KR101622454B1 - Predictive biomarkers for EGFR tyrosine kinase inhibitors as anticancer therapy - Google Patents

Abstract

The present invention provides an information providing method for predicting the prognosis of cancer treatment by EGFR tyrosine kinase inhibitor, a composition for predicting the prognosis of cancer treatment, and a kit. According to the present invention, since the prognosis of cancer treatment by EGFR tyrosine kinase inhibitor can be predicted by measuring the level of expression of one or more genes or proteins selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2, And can be usefully used for drug selection for treatment.

Description

{Predictive biomarkers for EGFR tyrosine kinase inhibitors as anticancer therapy for EGFR tyrosine kinase inhibitors}

The present invention relates to anticancer response predictive markers for EGFR tyrosine kinase inhibitors.

Globally, head and neck cancer is the sixth cause of cancer deaths, and about 650,000 new cases are diagnosed each year. Approximately 60% of squamous cell carcinoma of the head and neck are unresectable locally advanced and multifaceted. In multidisciplinary treatment, the majority (70%) of patients have local and / or local recurrence. Distant metastases are found in 10% of patients at diagnosis. Most recurrent or metastatic diseases are treated with single-agent chemotherapy, combined chemotherapy, or targeted therapy.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in squamous cell carcinoma of the head and is known to be associated with poor prognosis. The relationship between EGFR activation signaling pathway and tumor survival is well known by several other studies.

Target EGFR inhibitors are largely divided into monoclonal antibodies and small molecule EGFR tyrosine kinase inhibitors. The monoclonal antibody erbitux® (Cetuximab) is widely used as a therapeutic agent for metastatic head and neck cancer under the US FDA approval, and includes the small molecule EGFR tyrosine kinase inhibitors gefitinib, erlotinib, Dacomitinib and afatinib are currently in clinical trials for patients with metastatic head and neck tumors.

Cetuximab, among the EGFR-targeted antibody therapies, showed clinical anti-cancer effects in squamous cell carcinoma of the head and neck. When cetuximab was added to the 5FU and platinum preparations as a primary drug, the median overall survival was 10.1 months, which was improved compared to 7.4 months when only 5FU and platinum were used.

However, although EGFR is known to be an important therapeutic target for head and neck cancer, the general drug response rate is known to be 10-15%, which is significantly lower than that of the EGFR target therapy in lung cancer, which is 60-70%. This is because it is not known in which patient group the drug response is good. Therefore, it is required to find a biomarker that selects patients that can maximize the effect on EGFR tyrosine kinase inhibitor.

It is an object of the present invention to provide a prognostic marker for the cancer therapeutic effect of an EGFR tyrosine kinase inhibitor.

In order to achieve the above object, the present invention was to discover gene groups that differ between the drug response group and the drug non-response group for EGFR tyrosine kinase inhibitor by measuring gene expression in tumor tissues of head and neck cancer patients.

As a result, it was confirmed that gene expression of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and / or PTGS2 was highly expressed in the drug-nonreactive group for EGFR tyrosine kinase inhibitor. Therefore, it is predicted that the prognosis of cancer treatment by EGFR tyrosine kinase inhibitor is poor when the gene or protein expression level is high in the sample of the subject to be subjected to the prognosis prediction, and the diagnosis of cancer prognosis or the selection of an anticancer drug It can be utilized as information.

Therefore,

Measuring at least one gene or protein expression level selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2 from biological samples isolated from cancer patients;

Comparing the gene or protein expression level to a gene or protein expression level in a drug response group for an EGFR tyrosine kinase inhibitor; And

Determining that the prognosis of cancer treatment by the EGFR tyrosine kinase inhibitor is poor if the gene or protein expression level is higher than the gene or protein expression level in the drug response group for the EGFR tyrosine kinase inhibitor

The present invention provides an information providing method for predicting the prognosis of cancer treatment by an EGFR tyrosine kinase inhibitor.

In the present invention, " drug-reactive group " means a patient whose progression-free survival is longer than a specific period of time for a drug, and a patient whose progression-free survival period is less than the above- . The duration of the drug-resistant group and the non-drug-treated group may vary depending on the type of drug, the severity of the disease, and the state of the disease. For example, during the period of 3 months, 4 months, 5 months, And non-responders. In one embodiment of the present invention, drug-free and non-drug-free groups were divided into four months before and after progression-free survival.

When the expression level of the gene or protein is considered to be higher than that of the drug reaction group, it means that the statistical analysis is significantly higher than the average expression amount in the drug reaction group and the like.

In the present invention, when the expression level of at least one of IL6, IL8, PLA2G2A, ILlA, TNF, MMP3 and PTGS2 is higher than the gene or protein expression level in the drug response group for the EGFR tyrosine kinase inhibitor, the EGFR tyrosine kinase It is highly likely to exhibit drug resistance to inhibitors, and as a result, the prognosis of cancer treatment can be predicted to be poor. In the following examples, the tumor tissues of cancer patients were used to compare their gene levels. However, since these biomarkers can be detected even in the blood, comparison of protein levels is also possible.

In the present invention, a biological sample isolated from a cancer patient is a tumor tissue of a cancer patient, a cell or nucleic acid separated therefrom; Blood, serum, plasma or proteins isolated therefrom.

The method for measuring the expression level of a gene or protein in the above method can be carried out using a known technique, including a known process for separating mRNA or protein from a biological sample. The measurement of the level of expression of the gene is preferably a measure of the level of mRNA, and the measurement of the level of expression of the gene can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization (Sambrook et al. , Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Press (2001)).

For example, in the case of carrying out according to the RT-PCR protocol, first, total RNA is isolated from the sample-treated cells, and single-stranded cDNA is prepared using oligo dT primer and reverse transcriptase. Then, a single-stranded cDNA is used as a template and a PCR reaction is carried out using a gene-specific primer set. Then, the PCR amplification product is electrophoresed, and the band formed can be analyzed to measure changes in the expression level of the gene.

In addition, protein expression levels can be measured through a variety of immunoassay methods known in the art. But are not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), capture-ELISAs, sandwich assays, for example. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. In the present invention, the cancer may be head and neck cancer, particularly squamous cell head and neck cancer.

On the other hand, the EGFR tyrosine kinase inhibitor may be selected from gefitinib, erlotinib, dacomitinib and afatinib. In one embodiment of the present invention, the gene expression levels of inflammatory cytokines such as IL6, IL8, PLA2G2A, and PTGS2 were predictive of the prognosis of cancer treatment by Dakonitinib, but the type of EGFR tyrosine kinase inhibitor But is not limited thereto.

The present invention also provides a prognostic prediction of cancer treatment by EGFR tyrosine kinase inhibitors comprising a probe or primer having a sequence complementary to one or more genes selected from the group consisting of IL6, IL8, PLA2G2A, ILlA, TNF, MMP3 and PTGS2. ≪ / RTI >

The composition is used for predicting the prognosis of cancer treatment by confirming the expression level of the genes. The composition according to the present invention includes a probe or a primer having a sequence complementary to the gene.

The term " probe " refers to a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, which can hybridize specifically to the target nucleotide sequence, naturally occurring or artificially Lt; / RTI > The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.

The term " primer " refers to a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (i.e., four other nucleoside triphosphates and polymerase) it means. The suitable length of the primer is typically 15-30 nucleotides, although it varies with various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template.

The sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a perfectly complementary sequence to the nucleotide sequence which is a template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the gene sequence and acting as a primer. The design of such a primer can be easily carried out by a person skilled in the art with reference to the above-mentioned nucleotide sequence, for example, by using a program for primer design (for example, PRIMER 3 program).

The present invention also provides a composition for predicting the prognosis of cancer treatment by an EGFR tyrosine kinase inhibitor comprising an antibody that specifically binds to at least one protein selected from the group consisting of IL6, IL8, PLA2G2A, ILlA, TNF, MMP3 and PTGS2 to provide.

The composition is used for predicting the prognosis of cancer treatment by confirming the expression level of the proteins. The composition according to the present invention includes an antibody that specifically binds to the proteins.

Examples of the antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments (Fab '), F ; Diabody; Linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)); single chain antibody molecules; And multispecific antibodies formed from antibody fragments and the like.

Methods for producing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be prepared by injecting the mammal with one or more immunizing agents, if necessary, with an adjuvant. Usually, the immunizing agent and / or adjuvant is injected into the mammal several times by subcutaneous injection or intraperitoneal injection. Antibodies can be prepared by the hybridoma method as described in Kohler et al., Nature, 256: 495 (1975), or can be prepared by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,576) have.

Monoclonal antibodies can also be prepared, for example, by the methods described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 Can be isolated from the phage antibody library using the techniques described. The monoclonal antibody in the present invention specifically means that the part of the heavy chain and / or the light chain is identical with the corresponding sequence of the antibody belonging to the specific antibody class or subclass, Homology, the remainder of the chain (s) includes antibodies derived from other species or antibodies belonging to another antibody class or subclass, or "chimeric" antibodies that are identical or homologous to fragments of such antibodies (Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

The composition for predicting prognosis of cancer treatment according to the present invention may be included in the form of a kit.

The kit may comprise a probe, a primer or an antibody capable of measuring the level of one or more genes or protein expression levels or protein selected from the group consisting of IL6, IL8, PLA2G2A, ILIA, TNF, MMP3 and PTGS2, Is as defined above.

When the kit is applied to a PCR amplification process, a reagent such as a buffer, a DNA polymerase (for example, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis A thermostable DNA polymerase obtained from Pyrococcus furiosus (Pfu), a DNA polymerase cofactor and dNTPs, and where the kit is applied for immunoassay, the kit of the invention optionally comprises a secondary antibody and a label ≪ / RTI > Further, the kit according to the present invention may be manufactured from a number of separate packaging or compartments containing the reagent components described above.

In addition, the composition for predicting prognosis of cancer treatment of the present invention may be included in the form of a microarray.

In the microarray of the present invention, a probe, a primer, or an antibody capable of measuring the expression level of the gene or protein is used as a hybridizable array element and immobilized on a substrate. Preferred substrates may include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports have. The hybridization array elements are arranged and immobilized on the substrate, and such immobilization can be performed by chemical bonding methods or covalent bonding methods such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (e.g., ethylene glycol oligomer and diamine).

On the other hand, when the sample to be applied to the microarray of the present invention is a nucleic acid, it can be labeled and hybridized with an array element on a microarray. Hybridization conditions may vary, and detection and analysis of hybridization degree may be variously performed depending on the labeled substance.

According to the present invention, since the prognosis of cancer treatment by EGFR tyrosine kinase inhibitor can be predicted by measuring the level of expression of one or more genes or proteins selected from the group consisting of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2, And can be usefully used for drug selection for treatment.

FIG. 1 shows the expression levels of genes corresponding to inflammation-inducible cytokines between the drug-treated group (S, red) and the non-reactive group (R, blue) based on progression free survival (PFS) Lt; / RTI > (IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2)
FIG. 2 is a schematic diagram of the gene expression of inflammation-induced cytokines in the heatmap form between the drug-reactive group (S, red) and the non-reactive group (R, blue) based on the progression-free survival period of 4 months.

In order to find markers related to therapeutic prognosis according to the use of EGFR tyrosine kinase inhibitor in head and neck cancer, the present inventors carried out the expression of various genes by applying the NanoString method.

In this study, 48 patients with recurrent / metastatic squamous cell carcinoma of the head and neck were treated with dacomitinib, an EGFR tyrosine kinase inhibitor. They had a 3.9-month disease-free survival (PFS) and 8.2-month overall survival (OS), with 12 tumor tissues available for gene expression analysis. In the present study, the data in FIGS. 1 and 2 were analyzed by dividing the disease-free survival time of 4 months or longer into the drug-reactive group and the case of less than 4 months as the drug-nonreacting group. However, .

(Extraction of mRNA from tumor tissue)

MRNA was extracted from the 12 tumor tissues for gene expression analysis. Quantitative analysis was performed to quantitatively measure the degree of fragmentation of the mRNA using a bioanalyzer. The gene expression was measured with a qualitative and a quantitative analysis.

(Measurement and Analysis of Gene Expression Using Nanostrings)

NanoString® Technologies ( http://www.nanostring.com/) is a technology introduced in Nature Biotechnology 2008 (Nat Biotechnol. 2008 Mar; 26 (3): 317-25) It is a technology that quantitatively measures gene expression.

First, mRNA extracted from the sample tissue was hybridized with a capture probe, and hybridized mRNA-capture probe molecules were selected, inserted into a data analyzer, and gene expression levels were quantitatively measured. Six fluorescent substances are attached to the end of the capture probe, so that the expression value of a desired gene can be quantitatively measured by a combination thereof.

(Analysis of gene expression)

As a result of measuring gene expression through the NanoString (R) method, the results shown in Table 1 were obtained.

Unreactive group 1 Unreactive group  2 Beautification Excuse me 3 Beautification 4 Beautification Lie 5 Beautification Lie 6 Beautification Reply Reactive group  One Reactive group  2 Reactive group  3 Reactive group  4 Reactive group  5 IL1A 98.29 262.74 196.34 140.79 101.49 101.3 22.56 12.13 6.75 71.21 69.02 9.09 TNF 33.46 62.2 100.69 70.83 82.09 82.3 75.21 20.04 28.35 44.15 113.38 25.67 IL8 707.38 1677.26 503.45 197.8 252.23 373.53 185.01 93.86 93.17 158.09 36.97 48.66 IL6 417.21 146.92 105.72 2.59 5.97 56.98 73.7 1.05 2.03 52.7 2.46 2.67 PTGS2 241.02 4512.73 213.96 70.83 123.88 322.88 189.53 11.07 14.85 266.33 27.11 8.02 MMP3 151.62 229.5 178.72 109.7 122.38 240.58 43.62 7.38 8.78 121.06 39.44 13.9 PLA2G2A 1326.39 561.95 244.17 99.33 594 645.76 2451.8 45.35 15.53 431.54 69.02 96.79

Table 1 shows that the gene expression value has a positive value ranging from 0 to 5,000, and the higher the value, the higher the expression.

As a result of analyzing the data by the diagrams shown in FIGS. 1 and 2, gene expression such as IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2 as shown in FIG. 1, The disease duration was more than 4 months) and the drug non - response group (less than 4 months).

In order to see this as a picture, the heatmap of [Fig. 2] showed that gene expression of IL6, IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2 was highly expressed in the non-responding group to Dakonitinib there was.

In particular, IL6, IL8, PLA2G2A, IL1A, TNF and PTGS2 are proinflammatory cytokines, confirming that there is a correlation between drug resistance and proinflammatory cytokines in this study .

Proinflammatory cytokines have been reported to affect tumorigenesis, growth and metastasis (Klampfer L. Cytokines, inflammation and colon cancer, Current Cancer Drug Targets, 2011, 11, 451-464.) , There has been no report to date that an increase in inflammatory cytokines in the periphery of the tumor or serum is associated with the anticancer efficacy of EGFR inhibitors. This study confirmed that the increase of inflammatory cytokines in the tumor margin or serum could increase the resistance to anticancer drugs. This appears to be based on the increase in inflammatory cytokines that enhance survival signals of tumor cells and attenuate cellular apoptosis signals. Thus, increased expression of inflammatory cytokines can be used as useful biomarkers to predict the anticancer efficacy of EGFR inhibitors.

Claims (15)

Measuring at least one gene or protein expression level selected from the group consisting of IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2 from the biological sample isolated from the cancer patient;
Comparing the gene or protein expression level to a gene or protein expression level in a drug response group for an EGFR tyrosine kinase inhibitor; And
Determining that the prognosis of cancer treatment by the EGFR tyrosine kinase inhibitor is poor if the gene or protein expression level is higher than the gene or protein expression level in the drug response group for the EGFR tyrosine kinase inhibitor. A method of providing information to predict the prognosis of cancer treatment by.
The method according to claim 1,
Wherein the cancer is head and neck cancer.
The method according to claim 1,
Wherein the cancer is a squamous-head and neck cancer.
The method according to claim 1,
Wherein said EGFR tyrosine kinase inhibitor is selected from the group consisting of gefitinib, erlotinib, dacomitinib and afatinib.
The method according to claim 1,
RTI ID = 0.0 > IL6 < / RTI > gene or protein expression level.
A composition for predicting prognosis of cancer treatment by an EGFR tyrosine kinase inhibitor comprising a probe or primer having a sequence complementary to at least one gene selected from the group consisting of IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2.
The method according to claim 6,
Wherein said cancer is a head and neck cancer.
The method according to claim 6,
Wherein said cancer is squamous cervical cancer, EGFR tyrosine kinase inhibitor.
The method according to claim 6,
Wherein said EGFR tyrosine kinase inhibitor is selected from the group consisting of gefitinib, erlotinib, dacomitinib and afatinib for the prognosis of cancer treatment by EGFR tyrosine kinase inhibitor Composition.
The method according to claim 6,
A composition for predicting the prognosis of cancer therapy, which further comprises a probe or primer having a sequence complementary to the IL6 gene.
Wherein the antibody specifically binds to at least one protein selected from the group consisting of IL8, PLA2G2A, IL1A, TNF, MMP3 and PTGS2.
12. The method of claim 11,
Wherein said cancer is a head and neck cancer.
12. The method of claim 11,
Wherein said cancer is squamous cervical cancer, EGFR tyrosine kinase inhibitor.
12. The method of claim 11,
Wherein said EGFR tyrosine kinase inhibitor is selected from the group consisting of gefitinib, erlotinib, dacomitinib and afatinib for the prognosis of cancer treatment by EGFR tyrosine kinase inhibitor Composition.
12. The method of claim 11,
A composition for predicting the prognosis of cancer therapy, which further comprises an antibody that specifically binds to the IL6 protein.

Images