WO2013151026A1 - Diagnostic marker and diagnostic method for cancer cell resistant to pi3k inhibitor - Google Patents

Abstract

[Problem] To provide a diagnostic marker and a diagnostic method for determining whether or not cancer in a cancer patient is resistant to a PI3K inhibitor when the cancer patient is to be treated with the PI3K inhibitor. [Solution] It is elucidated that there is such a relationship that the expression amount of IGF1R gene is higher in a cancer cell having resistance to a PI3K inhibitor and is lower in a cancer cell that does not have resistant to the PI3K inhibitor (wherein the term "resistance" includes both acquired resistance and natural resistance within the scope thereof). The diagnostic marker comprises mRNA or cDNA for IGF1R or an IGF1R protein. The expression amount of IGF1R gene in a cancer cell or tissue collected from a cancer patient is employed as a measure. When the expression amount is low, it is determined that cancer in the patient does not have resistance to a PI3K inhibitor. From the result of this diagnosis, it is found that the administration of a PI3K inhibitor is effective for a patient in which the expression amount of IGF1R gene is low.

Description

PI3K inhibitor-resistant cancer cell diagnostic marker and diagnostic method

The present invention relates to a diagnostic marker and a diagnostic method for determining whether or not a cancer cell is resistant to a PI3K inhibitor.

Phosphatidylinositol 3-kinase (PI3K) are enzymes that phosphorylate the 3-position of the inositol phospholipids to localize in biological membranes, survival of carcinogenesis and cancer growth, play an important role for cancer, such as metastatic Therefore, it is considered as a powerful target for cancer treatment. In cancer cells, the PI3K pathway is activated, and inhibition of PI3K can suppress cancer growth (Non-patent Documents 1 and 2, etc.). Furthermore, an antitumor agent comprising such a PI3K inhibitor has been developed (Patent Document 1, etc.).
On the other hand, insulin-like growth factor 1 receptor (IGF1R) gene is a gene located upstream of PI3 gene, PI3K activity was correlated with the activation and positive IGF1R, activation of the PI3K pathway activity of IGF1R It is reported that activation of IGF1R indicates activation of the PI3K pathway (Patent Document 2). Furthermore, it is known that an inhibitor of IGF1R is an antitumor agent (Non-patent Document 3).

International Publication WO2002 / 088112 Special table 2010-538680

In general, because cancer is a diverse disease, one anticancer drug may not work for all cancers, and may or may not work depending on the cancer patient. Moreover, in many cases, there is a severe reality that the effect of the drug cannot be predicted at the time of prescribing the drug, that is, before the drug is administered, and the effect is not known until the drug is used. There can be a tragedy that people who work are good, but those who don't work suffer only side effects.
On the other hand, molecular target anticancer drugs such as PI3K inhibitors target target molecules with cancer abnormalities. It is considered. However, there is actually no significant difference in the effect of treatment with PI3K inhibitors in normal and abnormal cancers of the PI3K pathway, and genetic abnormalities such as PI3K are diagnostic markers for treatment suitability of PI3K inhibitors Don't be. Therefore, there is an urgent need to develop diagnostic markers that predict the therapeutic suitability of PI3K inhibitors.
Therefore, an object of the present application is to provide a diagnostic marker and a diagnostic method for determining whether a cancer cell is resistant (treatment resistance) to a PI3K inhibitor. Before prescribing an antitumor agent comprising a PI3K inhibitor to a patient by such a diagnostic marker and method, it is predicted in advance whether the drug will work, or an antitumor agent comprising a PI3K inhibitor is administered. It can be determined whether the patient's cancer has acquired drug resistance.

The present inventors produced anti-cancer drug-resistant cells comprising PI3K inhibitor (ZSTK474), compared gene expression of the resistant strain and the parent strain with GeneChip, and found the gene overexpressed in the resistant strain. Identified as drug resistance marker candidates. The gene was screened with siRNA to identify a gene (IGF1R gene) that is functionally involved in PI3K inhibitor resistance (data not shown). The expression of this IGF1R gene is commonly increased among the four acquired resistant strains (Example 1 described later), and it is found that PI3K inhibitors are not effective for cancer cells with high expression of the IGF1R gene. It was.
Furthermore, it was examined related sensitivity and IGF1R expression level for PI3K inhibitors of PI3K inhibitor untreated cancer cell lines, significant that higher cell expression of IGF1R gene PI3K inhibitor is unlikely effectiveness (natural resistance) Was found (Example 2 below).
That is, according to such studies by the present inventors, the expression level of the IGF1R gene is high in cancer cells resistant to both PI3K inhibitors (both acquired resistance and natural resistance), but not resistant to PI3K inhibitors. It was elucidated that there is a relationship that the expression level of the IGF1R gene is low in cancer cells.

That is, the present invention relates to a diagnostic marker for determining whether a cancer patient's cancer is resistant to a PI3K inhibitor, the diagnostic marker comprising IGF1R mRNA, cDNA or protein, It is a diagnostic marker determined by the expression level of the IGF1R gene in cancer cells or cancer tissues collected from patients.
Furthermore, the present invention is a diagnostic method for determining whether or not a cancer of a cancer patient is resistant to a PI3K inhibitor when treating the cancer patient with a PI3K inhibitor, This is a diagnostic method for determination based on the expression level of the IGF1R gene in cancer cells or cancer tissues collected from patients.
This diagnostic method may consist of the following steps.
(i) A step of collecting a tumor sample from a cancer patient and preparing a tissue section after formalin fixation and paraffin embedding. However, in the case of obtaining a cell sample, the tumor was transplanted subcutaneously into a nude mouse and removed. A step of creating a tissue section after fixing a piece (xenograft) with formalin and embedding in paraffin,
(ii) a process of preparing a control tissue section by treating a tumor sample as a control in the same manner as in (i),
(iii) a step of deparaffinizing the tissue section prepared in (i) and (ii), treating with a primary antibody specific for the IGF1R protein, staining with a secondary antibody, and quantifying the staining intensity; iv) By comparing the staining intensity obtained from the tumor sample of the cancer patient with the staining intensity obtained from the control tumor sample, it is determined whether or not the cancer of the cancer patient is resistant to the PI3K inhibitor. Judgment process

It is a Western blot which shows the expression of IGF1R protein in the cancer cell resistant to a PI3K inhibitor (Example 1). The upper row shows human brain tumor-derived cell line SF295, and the lower row shows human brain tumor-derived cell lines SNB-75, SNB-78 and human ovarian cancer cell line OVCAR3. It is a western blot which shows the expression of IGF1R protein in 39 types of cancer cells untreated with PI3K inhibitor (Example 2). It is a graph which shows the relationship between the tolerance with respect to PI3K inhibitor (ZSTK474) of 39 types of cancer cells untreated with PI3K inhibitor and the expression level of IGF1R protein. The vertical axis represents GI50 (drug concentration (M) required to suppress the growth of cancer cells by 50%), and the horizontal axis represents the expression level of the IGF1R gene (relative value, Example 2, FIG. 2). Show. The cell line marked with ○ indicates the reference cell line. It is a graph which shows the relationship between the tolerance with respect to PI3K inhibitor (NVP-BEZ235) of 39 types of cancer cells untreated with PI3K inhibitor and the expression level of IGF1R protein. The vertical axis represents GI50 (M), and the horizontal axis represents the expression level of the IGF1R gene (relative value, Example 2, FIG. 2). The cell line marked with ○ indicates the reference cell line. It is a graph which shows the relationship between the tolerance with respect to PI3K inhibitor (LY294002) of 39 types of cancer cells untreated with PI3K inhibitor and the expression level of IGF1R protein. The vertical axis represents GI50 (M), and the horizontal axis represents the expression level of the IGF1R gene (relative value, Example 2, FIG. 2). The cell line marked with ○ indicates the reference cell line. In vivo against PI3K inhibitor (ZSTK474) of 21 types of human cancer cell xenografts that have not been treated with PI3K inhibitor (xenograft tumor pieces = tumor pieces obtained by transplanting human-derived cancer cells subcutaneously into nude mice) It is a graph which shows the relationship between the treatment resistance of the cancer cell and the expression level of IGF1R protein in the living nude mouse). The vertical axis represents T / C (size of tumor in the drug-treated group (Treated) / size of tumor in the non-drug-treated group (Control)) (%), and the horizontal axis represents the expression of IGF1R protein in the transplanted tumor piece. Indicates the amount. The cell line marked with ○ indicates the reference cell line.

In cancer, gain-of-function mutations in the PIK3CA gene encoding the PI3Kα protein, which is the main PI3K isoform, and abnormalities in the PTEN gene, a phosphatase that reversely reacts with PI3K, occur frequently. In fact, PI3K inhibitors can suppress the growth of various cancers (Non-patent Document 1, etc.), but there is a significant difference in treatment responsiveness to PI3K inhibitors in both PIK3CA and PTEN abnormal and normal cancers. (Non-patent Document 2), PIK3CA and PTEN gene abnormalities do not serve as diagnostic markers for treatment responsiveness of PI3K inhibitors. Moreover, depending on the cancer cell, the PI3K inhibitor may or may not be effective, and even a cancer cell to which the PI3K inhibitor is effective may acquire drug resistance upon administration.
The diagnostic marker and diagnostic method of the present invention determine whether or not such cancer cells are resistant (both acquired resistance and natural resistance) to a PI3K inhibitor. Therefore, in cancer treatment with PI3K inhibitors, PI3K inhibition is achieved for patients with low IGF1R gene expression by measuring the expression level of IGF1R gene in cancer patients using the diagnostic marker and diagnosis method of the present invention. It is effective to administer the agent.

Examples of the PI3K inhibitor include the following compounds. These compounds have been confirmed to inhibit PI3K, while it has also been confirmed that inhibition of PI3K suppresses the growth of cancer cells (Non-patent Document 1, etc.).

As described above, the cancer to which the diagnostic marker and the diagnostic method of the present invention are applied is not particularly limited. For example, lung cancer, colon cancer, stomach cancer, breast cancer, ovarian cancer, brain tumor, kidney cancer, Examples include prostate cancer, liver cancer, pancreatic cancer, esophageal cancer, mesothelioma, and melanoma.
In order to apply the diagnostic marker and diagnostic method of the present invention, cancer cells or tissues (specimens) are collected from a subject (cancer patient). Tissue cells (cancer cells) at the target site of the subject may be collected by biopsy or the like, or if necessary, only cancer cells may be accurately collected by laser microdissection. Furthermore, when detecting by immunohistochemical method, the test subject's tumor tissue may be extract | collected and paraffin-embedded.

In the present invention, the expression level of the IGF1R gene in cancer cells is used as an index in order to determine whether the cancer is resistant (both acquired resistance and natural resistance) to the PI3K inhibitor.
Human IGF1R gene, the base sequence of SEQ ID NO: 1 (GenBank Accession NM_000875, NM_015883, its coding region is from 51 to 4154 th of the base sequence.), Or, a base sequence encoding the amino acid sequence of the following human IGF1R protein .
Human IGF1R protein consists of the amino acid sequence of SEQ ID NO: 2 (GenBank Accession AAI43722).

In the diagnosis of the present invention, if the expression level of the IGF1R gene is high, the cancer of the cancer patient is diagnosed as resistant to the PI3K inhibitor, and if the expression level is low, the cancer of the cancer patient Is not resistant to PI3K inhibitors. As a result of the diagnosis, it is effective to administer a PI3K inhibitor to a patient with a low expression level of the IGF1R gene. However, there is no specific threshold regarding the extent to which the expression level of the IGF1R gene in cancer is appropriate for treatment with a PI3K inhibitor. PI3K inhibitors are considered appropriate for cancers that do not show any expression of IGF1R, but for patients with other IGF1R expression to some extent, an appropriate threshold is set, depending on the patient's circumstances, etc. It can be said that it should be judged.

When using the diagnostic marker of the present invention to determine whether a cancer is resistant to a PI3K inhibitor (ie, whether it is therapeutically compatible with a PI3K inhibitor), this IGF1R gene The expression level may be measured as an absolute amount per cell or as a relative amount. This relative amount may be determined using the expression level of the reference gene or 18S ribosomal RNA. As an internal standard gene, for example, a gene that is expected to be expressed to the same extent in cancer cells resistant and non-resistant to PI3K inhibitors such as β-actin (GenBank Ac No. X00351) and GAPDH (NM_002046) May be used.
On the other hand, using a reference cell sample showing a high expression level of IGF1R gene and a reference cell sample showing a low expression level of IGF1R gene, the expression level of IGF1R gene in cancer cells or cancer tissues collected from cancer patients, Compared with the expression level of the IGF1R gene in these reference cell samples, the resistance of the patient to the PI3K inhibitor may be determined. In addition, when the expression level of the IGF1R gene is not more than a predetermined constant value, it is determined that the patient's cancer is not resistant to the PI3K inhibitor, and in other cases or a predetermined constant value If larger, the patient's cancer may be determined to be resistant to the PI3K inhibitor.
Such a reference cell sample may be selected from 39 types of human cancer cell lines (Table 3) examined for the IGF1R gene in Example 2. For example, as a reference indicating a high IGF1R gene expression level, the IGF1R expression level in Table 3 is 1.5 or higher, and as a reference indicating a low IGF1R gene expression level, the IGF1R expression level in Table 3 is 0.5 or lower. May be. Among these, NCI-H23 cells (ATCC No. CRL), which are available from the American Type Culture Collection (ATCC) and are relatively stable by each method and exhibit a high expression level of the IGF1R gene. PC-3 cells (ATCC No. CRL-1435) are preferably used as a reference cell sample exhibiting a low expression level of IGF1R gene.

By measuring the expression level of the IGF1R gene in the reference and the sample at the same time, or by collecting the reference cancer cell data in advance and measuring the expression level of the IGF1R gene in the sample under the same conditions, The expression level of the IGF1R gene in the specimen may be evaluated as a relative value to the expression level.
This diagnosis can be performed, for example, by calculating the following ratio X (%).
X = (AC) / (BC) × 100
(In the formula, A is the expression level of the IGF1R gene in the specimen, B is the expression level of the IGF1R gene of the reference cell sample showing a high expression level of the IGF1R gene, and C is the IGF1R gene of the reference cell sample showing a low expression level of the IGF1R gene. Represents the expression level of.
If X is, for example, 20% or less, or 10% or less, it is determined that the cancer of the sample is not resistant to the PI3K inhibitor (therapeutic compatibility with the PI3K inhibitor), and If X is otherwise, or higher than 50% or higher than 80%, the specimen's cancer is resistant to a PI3K inhibitor (therapeutic resistance that is not therapeutically compatible with a PI3K inhibitor, May be determined).
In addition, with experience in diagnosis using the diagnostic marker of the present invention, resistance to PI3K inhibitors can be obtained only by measuring the expression level of the IGF1R gene in a specimen without measuring such a standard for each test. It is also possible to determine to a certain extent whether or not (the presence or absence of therapeutic suitability).

Quantification of IGF1R gene expression can be performed by measuring the expression level of IGF1R mRNA or cDNA or the expression level of IGF1R protein.
(1) The expression level of IGF1R mRNA or cDNA is measured, for example, as follows:
Methods for quantifying mRNA expression include, for example, oligonucleotides consisting of IGF1R mRNA or its cDNA base sequence or a part of their complementary base sequences, which bind site-specifically to IGF1R mRNA or cDNA. And a method using a primer or probe containing an oligonucleotide to be used. The primer and probe may be variously modified for detecting and quantifying mRNA as long as the oligonucleotide forms a site-specific base pair with IGF1R mRNA or its cDNA.
Extraction of total RNA and mRNA from tissue cells (cancer cells) at the target site can be performed based on known methods. Reagent kits for extracting total RNA are commercially available from various manufacturers. For example, RNeasy mini kit (Qiagen) or TRIzol (Invitrogen) can be used.
For the detection of the expression level of IGF1R mRNA, any gene expression quantification method such as RT-PCR method, Northern blot method, DNA chip (manufactured by Affymetrix, etc.) can be used.

A real-time quantitative PCR method, which is one of RT-PCR methods, is preferable in that a very small amount of DNA can be detected with high sensitivity. In RT-PCR, mRNA of the IGF1R gene is reverse transcribed to produce first strand cDNA, which is used as a template for PCR amplification with primers specific to the gene. For the reverse transcription reaction, for example, High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) can be used. For the real-time quantitative PCR method, for example, TaqMan (registered trademark) Gene Expression Assays provided by Applied Biosystems can be used. Amplified products can be separated and quantified by electrophoresis or the like, but it is preferable that the quantification is performed accurately and conveniently using a real-time quantitative PCR instrument such as Applied Biosystems ABI PRISM 7000 Sequence Detection System.
In addition to real-time quantitative PCR, IGF1R mRNA can be quantified using various measurement methods (DNA array, Northern blot, ATAC-PCR method, etc.).
As a primer for PCR, a fragment consisting of about 10 to 30 base sequences sandwiching at least 50 bases, preferably 100 to 1,000 bases of a polynucleotide consisting of the base sequence of SEQ ID NO: 1 is usually used. .
As the probe, a fragment consisting of at least 15 consecutive nucleotide sequences of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is usually used. The probe base sequence is usually 15-30 bases, preferably 20-25 bases.
To measure the expression level of IGF1R mRNA or cDNA, when using a DNA array or Northern blot, when using the above probe, ATAC-PCR method, etc., when using the above primer or real-time quantitative PCR method Uses the above primer and the above probe.

(2) The expression level of IGF1R protein is measured, for example, as follows:
Examples of the method for quantifying IGF1R protein include a method using an antibody specific for IGF1R protein, and specifically, Western blotting, dot blotting, slot blotting, ELISA, and RIA. Can be detected. Moreover, the expression level can also be quantified by performing an immunohistochemical method.
As the ELISA / RIA sample, for example, the collected cell extract or serum is used as it is, or an appropriately diluted buffer is used. For the sample for Western blotting (electrophoresis), for example, the cell extract is used as it is or diluted appropriately with a buffer, and a sample buffer (Sigma) containing 2-mercaptoethanol for SDS-polyacrylamide gel electrophoresis. Used in combination with other products. As the sample for dot / slot blotting, for example, a collected cell extract or serum itself, or a solution diluted appropriately with a buffer solution, which is directly adsorbed on a membrane using a blotting apparatus or the like is used.
As antibodies reactive with IGF1R protein, e.g., Cell Signaling Technology, Inc. IGF-I Receptor β (111A9) Rabbit mAb and SantaCruz's IGF1Rβ (C20) may be a commercially available antibody of (sc-713) or the like, Moreover, you may use and make an appropriate antibody.
The antibody is directly labeled, or the antibody is used as a primary antibody for detection in cooperation with a labeled secondary antibody that specifically recognizes the primary antibody (recognizes an antibody derived from the animal from which the antibody was produced). It is done.
This label is preferably an enzyme (alkaline phosphatase or horseradish peroxidase, etc.), a fluorescent dye (Alexa680, IRDye800, etc.), or biotin (however, an operation for further binding an enzyme-labeled streptavidin to the biotin of the secondary antibody) Is mentioned. Various pre-labeled antibodies (or streptavidin) are commercially available as labeled secondary antibodies (or labeled streptavidin). In the case of RIA, an antibody labeled with a radioisotope such as ¹²⁵ I is used, and the measurement is performed using a liquid scintillation counter or the like.
The expression level of the antigen is quantified by measuring the activity of these labeled enzymes or the fluorescence intensity of the labeled fluorescent dye excited by laser light.

When the diagnostic marker and diagnostic method of the present invention are used clinically, the following immunohistochemical method (IHC method) is effective.
In the IHC method, a tumor sample of a subject is collected, and after formalin fixation and paraffin embedding, a tissue section is prepared. For those obtained cell samples, they are transplanted subcutaneously into nude mice, and tumor pieces (xenografts) are taken out and used.
This tumor section is deparaffinized, then treated with an appropriate primary antibody specific for IGF1R protein, stained with an appropriate secondary antibody, and the staining intensity is quantified. As the antibody that reacts with the IGF1R protein, the above-mentioned antibodies can be used. Alternatively, the stained section (slide) may be converted into a digital image using an Aperio Technologies Scanscope XT slide scanner or the like, and the staining intensity may be quantified using the company's Spectrum software or the like. Various tumor pieces may be prepared as a tissue microarray, and multiple samples may be measured simultaneously on one slide, or may be measured on separate slides. In that case, it is necessary to perform the staining step under the same conditions as the control sample to be compared. The treatment resistance of the PI3K inhibitor can be determined from the expression of the IGF1R protein in the obtained cancer derived from the subject. In addition, the treatment resistance of the PI3K inhibitor may be determined by comparing with the expression of control cells.

The kit used for quantifying the expression level of IGF1R mRNA or cDNA using the diagnostic marker of the present invention comprises a primer for amplifying cDNA of IGF1R gene and a heat-resistant DNA polymerase (such as Taq polymerase), and a detection Therefore, it comprises a probe to be paired with the amplification product. Examples of other consumable reagents that may be included in this kit include deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP), buffers, and the like.
The kit used for quantifying the expression level of the IGF1R protein using the diagnostic marker of the present invention includes, for example, a primary antibody specific for the IGF1R protein, a specific antibody specific to the primary antibody, and an appropriate amount. It consists of a secondary antibody labeled with an enzyme or chemical substance. Examples of other consumable reagents that may be included in this kit include enzymes, buffers, reaction reagents, and the like necessary for quantifying proteins.

The following examples illustrate the invention but are not intended to limit the invention.
Example 1
In this embodiment, prepared PI3K inhibitor (ZSTK474) resistant cancer cells was investigated the relationship between the development of tolerance and IGF1R gene for PI3K inhibitors in cancer cells that have acquired PI3K inhibitor-resistant.
Human brain tumor-derived cell lines SF295, SNB-75, SNB-78 and human ovarian cancer cell lines OVCAR3 (or more, the National Cancer Center Developmental Therapeutics available from the Program) of the 1 ~ 10μM ZSTK474 (Zenyaku Co., Ltd.) in the presence Under this condition, the cells were cultured for 1 year or longer to obtain cells that grew in the presence of 10 μM ZSTK474. Since normal cancer cells cannot grow in the presence of ZSTK474 (PI3K inhibitor), it can be said that these cells have acquired resistance to ZSTK474. These ZSTK474 resistant cancer cell lines are referred to as SF295R, SNB-75R, SNB-78R, and OVCAR3R, respectively.
Next, these cells are seeded into a 96-well plate, and the next day, 5 types of ZSTK474 of 10 ^-8 M, 10 ^-7 M, 10 ^-6 M, 10 ^-5 M and 10 ^-4 M are added thereto. After further incubation for 48 hours, the cells were fixed with trichloroacetic acid. Cell proliferation was measured by the sulfurhodamine B (SRB) assay (J Natl Cancer Inst. 1990 Jul 4; 82 (13): 1107-12.) And GI50 (drug concentration required to inhibit cell proliferation by 50%) Calculated. The results are shown in the table below.

Cancer cells that acquired resistance to PI3K inhibitors were each 12-120 times more resistant than the parent strain (drug-untreated).

Furthermore, in order to investigate whether the drug resistance acquired by long-term exposure to ZSTK474 is reversible, SF295R cells (ZSTK474 resistant cells) were removed in the absence of ZSTK474 for 3 days (DF_day3), 14 days (DF_day14), 3 The change in the degree of resistance after culturing for months (DF∞) was measured. As a result, it was found that the resistance level, which was originally about 38 times that of the parent strain, decreased with time, and DF∞ almost returned to the original sensitivity (that is, reversible resistance). The results are shown in Table 2.

Next, the various cancer cells were collected from the culture dish using a scraper, and total RNA was extracted from the cells using an RNeasy mini kit (manufactured by Qiagen) according to the user manual attached to the kit. Using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), 1 μg of this total RNA was reverse transcribed in 20 μl of a reverse transcription reaction solution to prepare first strand cDNA.
Using 1 μl of the obtained first strand cDNA, real-time monitoring of IGF1R cDNA amplification was performed using TaqMan (registered trademark: Applied Biosystems) Gene Expression Assays (Assay ID: Hs00609566_m1) and Applied Biosystems ABI PRISM 7000. The PCR reaction is performed under the following reaction conditions, and the sequence of the primers (18 bases each) used in the PCR amplification reaction is not disclosed, but amplicons containing probe sequences are amplified. The following IGF1R probe is used as a probe. As a control, the expression level of 18S ribosomal RNA was simultaneously measured, and the expression level of IGF1R in each sample was normalized with the expression level of 18S ribosomal RNA. Furthermore, a value obtained by normalizing the expression level of IGF1R in each resistant strain sample with the expression level of IGF1R in the SF295 parent strain obtained here was defined as the IGF1R expression level (relative value).
Probe for IGF1R:
5'-FAM- CCATCTTCGTGCCCAGACCTGAAAG-TAMRA-3 '
(* FAM represents 6-carboxyfluorescein, and TAMRA represents carboxytetramethylrhodamine. The base sequence of this probe corresponds to positions 2236 to 2260 of SEQ ID NO: 1.)
Reaction conditions:
95 ° C 10 minutes 1 cycle 95 ° C 15 seconds 60 ° C 1 minute 40 cycles

The results are shown in the table below. It can be seen that by culturing SF295R cells in the absence of ZSTK474, the expression of IGF1R mRNA also decreases as the degree of resistance decreases.

In addition, the above cancer cells were collected and Lysis buffer (10 mmol / L Tris-HCl (PH 7.4), 50 mmol / L NaCl, 0.5% w / v NP40, 0.1% w / v SDS, 50 mmol / L Sodium fluoride, 30 mmol / L sodium pyrophosphate, 50 mmol / L sodium orthavanadate, 5 mmol / L EDTA, 0.1 trypsin inhibitor unit / ml aprotinin, 1 mmol / L phenylmethylsulfonyl fluoride) and suspend the ice using Diagenode Bioruptor. Cells were disrupted by medium sonication.
The protein concentration of the cell extract was quantified using a protein assay kit manufactured by Pierce, and the cell extract corresponding to 10 μg protein was developed by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The results are shown in FIG.
That is, overexpression of IGF1R protein was observed in all resistant cell lines (Resistance) prepared from four types of cancer cells compared to the parent line (Parent). In addition, when SF295R cells were cultured in the absence of ZSTK474, the expression of IGF1R protein also decreased with a decrease in resistance.
From the above results, the resistance to PI3K inhibitor (acquired resistance) is related to the expression level of IGF1R, and a significant relationship was found that the higher the expression level of the IGF1R gene, the more resistant the PI3K inhibitor is. .

Example 2
In this example, the relationship between ZSTK474 resistance (natural resistance) and IGF1R gene expression in cancer cells not treated with a PI3K inhibitor was examined.
39 types of human cancer cell lines were prepared as non-treated PI3K inhibitor cancer cells, cell extracts were prepared from each cancer cell line in the same manner as in Example 1, and GI50 was measured.
Further, the expression level of IGF1R in each cell extract, a primary antibody (SantaCruz's IGF1Rβ (C20) (sc-713)), using a secondary antibody labeled with Alexa488 fluorescent dye, a Licor's Odyssey Measured and quantified. Odyssey supplied excitation light to the fluorescently labeled sample and detected the fluorescence emitted from the fluorescent dye. The fluorescence intensity of the IGF1R band in each detected sample was quantified from an image obtained by scanning the membrane labeled with Alexa488 fluorescent dye with IGF1R expression by Western blotting using Odyssey. Using a mixture of samples obtained from cancer cell lines at the same ratio as a reference sample, the final quantification of the expression level of IGF1R in each sample is the intermediate value of the quantification values obtained by three independent experiments. Value (relative value). The results are shown in the table below and FIG. In the table, * indicates a reference cell line.
This table also shows that the PI3K inhibitor may or may not work depending on the cancer cells of each patient, not the type of cancer cells.
From these expression levels, X (%) = (expression level of IGF1R gene in specimen−expression level of IGF1R gene in PC-3 cell) / (expression level of IGF1R gene in NCI-H23 cell−PC-3 cell) Expression level of IGF1R gene) × 100.

The same measurement was performed using other PI3K inhibitors NVP-BEZ235 and LY294002 instead of the PI3K inhibitor ZSTK474. The relationship between their resistance to PI3K inhibitors (GI50) and the expression level of the IGF1R gene is shown in FIGS. From these figures, it can be seen that the higher the expression of the IGF1R gene, the less effective the PI3K inhibitor is (that is, the resistance to the PI3K inhibitor). In addition, cell lines with an IGF1R expression level of X of 20% or less, particularly 10% or less, have a low GI50 and a low resistance to PI3K inhibitors.

Example 3
In this example, the expression of IGF1R protein in an in vivo tumor sample was detected using an immunohistochemical method (IHC method), and the relationship with ZSTK474 resistance (antitumor effect) in vivo was examined. Such an in vivo test can be said to be a condition closer to a clinical sample than an analysis result (Example 2) using human cancer cells cultured in a test tube.
Of the 39 human cancer cell lines of Example 2, 21 cell lines shown in Table 4 were transplanted subcutaneously into nude mice to form tumors (xenografts), and ZSTK474 (200 mg / kg) was given daily to nude mice ( (Monday to Saturday) Orally administered, and measured for the length (L) x width (W) of the tumor after 2 weeks of administration, and the tumor size (TV) was obtained by the following formula.
TV = L × ^W 2/2
Antitumor effect is expressed as T / C (Treated tumor size / Treated tumor size in control group) (%). The smaller T / C, the smaller the tumor regression due to drug treatment (That is, the antitumor effect is high).
On the other hand, a tumor sample before administration of ZSTK474 was extracted, fixed with formalin, and embedded in paraffin. These were prepared as a tissue microarray (TMA). TMA is a technique that allows multiple samples to be measured simultaneously on a single section (slide). Create a tumor sections (4 [mu] M thick) from TMA, after deparaffinization treatment with primary antibody (SantaCruz's IGF1Rβ (C20) (sc-713)), were stained with Dako Corporation Envision + kit. The sections (slides) were digitally imaged with a Scanscope XT slide scanner manufactured by Aperio Technologies, and the staining intensity (corresponding to the expression level of IGF1R) was quantified using the company's software (Color Deconvolution Algorithm).
Also, from these staining intensities, X (%) = (expression amount of IGF1R gene in specimen−expression amount of IGF1R gene in PC-3 cell) / (expression amount of IGF1R gene in NCI-H23 cell−PC-3 cell) Expression level of IGF1R gene) × 100.
The results are shown in the following table and FIG. In the table, * indicates a reference cell line.

As a result, the higher the expression of the IGF1R gene, the higher the T / C and the lower the antitumor effect (that is, the higher the resistance to ZSTK474). Moreover, it can be said that the cell line having an IGF1R expression level of X of 20% or less, particularly 10% or less, has a low T / C and a particularly low resistance to a PI3K inhibitor.

Claims (15)

1. A diagnostic marker for determining whether or not a cancer of a cancer patient is resistant to a PI3K inhibitor, the diagnostic marker comprising IGF1R mRNA, cDNA or protein, and cancer collected from the cancer patient A diagnostic marker that is determined by the expression level of the IGF1R gene in cells or cancer tissues.
2. Using a reference cell sample showing a high expression level of IGF1R gene and a reference cell sample showing a low expression level of IGF1R gene, the expression level of IGF1R gene in cancer cells or cancer tissues collected from cancer patients is used as a reference. The diagnostic marker of Claim 1 which determines the tolerance with respect to the PI3K inhibitor of the said patient's cancer compared with the expression level of the IGF1R gene of a cell sample.
3. The determination of whether a cancer patient's cancer is resistant to a PI3K inhibitor is performed at the ratio X (%) of the following formula,
   X = (AC) / (BC) × 100
   (Where A is the expression level of the IGF1R gene in the specimen, B is the expression level of the IGF1R gene of the reference cell sample showing a high expression level of the IGF1R gene, and C is the IGF1R gene of the reference cell sample showing a low expression level of the IGF1R gene) Represents the expression level of.
   The diagnostic marker according to claim 2, wherein if X is 20% or less, it is determined that the cancer is not resistant to the PI3K inhibitor.
4. As a reference cell sample showing a high expression level of IGF1R gene, NCI-H23 cell (ATCC No. CRL-5800) and as a reference cell sample showing a low expression level of IGF1R gene, PC-3 cell (ATCC No. CRL- The diagnostic marker according to claim 2 or 3, wherein 1435) is used.
5. The diagnostic marker according to claim 1, comprising IGF1R mRNA or cDNA, comprising: (i) from 10 to 30 nucleotide sequences sandwiching a polynucleotide portion of at least 50 nucleotides of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 A diagnostic marker using a pair of primers and / or (ii) a probe comprising at least 15 consecutive nucleotide sequences of a polynucleotide comprising the 51st to 4154th nucleotide sequence of SEQ ID NO: 1.
6. The diagnostic marker according to claim 1, comprising an IGF1R protein, wherein the diagnostic marker uses an antibody specific for the IGF1R protein.
7. A diagnostic method for determining whether or not a cancer of a cancer patient is resistant to a PI3K inhibitor, wherein the determination is based on the expression level of an IGF1R gene in cancer cells or cancer tissue collected from the cancer patient Diagnosis method.
8. Using a reference cell sample showing a high expression level of IGF1R gene and a reference cell sample showing a low expression level of IGF1R gene, the expression level of IGF1R gene in cancer cells or cancer tissues collected from cancer patients is used as a reference. The diagnostic method according to claim 7, wherein the resistance of the patient to the PI3K inhibitor is determined by comparing with the expression level of the IGF1R gene in the cell sample.
9. The determination of whether a cancer patient's cancer is resistant to a PI3K inhibitor is performed at the ratio X (%) of the following formula,
   X = (AC) / (BC) × 100
   (Where A is the expression level of the IGF1R gene in the specimen, B is the expression level of the IGF1R gene of the reference cell sample showing a high expression level of the IGF1R gene, and C is the IGF1R gene of the reference cell sample showing a low expression level of the IGF1R gene) Represents the expression level of.
   The diagnostic method according to claim 8, wherein if X is 20% or less, it is determined that the cancer is not resistant to the PI3K inhibitor.
10. NCI-H23 cells (ATCC No. CRL-5800) are used as a reference indicating high expression level of IGF1R gene, and PC-3 cells (ATCC No. CRL-1435) are used as reference indicating low expression level of IGF1R gene. The diagnostic method according to claim 8 or 9.
11. The diagnostic method according to claim 7, wherein the expression level of IGF1R mRNA or cDNA is used as an index indicating the expression level of the IGF1R gene, and (i) a polynucleotide of at least 50b of the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 A pair of primers consisting of 10 to 30 base sequences sandwiching the portion, and / or (ii) a probe consisting of at least 15 consecutive base sequences of a polynucleotide consisting of the 51st to 4154th base sequences of SEQ ID NO: 1 Diagnostic method used.
12. 8. The diagnostic method according to claim 7, wherein the expression level of the IGF1R protein is used as an index indicating the expression level of the IGF1R gene, and the protein is detected using an antibody specific for the IGF1R protein.
13. 8. The diagnostic method according to claim 7, comprising the following steps.
    (i) A step of collecting a tumor sample from a cancer patient and preparing a tissue section after formalin fixation and paraffin embedding. However, in the case of obtaining a cell sample, the tumor was transplanted subcutaneously into a nude mouse and removed. A step of creating a tissue section after fixing a piece (xenograft) with formalin and embedding in paraffin,
    (ii) a process of preparing a control tissue section by treating a tumor sample as a control in the same manner as in (i),
    (iii) a step of deparaffinizing the tissue section prepared in (i) and (ii), treating with a primary antibody specific for the IGF1R protein, staining with a secondary antibody, and quantifying the staining intensity; iv) By comparing the staining intensity obtained from the tumor sample of the cancer patient with the staining intensity obtained from the control tumor sample, it is determined whether or not the cancer of the cancer patient is resistant to the PI3K inhibitor. Judgment process
14. A diagnostic marker kit for determining whether a cancer of a cancer patient is resistant to a PI3K inhibitor, a primer for amplifying cDNA of an IGF1R gene and a heat resistant DNA polymerase, and / or the cDNA Alternatively, a kit comprising a probe to be paired with the amplification product.
15. A diagnostic marker kit for determining whether a cancer of a cancer patient is resistant to a PI3K inhibitor, a primary antibody specific for the IGF1R protein, a specific antibody for the primary antibody, and a label A kit comprising a secondary antibody.

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