 2.5 years, survival ranged from not achieved in the low-risk group to 14.4 months in the high-risk group.

Lymphoma refers to malignant tumors originating from the lymphatic system. Lymphoma is characterized by malignant neoplasia of lymphocytes - B-lymphocytes (B-cell lymphoma), T-lymphocytes (T-cell lymphoma) and natural killer cells (NK cell lymphoma). Lymphoma typically begins in the lymph nodes or collections of lymphatic tissue in organs including, but not limited to, the stomach or intestines. Lymphoma can affect the bone marrow and blood in some cases. Lymphoma can spread from one area to other parts of the body.

Treatments for various forms of lymphomas are described, for example, in US Pat. No. 7,468,363, the entire contents of which are incorporated herein by reference. Such lymphomas include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous B cell lymphoma, activated B cell lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL; including but not limited to FL grade I, FL grade II), follicle center cell lymphoma, transformed lymphoma, intermediate grade lymphocytic lymphoma, intermediate lymphocytic lymphoma (ILL), diffuse low-grade lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse lymphoma small cell cleaved cell (DSCCL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL) and mantle cell lymphoma, and low-grade follicular lymphoma.

Non-Hodgkin's lymphoma (NHL) is the fifth most common malignancy in both men and women in the United States, with an estimated 63,190 new cases and 18,660 deaths in 2007. Jemal A, et al., CA Cancer J Clin 2007; 57(1):43-66. The likelihood of developing NHL increases with age, and the incidence of NHL in the elderly has increased steadily over the past decade, raising concerns about the aging trend in the US population. Id. Clarke CA, et al., Cancer 2002; 94(7):2015-2023.

DLBCL accounts for approximately one third of non-Hodgkin's lymphomas. While some patients with DLBCL are cured with conventional chemotherapy, the rest die from the disease. Anticancer drugs cause rapid and persistent depletion of lymphocytes, possibly through direct induction of apoptosis in mature T and B cells. See K. Stahnke. et al., Blood 2001, 98:3066-3073. The absolute lymphocyte count (ALC) has been shown to be a prognostic factor in follicular non-Hodgkin's lymphoma, and recent results

- 56 040014 suggest that ALC at diagnosis is an important prognostic factor in DLBCL.

DLBCLs can be divided into distinct molecular subtypes according to their patterns of gene profiles: DLBCL from germinal center B-cell-like cells (GCB-DLBCL), DLBCL from activated B-cell-like cells (ABC-DLBCL), and primary mediastinal B-cell lymphoma ( PMBL) or unclassified type. These subtypes are characterized by distinct differences in survival, ability to respond to chemotherapy, and dependence on the signal transduction pathway, in particular the NF-kB pathway. See D. Kim et al., Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol. 25, no. 18S (June 20 Supplement), 2007: 8082. See Bea S, et al., Blood 2005; 106:3183-90; Ngo VN et al., Nature 2011; 470:115-9. Such differences have prompted the search for more effective and subtype-specific treatments for DLBCL. In addition to being divided into acute and chronic categories, neoplasias are also divided into progenitor or peripheral cell categories based on the cells that give rise to such a disorder. See, for example, US Patent Publication No. 2008/0051379, the entire disclosure of which is incorporated herein by reference. Progenitor cell neoplasias include ALL and lymphoblastic lymphomas and occur in lymphocytes prior to their differentiation into either T or B cells. Peripheral cell neoplasias are neoplasias arising in lymphocytes that have differentiated into either T or B cells. Such peripheral cell neoplasias include, but are not limited to, B-cell CLL, B-cell prolymphocytic leukemia, lymphoplasmic lymphoma, mantle cell lymphoma, follicular lymphoma, extranodal marginal zone B-cell lymphoma of mucosal lymphoid tissue, nodal marginal lymphoma zones, marginal zone lymphoma of the spleen, hairy cell leukemia, plasmacytoma, diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma. In more than 95 percent of CLL cases, clonal expansion occurs in the B cell line. See Cancer: Principles & Practice of Oncology ( ^3rd Edition) (1989) (pp. 1843-1847). Less than 5% of CLL tumor cells have a T cell phenotype. Regardless of these classifications, however, the pathological disruption of normal hematopoiesis is the hallmark of all leukemias.

PTCL consists of a group of rare and usually aggressive (fast growing) NHLs that develop from mature T cells. PTCLs together account for approximately 4 to 10% of all NHL cases, corresponding to an annual incidence of 2800-7200 patients per year in the United States. According to some estimates, the incidence of PTCL is increasing significantly, and the increased incidence may be due to an aging population. PTCL is further classified into various subtypes, each of which is generally considered to be a separate disease based on their distinct clinical differences. Most of these subtypes are rare; the three most common subtypes of PTCL are, without further specification, anaplastic large cell lymphoma, or ALCL, and angioimmunoblastic T-cell lymphoma, which together account for 70% of all PTCLs in the United States. ALCL may be cutaneous ALCL or systemic ALCL.

For most PTCL subtypes, the first-line regimen is usually combination chemotherapy such as CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), EPOCH (etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone), or other multidrug regimens. Patients relapsing or refractory to first-line therapy are usually treated with gemcitabine in combination with other chemotherapy agents, including vinorelbine (Navelbine®) and doxorubicin (Doxil®), in a regimen called GND, or other chemotherapy regimens such as as DHAP (dexamethasone, cytarabine, cisplatin) or ESHAP (etoposide, methylprednisolone, cytarabine and cisplatin).

Because most patients with PTCL may relapse, some oncologists have recommended high-dose chemotherapeutic agents followed by autologous stem cell transplantation in some patients who have responded well to initial chemotherapy. Recently, non-cytotoxic drugs approved for relapsed or refractory PTCL, such as pralatrexate (Folotin®), romidepsin (Istodax®), and belinostat (Beleodak®), have been associated with relatively low objective response rates (overall response rate, or ORR, 25-27 %) and a relatively short duration of response (8.2-9.4 months). Accordingly, the treatment of relapsed/refractory PTCL remains an important unmet medical need.

Multiple myeloma (MM) is a malignant tumor of plasma cells in the bone marrow. Normally, plasma cells produce antibodies and play a key role in immune function. However, uncontrolled growth of these cells leads to pain and fractures in the bones, anemia, infections and other complications. Multiple myeloma is the second most common hematological malignancy, although the exact causes of multiple myeloma remain unknown. Multiple myeloma causes high levels of proteins in

- 57 040014 blood, urine and organs, including, but not limited to, M-protein and other immunoglobulins (antibodies), albumin, and beta-2-microglobulin. M-protein, short for monoclonal protein, also known as paraprotein, is a particularly abnormal protein produced by myeloma plasma cells and can be found in the blood or urine of almost all patients with multiple myeloma.

Skeleton-related symptoms, including bone pain, are among the most clinically important symptoms of multiple myeloma. Malignant plasma cells release osteoclast-stimulating factors (including IL-1, IL-6, and TNF) that cause calcium to be leached from bones, causing lytic lesions; hypercalcemia is another symptom. Osteoclast stimulating factors, also referred to as cytokines, may prevent apoptosis or death of myeloma cells. Fifty percent of patients have radiologically detectable myeloma-related skeletal lesions at diagnosis. Other common clinical symptoms of multiple myeloma include polyneuropathy, anemia, increased blood viscosity, infections, and kidney failure.

It is well known that bone marrow stromal cells support the progression and resistance to chemotherapy of multiple myeloma disease. Disruption of interactions between multiple myeloma cells and stromal cells is an additional target for multiple myeloma chemotherapy.

Myelodysplastic syndrome (MDS) refers to a heterogeneous group of hematopoietic stem cell disorders. MDS can be characterized by an increase in the number of cells in the bone marrow with abnormal morphology and maturation (dysmyelopoiesis), inefficient production of blood cells, or hematopoiesis, leading to a reduced number of blood cells, or cytopenias, and a high risk of progression to acute myeloid leukemia resulting from ineffective production blood cells. See The Merck Manual 953 ( ^17th ed. 1999) and List et al., 1990, J Clin. oncol. 8:1424.

As a group of hematopoietic stem cell malignancies with significant morbidity and mortality, MDS is a highly heterogeneous disease, and symptom severity and disease progression can vary greatly among patients. The current standard clinical tool for evaluating risk stratification and treatment options is the Modified International Predictive Scoring System, or IPSS-R. The IPSS-R categorizes patients into five risk groups (very low, low, intermediate, high, very high) based on cytogenetics, percentage of blasts (undifferentiated blood cells) in the bone marrow, hemoglobin levels, and platelet and neutrophil counts. The WHO also proposes a stratification of patients with MDS by anomaly - deletion (5q).

According to the ACS, the annual incidence of MDS is approximately 13,000 patients in the United States, most of whom are 60 years of age or older. The estimated prevalence is over 60,000 patients in the United States. Approximately 75% of patients fall into the IPSS-R very low, low, and intermediate risk categories, or together are known as lower risk MDS patients.

Initial damage to hematopoietic stem cells can occur for reasons such as, but not limited to, cytotoxic chemotherapy, radiation, virus, chemical exposure, and genetic predisposition. The bone marrow is dominated by a clonal mutation that suppresses healthy stem cells. In the early stages of MDS, the main cause of cytopenias is an increase in programmed cell death (apoptosis). As the disease progresses to leukemia, a mutation in the gene rarely occurs, and the proliferation of leukemic cells suppresses healthy bone marrow. The course of the disease is different, where in some cases it behaves like an indolent disease, and in other cases it behaves aggressively with a very short clinical course of the disease, turning into an acute form of leukemia.

An international group of hematologists, the Franco-American-British (FAB) Joint Group, has classified MDS disorders into five subgroups, distinguishing them from AML. The Merck Manual 954 ( ^17th ed. 1999); Bennett JM, et al., All. Intern. Med. October 1985, 103(4): 620-5; and Besa E. C, Med. Clin. North Am. 1992 May, 76(3):599-617. An underlying trilinear dysplastic change in the patients' bone marrow cells was found in all subtypes.

There are two subgroups of refractory anemia characterized by 5% or less myeloblasts in the bone marrow: (1) refractory anemia (RA) and; (2) RA with ringed sideroblasts (RARS), defined morphologically as having 15% of erythroid cells with abnormal ringed sideroblasts, reflecting the abnormal accumulation of iron in the mitochondria. Both have a long clinical course and a low incidence of progression to acute leukemia. Besa E. C, Med. Clin. North Am. 1992 May, 76(3): 599-617.

There are two subgroups of non-responsive anemia with more than five percent of myeloblasts: (1) RA with excess blasts (RAEB), with a definition of 6-20% myeloblasts, and (2) RAEB in the transformation stage (RAEB-T), with 21-30 % myeloblasts. The higher the percentage of myeloblasts, the shorter the clinical course and the closer the disease to acute myelogenous leukemia. Transition of patients from early

- 58 040014 to later stages indicates that these subtypes are simply disease stages and not individual units. Elderly MDS patients with trilinear dysplasia and more than 30% myeloblasts progressing to acute leukemia are often considered to have a poor prognosis because their response rate to chemotherapy is lower than that of patients with de novo acute myeloid leukemia. The fifth type of MDS, the most difficult to classify, is CMML. This subtype can have any percentage of myeloblasts, but presents with monocytosis of 1000/dl or more. It may be associated with splenomegaly. This subtype overlaps with a myeloproliferative disorder and may have an intermediate clinical course. It differs from classical CML in that it is negatively characterized by the Ph chromosome.

MDS is primarily a disease of the elderly, with a median onset in the seventh decade of life. The median age of these patients is 65 years, where the age ranges from the beginning of the third decade of life to as old as 80 years or older. The syndrome can occur in any age group, including pediatric patients. Patients surviving cancer treatment with alkylating agents, with or without radiotherapy, have a high incidence of developing MDS or secondary acute leukemia. Approximately 60-70% of patients have no obvious effect or cause causing MDS and are classified as primary MDS patients.

The treatment of MDS is based on the stage and mechanism of the disease, where the particular phase of the disease development process predominates. Bone marrow transplantation is used for patients with poor prognosis or advanced MDS. Epstein and Slease, 1985, Surg. Ann. 17:125. An alternative therapy for MDS is the use of hematopoietic growth factors or cytokines to stimulate the development of blood cells in the recipient. Dexter, 1987, J. Cell Sci. 88:1; Moore, 1991, Annu. Rev. Immunol. 9:159; and Besa E. C, Med. Clin. North Am. 1992 May, 76(3):599-617. Treatment of MDS using immunomodulatory compounds is described in US Pat. No. 7,189,740, the entire contents of which are hereby incorporated by reference.

Possible therapies fall into three categories, including maintenance therapy, low intensity therapy, and high intensity therapy. Maintenance therapy includes the use of transfusions of erythrocytes and platelets, and hematopoietic cytokines, such as erythropoiesis-stimulating agents or colony-stimulating factors, to improve the number of blood cells. Low-intensity therapies include hypomethylating agents such as azacitidine (Vidaza®) and decitabine (Dacogen®), biological response modifiers such as lenalidomide (Revlimid®), and immunosuppressive drugs such as cyclosporine A or antithymocyte globulin. High-intensity therapies include chemotherapy agents such as idarubicin, azacitidine, fludarabine, and topotecan, and hematopoietic stem cell transplantation, or HSCT.

The US National Comprehensive Cancer Network, or NCCN, guidelines recommend subjecting lower-risk patients (IPSS-R groups of very low, low, intermediate risk) to maintenance or low-intensity therapy with the primary therapeutic goal of improving haematological parameters or HI. NCCN guidelines recommend subjecting higher-risk patients (high-risk, very-high-risk IPSS-R groups) to more aggressive treatment using high-intensity therapy. In some cases, high-risk patients are unable to tolerate chemotherapy and lower intensity regimens can be selected. Despite currently available treatments, there are no effective therapies for a large proportion of patients with MDS, and NCCN guidelines recommend clinical trials as additional therapeutic options. The treatment of MDS remains an important unmet need requiring the development of new therapies.

Accordingly, in some embodiments, provided herein are methods of treating a hematopoietic cancer in a subject using FTI, or selecting a patient with hematopoietic cancer for FTI treatment, wherein the patient with hematopoietic cancer is a carrier of KIR2DS2, or a carrier of KIR2DS5, or both; or where (i) the expression level of KIR2DS2 in a sample from a patient with hematopoietic malignancy is higher than the reference level of KIR2DS2 expression;

(ii) the expression level of KIR2DL2 in a sample from a patient with hematopoietic malignancy is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in a sample from a patient with hematopoietic malignancy is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in a sample from a patient with hematopoietic malignancy is lower than the reference level of KIR2DL5 expression;

(v) the level of GZMM expression in a sample from a patient with hematopoietic malignancy is higher than the reference level of GZMM expression;

- 59 040014 (vi) the ratio of KIR2DS2 expression level to KIR2DL2 expression level in a sample from a patient with hematopoietic malignancy is higher than the reference expression level ratio

KIR2DS2 to the expression level of KIR2DL2; or (vii) the ratio of KIR2DS5 expression level to KIR2DL5 expression level in a sample from a patient with hematopoietic malignancy is higher than the reference ratio of KIR2DS5 expression level to KIR2DL5 expression level;

or any combination of (i)-(vii) is present.

In some embodiments, provided herein are methods for treating hematopoietic cancer in a subject using FTI or selecting a patient with hematopoietic cancer for FTI treatment. Hematologic malignancies include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia , chronic myeloid leukemia and chronic lymphocytic leukemia), chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, polycythemia vera, NK cell leukemia, lymphoma, NK cell lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low and highly malignant forms), multiple myeloma, peripheral T- cellular lymphomas, cutaneous T-cell lymphoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, agnogenic myeloid metaplasia, familial erythrophagocytic lymphohistiocytosis, hairy cell leukemia, and myelodysplasia.

In some embodiments, the hematopoietic cancer to be treated by the methods provided herein may be AML, MDS, CMML, NK cell lymphoma, NK cell leukemia, CTCL, PTCL, CML. In some embodiments, the hematopoietic cancer is AML. In some embodiments, the hematopoietic cancer is MDS. In some embodiments, the MDS is a lower risk MDS. In some embodiments, the hematopoietic cancer is CMML. The CMML may be low risk CMML, intermediate risk CMML, or high risk CMML. The CMML may be myelodysplastic CMML or myeloproliferative CMML. In some embodiments, the CMML is wild-type NRAS/KRAS CMML. In some embodiments, the hematopoietic cancer is NK lymphoma. In some embodiments, the hematopoietic cancer is NK leukemia. In some embodiments, the hematopoietic cancer is CTCL. In some embodiments, the hematopoietic cancer is PTCL. In some embodiments, the PTCL is non-responsive or recurrent PTCL.

In some embodiments, provided herein are methods of treating MDS in a subject using FTI, or selecting an MDS patient for FTI treatment, wherein the MDS patient is a carrier of KIR2DS2, KIR2DS5, or HLA-C2, or any combination thereof; or where (i) the expression level of KIR2DS2 in a sample from an MDS patient is higher than the reference level of KIR2DS2 expression;

(ii) the level of expression of KIR2DL2 in the sample from the patient with MDS is lower than the reference level of expression of KIR2DL2;

(iii) the level of expression of KIR2DS5 in the sample from the patient with MDS is higher than the reference level of expression of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from an MDS patient is lower than the reference level of KIR2DL5 expression;

(v) the level of GZMM expression in the sample from the patient with MDS is higher than the reference level of GZMM expression;

(vi) the ratio of KIR2DS2 expression level to KIR2DL2 expression level in the MDS patient sample is higher than the reference ratio of KIR2DS2 expression level to KIR2DL2 expression level; or (vii) the ratio of KIR2DS5 expression level to KIR2DL5 expression level in a sample from an MDS patient is higher than the reference ratio of KIR2DS5 expression level to KIR2DL5 expression level;

or any combination of these is present.

In some embodiments, provided herein are methods of treating lower risk MDS in a subject using FTI, or selecting a lower risk MDS patient for FTI treatment, wherein the lower risk MDS patient is a carrier of KIR2DS2, KIR2DS5, or HLA- C2, or any combination thereof; or where (i) the expression level of KIR2DS2 in a sample from an MDS patient is higher than the reference level of KIR2DS2 expression;

(ii) the level of expression of KIR2DL2 in the sample from the patient with MDS is lower than the reference level of expression of KIR2DL2;

- 60 040014 (iii) the level of expression of KIR2DS5 in the sample from the patient with MDS is higher than the reference level of expression of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from an MDS patient is lower than the reference level of KIR2DL5 expression;

(v) the level of GZMM expression in the sample from the patient with MDS is higher than the reference level of GZMM expression;

(vi) the ratio of KIR2DS2 expression level to KIR2DL2 expression level in a sample from a lower risk MDS patient is higher than the reference ratio of KIR2DS2 expression level to KIR2DL2 expression level; or (vii) the ratio of KIR2DS5 expression level to KIR2DL5 expression level in a sample from a lower risk MDS patient is higher than the reference ratio of KIR2DS5 expression level to KIR2DL5 expression level;

or any combination of these is present.

In some embodiments, provided herein are methods of treating AML in a subject using FTI, or selecting an AML patient for FTI treatment, wherein the AML patient is a carrier of KIR2DS2, KIR2DS5, or HLA-C2, or any combination thereof; or where (i) the expression level of KIR2DS2 in a sample from an AML patient is higher than the reference level of KIR2DS2 expression;

(ii) the level of expression of KIR2DL2 in the sample from the patient with AML is lower than the reference level of expression of KIR2DL2;

(iii) the level of expression of KIR2DS5 in the sample from the patient with AML is higher than the reference level of expression of KIR2DS5;

(iv) the expression level of KIR2DL5 in a sample from an AML patient is lower than the reference level of KIR2DL5 expression;

(v) the level of GZMM expression in the sample from the patient with AML is higher than the reference level of GZMM expression;

(vi) the ratio of KIR2DS2 expression level to KIR2DL2 expression level in a sample from an AML patient is higher than the reference ratio of KIR2DS2 expression level to KIR2DL2 expression level; or (vii) the ratio of KIR2DS5 expression level to KIR2DL5 expression level in a sample from an AML patient is higher than the reference ratio of KIR2DS5 expression level to KIR2DL5 expression level;

or any combination of these is present.

In some embodiments, the AML patient is a post-remission induction patient. In some embodiments, the AML patient is a transplant patient. In some embodiments, the AML patient is over 60 years of age or otherwise ineligible for remission induction. In some embodiments, the AML patient is over 65, 70, or 75 years of age. In some embodiments, the AML patient is refractory to standard chemotherapy. In some embodiments, the AML patient is a relapsed patient.

In some embodiments, provided herein are methods for treating a solid tumor.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors may be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Solid tumors to be treated with the methods of the invention may be sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, malignant neoplasms of lymphoid tissue, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas, carcinoma sebaceous gland, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma and CNS tumors (such How glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioblastoma, neuroblastoma, retinoblastoma and brain metastases).

In some embodiments, provided herein are methods of treating a solid tumor, wherein the solid tumor is malignant melanoma, adrenal carcinoma, breast carcinoma, renal cell carcinoma, pancreatic carcinoma, non-small cell lung carcinoma (NSCLC), or carcinoma of unknown primary site.

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Drugs commonly administered to patients with various types or stages of solid tumors include, but are not limited to, Celebrex, etoposide, cyclophosphamide, docetaxel, apecitabine,

IFN, tamoxifen, IL-2, GM-CSF, or a combination thereof.

In some embodiments, the solid tumor to be treated by the methods provided herein may be thyroid cancer, head and neck cancer, urothelial cancer, salivary gland cancers, upper gastrointestinal cancers, bladder cancer, breast cancer. gland cancer, ovarian cancer, brain cancer, stomach cancer, prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, and pancreatic cancer. In some embodiments, the bladder cancer being treated by the methods presented herein may be transitional cell carcinoma.

In some embodiments, the solid tumor to be treated by the methods provided herein may be selected from the group consisting of carcinoma, melanoma, sarcoma, or chronic granulomatous disease.

In some embodiments, the pre-malignant conditions to be treated by the methods provided herein may be actinic cheilitis, Barrett's esophagus, atrophic gastritis, ductal carcinoma in situ, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucosal fibrosis, solar elastosis, cervical dysplasia, polyps, leukoplakia, erythroplakia, squamous intraepithelial lesion, premalignant disorder, or premalignant immunoproliferative disorder.

3.6. Illustrative FTIs and doses.

In some embodiments, methods for treating cancer in a subject include typing the KIR in the subject and administering a therapeutically effective amount of tipifarnib to the subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5, or a carrier of both KIR2DS2 and KIR2DS5. In some embodiments, the subject is also a carrier of HLA-C2. In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, a method for treating cancer in a subject comprises determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, and administering a therapeutically effective amount of tipifarnib to the subject, wherein (i) the level of expression of KIR2DS2 in the sample is higher than the reference level of expression of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression;

or any combination of (i)-(v) is present.

In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS214662.

In addition, a method for treating cancer in a subject includes determining the expression levels of KIR2DS2 and KIR2DL2 or KIR2DS5 and KIR2DL5 in a sample from the subject and administering a therapeutically effective amount of tipifarnib to the subject, where (i) the ratio of 2DS2/2DL2 in the sample is higher than the reference ratio of 2DS2/2DL2 ; or (ii) the 2DS5/2DL5 ratio in the sample is higher than the reference 2DS5/2DL5 ratio;

or both (i) and (ii) are present.

In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS214662.

In some embodiments, a method of treating a hematologic malignancy in a subject comprises typing KIR in the subject and administering a therapeutically effective amount of tipifarnib to the subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5, or a carrier of both KIR2DS2 and KIR2DS5. In some embodiments, the subject is also a carrier of HLA-C2. In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS214662.

In some embodiments, a method of treating a hematologic malignancy in a subject comprises determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, and administering a therapeutically effective amount of tipifarnib to the subject, where (i ) the level of expression of KIR2DS2 in the sample is higher than the reference level of expression of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression;

or any combination of (i)-(v) is present.

In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS214662.

In some embodiments, a method of treating a hematologic malignancy in a subject comprises determining expression levels of KIR2DS2 and KIR2DL2 or KIR2DS5 and KIR2DL5 in a sample from the subject, and administering a therapeutically effective amount of tipifarnib to the subject, wherein (i) the ratio of 2DS2/2DL2 in the sample is higher than the reference ratio 2DS2/2DL2; or (ii) the 2DS5/2DL5 ratio in the sample is higher than the reference 2DS5/2DL5 ratio;

or both (i) and (ii) are present.

In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS214662.

In some embodiments, a method for treating lower risk MDS in a subject comprises typing KIR in the subject and administering a therapeutically effective amount of tipifarnib to the subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5, or a carrier of both KIR2DS2 and KIR2DS5. In some embodiments, the subject is also a carrier of HLA-C2. In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, a method for treating lower risk MDS in a subject comprises determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, and administering a therapeutically effective amount of tipifarnib to the subject, wherein (i) the expression level of KIR2DS2 in the sample is higher than the reference level of KIR2DS2 expression;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression;

or any combination of (i)-(v) is present.

In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS214662.

Further, a method for treating lower risk MDS in a subject comprises determining expression levels of KIR2DS2 and KIR2DL2 or KIR2DS5 and KIR2DL5 in a sample from the subject and administering a therapeutically effective amount of tipifarnib to the subject wherein (i) the ratio of 2DS2/2DL2 in the sample is higher than the reference ratio 2DS2/2DL2; or (ii) the 2DS5/2DL5 ratio in the sample is higher than the reference 2DS5/2DL5 ratio; or both (i) and (ii) are present.

In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS214662.

In some embodiments, FTI is administered orally, parenterally, rectally, or topically. In some embodiments, FTI is administered orally.

In some embodiments, tipifarnib is administered orally, parenterally, rectally, or topically. In some embodiments, tipifarnib is administered orally.

In some embodiments, FTI is administered at a dose of 1-1000 mg/kg body weight. In some embodiments, FTI is administered twice a day. In some embodiments, FTI is administered at a dose of 200-1200 mg twice daily. In some embodiments, FTI is administered at a dose of 600 mg twice daily. In some embodiments, FTI is administered at a dose of 900 mg twice daily.

In some embodiments, tipifarnib is administered at a dose of 1-1000 mg/kg body weight. In some embodiments, tipifarnib is administered twice daily. In some embodiments, tipifarnib is administered at a dose of 200-1200 mg twice daily. In some embodiments, tipifarnib is administered at a dose of 600 mg twice daily. In some embodiments, tipifarnib is administered at a dose of 900 mg twice daily.

In some embodiments, FTI is administered in treatment cycles. In some embodiments, FTI is administered on alternating weeks. In some embodiments, FTI is administered on days 1-7 and 15-21 of 28 day treatment cycles. In some embodiments, FTI is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles.

In some embodiments, tipifarnib is administered in treatment cycles. In some embodiments, tipifarnib is administered on alternating weeks. In some embodiments, tipifarnib is administered on days 1-7 and 15-21 of 28 day treatment cycles. In some embodiments, tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles.

In some embodiments, FTI is administered for at least 3 cycles. In some embodiments, FTI is administered for at least 6 cycles. In some embodiments, FTI is administered for up to 12 cycles. In some embodiments, FTI is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles for at least three cycles.

In some embodiments, tipifarnib is administered for at least 3 cycles. In some embodiments, tipifarnib is administered for at least 6 cycles. In some embodiments, tipifarnib is administered for up to 12 cycles. In some embodiments, tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles for at least three cycles.

In some embodiments, a method of treating lower risk MDS in a subject comprises typing KIR in the subject and administering tipifarnib to the subject, wherein the subject is a carrier of KIR2DS2 or KIR2DS5, or a carrier of both KIR2DS2 and KIR2DS5, and wherein tipifarnib is administered orally at a dose of 900 mg twice per day on days 1-7 and 15-21 of the 28-day treatment cycles. In some embodiments, the subject is also a carrier of HLA-C2.

In some embodiments, a method for treating lower risk MDS in a subject comprises determining the expression level of a biomarker in a sample from the subject, where the biomarker is selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5, and GZMM, and administering tipifarnib to the subject, where (i) the level of expression of KIR2DS2 in the sample is higher than the reference level of expression of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression;

or any combination of (i)-(v) is present; and wherein tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles.

In addition, a method for treating lower risk MDS in a subject comprises determining expression levels of KIR2DS2 and KIR2DL2 or KIR2DS5 and KIR2DL5 in a sample from the subject, and administering tipifarnib to the subject where (i) the 2DS2/2DL2 ratio in the sample is higher than the reference 2DS2/ 2DL2; or (ii) the 2DS5/2DL5 ratio in the sample is higher than the reference 2DS5/2DL5 ratio;

or both (i) and (ii) are present; and wherein tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles.

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3.7. Sets.

In specific embodiments, provided herein is a kit for typing KIR in a subject. In some embodiments, the kit contains one or more probes that specifically bind to genomic DNA, cDNA, or mRNA of one or more KIR genes. The KIR genes may include KIR2DS2, KIR2DL2, KIR2DS5, KIRDL5, or any combination thereof. In some embodiments, the kits may further comprise an HLA typing agent. The HLA typing agent may be one or more probes that specifically bind to the genomic DNA, cDNA, or mRNA of one or more HLA genes. HLA genes may include HLA-C1, HLA-C2, or both.

In specific embodiments, the kit further comprises a wash solution. In specific embodiments, the kit further comprises genomic DNA isolation reagents or purification agents, detection agents, as well as positive and negative controls. In particular embodiments, the kit further comprises instructions for using the kit. In some embodiments, the kit further comprises an FTI or a pharmacological composition containing an FTI. The kit can be adapted for home use, clinical use, or research use.

In specific embodiments, provided herein is a kit for detecting the mRNA level of one or more biomarkers. One or more biomarkers are selected from a group that may include KIR2DS2, KIR2DL2, KIR2DS5, KIRDL5 and GZMM. In specific embodiments, the kit contains one or more probes that specifically bind to the mRNA of one or more biomarkers. In specific embodiments, the kit further comprises a wash solution. In specific embodiments, the kit further comprises reagents for hybridization assay, mRNA isolation, or purification, detection, as well as positive and negative controls. In particular embodiments, the kit further comprises instructions for using the kit. In some embodiments, the kit further comprises an FTI or a pharmacological composition containing an FTI. The kit can be adapted for home use, clinical use, or research use.

In specific embodiments, provided herein is a kit for detecting the protein level of one or more biomarkers. One or more biomarkers are selected from a group that may include KIR2DS2, KIR2DL2, KIR2DS5, KIRDL5, and GZMM. In specific embodiments, the kit contains an immersible probe coated with an antibody that recognizes a protein biomarker, wash solutions, assay reagents, protein isolation or purification agents, detection agents, as well as positive and negative controls. In particular embodiments, the kit further comprises instructions for using the kit. In some embodiments, the kit further comprises an FTI or a pharmacological composition containing an FTI. The kit can be adapted for home use, clinical use, or research use.

The kits provided herein may use, for example, an immersible probe, a membrane, a chip, a disc, a test strip, a filter, a microsphere, a glass slide, a multiwell plate, or an optical fiber. The solid support of the kit may be, for example, plastic, silicone, metal, resin, glass, membrane, particle, precipitate, gel, polymer, shell, sphere, polysaccharide, capillary, film, plate, or glass slide. The sample may be, for example, a blood sample, a bone marrow sample, a cell culture, a cell line, tissue, oral tissue, gastrointestinal tissue, an organ, an organelle, a body fluid, a urine sample, or a skin sample. The biological sample may be, for example, a lymph node biopsy, a bone marrow biopsy, or a peripheral blood tumor cell sample.

In some embodiments, the kits provided herein contain one or more containers and components for performing RT-PCR, qPCR, deep sequencing, NGS, or microarray analysis. In specific embodiments, the kits provided herein employ means for detecting biomarker expression by flow cytometry or immunofluorescence. In other embodiments, biomarker expression is measured by ELISA-based methods or other similar methods known in the art.

In specific embodiments, the kits provided herein contain protein isolation components. In another specific embodiment, the pharmaceutical or assay kit contains, in a container, an FTI or a pharmaceutical composition containing an FTI, and additionally contains, in one or more containers, components for performing flow cytometry or ELISA.

In some embodiments, provided herein are biomarker measurement kits that provide the materials needed to measure the presence of particular genes, or the occurrence of one or more of the gene products for a gene or subset of genes (e.g., one, two, three, four, five, or more genes) of the biomarkers presented in this

- 65 040014 document. Such kits may contain the materials and reagents needed to measure DNA, RNA, or protein. In some embodiments, such kits contain microarrays, where the microarray consists of oligonucleotides and/or DNA and/or RNA fragments that hybridize to one or more of the DNA or mRNA transcripts of one or more of the biomarker genes or subset of genes provided herein. , or any combination of them. In some embodiments, such kits may contain PCR primers for any of a DNA product, an RNA product, or a cDNA copy of an RNA product for genes or a subset of genes. In some embodiments, such kits may contain PCR primers as well as qPCR probes. In some embodiments, such kits may comprise a plurality of primers and a plurality of probes, where some of the probes have different fluorophores, so as to allow multiplexing of multiple products for a gene product or products of multiple genes. In some embodiments, the implementation of such kits may additionally contain materials and reagents for the synthesis of cDNA for RNA isolated from the sample. In some embodiments, such kits may contain antibodies specific for the protein products of a gene or subset of the biomarker genes provided herein. Such kits may additionally contain materials and reagents for isolating RNA and/or proteins from a biological sample. In some embodiments, such kits may include a computer program product embedded in computer-readable media for predicting whether a patient is clinically susceptible to FTI. In some embodiments, the kits may comprise a computer program product embedded in computer readable media along with instructions.

In some embodiments, provided herein are kits for measuring the expression of one or more nucleic acid sequences for a gene or subgroup of biomarker genes. In a specific embodiment, such kits measure the expression of one or more nucleic acid sequences associated with a gene or subgroup of biomarker genes provided herein. In accordance with this embodiment, the kits may contain materials and reagents necessary to measure the expression of a particular nucleic acid sequence of the gene products or subgroup of biomarker genes provided herein. For example, an RT-PCR microarray or kit may be prepared for a specific condition and contain only the reagents and materials needed to measure the levels of specific RNA transcript products for the genes or subset of biomarker genes provided herein to predict whether hematologic malignancy is tumor in a patient clinically sensitive to the compound. Alternatively, in some embodiments, the kits may contain materials and reagents, not limited to materials and reagents necessary to measure the expression of specific nucleic acid sequences for any of the specific biomarker genes provided herein. For example, in specific embodiments, the kits contain materials and reagents needed to measure expression levels of 1, 2, 3, 4, or 5 of the biomarkers provided herein, in addition to reagents and materials needed to measure expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more genes other than the biomarker genes provided herein. In other embodiments, the kits contain the reagents and materials needed to measure expression levels of at least 1, at least 2, at least 3, at least 4, at least 5 or more of the biomarker genes provided herein, and 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or more non-biomarker genes provided herein, or 1-10, 1-100, 1-150, 1-200, 1- 300, 1-400, 1-500, 1-1000, 25100, 25-200, 25-300, 25-400, 25-500, 25-1000, 100-150, 100-200, 100-300, 100- 400, 100-500, 100-1000 or 500-1000 non-genes of the biomarkers provided herein.

In the case of nucleic acid microarray kits, the kits typically contain probes attached to the surface of a solid support. In one such embodiment, the probes can be either oligonucleotides or longer probes, including probes ranging from 150 nucleotides in length to 800 nucleotides in length. Probes can be attached to a detectable label. In a specific embodiment, the probes are specific for one or more of the biomarker gene products provided herein. The microarray kits may contain instructions for performing the analysis and methods for interpreting and analyzing the data resulting from the analysis. In a specific embodiment, the kits contain instructions for predicting whether a patient's hematologic malignancy is clinically susceptible to FTI. The kits may also contain hybridization reagents and/or reagents necessary to detect the signal generated when the probe hybridizes to the target nucleic acid sequence. Typically, materials and reagents for microarray kits

- 66 040014 are in one or more containers. Each component of a kit is usually found in its own suitable container.

In specific embodiments, the nucleic acid microarray kit contains the materials and reagents needed to measure expression levels of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more identified biomarker genes provided herein, or combinations thereof, in addition to reagents and materials needed to measure expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more genes other than the biomarker genes provided herein. In other embodiments, the nucleic acid microarray kit contains reagents and materials necessary to measure expression levels of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least at least 45, at least 50 or more of the biomarker genes provided herein, or any combination thereof, and 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 , 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or more genes that are not biomarker genes provided in this document, or 1-10, 1-100, 1-150, 1-200, 1-300, 1-400, 1-500, 1-1000, 25-100, 25-200, 25-300, 25 -400, 25-500, 25-1000, 100150, 100-200, 100-300, 100-400, 100-500, 100-1000 or 500-1000 gene s that are not genes for the biomarkers presented in this document.

For quantitative PCR, kits may contain pre-selected primers specific to particular nucleic acid sequences. Quantitative PCR kits may also contain enzymes suitable for nucleic acid amplification (eg, polymerases such as Taq) and deoxynucleotides and buffers required for the amplification reaction mixture. Quantitative PCR kits may also contain probes specific for nucleic acid sequences associated with or indicative of a condition. Probes can be labeled with a fluorophore. Probes can also be labeled with a quencher molecule. In some embodiments, qPCR kits may also contain components suitable for RNA reverse transcription, including enzymes (e.g., reverse transcriptases such as AMV, MMLV, etc.) and primers for reverse transcription along with the deoxynucleotides and buffers necessary for the reverse transcription reaction. Each component of a quantitative PCR kit is usually found in its own suitable container. Thus, these kits typically contain separate containers suitable for each individual reagent, enzyme, primer, and probe. In addition, quantitative PCR kits may contain instructions for performing the assay and methods for interpreting and analyzing the data resulting from the assay. In a specific embodiment, the kits contain instructions for predicting whether a patient's hematologic malignancy is clinically susceptible to a compound.

In the case of antibody-based kits, the kit may contain, for example, (1) a first antibody that binds to a polypeptide or protein of interest; and optionally (2) a second, different antibody that binds either to the polypeptide or protein or to the first antibody and is conjugated to a detectable label (eg, a fluorescent label, a radioactive isotope, or an enzyme).

The first antibody may be attached to a solid support. In a specific embodiment, the polypeptide or protein of interest is the biomarker provided herein. Antibody kits may also contain immunoprecipitation beads. Each component of antibody-based kits is typically in its own suitable container. Thus, these kits typically contain separate containers suitable for each antibody. In addition, antibody-based kits may contain instructions for performing the assay and methods for interpreting and analyzing the data obtained from the assay. In a specific embodiment, the kits contain instructions for predicting whether a patient's hematologic malignancy is clinically susceptible to FTI.

In some embodiments, the kit provided herein contains the FTI provided herein or a pharmaceutical composition containing the FTI. The kits may further contain additional active ingredients, including, but not limited to, the active ingredients described herein, such as a DNA hypomethylating agent, a therapeutic antibody that specifically binds a cancer antigen, a hematopoietic growth factor, a cytokine, an anticancer agent, an antibiotic. , a cox-2 inhibitor, an immunomodulating agent, an antithymocyte globulin, an immunosuppressive agent, or a corticosteroid.

The kits provided herein may additionally contain devices that are used to administer FTI or other active ingredients. Examples of such devices include, but are not limited to, syringes, droppers, patches, and inhalers.

The kits may further contain cells or blood for transplantation, as well as pharmaceutically acceptable bases that can be used to administer one or more active ingredients. For example, if the active ingredient is in a solid form that needs to be reconstituted for parenteral administration, the kit may contain a hermetically sealed container with a suitable base in which the active ingredient can be dissolved to obtain a particulate-free sterile solution suitable for parenteral administration. Examples of pharmaceutically acceptable bases include, but are not limited to, water for injection USP; aqueous bases such as, but not limited to, sodium chloride injection, Ringer's solution for injection, dextrose solution for injection, dextrose and sodium chloride solution for injection, Ringer's solution with dextrose and lactate for injection; water-miscible bases such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous bases such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

In specific embodiments of the methods and kits provided herein, solid phase supports are used to purify proteins, label samples, or perform solid phase assays. Examples of solid phases suitable for the methods described herein include beads, particles, colloids, single surfaces, test tubes, multiwell plates, microtiter plates, glass slides, membranes, gels, and electrodes. When the solid phase is a particulate material (eg, beads), it is, in one embodiment, dispensed into the wells of multiwell plates to allow parallel processing of the solid phase supports.

The kit according to this description may contain an auxiliary reagent. In some embodiments, the auxiliary reagent may be a secondary antibody, a detection reagent, a detection buffer, an immobilization buffer, a dilution buffer, a wash buffer, or any combination thereof.

Secondary antibodies can be monoclonal or polyclonal antibodies. Secondary antibodies can be derived from any mammalian organism, including bovine, mice, rats, hamsters, goats, camels, chickens, rabbits, and others. Secondary antibodies may include, for example, an anti-human IgA antibody, an anti-human IgD antibody, an anti-human IgE antibody, an anti-human IgG antibody, or an anti-human IgM antibody. Secondary antibodies may be enzyme-conjugated (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), luciferase, etc.) or dyes (e.g., colorimetric dyes, fluorescent dyes, fluorescence resonance energy transfer (FRET) dyes, time-resolved (TR)-FRET FRET dyes, etc.). In some embodiments, the secondary antibody is a rabbit anti-human IgG polyclonal antibody that is conjugated to HRP.

Any detection reagent known in the art can be included in the kit as described herein. In some embodiments, the detection reagent is a colorimetric detection reagent, a fluorescent detection reagent, or a chemiluminescent detection reagent. In some embodiments, the colorimetric detection reagent comprises PNPP (p-nitrophenyl phosphate), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) or OPD (o-phenylenediamine). In some embodiments, the fluorescent detection reagent comprises QuantaBluTM or QuantaRedTM (Thermo Scientific, Waltham, MA). In some embodiments, the luminescent detection reagent comprises luminol or luciferin. In some embodiments, the detection reagent includes a triggering agent (eg, H ₂ O ₂ ) and an indicator (eg, isoluminol conjugate).

Any detection buffer known in the art can be included in the kit as described herein. In some embodiments, the detection buffer is a citrate phosphate buffer (eg, pH about 4.2).

Any stopping solution known in the art can be included in the kit as described herein. Stop solutions as described herein stop or delay further development of the detection reagent and associated assay signals. Stopping solutions may include, for example, low pH buffers (eg, glycine buffer, pH 2.0), chaotropic agents (eg, guanidinium chloride, sodium dodecyl sulfate (SDS)) or reducing agents (eg, dithiothreitol, mercaptoethanol), or t .P.

In some embodiments, the auxiliary reagent is an immobilization reagent, which may be any immobilization reagent known in the art.

- 68 040014 of this field, including reagents for covalent and non-covalent immobilization. Covalent immobilization reagents may include any chemical or biological reagent that can be used to covalently immobilize a peptide or nucleic acid on a surface. Covalent immobilization reagents may include, for example, a reactive group for the addition of a carboxyl to an amine (for example, carbodiimides such as EDC or DCC), amine-reactive groups (for example, N-hydroxysuccinimide (NHS) esters, imidoesters), reactive towards sulfhydryl, a crosslinker (e.g., maleimides, haloacetyls, pyridyl disulfides), carbonyl-reactive crosslinking groups (e.g., hydrazides, alkoxyamines), a photoreactive crosslinker (e.g., arylazides, diazirines), or a chemoselective ligation group (e.g., reactive pair of Staudinger). Reagents for non-covalent immobilization include any chemical or biological reagent that can be used to immobilize on the surface of a non-covalent peptide or nucleic acid, such as affinity tags (e.g., biotin) or binding reagents (e.g., streptavidin or antibodies against the tag, such as antibodies against His 6 or anti-Myc antibodies) (His6 is described as SEQ ID NO: 50) or anti-Myc antibodies.

Kits as described herein may include combinations of immobilization reagents. Such combinations include, for example, EDC and NHS, which can be used, for example, to immobilize a protein as described herein on a surface, such as a carboxylated dextran matrix (eg, on a BIAcoreTM CM5 chip or dextran bead). The immobilization reagent combinations can be stored in the form of premixed reagent combinations, or one or more immobilization reagents from the combination can be stored separately from the other immobilization reagents.

A wide variety of wash buffers are known in the art, such as tris(hydroxymethyl)aminomethane (Tris) based buffers (eg Tris buffered saline, TBS) or phosphate buffers (eg phosphate buffered saline, PBS). Wash buffers may include detergents such as ionic or non-ionic detergents. In some embodiments, the wash buffer is PBS buffer (eg, pH about 7.4) including Tween®20 (eg, about 0.05% Tween®20).

Any dilution buffers known in the art can be included in the kit as described herein. Reconstitution buffers may contain a carrier protein (eg, bovine serum albumin, BSA) and a detergent (eg, Tween®20). In some embodiments, the dilution buffer is PBS (eg, pH about 7.4) containing BSA (eg, about 1% BSA) and Tween®20 (eg, about 0.05% Tween®20).

In some embodiments, a kit as described herein contains a cleaning reagent for an automated assay system. The automated analysis system may include systems from any manufacturer. In some embodiments, automated analysis systems include, for example, BIO-FLASHTM, BEST 2000TM, DS2TM, ELx50 WASHER, ELx800 WASHER, and ELx800 READER. The cleaning agent may include any cleaning agent known in the art.

It should be noted that any combination of the above embodiments, for example with respect to one or more reagents such as, but not limited to, nucleic acid primers, solid support, and the like, is also contemplated in connection with any of the various methods and/or sets presented in this document.

4. Wild type K-Ras and N-Ras as biomarkers for the treatment of FTI.

The present document provides methods for selecting cancer patients for FTI treatment based in part on the discovery that Ras mutation status is associated with the clinical benefits of FTI and can be used to predict the ability of a cancer patient to respond to FTI treatment. Accordingly, provided herein are methods for predicting the ability of a cancer patient to respond to FTI treatment, methods for selecting a population of cancer patients for FTI treatment, and methods for treating cancer in a subject using a therapeutically effective amount of FTI based on the mutation status of Ras in a patient sample. .

4.1. Ras mutation status.

In some embodiments, provided herein is a method for treating cancer in a subject based on mutation status of K-Ras, N-Ras, or both. The method provided herein includes (a) determining the presence or absence of a Ras mutation in a sample from a subject, where the Ras mutation includes a K-Ras mutation or an N-Ras mutation, and then (b) administering a therapeutically effective amount of FTI to said subject, if it is determined that in the specified sample there is no K-Ras mutation or N-Ras mutation.

In some embodiments, the method provided herein comprises (a) determining the presence or absence of a K-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to said subject, if it is determined that said

- 69 040014 the sample lacks the K-Ras mutation. In some embodiments, the sample is determined to have wild-type K-Ras.

In some embodiments, the method provided herein comprises (a) determining the presence or absence of an N-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to said subject if said sample is determined to be free of the N mutation. -Ras. In some embodiments, the sample is determined to have wild-type N-Ras.

In some embodiments, the K-Ras mutation is a KA-Ras mutation. In some embodiments, the K-Ras mutation is a KB-Ras mutation. In some embodiments, the K-Ras mutation is a combination of a KA-Ras mutation and a KB-Ras mutation. The K-Ras mutation may include a mutation at a codon selected from the group consisting of G12, G13 and Q61 of KA-Ras, KB-Ras, or both. In some embodiments, the KA-Ras mutation may include a mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C, G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V. In some embodiments, the KB-Ras mutation may include a mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C, G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V.

In some embodiments, the Ras mutation is an N-Ras mutation. In some embodiments, the N-Ras mutation may include at least one mutation in a codon selected from the group consisting of G12, G13, G15, G60, and Q61. In some embodiments, the N-Ras mutation may include at least one mutation in a codon selected from the group consisting of G12, G13, and Q61. In some embodiments, the N-Ras mutation may include at least one mutation selected from the group consisting of G12C, G12D, G12F, G12S, G12A, G12V, G12R, G13C, G13R, G13A, G13D, G13V, G15W amino acid substitutions. , G60E, Q61P, Q61L, Q61R, Q61K, Q61H and Q61E.

In some embodiments, the sample is determined to have no amino acid substitution in G12, G13, and Q61 K-Ras, and no amino acid substitution in G12, G13, and Q61 of N-Ras. In some embodiments, the sample is determined to have no K-Ras mutation or no N-Ras mutation. In some embodiments, the sample is determined to have wild-type K-Ras and wild-type N-Ras.

In some embodiments, the method provided herein further comprises determining the presence or absence of an H-Ras mutation in a sample from a subject and administering a therapeutically effective amount of FTI to said subject if said sample is determined to have an H-Ras mutation.

In some embodiments, the H-Ras mutation is a mutation at a codon selected from the group consisting of G12, G13, and Q61. In some embodiments, the H-Ras mutation may be a mutation selected from the group consisting of G12R, G12V, G13C, G13R, Q61L, and Q61R amino acid substitutions.

In some embodiments, provided herein is a method of treating cancer in a subject based on K-Ras and N-Ras mutation status, comprising (a) determining the presence or absence of a K-Ras mutation and an N-Ras mutation in a sample from the subject and then ( b) administering a therapeutically effective amount of FTI to the subject if the sample does not have any K-Ras mutation or any N-Ras mutation. In some embodiments, the method includes administering a therapeutically effective amount of FTI to a subject if the sample has wild-type K-Ras and wild-type N-Ras. In some embodiments, the method further comprises determining the status of H-Ras mutations and then administering a therapeutically effective amount of FTI to the subject if the sample from the subject has no K-Ras mutation or no N-Ras mutation, but has an H-Ras mutation.

Provided herein are methods for predicting the ability of a cancer patient to respond to FTI treatment, methods for selecting a population of cancer patients for FTI treatment, and methods for treating cancer in a subject using a therapeutically effective amount of FTI based on the Ras mutation status in a sample from the patient. In some embodiments, the method includes determining the presence or absence of a Ras mutation in a sample from a subject prior to treatment. Tumors or malignancies that do not have a K-Ras mutation or an N-Ras mutation indicate that patients are likely to be responsive to FTI treatment. In some embodiments, patients are selected for FTI treatment based on the absence of a K-Ras mutation or an N-Ras mutation. In some embodiments, patients are selected for FTI treatment based on the absence of a K-Ras mutation and an N-Ras mutation. In some embodiments, patients are further selected based on the presence of an H-Ras mutation. Ras mutation status can be detected at the nucleic acid or protein level. In some embodiments, the Ras mutation status is determined by analysis of nucleic acids obtained from a sample. In some embodiments, the Ras mutation status is determined by analysis of a protein obtained from a sample.

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Technologies that can be used in the methods presented herein include in situ hybridization (Stoler, Clin. Lab. Med. 12:215-36 (1990), using radioactive isotope or fluorophore labeled probes, polymerase chain reaction (PCR); quantitative Southern blot, dot blot, and other techniques to quantify individual genes.In some embodiments, the probes or primers selected to evaluate gene amplification are highly specific to avoid detection of closely related homologous genes.Alternatively, antibodies can be used that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.Antibodies, in turn, can be labeled and assayed, where the duplex is bound to a surface, so that once the duplex is formed, the presence on the surface can be detected. antibodies associated with the duplex.

In some embodiments, the Ras mutation status is determined by analysis of nucleic acids obtained from a sample. Nucleic acids may be mRNA or genomic DNA molecules from the test subject.

Methods for determining Ras mutation status by nucleic acid analysis are well known in the art. In some embodiments, the methods include sequencing, polymerase chain reaction (PCR), DNA microarray analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, denaturing high performance liquid chromatography (DHPLC), or restriction fragment length polymorphism (RFLP) analysis. In some embodiments, the mutation status of Ras is determined using standard sequencing methods, including, for example, Sanger sequencing, next generation sequencing (NGS). In some embodiments, the Ras mutation status is determined using MS.

In some embodiments, the method includes determining the presence or absence of a Ras mutation by amplifying a Ras nucleic acid from a sample by PCR. For example, the PCR method and primer pairs that can be used are known to those skilled in the art (eg, Chang et al., Clinical Biochemistry, 43 (2010), 296-301; WO2015144184). For example, multiplex PCR can be used to amplify codons 12 and 13 from exon 2 and codon 61 from exon 3 of N-, H-, or K-Ras genes using two universal primer pairs for exons 2 and 3. For example, the following primers can be used:

SEQID NO Exon Primer sequence 21 2 5'-CYKRBKDRMRATGACKGARTAYAARCTKGTGGT-3' 22 2 5'-ACCTCTATDGTKGGRTCRTATTC-3' 23 3 5'-CAGGATTCYTACMGRAARCARGT-3' 24 4 5'-TTKATGGCAAAYACACAVAGRAAGC-3'

As used herein, letters are used according to the IUPAC designation, e.g. Y is pyrimidine, K is keto, e.g. G or C, R is purine, B is C, G or T, D is A, G or T, M is A, C, V means A, C or G.

After multiplex PCR amplification, products can be purified to remove primers and omitted deoxynucleotide triphosphates using the PCR-M™ Clean Up system (Viogenebiotek Co., Sunnyvale, CA, USA). The isolated DNA can then be semi-quantitated on a 1% agarose gel in 0.5 x TBE and visualized by staining with ethidium bromide. The products can then be subjected to primer extension analysis using primers as described in Chang et al., Clinical Biochemistry 43 (2010), 296-301, such as the following:

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SEQID NO RAS Primer sequence 25 TO 5'-AACTTGTGGTAGTTGGAGCT 26 TO 5'-ACTGAATATAAACTTGTGGTAGTTGGAGCTG 27 To 5'-TGAAAATGACTGAATATAAACTTGT GGTAGTTGGAGCTGGT 28 To 5'-GCCTGCCTGAAAATGAGTGAA TATAAACTTGTGGTAGTTGGAGCTGGTG 29 To 5'-GCAAGTAGTAATTGATGGAGAA ACCTGTCTCTTGGATATTTCTCGACACAGCAGGT thirty To 5'-GGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTTCTCGACACAGCAGGTC 31 To 5' - T 4 5 AT T C T C GAC AC AG CAG G T C A 32 N 5'-AACTGGTGGTGGTTGGAGCA-3' 33 N 5'- _{T7AACTGGTGGTGGTTGGAGCAG} -3' 34 N 5'- _Ti4 CAGTGCGCTTTTCCCCAACAC-3' 35 N 5'-T ₂₂ GTGGTGGTTGGAGCAGGTG-3' 36 N 5'-T _{29CTCATGGCACTGTACTCTTCTT} -3' 37 N 5'-T _{36CTCATGGCACTGTACTCTTCT} -3' 38 N 5'-T ₄₃ CTCTCATGGCACTGTACTCTTC-3' 39 H 5'-AGCTGGTGGTGGTGGGCGCC-3' 40 H 5'- _{T7AGCTGGTGGTGGTGGGCGCCG} -3' 41 H 5'- _{Ti4TGGTGGTGGTGGGCGCCGGC} -3' 42 H 5'-T ₂₂ GTGGTGGTGGGCGCCGGCG-3' 43 H 5'-T ₂₉ ACATCCTGGATACCGCCGGC-3' 44 H 5'-T ₃₆ ACATCCTGGATACCGCCGGCC-3' 45 H 5'- _{T43CGCATGGCGCTGTACTCCTC} -3'

Various probe concentrations can be used for any of codons 12, 13, or 61 (e.g., 0.03-0.6 µM) in reactions containing 1.5 µl of purified PCR products, as well as 4 µl of ABI PRISM SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA) containing AmpliTaq® DNA polymerase and fluorescently labeled dideoxynucleotide triphosphates (ddNTPs) (RGG labeled dideoxyadenosine triphosphate, TAMRA labeled dideoxycytidine triphosphate, ROX labeled dideoxythymidine triphosphate, and R110 labeled dideoxyguanosine triphosphate). Then, each 10 µl of the reaction mixture can be subjected to 25 cycles of one base extension consisting of a denaturation step at 96°C for 10 s and a hybridization and primer extension at 55°C for 35 s. After the extension cycle, unincorporated fluorescent ddNTPs can then be incubated with 1 µl shrimp alkaline phosphatase (United States Biochemical Co., Cleveland, USA) at 37°C for 1 hour, followed by enzyme inactivation at 75°C for 15 minutes. Primer extension reaction products can then be separated by automated capillary electrophoresis on a capillary electrophoresis platform, e.g. 14 µl Hi-Di™ formamide (Applied

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Biosystems) and 0.28 µl of GeneScan™-120LIZ® size standard (Applied Biosystems) were added to 6 µl of primer extension products. All samples can then, for example, be analyzed in an ABI analyzer

Prism 310 DNA Genetic Analyzer (Applied Biosystems) according to manufacturer's instructions using GeneScan™ 3.1 (Applied Biosystems).

Provided herein are methods for selecting cancer patients who are likely to benefit from FTI treatment, comprising determining the presence or absence of a Ras mutation by amplifying the Ras nucleic acid from patient tumor samples and sequencing the amplified nucleic acid.

Accordingly, Ras nucleic acid can be amplified using primers as described above and sequenced. For example, K-Ras, N-Ras, and H-Ras nucleic acid can be amplified by PCR as described above and then subcloned using, for example, the TOPO TA Cloning Kit for sequencing (Invitrogen).

In the above inventive method, the RAS nucleic acid can be obtained from patient tumor samples by any method known to the person skilled in the art. For example, any commercial kit, such as the Qlamp DNA mini kit or the RNeasy mini kit (Qiagen, Hilden, Germany), can be used to isolate genomic DNA or mRNA from a tumor sample. For example, if mRNA is isolated from a patient's tumor sample, cDNA synthesis can be performed prior to performing the methods as described herein, in accordance with any known technique in the art.

For example, the nucleic acid to be isolated from a tumor may, for example, be one of genomic DNA, total RNA, mRNA, or poly(A)+ mRNA. For example, if the mRNA is isolated from a patient's tumor sample, the mRNA (total mRNA or poly(A)+ mRNA) can be used to synthesize cDNA according to well-established methods in the prior art, such as those found in commercial cDNA synthesis kits, e.g., in the Superscript® III First Strand Synthesis Kit. The cDNA can then be further amplified, eg by PCR, and then subjected to sequencing, eg by Sanger sequencing or pyrosequencing to determine the nucleotide sequence, eg codons 12 and 13 of the RAS gene, eg H-RAS, N-RAS or KRAS. Alternatively, the PCR product can, for example, also be subcloned into a TA TOPO cloning vector for sequencing. Technologies other than sequencing to determine the absence or presence of Ras mutations can be used in the methods presented herein, such as, for example, single nucleotide primer extension (SNPE) (PLoS One. 2013 Aug 21; 8 (8): e72239); DNA microarray analysis, mass spectrometry (MS) (e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF) mass spectrometry), single nucleotide polymorphism (SNP) analysis, denaturing high performance liquid chromatography (DHPLC), or polymorphism analysis restriction fragment length (RFLP).

For example, single nucleotide polymorphism (SNP) analysis can be used to determine the mutation status of Ras in a sample. SNP analysis can be performed in the HT7900 from Applied Biosystems, followed by allelic discriminant analysis according to the manufacturer's protocol. Ras mutation status can also be determined by DHPLC or RFLP or any other methods known in the art. Bowen et al., Blood, 106:2113-2119 (2005); Bowen et al., Blood, 101:2770-2774 (2003); Nishikawa et al., Clin Chim Acta., 318:107-112 (2002); Lin SY et al., Am J Clin Pathol. 100:686-689 (1993); O'Leary JJ et al., J Clin Pathol. 51:576-582 (1998).

In some embodiments, the Ras mutation status is determined by analysis of a protein obtained from a sample. Mutant Ras protein can be detected by a variety of immunohistochemistry (IHC) methods or other immunoassay methods known in the art. IHC staining of tissue sections has been shown to be a reliable way to assess or detect the presence of proteins in a sample. Immunohistochemistry methods use an antibody as a probe and to visualize cellular antigens in situ, typically by chromogenic or fluorescent methods. Thus, antibodies or antisera, preferably polyclonal antisera and most preferably monoclonal antibodies that specifically target the mutant K-Ras or N-Ras, can be used to detect expression. As discussed in more detail below, antibodies can be detected by direct labeling of the antibodies themselves, for example using radioactive labels, fluorescent labels, hapten labels such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, an unlabeled primary antibody is used in combination with a labeled secondary antibody, including antisera, polyclonal antisera, or monoclonal antibodies specific for the primary antibody. Protocols and kits for immunohistochemistry are well known in the art and are commercially available. Automated systems for slide preparation and IHC are commercially available. The Ventana® BenchMark XT system is an example of such an automated system.

Standard immunological and immunoassay methods can be found in Basic and

- 73 040014

Clinical Immunology (Stites & Terr eds., 7th ed. 1991). In addition, immunoassays can be performed in any of several configurations, which are extensively reviewed in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, above. For a general overview of immunoassays, see also Methods in Cell Biology:

Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Ten, eds., 7th ed. 1991).

Assays for detecting K-Ras mutations or N-Ras mutations include non-competitive assays such as sandwich assays and competitive assays. Typically, an assay such as an ELISA assay can be used. ELISA assays are known in the art, for example, for analyzing a wide variety of tissues and samples, including blood, plasma, serum, or bone marrow.

A wide variety of immunoassay methods are available using this assay format, see, for example, US Pat. These include single site assays and dual site assays, or sandwich assays of non-competitive types, as well as traditional competitive binding assays. These assays also include direct binding of the labeled antibody to the mutant Ras protein target. Sandwich assays are conventional assays. There are a number of variations on the sandwich analysis method. For example, in a typical direct assay, an unlabeled antibody is immobilized on a solid substrate and the sample to be tested is brought into contact with the bound molecule. After a suitable incubation period for a period of time sufficient to allow the formation of an antibody-antigen complex, then a second antibody specific for the antigen labeled with a reporter molecule capable of producing a detectable signal is added and incubated, allowing time sufficient for the formation of another antibody complex. -antigen-labeled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observing the signal produced by the reporter molecule. The results can either be qualitative, by simply observing a visible signal, or they can be quantified by comparison with a control sample.

Direct assay options include simultaneous assays, in which both the sample and the labeled antibody are added simultaneously to the bound antibody. These methods are well known to those skilled in the art, including any minor modifications as will be readily apparent. In a typical direct sandwich assay, the first antibody with specificity for a biomarker is either covalently or passively bound to a solid surface. The solid surface may be glass or polymer, where the most common polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. Solid supports may be in the form of tubes, beads, microplate discs, or any other surface suitable for immunoassay. Coupling methods are well known in the art and typically consist of cross-linking covalent bonding or physical adsorption, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a sufficient period of time (e.g. 2-40 min or overnight if more convenient) and under suitable conditions (e.g. room temperature to 40°C, eg between 25 and 32°C inclusive) to allow binding of any subunit present in the antibody. After an incubation period, the solid phase with the antibody subunit is washed, dried, and incubated with a second antibody specific for a portion of the mutated Ras protein. The second antibody is linked to a reporter molecule, which is used to show the binding of the second antibody to the mutant Ras protein.

In some embodiments, flow cytometry (FACS) can be used to detect mutant K-Ras or N-Ras using an antibody that specifically targets the mutant K-Ras or N-Ras. The flow cytometer detects and records the intensity of the fluorochrome labeled antibody, which indicates the presence of the mutant K-Ras or N-Ras. Non-fluorescent cytoplasmic proteins can also be observed by staining permeabilized cells. The dye can be either a fluorescent compound capable of binding to specific molecules, or a fluorochrome labeled antibody to bind to a selected molecule.

An alternative method involves immobilizing the target Ras protein in the sample and then exposing the immobilized target to a mutant-specific antibody, which may or may not be labeled with a reporter molecule. Depending on the amount of target and the signal strength of the reporter molecule, the bound target can be detected by direct labeling using an antibody. Alternatively, a second labeled antibody specific for the first antibody is subjected to a target-first antibody complex to form a tertiary target-first antibody-second antibody complex. The complex is detected by a signal emitted from a labeled reporter molecule.

In the case of an enzyme immunoassay, the enzyme is conjugated to a second antibody, usually via glutaraldehyde or periodate. As is clearly understood, however, there are a wide variety of different conjugation methods readily available to those skilled in the art. Common enzymes including horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, and

- 74 040014 others are discussed in this document. The substrates to be used with specific enzymes are generally chosen to produce, after hydrolysis with the appropriate enzyme, a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to use fluorogenic substrates that form a fluorescent product, in contrast to the chromogenic substrates mentioned above. In all cases, the enzyme labeled antibody is added to the antibody-molecular marker complex, allowed to bind, and then the excess reagent is washed off. A solution containing the appropriate substrate is then added to the antibody-antigen-antibody complex. The substrate can react with the enzyme associated with the second antibody to form a qualitative visual signal that can be further quantified, usually spectrophotometrically, to provide an indication of the amount of mutant Ras protein that was present in the sample. Alternatively, fluorescent compounds such as fluorescein and rhodamine can be chemically attached to antibodies without changing their binding capacity. When activated by illumination with light of a specific wavelength, the fluorochrome labeled antibody absorbs light energy, inducing a state of excitation of the molecule, followed by emission of light of a characteristic color that can be visually detected using a light microscope. As with EIA, the fluorescently labeled antibody is allowed to bind to the first antibody-molecular marker complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of a suitable wavelength, the observed fluorescence indicates the presence of a molecular marker of interest. Immunofluorescence and EIA methods are both very well developed in the art and are discussed in this document.

In some embodiments, Ras mutation status determination is performed as an adjunct diagnostic for the treatment of FTI. Concomitant diagnosis can be performed at the clinical center where the subject is being treated. Concomitant diagnosis may also be performed at a center other than the clinical center where the subject is being treated.

As understood by one of skill in the art, provided herein are methods for predicting the ability of a cancer patient to respond to FTI treatment, methods for selecting a population of cancer patients for FTI treatment, and methods for treating cancer in a subject using a therapeutically effective amount of FTI, based on the Ras mutation status in a patient sample. Any of the methods described herein, or otherwise known in the art for determining the status of Ras mutations, can be used in these methods.

4.2. Samples.

In some embodiments, the methods provided herein include obtaining a sample from a subject. The sample used in the methods presented herein includes body fluids of the subject. Non-limiting examples of body fluids include blood (e.g. peripheral whole blood, peripheral blood), blood plasma, bone marrow, amniotic fluid, intraocular fluid, bile, lymph, menses, serum, urine, cerebrospinal fluid surrounding the brain and spinal cord, synovial fluid surrounding the joints of bones.

In one embodiment, the sample is a bone marrow sample. Methods for obtaining bone marrow samples are well known in the art, including, but not limited to, bone marrow biopsy and bone marrow aspiration. Bone marrow has a liquid part and a harder part. During a bone marrow biopsy, a sample of the hard part is taken. During aspiration of the bone marrow, a sample of the liquid part is taken. A bone marrow biopsy and a bone marrow aspiration may be performed at the same time and referred to as a bone marrow examination.

In some embodiments, the sample is a blood sample. The blood sample can be obtained using conventional methods as described, for example, in Innis et al., editors, PCR Protocols (Academic Press, 1990). Leukocytes can be isolated from blood samples using conventional methods or commercially available kits such as the RosetteSep kit (Stein Cell Technologies, Vancouver, Canada). Leukocyte subpopulations, such as mononuclear cells, NK cells, B cells, T cells, monocytes, granulocytes, or lymphocytes, can be further isolated using conventional methods, such as magnetic field activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California) or fluorescence-activated cell sorting (FACS) (Becton Dickinson, San Jose, California).

In one embodiment, the blood sample is from about 0.1 ml to about 10.0 ml, from about 0.2 ml to about 7 ml, from about 0.3 ml to about 5 ml, from about 0.4 ml to about 3.5 ml or from about 0.5 ml to about 3 ml. In another embodiment, the blood sample is approximately 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2 .5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 ml.

In some embodiments, the sample used in the present methods includes a biopsy (eg, a tumor biopsy). The biopsy may be from any organ or tissue, such as skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph nodes,

- 75 040014 hair, spleen, brain, mammary glands or other organs. Any biopsy technique known to those skilled in the art can be used to isolate a sample from a subject, such as open biopsy, closed biopsy, core biopsy, incisional biopsy, excisional biopsy, or fine needle aspiration biopsy.

In specific embodiments, the sample used in the methods presented herein includes a plurality of cells. Such cells may include any cell type, such as stem cells, blood cells (eg, PBMC), lymphocytes, NK cells, B cells, T cells, monocytes, granulocytes, immunocytes, or tumor cells or cancer cells. Specific cell populations can be generated using a combination of commercially available antibodies (eg, Quest Diagnostic (San Juan Capistrano, Calif.); Dako (Denmark)).

In specific embodiments, the implementation of the sample used in the methods presented herein, comes from diseased tissue, such as from an individual with a malignant tumor (eg, lymphoma, MDS or leukemia). In some embodiments, cells can be obtained from tumor cells, or cancer cells, or tumor tissues, such as tumor biopsies or tumor explants. In specific embodiments, the number of cells used in the methods provided herein may range from a single cell to about 10 ⁹ cells. In some embodiments, the number of cells used in the methods provided herein is approximately 1x10 ⁴ , 5x10 ⁴ , 1x105, 5x105, 1x10 ⁶ , 5x10 ⁶ , 1x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , or 5x10 ⁸ .

The number and type of cells collected from a subject can be monitored, for example, by measuring changes in cell morphology and surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining using tissue-specific or marker-specific cells antibodies), fluorescence activated cell sorting (FACS), magnetic field activated cell sorting (MACS), by checking cell morphology using light or confocal microscopy, and/or by measuring changes in gene expression using methods well known in the art, such as PCR and gene expression profiling. These methods can also be used to identify cells that are positive for one or more specific markers. Fluorescence activated cell sorting (FACS) is a well known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laser excitation of the fluorescent groups in the individual particles results in a small electrical charge allowing the electromagnetic separation of positive and negative particles from the mixture. In one embodiment, specific antibodies or ligands for cell surface markers are labeled with different fluorescent labels. The cells are passed through a cell sorter, allowing cells to be separated based on their ability to bind the antibodies used. FACS-sorted particles can be deposited directly into individual wells of 96-well or 384-well plates for ease of separation and cloning.

In specific embodiments, subsets of cells are used in the methods provided herein. Methods for sorting and isolating specific cell populations are well known in the art and may be based on cell size, morphology, or intracellular or extracellular markers. Such methods include, but are not limited to, flow cytometry, flow sorting, FACS, bead-based separation such as magnetic cell sorting, size-based separation (e.g., using a sieve, array of obstacles, or filter), microfluidic sorting, separation based on antibodies, precipitation, affinity adsorption, affinity extraction, density gradient centrifugation, laser capture microdissection, etc.

The sample may be a whole blood sample, a bone marrow sample, a partially purified blood sample, or a PBMC. The sample may be a tissue biopsy or a tumor biopsy. In some embodiments, the sample is a bone marrow sample from a cancer patient. In some embodiments, the sample is a PBMC from a cancer patient.

4.3 Malignant tumors.

Provided herein are methods for treating cancer in a subject using FTI and methods for selecting cancer patients for FTI treatment based on the absence of a K-Ras mutation and an N-Ras mutation. The cancer may be a hematopoietic cancer or a solid tumor. Also provided herein are methods for treating a pre-malignant condition in a subject using FTI, and methods for selecting patients with a pre-malignant condition for FTI treatment based on the absence of a K-Ras mutation and an N-Ras mutation.

In some embodiments, provided herein are methods for treating solid tumors using FTI based on the absence of a K-Ras mutation and an N-Ras mutation. Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors may be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Solid tumors to be treated with the methods of the invention may be sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, malignant neoplasms of lymphoid tissue, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, carcinoma sebaceous gland, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinomas, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma and CNS tumors (such How glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioblastoma, neuroblastoma, retinoblastoma and brain metastases). In some embodiments, the FTI is tipifarnib.

In some embodiments, provided herein are methods of treating a solid tumor using an FTI based on the absence of a K-Ras mutation and an N-Ras mutation, wherein the solid tumor is malignant melanoma, adrenal carcinoma, breast carcinoma, renal cell carcinoma, pancreatic carcinoma. gland, non-small cell lung carcinoma (NSCLC), or carcinoma of unknown primary site. In some embodiments, the FTI is tipifarnib. Drugs commonly administered to patients with various types or stages of solid tumors include, but are not limited to, Celebrex, etoposide, cyclophosphamide, docetaxel, apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combination thereof.

In some embodiments, the solid tumor to be treated by the methods provided herein may be thyroid cancer, head and neck cancer, urothelial cancer, salivary gland cancers, upper gastrointestinal cancers, bladder cancer, breast cancer. gland cancer, ovarian cancer, brain cancer, stomach cancer, prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, and pancreatic cancer. In some embodiments, the bladder cancer being treated by the methods presented herein may be transitional cell carcinoma. In some embodiments, the FTI is tipifarnib.

In some embodiments, the solid tumor to be treated by the methods provided herein may be selected from the group consisting of carcinoma, melanoma, sarcoma, or chronic granulomatous disease.

In some embodiments, the pre-malignant conditions to be treated by the methods provided herein may be actinic cheilitis, Barrett's esophagus, atrophic gastritis, ductal carcinoma in situ, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucosal fibrosis, solar elastosis, cervical dysplasia, polyps, leukoplakia, erythroplakia, squamous intraepithelial lesion, premalignant disorder, or premalignant immunoproliferative disorder.

In some embodiments, provided herein are methods for treating hematopoietic cancer in a subject using FTI or selecting patients with cancers for FTI treatment based on the absence of a K-Ras mutation and an N-Ras mutation.

Hematologic malignancies are malignant tumors of the blood or bone marrow. Examples of hematologic (or hematogenous) malignancies include myeloproliferative neoplasia (MPN), myelodysplastic syndrome (MDS), leukemia, and lymphoma. In some embodiments, the cancer is acute myeloid leukemia (AML), natural killer cell lymphoma (NK lymphoma), natural killer cell leukemia (NK leukemia), cutaneous T-cell lymphoma (CTCL), juvenile myelomonocytic leukemia (JMML), peripheral T-cell lymphoma (PTCL), chronic myeloid leukemia (CML), or chronic myelomonocytic leukemia (CMML). In some embodiments, the cancer is CMML. In some embodiments, the cancer is JMML.

In some embodiments, provided herein are methods for treating CMML in a subject using FTI or selecting patients with CMML for FTI treatment based on

- 77 040014 no K-Ras mutation and no N-Ras mutation. CMML is classified as myelodysplastic/myeloproliferative neoplasia in the 2008 World Health Organization classification of tumors of the hematopoietic system. CMML may be myelodysplastic CMML or myeloproliferative CMML. Patients with CMML have a high number of monocytes in the blood (at least 1000 per mm ³ ). Two classes - myelodysplastic and myeloproliferative - are distinguished on the basis of the level of the number of leukocytes (threshold 13 g/l). Often the number of monocytes is much higher, which leads to the fact that the total number of leukocytes also becomes very high. Normally, abnormal cells are present in the bone marrow, but the number of blast cells is less than 20%. In approximately 15-30% of patients with CMML, the process continues until the development of acute myeloid leukemia. The diagnosis of CMML is based on a combination of morphological, histopathological, and chromosomal abnormalities in the bone marrow. The Mayo predictive model classified patients with CMML into three risk groups based on: increased absolute monocyte count, presence of circulating blasts, hemoglobin <10 g/dl, and platelets <100 x 10 ⁹ /l. Median survival was 32 months, 18.5 months, and 10 months in the low, intermediate, and high risk groups, respectively. The Groupe Francophone (GFM) index divided patients with CMML into three risk groups based on: age >65 years, WBC >15x10 ⁹ /l, anemia, platelets <100x10 ⁹ /l, and ASXL1 mutation status. After tracking with a median of 2.5 years, survival ranged from not achieved in the low-risk group to 14.4 months in the high-risk group.

In some embodiments, provided herein are methods of treating CMML in a subject by determining the presence or absence of a K-Ras mutation and an NRas mutation in a sample from the subject and then administering a therapeutically effective amount of FTI to the subject if the sample is determined to be free of the K-Ras mutation and no N-Ras mutation. In some embodiments, the FTI is tipifarnib. In some embodiments, the sample is determined to have wild-type K-Ras and wild-type N-Ras.

In some embodiments, provided herein are methods of treating CMML in a subject by determining the presence or absence of a K-Ras mutation in a sample from the subject and then administering a therapeutically effective amount of FTI to the subject if the sample is determined to be free of the K-Ras mutation. In some embodiments, the FTI is tipifarnib. In some embodiments, the sample is determined to have wild-type K-Ras.

In some embodiments, provided herein are methods of treating CMML in a subject by determining the presence or absence of an N-Ras mutation in a sample from the subject and then administering a therapeutically effective amount of FTI to the subject if the sample is determined to be free of the N-Ras mutation. In some embodiments, the FTI is tipifarnib. In some embodiments, the sample is determined to have wild-type N-Ras.

In some embodiments, provided herein are methods of treating CMML in a subject by determining the presence or absence of a K-Ras mutation and an NRas mutation in a sample from the subject and then administering tipifarnib to the subject if the sample is determined to have wild-type K-Ras and N-Ras wild type.

In some embodiments, provided herein are methods for treating MDS in a subject using FTI or selecting MDS patients for FTI treatment. MDS belongs to a heterogeneous group of hematopoietic stem cell disorders. MDS can be characterized by an increase in the number of cells in the bone marrow with abnormal morphology and maturation (dysmyelopoiesis), inefficient production of blood cells, or hematopoiesis leading to a reduced number of blood cells, or cytopenias (including anemia, leukopenia, and thrombocytopenia), and a high risk of progression to acute myeloid leukemia resulting from inefficient production of blood cells. See The Merck Manual 953 ( ^17th ed. 1999) and List et al., 1990, J Clin. oncol. 8:1424.

MDS can be divided into a number of subtypes, depending on at least 1) whether there is an increased number of blast cells in the bone marrow or blood and what percentage of the bone marrow or blood consists of these blast cells; 2) whether abnormal growth of the bone marrow (dysplasia) is indicated for only one type of blood cell (unilinear dysplasia) or for more than one type of blood cell (multilinear dysplasia); and 3) whether chromosomal abnormalities are present in bone marrow cells, and if present, what type or types of abnormalities. MDS can also be classified based on surface markers of malignant tumor cells. According to the World Health Organization, subtypes of MDS include refractory cytopenia with unilinear dysplasia (RCUD), also known as refractory anemia, refractory neutropenia, or refractory thrombocytopenia; non-responsive anemia with annular sideroblasts (RARS); non-responsive cytopenia with multilinear dysplasia (RCMD), which includes RCMD-RS if both multilinear dysplasia and annular sideroblasts are present; refractory anemia-1 with blast excess (RAEB-1) and refractory anemia-2 with blast excess (RAEB-2) (these subtypes mean that patients have at least 5%

- 78 040014 (RAEB-1) or at least 10% (RAEB-2) but less than 20% of blast cells in the bone marrow);

MDS associated with an isolated chromosome 5 anomaly [del(5q)]; and unclassifiable MDS (MDS-U).

As a group of hematopoietic stem cell malignancies with significant morbidity and mortality, MDS is a highly heterogeneous disease, and symptom severity and disease progression can vary greatly among patients. The current standard clinical tool for evaluating risk stratification and treatment options is the Modified International Predictive Scoring System, or IPSS-R. The IPSS-R categorizes patients into five risk groups (very low, low, intermediate, high, very high) based on cytogenetics, the percentage of blasts (undifferentiated blood cells) in the bone marrow, hemoglobin levels, and platelet and neutrophil counts. The WHO also proposes a stratification of patients with MDS by anomaly - deletion (5q).

According to the ACS, the annual incidence of MDS is approximately 13,000 patients in the United States, most of whom are 60 years of age or older. The estimated prevalence is over 60,000 patients in the United States. Approximately 75% of patients fall into the IPSS-R very low, low, and intermediate risk categories, or together are known as lower risk MDS patients.

Initial damage to hematopoietic stem cells can occur for reasons such as, but not limited to, cytotoxic chemotherapy, radiation, virus, chemical exposure, and genetic predisposition. The bone marrow is dominated by a clonal mutation that suppresses healthy stem cells. In the early stages of MDS, the main cause of cytopenias is an increase in programmed cell death (apoptosis). As the disease progresses to leukemia, a mutation in the gene rarely occurs, and the proliferation of leukemic cells suppresses healthy bone marrow. The course of the disease is different, where in some cases it behaves like an indolent disease, and in other cases it behaves aggressively with a very short clinical course of the disease, turning into an acute form of leukemia.

An international group of hematologists, the Franco-American-British (FAB) Joint Group, has classified MDS disorders into five subgroups, distinguishing them from AML. The Merck Manual 954 ( ^17th ed. 1999); Bennett JM, et al., All. Intern. Med. October 1985, 103(4): 620-5; and Besa E. C, Med. Clin. North Am. 1992 May, 76(3):599-617. An underlying trilinear dysplastic change in the patients' bone marrow cells was found in all subtypes.

There are two subgroups of refractory anemia, characterized by five percent or less myeloblasts in the bone marrow: (1) refractory anemia (RA) and; (2) RA with ringed sideroblasts (RARS), defined morphologically as having 15% of erythroid cells with abnormal ringed sideroblasts, reflecting the abnormal accumulation of iron in the mitochondria. Both have a long clinical course and a low incidence of progression to acute leukemia. Besa E. C, Med. Clin. North Am. 1992 May, 76(3): 599-617.

There are two subgroups of non-responsive anemia with more than five percent myeloblasts: (1) RA with excess blasts (RAEB), with a definition of 6-20% myeloblasts, and (2) RAEB in the transformation stage (RAEB-T), with 21-30 % myeloblasts. The higher the percentage of myeloblasts, the shorter the clinical course and the closer the disease to acute myelogenous leukemia. The progression of patients from early to later stages indicates that these subtypes are simply disease stages and not distinct units. Elderly MDS patients with trilinear dysplasia and more than 30% myeloblasts progressing to acute leukemia are often considered to have a poor prognosis because their response rate to chemotherapy is lower than that of patients with de novo acute myeloid leukemia. The fifth type of MDS, the most difficult to classify, is CMML. This subtype can have any percentage of myeloblasts, but presents with monocytosis of 1000/dl or more. It may be associated with splenomegaly. This subtype overlaps with myeloproliferative disorder and may have an intermediate clinical course. It differs from classical CML in that it is negatively characterized by the Ph chromosome.

MDS is primarily a disease of the elderly, with a median onset in the seventh decade of life. The median age of these patients is 65 years, where the age ranges from the beginning of the third decade of life to as old as 80 years or older. The syndrome can occur in any age group, including pediatric patients. Patients surviving cancer treatment with alkylating agents, with or without radiotherapy, have a high incidence of MDS or secondary acute leukemia. Approximately 60-70% of patients have no obvious effect or cause causing MDS and are classified as primary MDS patients.

In some embodiments, provided herein are methods for treating MPN in a subject using FTI or selecting MPN patients for FTI treatment. MPN is a group of diseases that affect the formation of blood cells. In all forms of MPN, stem cells in the bone marrow develop genetic defects (called acquired defects) that cause abnormal growth and survival. This leads to an unusually high number of blood cells in the bone marrow (bone marrow with excess cellular content) and in the bloodstream. Sometimes in MPN, abnormal stem cells cause scarring in the bone marrow called myelofibrosis. Myelofibrosis can lead to low levels of blood cells, especially low levels of red blood cells (anemia). In MPN, abnormal stem cells can also grow in the spleen, causing enlargement of the spleen (splenomegaly), and in other areas outside the bone marrow, causing enlargement of other organs.

There are several types of chronic MPN, based on the cells affected. The three classic types of MPN include polycythemia vera (PV), in which too much RBC is present; essential thrombocythemia (ET), in which there are too many platelets; primary myelofibrosis (PMF), in which fibers and blast cells (abnormal stem cells) accumulate in the bone marrow. Other types of MPN include: chronic myeloid leukemia, in which there are too many white blood cells; chronic neutrophilic leukemia, in which there are too many neutrophils; chronic eosinophilic leukemia, not otherwise specified, in which there are too many eosinophils (hypereosinophilia); mastocytosis, also called mast cell disease, in which there are too many mast cells, which are a type of immune system cell found in tissues such as the skin and tissues of the digestive organs, rather than in the bloodstream; myeloid and lymphoid neoplasias with eosinophilia and abnormalities of the PDGFRA, PDGFRB and FGFR1 genes; and other unclassifiable myeloproliferative neoplasias.

In some embodiments, provided herein are methods for treating leukemia in a subject using FTI or selecting patients with leukemia for FTI treatment. Leukemia refers to malignant neoplasia of hematopoietic tissues. Various forms of leukemia are described, for example, in US patent No. 7393862 and provisional patent application US No. 60/380842, filed May 17, 2002, the entire contents of which are incorporated herein by reference. Although viruses have been reported to cause several forms of leukemia in animals, the causes of leukemia in humans remain largely unknown. The Merck Manual, 944-952 ( ^17th ed. 1999). Transformation to malignancy typically occurs in a single cell in two or more stages, followed by proliferation and clonal expansion. In some leukemias, specific chromosomal translocations have been identified with corresponding leukemic cell morphology and specific clinical features (eg, translocations 9 and 22 in chronic myelocytic leukemia, and 15 and 17 in acute promyelocytic leukemia). Acute leukemias are predominantly undifferentiated cell populations, while chronic leukemias are more mature cell forms.

Acute leukemias are divided into lymphoblastic (ALL) and non-lymphoblastic (ANLL) types. The Merck Manual, 946-949 ( ^17th ed. 1999). They can be further subdivided according to their morphological and cytochemical appearance according to the Franco-American-British (FAB) classification or according to their type and degree of differentiation. The use of B- and T-cell-specific and myeloid antigen-specific monoclonal antibodies best aids classification. ALL is predominantly a disease of childhood, which is determined by the results of laboratory tests and examination of the bone marrow. ANLL, also known as acute myelogenous leukemia or AML, occurs at any age and is the more common acute leukemia in adults; it is the form usually associated with radiation as an etiological factor. In some embodiments, provided herein are methods for treating patients with AML using FTI, or methods for selecting patients for treatment with FTI.

Standard treatments for patients with AML typically include 2 phases of chemotherapy (chemo): remission induction (or induction) and consolidation (post-remission therapy). The goal of the first part of treatment (induction of remission) is to get rid of as many leukemic cells as possible. The intensity of treatment may depend on the age and health of the individual. Intensive chemotherapy is often given to people under the age of 60. Slightly older patients in good general health may benefit from similar or slightly less intense treatment. People who are much older or have poor general health are not suitable for intensive chemotherapeutic agents.

In younger patients, such as those under 60 years of age, induction often involves treatment with 2 chemotherapeutic agents, cytarabine (ara-C) and an anthracycline drug such as daunorubicin (daunomycin) or idarubicin. Sometimes a third drug, cladribine (leustatin, 2-CdA), is also administered. Chemotherapy is usually given in a hospital and continues for about a week. In rare cases where the leukemia has spread to the brain or spinal cord, cerebrospinal fluid (CSF) chemotherapy may also be given. Radiotherapy can also be used.

Induction is considered successful if remission is achieved. However, AML in some patients may be refractory to induction. Patients who respond to induction are then subjected to further treatment to kill the remaining leukemic cells and help prevent recurrence, which is called consolidation. For younger patients, the main options for consolidation therapy are multiple cycles of high-dose cytarabine (ara-C) chemotherapy (known in some cases as HiDAC); transplantation of allogeneic (donor) stem cells; and transplantation of autologous stem cells.

Chronic leukemias are described as being lymphocytic (CLL) or myelocytic (CML). The Merck Manual, 949-952 ( ^17th ed. 1999). CLL is characterized by the appearance of mature lymphocytes in the blood, bone marrow, and lymphoid organs. The hallmark of CLL is persistent absolute lymphocytosis (>5000/μl) and an increase in the number of lymphocytes in the bone marrow. In most patients with CLL, there is also a clonal expansion of lymphocytes with B-cell characteristics. CLL is a disease of middle or older age. In CML, a characteristic feature is the predominance of granulocytes at all stages of differentiation in the blood, bone marrow, liver, spleen, and other organs. In a symptomatic patient at diagnosis, the total leukocyte count (WBC) is usually around 200,000/mcL, but can be as high as 1,000,000/mcL. CML is relatively easy to diagnose due to the presence of the Philadelphia chromosome. It is well known that bone marrow stromal cells support the progression of CLL disease and resistance to chemotherapy. Disruption of interactions between CLL cells and stromal cells is an additional target of CLL chemotherapy.

In addition, other forms of CLL include prolymphocytic leukemia (PLL), large granular lymphocyte leukemia (LGL), hairy cell leukemia (HCL). Cancer cells in PLL are similar to normal cells called prolymphocytes, immature forms of B-lymphocytes (B-PLL) or T-lymphocytes (T-PLL). Both B-PLL and T-PLL tend to be more aggressive than the normal type of CLL. Malignant cells in LGL are large and have features of either T cells or NK cells. Most LGL leukemias are slow growing, however, a small number are more aggressive. HCL is another lymphocyte malignancy that tends to progress slowly and accounts for approximately 2% of all leukemias.

Malignant cells are of the B-lymphocyte type, but differ from the malignant cells seen in CLL.

Juvenile myelomonocytic leukemia (JMML) is a severe chronic leukemia that mainly affects children 4 years of age and younger. The median age of patients at diagnosis is 2 years. The World Health Organization classifies JMML as a mixed myelodysplastic and myeloproliferative disorder. The JMML covers the diagnoses formerly referred to as juvenile chronic myeloid leukemia (JCML), childhood chronic myelomonocytic leukemia, and monosomy 7 neonatal syndrome.

Lymphoma refers to malignant tumors originating from the lymphatic system. Lymphoma is characterized by malignant neoplasia of lymphocytes - B-lymphocytes (B-cell lymphoma), T-lymphocytes (T-cell lymphoma) and natural killer cells (NK cell lymphoma). Lymphoma typically begins in the lymph nodes or collections of lymphatic tissue in organs including, but not limited to, the stomach or intestines. Lymphoma can affect the bone marrow and blood, in some cases. Lymphoma can spread from one area to other parts of the body.

Treatments for various forms of lymphomas are described, for example, in US Pat. No. 7,468,363, the entire contents of which are incorporated herein by reference. Such lymphomas include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous B cell lymphoma, activated B cell lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL; including but not limited to FL grade I, FL grade II), follicle center cell lymphoma, transformed lymphoma, intermediate grade lymphocytic lymphoma, intermediate lymphocytic lymphoma (ILL), diffuse low-grade lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse lymphoma small cell cleaved cell (DSCCL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma (CTCL) and mantle cell lymphoma, and low-grade follicular lymphoma.

Non-Hodgkin's lymphoma (NHL) is the fifth most common malignancy in both men and women in the United States, with an estimated 63,190 new cases and 18,660 deaths in 2007. Jemal A, et al., CA Cancer J Clin 2007; 57(1):43-66. The likelihood of developing NHL increases with age, and the incidence of NHL in the elderly has increased steadily over the past decade, raising concerns about the aging trend in the US population. Id. Clarke CA, et al., Cancer 2002; 94(7):2015-2023.

DLBCL accounts for approximately one third of non-Hodgkin's lymphomas. While some patients with DLBCL are cured with conventional chemotherapy, the rest die from the disease. Anticancer drugs cause rapid and persistent depletion of lymphocytes, possibly through direct induction of apoptosis in mature T and B cells. See K.

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Stahnke. et al., Blood 2001, 98:3066-3073. The absolute lymphocyte count (ALC) has been shown to be a prognostic factor in follicular non-Hodgkin's lymphoma, and recent results suggest that ALC at diagnosis is an important prognostic factor in DLBCL.

DLBCL can be divided into distinct molecular subtypes according to their gene profiling patterns: DLBCL from germinal center B-cell-like cells (GCB-DLBCL), DLBCL from activated B-cell-like cells (ABC-DLBCL), and primary mediastinal B-cell lymphoma (PMBL) or unclassified type. These subtypes are characterized by distinct differences in survival, ability to respond to chemotherapy, and dependence on the signal transduction pathway, in particular the NF-kB pathway. See D. Kim et al., Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol. 25, no. 18S (June 20 Supplement), 2007: 8082. See Bea S, et al., Blood 2005; 106:3183-90; Ngo V.N. et al., Nature 2011; 470:115-9. Such differences have prompted the search for more effective and subtype-specific treatments for DLBCL. In addition to being divided into acute and chronic categories, neoplasias are also divided into progenitor or peripheral cell categories based on the cells that give rise to such a disorder. See, for example, US Patent Publication No. 2008/0051379, the entire disclosure of which is incorporated herein by reference. Progenitor cell neoplasias include ALL and lymphoblastic lymphomas and occur in lymphocytes prior to their differentiation into either T or B cells. Peripheral cell neoplasias are neoplasias arising in lymphocytes that have differentiated into either T or B cells. Such peripheral cell neoplasias include, but are not limited to, B-cell CLL, B-cell prolymphocytic leukemia, lymphoplasmic lymphoma, mantle cell lymphoma, follicular lymphoma, extranodal marginal zone B-cell lymphoma of mucosal lymphoid tissue, nodal marginal lymphoma zones, marginal zone lymphoma of the spleen, hairy cell leukemia, plasmacytoma, diffuse large B-cell lymphoma (DLBCL) and Burkitt's lymphoma. In more than 95 percent of CLL cases, clonal expansion occurs in the B cell line. See Cancer: Principles & Practice of Oncology (3rd Edition) (1989) (pp. 1843-1847). Less than 5% of CLL tumor cells have a T cell phenotype. Regardless of these classifications, however, the pathological disruption of normal hematopoiesis is the hallmark of all leukemias.

PTCL consists of a group of rare and usually aggressive (fast growing) NHLs that develop from mature T cells. PTCL together account for approximately 4 to 10% of all NHL cases, corresponding to an annual incidence of 2800-7200 patients per year in the United States. According to some estimates, the incidence of PTCL is increasing significantly, and the increased incidence may be due to an aging population. PTCL is further classified into various subtypes, each of which is generally considered to be a separate disease based on their distinct clinical differences. Most of these subtypes are rare; the three most common subtypes of PTCL are, without further specification, anaplastic large cell lymphoma, or ALCL, and angioimmunoblastic T-cell lymphoma, which together account for 70% of all PTCLs in the United States. ALCL may be cutaneous ALCL or systemic ALCL.

For most PTCL subtypes, the first-line regimen is usually combination chemotherapy such as CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), EPOCH (etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone), or other multidrug regimens. Patients relapsing or refractory to first-line therapy are usually treated with gemcitabine in combination with other chemotherapy agents, including vinorelbine (Navelbine®) and doxorubicin (Doxil®), in a regimen called GND, or other chemotherapy regimens such as as DHAP (dexamethasone, cytarabine, cisplatin) or ESHAP (etoposide, methylprednisolone, cytarabine and cisplatin).

Because most patients with PTCL may relapse, some oncologists have recommended high-dose chemotherapeutic agents followed by autologous stem cell transplantation in some patients who have responded well to initial chemotherapy. Recently, non-cytotoxic drugs approved for relapsed or refractory PTCL, such as pralatrexate (Folotin®), romidepsin (Istodax®), and belinostat (Beleodak®), have been associated with relatively low objective response rates (overall response rate, or ORR, 25-27 %) and a relatively short duration of response (8.2-9.4 months). Accordingly, the treatment of relapsed/refractory PTCL remains an important unmet medical need.

Multiple myeloma (MM) is a malignant tumor of plasma cells in the bone marrow. Normally, plasma cells produce antibodies and play a key role in immune function. However, uncontrolled growth of these cells leads to pain and fractures in the bones, anemia, infections and other complications. Multiple myeloma is the second most common hematological malignancy, although the exact causes of multiple myeloma remain unknown. Multiple myeloma causes high levels of proteins in the blood, urine, and organs, including, but not limited to, M-protein and other immunoglobulins (antibodies), albumin, and beta-2-microglobulin. M-protein, short for monoclonal protein, also known as paraprotein, is a particularly abnormal protein produced by myeloma plasma cells and can be found in the blood or urine of almost all patients with multiple myeloma.

Skeleton-related symptoms, including bone pain, are among the most clinically important symptoms of multiple myeloma. Malignant plasma cells release osteoclast-stimulating factors (including IL-1, IL-6, and TNF) that cause calcium to be leached from bones, causing lytic lesions; hypercalcemia is another symptom. Osteoclast stimulating factors, also referred to as cytokines, may prevent apoptosis or death of myeloma cells. Fifty percent of patients have radiologically detectable myeloma-related skeletal lesions at diagnosis. Other common clinical symptoms of multiple myeloma include polyneuropathy, anemia, increased blood viscosity, infections, and kidney failure.

It is well known that bone marrow stromal cells support the progression and resistance to chemotherapy of multiple myeloma disease. Disruption of interactions between multiple myeloma cells and stromal cells is an additional target for multiple myeloma chemotherapy.

In some embodiments, provided herein are methods for predicting the ability of an MDS patient to respond to FTI treatment, methods for selecting a population of MDS patients for FTI treatment, and methods for treating MDS in a subject using a therapeutically effective amount of FTI based on the mutation status of Ras in a patient sample. . In some embodiments, provided herein are methods for predicting the ability of an MPN patient to respond to FTI treatment, methods for selecting a population of MDS patients for FTI treatment, and methods for treating MPN in a subject using a therapeutically effective amount of FTI, based on the mutation status of Ras in a patient sample. . In some embodiments, provided herein are methods for predicting the ability of an AML patient to respond to FTI treatment, methods for selecting a population of AML patients for FTI treatment, and methods for treating AML in a subject using a therapeutically effective amount of FTI based on the mutation status of Ras in a patient sample. . In some embodiments, provided herein are methods for predicting the ability of a JMML patient to respond to FTI treatment, methods for selecting a population of JMML patients for FTI treatment, and methods for treating JMML in a subject using a therapeutically effective amount of FTI, based on the mutation status of Ras in a patient sample. .

In some embodiments, provided herein are methods for predicting the ability of a CMML patient to respond to FTI treatment, methods for selecting a population of CMML patients for FTI treatment, and methods for treating CMML in a subject using a therapeutically effective amount of FTI based on the mutation status of Ras in a patient sample. . In some embodiments, provided herein is a method for treating CMML in a subject based on K-Ras, N-Ras, or both mutation status. The method provided herein includes (a) determining the presence or absence of a Ras mutation in a sample from a subject, where the Ras mutation includes a K-Ras mutation or an N-Ras mutation, and then (b) administering a therapeutically effective amount of FTI to said subject, if it is determined that in the specified sample there is no K-Ras mutation or N-Ras mutation. In some embodiments, the sample is determined to have no K-Ras mutation or no N-Ras mutation. In some embodiments, the sample is determined to have wild-type KRas. In some embodiments, the sample is determined to have wild-type N-Ras. In some embodiments, the sample is determined to have wild-type K-Ras and wild-type N-Ras. In some embodiments, the FTI is tipifarnib.

In some embodiments, provided herein are methods for predicting the ability of a patient with CMML to respond to tipifarnib, methods for selecting a population of patients with CMML for treatment with tipifarnib, and methods for treating CMML in a subject using a therapeutically effective amount of tipifarnib based on the status of K-Ras and N- Ras in a sample from a patient. In some embodiments, provided herein is a method for treating CMML in a subject based on K-Ras, N-Ras, or both mutation status. The method provided herein comprises (a) determining the presence or absence of a Ras mutation in a sample from a subject having CMML, where the Ras mutation includes a K-Ras mutation or an N-Ras mutation, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if the sample is determined to be free of a K-Ras mutation or an N-Ras mutation. In some embodiments, the sample is determined to have no K-Ras mutation or no N-Ras mutation. In some embodiments, the sample is determined to have wild-type K-Ras. In some embodiments, the sample is determined to have wild-type N-Ras. In some embodiments, the sample is determined to have wild-type K-Ras and wild-type N-Ras.

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4.4. Illustrative FTIs and doses.

In some embodiments, provided herein is a method for treating cancer in a subject based on mutation status of K-Ras, N-Ras, or both. The method provided herein includes (a) determining the presence or absence of a Ras mutation in a sample from a subject, where the Ras mutation includes a K-Ras mutation or an N-Ras mutation, and then (b) administering a therapeutically effective amount of tipifarnib to said subject, if it is determined that in the specified sample there is no K-Ras mutation or N-Ras mutation. In some embodiments, the sample is determined to have wild-type K-Ras. In some embodiments, the sample is determined to have wild-type N-Ras. In some embodiments, the sample is determined to have wild-type K-Ras and wild-type N-Ras. In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, provided herein is a method of treating a hematologic malignancy in a subject based on mutation status of K-Ras, N-Ras, or both. The method provided herein includes (a) determining the presence or absence of a Ras mutation in a sample from a subject, where the Ras mutation includes a K-Ras mutation or an N-Ras mutation, and then (b) administering a therapeutically effective amount of tipifarnib to said subject, if it is determined that in the specified sample there is no K-Ras mutation or N-Ras mutation. In some embodiments, the sample is determined to have wild-type K-Ras. In some embodiments, the sample is determined to have wild-type N-Ras. In some embodiments, the sample is determined to have wild-type K-Ras and wild-type N-Ras. In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, provided herein is a method for treating CMML in a subject based on mutation status of K-Ras, N-Ras, or both. The method provided herein includes (a) determining the presence or absence of a Ras mutation in a sample from a subject, where the Ras mutation includes a K-Ras mutation or an N-Ras mutation, and then (b) administering a therapeutically effective amount of tipifarnib to said subject, if it is determined that in the specified sample there is no K-Ras mutation or N-Ras mutation. In some embodiments, the sample is determined to have wild-type K-Ras. In some embodiments, the sample is determined to have wild-type NRas. In some embodiments, the sample is determined to have wild-type K-Ras and wild-type N-Ras. In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, FTI is administered orally, parenterally, rectally, or topically. In some embodiments, FTI is administered orally. In some embodiments, tipifarnib is administered orally, parenterally, rectally, or topically. In some embodiments, tipifarnib is administered orally.

In some embodiments, FTI is administered at a dose of 1-1000 mg/kg body weight. In some embodiments, FTI is administered twice a day. In some embodiments, FTI is administered at a dose of 200-1200 mg twice daily. In some embodiments, FTI is administered at a dose of 600 mg twice daily. In some embodiments, FTI is administered at a dose of 900 mg twice daily. In some embodiments, tipifarnib is administered at a dose of 1-1000 mg/kg body weight. In some embodiments, tipifarnib is administered twice daily. In some embodiments, tipifarnib is administered at a dose of 200-1200 mg twice daily. In some embodiments, tipifarnib is administered at a dose of 600 mg twice daily. In some embodiments, tipifarnib is administered at a dose of 900 mg twice daily.

In some embodiments, FTI is administered at a dose of 1-1000 mg/kg body weight. In some embodiments, FTI is administered twice a day. In some embodiments, FTI is administered at a dose of 200-1200 mg twice daily. In some embodiments, FTI is administered at a dose of 600 mg twice daily. In some embodiments, FTI is administered at a dose of 900 mg twice daily. In some embodiments, tipifarnib is administered in treatment cycles. In some embodiments, tipifarnib is administered on alternating weeks. In some embodiments, tipifarnib is administered on days 1-7 and 15-21 of 28 day treatment cycles. In some embodiments, tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles.

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In some embodiments, FTI is administered for at least 3 cycles. In some embodiments, FTI is administered for at least 6 cycles. In some embodiments, FTI is administered for up to 12 cycles. In some embodiments, FTI is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles for at least three cycles. In some embodiments, tipifarnib is administered for at least 3 cycles. In some embodiments, tipifarnib is administered for at least 6 cycles. In some embodiments, tipifarnib is administered for up to 12 cycles. In some embodiments, tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles for at least three cycles.

In some embodiments, provided herein are methods of treating CMML in a subject using a therapeutically effective amount of tipifarnib based on the status of K-Ras mutations in a sample from the patient. In some embodiments, provided herein is a method of treating CMML in a subject, comprising (a) determining the presence of wild-type K-Ras in a sample from the subject, and then (b) administering tipifarnib to the subject at a dose of 900 mg twice daily on days 1-7, and 15-21 of 28-day treatment cycles.

In some embodiments, provided herein are methods of treating CMML in a subject using a therapeutically effective amount of tipifarnib based on the N-Ras mutation status in a sample from the patient. In some embodiments, provided herein is a method of treating CMML in a subject, comprising (a) determining the presence of wild-type N-Ras in a sample from the subject, and then (b) administering tipifarnib to the subject at a dose of 900 mg twice daily on days 1-7, and 15-21 of 28-day treatment cycles.

In some embodiments, provided herein are methods of treating CMML in a subject using a therapeutically effective amount of tipifarnib based on the status of K-Ras and N-Ras mutations in a sample from the patient. In some embodiments, provided herein is a method of treating CMML in a subject, comprising (a) determining the presence of wild-type K-Ras and wild-type N-Ras in a sample from the subject, and then (b) administering tipifarnib to the subject at a dose of 900 mg twice daily on days 1-7 and 15-21 of the 28-day treatment cycles.

5. Mutant H-Ras as biomarkers for the treatment of FTI.

5.1. H-ras mutation status.

The H-ras protein is involved in the regulation of cell division in response to stimulation by growth factors. Growth factors act by binding to cell surface receptors penetrating the plasma membrane of cells. Once activated, the receptors stimulate a signal transduction event in the cytoplasm, the process by which proteins and second messengers transmit signals from the cell's external environment to the cell's nucleus and instruct the cell to grow or divide. H-ras is localized to the plasma membrane and is an early participant in many signaling pathways. H-ras acts as an on/off molecular switch - when turned on, it attracts and activates the proteins needed to signal from the receptor. In specific tumors, mutations in H-ras or its upstream effectors cause it to be constantly turned on, resulting in permanent activation of downstream growth and proliferation signals that drive tumor cell growth. FTIs act to prevent abnormal growth and proliferation of cells dependent on these signaling pathways by inhibiting protein farnesylation and subsequent localization to the H-ras membrane, thus turning off the H-ras.

FTIs such as tipifarnib prevent protein farnesylation, a type of protein modification known as prenylation, which, together with other protein modifications, allows H-ras to be localized to the membrane where it can receive and transmit extracellular signals involved in the initiation and development of malignant tumors. FTIs such as tipifarnib can block farnesylation and subsequent H-ras membrane localization and inhibit oncogenic H-ras-driven transformation in vitro and in vivo. While K-ras and N-ras similarly use protein farnesylation, they may also use a related prenylation pathway that also results in membrane localization. At the same time, H-ras membrane localization depends solely on protein farnesylation.

In some embodiments, the cancer being treated by the methods presented herein may have H-ras mutations. In some embodiments, the cancer being treated by the methods provided herein may be a solid tumor with an H-ras mutation. A solid tumor with a Hras mutation can be any of the solid tumors described above. In some embodiments, the solid tumor may be thyroid cancer, head and neck cancer, urothelial carcinomas, bladder cancer, or H-ras mutated salivary gland cancers. The methods provided herein or otherwise known in the art can be used to determine the mutation status of the ras gene. In some embodiments, the mutation status of the ras gene can be determined in an NGS-based assay. In some embodiments, the mutation status of the ras gene can be determined by qualitative analysis based on

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PCR. Qualitative PCR-based assays may be technically similar to assays based on

PCR already developed and approved by the FDA for K-ras. In some embodiments, the mutation status of the ras gene can be determined in the form of an accompanying diagnosis for FTI treatment, such as treatment with tipifarnib. Concomitant diagnosis can be performed at the clinical center where the patient is being treated with tipifarnib, or at a separate center.

Provided herein are methods for selecting cancer patients for FTI treatment based on the presence of an H-Ras mutation. These methods are based in part on the discovery that an H-Ras mutation is associated with a clinical benefit of FTI treatment and thus can be used to predict the ability of a cancer patient to respond to FTI treatment. Accordingly, provided herein are methods for predicting the ability of a cancer patient to respond to FTI treatment, methods for selecting a population of cancer patients for FTI treatment, and methods for treating cancer in a subject using a therapeutically effective amount of FTI based on the presence of an H-Ras mutation in a sample. from the patient. The cancer may be a hematopoietic cancer or a solid tumor. In some embodiments, the cancer is a solid tumor.

In some embodiments, provided herein is a method for treating cancer in a subject based on the presence of an H-Ras mutation. The method provided herein includes (a) determining the presence or absence of an H-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to the subject if the sample is determined to have an H-Ras mutation.

The sample may be a tumor sample. In some embodiments, the methods include (a) determining whether a patient with cancer has an H-Ras mutation and then (b) administering a therapeutically effective amount of FTI to the subject.

In some embodiments, the H-Ras mutation is a mutation at a codon selected from the group consisting of G12, G13, and Q61. In some embodiments, the H-Ras mutation may be a mutation selected from the group consisting of G12R, G12V, G13C, G13R, Q61L, and Q61R amino acid substitutions. In some embodiments, the mutation may be a mutation at a different codon that results in activation of the H-Ras protein.

In some embodiments, the methods provided herein further comprise (a) determining the presence or absence of a K-Ras mutation and an N-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to the subject if the sample does not have a K mutation. -Ras or N-Ras mutations. In some embodiments, the method includes administering a therapeutically effective amount of FTI to a subject if the sample has wild-type K-Ras and wild-type N-Ras.

In some embodiments, the K-Ras mutation is a K _A -Ras mutation. In some embodiments, the K-Ras mutation is a KB-Ras mutation. In some embodiments, the K-Ras mutation is a combination of a K _A -Ras mutation and a KB-Ras mutation. The K-Ras mutation may include a mutation at a codon selected from the group consisting of G12, G13 and Q61 K _A -Ras, K _B -Ras, or both. In some embodiments, the K _A -Ras mutation may include a mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C, G13D, G13R, G13S, G13N, Q61K, Q61H, Q61L, Q61P, Q61R and A146V. In some embodiments, the KB-Ras mutation may include a mutation selected from the group consisting of the amino acid substitutions G12C, G12D, G12A, G12V, G12S, G12F, G12R, G12N, G13C, G13D, G13R, G13S, G13N, Q61K, Q61H , Q61L, Q61P, Q61R and A146V.

In some embodiments, the N-Ras mutation may include at least one mutation in a codon selected from the group consisting of G12, G13, G15, G60, and Q61. In some embodiments, the N-Ras mutation may include at least one mutation in a codon selected from the group consisting of G12, G13, and Q61. In some embodiments, the N-Ras mutation may include at least one mutation selected from the group consisting of amino acid substitutions G12C, G12D, G12F, G12S, G12A, G12V, G12R, G13C, G13R, G13A, G13D, G13V, G15W, G60E, Q61P, Q61L, Q61R, Q61K, Q61H and Q61E.

In some embodiments, the sample is determined to have no amino acid substitution in G12, G13 and Q61 K-Ras, and no amino acid substitution in G12, G13 and Q61 N-Ras. In some embodiments, the sample is determined to have no K-Ras mutation or no N-Ras mutation. In some embodiments, the sample is determined to have wild-type K-Ras and wild-type N-Ras.

In some embodiments, the method provided herein comprises (a) determining the presence or absence of an H-Ras mutation, a K-Ras mutation, and an N-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to the subject if the sample was determined to have an H-Ras mutation, but not a K-Ras mutation or an N-Ras mutation. The sample may be a tumor sample. In some embodiments, the methods include (a) determining

- 86 040014 having a cancer patient with an H-Ras and wild-type K-Ras and wild-type N-Ras mutation, and then (b) administering a therapeutically effective amount of FTI to the subject. In some embodiments, the FTI is tipifarnib.

Provided herein are methods for treating cancer in a subject with FTI and methods for selecting cancer patients for FTI treatment based on the presence of an H-Ras mutation. The cancer may be a hematopoietic cancer or a solid tumor. Also provided herein are methods for treating a pre-malignant condition in a subject using FTI, and methods for selecting patients with a pre-malignant condition for FTI treatment based on H-Ras mutation status.

In some embodiments, provided herein are methods of treating cancer in a subject using FTI or selecting patients with cancer for FTI treatment based on the presence of an H-Ras mutation. The cancer may be a hematopoietic cancer or a solid tumor. The cancer may be human papillomavirus related (HPV+ or HPV positive) or non-HPV related (HPV- or HPV negative).

Provided herein are methods for predicting the ability of a cancer patient to respond to FTI treatment, methods for selecting a population of cancer patients for FTI treatment, and methods for treating cancer in a subject using a therapeutically effective amount of FTI based on the presence of an H-Ras mutation in a sample from patient. H-Ras mutation status can be detected at the nucleic acid or protein level. In some embodiments, the H-Ras mutation status is determined by analysis of nucleic acids obtained from a sample. In some embodiments, the H-Ras mutation status is determined by analysis of the protein obtained from the sample.

In some embodiments, the H-Ras mutation status is determined by analysis of nucleic acids obtained from a sample. Nucleic acids may be mRNA or genomic DNA molecules from the test subject. Methods for determining Ras mutation status by nucleic acid analysis are well known in the art. In some embodiments, the methods include sequencing, polymerase chain reaction (PCR), DNA microarray analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, denaturing high performance liquid chromatography (DHPLC), or restriction fragment length polymorphism (RFLP) analysis. In some embodiments, the Ras mutation status is determined using standard sequencing methods, including, for example, Sanger sequencing, next generation sequencing (NGS). In some embodiments, the Ras mutation status is determined using MS.

In some embodiments, the H-Ras mutation status is determined by analysis of a protein obtained from a sample. Mutant H-Ras protein can be detected by a variety of immunohistochemistry (IHC) methods, immunoblot analysis, enzyme-linked immunosorbent assay (ELISA), or other immunoassay methods known in the art.

As one of skill in the art would appreciate, any of the methods described herein or otherwise known in the art for Ras mutation analysis can be used to determine the presence or absence of an H-Ras mutation.

5.2. Samples.

In some embodiments, the methods provided herein include obtaining a sample from a subject. In some embodiments, the sample is a tumor sample. In some embodiments, the sample used in the present methods includes a biopsy (eg, a tumor biopsy). The biopsy may be from any organ or tissue, such as skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph nodes, hair, spleen, brain, breast, or other organs. Any biopsy technique known to those skilled in the art can be used to isolate a sample from a subject, such as open biopsy, closed biopsy, core biopsy, incisional biopsy, excisional biopsy, or fine needle aspiration biopsy.

The sample used in the methods presented herein includes body fluids of the subject.

Non-limiting examples of body fluids include blood (e.g. peripheral whole blood, peripheral blood), blood plasma, bone marrow, amniotic fluid, intraocular fluid, bile, lymph, menses, serum, urine, cerebrospinal fluid surrounding the brain and spinal cord, synovial fluid surrounding the joints of bones.

In one embodiment, the sample is a bone marrow sample. Methods for obtaining bone marrow samples are well known in the art, including, but not limited to, bone marrow biopsy and bone marrow aspiration. Bone marrow has a liquid part and a harder part. In a bone marrow biopsy, a sample of the hard part is taken. During aspiration of the bone marrow, a sample of the liquid part is taken. Bone marrow biopsy and bone marrow aspiration can be performed at

- 87 040014 the same time and designate as a bone marrow study.

In some embodiments, the sample is a blood sample. The blood sample can be obtained using conventional methods as described, for example, in Innis et al., editors, PCR Protocols (Academic Press, 1990). Leukocytes can be isolated from blood samples using conventional methods or commercially available kits such as the RosetteSep kit (Stein Cell Technologies, Vancouver, Canada). Leukocyte subpopulations, such as mononuclear cells, NK cells, B cells, T cells, monocytes, granulocytes, or lymphocytes, can be further isolated using conventional methods, such as magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, Calif.) or fluorescence-activated cell sorting (FACS) (Becton Dickinson, San Jose, California).

In specific embodiments, the sample used in the methods presented herein includes a plurality of cells. Such cells may include any cell type, such as stem cells, blood cells (eg, PBMCs), lymphocytes, NK cells, B cells, T cells, monocytes, granulocytes, immunocytes, or tumor cells or cancer cells. Specific cell populations can be generated using a combination of commercially available antibodies (eg, Quest Diagnostic (San Juan Capistrano, Calif.); Dako (Denmark)).

In specific embodiments, the sample used in the methods provided herein comes from diseased tissue, such as from an individual having a malignant tumor (eg, head and neck cancer, salivary gland tumor, or thyroid tumor). In some embodiments, cells can be obtained from tumor cells or cancer cells, or tumor tissues such as tumor biopsies or tumor explants. In specific embodiments, the number of cells used in the methods provided herein may range from a single cell to about 109 cells. In some embodiments, the number of cells used in the methods provided herein is approximately 1x10 ⁴ , 5x10 ⁴ , 1x105, 5x105, 1x10 ⁶ , 5x10 ⁶ , 1x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , or 5x10 ⁸ .

The number and type of cells collected from a subject can be monitored, for example, by measuring changes in cell morphology and surface markers using standard cell detection techniques such as flow cytometry, cell sorting, immunocytochemistry (e.g., staining using tissue-specific or marker-specific cells antibodies) fluorescence activated cell sorting (FACS), magnetic field activated cell sorting (MACS), by checking cell morphology using light or confocal microscopy, and/or by measuring changes in gene expression using methods well known in the art, such as PCR and gene expression profiling. These methods can also be used to identify cells that are positive for one or more specific markers.

Fluorescence activated cell sorting (FACS) is a well known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laser excitation of the fluorescent groups in the individual particles results in a small electrical charge allowing the electromagnetic separation of positive and negative particles from the mixture. In one embodiment, specific antibodies or ligands for cell surface markers are labeled with different fluorescent labels. The cells are passed through a cell sorter, allowing cells to be separated based on their ability to bind the antibodies used. FACS-sorted particles can be deposited directly into individual wells of 96-well or 384-well plates to facilitate separation and cloning.

In specific embodiments, subsets of cells are used in the methods provided herein. Methods for sorting and isolating specific cell populations are well known in the art and may be based on cell size, morphology, or intracellular or extracellular markers. Such methods include, but are not limited to, flow cytometry, flow sorting, FACS, bead-based separation such as magnetic cell sorting, size-based separation (e.g., using a sieve, array of obstacles, or filter), microfluidic sorting, separation based on antibodies, precipitation, affinity adsorption, affinity extraction, density gradient centrifugation, laser capture microdissection, etc.

5.3. Malignant tumors.

In some embodiments, provided herein are methods of treating hematopoietic cancer in a subject using FTI or selecting patients with cancers for FTI treatment based on the presence of an H-Ras mutation. In some embodiments, the hematopoietic cancer is HPV negative. In some embodiments, the methods include (a) determining in a patient with a negative

- 88 040014 for HPV hematopoietic malignancy of having an H-Ras mutation, and then (b) administering a therapeutically effective amount of tipifarnib to the patient.

Hematologic malignancies are malignant tumors of the blood or bone marrow. Examples of hematologic (or hematogenous) malignancies include myeloproliferative neoplasia (MPN), myelodysplastic syndrome (MDS), leukemia, and lymphoma. In some embodiments, the cancer is acute myeloid leukemia (AML), natural killer cell lymphoma (NK lymphoma), natural killer cell leukemia (NK leukemia), cutaneous T-cell lymphoma (CTCL), juvenile myelomonocytic leukemia (JMML), peripheral T-cell lymphoma (PTCL), chronic myeloid leukemia (CML), or chronic myelomonocytic leukemia (CMML). In some embodiments, the cancer is CMML. In some embodiments, the cancer is JMML.

In some embodiments, provided herein are methods for treating a solid tumor using FTI based on the presence of an H-Ras mutation. In some embodiments, the solid tumor is HPV negative. In some embodiments, the FTI is tipifarnib. In some embodiments, the methods comprise (a) determining the presence of an H-Ras mutation in an HPV negative solid tumor patient and then (b) administering a therapeutically effective amount of tipifarnib to the patient.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors may be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Solid tumors to be treated with the methods of the invention may be sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, malignant neoplasms of lymphoid tissue, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma , pheochromocytoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma oma and CNS tumors (such as glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases).

In some embodiments, provided herein are methods of treating a solid tumor using an FTI based on the presence of an H-Ras mutation, wherein the solid tumor is thyroid cancer, head and neck cancer, salivary gland cancers, malignant melanoma, adrenal carcinoma, breast carcinoma. cancer, renal cell carcinoma, pancreatic carcinoma, non-small cell lung carcinoma (NSCLC), or carcinoma of unknown primary site. In some embodiments, the FTI is tipifarnib. Drugs commonly administered to patients with various types or stages of solid tumors include, but are not limited to, Celebrex, etoposide, cyclophosphamide, docetaxel, apecitabine, IFN, tamoxifen, IL-2, GM-CSF, or a combination thereof.

In some embodiments, the solid tumor to be treated by the methods provided herein may be thyroid cancer, head and neck cancer, urothelial cancer, salivary gland cancers, upper gastrointestinal cancers, bladder cancer, breast cancer. gland cancer, ovarian cancer, brain cancer, stomach cancer, prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, and pancreatic cancer. In some embodiments, the bladder cancer being treated by the methods presented herein may be transitional cell carcinoma. In some embodiments, the FTI is tipifarnib.

In some embodiments, the solid tumor to be treated by the methods provided herein may be selected from the group consisting of carcinoma, melanoma, sarcoma, or chronic granulomatous disease.

In some embodiments, the pre-malignant conditions to be treated by the methods presented herein may be actinic cheilitis, Barrett's esophagus, atrophic gastritis, ductal carcinoma in situ, dyskeratosis congenita, sideropenic dysphagia, lichen planus, submucosal fibrosis of the oral cavity, solar elaboration. 89 040014 stosis, cervical dysplasia, polyps, leukoplakia, erythroplakia, squamous intraepithelial lesion, premalignant disorder, or premalignant immunoproliferative disorder.

In some embodiments, provided herein are methods of treating a solid tumor in a subject using FTI and methods of selecting patients with a solid tumor for FTI treatment based on the presence of an H-Ras mutation in the subject, wherein the solid tumor is thyroid cancer, head and neck cancer. or malignant tumor of the salivary glands. In some embodiments, the solid tumor is thyroid cancer. In some embodiments, the solid tumor is head and neck squamous cell carcinoma (HNSCC). In some embodiments, the solid tumor is salivary gland cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, with approximately 650,000 cases and 200,000 deaths per year worldwide and approximately 54,000 new cases per year in the US. It is also the most common malignant tumor in Central Asia.

HNSCC has 2 distinct etiologies and corresponding tumor types. The first subtype is associated with tobacco smoking and alcohol use and is not associated with human papillomavirus (HPV- or HPV-negative). The second subtype is associated with high-risk HPV infection (HPV+ or HPV positive). The second subtype is largely limited to malignant tumors of the oropharynx. HPV+ tumors are a single entity with a better prognosis and may require excellent treatment.

A significant proportion of HNSCC, in particular malignant tumors of the oropharynx, is caused by HPV infection. High-risk HPV subtype 16 accounts for 85% of all HPV+ tumors in HNSCC. P16 can be used as a surrogate marker for HPV infection in HNSCC, particularly in the oropharynx. More accurate HPV testing is available and based on E6/E7 detection (Liang C, et al., Cancer Res. 2012;72:5004-5013).

HPV+ HNSCC showed significantly lower levels of EGFR expression than HPVHNSCC. EGFR amplification occurs only in HPV-HNSCC. High EGFR gene copy number and protein expression are associated with poor clinical outcome in advanced HNSCC.

Current first-line therapy for relapsed/metastatic HNSCC includes dual platinum-based chemotherapy (eg, cisplatin/5-FU or carboplatin/paclitaxel), optionally in combination with anti-EGFR antibody therapy (eg, cetuximab, panitumumab, afatinib). Second line therapy includes taxanes, methotrexate and/or cetuximab. Therapy with an anti-EGFR antibody such as cetuximab (chimeric IgG1) or panitumumab can be used as monotherapy, with chemotherapy (eg, platinum/5-FU, cisplatin), or with radiotherapy. Despite high levels of EGFR expression in HNSCC, the response rate to cetuximab alone is only 13% with an SD rate of 33%, and there is currently no predictive biomarker available.

Drugs in development for HNSCC include drugs targeting the PI3K pathway: VKM120 (buparlisib) + cetuximab, BYL719 + cetuximab, temsirolimus + cetuximab, rigosertib + cetuximab; drugs targeting the MET pathway: tivantinib + cetuximab, ficlatuzumab + cetuximab; drugs targeting the EGFR/HER3 pathway: afatinib + cetuximab + paclitaxel, patritumab; drugs targeting the FGFR pathway: BGJ398; drugs targeting the CDK4/6-cell cycle pathway: palbociclib, LEE011; RTK inhibitor: anlotinib and chemotherapy: oral azacitidine. More recent potential therapies for HNSCC include immunotherapy such as anti-PD1 or anti-PDL1 antibodies.

While high cure rates have been achieved for localized and loco-regional disease using surgery, radiation, chemoradiotherapy, and induction chemotherapy, the survival rate for recurrent/metastatic diseases remains very low and the best possible therapies are needed.

In some embodiments, provided herein is a method for treating HNSCC in a subject based on the presence of an H-Ras mutation. In some embodiments, the HNSCC may be HPV negative HNSCC. In some embodiments, the HNSCC may be recurrent/repeated HNSCC. In some embodiments, the HNSCC may be a metastatic HNSCC. The method provided herein includes (a) determining the presence or absence of an H-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to the subject if the sample is determined to have an H-Ras mutation. The sample may be a tumor sample. In some embodiments, the methods include (a) determining the presence of an HRas mutation in an HNSCC patient and then (b) administering a therapeutically effective amount of FTI to the subject. In some embodiments, the FTI is tipifarnib.

- 90 040014

In some embodiments, provided herein is a method for treating salivary gland cancer in a subject based on the presence of an H-Ras mutation. In some embodiments, the salivary gland cancer may be an advanced salivary gland cancer. In some embodiments, the salivary gland cancer may be a metastatic salivary gland cancer. The method provided herein includes (a) determining the presence or absence of an H-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to the subject if the sample is determined to have an H-Ras mutation. The sample may be a tumor sample. In some embodiments, the methods include (a) determining the presence of an H-Ras mutation in a cancer patient, and then (b) administering a therapeutically effective amount of FTI to the subject. In some embodiments, the FTI is tipifarnib.

In some embodiments, provided herein is a method for treating thyroid cancer in a subject based on the presence of an H-Ras mutation. In some embodiments, the thyroid cancer may be recurrent/recurrent thyroid cancer. In some embodiments, the thyroid cancer may be metastatic thyroid cancer. In some embodiments, the thyroid cancer may be advanced thyroid cancer. The method provided herein includes (a) determining the presence or absence of an H-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of FTI to the subject if the sample is determined to have an H-Ras mutation. The sample may be a tumor sample. In some embodiments, the methods include (a) determining the presence of an HRas mutation in an HNSCC patient and then (b) administering a therapeutically effective amount of FTI to the subject. In some embodiments, the FTI is tipifarnib.

5.4. Illustrative FTIs and doses.

In some embodiments, provided herein are methods of treating cancer in a subject using tipifarnib or selecting patients with cancer for treatment with tipifarnib based on the presence of an H-Ras mutation. In some embodiments, the methods include treating a subject with another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, FTI is administered orally, parenterally, rectally, or topically. In some embodiments, FTI is administered orally. In some embodiments, tipifarnib is administered orally, parenterally, rectally, or topically. In some embodiments, tipifarnib is administered orally.

In some embodiments, FTI is administered at a dose of 1-1000 mg/kg body weight. In some embodiments, FTI is administered twice a day. In some embodiments, FTI is administered at a dose of 200-1200 mg twice daily. In some embodiments, FTI is administered at a dose of 600 mg twice daily. In some embodiments, FTI is administered at a dose of 900 mg twice daily. In some embodiments, tipifarnib is administered at a dose of 1-1000 mg/kg body weight. In some embodiments, tipifarnib is administered twice daily. In some embodiments, tipifarnib is administered at a dose of 200-1200 mg twice daily. In some embodiments, tipifarnib is administered at a dose of 600 mg twice daily. In some embodiments, tipifarnib is administered at a dose of 900 mg twice daily.

In some embodiments, FTI is administered in treatment cycles. In some embodiments, FTI is administered on alternating weeks. In some embodiments, FTI is administered on days 1-7 and 15-21 of 28 day treatment cycles. In some embodiments, FTI is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles. In some embodiments, tipifarnib is administered in treatment cycles. In some embodiments, tipifarnib is administered on alternating weeks. In some embodiments, tipifarnib is administered on days 1-7 and 15-21 of 28 day treatment cycles. In some embodiments, tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles.

In some embodiments, FTI is administered for at least 3 cycles. In some embodiments, FTI is administered for at least 6 cycles. In some embodiments, FTI is administered for up to 12 cycles. In some embodiments, FTI is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles for at least three cycles. In some embodiments, tipifarnib is administered for at least 3 cycles. In some embodiments, tipifarnib is administered for at least 6 cycles. In some embodiments, tipifarnib is administered for up to 12 cycles. In some embodiments, tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles for at least three cycles.

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In some embodiments, provided herein is a method for treating HNSCC in a subject using tipifarnib based on the presence of an H-Ras mutation. In some embodiments, the HNSCC may be HPV negative HNSCC. In some embodiments, the HNSCC may be recurrent/repeated HNSCC. In some embodiments, the HNSCC may be a metastatic HNSCC. The method provided herein includes (a) determining the presence or absence of an H-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if the presence of an H-Ras mutation is determined in the sample. The sample may be a tumor sample. In some embodiments, the methods include (a) determining the presence of an H-Ras mutation in an HNSCC patient and then (b) administering a therapeutically effective amount of tipifarnib to the subject.

In some embodiments, provided herein is a method of treating salivary gland cancer in a subject using tipifarnib based on the presence of an H-Ras mutation. In some embodiments, the salivary gland cancer may be an advanced salivary gland cancer. In some embodiments, the salivary gland cancer may be a metastatic salivary gland cancer. The method provided herein includes (a) determining the presence or absence of an H-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if the presence of an H-Ras mutation is determined in the sample. The sample may be a tumor sample. In some embodiments, the methods include (a) determining the presence of an H-Ras mutation in a patient with salivary gland cancer, and then (b) administering a therapeutically effective amount of tipifarnib to the subject. In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, provided herein is a method for treating thyroid cancer in a subject using tipifarnib based on the presence of an HRas mutation. In some embodiments, the thyroid cancer may be recurrent/recurrent thyroid cancer. In some embodiments, the thyroid cancer may be metastatic thyroid cancer. In some embodiments, the thyroid cancer may be advanced thyroid cancer. The method provided herein includes (a) determining the presence or absence of an H-Ras mutation in a sample from a subject, and then (b) administering a therapeutically effective amount of tipifarnib to the subject if the presence of an H-Ras mutation is determined in the sample. The sample may be a tumor sample. In some embodiments, the methods include (a) determining the presence of an H-Ras mutation in an HNSCC patient and then (b) administering a therapeutically effective amount of tipifarnib to the subject. In some embodiments, the methods include administering to the subject another FTI as described herein or otherwise known in the art. In some embodiments, the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP609,754, R208176, AZD3409, and BMS-214662.

In some embodiments, the methods include (a) determining the presence of an H-Ras mutation in an HNSCC patient and then (b) administering tipifarnib to the subject, wherein tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 -daily cycles of treatment. In some embodiments, the HNSCC patient has relapsed/refractory HNSCC. In some embodiments, the HNSCC patient is HPV negative HNSCC.

In some embodiments, the methods comprise (a) determining the presence of an H-Ras mutation in a patient with salivary gland cancer, and then (b) administering tipifarnib to the subject, wherein tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15- 21 of the 28-day treatment cycles.

In some embodiments, the methods include (a) determining the presence of an H-Ras mutation in a thyroid cancer patient and then (b) administering tipifarnib to the subject, wherein tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 from 28-day treatment cycles.

In some embodiments, the methods further comprise administering a second drug to a patient having a solid tumor with an H-Ras mutation. In some embodiments, the second drug is a chemotherapy agent such as cisplatin, 5-FU, carboplatin, paclitaxel, or platinum-based dual chemotherapy (eg, cisplatin/5-FU or carboplatin/paclitaxel). In some embodiments, the second drug is an anti-EGFR antibody therapy (eg, cetuximab, panitumumab, afatinib). In some embodiments, the second drug is taxanes, methotrexate, and/or cetuximab. In some embodiments, the implementation of the second drug is a radiotherapy. In some embodiments, the second drug includes drugs that target the PI3K pathway: BKM120 (buparlisib) + cetuximab, BYL719 + cetuximab, temsirolimus + cetuximab, rigosertib + cetuximab; drugs targeting the MET pathway: tivantinib + cetuximab, ficlatuzumab + cetuximab; drugs targeting the EGFR/HER3 pathway afatinib+cetuximab+paclitaxel, patritumab; drugs targeting the FGFR pathway: BGJ398; drugs targeting the CDK4/6 cell cycle pathway: palbociclib, LEE011; RTK inhibitor: anlotinib and chemotherapy: oral azacitidine. In some embodiments, the second drug is an immunotherapy, such as anti-PD1 or anti-PDL1 antibodies.

Some embodiments of the claimed invention are as follows.

1. A method of treating a solid tumor in a subject, comprising (a) determining the presence or absence of an H-Ras mutation in a sample from said subject, and then (b) administering a therapeutically effective amount of a farnesyl transferase inhibitor (FTI) to said subject, if said sample is determined to have H-Ras mutation.

2. The method of claim 1, wherein said H-Ras mutation contains an amino acid substitution at a codon selected from the group consisting of G12, G13, Q61, and any combination thereof.

3. The method of claim 1 or 2, further comprising determining the presence or absence of a K-Ras mutation or an N-Ras mutation, wherein said sample does not have a K-Ras mutation or an N-Ras mutation.

4. The method of claim 3, wherein said sample has wild-type K-Ras and wild-type N-Ras.

5. The method according to any one of claims 1 to 4, wherein said sample is a tissue biopsy.

6. The method according to any one of claims 1 to 4, wherein said sample is a tumor biopsy.

7. The method according to any one of claims 1 to 6, wherein determining the presence or absence of a Ras mutation comprises analyzing nucleic acids obtained from said sample.

8. The method of claim 7, wherein said Ras mutation is determined by sequencing, polymerase chain reaction (PCR), DNA microarray analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, denaturing high performance liquid chromatography (DHPLC), or restriction fragment length polymorphism (RFLP).

9. The method of claim 8, wherein the Ras mutation is determined by PCR.

10. The method of claim 8, wherein the Ras mutation is determined by sequencing.

11. The method of any one of claims 1-6, wherein determining the presence or absence of a Ras mutation comprises analyzing proteins derived from said sample.

12. The method of any one of claims 1 to 11, wherein said cancer is a hematological cancer or a solid tumor.

13. The method of claim 12, wherein said cancer is HPV negative.

14. The method of claim 12 wherein said cancer is in advanced stages or is metastatic.

15. The method of claim 12, wherein said cancer is a solid tumor selected from the group consisting of hepatocellular carcinoma, head and neck cancer, salivary gland tumor, thyroid tumor, urothelial cancer, breast cancer, melanoma, gastric cancer , pancreatic cancer and lung cancer.

16. The method of claim 13 wherein said cancer is head and neck cancer.

17. The method of claim 16 wherein said head and neck cancer is head and neck squamous cell carcinoma (HNSCC).

18. The method of claim 17 wherein the HNSCC is HPV negative HNSCC.

19. The method of claim 17 or 18, wherein said HNSCC is relapsed/refractory HNSCC.

20. The method of claim 13, wherein said cancer is a salivary gland tumor.

21. The method of claim 20 wherein said salivary gland tumor is an advanced salivary gland tumor.

22. The method of claim 13 wherein said cancer is a thyroid tumor.

23. The method of claim 21, wherein said thyroid tumor is a malignant thyroid tumor or an advanced thyroid tumor.

24. The method of claim 12 wherein the cancer is a hematologic cancer selected from the group consisting of myeloproliferative neoplasia (MPN), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), lymphoma natural killer cells (NK lymphoma), natural killer cell leukemia (NK leukemia), skin T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and chronic myeloid leukemia (CML).

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25. A method of treating a thyroid cancer in a subject, comprising (a) determining the presence or absence of an H-Ras mutation in a thyroid tumor sample from said subject, and then (b) administering a therapeutically effective amount of tipifarnib to said subject, if it is determined that said sample has an H-Ras mutation.

26. A method of treating HNSCC in a subject, comprising (a) determining the presence or absence of an H-Ras mutation in an HNSCC sample from said subject, and then (b) administering a therapeutically effective amount of tipifarnib to said subject if said sample is determined to have an H-Ras mutation .

27. A method of treating a salivary gland tumor in a subject, comprising (a) determining the presence or absence of an H-Ras mutation in a sample of said salivary gland tumor in said subject, and then (b) administering a therapeutically effective amount of tipifarnib to said subject, if said sample is determined to be has an H-Ras mutation.

28. A method of treating cancer in a subject, comprising (a) determining the presence or absence of a Ras mutation in a sample from said subject, wherein said Ras mutation contains a K-Ras mutation or an N-Ras mutation, and then (b) administering a therapeutically effective amount of an inhibitor farnesyl transferase (FTI) to said subject if it is determined that said sample lacks said K-Ras mutation or said N-Ras mutation.

29. The method of claim 28, wherein said Ras mutation comprises a K-Ras mutation.

30. The method of claim 28, wherein said Ras mutation comprises an N-Ras mutation.

31. The method of claim 28, wherein said Ras mutation comprises a K-Ras mutation and an N-Ras mutation.

32. The method of any one of claims 28-31, wherein said K-Ras mutation contains an amino acid substitution at a codon selected from the group consisting of G12, G13, Q61, and any combination thereof.

33. The method of any one of claims 28-31, wherein said N-Ras mutation contains an amino acid substitution at a codon selected from the group consisting of G12, G13, Q61, and any combination thereof.

34. The method according to any one of claims 28-33, wherein said Ras mutation contains an amino acid substitution at G12, G13 and Q61 in the K-Ras mutation; and an amino acid substitution at G12, G13 and Q61 in the N-Ras mutation.

35. The method of any one of claims 28-33, wherein said sample does not have a K-Ras mutation or an N-Ras mutation.

36. The method of claim 35, wherein said sample has wild-type K-Ras.

37. The method of claim 35, wherein said sample has wild-type N-Ras.

38. The method of claim 35, wherein said sample has wild-type K-Ras and wild-type N-Ras.

39. The method of any one of claims 28-38, wherein said Ras mutation further comprises an HRas mutation and said sample is determined to have said H-Ras mutation.

40. The method of claim 39, wherein said H-Ras mutation contains an amino acid substitution at a codon selected from the group consisting of G12, G13, Q61, and any combination thereof.

41. The method according to any one of claims 28-40, wherein said sample is a tissue biopsy.

42. The method according to any one of claims 28-40, wherein said sample is a tumor biopsy.

43. The method of any one of claims 28-40, wherein said sample is a blood sample, a serum sample, or a bone marrow sample.

44. The method according to any one of claims 28-43, wherein determining the presence or absence of a Ras mutation comprises analyzing nucleic acids obtained from said sample.

45. The method of claim 44, wherein said Ras mutation is determined by sequencing, polymerase chain reaction (PCR), DNA microarray analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, denaturing high performance liquid chromatography (DHPLC), or restriction fragment length polymorphism (RFLP).

46. The method of claim 45, wherein the Ras mutation is determined by PCR.

47. The method of claim 45, wherein the Ras mutation is determined by sequencing.

48. The method of any one of claims 28-43, wherein determining the presence or absence of a Ras mutation comprises analyzing proteins derived from said sample.

49. The method according to any one of claims 28-48, wherein said malignant tumor is myeloproliferative neoplasia (MPN), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), natural killer cell lymphoma (lymphoma NK), natural killer cell leukemia (NK leukemia), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), chronic myeloid leukemia (CML), or solid tumor.

50. The method of claim 49 wherein said cancer is MDS.

51. The method of claim 50, wherein said MDS is a lower risk MDS.

52. The method of claim 49, wherein said cancer is CMML.

53. The method of claim 52, wherein said CMML is low risk CMML or CMML

- 94 040014 with intermediate risk.

54. The method of claim 52, wherein said CMML is myelodysplastic CMML or myeloproliferative CMML.

55. The method of claim 49, wherein said cancer is AML.

56. The method of claim 55, wherein said subject is a subject after induction of remission or after transplantation.

57. The method of claim 55 wherein said subject is over 60 years of age or is otherwise unsuitable for remission induction.

58. The method of claim 49, wherein said cancer is a solid tumor selected from the group consisting of hepatocellular carcinoma, head and neck cancer, salivary gland tumor, thyroid tumor, breast cancer, melanoma, gastric cancer, pancreatic cancer gland and lung cancer.

59. A method of treating CMML in a subject, comprising (a) determining that said subject has wild type K-Ras and then (b) administering a therapeutically effective amount of tipifarnib to said subject.

60. A method of treating CMML in a subject, comprising (a) determining that said subject has wild-type N-Ras and then (b) administering a therapeutically effective amount of tipifarnib to said subject.

61. A method of treating CMML in a subject, comprising (a) determining that said subject has wild-type K-Ras and wild-type N-Ras, and then (b) administering a therapeutically effective amount of tipifarnib to said subject.

62. A method of treating cancer in a subject, comprising (a) typing KIR in said subject, wherein said subject is a carrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeutically effective amount of FTI to said subject.

63. The method of claim 62, wherein said subject is a KIR2DS2 carrier.

64. The method of claim 62, wherein said subject is a carrier of KIR2DS5.

65. The method of any one of claims 62-64, further comprising HLA typing in said subject, wherein said subject is an HLA-C2 carrier.

66. The method of claim 65, wherein said subject is a carrier of KIR2DS2 and HLA-C2.

67. The method of claim 65 or 66, wherein said HLA-C2 carrier is homozygous for HLA-C2.

68. The method of any one of claims 62-67, wherein the KIR typing comprises determining the presence of a KIR gene in a sample from said subject, wherein said KIR gene is selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, and KIR2DL5.

69. The method according to any one of claims 62-67, wherein KIR typing and HLA typing are performed by sequencing, polymerase chain reaction (PCR), DNA microarray analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, immunoblot analysis, or enzyme-linked immunosorbent assay (ELISA).

70. The method of claim 69, wherein the KIR typing is performed by SNP analysis using MS.

71. The method according to any one of claims 68-70, wherein said sample is a blood sample or a bone marrow sample.

72. The method of any one of claims 68-70, wherein said sample is peripheral blood mononuclear cells (PBMCs).

73. The method of any one of claims 68-70, wherein said sample is enriched in NK cells.

74. A method for treating a cancer in a subject, comprising (a) determining the expression level of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5 and GZMM in a sample from the specified subject, where (i) the expression level of KIR2DS2 in the sample is higher, than the reference level of expression of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression; and (b) administering a therapeutically effective amount of FTI to said subject.

75. A method of treating cancer in a subject, comprising (a) determining the expression levels of KIR2DS2 and KIR2DL2, or KIR2DS5 and KIR2DL5 in a sample from the specified subject, where (i) the ratio of the expression level of KIR2DS2 to the expression level of KIR2DL2 in the sample is higher than the reference ratio; or (ii) the ratio of the level of expression of KIR2DS5 to the level of expression of KIR2DL5 in the sample is higher than the reference ratio; and (b) administering a therapeutically effective amount of FTI to said subject.

- 95 040014

76. The method according to claim 74, including determining the expression level of KIR2DS2, where the expression level

KIR2DS2 in the sample is higher than the reference level of KIR2DS2 expression.

77. The method according to claim 74, including determining the expression level of KIR2DL2, where the expression level

KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression.

78. The method of claim 74, comprising determining the level of expression of KIR2DS5, where the level of expression of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression.

79. The method of claim 74, comprising determining the level of expression of KIR2DL5, where the level of expression of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression.

80. The method of claim 74, comprising determining the level of GZMM expression, where the level of GZMM expression in the sample is higher than a reference level of GZMM expression.

81. The method according to claim 75, comprising determining the levels of expression of KIR2DS2 and KIR2DL2, where the ratio of the expression level of KIR2DS2 to the level of expression of KIR2DL2 in the sample is higher than the reference ratio.

82. The method according to claim 75, comprising determining the levels of expression of KIR2DS5 and KIR2DL5, where the ratio of the expression level of KIR2DS5 to the expression level of KIR2DL5 in the sample is higher than the reference ratio.

83. The method according to any one of claims 74-82, wherein said sample is a blood sample or a bone marrow sample.

84. The method according to any one of claims 74-82, wherein said sample is a PBMC.

85. The method of any one of claims 74-82, wherein said sample is enriched in NK cells.

86. The method according to any one of claims 74-85, wherein said biomarker expression level is measured by determining the protein level of said biomarker.

87. The method of claim 86, wherein the protein level of said biomarker is determined using immunohistochemistry (IHC), immunoblot analysis, flow cytometry (FACS), or ELISA.

88. The method of claim 86, comprising contacting the sample with an antibody that immunospecifically binds to said biomarker protein.

89. The method according to any one of claims 74-85, wherein said biomarker expression level is measured by determining the RNA level of said biomarker.

90. The method of claim 89, wherein the mRNA level of said biomarker is measured using qPCR, RT-PCR, RNA sequencing, microarray analysis, SAGE, MassARRAY, or FISH.

91. The method according to any one of claims 62-90, wherein said malignant tumor is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), natural killer cell lymphoma (NK lymphoma), natural cell leukemia killer (NK leukemia), cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), chronic myeloid leukemia (CML), or a solid tumor.

92. The method of claim 91 wherein said cancer is AML.

93. The method of claim 92, wherein said subject is a subject after induction of remission or after transplantation.

94. The method of claim 92, wherein said subject is over 60 years of age or is otherwise unsuitable for remission induction.

95. The method of claim 91 wherein said cancer is MDS.

96. The method of claim 95, wherein said MDS is low risk MDS, intermediate risk MDS, or high risk MDS.

97. The method of claim 95, wherein said MDS is a lower risk MDS.

98. The method of claim 91 wherein said cancer is CMML.

99. The method of claim 98, wherein said CMML is low risk CMML or intermediate risk CMML.

100. The method of claim 98, wherein said CMML is myelodysplastic CMML or myeloproliferative CMML.

101. The method of claim 98, wherein said CMML is wild-type NRAS/KRAS CMML.

102. The method of claim 91 wherein said cancer is NK lymphoma or NK leukemia.

103. The method of claim 91 wherein said cancer is CTCL or PTCL.

104. The method of claim 91, wherein said cancer is a solid tumor selected from the group consisting of hepatocellular carcinoma, head and neck cancer, salivary gland tumor, thyroid tumor, breast cancer, melanoma, gastric cancer, pancreatic cancer gland and lung cancer.

105. A method of treating MDS in a subject, comprising (a) typing a KIR in said subject, wherein said subject is a carrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeutically effective amount of tipifarnib to said subject.

106. The method of claim 105, further comprising HLA typing in said subject, wherein said subject is an HLA-C2 carrier.

- 96 040014

107. The method of claim 106, wherein said subject is a carrier of KIR2DS2 and HLA-C2.

108. The method of claim 106 or 107, wherein said HLA-C2 carrier is homozygous for HLA-C2.

109. A method for treating MDS in a subject, comprising (a) determining the expression level of a biomarker selected from the group consisting of KIR2DS2,

KIR2DL2, KIR2DS5, KIR2DL5 and GZMM, in a sample from the specified subject, where (i) the level of expression of KIR2DS2 in the sample is higher than the reference level of expression of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression; and (b) administering a therapeutically effective amount of tipifarnib to said subject.

110. A method of treating MDS in a subject, comprising (a) determining the expression levels of KIR2DS2 and KIR2DL2, or KIR2DS5 and KIR2DL5 in a sample from the specified subject, where (i) the ratio of the expression level of KIR2DS2 to the expression level of KIR2DL2 in the sample is higher than the reference ratio; or (ii) the ratio of the level of expression of KIR2DS5 to the level of expression of KIR2DL5 in the sample is higher than the reference ratio; and (b) administering a therapeutically effective amount of tipifarnib to said subject.

111. A method for selecting a cancer patient for FTI treatment, comprising (a) typing a KIR in said cancer patient, wherein said cancer patient is a carrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeutically effective amount of FTI to said cancer patient. tumor.

112. The method of claim 111, further comprising HLA typing in said subject, wherein said subject is an HLA-C2 carrier.

113. The method of claim 112, wherein said subject is a carrier of KIR2DS2 and HLA-C2.

114. The method of claim 112 or 113, wherein said HLA-C2 carrier is homozygous for HLA-C2.

115. A method for selecting a patient with a malignant tumor for FTI treatment, comprising (a) determining the expression level of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5 and GZMM, in a sample from the specified subject, where (i) the expression level of KIR2DS2 in the sample is higher than the reference level of expression of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression; and (b) administering a therapeutically effective amount of FTI to said subject.

116. A method for selecting a patient with a malignant tumor for the treatment of FTI, comprising (a) determining the expression levels of KIR2DS2 and KIR2DL2, or KIR2DS5 and KIR2DL5 in a sample from the specified subject, where (i) the ratio of the expression level of KIR2DS2 to the expression level of KIR2DL2 in the sample is higher than reference ratio; or (ii) the ratio of the level of expression of KIR2DS5 to the level of expression of KIR2DL5 in the sample is higher than the reference ratio; and (b) administering a therapeutically effective amount of FTI to said subject.

117. A method of inhibiting NK cell activity in a subject, comprising (a) typing KIR in said subject, wherein said subject is a carrier of KIR2DS2 or KIR2DS5, and (b) administering a therapeutically effective amount of FTI to said subject.

118. The method of claim 117, further comprising HLA typing in said subject, wherein said subject is an HLA-C2 carrier.

119. A method for inhibiting NK cell activity in a subject, comprising (a) determining the expression level of a biomarker selected from the group consisting of KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5 and GZMM in a sample from the specified subject, where (i) the expression level of KIR2DS2 in the sample higher than the reference level of expression of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression; or (v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression; And

- 97 040014 (b) administering a therapeutically effective amount of FTI to said subject.

120. A method for inhibiting NK cell activity in a subject, comprising (a) determining the expression levels of KIR2DS2 and KIR2DL2 or KIR2DS5 and KIR2DL5 in a sample from the specified subject, where (i) the ratio of the expression level of KIR2DS2 to the expression level of KIR2DL2 in the sample from the specified subject is higher than reference ratio; or (ii) the ratio of the level of expression of KIR2DS5 to the level of expression of KIR2DL5 in the sample from the specified subject is higher than the reference ratio; and (b) administering a therapeutically effective amount of FTI to said subject.

121. The method according to any one of claims 1-120, wherein the FTI is selected from the group consisting of tipifarnib, arglabin, peryl alcohol, lonafarnib (SCH-66336), L778123, L739749, FTI-277, L744832, CP-609,754, R208176, AZD3409 and BMS-214662.

122. The method of claim 121 wherein the FTI is tipifarnib.

123. The method of any one of claims 1-122, wherein the FTI is administered orally, parenterally, rectally, or topically.

124. The method of any one of claims 1-123, wherein the FTI is administered at a dose of 1-1000 mg/kg body weight.

125. The method of any one of claims 1-124, wherein the FTI is administered twice a day.

126. The method of claim 125 wherein FTI is administered at a dose of 200-1200 mg twice daily.

127. The method of claim 125 wherein FTI is administered at a dose of 600 mg twice daily.

128. The method of claim 125, wherein FTI is administered at a dose of 900 mg twice daily.

129. The method of any one of claims 1-128, wherein the FTI is administered over a period of one to seven days.

130. The method of any one of claims 1-129, wherein the FTI is administered on alternating weeks.

131. The method of any one of claims 1-130, wherein the FTI is administered on days 1-7 and 15-21 of 28 day treatment cycles.

132. The method of claim 131, wherein the FTI is administered for at least 3 cycles.

133. The method of claim 131, wherein the FTI is administered for at least 6 cycles.

134. The method of claim 131, wherein said treatment cycle is continued up to 12 months.

135. The method of claim 132 wherein tipifarnib is administered orally at a dose of 900 mg twice daily on days 1-7 and 15-21 of 28 day treatment cycles.

136. The method of any one of claims 1-135, wherein the FTI is administered before, during, or after irradiation.

137. The method according to any one of claims 1-135, further comprising administering a therapeutically effective amount of the second active substance or maintenance therapy.

138. The method of claim 137, wherein said second active ingredient is a DNA hypomethylating agent, a therapeutic antibody that specifically binds a cancer antigen, a hematopoietic growth factor, a cytokine, an anticancer agent, an antibiotic, a cox-2 inhibitor, an immunomodulating agent, an antithymocyte globulin, an immunosuppressive agent, a corticosteroid, or a pharmacological derivative thereof.

139. The method of claim 137, wherein said second active ingredient is an anti-PD1 antibody or an anti-PDL1 antibody.

140. A kit for predicting the ability of a cancer patient to respond to FTI treatment, comprising at least one agent for typing KIR in said cancer patient and an auxiliary agent where said cancer patient is predicted to be capable of responding to FTI treatment if said cancer patient is a carrier of KIR2DS2 or KIR2DS5.

141. The kit of claim 140, further comprising an agent for typing HLA in said cancer patient, wherein said cancer patient is an HLA-C2 carrier.

142. A kit for predicting the ability of a patient with a malignant tumor to respond to FTI treatment, containing at least one agent for determining the expression of at least one biomarker in a sample from a patient with a malignant tumor and an auxiliary agent, where the specified biomarker is selected from the group consisting of KIR2DS2 , KIR2DL2, KIR2DS5, KIR2DL5 and GZMM; where predict that the specified patient with a malignant tumor is able to respond to treatment with FTI, if (i) the level of expression of KIR2DS2 in the sample is higher than the reference level of expression of KIR2DS2;

(ii) the expression level of KIR2DL2 in the sample is lower than the reference level of KIR2DL2 expression;

(iii) the expression level of KIR2DS5 in the sample is higher than the reference level of KIR2DS5 expression;

(iv) the expression level of KIR2DL5 in the sample is lower than the reference level of KIR2DL5 expression;

(v) the level of GZMM expression in the sample is higher than the reference level of GZMM expression;

(vi) the ratio of the level of expression of KIR2DS2 to the level of expression of KIR2DL2 in the sample is higher than the reference ratio; or (vii) the ratio of KIR2DS5 expression level to KIR2DL5 expression level in the sample is higher than the reference ratio.

- 98 040014

143. A kit for predicting the ability of a patient with a malignant tumor to respond to FTI treatment, containing one or more agents for determining the level of expression of KIR2DS2 and

KIR2DL2 in a sample from a cancer patient, and an adjuvant; where the indicated patient is predicted to be FTI responsive if

i) the level of expression of KIR2DS2 in the sample of the specified patient is higher than the reference level of expression of KIR2DS2;

(ii) the level of expression of KIR2DL2 in the sample from the specified patient is lower than the reference level of expression of KIR2DL2; or (iii) the ratio of the level of expression of KIR2DS2 to the level of expression of KIR2DL2 in the sample from the specified patient is higher than the reference ratio.

144. The kit of any one of claims 140-143, further comprising a label indicating that the kit is used to predict the ability of a cancer patient to respond to FTI treatment.

145. The kit of any one of claims 140-143, wherein said patient is an MDS patient and said FTI is tipifarnib.

6. Examples.

It is understood that modifications that do not substantially affect the operation of the various embodiments of this invention are also presented within the definition of the invention provided herein.

Accordingly, the following examples are intended to illustrate and not limit the present invention. The entire contents of all references cited herein are incorporated by reference.

Example I

Identification of immunological biomarkers associated with the clinical benefit of tipifarnib

Gene expression profiling analyzes in bone marrow samples from two AML studies, as well as IC ₅₀ data for tipifarnib in multiple cell lines, revealed that many immune-related genes, especially NK cell-related genes, were associated with better prognosis for tipifarnib. . While some of these genes appear to be non-specific for tipifarnib treatment, others, including KIR2DS2, KIR2DL2, KIR2DS5, KIR2DL5 and GZMM, were specifically associated with the clinical benefits of tipifarnib, but not other non-FTI chemotherapeutic agents. such as cytarabine and mitoxantrone.

The present study utilized microarray data obtained from a global gene expression analysis of bone marrow samples collected in two clinical trials investigating the efficacy and safety of tipifarnib FTI. One clinical study was performed on adult patients aged 65 years or older with previously untreated AML, and another was conducted on recurrent and refractory AML. The clinical results of these studies have been published previously (Lancet et al., Blood 109:1387-1394 (2007); Harousseau et al., Blood 9:9 (2007); Raponi et al., Clin. Cancer Res. 13:2254-2260 (2007)), and gene profiling data has been publicly available from NCBI's Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.qov/qeo) and is available under accession serial numbers from GEO Series GSE8970 and GSE5122.

As described in Raponi et al., Blood 111:2589-96 (2008), VM samples were collected from consenting patients prior to tipifarnib treatment and mononuclear cells were processed at the same center. Total RNA was isolated from cell samples, subjected to quality control, and further processed for microarray analysis. DNA was isolated from the same sample. Samples were subjected to global gene expression and/or quantitative polymerase chain reaction (QPCR) analysis of specific genes).

Response to tipifarnib was recorded in the clinical study report and defined as patients having complete remission (CR), partial remission (PR), or improvement in hematological parameters (HI). Patients with PR and HI were included as responders because they had previously been shown to have a similar survival advantage to those who achieved CR. Briefly, CR was defined as VM showing less than 5% of myeloblasts with normal maturation of all cell lines, an absolute neutrophil count (ANC) of at least 10 ⁹ /l (1000/μl), and a platelet count of 100x10 ⁹ /l (100000/ µl). PR was defined as the presence of ANC and platelet recovery to the above levels, but with 5-19% blast cells in the VM, and more than a 50% decrease in the percentage of blast cells in the VM from baseline. HI was defined as the same as PR except for ANC recovery to 0.5-1x10 ⁹ /l (500-1000/μl) and platelet count to 20-100x109/l (20000-100000/μl). Progressive disease (PD) was defined as any of the following: greater than 50% increase in the percentage of blast cells in the VM from baseline (greater than 5% blast cells if baseline percentage is less than 5%, greater than 10% blast cell count if the initial percentage is 5-10%, and more

- 99 040014 than 20% of blast cells, if the initial percentage is 10-20%); more than 50% increase in the percentage of circulating blast cells; the appearance of new circulating blast cells in at least 2 consecutive cases; or development of extramedullary leukemia. Stable disease (SD) was defined as any response that did not meet the criteria for CR, PR, HI, or PD.

Kaplan-Meier curves were used to investigate the relationship between biomarker values and clinical benefit. The identification of multiple NK cell-associated genes, the long duration of response observed in some patients on tipifarnib treatment, and the role of RAS-mediated signaling in NK cells support that the ability of AML patients to respond to tipifarnib treatment may result from immunocyte infiltration into the bone marrow. .

1. Correlation of KIR2DS5 expression level with the clinical benefit of tipifarnib.

As shown in FIG. 1A, KIR2DS5 expression level is associated with outcome in AML patients treated with tipifarnib. When categorizing patients based on different clinical responses, PD patients were found to cluster at the lower end of the KIR2DS5 expression range; CR patients clustered at the upper end of the KIR2DS5 expression range; and patients with HI and SD clustered between the CR and PD groups.

In addition, a strong correlation was identified between the expression level of KIR2DS5 and PFS in AML patients treated with tipifarnib (FIG. 1B). Patients whose KIR2DS5 expression levels were in the highest (fourth) quartile had a statistically significant longer PFS compared to the rest of the patients. The correlation supports that AML patients can be selected based on KIR2DS5 expression levels for tipifarnib treatment to increase the likelihood of being able to respond to treatment.

2. Specific correlation between the ratio of KIR2DS2 to KIR2DL2 expression and the clinical benefit of tipifarnib.

As shown in FIG. 2A and B, the ratio of KIR2DS2 expression level to KIR2DL2 expression level (2DS2/2DL2 ratio) was highly correlated with both PFS (FIG. 2A) and OS (FIG. 2B) of AML patients treated with tipifarnib. As shown, patients with a 2DS2/2DL2 ratio in the highest (fourth) quartile had statistically significant longer PFS (median=431 days) and OS (median=750 days) compared to the rest of the patient population.

In addition, the correlation between 2DS2/2DL2 ratio and clinical benefit was tipifarnib specific and was not observed for other non-FTI chemotherapeutic agents such as cytarabine and mitoxantrone (chemotherapy data from Metzeler KH et al., Blood, 112:4193 -201 (2008)). As shown in FIG. 3A and B, as well as in the table below, the survival rate of AML patients treated with high dose cytarabine and mitoxantrone did not differ between patients with a 2DS2/2DL2 ratio in the highest (fourth) quintile and the remaining patients.

Study 5~ ^th 0/ ^1-4th Q/all, median (days) Conditions Treatment HR I/5~ ^th Q GSE12417-GPL96 233/321/294 First line High dose cytarabine plus mitoxantrone 1.39 163/33 GSE12417-GPL570 308/624/538 First line High dose cytarabine plus mitoxantrone/intense chemotherapy in 17 patients 1.38 79/16 GSE8970 (STER-20) 754/179/233 First line, seniors tipifarnib 0.21 34/7

The specific correlation between the 2DS2/2DL2 ratio and the clinical benefit of tipifarnib supports that AML patients can be selected based on the 2DS2/2DL2 ratio for tipifarnib treatment to increase overall response to treatment.

3. Specific correlation between the ratio of KIR2DS5 to KIR2DL5 expression and the clinical benefit of tipifarnib.

As shown in FIG. 4A, the ratio of KIR2DS5 expression level to KIR2DL5A expression level (2DS5/2DL5 ratio) was strongly correlated with both PFS and OS of AML patients treated with tipifarnib. As shown, patients with a 2DS5/2DL5A ratio in the highest (fourth) quartile had a statistically significant longer PFS and OS compared to the rest of the patient population.

In addition, the correlation between 2DS5/2DL5 ratio and clinical benefit was tipifarnib specific and was not observed for other non-FTI chemotherapeutic agents such as cytarabine and mitoxantrone (chemotherapy data from Metzeler KH et al., Blood ,

112:4193-201 (2008)) (FIG. 4B). As shown in FIG. 4B, survival rates of AML patients treated with high dose cytarabine and mitoxantrone did not differ between patients with different 2DS5/2DL5 ratios.

The specific correlation between the 2DS5/2DL5 ratio and the clinical benefit of tipifarnib supports that AML patients can be selected based on the 2DS5/2DL5 ratio for tipifarnib treatment to increase overall response to treatment.

4. Specific correlation of GZMM expression level with the clinical benefit of tipifarnib.

As shown in FIG. 5, GZMM expression level is associated with outcome for AML patients treated with tipifarnib. When categorizing patients based on different clinical responses, PD patients were found to cluster at the lower end of the GZMM expression range and have the lowest median GZMM expression among the four groups (CR, HI, SD, and PD). CR patients clustered at the upper end of the GZMM expression range and had the highest median GZMM expression among the four groups.

In addition, a strong correlation was also identified between the expression level of GZMM and OS and PFS in AML patients treated with tipifarnib (FIG. 6A). Patients whose GZMM expression levels were in the highest (fourth) quartile had statistically significant longer OS and PFS compared to the rest of the patients. The correlation between GZMM expression level and clinical benefit was tipifarnib specific and was not observed for other non-FTI chemotherapeutic agents such as cytarabine and mitoxantrone (FIG. 6B). The specific correlation supports that AML patients can be selected based on GZMM expression levels for tipifarnib treatment to increase overall response to treatment.

5. Specific correlation of KIR2DS2 expression level with the clinical benefit of tipifarnib.

As shown in FIG. 7A, KIR2DS2 expression level is associated with outcome in AML patients treated with tipifarnib. A strong correlation was identified between KIR2DS2 expression level and OS in AML patients treated with tipifarnib (FIG. 7A). Patients whose KIR2DS2 expression levels were in the highest ( ^4th ) quartile had a statistically significant longer OS compared to the rest of the patients (Fig. 7A and Fig. 7B, upper left panel).

As shown in FIG. 7B and in the table below, KIR2DS2 expression strongly correlated with clinical benefit, including complete response rates and survival endpoints. Patients in the upper (fourth) quartile of KIR2DS2 expression had a median survival of 564 days, while patients in 1-3 quartiles of KIR2DS2 expression had a median survival of 153 days. In contrast, no correlation was identified between the expression of NK cell markers, including KIR2DS2, and the clinical benefit derived from chemotherapy treatment in a subgroup of 51 previously untreated and elderly (>65 years) AML patients enrolled in the German AML Cooperative Group study. 1999 study (AMLCG 1999) (Fig. 7B, right panel). Of 34 previously untreated, poor-risk, and elderly patients with AML who were treated with tipifarnib in prior phase 2 clinical trials, 25 had prior MDS. This specific correlation supports that cancer patients can be selected based on KIR2DS2 expression levels for tipifarnib treatment to increase the likelihood of being able to respond to AML and MDS treatment.

Treatment (p) Median overall survival (days) KIR2DS2, lower 1st to 3rd quartiles, median survival (days) KIR2DS2, upper 4~ ^and quartile, (upper) median survival (days) Risk ratio Tipifarnib (34) 233 153 564 0.303 Chemotherapy (51) 240 176 284 0.83

Example II.

A. Stratification of patients with AML for tipifarnib clinical trials.

Clinical studies can be conducted that include KIR typing as part of the patient inclusion criteria. For example, a study could be conducted for the treatment of tipifarnib in patients with AML who are over 60 years of age or are otherwise unsuitable for standard chemotherapy or who have refractory or recurrent AML.

Before a patient with AML is admitted to clinical studies, a bone marrow or blood sample is obtained from the patient. The sample is then subjected to microarray analysis. DNA was isolated from a Trizol-treated bone marrow sample according to the manufacturer's instructions (Qiagen). Samples are subjected to global gene expression and quantitative polymerase chain reaction (QPCR) analysis of specific genes including KIR2DS2, KIR2DL2, KIR2DS5 and KIR2DL5. Among other things, microarray analysis provides the genotype of the patient's KIR genes. If the patient is identified as a carrier of the KIR2DS2 gene or a carrier of the KIR2DS5 gene, the patient is then admitted to the tipifarnib study. Illustrative inclusion criteria may be as follows.

Pathological confirmation of the diagnosis of AML (>20% blast cells in the bone marrow).

General condition according to ECOG 0 or 1.

Patients must be able to give informed consent with SGOT and SGPT < 2.5x limit of normal (Grade 1).

Serum creatinine <1.5x normal range (Grade 1).

AML (any of the following).

Newly diagnosed AML in adults >70 years of age.

Newly diagnosed AML resulting from MDS in adults >60 years of age.

Biopsy-proven recurrent or refractory AML.

Hyperleukocytosis with >30,000 leukemic blast cells/µl.

Carrier of KIR2DS2, or KIR2DS5, confirmed by bone marrow biopsy.

An exemplary dosing regimen may be as follows.

Patients are given 600 mg of tipifarnib orally (PO) twice daily (B.I.D.) on days 1-21. Courses are repeated every 28 days in the absence of disease progression or unacceptable toxicity.

Complete remission rate (CR) and partial remission rate (PR) may be indicators of the primary outcome of the study.

B. MDS patients for tipifarnib clinical trials with immunocyte markers as secondary endpoints.

Clinical studies can be conducted that include KIR typing as part of the patient inclusion criteria. For example, a study could be conducted to treat tipifarnib in AML patients with MDS, or specifically, lower risk MDS. The primary endpoint of the study is transfusion independence according to the criteria of the International Working Group for Myelodysplastic/Myeloproliferative Neoplasias in Adults or a related response scoring system. Secondary endpoints may include analysis of immunocyte markers, especially NK cell markers such as KIR2DS2, KIR2DS5, KIR2DL2, KIR2DL5 and GZMM.

When a lower risk MDS patient is admitted to clinical studies, a bone marrow sample or blood sample is obtained from the patient. The sample is then subjected to microarray analysis. DNA was isolated from a Trizol treated bone marrow sample according to the manufacturer's instructions (Qiagen). Samples are subjected to global gene expression and quantitative polymerase chain reaction (QPCR) analysis of specific genes such as KIR2DS2, KIR2DS5, KIR2DL2, KIR2DL5 and GZMM. Among other things, microarray analysis provides the genotype of the patient's KIR genes.

An exemplary dosing regimen may be: Patients are administered 600 mg of tipifarnib orally (PO) twice a day (B.I.D.) on days 1-21. Courses are repeated every 28 days in the absence of disease progression or unacceptable toxicity.

A companion diagnostic test can also be used to help select patients in tipifarnib clinical trials in a lower-risk MDS patient population. You can use genetic assays that detect the presence or absence of KIR genes in NK cells, as described herein or otherwise known in the art. Assays described herein (eg, PCR-based assay) or otherwise known in the art can also be used to determine biomarker expression levels, and an optimal biomarker cut-off criterion can be determined to select patients for subsequent clinical studies.

Example III.

Individualized therapeutic solutions for patients with CMML.

The following methods can be taken to determine if a patient with CMML is suitable for FTI treatment, such as tipifarnib treatment.

A VM sample is taken from the patient prior to treatment and the mononuclear cells are processed at the same center. Total RNA is isolated from cell samples using a Trizol kit (Qiagen, Santa Clarita, CA). RNA quality is determined by assessing the presence of ribosomal RNA bands in an Agilent Bioanalyzer (Agilent, Palo Alto, CA). Good quality samples are further processed for microarray analysis.

For each sample, 1 g of total RNA (as estimated by OD260) was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Samples can then be incubated at 25°C for 10 min and then at 37°C for 30 min for optimal

- 102 040014 RNA transformations. QPCR was performed using an ABI Prism 7900HT sequence detection system (Applied Biosystems) where all samples were run in triplicate. Each reaction contains 5 µl of Taqman Universal Preset PCR Reaction Mix containing uracil-N-glycosylase (Applied Biosystems), 4.5 µl of cDNA template and 0.5 µl of 20x Gene Expression Assay Reaction Mix (Applied Biosystems) or 9 pmol both forward and reverse primers, and 2.5 pmol of probe in a total reaction volume of 10 μl. All sets of primers and fluorogenic probes with fluorescein amidite (FAM) are chosen to generate amplicons of less than 100 nucleotides, allowing the amplification of transcripts from degraded RNA samples. Primers and probes are designed to specifically amplify KIR2DS2 and KIR2DL2. All primer sets cross exon boundaries and thus specifically amplify mRNA transcripts rather than genomic DNA.

The KIR2DS2/KIR2DL2 expression ratio is then calculated using methods known in the art (eg, Ma et al., Cancer Cell, 5:607-616 (2004), the entire content of which is hereby incorporated by reference). The raw Ct values are normalized by subtracting the mean Ct for the set of samples, dividing by the standard deviation, and then calculating the differences in the normalized Ct values for each gene.

As described herein, the reference ratio of expression of KIR2DS2/KIR2DL2 can be determined through statistical analysis. As shown in FIG. 2A and 2B (Example I. II above), for example, the reference expression ratio can be an expression ratio that distinguishes patients with a 2DS2/2DL2 ratio in the highest ( ^4th ) quartile from other patients. Accordingly, if it is determined that the CMML patient's 2DS2/2DL2 ratio is higher than the reference ratio (namely, the CMML patient's 2DS2/2DL2 ratio is in the highest (fourth) quartile) and the patient has no other barriers to being treated with tipifarnib, then prescribe treatment with tipifarnib. On the other hand, if the CMML patient's 2DS2/2DL2 ratio is determined to be lower than the reference ratio, tipifarnib treatment is not recommended.

If tipifarnib treatment is prescribed to a patient with CMML, the patient with CMML may be simultaneously subjected to other treatment, such as exposure to ionizing radiation or a second active agent, or supportive care, as deemed appropriate by the oncologist. The second active ingredient may be a DNA hypomethylating agent such as azacitidine or decitabine.

Example IV.

Lower IC ₅₀ for tipifarnib in myeloid and lymphoid cell lines with wild-type KRAS/N-RAS status.

As shown in the table below, analyzes of tipifarnib IC ₅₀ data in multiple myeloid and lymphoid cell lines revealed that cell lines carrying mutations at codon 12, 13 and/or 61 of N-RAS or K-RAS are resistant to tipifarnib, in while cell lines not carrying these mutations, including cells having wild-type N-RAS and K-RAS, are more sensitive to tipifarnibom treatment.

Tipifarnib IC ₅₀ for myeloid and lymphoid cell lines.

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cell line NRAS KRAS Tipifarnib IC50 (log µM) ML-2 wt A146T -4.29 SIG-M5 wt wt -4.23 QIMR-WIL wt wt -4, 12 QMS wt wt -1,89 GDM-1 wt wt -1, 04 HEL wt wt -0.93 NKM-1 wt wt -0.26 CESS wt wt 0.70 OCI-AML2 wt wt 0.71 KG-1 wt wt 1, 12 MONO-MAC-6 wt wt 1.21 CTV-1 wt wt 1.52 HL-60 Q61L wt 1.94 KMOE-2 Q61R wt 2.14 K052 G13R wt 2.77 NOMO-1 wt G13D 6, 84 THP-1 G12D wt 7, 67 P31-FUJ G12C wt 7.76

Data from Genomics of Drug Sensitivity in Cancer (GDSC).

Example V.

Long-term responses in MDS/CMML patients with wild-type N-RAS/K-RAS tumors after tipifarnib treatment.

Twenty-one patients with MDS were treated with tipifarnib in a phase 1 dose escalation study. Tipifarnib was administered twice daily (scheduled 3 weeks on/1 week off, for 8 weeks) (initial dose 300 mg po twice daily; total 600 mg).

Objective responses of 3 HI, 2PR and 1 CR were observed in 6 of 20 (30%) evaluable patients. As shown in Table. 2 below, MDS patients with N-RAS and wild-type K-RAS are more likely to have sustained responses (Kurzrock et al., Blood, 102 (13):4527-34 (2003)).________________________

Diagnosis Answer Duration (months) Total daily dose, mg Ras mutation RAE B HI 16 300* No CMML HI 2 600 Yes (K-RAS) CMML PR 6 600 Yes (N-RAS) CMML PR 16+ 800 No RAE B HI 3 900 No RAEB-T CR 9+ 800 No

*The patient was started on a total daily dose of 600 mg/day, but the dose was reduced after 2 weeks

RAEB: refractory anemia with excess blasts

Example VI.

Prolonged PFS and higher response rates in AML patients with wild-type N-RAS status following tipifarnib treatment with CTER-20 was a phase 2 clinical trial of tipifarnib in previously untreated elderly or untreated AML patients. (Raponi et al., Blood 111:2589-96 (2008)).

N-RAS gene status was determined for 32 patients. As shown in FIG. 8, a trend for better PFS was observed in AML patients with wild-type N-RAS (PFS=157 days) compared to mutant N-RAS patients (PFS=65 days). As shown in FIG. 9, patients with wild-type N-RAS (43% ORR) have a higher response rate compared to patients with mutant N-RAS (27% ORR). Accordingly, patients with AML can be selected based on the mutation status of the RAS gene for treatment.

- 104 040014 tipifarnib to increase the ability to respond to treatment.

Example VII.

Clinical studies of tipifarnib in patients with CMML stratified based on RAS mutation status.

This example describes a phase 2 clinical trial of tipifarnib with the primary objective of evaluating the antitumor activity of tipifarnib, in terms of objective response rates (ORR) using the International Working Group for Myelodysplastic/Myeloproliferative Neoplasia (MDS/MPN IWG) criteria, for tipifarnib in subjects with chronic myelomonocytic leukemia (CMML) and in subjects with CMML whose disease is characterized by wild-type KRAS/NRAS. Secondary goals include evaluating the effect of tipifarnib on CR rates, cytogenetic complete remission, partial remission, bone marrow response, and clinical benefit; response time; frequency of PFS within 1 year; survival rate at 1 year; adverse event profile (AE) according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03 (NCI CTCAE v 4.03).

This phase 2 study investigates the antitumor activity of tipifarnib against ORR in subjects with CMML. Up to 20 eligible subjects were enrolled and retrospectively stratified into one of two strata (approximately 10 subjects per stratum) based on the subject's KRAS and/or NRAS mutation status. The first stratum can enroll subjects with KRAS and wild-type NRAS tumors. The second stratum can enroll subjects with KRAS mutant, NRAS mutant, or double mutant tumor status.

Subjects can be treated with tipifarnib given at a starting dose of 900 mg, orally with food, twice a day (b.i.d.) for 7 days on alternating weeks (days 1-7 and 15-21) in 28-day cycles. At the discretion of the investigator, the dose of tipifarnib may be increased to 1200 mg b.i.d. if the subject does not experience dose-limiting toxicity at the 900 mg dose level. Subjects who develop a serious adverse event (SAE) or a >grade 2 adverse event (TEAE) that appears to be related to tipifarnib and lasts >14 days during treatment should not be subjected to a dose increase. Stepwise dose reductions of 300 mg are also allowed to control treatment-related toxicity that occurs during treatment.

In the absence of uncontrolled toxicity, subjects may continue to be treated with tipifarnib until disease progression. If a complete response is observed, tipifarnib therapy may be maintained for at least 6 months after the onset of response.

Disease assessment (bone marrow, hematology, and quality of life assessments) can be performed at screening and at day 22 (±5 days) examination during cycles 2, 4, 6 and approximately every 12 weeks thereafter (cycles 9, 12, 15, etc.). Hematologic evaluations, including peripheral blood evaluations and review of required transfusions, can be performed at screening and at least monthly until disease progression. Bone marrow aspiration/biopsy screening is not necessary to initiate treatment in subjects who have a bone marrow aspiration/biopsy confirming their diagnosis within 4 weeks prior to day 1 of cycle 1 and can provide specimens to complete study objectives. If bone marrow aspiration is inadequate to evaluate the disease on schedule, a bone marrow biopsy may be performed. Additional disease or haematological evaluations may be performed as deemed necessary by the investigator. The time period for disease assessments and hematological assessments is kept as long as possible, regardless of the potential length of the treatment cycle.

Example VIII.

Individualized therapeutic solutions for patients with CMML.

The following methods can be taken to determine if a patient with CMML is suitable for FTI treatment, such as tipifarnib treatment.

DNA can be previously isolated from bone marrow cells (mononuclear cells or buffy coat) or peripheral blood of a patient exhibiting CMML. N-Ras and K-Ras mutation status is determined by DNA sequencing using a chain termination method adapted for fluorescent primers in an ABI 3100 sequencer (Applied Biosystems, Foster City, CA). If negative results are obtained for direct sequencing, the PCR products are cloned (original TA Cloning Kit; Invitrogen, Groningen, the Netherlands) and sequenced.

If a CMML patient is determined not to have any mutations in K-Ras or N-Ras codons 12, 13, and 61, or if a CMML patient is determined to have wild-type K-Ras and N-Ras, and if the patient does not have other obstacles to exposure to tipifarnib treatment, treatment with tipifarnib is prescribed. On the other hand, if a CMML patient is determined to have either an N-Ras or K-Ras mutation resulting in either N-Ras or K-Ras activation, tipifarnib treatment is not recommended.

If tipifarnib treatment is prescribed to a patient with CMML, the patient with CMML may be simultaneously subjected to other treatment, such as exposure to ionizing radiation or a second active agent, or supportive care, as deemed appropriate by the oncologist. The second active ingredient may be a DNA hypomethylating agent such as azacitidine or decitabine.

Example IX.

Clinical studies of tipifarnib in patients with solid tumors stratified based on HRAS mutation status.

A phase 2 clinical trial has begun for the use of tipifarnib in the treatment of advanced tumors with a known HRAS mutation. The clinical trial design includes enrollment of 2 cohorts of 18 patients each. Cohort 1 enrolled subjects with thyroid malignancies with HRAS mutations, regardless of thyroid histology. Cohort 2 included any subjects with non-hematologic HRAS mutant tumors other than thyroid cancer who met the inclusion criteria.

This clinical trial is designed to include two stages where the first stage included 11 evaluable patients and the second stage included 7 additional evaluable patients, and the cohort is not advanced to the second stage if one objective response is observed or no objective responses are observed in the first cohort. stages. A clinical trial is considered positive if at least 4 responders are seen in a cohort of 18 subjects. The primary endpoint is the objective response rate, and tumor response is assessed according to the Solid Tumor Response Evaluation Criteria version 1.1 (response to be confirmed).

As per protocol, tipifarnib is administered to registered patients at a baseline dose of 900 mg, orally with food, twice daily (b.i.d.) for 7 days on alternating weeks (days 1-7 and 15-21) in 28-day cycles. At the discretion of the investigator, the dose of tipifarnib may be increased to 1200 mg b.i.d. if the subject does not experience dose-limiting toxicity at the 900 mg dose level. Subjects who develop a serious adverse event (SAE) or a >grade 2 adverse event (TEAE) that appears to be related to tipifarnib and lasts >14 days during treatment should not be subjected to a dose increase. Stepwise dose reductions of 300 mg are also allowed to control treatment-related toxicity that occurs during treatment.

Four (4) eligible patients were enrolled in the first cohort (patients with thyroid cancers with HRAS mutations) and eleven (11) eligible patients were enrolled in the second cohort (patients with non-hematologic tumors other than thyroid cancer with HRAS mutations) . In the second cohort, three (3) of these patients had recurrent/refractory head and neck carcinoma, and two (2) of these three (3) experienced confirmed objective partial responses (PR), and the third patient experienced disease stabilization for more than six months (> 8 months). All three patients with head and neck carcinoma are HPV negative. All patients with head and neck tumors remained in the study. Responses were observed for two patients with PR after 3 cycles of treatment, six cycles for one, and three for the other. In addition, five (5) reported patients had salivary gland malignancies with HRAS mutations, and three (3) of them experienced disease stabilization for more than six months (>7, 9, and >11 months). Cohort 2 was moved to the second phase of the study to enroll seven additional patients for the study protocol.

X example.

Individualized therapeutic solutions for patients with solid tumors.

The following methods can be adopted to determine if a patient having a solid tumor is suitable for FTI treatment, such as tipifarnib treatment. The patient may have a thyroid tumor. The patient may have a salivary gland tumor. The patient may also have head and neck swelling. The head and neck tumor may be a squamous cell carcinoma of the head and neck.

DNA can be isolated from a tumor sample from a patient. H-Ras mutation status is determined by DNA sequencing using a chain termination method adapted for fluorescent primers in an ABI 3100 sequencer (Applied Biosystems, Foster City, CA). If negative results are obtained for direct sequencing, the PCR products are cloned (original TA Cloning Kit; Invitrogen, Groningen, the Netherlands) and sequenced.

If a patient with a solid tumor is determined to have a mutation at H-Ras codons 12, 13 and 61, or another mutation leading to H-Ras activation, and if the patient has no other barriers to being treated with tipifarnib, treatment with tipifarnib is prescribed. On the other hand, if the patient is determined not to have any mutations leading to H-Ras activation, or has wild-type HRas, tipifarnib treatment is not recommended.

If tipifarnib treatment is prescribed to a patient, the patient may be simultaneously subjected to other treatment, such as exposure to ionizing radiation or a second active agent, or supportive care, as deemed appropriate by the oncologist. For a patient with head and neck squamous cell carcinoma, the additional treatment may be anti-EGFR antibody treatment, anti-PD1/PDL1 antibody treatment.

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This description contains a listing of sequences, which is filed in electronic form in ASCII format and the entire content of which, therefore, is given as a reference. The specified ASCII copy, created on September 29, 2016, is named 14168-011-999 SL.txt and is 42868 bytes in size.

Claims (26)

CLAIM 1. A method of treating H-Ras mutated head and neck squamous cell carcinoma (HNSCC) in a subject, comprising administering a therapeutically effective amount of tipifarnib to said subject, wherein said HNSCC is at an advanced stage, is metastatic, recurrent or non-responsive. 2. The method of claim 1 wherein said HNSCC is human papillomavirus (HPV) negative. 3. The method of claim 1, wherein said H-Ras mutation contains an amino acid substitution at a codon selected from the group consisting of G12, G13, Q61, and any combination thereof. 4. The method of claim 3, wherein said H-Ras mutation of said subject contains an amino acid substitution at codon G12. 5. The method of claim 3, wherein said H-Ras mutation of said subject contains an amino acid substitution at codon G13. 6. The method of claim 3, wherein said H-Ras mutation of said subject contains an amino acid substitution at codon Q61. 7. A method according to any one of claims 1 to 6, comprising determining the presence of an H-Ras mutation in a sample from said subject. 8. The method of claim 7, wherein said sample is a tissue biopsy or a tumor biopsy. 9. The method of claim 7 or 8, wherein said H-Ras mutation is determined by a method selected from the group consisting of sequencing, polymerase chain reaction (PCR), DNA microarray analysis, mass spectrometry (MS), single nucleotide polymorphism (SNP) analysis, denaturing high performance liquid chromatography (DHPLC) or restriction fragment length polymorphism (RFLP) analysis. 10. The method according to any one of claims 1-9, wherein tipifarnib is administered at a dose of 1-1000 mg/kg of body weight. 11. The method of any one of claims 1 to 10 wherein tipifarnib is administered twice daily. 12. The method of claim 11 wherein tipifarnib is administered at a dose of 300 mg twice daily. 13. The method of claim 11 wherein tipifarnib is administered at a dose of 600 mg twice daily. 14. The method of claim 11 wherein tipifarnib is administered at a dose of 900 mg twice daily. 15. The method of any one of claims 1 to 14 wherein tipifarnib is administered over a period of one to seven days. 16. The method of any one of claims 1-15, wherein tipifarnib is administered on days 1-7 and 15-21 of a 28 day treatment cycle. 17. The method of claim 16 wherein tipifarnib is administered for at least 3 cycles. 18. The method of claim 16 wherein tipifarnib is administered for at least 6 cycles. 19. The method of claim 16 wherein tipifarnib is administered for up to 12 months. 20. The method of claim 12 wherein tipifarnib is administered at a dose of 300 mg twice daily for 3 out of 4 weeks in repeated 4 week cycles. 21. The method of claim 13 wherein tipifarnib is administered at a dose of 600 mg twice daily on days 1-7 and 15-21 of a 28 day treatment cycle. 22. The method of claim 14 wherein tipifarnib is administered at a dose of 900 mg twice daily on days 1-7 and 15-21 of a 28 day treatment cycle. 23. The method of any one of claims 1-22, wherein tipifarnib is administered before, during, or after irradiation. 24. The method according to any one of claims 1 to 23, wherein tipifarnib is administered with a therapeutically effective amount of the second active ingredient or maintenance therapy. 25. The method of claim 24, wherein said second active ingredient is selected from the group consisting of an anti-EGFR antibody, cisplatin, carboplatin, taxane, gemcitabine, methotrexate, an anti-PD1 antibody, and an anti-PDL1 antibody. 26. The method of claim 24 wherein said second active ingredient is an anti-PD1 antibody and an anti-PDL1 antibody.