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  • C12Q2600/158—Expression markers

Definitions

• sequencelisting.txt and its size is 320kb (328,378 bytes). The entire contents of the Sequence Listing in the sequencelisting.txt file are incorporated herein by this reference.
• This disclosure relates to methods for the treatment of various cancers.
• the disclosure provides methods for treatment of various cancers by administering to a patient a selective inhibitor of Aurora A kinase if said patient is identified as a likely responder to the treatment by assessing the patient's genetic profile.
• One or more genes is/are mutated, amplified, deleted, overexpressed or imderexpressed. Chromosome portions can be lost or moved from one location to another.
• Some cancers have characteristic patterns by which genotypes or phenotypes are altered.
• genes have mutations which are associated with cancer. Some genes have multiple sites where mutations can occur. Many cancers have mutations in and/or mis-expression of more than one gene. Gene mutations can facilitate tumor progression, tumor growth rate or whether a tumor will metastasize. Some mutations can affect whether a tumor cell will respond to therapy.
• the cell division cycle also involves various protein kinases that are frequently overexpressed in cancer cells.
• Aurora A kinase for example, is a key mitotic regulator that is implicated in the pathogenesis of several tumor types.
• the Aurora kinases first identified in yeast (Ipll), Xenopus (Eg2) and Drosophila (Aurora), are critical regulators of mitosis. (Embo J (1998) 17, 5627-5637; Genetics (1993) 135, 677-691; Cell (1995) 81, 95-105; J Cell Sci (1998) l l l(Pt 5), 557-572).
• three isoforms of Aurora kinase exist, including Aurora A, Aurora B and Aurora C.
• Aurora A and Aurora B play critical roles in the normal progression of cells through mitosis, whereas Aurora C activity is largely restricted to meiotic cells.
• Aurora A and Aurora B are structurally closely related. Their catalytic domains lie in the C-terminus, where they differ in only a few amino acids. Greater diversity exists in their non-catalytic N-terminal domains. It is the sequence diversity in this region of Aurora A and Aurora B that dictates their interactions with distinct protein partners, allowing these kinases to have unique subcellular localizations and functions within mitotic cells.
• the Aurora A gene (AURKA) localizes to chromosome 20ql3.2 which is commonly amplified or overexpressed at a high incidence in a diverse array of tumor types.
• AURKA Aurora A gene
• Embo J (1998) 17, 3052-3065; Int J Cancer (2006) 118, 357-363; J Cell Biol (2003) 161, 267-280; Mol Cancer Ther (2007) 6, 1851-1857; J Natl Cancer Inst (2002) 94, 1320-1329).
• Increased Aurora A gene expression has been correlated to the etiology of cancer and to a worsened prognosis.
• Aurora A kinase Overexpression of Aurora A kinase is suspected to result in a stoichiometric imbalance between Aurora A and its regulatory partners, leading to chromosomal instability and subsequent transforming events.
• the potential oncogenic role of Aurora A has led to considerable interest in targeting this kinase for the treatment of cancer.
• Aurora A plays an essential role in mitotic entry and normal progression of cells through mitosis. ⁇ Nat Rev Mol Cell Biol (2003) 4, 842-854; Curr Top Dev Biol (2000) 49, 331-42; Nat Rev Mol Cell Biol (2001) 2(1), 21-32).
• Aurora A kinase is first expressed in the G2 stage where it localizes to centrosomes and functions in centrosome maturation and separation as well as in the entry of cells into mitosis.
• Aurora A kinase predominantly localizes to centrosomes and the proximal portion of incipient mitotic spindles.
• Bipolar spindles that are formed in the absence of Aurora A kinase function frequently show chromosome alignment and segregation defects, including chromosome congression defects at metaphase, lagging chromosomes at anaphase, and telophase bridges.
• the present disclosure relates to prognosis, planning for treatment and treatment of tumors by measurement of at least one characteristic of a marker provided herein.
• Markers were identified in tumor biopsies and blood samples from 47 patients enrolled in a phase 1/2 clinical trial of single agent alisertib by associating their characteristics, e.g., size, sequence, composition, activity or amount, with outcome of subsequent treatment of the patient with alisertib therapy.
• the markers are predictive of whether there will be a favorable outcome (e.g., good response and/or long time-to-progression) after treatment of patients with an Aurora A Kinase inhibitor, such as alisertib.
• Testing patient samples comprising tumor cells to determine the presence, amounts or changes of genetic markers identifies particular patients who are expected to have a favorable outcome with treatment, e.g., with an Aurora A Kinase inhibitor, e.g., alisertib, and whose disease may be managed by standard or less aggressive treatment, as well as those patients who are expected to have an unfavorable outcome with the treatment and may require an alternative treatment to, a combination of treatments and/or more aggressive treatment with an Aurora A Kinase inhibitor to ensure a favorable outcome and/or successful management of the disease.
• an Aurora A Kinase inhibitor e.g., alisertib
• the disclosure provides methods for determining prognosis and treatment or disease management strategies.
• the characteristic e.g., size, sequence, composition, activity or amount of markers in a sample comprising tumor cells is measured.
• the tumor is a liquid, e.g., hematological tumor, e.g., a leukemia, a lymphoma or a myeloma.
• the tumor is a solid tumor, e.g., non- hematological tumor, e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer, bladder cancer or melanoma.
• non- hematological tumor e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer
• small cell lung cancer non-small cell lung cancer
• gastric cancer e.g., renal cancer, pancreatic cancer, bladder cancer or melanoma.
• the characteristic e.g., size, sequence, composition, activity or amount of marker DNA, the size, sequence, composition or amount of marker RNA and/or the size, sequence, composition, activity or amount of marker protein corresponding to a marker gene with one or more mutation, e.g., somatic mutation, described herein is measured.
• Useful information leading to the prognosis or treatment or disease management strategies is obtained when assays reveal information about a marker gene, e.g., whether the gene is mutated, or not, the identity of the mutation, and/or whether the RNA or protein amount of a mutated gene or genes indicates overexpression or underexpression.
• the strategy is determined for therapy with Aurora A Kinase inhibitors, e.g., alisertib, a pharmaceutically acceptable salt or a pharmaceutical composition thereof (MLN8237).
• a marker gene useful to test for determination of prognosis or treatment or disease management strategy is selected from the group consisting of the marker genes listed below in Table 8.
• a marker gene useful to test for determination of prognosis or treatment or disease management strategy is selected from the group consisting of genes within the Wnt signaling pathway or the Hippo signaling pathway.
• a marker gene useful to test for determination of prognosis or treatment or disease management strategy is selected from the group consisting of LEF1, MAP2K7, APC, FZD2, PRKCA, RORA, CAMK2G, JUN, XPOl, ROR2, CCND1, CTNNB1, AMOT, DVL2, LATS1, LATS2, MOB IB, NPHP4, TJP1, TJP2, WWC1, WWC1, WWTR1 and YAP1.
• Each marker gene includes mutations or alterations whose presence in DNA or whose effects, e.g., on marker RNA and/or protein characteristics, e.g., amounts, size, sequence or composition, can provide information for determination of prognosis or treatment or disease management.
• a gene or a mutant or modified form thereof useful as a marker has a DNA, an RNA and/or protein characteristic, e.g., size, sequence, composition or amount, e.g., in a sample comprising tumor cells, which is different than a normal DNA, RNA and/or protein. Described herein are examples of modifications of these genes, referred to as "marker genes" whose mutation or amounts can provide such information.
• the mutation of a marker gene of the present disclosure provides information about outcome after treatment, e.g., with an Aurora A Kinase inhibitor, e.g., alisertib.
• a characteristic e.g., size, sequence, composition, activity or amount of one or more of identified markers in a tumor
• the characteristic e.g., size, sequence, composition, activity or amount of one or more of the identified markers or marker sets in a cancer, it is also possible to determine which therapeutic agent, combination of agents, dosing and/or administration regimen is less likely to provide a favorable outcome upon treatment.
• the characteristic e.g., size, sequence, composition, activity or amount of one or more of the identified markers, it is therefore possible to eliminate ineffective or inappropriate therapeutic agents or regimens.
• these determinations can be made on a patient-by -patient basis. Thus, one can determine whether or not a particular therapeutic regimen is likely to benefit a particular patient or type of patient, and/or whether a particular regimen should be started or avoided, continued, discontinued or altered.
• the present disclosure is directed to methods of identifying and/or selecting a cancer patient for treatment with a therapeutic regimen, e.g., a therapeutic regimen comprising an Aurora A Kinase inhibitor, such as alisertib treatment, if the patient is expected to demonstrate a favorable outcome upon administration of the therapeutic regimen.
• a therapeutic regimen e.g., a therapeutic regimen comprising an Aurora A Kinase inhibitor, such as alisertib treatment
• the method may further comprise treating with the therapeutic regimen a cancer patient who is identified for a favorable outcome.
• methods of identifying a patient for alternative therapy or supplemental therapy if the patient is expected to have an unfavorable outcome upon administration of such a therapeutic regimen.
• These methods typically include measuring, determining, receiving, storing or transmitting information about the characteristic, e.g., size, sequence, composition, activity or amount of one or more markers or mutation of marker genes in a patient's tumor (e.g., in a patient's cancer cells, e.g., non- hematological cancer cells, e.g., solid tumor cells), optionally comparing that to the characteristic, e.g., size, sequence, composition, activity or amount of a reference marker, and in a further embodiment, identifying or advising whether the result from the sample corresponds to a favorable outcome of a treatment regimen, e.g., an Aurora A Kinase inhibitor, e.g., alisertib regimen.
• a treatment regimen e.g., an Aurora A Kinase inhibitor, e.g., alisertib regimen.
• therapeutic methods include therapeutic methods which further include the step of beginning, continuing, or commencing a therapy accordingly where the presence of a mutation in a marker gene or the characteristic, e.g., size, sequence, composition, activity or amount of a patient's marker or markers indicates that the patient is expected to demonstrate a favorable outcome with the therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib therapeutic regimen.
• a marker gene or the characteristic e.g., size, sequence, composition, activity or amount of a patient's marker or markers indicates that the patient is expected to demonstrate a favorable outcome with the therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib therapeutic regimen.
• the methods include therapeutic methods which further include the step of stopping, discontinuing, altering or halting a therapy accordingly where the presence of a mutation in a marker gene or the characteristic, e.g., size, sequence, composition, activity or amount of a patient's marker indicates that the patient is expected to demonstrate an unfavorable outcome with the treatment, e.g., with an Aurora A Kinase inhibitor, such as alisertib treatment regimen, e.g., as compared to a patient identified as having a favorable outcome receiving the same therapeutic regimen.
• an Aurora A Kinase inhibitor such as alisertib treatment regimen
• methods are provided for analysis of a patient not yet being treated with a therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib, and identification and prediction of treatment outcome based upon the presence of a mutation in a marker gene or characteristic, e.g., size, sequence, composition, activity or amount of one or more of a patient's markers described herein.
• a therapy e.g., an Aurora A Kinase inhibitor, such as alisertib
• Such methods can include not being treated with the therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib therapy; being treated with therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib therapy in combination with one more additional therapies; being treated with an alternative therapy to an Aurora A Kinase inhibitor, such as alisertib therapy; or being treated with a more aggressive dosing and/or administration regimen of a therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib, e.g., as compared to the dosing and/or administration regimen of a patient identified as having a favorable outcome to a standard Aurora A Kinase inhibitor, such as alisertib therapy.
• the provided methods of the disclosure can eliminate ineffective or inappropriate use of therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib therapy regimens.
• Additional methods include methods to determine the activity of an agent, the efficacy of an agent, or identify new therapeutic agents or combinations. Such methods include methods to identify an agent as useful, e.g., as an Aurora A Kinase inhibitor, such as alisertib, for treating a cancer, e.g., a non-hematological cancer, i.e., a solid tumor cancer (e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer, bladder cancer or melanoma), based on its ability to affect the presence of a mutation in a marker gene or characteristic, e.g., size, sequence, composition, activity or amount of a marker or markers of the disclosure.
• a cancer e.g., a non-hematological cancer, i.e., a solid tumor cancer (e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer,
• an inhibitor which decreases or increases the presence of a mutation in a marker gene or characteristic e.g., size, sequence, composition, activity or amount of a marker or markers provided in a manner that indicates favorable outcome of a patient having cancer would be a candidate agent for the cancer.
• an agent which is able to decrease the viability of a tumor cell comprising a marker indicative of an unfavorable outcome would be a candidate agent for the cancer.
• the present disclosure is also directed to methods of treating a cancer patient with a therapeutic regimen, e.g., an Aurora A Kinase inhibitor, such as alisertib therapeutic regimen (e.g., alone, or in combination with an additional agent such as a chemotherapeutic agent), which includes the step of selecting for treatment a patient whose marker characteristic, e.g., size, sequence, composition, activity or amount indicates that the patient is expected to have a favorable outcome with the therapeutic regimen, and treating the patient with the therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib therapy.
• a therapeutic regimen e.g., an Aurora A Kinase inhibitor, such as alisertib therapeutic regimen
• a therapeutic regimen e.g., an Aurora A Kinase inhibitor, such as alisertib therapeutic regimen
• an additional agent such as a chemotherapeutic agent
• the method can include the step of selecting a patient whose marker characteristic, e.g., size, sequence, composition, activity or amount or amounts indicates that the patient is expected to have a favorable outcome and administering a therapy other than an Aurora A Kinase inhibitor therapy that demonstrates similar expected progression-free survival times as the Aurora A Kinase inhibitor, such as alisertib therapy.
• a marker characteristic e.g., size, sequence, composition, activity or amount or amounts indicates that the patient is expected to have a favorable outcome
• administering a therapy other than an Aurora A Kinase inhibitor therapy that demonstrates similar expected progression-free survival times as the Aurora A Kinase inhibitor, such as alisertib therapy.
• Additional methods of treating a cancer patient include selecting patients that are unlikely to experience a favorable outcome upon treatment with a cancer therapy (e.g., an Aurora A Kinase inhibitor, such as alisertib therapy). Such methods can further include one or more of: administering a higher dose or increased dosing schedule of a therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib as compared to the dose or dosing schedule of a patient identified as having a favorable outcome with standard therapy; administering a cancer therapy other than an Aurora A Kinase inhibitor, such as alisertib therapy; administering an Aurora A Kinase inhibitor, such as alisertib in combination with an additional agent. Further provided are methods for selection of a patient having aggressive disease which is expected to demonstrate more rapid time to progression.
• a cancer therapy e.g., an Aurora A Kinase inhibitor, such as alisertib therapy.
• Additional methods include a method to evaluate whether to treat or pay for the treatment of cancer, e.g., non-hematological cancer, i.e., solid tumor cancer (e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer, bladder cancer or melanoma) by reviewing the amount of a patient's marker or markers for indication of outcome to a cancer therapy, e.g., an Aurora A Kinase inhibitor, such as alisertib therapy regimen, and making a decision or advising on whether payment should be made.
• a cancer therapy e.g., an Aurora A Kinase inhibitor, such as alisertib therapy regimen
• the disclosure provides a method for determining whether to treat a patient having cancer with an Aurora A kinase inhibitor, the method comprising the steps of: (a) obtaining a cancer cell sample from the patient; (b) determining whether any of the WNT pathway genes listed in Table 9 and/or any of the Hippo pathway genes listed in Table 10 contain mutations in comparison to each of the genes' respective wild type sequence; and determining whether to treat the patient with the Aurora A kinase inhibitor based on the mutation analysis in step b), wherein if at least one gene from Table 9 and/or 10 is found to be mutated, the patient may favorably respond to the drug.
• the disclosure provides a method for identifying a patient having cancer as a candidate for treatment with an Aurora A kinase inhibitor, the method comprising the steps of: (a) obtaining a cancer cell sample from the patient; (b) determining whether any of the WNT pathway genes listed in Table 9 and/or any of the Hippo pathway genes listed in Table 10 contain mutations in comparison to each of the genes' respective wild type sequence; and identifying the patient as a candidate for treatment with the Aurora A kinase inhibitor if the mutation analysis in step b) indicates the presence of a mutation or the presence of several mutations in at least one gene from Table 9 and/or 10.
• the disclosure provides a method for treating a patient having cancer, the method comprising the steps of: (a) obtaining a cancer cell sample from the patient; (b) determining whether any of the WNT pathway genes listed in Table 9 and/or any of the Hippo pathway genes listed in Table 10 contain mutations in comparison to each of the genes' respective wild type sequence; and treating the subject with an Aurora A Kinase inhibitor if the mutation analysis in b) indicates the presence of a mutation or the presence of several mutations in at least one gene from Table 9 and/or 10.
• FIGURE 1 The left pie chart represents the tumor types of the 47 patients in the clinical trial. A total or five tumor types were treated among the patients, namely breast cancer, head and neck cancer (H&N), small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) and gastric cancer. The right pie chart indicates the tumour types of responders to treatment with alisertib.
• FIGURE 2 The mutational landscape of the 47 patients in the clinical trial.
• the patients (represented by the columns) are sorted by best tumor size change from responders to non-responders. Line segments in the heatmap indicate mutated genes.
• FIGURE 3 The heatmaps of mutated genes of the WNT and Hippo signaling pathways.
• each column represents a patient
• each row represents a mutated gene
• each type of tumor is represented by the markings in the cells.
• the top waterfall plots indicate the distribution of the best tumor size change and each bar corresponds to a column (patient) of the heatmap.
• FIGURE 4 In each plot, the left box and right box represent wild type (WT) patients and mutant patients, respectively. The y-axis indicates the best tumor size change (%). Faint grey dots represent patients that responded to treatment with alisertib. As can be seen from these plots, patients with mutations in their WNT or Hippo signaling pathways respond more favorably to treatment with alisertib. DETAILED DESCRIPTION OF THE DISCLOSURE
• non-hematological cancer patients e.g., patients with solid tumors (e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer, bladder cancer or melanoma) who would benefit from particular cancer inhibition therapies as well as those who would benefit from a more aggressive and/or alternative cancer inhibition therapy, e.g., alternative to a cancer therapy or therapies the patient has received, thus resulting in appropriate preventative measures.
• non-hematological cancer patients e.g., patients with solid tumors (e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer, bladder cancer or melanoma)
• solid tumors e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer
• the present disclosure is based, in part, on the recognition that mutation of a marker gene can be associated with sensitivity of a cell comprising the mutated gene to an Aurora A Kinase inhibitor, e.g., alisertib.
• the marker gene is involved in the WNT/ ⁇ -catenin signaling pathway, e.g., a gene whose mutation enables activation of the pathway.
• the WNT/ ⁇ -catenin signaling pathway is a well described oncogenic pathway.
• the marker gene is involved in the Hippo signaling pathway. Hippo pathway components are largely known as tumor suppressors. Both the WNT/ ⁇ -catenin and Hippo signaling pathways have functional interactions with Aurora A.
• Aurora A inhibition with, for example, alisertib, induces cell mitotic defects and results in tetraploidy -induced cell cycle arrest.
• Hippo pathway genetically and functionally suppresses the WNT/ ⁇ -catenin signaling pathway.
• Silencing of the Aurora A gene also results in down-regulation of WNT/ ⁇ -catenin dependent signaling.
• a marker gene can exhibit one or more mutations, e.g., somatic mutations, whose presence can affect expression or activity of the encoded gene product.
• the mutation is an activating mutation.
• the mutation affects the expression of the marker gene.
• a mutation can result in an altered interaction of the encoded gene product with a cellular binding partner.
• the identification and/or measurement of the mutation in the marker gene can be used to determine whether a favorable outcome can be expected by treatment of a tumor, e.g. , with an Aurora Kinase inhibitor, e.g., alisertib therapy or whether an alternative therapy to and/or a more aggressive therapy with, e.g. , an Aurora Kinase inhibitor, e.g., alisertib may enhance the response.
• an Aurora Kinase inhibitor e.g., alisertib therapy
• an alternative therapy to and/or a more aggressive therapy e.g.
• an Aurora Kinase inhibitor e.g., alisertib
• the compositions and methods provided herein can be used to determine whether a patient is expected to have a favorable outcome to an Aurora Kinase inhibitor, e.g., alisertib therapeutic agent dosing or administration regimen.
• the present disclosure provides, without limitation: 1) methods and compositions for determining whether an Aurora Kinase inhibitor, e.g., alisertib therapy regimen will or will not be effective to achieve a favorable outcome and/or manage the cancer; 2) methods and compositions for monitoring the effectiveness of an Aurora Kinase inhibitor, e.g., alisertib therapy (alone or in a combination of agents) and dosing and administrations used for the treatment of tumors; 3) methods and compositions for treatments of tumors comprising, e.g.
• an Aurora Kinase inhibitor e.g., alisertib therapy regimen
• methods and compositions for identifying specific therapeutic agents and combinations of therapeutic agents as well as dosing and administration regimens that are effective for the treatment of tumors in specific patients e.g., alisertib therapy regimen
• methods and compositions for identifying disease management strategies e.g., alisertib therapy regimen
• compositions and methods are provided to identify mutations in marker genes in hematological (e.g. , multiple myeloma, leukemias, lymphoma, etc.) or solid (e.g. , breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer, bladder cancer or melanoma) tumors to predict response to treatment, time-to-progression and survival upon treatment.
• hematological e.g. , multiple myeloma, leukemias, lymphoma, etc.
• solid e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer, bladder cancer or melanoma
• Markers were identified based on genetic profiles of tumor cells which exhibit sensitivity to treatment to alisertib. Observed sensitivity generally is consistent among tumor cells tested by more than one method.
• a "favorable” outcome or prognosis refers to long term survival, long time-to- progression (TTP), and/or good response. Conversely, an “unfavorable” outcome or prognosis refers to short term survival, short time-to-progression (TTP) and/or poor response.
• a "marker” as used herein includes a material associated with a marker gene which has been identified as having a mutation in tumor cells of a patient and furthermore that mutation is characteristic of a patient whose outcome is favorable or unfavorable with treatment e.g., by an Aurora A Kinase inhibitor, such as alisertib.
• Examples of a marker include a material, e.g., a chromosome locus, DNA for a gene, RNA for a gene or protein for a gene.
• a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein which demonstrates a characteristic, e.g., size, sequence, composition or amount indicative of a short term survival patient; alternatively a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein which demonstrates a mutation or characteristic, e.g., size, sequence, composition or amount indicative of a long term survival patient.
• a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein which demonstrates a mutation or characteristic, e.g., size, sequence, composition or amount indicative of a long term survival patient.
• a marker in another example, includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein whose mutation or characteristic, e.g., size, sequence, composition or amount is indicative of a patient with a poor response to treatment; alternatively a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein whose mutation or characteristic, e.g., size, sequence, composition or amount is indicative of a patient with a good response to treatment.
• a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein whose mutation or characteristic, e.g., size, sequence, composition or amount is indicative of a patient with a good response to treatment.
• a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein whose mutation or characteristic, e.g., size, sequence, composition or amount is indicative of a patient whose disease has a short time-to-progression (TTP) upon treatment; alternatively a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein whose mutation or characteristic, e.g., size, sequence, composition or amount is indicative of a patient whose disease has a long TTP upon treatment.
• TTP time-to-progression
• a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein whose mutation or characteristic, e.g., size, sequence, composition or amount is indicative of a patient whose disease has a short term survival upon treatment; alternatively a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein whose mutation or characteristic, e.g., size, sequence, composition or amount is indicative of a patient whose disease has a long term survival upon treatment.
• a marker includes a marker gene material, e.g., a chromosome locus, DNA, RNA or protein whose mutation or characteristic, e.g., size, sequence, composition or amount is indicative of a patient whose disease has a long term survival upon treatment.
• marker is intended to include each and every one of these possibilities, and further can include each single marker individually as a marker; or alternatively can include one or more, or all of the characteristics collectively when reference is made to "markers” or “marker sets.”
• a "marker nucleic acid” is a nucleic acid (e.g. , genomic DNA, mRNA, cDNA) encoded by or corresponding to a marker gene of the disclosure.
• marker nucleic acids include DNA, e.g., sense and anti-sense strands of genomic DNA (e.g., including any introns occurring therein), comprising the entire or a partial sequence, e.g., one or more of the exons of the genomic DNA, up to and including the open reading frame of any of the marker genes or the complement of such a sequence.
• the marker nucleic acids also include RNA comprising the entire or a partial sequence of any marker or the complement of such a sequence, wherein all thymidine residues are replaced with uridine residues, RNA generated by transcription of genomic DNA (i.e. prior to splicing), RNA generated by splicing of RNA transcribed from genomic DNA, and proteins generated by translation of spliced RNA (i.e. including proteins both before and after cleavage of normally cleaved regions such as transmembrane signal sequences).
• a "marker nucleic acid” may also include a cDNA made by reverse transcription of an RNA generated by transcription of genomic DNA (including spliced RNA).
• a marker nucleic acid also includes sequences which differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a protein which corresponds to a marker, e.g., a mutated marker, of the disclosure, and thus encode the same protein, e.g., mutated protein.
• allelic variant refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. Such naturally occuring allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
• Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals, e.g., in cells, e.g., germline cells, of individuals without cancer. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Detection of any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of naturally occurring allelic variation and that do not alter the functional activity of a wild type marker gene is intended to be within the scope of the wild type version of a marker described herein.
• a "marker protein” is a protein encoded by or corresponding to a marker, e.g., a mutant nucleic acid, of the disclosure.
• protein and “polypeptide' are used interchangeably.
• a protein of a marker specifically can be referred to by its name or amino acid sequence, but it is understood by those skilled in the art, that mutations, deletions and/or post-translational modifications can affect protein structure, appearance, cellular location and/or behavior. Unless indicated otherwise, such differences are not distinguished herein, and a marker described herein is intended to include any or all such varieties.
• a "marker gene” refers to a gene which can have a mutation such that its DNA, RNA and/or protein has a characteristic, e.g., size, sequence, composition or amount(s) which provide information about prognosis (i.e., are "informative") upon treatment.
• Marker genes described herein as linked to outcome after treatment with an Aurora A Kinase inhibitor, such as alisertib (e.g. , MLN8237) are examples of marker genes and are provided in Tables 8, 9 and 10.
• a marker gene listed in Tables 8, 9 and 10 can have isoforms which are either ubiquitous or have restricted expression. These sequences are not intended to limit the marker gene identity to that isoform or precursor.
• LEF1 refers to the gene associated with GenBank Accession No. NM 016269.4 (SEQ ID NO: l), encoding GenPept Accession No. NP 057353.1 (SEQ ID NO:2).
• Other names for LEF1 include TCF1 -alpha and T cell-specific transcription factor 1 -alpha.
• the protein encodes a transcription factor belonging to a family of proteins that share homology with the high mobility group protein- 1.
• the protein encoded by this gene can bind to a functionally important site in the T-cell receptor-alpha enhancer, thereby conferring maximal enhancer activity. This transcription factor is involved in the WNT signaling pathway, and it may function in hair cell differentiation and follicle morphogenesis.
• LEF1 as marker gene may be organ-specific, i.e., it can be a marker of breast neoplasms, colorectal neoplasms, insulin resistance, and other types of diseases particularly cancers.
• MAP3K7 refers to the gene associated with GenBank Accession No. NM 145331.2 (SEQ ID NO:3), encoding GenPept Accession No. NP 663304.1 (SEQ ID NO:4).
• Other names for MAP3K7 include transforming growth factor-beta-activated kinase 1, TGF-beta activated kinase 1, and TGF-beta-activated kinase 1.
• the protein encoded by this gene is a member of the serine/threonine protein kinase family.
• This kinase mediates the signaling transduction induced by TGF beta and morphogenetic protein (BMP), and controls a variety of cell functions including transcription regulation and apoptosis.
• TGF beta and morphogenetic protein BMP
• this protein forms a kinase complex including TRAF6, MAP3K7P 1/TAB 1 and M AP3 K7P2/T AB2 ; this complex is required for the activation of nuclear factor kappa B.
• This kinase can also activate MAPK8/JNK, MAP2K4/MKK4, and thus plays a role in the cell response to environmental stresses.
• MAP3K7 as marker gene may be disease-specific, i.e., it can be a marker of arthritis, rheumatoid, endometrial neoplasms, and renal cancers.
• APC refers to the gene associated with GenBank Accession No. NM 000038.5 (SEQ ID NO:5), encoding GenPept Accession No. NP 000029.2 (SEQ ID NO:6).
• Other names for APC include protein phosphatase 1, regulatory subunit 46, adenomatosis polyposis coli tumor suppressor, adenomatous polyposis coli protein, deleted in polyposis 2.5, and adenomatosis polyposis coli. This gene encodes a tumor suppressor protein that acts as an antagonist of the Wnt signaling pathway. It is also involved in other processes including cell migration and adhesion, transcriptional activation, and apoptosis.
• FAP familial adenomatous polyposis
• MCR mutation cluster region
• FZD2 refers to the gene associated with GenBank Accession No. NM 001466.3 (SEQ ID NO:7), encoding GenPept Accession No. NP 001457.1 (SEQ ID NO:8).
• Other names for FZD2 include frizzled homolog 2 (Drosophila), frizzled homolog 2, frizzled-2, frizzled (Drosophila) homolog 2, frizzled 2, seven transmembrane spanning receptor, and frizzled family receptor 2.
• This intronless gene is a member of the frizzled gene family. Members of this family encode seven-transmembrane domain proteins that are receptors for the wingless type MMTV integration site family of signaling proteins.
• This gene encodes a protein that is coupled to the beta- catenin canonical signaling pathway. Competition between the wingless-type MMTV integration site family, member 3A and wingless-type MMTV integration site family, member 5A gene products for binding of this protein is thought to regulate the beta-catenin-dependent and -independent pathways.
• Use of FZD2 as marker gene may be similar to MAP3K7, covering arthritis, rheumatoid, colorectal neoplasms, and endometrial neoplasms.
• PRKCA refers to the gene associated with GenBank Accession No. XM 011524990.1 (SEQ ID NO:9), encoding GenPept Accession No. XP 011523292.1 (SEQ ID NO: 10).
• Other names for PRKCA include aging-associated gene 6, and protein kinase C alpha type, PKC-A.
• PKC Protein kinase C
• PKC is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members
• PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells.
• the protein encoded by this gene is one of the PKC family members.
• This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control. Knockout studies in mice suggest that this kinase may be a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes..
• Use of PRKCA as marker gene may be organ-specific; i.e. it can be a marker of Alzheimer disease and amyotrophic lateral.
• RORA refers to the gene associated with GenBank Accession No. NM 002943.3 (SEQ ID NO: 11), encoding GenPept Accession No. NP 002934.1 (SEQ ID NO: 12).
• Other names for RORA include retinoic acid receptor-related orphan receptor alpha, ROR-alpha, transcription factor RZR-alpha, thyroid hormone nuclear receptor alpha variant 4, nuclear receptor RZR-alpha, retinoid-related orphan receptor alpha, nuclear receptor ROR-alpha, and nuclear receptor subfamily 1 group F member 1.
• the protein encoded by this gene is a member of the NR1 subfamily of nuclear hormone receptors.
• RORA as marker gene may various diseases; i.e. it can be a marker of anoxia, bipolar disorder, cancers, particularly non-small cell lung cancer.
• CAMK2G refers to the gene associated with GenBank Accession No. NM 172171.2 (SEQ ID NO: 13), encoding GenPept Accession No. NP 751911.1 (SEQ ID NO: 14).
• Other names for CAMK2G include calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma, calcium/calmodulin-dependent protein kinase type II subunit gamma, and caMK-II subunit gamma.
• the product of this gene is one of the four subunits of an enzyme which belongs to the serine/threonine protein kinase family, and to the Ca(2+)/calmodulin-dependent protein kinase subfamily. Calcium signaling is crucial for several aspects of plasticity at glutamatergic synapses. In mammalian cells the enzyme is composed of four different chains: alpha, beta, gamma, and delta. The product of this gene is a gamma chain. Many alternatively spliced transcripts encoding different isoforms have been described but the full-length nature of all the variants has not been determined.
• Use of CAMK2G as marker gene may be neuro-degenerative diseases, cardiovascular diseases, and cancer; i.e. it can be a marker of Alzheimer disease, arrhythmias, colorectal neoplasms, and glioblastoma.
• JUN refers to the gene associated with GenBank Accession No. NM 002228.3 (SEQ ID NO: 15), encoding GenPept Accession No. NP 002219.1 (SEQ ID NO: 16).
• Other names for JUN include v-jun avian sarcoma virus 17 oncogene homolog, activator protein 1, Jun activation domain binding protein, v-jun sarcoma virus 17 oncogene homolog, enhancer-binding protein API, jun oncogene, p39, proto-oncogene c-Jun, transcription factor AP-1, and v-jun sarcoma virus 17 oncogene homolog (avian).
• This gene is the putative transforming gene of avian sarcoma virus 17. It encodes a protein which is highly similar to the viral protein, and which interacts directly with specific target DNA sequences to regulate gene expression. This gene is intronless and is mapped to Ip32-p31, a chromosomal region involved in both translocations and deletions in human malignancies.
• Use of JUN as marker gene may be cancer and auto-immune diseases; i.e. it can be a marker of breast neoplasms, colorectal neoplasms, Crohn disease, and asthma.
• XPOl refers to the gene associated with GenBank Accession No. NM 003400.3 (SEQ ID NO: 17), encoding GenPept Accession No. NP 003391.1 (SEQ ID NO: 18).
• Other names for XPOl include exportin 1 (CRM1, yeast, homolog), exportin-1 (required for chromosome region maintenance), exportin 1 (CRM1 homolog, yeast), exportin-1, chromosome region maintenance 1 protein homolog, and chromosome region maintenance 1 homolog.
• This cell- cycle-regulated gene encodes a protein that mediates leucine-rich nuclear export signal (NES)- dependent protein transport. The protein specifically inhibits the nuclear export of Rev and U snRNAs.
• XPOl as marker gene may be related to cancer; i.e. it can be a marker of breast neoplasms, endometrial neoplasms, and ovarian neoplasms.
• ROR2 refers to the gene associated with GenBank Accession No. NM 004560.3 (SEQ ID NO: 19), encoding GenPept Accession No. NP 004551.2 (SEQ ID NO:20).
• Other names for ROR2 include neurotrophic tyrosine kinase receptor-related 2, and tyrosine -protein kinase transmembrane receptor ROR2.
• the protein encoded by this gene is a receptor protein tyrosine kinase and type I transmembrane protein that belongs to the ROR subfamily of cell surface receptors. The protein may be involved in the early formation of the chondrocytes and may be required for cartilage and growth plate development.
• Mutations in this gene can cause brachydactyly type B, a skeletal disorder characterized by hypoplasia/aplasia of distal phalanges and nails.
• mutations in this gene can cause the autosomal recessive form of Robinow syndrome, which is characterized by skeletal dysplasia with generalized limb bone shortening, segmental defects of the spine, brachydactyly, and a dysmorphic facial appearance.
• Use of ROR2 as marker gene may be related to cancer; i.e. it can be a marker of breast neoplasms, non-small cell lung cancer, and colorectal neoplasms.
• CCND1 refers to the gene associated with GenBank Accession No. NM 053056.2 (SEQ ID NO:21), encoding GenPept Accession No. NP 444284.1 (SEQ ID NO:22).
• Other names for CCND1 include B-cell CLL/lymphoma 1, BCL-1 oncogene, PRAD1 oncogene, B- cell lymphoma 1 protein, and Gl/S-specific cyclin-Dl, and cyclin Dl (PRAD1: parathyroid adenomatosis 1).
• the protein encoded by this gene belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance throughout the cell cycle.
• Cyclins function as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. This cyclin forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle Gl/S transition. This protein has been shown to interact with tumor suppressor protein Rb and the expression of this gene is regulated positively by Rb. Mutations, amplification and overexpression of this gene, which alters cell cycle progression, are observed frequently in a variety of tumors and may contribute to tumorigenesis. Use of CCND1 as marker gene may be related to many diseases; i.e. it can be a marker of various types of cancer and neuro-degenerative diseases.
• CTNNBl refers to the gene associated with GenBank Accession No. NM 001098209.1 (SEQ ID NO:23), encoding GenPept Accession No. NP 001091679.1 (SEQ ID NO:24).
• Other names for CTNNBl include catenin beta-1, catenin (cadherin-associated protein), and beta 1 (88kD).
• the protein encoded by this gene is part of a complex of proteins that constitute adherens junctions (AJs). AJs are necessary for the creation and maintenance of epithelial cell layers by regulating cell growth and adhesion between cells.
• the encoded protein also anchors the actin cytoskeleton and may be responsible for transmitting the contact inhibition signal that causes cells to stop dividing once the epithelial sheet is complete. Finally, this protein binds to the product of the APC gene, which is mutated in adenomatous polyposis of the colon. Mutations in this gene are a cause of colorectal cancer (CRC), pilomatrixoma (PTR), medulloblastoma (MDB), and ovarian cancer. Three transcript variants encoding the same protein have been found for this gene.
• CTNNBl as marker gene may be related to many diseases; i.e. it can be a marker of various types of cancer particularly colorectal neoplasms and immune diseases.
• AMOT refers to the gene associated with GenBank Accession No. NM 001113490.1 (SEQ ID NO:25), encoding GenPept Accession No. NP 001106962.1 (SEQ ID NO:26).
• Other names for AMOT include angiomotin pl30 isoform, and angiomotin p80 isoform. This gene belongs to the motin family of angiostatin binding proteins characterized by conserved coiled-coil domains and C-terminal PDZ binding motifs.
• the encoded protein is expressed predominantly in endothelial cells of capillaries as well as larger vessels of the placenta where it may mediate the inhibitory effect of angiostatin on tube formation and the migration of endothelial cells toward growth factors during the formation of new blood vessels.
• Alternative splicing results in multiple transcript variants encoding different isoforms.
• Use of AMOT as marker gene may be related to cancer; i.e. it can be a marker of breast neoplasms, endometrial neoplasms, and leukemia.
• DVD2 refers to the gene associated with GenBank Accession No. NM 004422.2 (SEQ ID NO:27), encoding GenPept Accession No.
• DVL2 includes dishevelled 2 (homologous to Drosophila dsh), segment polarity protein dishevelled homolog DVL-2, dishevelled, dsh homolog 2, and dishevelled, dsh homolog 2 (Drosophila).
• This gene belongs to the motin family of angiostatin binding proteins characterized by conserved coiled-coil domains and C-terminal PDZ binding motifs. This gene encodes a member of the dishevelled (dsh) protein family.
• the vertebrate dsh proteins have approximately 40% amino acid sequence similarity with Drosophila dsh.
• This gene encodes a 90-kD protein that undergoes posttranslational phosphorylation to form a 95-kD cytoplasmic protein, which may play a role in the signal transduction pathway mediated by multiple Wnt proteins.
• the mechanisms of dishevelled function in Wnt signaling are likely to be conserved among metazoans.
• Use of DVL2 as marker gene may be related to cancer and psychiatry diseases; i.e. it can be a marker of breast neoplasms, non- small cell cancer, bipolar disorder, and tobacco use disorder.
• LATSl refers to the gene associated with GenBank Accession No. NM 004690.3 (SEQ ID NO:29), encoding GenPept Accession No. NP 004681.1 (SEQ ID NO:30).
• Other names for LATSl include WARTS protein kinase, LATS (large tumor suppressor, Drosophila) homolog 1, large tumor suppressor homolog 1, serine/threonine-protein kinase LATSl, h-warts, LATS, large tumor suppressor, homolog 1, LATS, and large tumor suppressor, homolog 1
• This gene belongs to the motin family of angiostatin binding proteins characterized by conserved coiled-coil domains and C-terminal PDZ binding motifs. This gene encodes a member of the dishevelled (dsh) protein family. The vertebrate dsh proteins have approximately 40% amino acid sequence similarity with Drosophila dsh. This gene encodes a 90-kD protein that undergoes posttranslational phosphorylation to form a 95-kD cytoplasmic protein, which may play a role in the signal transduction pathway mediated by multiple Wnt proteins. The mechanisms of dishevelled function in Wnt signaling are likely to be conserved among metazoans.
• Use of LATSl as marker gene may be related to cancer and psychiatry diseases; i.e. it can be a marker of breast neoplasms, non- small cell cancer, bipolar disorder, and tobacco use disorder.
• LATS2 refers to the gene associated with GenBank Accession No. XM 005266342.1 (SEQ ID NO:31), encoding GenPept Accession No. XP 005266399.1 (SEQ ID NO:32).
• LATS2 include serine/threonine kinase KPM, warts-like kinase, LATS (large tumor suppressor, Drosophila) homolog 2, kinase phosphorylated during mitosis protein, large tumor suppressor homolog 2, serine/threonine-protein kinase kpm, serine/threonine-protein kinase LATS2, LATS, large tumor suppressor, homolog 2, LATS, and large tumor suppressor, homolog 2 (Drosophila).
• This gene encodes a serine/threonine protein kinase belonging to the LATS tumor suppressor family. The protein localizes to centrosomes during interphase, and early and late metaphase.
• LATS2 as marker gene may be related to cancer and immune diseases; i.e. it can be a marker of breast neoplasms and asthma.
• MOB1B refers to the gene associated with GenBank Accession No. NM_001244766.1 (SEQ ID NO:33), encoding GenPept Accession No. NP 001231695.1 (SEQ ID NO:34).
• Other names for MOB IB include MOB1 Mps One Binder homolog B, MOB1 Mps One Binder homolog B (yeast), MOB1, Mps One Binder kinase activator-like 1A (yeast), moblA, mps one binder kinase activator-like 1A, mobl homolog 1A, MOB1, and Mps One Binder kinase activator-like 1A.
• the protein encoded by this gene is similar to the yeast Mobl protein.
• Yeast Mobl binds Mpslp, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Three transcript variants encoding different isoforms have been found for this gene.
• Use of MOB IB as marker gene may be related to cancer; i.e. it can be a marker of breast neoplasms, endometrial neoplasms, and lung neoplasms.
• NPHP4 refers to the gene associated with GenBank Accession No. NM 015102.4 (SEQ ID NO:35), encoding GenPept Accession No. NP 055917.1 (SEQ ID NO:36).
• Other names for NPHP4 include nephroretinin, nephrocystin-4, and POC10 centriolar protein homolog. This gene encodes a protein involved in renal tubular development and function. This protein interacts with nephrocystin, and belongs to a multifunctional complex that is localized to actin- and microtubule-based structures.
• NPHP4 as marker gene may be related to cancer and eye diseases; i.e. it can be a marker of breast neoplasms, endometrial neoplasms, and retinitis.
• TJP1 refers to the gene associated with GenBank Accession No. NM 003257.4 (SEQ ID NO:37), encoding GenPept Accession No. NP 003248.3 (SEQ ID NO:38).
• Other names for TJP1 include tight junction protein ZO-1, zona occludens 1, and zonula occludens 1 protein. This gene encodes a protein located on a cytoplasmic membrane surface of intercellular tight junctions. The encoded protein may be involved in signal transduction at cell-cell junctions.
• TJP1 as marker gene may be related to cancer and immune diseases; i.e. it can be a marker of breast neoplasms, hepatocellular neoplasms, and asthma.
• TJP2 refers to the gene associated with GenBank Accession No. NM 004817.3 (SEQ ID NO:39), encoding GenPept Accession No. NP 004808.2 (SEQ ID NO:40).
• Other names for TJP2 include Friedreich ataxia region gene X104 (tight junction protein ZO-2), deafness, autosomal dominant 51, zonula occludens protein 2, tight junction protein ZO-2, and zona occludens 2. This gene encodes a zonula occluden that is a member of the membrane-associated guanylate kinase homolog family.
• the encoded protein functions as a component of the tight junction barrier in epithelial and endothelial cells and is necessary for proper assembly of tight junctions. Mutations in this gene have been identified in patients with hypercholanemia, and genomic duplication of a 270 kb region including this gene causes autosomal dominant deafness-51.
• TJP2 as marker gene may be related to cancer; i.e. it can be a marker of breast neoplasms.
• WWC1 refers to the gene associated with GenBank Accession No. NM 001161661.1 (SEQ ID NO:41), encoding GenPept Accession No. NP 001155133.1 (SEQ ID NO:42).
• Other names for WWC1 include kidney and brain protein, WW, C2 and coiled-coil domain containing 1, HBeAg-binding protein 3, protein WWC1, protein KIBRA, protein phosphatase 1, and regulatory subunit 168.
• the protein encoded by this gene is a cytoplasmic phosphoprotein that interacts with PRKC-zeta and dynein light chain- 1. Alleles of this gene have been found that enhance memory in some individuals. Three transcript variants encoding different isoforms have been found for this gene.
• Use of WWC1 as marker gene may be related to cancer and neurodegenerative diseases; i.e. it can be a marker of breast neoplasms and Alzheimer disease.
• WWTR1 refers to the gene associated with GenBank Accession No. NM 001168278.1 (SEQ ID NO:43), encoding GenPept Accession No. NP 001161750.1 (SEQ ID NO:44).
• Other names for WWTR1 include transcriptional coactivator with PDZ-binding motif, transcriptional co-activator with PDZ-binding motif, and WW domain-containing transcription regulator protein 1. The molecular function of this gene is relatively unknown.
• Use of WWTR1 as marker gene may be related to cancer; i.e. it can be a marker of breast neoplasms, endometrial neoplasms, lung neoplasms, and ovarian neoplasms.
• YAPl refers to the gene associated with GenBank Accession No. NM 001282101.1 (SEQ ID NO:45), encoding GenPept Accession No. NP 001269030.1 (SEQ ID NO:46).
• Other names for YAPl include Yes-associated protein 1, 65kDa, yes-associated protein 2, yorkie homolog, 65 kDa Yes-associated protein, protein yorkie homolog, transcriptional coactivator YAPl, and yes-associated protein YAP65 homolog. This gene encodes a downstream nuclear effector of the Hippo signaling pathway which is involved in development, growth, repair, and homeostasis.
• YAP l as marker gene may be related to cancer; i.e. it can be a marker of breast neoplasms.
• an "informative" characteristic e.g., size, sequence, composition or amount of a marker refers to a characteristic, e.g., size, sequence, composition or amount whose difference is correlated to prognosis or outcome.
• the informative characteristic e.g., size, sequence, composition or amount of a marker can be obtained by analyzing either nucleic acid, e.g., DNA or RNA, or protein corresponding to the marker gene.
• the characteristic e.g., size (e.g., length or molecular weight), sequence (e.g., nucleic acid sequence or protein sequence), composition (e.g., base or amino acid composition or peptide digest or gene fragment pattern) or amount (e.g.
• a marker in a sample from a patient is "informative" if it is different than the wild type or allelic variant of the substance being analyzed.
• an amount is "informative” if it is greater than or less than a reference amount by a degree greater than the standard error of the assay employed to assess expression.
• the informative expression level of a marker can be determined upon statistical correlation of the measured expression level and the outcome, e.g. , good response, poor response, long time-to-progression, short time-to-progression, short term survival or long term survival.
• a marker e.g., a chromosome locus marker, or a marker gene that has differential characteristic, e.g., size, sequence, composition or amounts will have typical ranges of amounts that are predictive of outcome.
• An informative characteristic, e.g., size, sequence, composition or amount is a characteristic, e.g., size, sequence, composition or amount that falls within the range of characteristic, e.g., size, sequence, composition or amounts determined for the outcome.
• a set of markers may together be "informative" if the combination of their characteristics, e.g., sizes, sequences, compositions or amounts either meets or is above or below a pre-determined score for the combination of markers, e.g., chromosome locus markers, or marker genes, in a set as determined by methods provided herein.
• Measurement of only one characteristic, e.g., marker, of a marker gene i.e., DNA, RNA or protein
• can provide a prognosis i.e., indicate outcome.
• Measurement of more than one characteristic, e.g., marker, of a marker gene can provide a prognosis when the informative amounts of the two characteristics are consistent with each other, i.e., the biologies of the results are not contradictory.
• Examples of consistent results from measurement of multiple characteristics of a marker gene can be identification of a nonsense mutation or deletion in a DNA or RNA and a low amount or low molecular weight of encoded protein, or a mutation in a region which encodes a binding pocket or active site of a protein and low activity of the encoded protein.
• a different example can occur when a protein is in a pathway with a feedback loop controlling its synthesis based on its activity level.
• a low amount or activity of protein can be associated with a high amount of its mutated mRNA as a tissue, due to the marker gene mutation, thus is starved for the protein activity and repeatedly signals the production of the protein.
• gene deletion refers to an amount of DNA copy number less than 2 and "amplification” refers to an amount of DNA copy number greater than 2.
• a “diploid” amount refers to a copy number equal to 2.
• the term “diploid or amplification” can be interpreted as “not deletion” of a gene copy. In a marker whose alternative informative amount is gene deletion, amplification generally would not be seen. Conversely, the term “diploid or deletion” can be interpreted as “not amplification” of copy number. In a marker whose alternative informative amount is amplification, gene deletion generally would not be seen.
• sequence deletion can occur within a gene as a result of marker gene mutation and can result in absence of transcribed protein or a shortened mRNA or protein. Such a deletion may not affect copy number.
• long term survival and “short term survival” refer to the length of time after receiving a first dose of treatment that a cancer patient is predicted to live.
• a “long term survivor” refers to a patient expected have a slower rate of progression or later death from the tumor than those patients identified as short term survivors.
• Enhanced survival or “a slower rate of death” are estimated life span determinations based upon characteristic, e.g., size, sequence, composition or amount of one or more of markers described herein, e.g., as compared to a reference standard such that 70%, 80%, 90% or more of the population will be alive a sufficient time period after receiving a first dose of treatment.
• a “faster rate of death” or “shorter survival time” refer to estimated life span determinations based upon characteristic, e.g., size, sequence, composition or amount of one or more of markers described herein, e.g., as compared to a reference standard such that 50%, 40%, 30%, 20%, 10% or less of the population will not live a sufficient time period after receiving a first dose of treatment.
• the sufficient time period is at least 6, 12, 18, 24 or 30 months measured from the first day of receiving a cancer therapy.
• a cancer is "responsive" to a therapeutic agent or there is a "good response" to a treatment if its rate of growth is inhibited as a result of contact with the therapeutic agent, compared to its growth in the absence of contact with the therapeutic agent.
• Growth of a cancer can be measured in a variety of ways, for instance, the characteristic, e.g., size of a tumor or the expression of tumor markers appropriate for that tumor type may be measured.
• the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (Eisenhauer et al. (2009) E. J. Cane. 45:228-247) can be used to support the identification of markers associated with solid tumors and response of solid tumors to an Aurora A Kinase inhibitor.
• Criteria take into account analysis methods such as Positron Emission Tomography (PET), e.g., for identifying sites with measurable altered metabolic activity (e.g., at tumor sites) or to trace specific markers into tumors in vivo, immunohistochemistry, e.g., to identify tumor cells by detecting binding of antibodies to specific tumor markers, and flow cytometry, e.g., to characterize cell types by differential markers and fluorescent stains, in addition to traditional methods such as histology to identify cell composition (e.g., blast counts in a blood smear or a bone marrow biopsy, presence and number of mitotic figures) or tissue structure (e.g., disordered tissue architecture or cell infiltration of basement membrane).
• PET Positron Emission Tomography
• the quality of being responsive to an Aurora A Kinase inhibitor, such as alisertib therapy can be a variable one, with different cancers exhibiting different levels of "responsiveness" to a given therapeutic agent, under different conditions.
• a breast cancer is responsive to Aurora A Kinase inhibition, e.g., alisertib therapy, if the breast cancer patient has mutations in marker genes LEF1, MAP3K7, FZD2, LATS1 and/or WWC1.
• a gastric cancer is responsive to Aurora A Kinase inhibition, e.g., alisertib therapy, if the gastric cancer patient has mutations in marker genes FZD2 and/or LATS2.
• a head and neck cancer is responsive to Aurora A Kinase inhibition, e.g., alisertib therapy, if the head and neck cancer patient has mutations in marker genes MAP3K7, JU , ROR2, CCND1, LATS1, MOB IB and/or NPHP4.
• a non-small cell lung cancer is responsive to Aurora A Kinase inhibition, e.g., alisertib therapy, if the non-small cell lung cancer patient has mutations in marker genes XPOl and/or TJP1.
• a small cell lung cancer is responsive to Aurora A Kinase inhibition, e.g., alisertib therapy, if the small cell lung cancer patient has mutations in marker genes LEF1, APC, PRKCA, RORA, CAMK2G, CTNNB1, AMOT, DVL2, TJP1, TJP2, WWTR1 and/or YAP1.
• measures of responsiveness can be assessed using additional criteria beyond growth size of a tumor, including patient quality of life, degree of metastases, etc.
• clinical prognostic markers and variables can be assessed (e.g., M protein in myeloma, PSA levels in prostate cancer) in applicable situations.
• a cancer is "non-responsive” or has a "poor response" to a therapeutic agent or there is a poor response to a treatment if its rate of growth is not inhibited, or inhibited to a very low degree, as a result of contact with the therapeutic agent when compared to its growth in the absence of contact with the therapeutic agent.
• growth of a cancer can be measured in a variety of ways, for instance, the size of a tumor or the expression of tumor markers appropriate for that tumor type may be measured.
• the response definitions used to support the identification of markers associated with non- response of tumors to therapeutic agents, guidelines such as those described above can be used.
• the quality of being non-responsive to a therapeutic agent can be a highly variable one, with different cancers exhibiting different levels of "non-responsiveness" to a given therapeutic agent, under different conditions. Still further, measures of non-responsiveness can be assessed using additional criteria beyond growth size of a tumor, including patient quality of life, degree of metastases, etc. In addition, clinical prognostic markers and variables can be assessed (e.g., M protein in myeloma, PSA levels in prostate cancer) in applicable situations.
• long time-to-progression As used herein, “long time-to-progression, “long TTP” and “short time-to-progression,” “short TTP” refer to the amount of time until when the stable disease brought by treatment converts into an active disease. On occasion, a treatment results in stable disease which is neither a good nor a poor response, e.g., MR, the disease merely does not get worse, e.g., become a progressive disease, for a period of time. This period of time can be at least 4-8 weeks, at least 3-6 months or more than 6 months.
• Treatment shall mean the use of a therapy to prevent or inhibit further tumor growth, as well as to cause shrinkage of a tumor, and to provide longer survival times. Treatment is also intended to include prevention of metastasis of tumor.
• a tumor is "inhibited” or “treated” if at least one symptom (as determined by responsiveness/non-responsiveness, time to progression, or indicators known in the art and described herein) of the cancer or tumor is alleviated, terminated, slowed, minimized, or prevented. Any amelioration of any symptom, physical or otherwise, of a tumor pursuant to treatment using a therapeutic regimen (e.g. , Aurora A Kinase inhibitor, such as alisertib regimen) as further described herein, is within the scope of the disclosure.
• a therapeutic regimen e.g. , Aurora A Kinase inhibitor, such as alisertib regimen
• agents are defined broadly as anything that cancer cells, including tumor cells, may be exposed to in a therapeutic protocol.
• agents include, but are not limited to, an Aurora A Kinase inhibitor, such as alisertib agents, as well as chemotherapeutic agents as known in the art and described in further detail herein.
• probe refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example a marker of the disclosure. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, enzymatic reporter reagents and organic monomers.
• a "normal" characteristic, e.g., size, sequence, composition or amount of a marker may refer to the characteristic, e.g., size, sequence, composition or amount in a "reference sample.”
• a reference sample can be a matched normal, e.g., germline, sample from the same patient from whom the tumor, e.g., with a somatic mutation, is derived.
• a reference sample can be a sample from a healthy subject not having the marker-associated disease or a reference characteristic e.g., the average characteristic, e.g., size, sequence, composition or amount of the wild type marker in several healthy subjects.
• a reference sample characteristic e.g., size, sequence, composition or amount may be comprised of a characteristic, e.g., size, sequence, composition or amount of one or more markers from a reference database.
• a "normal" characteristic e.g., size, sequence, composition or level of expression of a marker is the characteristic, e.g., size, sequence, composition or amount of the marker, e.g., marker gene in non-tumor cells in a similar environment or response situation from the same patient from whom the tumor is derived.
• the normal amount of DNA copy number is 2 or diploid, with the exception of X-linked genes in males, where the normal DNA copy number is 1.
• “Over-expression” and “under-expression” of a marker gene refer to expression of the marker gene of a patient at a greater or lesser level (e.g. more than three-halves-fold, at least two-fold, at least three-fold, greater or lesser level etc.), respectively, than normal level of expression of the marker gene, e.g., as measured by mRNA or protein, in a test sample that is greater than the standard error of the assay employed to assess expression.
• a "significant" expression level may refer to a level which either meets or is above or below a pre-determined score for a marker gene set as determined by methods provided herein.
• “Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
• a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
• the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% or all of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
• Homologous refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue (i.e., by percent identity).
• a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'- TATGGC-3' share homology with 50% identity.
• the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
• all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
• antibody broadly encompass naturally -occurring forms of antibodies, e.g., polyclonal antibodies (e.g., IgG, IgA, IgM, IgE) and monoclonal and recombinant antibodies such as single-chain antibodies, two-chain and multi-chain proteins, chimeric, CDR-grafted, human and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments (e.g., dAbs, scFv, Fab, F(ab)' 2 , Fab') and derivatives have at least an antigenic binding site.
• Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
• the term "antibody” also includes synthetic and genetically engineered variants.
• a "kit” is any article of manufacture (e.g., a package or container) comprising at least one reagent, e.g. a probe, for specifically detecting a marker or marker set of the disclosure.
• the article of manufacture may be promoted, distributed, sold or offered for sale as a unit for performing, e.g., in vitro, the methods of the present disclosure, e.g., on a sample having been obtained from a patient.
• the reagents included in such a kit can comprise probes/primers and/or antibodies for use in detecting short term and long term survival marker expression.
• a kit of the present disclosure can contain instructions which describe a suitable detection assay. Such a kit can be conveniently used, e.g.
• a clinical or a contract testing setting in a clinical or a contract testing setting, to generate information, e.g., on expression levels, characteristic, e.g., size, sequence or composition of one or more marker, to be recorded, stored, transmitted or received to allow for diagnosis, evaluation or treatment of patients exhibiting symptoms of cancer, in particular patients exhibiting the possible presence of a cancer capable of treatment with Aurora A Kinase inhibition therapy, including, e.g. , hematological cancers e.g., myelomas (e.g.
• multiple myeloma multiple myeloma
• lymphomas e.g., non-hodgkins lymphoma
• leukemias e.g., acute myelogenous leukemia
• solid tumors e.g., breast cancer, ovarian cancer, prostate cancer, head and neck cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, renal cancer, pancreatic cancer, bladder cancer or melanoma, etc.
• the present methods and compositions are designed for use in diagnostics and therapeutics for a patient suffering from cancer.
• a cancer or tumor is treated or diagnosed according to the present methods.
• Cancer or “tumor” is intended to include any neoplastic growth in a patient, including an initial tumor and any metastases.
• the cancer can be of the hematological or solid tumor type.
• Hematological tumors include tumors of hematological origin, including, e.g. , myelomas (e.g. , multiple myeloma), leukemias (e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, other leukemias), lymphomas (e.g. , B-cell lymphomas, non-Hodgkin's lymphoma) and myelodysplastic syndrome.
• myelomas e.g. , multiple myeloma
• leukemias e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, other leukemias
• lymphomas e.g. , B-cell lymphomas, non-Hodgkin's lymphoma
• myelodysplastic syndrome e.
• Solid tumors can originate in organs, and include cancers such as in skin, lung, brain, breast, prostate, ovary, colon, kidney, pancreas, liver, esophagus, stomach, intestine, bladder, uterus, cervix, testis, adrenal gland, etc.
• the cancer can comprise a cell in which a marker gene has a mutation.
• cancer cells, including tumor cells refer to cells that divide at an abnormal (increased) rate or whose control of growth or survival is different than for cells in the same tissue where the cancer cell arises or lives.
• Cancer cells include, but are not limited to, cells in carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosar
• lymphomas e.g. , follicular lymphoma, mantle cell lymphoma, diffuse large Bcell lymphoma, malignant lymphoma,
• plasmocytoma reticulum cell sarcoma, or Hodgkins disease
• tumors of the nervous system including glioma, meningoma, medulloblastoma, schwannoma or epidymoma.
• proliferative disorders or “proliferative diseases” includes, but is not limited to, cancerous hyperproliferative disorders (e.g., brain, lung, squamous cell, bladder, gastric, pancreatic, breast, head and neck, renal, liver, kidney, ovarian, prostate, colorectal, colon, epidermoid, esophageal, testicular, gynecological or thyroid cancer, acute myeloid leukemia, multiple myeloma, mesothelioma, Non-small cell lung carcinoma (NSCLC), Small cell lung carcinoma (SCLC), neuroblastoma, and acute lymphoblastic leukemia (ALL)); non-cancerous hyperproliferative disorders (e.g., benign hyperplasia of the skin (e.g., psoriasis), restenosis, and benign prostatic hypertrophy (BPH)); and diseases related to vasculogenesis or angiogenesis (e.g., cancerous hyperproliferative disorders (e
• noninvasive refers to a procedure which inflicts minimal harm to a subject.
• a noninvasive sampling procedure can be performed quickly, e.g., in a walk-in setting, typically without anaesthesia and/or without surgical implements or suturing.
• noninvasive samples include blood, serum, saliva, urine, buccal swabs, throat cultures, stool samples and cervical smears.
• Noninvasive diagnostic analyses include x-rays, magnetic resonance imaging, positron emission tomography, etc.
• Aurora A kinase refers to a serine/threonine kinases involved in mitotic progression. Aurora A kinase is also known as AIK, ARK1, AURA, BTAK, STK6, STK7, STK15, AURORA2, MGC34538, and AURKA. A variety of cellular proteins that play a role in cell division are substrates for phosphorylation by the Aurora A kinase enzyme, including, without limitation, p53, TPX-2, XIEg5 (in Xenopus), and D-TACC (in Drosophila). The Aurora A kinase enzyme is also itself a substrate for autophosphorylation, e.g., at Thr288. Preferably, the Aurora A kinase is a human Aurora A kinase.
• inhibitor of Aurora A kinase or "Aurora A kinase inhibitor” is used to signify a compound that is capable of interacting with Aurora A kinase and inhibiting its enzymatic activity.
• Inhibiting Aurora A kinase enzymatic activity means reducing the ability of Aurora A kinase to phosphorylate a substrate peptide or protein. In various embodiments, such reduction of Aurora A kinase activity is at least about 75%, at least about 90%, at least about 95%, or at least about 99%.
• the concentration, e.g., the IC 50 , of Aurora A kinase inhibitor required to reduce an Aurora A kinase enzymatic activity is less than about 1 ⁇ , less than about 500 nM, less than about 100 nM, or less than about 50 nM. In one embodiment, the concentration that is required to inhbit the enzymatic activity of Aurora A kinase is lower than the concentration of the inhibitor that is required to inhibit the enzymatic activity of Aurora B kinase. In an embodiment, the concentration of Aurora A kinase inhibitor required to reduce an Aurora A kinase enzymatic activity is 50 nM to 100 nM.
• the concentration of Aurora A kinase inhibitor required to reduce an Aurora A kinase enzymatic activity is 100 nM to 500 nM. In an embodiment, the concentration of Aurora A kinase inhibitor required to reduce an Aurora A kinase enzymatic activity is 50 nM to 500 nM. In an embodiment, the concentration of Aurora A kinase inhibitor required to reduce an Aurora A kinase enzymatic activity is 50 nM to 1 ⁇ . In an embodiment, the concentration of Aurora A kinase inhibitor required to reduce an Aurora A kinase enzymatic activity is 100 nM to 1 ⁇ .
• the concentration of Aurora A kinase inhibitor required to reduce an Aurora A kinase enzymatic activity is 500 nM to 1 ⁇ . In various embodiments, the concentration of an Aurora A kinase inhibitor that is required to reduce Aurora A kinase enzymatic activity is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold lower than the concentration of the inhibitor that is required to reduce Aurora B kinase enzymatic activity.
• the concentration of an Aurora A kinase inhibitor that is required to reduce Aurora A kinase enzymatic activity is 2-fold to 10-fold 5-fold to 50-fold, 10-fold to 100-fold, 20-fold to 200-fold or 50-fold to 500-fold lower than the concentration of the inhibitor that is required to reduce Aurora B kinase enzymatic activity.
• the term "selective inhibitor of Aurora A kinase” or “selective Aurora A kinase inhibitor” refers to an inhibitor that exhibits an Aurora A kinase inhibitor phenotype at effective antitumor concentrations.
• the selective Aurora A kinase inhibitor causes a transient mitotic delay, as measured by quantification of pHisH3, when administered to mice at a dose where the free fraction adjusted concentration (C ave ) in plasma is equivalent to the free fraction adjusted concentration achieved in plasma in humans at the maximum tolerated dose (MTD).
• free fraction adjusted concentration refers to the plasma concentration of free drug (not protein bound).
• the term “comprises” means “includes, but is not limited to.”
• the term "patient”, as used herein, means an animal, preferably a mammal, more preferably a human. In some embodiments, the patient has been treated with an agent, e.g., an Aurora A kinase selective inhibitor, prior to initiation of treatment according to the method of the disclosure. In some embodiments, the patient is a patient at risk of developing or experiencing a recurrence of a proliferative disorder.
• aliphatic or "aliphatic group”, as used herein, means a substituted or unsubstituted straight-chain, branched or cyclic C ⁇ . ⁇ 2 hydrocarbon, which is completely saturated or which contains one or more units of unsaturation, but which is not aromatic.
• suitable aliphatic groups include substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl, alkynyl groups and hybrids thereof, such as (cylcoalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
• alkyl refers to a straight and branched chain aliphatic group having from 1 to 12 carbon atoms.
• alkyl will be used when the carbon atom attaching the aliphatic group to the rest of the molecule is a saturated carbon atom.
• an alkyl group may include unsaturation at other carbon atoms.
• alkyl groups include, without limitation, methyl, ethyl, propyl, allyl, propargyl, butyl, pentyl, and hexyl.
• alkenyl will be used when the carbon atom attaching the aliphatic group to the rest of the molecule forms part of a carbon-carbon double bond.
• Alkenyl groups include, without limitation, vinyl, 1-propenyl, 1-butenyl, 1-pentenyl, and 1-hexenyl.
• alkynyl will be used when the carbon atom attaching the aliphatic group to the rest of the molecule forms part of a carbon-carbon triple bond.
• Alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, 1-pentynyl, and
• cycloaliphatic used alone or as part of a larger moiety, refers to a saturated or partially unsaturated cyclic aliphatic ring system having from 3 to about 14 members, wherein the aliphatic ring system is optionally substituted.
• the cycloaliphatic is a monocyclic hydrocarbon having 3-8 or 3-6 ring carbon atoms.
• Nonlimiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl.
• the cycloaliphatic is a bridged or fused bicyclic hydrocarbon having 6-12, 6-10, or 6-8 ring carbon atoms, wherein any individual ring in the bicyclic ring system has 3-8 members.
• two adjacent substituents on the cycloaliphatic ring taken together with the intervening ring atoms, form an optionally substituted fused 5- to 6-membered aromatic or 3- to 8-membered non-aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S.
• cycloaliphatic includes aliphatic rings that are fused to one or more aryl, heteroaryl, or heterocyclyl rings.
• Nonlimiting examples include indanyl, 5,6,7,8-tetrahydro- quinoxalinyl, decahydronaphthyl, or tetrahydronaphthyl, where the radical or point of attachment is on the aliphatic ring.
• cycloaliphatic may be used interchangeably with the terms “carbocycle”, “carbocyclyl”, “carbocyclo”, or “carbocyclic”.
• aryl and "ar-”, used alone or as part of a larger moiety, e.g., "aralkyl”,
• aryloxy refers to a C 6 to C M aromatic hydrocarbon, comprising one to three rings, each of which is optionally substituted.
• the aryl group is a C 6- io aryl group.
• Aryl groups include, without limitation, phenyl, naphthyl, and anthracenyl.
• aryl includes groups in which an aromatic ring is fused to one or more heteroaryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the aromatic ring.
• fused ring systems include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, fluorenyl, indanyl, phenanthridinyl, tetrahydr
• aryl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic.
• aryl may be used interchangeably with the terms “aryl group”, “aryl moiety”, and “aryl ring”.
• an "aralkyl” or “arylalkyl” group comprises an aryl group covalently attached to an alkyl group, either of which independently is optionally substituted.
• the aralkyl group is
• heteroaryl and “heteroar-”, used alone or as part of a larger moiety, e.g., heteroaralkyl, or “heteroaralkoxy”, refer to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14 ⁇ electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to four heteroatoms.
• heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
• Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
• heteroaryl taken together with the intervening ring atoms, form an optionally substituted fused 5- to 6-membered aromatic or 4- to 8-membered non- aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S.
• heteroaryl and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
• Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4i/-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one.
• a heteroaryl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic.
• heteroaryl may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.
• heterooaralkyl refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.
• heterocycle As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and
• heterocyclic ring are used interchangeably and refer to a stable 3- to 7-membered monocyclic, or to a fused 7- to 10-membered or bridged 6- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
• nitrogen includes a substituted nitrogen.
• the nitrogen may be N (as in 3,4-dihydro-2i/- pyrrolyl), NH (as in pyrrolidinyl) or 3 ⁇ 4R (as in -substituted pyrrolidinyl).
• a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted.
• saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
• two adjacent substituents on a heterocyclic ring taken together with the intervening ring atoms, for an optionally substituted fused 5- to 6-membered aromatic or 3- to 8- membered non-aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S.
• heterocycle used interchangeably herein, and include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3i/-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring.
• a heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic.
• heterocyclylalkyl refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
• partially unsaturated refers to a ring moiety that includes at least one double or triple bond between ring atoms.
• the term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
• haloaliphatic refers to an aliphatic, alkyl, alkenyl or alkoxy group, as the case may be, which is substituted with one or more halogen atoms.
• halogen or halo means F, CI, Br, or I.
• fluoroaliphatic refers to a haloaliphatic wherein the halogen is fluoro.
• alkylene refers to a bivalent alkyl group.
• An "alkylene chain” is a polymethylene group, i.e., -(CH 2 ) n -, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3.
• a substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
• An alkylene chain also may be substituted at one or more positions with an aliphatic group or a substituted aliphatic group.
• substituted means that a hydrogen radical of the designated moiety is replaced with the radical of a specified substituent, provided that the substitution results in a stable or chemically feasible compound.
• substituents refers to a number of substituents that equals from one to the maximum number of substituents possible based on the number of available bonding sites, provided that the above conditions of stability and chemical feasibility are met.
• an optionally substituted group may have a substituent at each substitutable position of the group, and the substituents may be either the same or different.
• An aryl (including the aryl moiety in aralkyl, aralkoxy, aryloxyalkyl and the like) or heteroaryl (including the heteroaryl moiety in heteroaralkyl and heteroaralkoxy and the like) group may contain one or more substituents. Examples of suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl group
• An aryl (including the aryl moiety in aralkyl, aralkoxy, aryloxyalkyl and the like) or heteroaryl (including the heteroaryl moiety in heteroaralkyl and heteroaralkoxy and the like) group may contain one or more substituents. Examples of suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl group
• Each R + is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group, or two R + on the same nitrogen atom, taken together with the nitrogen atom, form a 5-8 membered aromatic or non-aromatic ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms selected from N, O, and S.
• Each R* independently is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group.
• Each R° is an optionally substituted aliphatic or aryl group.
• An aliphatic group or a non-aromatic heterocyclic ring may be substituted with one or more substituents.
• each R* is as defined above.
• structures depicted herein are meant to include compounds which differ only in the presence of one or more isotopically enriched atoms.
• compounds having the present structure except for the replacement of a hydrogen atom by a deuterium or tritium, or the replacement of a carbon atom by a 13 C- or 14 C-enriched carbon are within the scope of the disclosure.
• any molecule capable of selectively inhibiting the enzymatic activity of Aurora A kinase may be used in the methods, pharmaceutical compositions, and kits of the present disclosure.
• the selective Aurora A kinase inhibitor is a small molecular weight compound.
• selective inhibitors of Aurora A kinase include the compounds described herein, as well as compounds disclosed in, for example, US Publication No. 2008/0045501, US Patent No. 7,572,784, WO 05/111039, WO 08/021038, US Patent No. 7,718,648, WO 08/063525, US Publication No. 2008/0167292, US Patent No. 8,026,246, WO 10/134965, US Publication No.
• compositions, and kits of the disclosure are pharmaceutically acceptable salts of any of the compounds, and solvated and hydrated forms of such salts.
• selective Aurora A kinase inhibitors can be prepared in a number of ways well known to one skilled in the art of organic synthesis, including, but not limited to, the methods of synthesis described in detail in the references referred to herein.
• Aurora A kinase inhibitors can be assayed in vitro or in vivo for their ability to selectively bind to and/or inhibit an Aurora A kinase.
• In vitro assays include assays to determine selective inhibition of the ability of an Aurora A kinase to phosphorylate a substrate protein or peptide.
• Alternate in vitro assays quantitate the ability of the compound to selectively bind to an Aurora A kinase.
• Selective inhibitor binding may be measured by radiolabelling the inhibitor prior to binding, isolating the inhibitor/ Aurora A kinase complex and determining the amount of radiolabel bound.
• selective inhibitor binding may be determined by running a competition experiment in which new inhibitors are incubated with Aurora A kinase bound to a known radioligand. The compounds also can be assayed for their ability to affect cellular or physiological functions mediated by Aurora A kinase activity.
• inhibitors can also be assayed in vitro and in vivo for their ability to selectively bind to and/or inhibit an Aurora B kinase, using assays analogous to those described above for Aurora A kinase.
• Inhibitors can be assayed in vitro and in vivo for their ability to inhibit Aurora A kinase in the absence of Aurora B kinase inhibition, by immunofluore scent detection of pHisH3. (Proc. Natl. Acad. Sci. (2007) 104, 4106). Assays for each of these activities are known in the art.
• the selective Aurora A kinase inhibitor is a compound represented by formula (7):
• Ring A is a substituted or unsubstituted 5- or 6-membered aryl, heteroaryl, cycloaliphatic, or heterocyclyl ring;
• Ring B is a substituted or unsubstituted aryl, heteroaryl, cycloaliphatic, or heterocyclyl ring
• Ring C is a substituted or unsubstituted aryl, heteroaryl, heterocyclyl, or cycloaliphatic ring
• R e is hydrogen, -OR 5 , -N(R 4 ) 2 , -SR 5 , or a Ci -3 aliphatic optionally substituted with R 3 or R 7 ; each of R x and R y independently is hydrogen, fluoro, or an optionally substituted Ci -6 aliphatic; or
• R x and R y taken together with the carbon atom to which they are attached, form an optionally substituted 3- to 6-membered cycloaliphatic ring;
• each R 3 independently is selected from the group consisting of
• each R 4 independently is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group; or two R 4 on the same nitrogen atom, taken together with the nitrogen atom, form an optionally substituted 5- to 6-membered heteroaryl or 4- to 8-membered heterocyclyl ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms selected from
• each R 5 independently is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group
• each R 7 independently is an optionally substituted aryl, heterocyclyl, or heteroaryl group.
• Ring A is a substituted or unsubstituted 5- or 6-membered aryl, heteroaryl, cycloaliphatic, or heterocyclyl ring.
• Ring A include furano, dihydrofurano, thieno, dihydrothieno, cyclopenteno, cyclohexeno, 2H-pyrrolo, pyrrolo, pyrrolino, pyrrolidino, oxazolo, thiazolo, imidazolo, imidazolino, imidazolidino, pyrazolo, pyrazolino, pyrazolidino, isoxazolo, isothiazolo, oxadiazolo, triazolo, thiadiazolo, 2H-pyrano, 4H-pyrano, benzo, pyridino, piperidino, dioxano, morpholino, dithiano, thiomorpholino, pyridazin
• Ring A examples include, without limitation, substituted or unsubstituted rings selected from the group consisting of furano, thieno, pyrrolo, oxazolo, thiazolo, imidazolo, pyrazolo, isoxazolo, isothiazolo, triazolo, benzo, pyridino, pyridazino, pyrimidino, and pyrazino.
• Ring A may be substituted or unsubstituted.
• Each substitutable unsaturated ring carbon atom in Ring A is unsubstituted or substituted with -R b .
• Each substitutable ring nitrogen atom in Ring A is unsubstituted or is substituted with -R 9b , and one ring nitrogen atom in Ring A optionally is oxidized.
• Each R 9b independently is -C(0)R 5 , -C(0)N(R 4 ) 2 , -C0 2 R 6 , -S0 2 R 6 , -S0 2 N(R 4 ) 2 , or a C H aliphatic optionally substituted with R 3 or R 7 .
• Each R b independently is R 2b , an optionally substituted aliphatic, or an optionally substituted aryl, heterocyclyl, or heteroaryl group; or two adjacent R b , taken together with the intervening ring atoms, form an optionally substituted fused 4- to 8-membered aromatic or non-aromatic ring having 0-3 ring heteroatoms selected from the group consisting of O, N, and S.
• Ring A is substituted by 0-2 substituents R b .
• each R b independently is selected from the group consisting of -halo, Ci -3 aliphatic, Ci fluoroaliphatic, and -OR 5 , where R 5 is hydrogen or Ci -3 aliphatic.
• Ring A is substituted with 0, 1, or 2 substituents, preferably 0 or 1 substituents, independently selected from the group consisting of chloro, fluoro, bromo, methyl, trifluoromethyl, and methoxy.
• Ring B is a substituted or unsubstituted mono- or bicyclic aryl or heteroaryl ring selected from the group consisting of furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolizinyl, indolyl, isoindolyl, indazolyl,
• Each substitutable unsaturated ring carbon atom in Ring B is unsubstituted or substituted with -R c .
• Each substitutable ring nitrogen atom in Ring B is unsubstituted or is substituted with -R 9c , and one ring nitrogen atom in Ring B optionally is oxidized.
• Each R 9c independently
• Ring B is -C(0)R 5 , -C(0)N(R 4 ) 2 , -C0 2 R 6 , -S0 2 R 6 , -S0 2 N(R 4 ) 2 , or a d -4 aliphatic optionally substituted with R 3 or R 7 .
• Ring B may be unsubstituted or may be substituted on any one or more of its component rings, wherein the substituents may be the same or different. In some embodiments, Ring B is substituted with 0-2 independently selected R c and 0-3 independently selected R 2c or Ci -6 aliphatic groups.
• the variables R 3 , R 4 , R 5 , R 6 , and R 7 are as defined above for Ring A, and R c and R c are defined below.
• Each R c independently is R 2c , an optionally substituted Ci -6 aliphatic, or an optionally substituted aryl, heteroaryl, or heterocyclyl group.
• Each R 2c independently is -halo, -N0 2 , -CN, -
• Ring B is a monocyclic 5- or 6-membered aryl or heteroaryl ring, substituted with 0-2 independently selected R c and 0-2 independently selected R 2c or Ci.6 aliphatic groups. In certain such embodiments, Ring B is a substituted or unsubstituted phenyl or pyridyl ring.
• Ring B is substituted with 0-2 substituents R c .
• each R c independently is selected from the group consisting of -halo, Ci -3 aliphatic, Ci -3 haloaliphatic, and -OR 5 , where R 5 is hydrogen or Ci -3 aliphatic.
• R 5 is hydrogen or Ci -3 aliphatic.
• Ring B is substituted with 0, 1, or 2 substituents, independently selected from the group consisting of chloro, fluoro, bromo, methyl, trifluoromethyl, and methoxy.
• Each substitutable unsaturated ring carbon atom in Ring C is unsubstituted or substituted with -R d .
• Each substitutable ring nitrogen atom in Ring C is unsubstituted or is substituted with -R 9d , and one ring nitrogen atom in Ring C optionally is oxidized.
• Each R 9d independently
• Ring C is -C(0)R 5 , -C(0)N(R 4 ) 2 , -C0 2 R 6 , -S0 2 R 6 , -S0 2 N(R 4 ) 2 , or a d -4 aliphatic optionally substituted with R 3 or R 7 .
• Ring C may be unsubstituted or may be substituted on any one or more of its component rings, wherein the substituents may be the same or different. In some embodiments, Ring C is substituted with 0-2 independently selected R d and 0-3 independently selected R 2d or Ci -6 aliphatic groups.
• the variables R 3 , R 4 , R 5 , R 6 , and R 7 are as described above for Rings A and B.
• the variables R d and R 2d are described below.
• Each R d independently is R 2d , an optionally substituted aliphatic, or an optionally substituted aryl, heteroaryl, or heterocyclyl group.
• Ring C is a monocyclic 5- or 6-membered aryl or heteroaryl ring, which is substituted with 0-2 independently selected substituents R d and 0-2 independently selected R 2d or Ci_s aliphatic groups.
• Ring C is an optionally substituted heteroaryl ring selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, and oxazolyl.
• Ring C is a substituted or unsubstituted phenyl ring.
• Ring C is a monocyclic 5- or 6-membered aryl or heteroaryl ring, which is substituted with 0, 1, or 2 substituents R d , as defined above.
• Ring C is a monocyclic 5- or 6-membered heterocyclyl or cycloaliphatic ring, which is substituted with 0-2 independently selected substituents R d and 0-2 independently selected R 2d or Ci -6 aliphatic groups.
• the selective Aurora A kinase inhibitor is a compound represented by formula (II):
• R e is hydrogen or a Ci -3 aliphatic optionally substituted with R 3 or R 7 ;
• Ring A is substituted with 0-3 R b ;
• each R b independently is selected from the group consisting of Ci -6 aliphatic, R 2b ,
• each R ⁇ independently is an optionally substituted aryl, heterocyclyl, or heteroaryl group; Ring B is substituted with 0-2 independently selected R c and 0-2 independently selected R 2c or Ci-s aliphatic groups; each R c independently is selected from the group consisting of Ci -6 aliphatic, R 2c , R3 ⁇ 4 . T i. R 2 Cj and . x i. R 7c.
• each R 7c independently is an optionally substituted aryl, heterocyclyl, or heteroaryl group
• T 1 is a Ci-s alkylene chain optionally substituted with R 3 or R 3b , wherein T 1 or a portion thereof optionally forms part of a 3- to 7-membered ring;
• Ring C is substituted with 0-2 independently selected R d and 0-3 independently selected R 2d or Ci-s aliphatic groups;
• each R d independently is selected from the group consisting of Ci -6 aliphatic, R 2d ,
• T 2 is a Ci-6 alkylene chain optionally substituted with R 3 or R 3b , wherein the alkylene chain optionally is interrupted
• T 3 is a Ci-s alkylene chain optionally substituted with R 3 or R 3b , wherein the alkylene chain optionally is interrupted
• each R 7d independently is an optionally substituted aryl, heterocyclyl, or heteroaryl group
• each R 3 independently is selected from the group consisting
• each R 3b independently is a Ci -3 aliphatic optionally substituted with R 3 or R 7 , or two substituents
• R 3b on the same carbon atom, taken together with the carbon atom to which they are attached, form a 3- to 6-membered carbocyclic ring;
• each R 4 independently is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group; or two R 4 on the same nitrogen atom, taken together with the nitrogen atom, form an optionally substituted 5- to 8-membered heteroaryl or heterocyclyl ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms selected from N, O, and S;
• each R 5 independently is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group
• each R 6 independently is an optionally substituted aliphatic or aryl group
• each R 7 independently is an optionally substituted aryl, heterocyclyl, or heteroaryl group.
• Table 1 provides the chemical names for specific examples of compounds of formula (II).
• the selective Aurora A kinase inhibitor is represented by formula (III):
• R a is selected from the group consisting of Ci -3 aliphatic, Ci -3 fluoroaliphatic, -R 1 , -T-R 1 , -R 2 , and -T-R 2 ;
• T is a Ci alkylene chain optionally substituted with fluoro
• R 1 is an optionally substituted aryl, heteroaryl, or heterocyclyl group
• R 3 is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group; each R 4 independently is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group; or two R 4 on the same nitrogen atom, taken together with the nitrogen atom form an optionally substituted 5- to 6-membered heteroaryl or 4- to 8-membered heterocyclyl ring having, in addition to the nitrogen atom, 0-2 ring heteroatoms selected from N, O, and S;
• R 5 is hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, or heterocyclyl group
• R b is selected from the group consisting of fluoro
• R 1 is a 5- or 6-membered aryl, heteroaryl, or heterocyclyl ring optionally substituted with one or two substituents independently selected from the group consisting of halo, Ci -3 aliphatic, and Ci -3 fluoroaliphatic.
• R 1 is a phenyl, furyl, pyrrolidinyl, or thienyl ring optionally substituted with one or two substituents independently selected from the group consisting of halo, Ci -3 aliphatic, and Ci -3 fluoroaliphatic.
• R 3 is hydrogen, Ci -3 aliphatic, Ci -3 fluoroaliphatic, or -CH 2 -OCH 3 .
• R 5 is hydrogen, Ci -3 aliphatic, or Ci -3 fluoroaliphatic.
• R a is halo, Ci -3 aliphatic,
• R a is selected from the group consisting of chloro, fluoro, Ci -3 aliphatic,
• Table 2 provides the chemical names for specific examples of compounds of formula ⁇ III).
• the compound of formula (III) is 4- ⁇ [9-chloro-7-(2-fluoro-6- methoxyphenyl)-5i/-pyrimido[5,4-rf
• the compound of formula (I) is sodium 4- ⁇ [9-chloro-7-(2-fluoro-6-methoxyphenyl)-5i/-pyrimido[5,4- i3 ⁇ 4 [2]benzazepin-2-yl]amino ⁇ -2-methoxybenzoate.
• the compound of formula (III) is sodium 4- ⁇ [9-chloro-7-(2-fluoro-6-methoxyphenyl)-5i/-pyrimido[5,4-d] [2]benzazepin-2- yl]amino ⁇ -2-methoxybenzoate monohydrate.
• the compound of formula (III) is sodium 4- ⁇ [9-chloro-7-(2-fluoro-6-methoxyphenyl)-5i/-pyrimido[5,4-d] [2]benzazepin-2- yl]amino ⁇ -2-methoxybenzoate or a crystakline form thereof, as described in US Publication No. 2008/0167292, US Patent No. 8,026,246, and US Publication No. 2011/0245234, hereby incorporated by reference in their entirety.
• the present disclosure provides a method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of anyone of the compounds of formulas (I),
• the disclosure provides anyone of the compounds of formulas (I), (II) or (III), or a pharmaceutically acceptable salt thereof for use in treating cancer.
• the disclosure provides the use of anyone of the compounds of formulas (I), (II) or
• the disclosure provides the use of a therapeutically effective amount of anyone of the compounds of formulas (I),
• the disclosure provides a method of determining whether to treat a patient with cancer with a therapeutically effective amount of anyone of the compounds of formulas (I), (II) or (III), or a pharmaceutically acceptable salt thereof, based on identifying the patient with cancer as being likely to respond to the treatment based upon the presence of mutations in the patient's cell sample(s).
• the disclosure provides a method of determining whether to treat a patient with cancer with a therapeutically effective amount of an Aurora A Kinase inhibitor, e.g., alisertib, or a pharmaceutically acceptable salt thereof based on identifying the patient with cancer as being likely to respond to the treatment based upon the presence of mutations in the patient's cell sample(s).
• the mutations in the patient's cell samples are mutations of genes in the Wnt/p-catenin signaling pathway or the Hippo signaling pathway.
• the present disclosure provides a method of treating cancer, comprising administering a therapeutically effective amount of an Aurora A Kinase inhibitor, e.g., alisertib, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, to a cancer patient whose tumor sample is characterized by having a mutation in a gene selected from the group consisting of LEF1, MAP3K7, APC, FZD2, PRKCA, RORA, CAMK2G, JUN, XPOl, ROR2, CCND1, CTNNB1, AMOT, DVL2, LATS1, LATS2, MOB IB, NPHP4, TJP1, TJP2, WWC1, WWTR1 and YAP 1.
• an Aurora A Kinase inhibitor e.g., alisertib
• the disclosure provides a compound of anyone of formulas (I), (II) or
• (III) or a pharmaceutically acceptable salt or a pharmaceutical composition thereof, for use in treating cancer in a patient with a mutation in a gene selected from the group consisting of LEF1, MAP3K7, APC, FZD2, PRKCA, RORA, CAMK2G, JUN, XPOl, ROR2, CCND 1, CTNNB1, AMOT, DVL2, LATS1, LATS2, MOB IB, NPHP4, TJP1, TJP2, WWC1, WWTR1 and YAP1.
• Described herein is the assessment of outcome for treatment of a tumor through measurement of the amount of pharmacogenomic markers.
• kits and methods disclosed herein can identify the potential for unfavorable outcome and allow their prevention, and thus a savings in morbidity, mortality and treatment costs through adjustment in the therapeutic regimen, cessation of therapy or use of alternative therapy.
• biological sample is intended to include a patient sample, e.g, tissue, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject and can be obtained from a patient or a normal subject.
• a patient sample e.g., tissue, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject and can be obtained from a patient or a normal subject.
• hematological tumors of the bone marrow e.g., myeloma tumors
• primary analysis of the tumor can be performed on bone marrow samples.
• some tumor cells e.g., clonotypic tumor cells, circulating endothelial cells
• noninvasive samples e.g., for in vitro measurement of markers to determine outcome of treatment, can include peripheral blood samples. Accordingly, cells within peripheral blood can be tested for marker amount.
• a control, reference sample for normal characteristic e.g., size, sequence, composition or amount
• a typical tumor sample is a biopsy of the tumor.
• a control reference sample for normal characteristic e.g., size, sequence, composition or amount can be obtained from blood of the patient.
• Blood collection containers can comprise an anti-coagulant, e.g., heparin or ethylene- diaminetetraacetic acid (EDTA), sodium citrate or citrate solutions with additives to preserve blood integrity, such as dextrose or albumin or buffers, e.g., phosphate.
• an anti-coagulant e.g., heparin or ethylene- diaminetetraacetic acid (EDTA)
• EDTA ethylene- diaminetetraacetic acid
• sodium citrate or citrate solutions with additives to preserve blood integrity
• dextrose or albumin or buffers e.g., phosphate.
• a DNA stabilizer e.g., an agent that inhibits DNAse
• an RNA stabilizer e.g., an agent that inhibits RNAse
• a protein stabilizer e.g., an agent that inhibits proteases
• An example of a blood collection container is PAXGENE® tubes (PREANALYTIX, Valencia, CA), useful for RNA stabilization upon blood collection.
• Peripheral blood samples can be modified, e.g., fractionated, sorted or concentrated (e.g., to result in samples enriched with tumor or depleted of tumor (e.g., for a reference sample)).
• modified samples include clonotypic myeloma cells, which can be collected by e.g. , negative selection, e.g., separation of white blood cells from red blood cells (e.g. , differential centrifugation through a dense sugar or polymer solution (e.g. ,
• FICOLL® solution (Amersham Biosciences division of GE healthcare, Piscataway, NJ) or
• HISTOPAQUE®-1077 solution Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co., St. Louis, MO
• a selection agent e.g. , a reagent which binds to a tumor cell or myeloid progenitor marker, such as CD34, CD38, CD138, or CD 133
• direct isolation e.g. , the application of a magnetic field to solutions of cells comprising magnetic beads (e.g. , from Miltenyi Biotec, Auburn, CA) which bind to the B cell markers) or fluorescent-activated cell sorting).
• a tumor cell line e.g., OCI-Ly3, OCI-Ly 10 cell (Alizadeh et al. (2000) Nature 403:503-511), a RPMI 6666 cell, a SUP-B15 cell, a KG-1 cell, a CCRF-SB cell, an 8ES cell, a Kasumi-1 cell, a Kasumi-3 cell, a BDCM cell, an HL-60 cell, a Mo-B cell, a JM1 cell, a GA-10 cell or a B-cell lymphoma (e.g. , BC-3) or a cell line or a collection of tumor cell lines (see e.g., OCI-Ly3, OCI-Ly 10 cell (Alizadeh et al. (2000) Nature 403:503-511), a RPMI 6666 cell, a SUP-B15 cell, a KG-1 cell, a CCRF-SB cell, an 8ES cell, a Kasumi-1
• the sample e.g. , tumor, e.g., biopsy or bone marrow, blood or modified blood, (e.g. , comprising tumor cells) and/or the reference, e.g., matched control (e.g., germline), sample can be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of the marker in the sample.
• a variety of well-known post-collection preparative and storage techniques e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.
• a mutation in a marker can be identified by sequencing a nucleic acid, e.g., a DNA, RNA, cDNA or a protein correlated with the marker gene.
• a nucleic acid e.g., a DNA, RNA, cDNA or a protein correlated with the marker gene.
• a primer can be designed to bind to a region comprising a potential mutation site or can be designed to complement the mutated sequence rather than the wild type sequence.
• Primer pairs can be designed to bracket a region comprising a potential mutation in a marker gene.
• a primer or primer pair can be used for sequencing one or both strands of DNA corresponding to the marker gene.
• a primer can be used in conjunction with a probe to amplify a region of interest prior to sequencing to boost sequence amounts for detection of a mutation in a marker gene.
• regions which can be sequenced include an entire gene, transcripts of the gene and a fragment of the gene or the transcript, e.g., one or more of exons or untranslated regions.
• mutations to target for primer selection and sequence or composition analysis can be found in public databases which collect mutation information, such as COSMIC and dbGaP.
• marker genes such as LEF1, MAP2K7, APC, FZD2, PRKCA, RORA, CAMK2G, JUN, XPOl, ROR2, CCND1, CTNNB1, AMOT, DVL2, LATS1, LATS2, MOB IB, NPHP4, TJP1, TJP2, WWC1, WWC1, WWTR1 and YAP1, etc. are listed in Tables 9-10 in the Examples as examples of mutations that can be associated with sensitivity to Aurora A Kinase inhibition, e.g., inhibition by alisertib.
• Sequencing methods are known to one skilled in the art. Examples of methods include the Sanger method, the SEQUENOMTM method and Next Generation Sequencing (NGS) methods.
• the Sanger method comprising using electrophoresis, e.g., capillary electrophoresis to separate primer- elongated labeled DNA fragments, can be automated for high-throughput applications.
• the primer extension sequencing can be performed after PCR amplification of regions of interest.
• Software can assist with sequence base calling and with mutation identification.
• SEQUENOMTM MASSARRAY® sequencing analysis (San Diego, CA) is a mass-spectrometry method which compares actual mass to expected mass of particular fragments of interest to identify mutations.
• NGS technology also called “massively parallel sequencing” and “second generation sequencing” in general provides for much higher throughput than previous methods and uses a variety of approaches (reviewed in Zhang et al. (2011) J. Genet. Genomics 38:95-109 and Shendure and Hanlee (2008) Nature Biotech. 26: 1135- 1145).
• NGS methods can identify low frequency mutations in a marker in a sample.
• Some NGS methods see, e.g., GS-FLX Genome Sequencer (Roche Applied Science, Branford, CT), Genome analyzer (Illumina, Inc.
• SOLIDTM analyzer (Applied Biosystems, Carlsbad, CA), Polonator G.007 (Dover Systems, Salem, NH), HELISCOPETM (Helicos Biosciences Corp., Cambridge, MA)
• SOLIDTM analyzer Applied Biosystems, Carlsbad, CA
• Polonator G.007 Denssion Inhibition of PCR products
• HELISCOPETM Helicos Biosciences Corp., Cambridge, MA
• primer pairs can be used in PCR reactions to amplify regions of interest. Amplified regions can be ligated into a concatenated product.
• Clonal libraries are generated in the flow cell from the PCR or ligated products and further amplified ("bridge” or “cluster” PCR) for single-end sequencing as the polymerase adds a labeled, reversibly terminated base that is imaged in one of four channels, depending on the identity of the labeled base and then removed for the next cycle.
• Software can aid in the comparison to genomic sequences to identify mutations.
• composition of proteins and nucleic acids can be determined by many ways known in the art, such as by treating them in ways that cleave, degrade or digest them and then analyzing the components. Mass spectrometry, electrophoresis and chromatography can separate and define components for comparison. Mutations which cause deletions or insertions can be identified by size or charge differences in these methods. Protein digestion or restriction enzyme nucleic acid digestion can reveal different fragment patterns after some mutations. Antibodies that recognize particular mutant amino acids in their structural contexts can identify and detect these mutations in samples (see below).
• DNA e.g., genomic DNA corresponding to the wild type or mutated marker can be analyzed both by in situ and by in vitro formats in a biological sample using methods known in the art.
• DNA can be directly isolated from the sample or isolated after isolating another cellular component, e.g., RNA or protein.
• Kits are available for DNA isolation, e.g., QIAAMP® DNA Micro Kit (Qiagen, Valencia, CA). DNA also can be amplified using such kits.
• mRNA corresponding to the marker can be analyzed both by in situ and by in vitro formats in a biological sample using methods known in the art.
• Many expression detection methods use isolated RNA.
• any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from tumor cells (see, e.g., Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999).
• large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Patent No. 4,843,155).
• RNA can be isolated using standard procedures (see e.g., Chomczynski and Sacchi (1987) Anal. Biochem.162: 156-159), solutions (e.g., trizol, TRI REAGENT® (Molecular Research Center, Inc., Cincinnati, OH; see U.S. Patent No. 5,346,994) or kits (e.g., a QIAGEN® Group RNEASY® isolation kit (Valencia, CA) or LEUKOLOCKTM Total RNA Isolation System, Ambion division of Applied Biosystems, Austin, TX).
• solutions e.g., trizol, TRI REAGENT® (Molecular Research Center, Inc., Cincinnati, OH; see U.S. Patent No. 5,346,994)
• kits e.g., a QIAGEN® Group RNEASY® isolation kit (Valencia, CA) or LEUKOLOCKTM Total RNA Isolation System, Ambion division of Applied Biosystems, Austin, TX).
• RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (Chirgwin et al. (1979) Biochemistry 18:5294-99).
• Poly(A)+RNA is selected by selection with oligo- dT cellulose (see Sambrook et al. (1989) Molecular Cloning— A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
• separation of RNA from DNA can be accomplished by organic extraction, for example, with hot phenol or
• RNAse inhibitors may be added to the lysis buffer.
• mRNAs transfer RNA (tRNA) and ribosomal RNA (rRNA).
• Most mRNAs contain a poly(A) tail at their 3' end. This allows them to be enriched by affinity chromatography, for example, using oligo(dT) or poly(U) coupled to a solid support, such as cellulose or SEPHADEX.RTM. medium (see Ausubel et al. (1994) Current Protocols In Molecular Biology, vol. 2, Current Protocols Publishing, New York).
• poly(A)+mRNA is eluted from the affinity column using 2 mM
• the characteristic of a marker of the disclosure in a biological sample involves obtaining a biological sample (e.g., a bone marrow sample, a tumor biopsy or a reference sample) from a test subject may be assessed by any of a wide variety of well known methods for detecting or measuring the characteristic, e.g., of a nucleic acid (e.g., RNA, mRNA, genomic DNA, or cDNA) and/or translated protein.
• Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. These methods include gene array /chip technology, RT-PCR, TAQMAN® gene expression assays (Applied Biosystems, Foster City, CA), e.g., under GLP approved laboratory conditions, in situ hybridization,
• RNA, mRNA, protein, cDNA, or genomic DNA for example, in a biological sample in vitro as well as in vivo.
• in vivo techniques for detection of a polypeptide or nucleic acid corresponding to a marker of the disclosure include introducing into a subject a labeled probe to detect the biomarker, e.g., a nucleic acid complementary to the transcript of a biomarker or a labeled antibody, Fc receptor or antigen directed against the polypeptide, e.g., wild type or mutant marker.
• the antibody can be labeled with a radioactive isotope whose presence and location in a subject can be detected by standard imaging techniques.
• ELISAs enzyme linked immunosorbent assays
• Western blots protein array
• expression of a marker is assessed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore -labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically with a marker protein or fragment thereof, e.g., a protein or fragment comprising a region which can be mutated or a portion comprising a mutated sequence, or a mutated residue in its structural context, including a marker protein which has undergone all or a portion of its normal post-translational modification.
• an antibody e.g., a radio-labeled, chromophore-labeled, fluorophore -
• An antibody can detect a protein with an amino acid sequence selected from the group of proteins disclosed by GenPept Accession numbers within Tables 9 and 10 herein.
• an antibody can detect a mutated protein with a variant amino acid sequence selected from the group of proteins disclosed by GenPept Accession numbers within Tables 9 and 10 herein.
• Residues listed as mutated in public databases such as COSMIC of dbGaP can be prepared in immunogenic compositions for generation of antibodies that will specifically recognize and bind to the mutant residues.
• Another method can employ pairs of antibodies, wherein one of the pair would bind a marker protein upstream, i.e. N-terminal to the region of expected mutation, e.g., nonsense or deletion and the other of the pair would bind the protein downstream.
• Wild type protein would bind both antibodies of the pair, but a protein with a nonsense or deletion mutation would bind only the N-terminal antibody of the pair.
• An assay such as a sandwich ELISA assay could detect a loss of quantity of the wild type protein in the tumor sample, e.g., in comparison to the reference sample, or a standard ELISA would comparison of the levels of binding of the antibodies to infer that a mutation is present in a tumor sample.
• Indirect methods for determining the amount or functionality of a protein marker also include measurement of the activity of the protein.
• a sample, or a protein isolated from the sample or expressed from nucleic acid isolated, cloned or amplified from the sample can be assessed for marker protein activity.
• Biomarker activity can be measured by its ability to associate with binding partners, e.g., in a cell-free assay or in a cell-based assay.
• biomarker activity can be measured by its activity in signal transduction, e.g., in a cell-free assay or in a cell-based assay.
• expression of a marker is assessed by preparing mRNA/cDNA (i.e., a transcribed polynucleotide) from cells in a patient sample, and by hybridizing the mRNA/cDNA with a reference polynucleotide which is a complement of a marker nucleic acid, or a fragment thereof.
• cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression of one or more markers likewise can be detected using quantitative PCR to assess the level of expression of the marker(s).
• an inactivating mutation in a marker gene can result in an altered level of mRNA in a cell.
• the level can be upregulated due to feedback signaling protein production in view of nonfunctional or absent protein or downregulated due to instability of an altered mRNA sequence.
• any of the many known methods of detecting mutations or variants (e.g. single nucleotide polymorphisms, deletions, etc., discussed above) of a marker of the disclosure may be used to detect occurrence of a mutation in a marker gene in a patient.
• the level or amount of expression refers to the absolute amount of expression of an mRNA encoded by the marker or the absolute amount of expression of the protein encoded by the marker.
• the absolute amount of expression refers to the absolute amount of expression of an mRNA encoded by the marker or the absolute amount of expression of the protein encoded by the marker.
• Expression amount can be normalized by correcting the absolute expression level of a marker upon comparing its expression to the expression of a control marker that is not a marker, e.g., in a housekeeping role that is constitutively expressed. Suitable markers for normalization also include housekeeping genes, such as the actin gene or beta-2 microglobulin. Reference markers for data normalization purposes include markers which are ubiquitously expressed and/or whose expression is not regulated by oncogenes. Constitutively expressed genes are known in the art and can be identified and selected according to the relevant tissue and/or situation of the patient and the analysis methods. Such normalization allows one to compare the expression level in one sample, to another sample, e.g., between samples from different times or different subjects.
• the expression level can be provided as a relative expression level.
• the baseline of a genomic DNA sample e.g., diploid copy number
• the amount of the marker or marker set is determined for at least 1, or 2, 3, 4, 5, or more samples, e.g., 7, 10, 15, 20 or 50 or more samples in order to establish a baseline, prior to the determination of the expression level for the sample in question.
• the mean amount or level of each of the markers or marker sets assayed in the larger number of samples is determined and this is used as a baseline expression level for the biomarkers or biomarker sets in question.
• the amount of the marker or marker set determined for the test sample (e.g., absolute level of expression) is then divided by the baseline value obtained for that marker or marker set. This provides a relative amount and aids in identifying abnormal levels of marker protein activity.
• Probes based on the sequence of a nucleic acid molecule of the disclosure can be used to detect transcripts or genomic sequences corresponding to one or more markers of the disclosure.
• the probe can comprise a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
• Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.
• DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to naturally occuring allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. In addition, it will be appreciated that DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level of that gene (e.g., by affecting regulation or degradation).
• Primers or nucleic acid probes comprise a nucleotide sequence complementary to a specific marker or a mutated region thereof and are of sufficient length to selectively hybridize with a marker gene or nucleic acid associated with a marker gene. Primers and probes can be used to aid in the isolation and sequencing of marker nucleic acids.
• the primer or nucleic acid probe e.g., a substantially purified oligonucleotide, comprises a region having a nucleotide sequence which hybridizes under stringent conditions to about 6, 8, 10, 12, or 15, 20, 25, 30, 40, 50, 60, 75, 100 or more consecutive nucleotides of a marker gene.
• the primer or nucleic acid probe is capable of hybridizing to a marker nucleic acid comprising a nucleotide sequence disclosed by GenBank Accession number within Tables 9 and 10 herein, or a complement of any of the foregoing.
• a primer or nucleic acid probe comprising a nucleotide sequence of at least about 15 consecutive nucleotides, at least about 25 nucleotides or having from about 15 to about 20 nucleotides, 10 to 50 consecutive nucleotides, 12 to 35 consecutive nucleotides, 15 to 50 consecutive nucleotides, 20 to 100 consecutive nucleotides set forth in any of the nucleotide sequences disclosed by GenBank Accession number within Tables 9 and 10 herein, or a complement of any of the foregoing are provided by the disclosure.
• Primers or nucleic acid probes having a sequence of more than about 25 nucleotides are also within the scope of the disclosure.
• a primer or nucleic acid probe can have a sequence at least 70%, at least 75%, 80% or 85%, or at least, 90%, 95% or 97% identical to the nucleotide sequence of any nucleotide sequence disclosed by GenBank Accession number within Tables 9 and 10 herein, or a complement of any of the foregoing.
• Nucleic acid analogs can be used as binding sites for hybridization.
• An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., Nature 363:566 568 (1993); U.S. Pat. No. 5,539,083).
• Primers or nucleic acid probes can be selected using an algorithm that takes into account binding energies, base composition, sequence complexity, cross-hybridization binding energies, and secondary structure (see Friend et al, International Patent Publication WO 01/05935, published Jan. 25, 2001; Hughes et al., Nat. Biotech. 19:342-7 (2001).
• Useful primers or nucleic acid probes of the disclosure bind sequences which are unique for each transcript, e.g., target mutated regions and can be used in PCR for amplifying, detecting and sequencing only that particular nucleic acid, e.g., transcript or mutated transcript.
• markers genes e.g., LEF1, MAP2K7, APC, FZD2, PRKCA, RORA, CAMK2G, JUN, XPOl, ROR2, CCNDl, CTNNBl, AMOT, DVL2, LATSl, LATS2, MOB IB, NPHP4, TJP1, TJP2, WWC1, WWC1, WWTR1 and YAP1, etc... are found in Tables in the Examples (Tables 9-10). Other mutations are described in reference articles cited herein and in public databases described herein.
• primers and nucleic acid probes for the markers disclosed herein or related markers with similar characteristics, e.g., markers on the chromosome loci, or mutations in different regions of the same marker gene described herein, using the skill in the art, e.g., adjusting the potential for primer or nucleic acid probe binding to standard sequences, mutants or allelic variants by manipulating degeneracy or GC content in the primer or nucleic acid probe.
• Computer programs that are well known in the art are useful in the design of primers with the required specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences, Plymouth, MN). While perfectly complementary nucleic acid probes and primers can be used for detecting the markers described herein and mutants,
• an oligonucleotide primer may have a non-complementary fragment at its 5' end, with the remainder of the primer being complementary to the target region.
• non-complementary nucleotides may be interspersed into the nucleic acid probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
• An indication of treatment outcome can be assessed by studying the amount of 1 marker, 2 markers, 3 markers or 4 markers, or more, e.g. , 5, 6, 7, 8, 9, 10, 15, 20, or 25 markers, or mutated portions thereof e.g., marker genes which participate in or interact with the Wnt/p-catenin signaling pathway, marker genes which participate in or interact with the Hippo pathway, or marker genes which participate in or interact with tumor suppressors. Markers can be studied in combination with another measure of treatment outcome, e.g. , biochemical markers (e.g. , M protein, proteinuria) or histology markers (e.g., blast count, number of mitotic figures per unit area).
• biochemical markers e.g. , M protein, proteinuria
• histology markers e.g., blast count, number of mitotic figures per unit area.
• Any marker e.g., marker gene or combination of marker, e.g. , marker genes of the disclosure, or mutations thereof as well as any known markers in combination with the markers, e.g. , marker genes of the disclosure, may be used in the compositions, kits, and methods of the present disclosure.
• markers are selected for as great as possible difference between the characteristic, e.g., size, sequence, composition or amount of the marker in samples comprising tumor cells and the characteristic, e.g., size, sequence, composition or amount of the same marker in control cells.
• RNA or protein amount can be at least 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 100-, 500-, 1000-fold or greater.
• "Low" RNA or protein amount can be that expression relative to the overall mean across tumor samples (e.g. , hematological tumor, e.g., myeloma) is low.
• amount of DNA e.g., copy number
• the amount is 0, 1, 2, 3, 4, 5, 6, or more copies.
• a deletion causes the copy number to be 0 or 1; an amplification causes the copy number to be greater than 2.
• the difference can be qualified by a confidence level, e.g., p ⁇ 0.05, p ⁇ 0.02, p ⁇ 0.01 or lower p-value.
• Measurement of more than one marker e.g. , a set of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 25 or more markers can provide an expression profile or a trend indicative of treatment outcome.
• the marker set comprises no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 25 markers.
• the marker set comprises 1-5, 1-10, 1-15, 1-20, 1-25, 2-5, 2-10, 2-15, 2-20, 2-25, 3-5, 3-10, 3-15, 3-20, 3-25, 4-10, 4-15, 4-20, 4-25, 5-10, 5-15, 5-20, 5-25, 6-10, 6-15, 6- 20, 6-25, 7-10, 7-15, 7-20, 7-25, 8-10, 8-15, 8-20, 8-25, 9-15, 9-20, 9-25, 10-15, 10-20, 10-25, 11-15, 11-20, 11-25, 12-15, 12-20, 12-25, 13-15, 13-20, 13-20, 14-20, 14-25, 15-20, 15-25, 16-20, 16-25, 17- 20, 17-25, 18-20, 18-25, 19-25, 20-25, 21-25, 22-25, 23-25 or 24-25 markers.
• the marker set includes a plurality of chromosome loci, a plurality of marker genes, or a plurality of markers of one or more marker genes (e.g., nucleic acid and protein, genomic DNA and mRNA, or various combinations of markers described herein).
• Analysis of treatment outcome through assessing the amount of markers in a set can be accompanied by a statistical method, e.g., a weighted voting analysis which accounts for variables which can affect the contribution of the amount of a marker in the set to the class or trend of treatment outcome, e.g., the signal-to-noise ratio of the measurement or hybridization efficiency for each marker.
• a marker set e.g., a set of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 25 or more markers, can comprise a primer, probe or primers to analyze at least one marker DNA or RNA described herein, e.g., LEF1, MAP3K7, APC, FZD2, PRKCA, RORA, CAMK2G, JUN, XPOl, ROR2, CCNDl, CTNNB1, AMOT, DVL2, LATS1, LATS2, MOB IB, NPHP4, TJP1, TJP2, WWCl, WWTRl and/or YAPl, or a complement of any of the foregoing.
• a marker set e.g., a set of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 25 or more markers
• a marker set can comprise LEF1, MAP3K7, APC, FZD2, PRKCA, RORA, CAMK2G, JUN, XPOl, ROR2, CCNDl, CTNNB1, AMOT, DVL2, LATS1, LATS2, MOB IB, NPHP4, TJP1, TJP2, WWCl, WWTRl and/or YAPl.
• a marker set for breast cancer comprises LEF1, MAP3K7, FZD2, LATS1 and/or WWCl.
• a marker set for gastric cancer comprises FZD2 and/or LATS2.
• a marker set for head and neck cancer comprises MAP3K7, JUN, ROR2, CCNDl, LATS1, MOB IB and/or NPHP4.
• a marker set for non-small cell lung cancer comprises XPOl and/or TJP1.
• a marker set for small cell lung cancer comprises LEF1, APC, PRKCA, RORA, CAMK2G, CTNNB1, AMOT, DVL2, TJP1, TJP2, WWTRl and/or YAPl. Selected marker sets can be assembled from the markers provided herein or selected from among markers using methods provided herein and analogous methods known in the art.
• a way to qualify a new marker for use in an assay of the disclosure is to correlate DNA copy number in a sample comprising tumor cells with differences in expression (e.g., fold-change from baseline) of a marker, e.g., a marker gene.
• a useful way to judge the relationship is to calculate the coefficient of determination r2, after solving for r, the Pearson product moment correlation coefficient and/or preparing a least squares plot, using standard statistical methods.
• a correlation can analyze DNA copy number versus the level of expression of marker, e.g., a marker gene.
• a gene product can be selected as a marker if the result of the correlation (r2, e.g., the linear slope of the data in this analysis), is at least 0.1- 0.2, at least 0.3-0.5, or at least 0.6-0.8 or more.
• Markers can vary with a positive correlation to response, TTP or survival (i.e., change expression levels in the same manner as copy number, e.g., decrease when copy number is decreased). Markers which vary with a negative correlation to copy number (i.e., change expression levels in the opposite manner as copy number levels, e.g., increase when copy number is decreased) provide inconsistent determination of outcome.
• Another way to qualify a new marker for use in the assay would be to assay the expression of large numbers of markers in a number of subjects before and after treatment with a test agent.
• the expression results allow identification of the markers which show large changes in a given direction after treatment relative to the pre-treatment samples.
• markers can be combined together in a set by using such methods as principle component analysis, clustering methods (e.g., k-means, hierarchical), multivariate analysis of variance (MANOVA), or linear regression techniques.
• principle component analysis e.g., clustering methods (e.g., k-means, hierarchical), multivariate analysis of variance (MANOVA), or linear regression techniques.
• clustering methods e.g., k-means, hierarchical
• MANOVA multivariate analysis of variance
• linear regression techniques e.g., linear regression techniques.
• An expression profile e.g., a composite of the expression level differences from baseline or reference of the aggregate marker set would indicate at trend, e.g., if a majority of markers show a particular result, e.g., a significant difference from baseline or reference, e.g., 60%, 70%, 80%, 90%, 95% or more markers; or more markers, e.g., 10% more, 20% more, 30% more, 40% more, show a significant result in one direction than the other direction.
• a probe set can comprise probes for assessing characteristics of markers selected from the group consisting of LEF1, MAP3K7, APC, FZD2, PRKCA, RORA, CAMK2G, JUN, XPOl, ROR2, CCND1, CTNNB1, AMOT, DVL2, LATS1, LATS2, MOB IB, NPHP4, TJP1, TJP2, WWC1, WWTR1 and YAP1.
• a probe set for breast cancer comprises probes for assessing characteristics of LEF1, MAP3K7, FZD2, LATS1 and/or WWC1.
• a probe set for gastric cancer comprises probes for assessing characteristics of FZD2 and/or LATS2.
• a probe set for head and neck cancer comprises probes for assessing characteristics of MAP3K7, JUN, ROR2, CCND1, LATS1, MOB IB and/or NPHP4.
• a probe set for non-small cell lung cancer comprises probes for assessing characteristics XPOl and/or TJP1.
• a probe set for small cell lung cancer comprises probes for assessing characteristics of LEF1, APC, PRKCA, RORA, CAMK2G, CTNNB1, AMOT, DVL2, TJP1, TJP2, WWTR1 and/or YAP1.
• the marker or set of markers of the disclosure is selected such that a significant result is obtained in at least about 20%, at least about 40%, 60%, or 80%, or in substantially all patients treated with the test agent.
• the marker or set of markers of the disclosure can be selected such that a positive predictive value (PPV) of greater than about 10% is obtained for the general population and additional confidence in a marker can be inferred when the PPV is coupled with an assay specificity greater than 80%.
• a general principle of prognostic assays involves preparing a sample or reaction mixture that may contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture.
• These assays can be conducted in a variety of ways.
• one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction.
• a sample from a subject which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support.
• the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.
• One example of such an embodiment includes use of an array or chip which contains a predictive marker or marker set anchored for expression analysis of the sample.
• biotinylated assay components can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
• biotinylation kit N- hydroxy-succinimide
• Pierce Chemicals Pierce Chemicals, Rockford, IL
• streptavidin-coated 96 well plates Piereptavidin-coated 96 well plates
• suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the marker or probe belongs.
• Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
• suitable carriers for binding antibody or antigen One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present disclosure.
• protein isolated from cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose.
• the support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody.
• the solid phase support can then be washed with the buffer a second time to remove unbound antibody.
• the amount of bound label on the solid support can then be detected by conventional means.
• the non-immobilized component is added to the solid phase upon which the second component is anchored.
• uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase.
• the detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.
• the probe when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.
• labeled with regard to the probe (e.g., nucleic acid or antibody), is intended to encompass direct labeling of the probe by coupling (i.e., physically linking) a detectable substance to the probe, as well as indirect labeling of the probe by reactivity with another reagent that is directly labeled.
• An example of indirect labeling includes detection of a primary antibody using a fluorescently labeled secondary antibody.
• marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (FET, see, for example, Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos, et al, U.S. Patent No. 4,868,103).
• FET fluorescence energy transfer
• a fluorophore label on the first, 'donor' molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy.
• the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
• determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705).
• BIOA Biomolecular Interaction Analysis
• surface plasmon resonance is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIACORETM).
• analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase.
• the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation.
• differential centrifugation marker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A.P.
• Standard chromatographic techniques also can be utilized to separate complexed molecules from uncomplexed ones.
• gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components.
• the relatively different charge properties of the marker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example through the utilization of ion-exchange chromatography resins.
• Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N.H. (1998) J.
• Gel electrophoresis may also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In some embodiments, non-denaturing gel matrix materials and conditions in the absence of reducing agent are used in order to maintain the binding interaction during the electrophoretic process. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.
• the isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction and TAQMAN® gene expression assays (Applied Biosystems, Foster City, CA) and probe arrays.
• One diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected.
• Nucleic acids comprising mutations of marker genes can be used as probes or primers.
• the nucleic acid probes or primers of the disclosure can be single stranded DNA (e.g., an oligonucleotide), double stranded DNA (e.g., double stranded oligonucleotide) or RNA.
• Primers of the disclosure refer to nucleic acids which hybridize to a nucleic acid sequence which is adjacent to the region of interest and is extended or which covers the region of interest.
• a nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of 10 to 50 consecutive nucleotides, 15 to 45 consecutive nucleotides, 15 to 75 consecutive nucleotides, 20 to 100 consecutive nucleotides, 25 to 250 consecutive nucleotides, or at least 7, 15, 20, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250 or 500 or more consecutive nucleotides of the marker and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a marker of the present disclosure.
• the exact length of the nucleic acid probe will depend on many factors that are routinely considered and practiced by the skilled artisan.
• Nucleic acid probes of the disclosure may be prepared by chemical synthesis using any suitable methodology known in the art, may be produced by recombinant technology, or may be derived from a biological sample, for example, by restriction digestion. Other suitable probes for use in the diagnostic assays of the disclosure are described herein.
• the probe can comprise a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, an enzyme co-factor, a hapten, a sequence tag, a protein or an antibody.
• the nucleic acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone.
• nucleic acid label is incorporated using SUPERTM Modified Base Technology (Nanogen, Bothell, WA, see U.S. Patent No. 7,045,610).
• the level of expression can be measured as general nucleic acid levels, e.g., after measuring the amplified DNA levels (e.g. using a DNA intercalating dye, e.g., the SYBR green dye (Qiagen Inc., Valencia, CA) or as specific nucleic acids, e.g., using a probe based design, with the probes labeled.
• TAQMAN® assay formats can use the probe-based design to increase specificity and signal-to-noise ratio.
• Such primers or probes can be used as part of a diagnostic test kit for identifying cells or tissues which express the protein, such as by measuring amounts of a nucleic acid molecule transcribed in a sample of cells from a subject, e.g., detecting transcript, mRNA levels or determining whether a gene encoding the protein has been mutated or deleted. Hybridization of an RNA or a cDNA with the nucleic acid probe can indicate that the marker in question is being expressed.
• the disclosure further encompasses detecting nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a marker protein (e.g., protein having the sequence disclosed by GenPept Accession number within Tables 9 and 10 herein), and thus encode the same protein.
• a marker protein e.g., protein having the sequence disclosed by GenPept Accession number within Tables 9 and 10 herein
• DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation.
• An allele is one of a group of genes which occur alternatively at a given genetic locus.
• Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene.
• Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals, e.g., normal samples from individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Detecting any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the disclosure.
• DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level of that gene (e.g., by affecting regulation or degradation).
• hybridizes is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
• the conditions are such that sequences at least about 70%, at least about 80%, at least about 85%, 90% or 95% identical to each other remain hybridized to each other for subsequent amplification and/or detection.
• Stringent conditions vary according to the length of the involved nucleotide sequence but are known to those skilled in the art and can be found or determined based on teachings in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6.
• stringent hybridization conditions for hybrids that are at least 10 basepairs in length includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65-70°C.
• SSC sodium chloride/sodium citrate
• a non-limiting example of highly stringent hybridization conditions for such hybrids includes hybridization in IX SSC, at about 65-70°C (or hybridization in IX SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C.
• a non-limiting example of reduced stringency hybridization conditions for such hybrids includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45°C) followed by one or more washes in 2X SSC, at about 50-60°C. Ranges intermediate to the above-recited values, e.g., at 65-70°C or at 42-50°C are also intended to be encompassed by the present disclosure.
• stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.
• a further example of stringent hybridization buffer is hybridization in 1 M NaCl, 50 mM 2-( ⁇ 1 ⁇ 1 ⁇ )6 ⁇ 1 ⁇ 3 ⁇ 68 ⁇ 1 ⁇ 3 ⁇ acid (MES) buffer (pH 6.5), 0.5% sodium sarcosine and 30% formamide.
• SSC sodium chloride/sodium citrate
• MES 2-( ⁇ 1 ⁇ 1 ⁇ )6 ⁇ 1 ⁇ 3 ⁇ 68 ⁇ 1 ⁇ 3 ⁇ acid
• SSPE lxSSPE is 0.15M NaCl, lOmM Na3 ⁇ 4P0 4 , and 1.25mM EDTA, pH 7.4
• IxSSC 0.15M NaCl and 15mM sodium citrate
• T m melting temperature
• additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents ⁇ e.g., BSA or salmon or herring sperm carrier DNA), detergents ⁇ e.g., SDS), chelating agents ⁇ e.g., EDTA), Ficoll, polyvinylpyrrolidone (PVP) and the like.
• blocking agents ⁇ e.g., BSA or salmon or herring sperm carrier DNA
• detergents ⁇ e.g., SDS
• chelating agents ⁇ e.g., EDTA
• Ficoll polyvinylpyrrolidone (PVP) and the like.
• an additional non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH 2 P0 4 , 7% SDS at about 65°C, followed by one or more washes at 0.02M Na3 ⁇ 4P0 4 , 1% SDS at 65°C, see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81: 1991-1995, (or alternatively 0.2X SSC, 1% SDS).
• a primer or nucleic acid probe can be used alone in a detection method, or a primer can be used together with at least one other primer or nucleic acid probe in a detection method.
• Primers can also be used to amplify at least a portion of a nucleic acid.
• Nucleic acid probes of the disclosure refer to nucleic acids which hybridize to the region of interest and which are not further extended.
• a nucleic acid probe is a nucleic acid which specifically hybridizes to a mutant region of a biomarker, and which by hybridization or absence of hybridization to the DNA of a patient or the type of hybrid formed can be indicative of the presence or identity of the mutation of the biomarker or the amount of marker activity.
• the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose.
• the nucleic acid probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an AFFYMETRIX® gene chip array or a SNP chip (Santa Clara, CA) or customized array using a marker set comprising at least one marker indicative of treatment outcome.
• a skilled artisan can readily adapt known RNA and DNA detection methods for use in detecting the amount of the markers of the present disclosure.
• the high density microarray or branched DNA assay can benefit from a higher concentration of tumor cell in the sample, such as a sample which had been modified to isolate tumor cells as described in earlier sections.
• a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g., at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) of a marker nucleic acid.
• polynucleotides complementary to or homologous with the marker are differentially detectable on the substrate (e.g., detectable using different chromophores or fluorophores, or fixed to different selected positions), then the levels of expression of a plurality of markers can be assessed simultaneously using a single substrate (e.g., a "gene chip" microarray of polynucleotides fixed at selected positions).
• a method of assessing marker expression is used which involves hybridization of one nucleic acid with another, the hybridization can be performed under stringent hybridization conditions.
• An alternative method for determining the amount of RNA corresponding to a marker of the present disclosure in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh e/ a/., 1989, Proc. Natl. Acad. Sci.
• amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between.
• amplification primers are from about 10 to about 30 nucleotides in length and flank a region from about 50 to about 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
• RNA does not need to be isolated from the cells prior to detection.
• a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to RNA that encodes the marker.
• a polypeptide corresponding to a marker is detected.
• an agent for detecting a polypeptide of the disclosure is an antibody capable of binding to a polypeptide corresponding to a marker of the disclosure.
• the antibody has a detectable label.
• Antibodies can be polyclonal, or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
• a variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked
• ELISA immunoabsorbant assay
• Another method for determining the level of a polypeptide corresponding to a marker is mass spectrometry.
• intact proteins or peptides e.g., tryptic peptides can be analyzed from a sample, e.g., a blood sample, a lymph sample or other sample, containing one or more polypeptide markers.
• the method can further include treating the sample to lower the amounts of abundant proteins, e.g., serum albumin, to increase the sensitivity of the method.
• liquid chromatography can be used to fractionate the sample so portions of the sample can be analyzed separately by mass spectrometry.
• the steps can be performed in separate systems or in a combined liquid chromatography /mass spectrometry system (LC/MS, see for example, Liao, et al. (2004) Arthritis Rheum. 50:3792-3803).
• the mass spectrometry system also can be in tandem (MS/MS) mode.
• the charge state distribution of the protein or peptide mixture can be acquired over one or multiple scans and analyzed by statistical methods, e.g. using the retention time and mass-to-charge ratio (m/z) in the LC/MS system, to identify proteins expressed at statistically significant levels differentially in samples from patients responsive or non-responsive to Aurora A Kinase inhibition therapy.
• mass spectrometers which can be used are an ion trap system (ThermoFinnigan, San Jose, CA) or a quadrupole time-of -flight mass spectrometer (Applied Biosystems, Foster City, CA).
• the method can further include the step of peptide mass fingerprinting, e.g. in a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry method.
• MALDI-TOF matrix-assisted laser desorption ionization with time-of-flight
• the method can further include the step of sequencing one or more of the tryptic peptides. Results of this method can be used to identify proteins from primary sequence databases, e.g., maintained by the National Center for Biotechnology Information, Bethesda, MD, or the Swiss Institute for
• Electronic apparatus including readable arrays comprising at least one predictive marker of the present disclosure is also contemplated for use in conjunction with the methods of the disclosure.
• electronic apparatus readable media refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus.
• electronic apparatus is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information.
• Examples of electronic apparatus suitable for use with the present disclosure and monitoring of the recorded information include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.
• LAN local area network
• WAN wide area network
• Extranet Internet
• PDAs personal digital assistants
• recording refers to a process for storing or encoding information on the electronic apparatus readable medium.
• microarray systems are well known and used in the art for assessment of samples, whether by assessment gene expression (e.g., DNA detection, RNA detection, protein detection), or metabolite production, for example.
• Microarrays for use according to the disclosure include one or more probes of predictive marker(s) of the disclosure characteristic of response and/or non-response to a therapeutic regimen as described herein.
• the microarray comprises one or more probes corresponding to one or more of markers selected from the group consisting of markers which demonstrate increased expression in short term survivors, and genes which demonstrate increased expression in long term survivors in patients.
• a number of different microarray configurations and methods for their production are known to those of skill in the art and are disclosed, for example, in U.S. Pat.
• a tissue microarray can be used for protein identification (see Hans et al. (2004)Blood 103:275-282).
• a phage-epitope microarray can be used to identify one or more proteins in a sample based on whether the protein or proteins induce auto-antibodies in the patient (Bradford et al. (2006) Urol. Oncol. 24:237-242).
• a microarray thus comprises one or more probes corresponding to one or more markers identified herein, e.g., those indicative of treatment outcome, e.g., to identify wild type marker genes, normal allelic variants and mutations of marker genes.
• the microarray can comprise probes corresponding to, for example, at least 2, at least 3, at least 4, at least 5, 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, at least 75, or at least 100, biomarkers and/or mutations thereof indicative of treatment outcome.
• the microarray can comprise probes corresponding to one or more biomarkers as set forth herein.
• the microarray may comprise complete marker sets as set forth herein and which may be selected and compiled according to the methods set forth herein.
• the microarray can be used to assay expression of one or more predictive markers or predictive marker sets in the array.
• the array can be used to assay more than one predictive marker or marker set expression in a sample to ascertain an expression profile of markers in the array. In this manner, up to about 44,000 markers can be simultaneously assayed for expression. This allows an expression profile to be developed showing a battery of markers specifically expressed in one or more samples. Still further, this allows an expression profile to be developed to assess treatment outcome.
• the array is also useful for ascertaining differential expression patterns of one or more markers in normal and abnormal (e.g., sample, e.g., tumor) cells. This provides a battery of markers that could serve as a tool for ease of identification of treatment outcome of patients. Further, the array is useful for ascertaining expression of reference markers for reference expression levels. In another example, the array can be used to monitor the time course of expression of one or more markers in the array.
• the disclosure allows the quantification of marker expression.
• predictive markers can be grouped on the basis of marker sets or outcome indications by the amount of the marker in the sample. This is useful, for example, in ascertaining the outcome of the sample by virtue of scoring the amounts according to the methods provided herein.
• the array is also useful for ascertaining the effect of the expression of a marker on the expression of other predictive markers in the same cell or in different cells. This provides, for example, a selection of alternate molecular targets for therapeutic intervention if patient is predicted to have an unfavorable outcome.
• the markers and marker sets of the present disclosure assess the likelihood of favorable outcome in cancer patients. Using this prediction, cancer therapies can be evaluated to design a therapy regimen best suitable for patients in either category.
• Therapeutic agents for use in the methods of the disclosure include a class of therapeutic agents known as Aurora A Kinase inhibitors, as described herein.
• the agents disclosed herein may be administered by any route, including intradermally, subcutaneously, orally, intraarterially or intravenously. In one embodiment, administration will be by the intravenous route. Parenteral administration can be provided in a bolus or by infusion.
• the concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound(s) employed, and the route of administration.
• the agent may be administered in a single dose or in repeat doses. Treatments may be administered daily or more frequently depending upon a number of factors, including the overall health of a patient, and the formulation and route of administration of the selected compound(s).
• a pharmaceutically acceptable salt of Aurora A kinase is utilized in these compositions, the salt preferably is derived from an inorganic or organic acid or base.
• suitable salts see, e.g., Berge et al, J. Pharm. Sci. 66: 1-19 (1977) and Remington: The Science and Practice of Pharmacy, 20th Ed, ed. A. Gennaro, Lippincott Williams & Wilkins, 2000.
• Nonlimiting examples of suitable acid addition salts include the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenyl-propionate, picrate, pivalate, propionate,
• Suitable base addition salts include, without limitation, ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine, V-methyl-D-glucamine, /-butylamine, ethylene diamine, ethanolamine, and choline, and salts with amino acids such as arginine, lysine, and so forth.
• alkali metal salts such as sodium and potassium salts
• alkaline earth metal salts such as calcium and magnesium salts
• salts with organic bases such as dicyclohexylamine, V-methyl-D-glucamine, /-butylamine, ethylene diamine, ethanolamine, and choline
• amino acids such as arginine, lysine, and so forth.
• basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
• lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
• dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates
• long chain halides such as
• pharmaceutically acceptable carrier is used herein to refer to a material that is compatible with a recipient subject, preferably a mammal, more preferably a human, and is suitable for delivering an active agent to the target site without terminating the activity of the agent.
• the toxicity or adverse effects, if any, associated with the carrier preferably are commensurate with a reasonable risk/benefit ratio for the intended use of the active agent.
• carrier includes any and all solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
• Remington The Science and Practice of Pharmacy, 20th Ed, ed. A. Gennaro, Lippincott Williams & Wilkins, 2000 discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof.
• any conventional carrier medium is incompatible with the compounds of the disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this disclosure.
• materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as disodium hydrogen phosphate, potassium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium hydroxide and aluminum hydroxide, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, pyrogen-free water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose, sucrose, starches such as corn starch and potato starch, cellulose and
• compositions of the disclosure can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others.
• Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions.
• Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
• compositions of this disclosure are formulated for pharmaceutical administration to a mammal, preferably a human being.
• Such pharmaceutical compositions of the present disclosure may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
• parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
• the compositions are administered orally, intravenously, or subcutaneously.
• the formulations of the disclosure may be designed to be short-acting, fast-releasing, or long-acting.
• compounds can be administered in a local rather than systemic means, such as administration (e.g., by injection) at a tumor site.
• Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art such as,
• Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3- butanediol.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
• sterile, fixed oils are examples of the acceptable vehicles and solvents that may be employed.
• injectables conventionally employed as a solvent or suspending medium.
• any bland fixed oil can be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid are used in the preparation of injectables.
• the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
• Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.
• the rate of compound release can be controlled.
• biodegradable polymers include poly(orthoesters) and poly(anhydrides).
• Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.
• compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this disclosure with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
• suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
• the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and g
• Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
• the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
• the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
• Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
• the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
• buffering agents include polymeric substances and waxes.
• Dosage forms for topical or transdermal administration of a compound of this disclosure include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
• the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
• Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this disclosure.
• the present disclosure contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body.
• Such dosage forms can be made by dissolving or dispensing the compound in the proper medium.
• Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
• the selective inhibitor of Aurora A kinase can be administered by any method known to one skilled in the art.
• the selective inhibitor of Aurora A kinase can be administered in the form of a composition, in one embodiment a pharmaceutical composition of the selective inhibitor of Aurora A kinase and a pharmaceutically acceptable carrier, such as those described
• the pharmaceutical composition is suitable for oral administration.
• the pharmaceutical composition is a tablet for oral administration, such as an enteric coated tablet. Such tablets are described in US Publication No. 2010/0310651, which is hereby incorporated by reference in its entirety.
• the pharmaceutical composition is a liquid dosage form for oral administration. Such liquid dosage forms are described in US Publication No. 2011/0039826, hereby incorporated by reference.
• these compositions optionally further comprise one or more additional therapeutic agents.
• the expressions "therapeutically effective” and “therapeutic effect” refer to a benefit including, but not limited to, the treatment or prophylaxis or amelioration of symptoms of a proliferative disorder discussed herein.
• the therapeutically effective amount or the amount of agent required to provide a therapeutic effect will vary depending upon the intended application ⁇ in vitro or in vivo), or the subject and disease condition being treated (e.g., nature of the severity of the condition to be treated, the particular inhibitor, the route of administration and the age, weight, general health, and response of the individual patient), which can be readily determined by a person of skill in the art.
• an amount of a selective inhibitor of Aurora A kinase is therapeutically effective if it is sufficient to effect the treatment or prophylaxis or amelioration of symptoms of a proliferative disorder discussed herein.
• compositions for use in the method of the disclosure may be formulated in unit dosage form for ease of administration and uniformity of dosage.
• unit dosage form refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment.
• a unit dosage form for parenteral administration may be in ampoules or in multi-dose containers.
• the treatment period during which an agent is administered is then followed by a non-treatment period of particular time duration, during which the therapeutic agents are not administered to the patient.
• This non-treatment period can then be followed by a series of subsequent treatment and non-treatment periods of the same or different frequencies for the same or different lengths of time.
• the treatment and non-treatment periods are alternated. It will be understood that the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the treatment may be stopped. Alternatively, the period of treatment in cycling therapy may continue until the patient has achieved a complete response or a partial response, at which point the period of treatment may continue for a particular number of cycles. In some embodiments, the length of the period of treatment may be a particular number of cycles, regardless of patient response. In some other embodiments, the length of the period of treatment may continue until the patient relapses.
• the frequency with which any of these therapeutic agents can be administered can be once or more than once over a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 20 days, about 28 days, about a week, about 2 weeks, about 3 weeks, about 4 weeks, about a month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, about every year, about every 2 years, about every 3 years, about every 4 years, or about every 5 years.
• an agent may be administered daily, weekly, biweekly, or monthly for a particular period of time.
• An agent may be dosed daily over a 14 day time period, or twice daily over a seven day time period.
• a certain amount of the selective Aurora A kinase can be administered daily for 7 days.
• an agent may be administered daily, weekly, biweekly, or monthly for a particular period of time followed by a particular period of non-treatment.
• a certain amount of the Aurora A kinase inhibitor can be administered daily for 14 days followed by seven days of non-treatment, and repeated for two more cycles of daily administration for 14 days followed by seven days of non-treatment.
• a certain amount of the selective Aurora A kinase inhibitor can be administered twice daily for seven days followed by 14 days of non-treatment, which may be repeated for one or two more cycles of twice daily administration for seven days followed by 14 days of non-treatment.
• a certain amount of the selective Aurora A kinase inhibitor is administered daily over a period of seven days.
• a certain amount of the Aurora A inhibitor is administered daily over a period of six days, or five days, or four days, or three days.
• a certain amount of the selective Aurora A kinase inhibitor is administered twice daily over a period of seven days, followed by a treatment-free period of 7, 14 or 21 days.
• alisertib is administered twice daily at a dose of 50mg for 7 days, followed by a 14 day treatment-free interval, in 21-day cycles.
• Suitable daily dosages of selective inhibitors of Aurora A kinase can generally range, in single or divided or multiple doses, from about 10% to about 120% of the maximum tolerated dose as a single agent. In certain embodiments, the suitable dosages are from about 20% to about 100% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages are from about 25% to about 90% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages are from about 30% to about 80% of the maximum tolerated dose as a single agent. In some other embodiments, the suitable dosages are from about 40% to about 75% of the maximum tolerated dose as a single agent.
• the suitable dosages are from about 45% to about 60% of the maximum tolerated dose as a single agent. In other embodiments, suitable dosages are about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, or about 120% of the maximum tolerated dose as a single agent.
• Suitable daily dosages of alisertib can generally range, in single or divided or multiple doses, from about 20 mg to about 120 mg per day. Other suitable daily dosages of alisertib can generally range, in single or divided or multiple doses, from about 30 mg to about 90 mg per day. Other suitable daily dosages of alisertib can generally range, in single or divided or multiple doses, from about 40 mg to about 80 mg per day. In some embodiments, the suitable dosages are from about 10 mg twice daily to about 50 mg twice daily. In some other embodiments, the suitable dosages are from about 15 mg twice daily to about 45 mg twice daily. In some other embodiments, the suitable dosages are from about 20 mg twice daily to about 40 mg twice daily.
• the suitable dosages are from about 25 mg twice daily to about 40 mg twice daily.
• suitable dosages are about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, or about 120 mg per day.
• suitable dosages are about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, or about 60 mg twice daily.
• the suitable dosage of alisertib is about 30 mg twice daily. In some embodiments, the suitable dosage of alisertib is about 35 mg twice daily. In some embodiments, the suitable dosage of alisertib is about 40 mg twice daily. In some
• the suitable dosage of alisertib is about 50 mg twice daily.
• a suitable dosage of a selective inhibitor of Aurora A kinase may be taken at any time of the day or night. In some embodiments, a suitable dosage of a selective inhibitor of Aurora A kinase is taken in the morning. In some other embodiments, a suitable dosage of a selective inhibitor of Aurora A kinase is taken in the evening. In some other embodiments, a suitable dosage of a selective inhibitor of Aurora A kinase is taken both in the morning and the evening. It will be understood that a suitable dosage of a selective inhibitor of Aurora A kinase may be taken with or without food. In some embodiments a suitable dosage of a selective inhibitor of Aurora A kinase is taken with a meal. In some embodiments a suitable dosage of a selective inhibitor of Aurora A kinase is taken while fasting.
• a first treatment period in which a first amount of the selective inhibitor of Aurora A kinase is administered can be followed by another treatment period in which a same or different amount of the same or a different selective inhibitor of Aurora A kinase is administered.
• a wide variety of therapeutic agents may have a therapeutically relevant added benefit in combination with the Aurora A kinase of the present disclosure.
• Combination therapies that comprise the Aurora A kinase of the present disclosure with one or more other therapeutic agents can be used, for example, to: 1) enhance the therapeutic effect(s) of the methods of the present disclosure and/or the one or more other therapeutic agents; 2) reduce the side effects exhibited by the methods of the present disclosure and/or the one or more other therapeutic agents; and/or 3) reduce the effective dose of the Aurora A kinase of the present disclosure and/or the one or more other therapeutic agents.
• such therapeutic agents may combine with the Aurora A kinase of the present disclosure to inhibit undesirable cell growth, such as inappropriate cell growth resulting in undesirable benign conditions or tumor growth.

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