JP6635374B2 - Irinotecan sensitivity prediction method using phosphorylated RB protein as an index in cancer - Google Patents

Description

  The present invention relates to a method for predicting irinotecan sensitivity using phosphorylated RB protein as an index in cancer.

  Cancer is one of the leading causes of illness and its life expectancy is greatly reduced when it occurs. Above all, advanced cancers and metastatic cancers progress rapidly, so that it is required to accurately grasp the state of cancer lesions and to take appropriate measures promptly. Various treatments are currently being attempted for such advanced and metastatic cancers, including surgery, radiation therapy, chemotherapy, and immunotherapy. As a treatment method adopted in many medical sites, there is chemotherapy using an antitumor agent.

  In particular, the antineoplastic drug irinotecan (irinotecan hydrochloride) is a central drug that has been used for approximately 20 years in clinical settings where chemotherapy for advanced cancer is performed, and is used for colorectal cancer (colorectal cancer and colorectal cancer). ), Stomach cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, pediatric malignant solid tumors, and various other cancers. In particular, in the treatment of metastatic cancer such as metastatic colorectal cancer, it is one of the first-line drugs. By prescribing irinotecan to patients suffering from the above-mentioned various cancers, it is expected that the cancer-caused condition does not progress for a longer period, and that it is possible to obtain a longer overall survival time. In the treatment of various cancers including metastatic colorectal cancer and the like, it has become one of the antitumor drugs that have become a reliable source (Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3). However, not all patients prescribed irinotecan enjoy satisfactory clinical outcomes.

  Above all, advanced cancers require rapid and effective treatment because the disease progresses quickly. For such treatment, it is required to appropriately select a more effective antitumor drug. The use of less effective anti-tumor drugs in the treatment of advanced cancer may not produce sufficient therapeutic effects and may lead to exacerbation of the condition, such as resistance to the drug or metastasis to other organs, resulting in a survival time Disadvantages, such as shortening the time.

  Therefore, the effectiveness of the antitumor drug on the cancer tissue is grasped in advance so that the highly effective antitumor drug adapted to the condition of the cancer tissue in the advanced cancer patient can be appropriately selected and used for the treatment. That is very useful. In particular, since chemotherapy is a treatment that exerts a cancer growth inhibitory effect by acting on a susceptibility factor, if the factor is identified as a susceptibility marker, it can be used for patients with early diagnosis and susceptibility using it as an index. Early treatment and increase the likelihood of a cure or long-term survival. In addition, in patients with low or no sensitivity, it is possible to avoid unnecessary treatment and to provide an opportunity to early select another more appropriate treatment.

  Therefore, for various antitumor drugs used for cancer treatment, markers for predicting the efficacy of cancer treatment using the drugs have been investigated and used for selection of therapeutic drugs. Also for irinotecan, it is desired to identify a marker for predicting the efficacy of cancer treatment and establish a method for using it.

  It has been reported that irinotecan has a topoisomerase I (Top I) inhibitory effect and has an activity of preventing Top I from religating a nicked DNA strand during DNA replication (Non-patent Document 4). Non-Patent Document 5). Therefore, it was assumed that the high expression level of Top I was related to the drug efficacy of irinotecan, and that the expression level of Top I was an effect predictor (Non-patent Document 6). And in a large prospective study, the UK MRC FOCUS Trial for Advanced Colorectal Cancer Patients, moderate or high Top I expression was more pronounced in patients using irinotecan in addition to 5-fluorouracil. It has been reported that it was associated with long-term survival (Non-Patent Document 7).

  However, in the CAIRO test, which was also a prospective study performed on patients with advanced colorectal cancer, no correlation was found between irinotecan responsiveness and Top I expression (Non-Patent Document 8, Non-Patent Document 8). 9). Furthermore, there is another report that TopI expression was maintained in cells that acquired resistance to SN38 (active metabolite of irinotecan) (Non-Patent Document 10).

  Thus, there are multiple conflicting reports on the relationship between irinotecan responsiveness in chemotherapy for advanced cancer and Top I expression, and Top I expression predicts the clinical efficacy of irinotecan for advanced cancer Is not a marker for this. Despite various attempts and studies, a marker for irinotecan sensitivity in cancer has not been clarified yet.

  Irinotecan is used in the treatment of various cancers such as colorectal cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, and pediatric malignant solid tumor. Despite widespread use and continued research and exploration, the search for susceptibility markers to predict the efficacy of irinotecan in these cancers and to enhance the therapeutic potential of irinotecan has yet to be fully accomplished. I can't say that. And there is still a strong need for identification of the sensitivity marker and establishment of a method for predicting or monitoring the response of the patient to irinotecan in cancer using the sensitivity marker.

Fuchs C, et al. Cancer Treat Rev 2006; 32: 491-503. Colucci G, et al. J Clin Oncol 2005; 23: 4866-75. Peeters M, et al. J Clin Oncol 2010; 28: 4706-13. Hsiang YH, et al. J Biol Chem 1985; 260: 14873-8. Hsiang Y, et al. Cancer Res 1988; 48: 1722-6. Gilbert DC, et al. Br J Cancer 2012; 106: 18-24. Braun MS, et al. J Clin Oncol 2008; 26: 2690-8. Koopman M, et al. Lancet 2007; 370: 135-42. Koopman M, et al. Eur J Cancer Suppl 2009; 7: 321-2. Gongora C, et al. Cancer Biol Ther 2008; 7: 822-32.

  The problems to be solved by the present invention include colorectal cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell cancer, non-Hodgkin's lymphoma, pediatric malignant solid tumor, etc. To predict, monitor, or diagnose a response to irinotecan in various cancers, and to provide a kit for use in the method.

  The present inventors have established a sensitivity marker for irinotecan, and at the same time, irinotecan is originally effective for colorectal cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell cancer In the treatment of cancer patients such as non-Hodgkin's lymphoma and pediatric malignant solid tumor, intensive research has been conducted to provide an early diagnosis of irinotecan susceptibility in the cancer and to provide an opportunity for cancer cure by more appropriate treatment.

  Among them, the present inventors have revealed that the sensitivity of irinotecan in colorectal cancer has no relationship with the expression level of Top I, which has been previously pointed out as a potential irinotecan sensitivity marker. . On the other hand, the sensitivity of irinotecan in colorectal cancer was found to correlate with the phosphorylation status of RB (retinoblastoma gene), a central molecule that controls the cell cycle and cell proliferation of cancer cells, at specific sites. The present inventors have newly revealed that the sensitivity of cancer such as colorectal cancer to irinotecan can be appropriately predicted by evaluating the phosphorylation state of a specific site of RB protein in such cancer tissues as a marker.

  In addition, the present inventors have developed various types of irinotecan such as colorectal cancer (colorectal cancer), stomach cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, pediatric malignant solid tumor, etc. To evaluate whether the susceptibility of cancer to cancer can be predicted by evaluating the histopathology of the patient, we evaluated the phosphorylation status of a specific site of RB protein in the cancer lesion tissue of colorectal cancer patients. Similarly, in retrospective studies by immunohistochemical staining using clinical samples in cases, it was newly found that irinotecan sensitivity can be well evaluated by the phosphorylation status of specific sites of RB protein. Furthermore, by investigating the mechanism using siRNA, it was also found that in the colorectal cancer cells, the sensitivity of irinotecan is affected by controlling the phosphorylation state of the RB protein, and the identification of the RB protein in the colorectal cancer tissues of patients The mechanism of action confirms that the sensitivity to irinotecan can be predicted by evaluating the phosphorylation status of the site.

  As a result of the above various studies, irinotecan in cancer patients such as colorectal cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, and pediatric malignant solid tumor Sensitivity can now be properly predicted, monitored, and diagnosed by assessing the phosphorylation status of RB protein in cancer tissues of patients, and it has now become clear that colorectal cancer (colorectal and rectal cancer), gastric cancer, A rational, highly effective, new method that uses phosphorylated RB protein in various cancers such as pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, pediatric malignant solid tumor, etc. The invention of a novel method for predicting irinotecan sensitivity was completed.

  A first means of the present invention for solving the above-mentioned problems is a first step of measuring the amount of phosphorylated RB protein in an extracted specimen sample obtained from a region containing cancerous tissue of a patient and a control sample. And a second step of comparing the amount of phosphorylated RB protein measured in the excised specimen sample and the control sample. A method for predicting or monitoring the response to irinotecan in a patient's cancer. Various cancers such as colorectal cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, and pediatric malignant solid tumor are targeted. Then, according to the above method, the amount of the phosphorylated RB protein measured in the extirpated sample obtained from the region containing the cancer tissue of the patient is significantly greater than the amount of the phosphorylated RB protein measured in the control sample. If it is confirmed that the cancer tissue is sensitive to irinotecan, it is expected to show responsiveness when irinotecan is administered, if there is no significant difference and the expression of phosphorylated RB protein is insufficient On the other hand, the cancer tissue has no sensitivity to irinotecan, and is expected to show no response even when irinotecan is administered.

  A second means of the present invention for solving the above-mentioned problem is characterized in that the phosphorylation site of the phosphorylated RB protein is Ser780 and / or Ser807 / 811 of the RB protein. This is the method of the first means.

  A third means of the present invention for solving the above problems is that the amount of the phosphorylated RB protein is selected from the group consisting of Western blotting, immunohistochemical staining, immunofluorescence, immunoassay, ELISA or mass spectrometry. A method according to the first or second means of the present invention, characterized in that the measurement is performed using a measurement method performed.

  A fourth means of the present invention for solving the above problems is that the amount of the phosphorylated RB protein is measured using a phosphorylation site-specific anti-RB antibody or a phosphorylation site-specific anti-RB antibody fragment. A method according to any one of the first to third means of the present invention, characterized in that:

  A fifth means of the present invention for solving the above-mentioned problem is that the phosphorylation site-specific anti-RB antibody detects phosphorylation of Ser780 and / or Ser807 / 811 of RB protein. This is the method of the fourth means of the present invention.

  The sixth means of the present invention for solving the above-mentioned problem further comprises the following steps: Western blotting, immunohistochemistry, and the extraction of BCRP (BREAST CANCER RESISTANCE PROTEIN) from an extirpated sample obtained from a region containing cancer tissue of a patient. Any one of the first to fifth means of the present invention, comprising a step of measuring using a measuring method selected from the group consisting of staining, immunofluorescence, immunoassay, ELISA or mass spectrometry. It is a method of description.

  A seventh means of the present invention for solving the above-mentioned problem is any one of the first to sixth means of the present invention, which comprises a phosphorylation site-specific anti-RB antibody or a phosphorylation site-specific anti-RB antibody fragment. A kit for predicting or monitoring a response to irinotecan in a cancer of a patient according to the method described in 1 or 2.

  An eighth means of the present invention for solving the above-mentioned problem is that the phosphorylation site-specific anti-RB antibody or the phosphorylation site-specific anti-RB antibody fragment comprises a RB protein Ser780 and / or Ser807 / 811 phosphorylation. It is a kit of the seventh means of the present invention, which detects oxidation.

  A further means of the present invention for solving the above-mentioned problem is a first step of measuring the amount of phosphorylated RB protein in an extracted specimen sample obtained from a region containing a cancer tissue of a patient and a control sample. A second step of comparing the amount of phosphorylated RB protein measured in the excised specimen sample and the control sample, and a response to irinotecan in the cancer of the patient. Further, a further means of the present invention for solving the above-mentioned problems is characterized in that the phosphorylation site of the phosphorylated RB protein is Ser780 and / or Ser807 / 811 of the RB protein. This is the method. Further, a further means of the present invention for solving the above-mentioned problems is that the amount of the phosphorylated RB protein is selected from the group consisting of Western blotting, immunohistochemical staining, immunofluorescence, immunoassay, ELISA or mass spectrometry. The diagnostic method according to any one of the above means, wherein the measurement is performed using a selected measurement method. Further, a further means of the present invention for solving the above problems is that the amount of the phosphorylated RB protein is measured using a phosphorylation site-specific anti-RB antibody or a phosphorylation site-specific anti-RB antibody fragment. A diagnostic method according to any one of the above means. Further, a further means of the present invention for solving the above problems is that the phosphorylation site-specific anti-RB antibody detects phosphorylation of Ser780 and / or Ser807 / 811 of RB protein. A diagnostic method according to any one of the above means. Further, a further means of the present invention for solving the above-mentioned problems is that the expression of BCRP (breast cancer resistance protein) can be further analyzed by Western blotting, immunohistochemistry, The diagnostic method of any one of the above means, comprising a step of measuring using a measuring method selected from the group consisting of chemical staining, immunofluorescence, immunoassay, ELISA or mass spectrometry.

  Response of the patient of the present invention to irinotecan in various cancers such as colorectal cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, and pediatric malignant solid tumor By using a method and a kit for predicting or monitoring, the phosphorylation state of a specific site of RB protein in a pathological tissue of a patient suffering from those cancers was measured, and a specific hyperphosphorylation was confirmed in the tissue. If irinotecan is susceptible to irinotecan and specific hyperphosphorylation has not occurred, it is evaluated as having irinotecan resistance, so that the efficacy of irinotecan in the treatment of these cancers can be reasonably predicted. , Monitoring and diagnosing effects. Furthermore, by confirming the expression of BCRP, it became possible to eliminate false positives in the evaluation of irinotecan efficacy. And, by using the method and the kit of the present invention, colorectal cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell cancer, non-Hodgkin's lymphoma, pediatric malignant solid tumor It has become possible to examine in advance the positive rate of phosphorylation at the serine site of RB protein in the tissues of patients suffering from various cancers such as tumors. And, for patients suffering from susceptible cancer, it provides an opportunity to prescribe irinotecan appropriately earlier than before, and for patients suffering from insensitive cancer, useless irinotecan Avoiding prescriptions and providing the opportunity to choose alternative treatments earlier, greatly improving the probability of successful treatment and cure of those cancers and greatly improving the quality of life of cancer patients .

FIG. 2 shows the results of Western blot analysis confirming the expression of Top I (topoisomerase-I) in various human colon cancer cell lines. 1 is a graph showing the relationship between the doubling time in various human colon cancer cell lines and the IC50 value of SN38 in the colon cancer cell lines. FIG. 9 shows the results of Western blot analysis confirming the expression of phosphorylated RB protein and RB protein in various human colon cancer cell lines. FIG. 9 shows the results of Western blot analysis confirming the expression of BCRP (breast cancer resistance protein) in various human colon cancer cell lines. It is a typical example of the result of immunohistochemical staining of phosphorylated RB protein in a patient suffering from colorectal cancer. 1 is a representative example of the results of immunohistochemical staining of Ki-67 (MIB-1) in a patient suffering from colorectal cancer. It is a figure which shows the labeling index (LI) of Ki-67 (MIB-1) in the patient who suffered from colorectal cancer. FIG. 9 shows the results of Western blot analysis confirming suppression of expression of CDK2, CDK4, CDK6, and CDK4 / 6 in various human colon cancer cell lines treated with siRNA against cyclin-dependent kinase. FIG. 2 shows the results of Western blot analysis confirming the expression of phosphorylated RB protein and RB protein in various human colon cancer cell lines treated with siRNA against cyclin-dependent kinase. Fig. 9 shows analysis results of cell cycle progression of SW620 cells and SW480 cells by treating SN38 for 72 hours. Data show mean ± SD. ** indicates p <0.01. FIG. 9 shows analysis results of cell cycle progression of SW620 cells and SW480 cells treated with siRNA against cyclin-dependent kinase by treatment with SN38 at 5 nM. Data show mean ± SD. * Indicates p <0.05, ** indicates p <0.01. FIG. 9 shows analysis results of cell viability when SW620 cells were treated with SN38 at various concentrations by siNC or siCDK4 / 6 treatment. Data show mean ± SD. * Indicates p <0.05, ** indicates p <0.01. FIG. 10 shows the results of measuring the IC50 value of SN38 by treating SW620 cells with siNC or siCDK4 / 6. Data show median ± SD. * Indicates p <0.05.

  The present invention relates to the treatment of patients suffering from various cancers such as colorectal cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, and pediatric malignant solid tumor. A first step of measuring the amount of phosphorylated RB protein in the excised specimen sample obtained from the region containing the cancer tissue and the control sample, and the phosphorylated RB measured in the excised specimen sample and the control sample. A method of predicting or monitoring the response to irinotecan in a patient's cancer, and a phosphorylation site-specific anti-RB antibody or phosphorylation site-specific anti-RB antibody fragment comprising a second step of comparing the amount of protein The present invention provides a kit for predicting or monitoring a response of a patient to irinotecan in cancer by the method.

  As used herein, "RB" refers to a retinoblastoma gene that is a tumor suppressor gene. The RB protein refers to a protein of a transcription and translation product of the retinoblastoma gene. As used herein, “BCRP” refers to breast cancer resistance protein. As used herein, “CDK” refers to cyclin-dependent kinase.

  As used herein, the term “treatment” refers to administration of a pharmaceutical composition according to the present invention to a subject, whereby the tumor is killed or its cell number is reduced, tumor growth is suppressed, It means that various symptoms caused by are improved. In the present invention, the term "prevention" refers to preventing the number of decreased tumors from increasing due to re-growth, and preventing re-growth of tumors whose growth has been suppressed.

[About antibodies]
In the present invention, antibodies that can be used to detect phosphorylation of RB protein include, for example, a phosphorylation site-specific anti-RB antibody that detects phosphorylation of Ser780 and / or Ser807 / 811 of RB protein or a fragment thereof. Can be used. Further, as an antibody capable of detecting BCRP, a BCRP-specific antibody or a fragment thereof can be used. As the antibody, a monoclonal antibody, a polyclonal antibody, an antibody fragment having binding specificity, a phage antibody, or the like can also be used. A product obtained by modifying a gene encoding an antibody so as to improve specificity can also be used.

  These antibodies and antibody fragments are, according to known methods, a mouse or rat, a pig, a cow, a horse, a rabbit, a goat, a sheep, a host animal selected from birds, etc., a protein or peptide having an appropriate antigen or epitope. And hybridoma technology (eg, Nature, 1975, 256: 495-497, Immunology Today 1983, 4:72, PNAS 1983, 80: 2026-2030, Monoclonal Antibodies and Cancer Therapy, 1985, 77-96, etc.), using any technique for producing antibody molecules, such as production as a monoclonal antibody, for example, by using a method for preparing and isolating the antibody molecule (for example, Antibodies: A Laboratory Manual, 1988, Cold Method). Spring Harbor Laboratory, Monoclonal Antibodies: Principles and Practice, 1986, Academic Press, etc.) can be used. Those fragmented according to a known method can also be used.

  In addition, commercially available antibodies can also be used. Antibodies that can be used to detect phosphorylation of RB protein include, for example, Anti-Rb (phospho S780) antibody (ab131264) and Anti-Rb (phospho S780) antibody (ab47763) (Abcam, Cambridge, UK) Anti-RB1 / Retinoblastoma / RB Antibody (phospho-Ser780) LS-C96958 (LifeSpan BioSciences, Inc., Washington, USA), Rb Antibody (Phospho-S780) (OAAB16109) (Aviva Systems Biology Corporation, California, USA) ), Phospho-Rb pSer780 Antibody (Catalog #: PA5-12681) (Thermo Fisher Scientific Inc., Mass., USA), ANTI-PHOSPHO-RB (S780) antibody produced in rabbit (Product Number: SAB1305093) (Sigma-Aldrich) Co. LLC., Mass., USA, Rabbit Polyclonal Retinoblastoma (Ser780) Antibody (Phospho-specific) (TA325793) (OriGene Technologies, MD, USA), Phospho-RB1-S780 Antibody (AP0444) (NeoScientific, NEO) Group Inc., Massachusetts Retinoblastoma (Phospho-Ser780) antibody (orb223093) (Biorbyt Ltd., Cambridge, UK) and Retinoblastoma (phospho-Ser780) Antibody (abx011461) (Abbexa Ltd., Cambridge, UK) can do. Antibodies that can detect BCRP include # 9308 Phospho-Rb (Ser807 / 811) Antibody (Cell Signaling Technology, Inc., Mass., USA), p-Rb antibody (Ser 807/811): sc -16670 (Santa Cruz Biotechnology, Inc., Texas, USA) can be used.

  When used in a method selected from the group consisting of Western blotting, immunohistochemical staining, immunofluorescence, immunoassay, ELISA or mass spectrometry, these antibodies and fragments thereof are appropriately labeled with a compound, a fluorescent substance, or the like. Can be prepared and used. Further, in order to increase the detection sensitivity, a labeled secondary antibody such as an HRP-labeled secondary antibody can be used.

[About the process of measuring and comparing the amount of phosphorylated RB protein]
After obtaining written informed consent from the area containing cancerous tissue in the colon of the patient, Western blotting, immunohistochemistry, The amount of phosphorylated RB protein is measured by chemical staining, immunofluorescence, immunoassay, ELISA or mass spectrometry. When subjected to immunohistochemical staining, after appropriately performing a fixing treatment such as a formalin fixing treatment, arbitrarily embedding in paraffin, preparing a section, and performing a staining treatment step using a known method. . The staining procedure may be performed as described in the Materials and Methods section of the Examples. Further, counterstaining with hematoxylin is performed as appropriate to ensure identification of the stained site.

  In addition, Ventana XT System Discovery, Ventana Benchmark ULTRA (Roche Diagnostics Co., Ltd.), fully automatic antibody and in situ staining device Lica BOND, fully automatic staining device Autostainer XL / Multistainer ST5020 (Leica Microsystems), etc. Of the present invention can be carried out using an arbitrary fully automatic staining apparatus and an automatic staining processing apparatus, a microscope, and an automatic image processing system.

  Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention should not be construed as being limited by the description.

<Materials and methods>
[1. Obtaining cells)
HCT116, SW480 and SW620 cells were obtained from the American Type Culture Collection (ATCC, USA). CCK-81 cells, CoCM-1 cells and SW837 cells were obtained from Human Science Research Resource Bank (HSRRB, Japan). LIM1215 cells were obtained from the European Collection of Cell Culture (ECACC, UK). Caco-2 cells were obtained from RIKEN BioResource Center (RIKEN BRC, Japan).
[2. Cell culture)
Each of the above cells was cultured in a DMEM medium supplemented with 10% FBS, 4 mM glutamine, 50 U / mL penicillin and 100 μg / mL streptomycin at 37 ° C. under a saturated steam pressure of 5% CO ₂ .

[3. reagent〕
SN-38 (Sigma, Missouri, USA) was dissolved in dimethyl sulfoxide (DMSO), dispensed, and stored at -20 ° C until use.

[4. Cell viability test)
The cell viability was determined using Cell Counting Kit-8 (Dojindo Laboratories, Inc., Japan) according to the instructions attached to the kit, and the IC50 value was calculated.
SN38 adjusted to each predetermined concentration was added to the DMEM medium, and each cell was cultured for 72 hours. Thereafter, the solution WST-8 attached to the kit was added to the above DMEM medium, and the cells were further cultured for 4 hours. The measurement was performed at a wavelength of 450 nm using a multiwell plate reader (DS Pharma Biomedical, Japan). The number of cells was counted by the procedure specified by the manufacturer using ViaCount Assay (Guava Technologies, CA, USA), and the cell doubling time of each cell line was calculated.

[5. Protein preparation and Western blot analysis)
Cells were buffered (50 mM Tris-HCl, 1% SDS, 1 mM DTT, 2 μg / mL leupeptin, 2 μg / mL aprotinin, 0.5 mM phenylmethylsulfonyl fluoride and a phosphatase inhibitor cocktail (Nacalai Tesque, Japan). ) To obtain a lysate, which was subjected to ultrasonic treatment, and further centrifuged at 15,000-rpm and 4 ° C to collect a supernatant.

  The resulting supernatant is boiled for 5 minutes and 12% polyacrylamide gel (for detection of CDK2, CDK4, CDK6 and GAPDH) or (phosphorylated RB protein, RB protein, topoisomerase I, BCRP and α -Loaded on a 7% polyacrylamide gel (for detection of tubulin), electrophoresed and then transferred to PVDF membrane (Millipore, Mass., USA).

The following primary antibodies were used for Western blot analysis.
Mouse anti-human RB antibody (BD Biosciences, CA, USA), mouse anti-human phosphorylated RB (Ser780) antibody (Calbiochem, CA, USA), mouse anti-human CDK6 antibody (Cell Signaling Technology, Inc., Mass., US), rabbit anti-human phosphorylated RB (Ser780) antibody (Cell Signaling Technology, Inc., Mass., USA), rabbit anti-human phosphorylated RB (Ser807 / 811) antibody (Cell Signaling Technology, Inc., Mass., USA) , Rabbit anti-human CDK2 antibody (Santa Cruz Biotechnology, California, USA), rabbit anti-human CDK4 antibody (Santa Cruz Biotechnology, California, USA), rabbit anti-human topoisomerase I antibody (Abcam, Cambridge, UK), mouse Anti-human BCRP antibody (Abcam, Cambridge, UK); and Mouse anti-human GAPDH antibody (HyTest, Turku, Finland).

  Blots were incubated with an HRP-labeled secondary antibody (GE Healthcare, NJ, USA). Signals were detected using Chemi-Lumi One (Nacalai Tesque, Japan) or Western Chemiluminescent HRP substrate (Merck Millipore).

[6. Small interfering RNA introduction)
As small interfering RNAs (siRNAs), CDK2 (UAAGUACGAACAGGGACUCca), CDK4 (UGUGGGUUAAAAGUCAGCAtt), CDK6 (UUCUACGAAACAUUUCUGCaa) and Silencer Select Negative Control # 2 siRNA were obtained from Ambion (California, USA). Cells were transfected with 50 nM of each siRNA using Lipofectamine RNAiMAX reagent (Invitrogen, CA, USA). Twenty-four hours after transfection, the culture solution was further incubated for 72 hours with DMEM supplemented with 5 nM SN38, DMEM supplemented with 0.1% DMSO or DMEM alone. Thereafter, the cells were collected, the cell cycle was confirmed, and the cells were analyzed by Western blotting.

[5. Cell cycle analysis)
After exposing various cells to SN38 at predetermined concentrations for 72 hours, the cells were collected. The collected cells were permeabilized with 0.1% Triton-X100, and nuclei were stained with propidium iodide (PI). DNA levels were measured on a FACSCalibur (Becton-Dickinson, NJ, USA) and analyzed by ModFit LT (Verity Software House, ME, USA).

[7. Human tissue sample)
Primary tumor samples were obtained from 23 colorectal cancer patients in stage IV of the clinical stage who underwent colorectal resection at Kyoto Prefectural University of Medicine gastroenterological surgery between 2008 and 2013. Each sample was embedded in paraffin after formalin fixation for 24 hours.

  The criteria for the selection of patients are that they do not have other tumors and that they have not received chemotherapy or radiation therapy before surgery. Written informed consent was obtained in all patients. Relevant clinical pathology data and survival data were obtained from hospital databases.

  After surgery, 22 patients received second or third-line irinotecan chemotherapy for metastasis or recurrence, and only one patient received irinotecan as the first line therapy. All patients were treated with the following regimens containing irinotecan: FOLFIRI (5-FU + leucovorin + irinotecan), FOLFIRI + BEV (FOLFIRI + bevacizumab), FOLFIRI + C-mab (FOLFIRI + cetuximab), FOLFIRI + P-mab FOLFIRI + panitumumab), CPT-11 + C-mab (irinotecan + cetuximab), IRIS (irinotecan + S-1), and IRIS + BEV (IRIS + bevacizumab).

  After about 5 courses of treatment with a regimen containing irinotecan, X-ray tomography (CT) was performed. The therapeutic effect was evaluated using RECIST v1.1 (Eisenhauer EA., Et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45: 228 -47.).

  Five patients were classified as partial response (PR), 10 patients were classified as stable (SD), and 8 patients were classified as advanced (SD). Patients with PR were classified into the treatment response group, and patients with SD and PD were classified into the treatment non-response group. The disease stage was determined based on The Union for International Cancer Control (Sobin L, et al. TNM classification of malignant tumors. 7th ed. Hoboken: Wiley, 2009.).

[8. (Immunohistochemical staining)
Paraffin-embedded sections (3 mm thick) of tumor tissue were subjected to immunohistochemical staining for detection of phosphorylated RB protein (Ser780) and Ki-67 (MIB-1) using the avidin-biotin-peroxidase method. did.

  As a first step, paraffin-embedded sections were deparaffinized with xylene and hydrated with a descending alcohol series. The sections were then incubated with 0.3% aqueous hydrogen peroxide for 30 minutes to inactivate endogenous peroxidase activity.

  Next, the sections were incubated with the protein blocking agent and various antibodies. The sections were then incubated for 1 hour at 37 ° C. for mouse anti-phosphorylated RB (Ser780) antibody (Abcam) and 1 hour at room temperature for Ki-67 (Santa Cruz Biotechnology). An avidin-biotin-peroxidase complex system (Vectastain Elite ABC kit; Vector Laboratories, CA, USA) was used for color development of the diaminobenzidine solution (3,3'-diaminobenzidine, tetrahydrochloride: DAB). The sections were also counterstained with hematoxylin.

  In the final step, the slices were dehydrated using alcohol, subjected to a clearing treatment with xylene, and then sealed.

  Expression by immunohistochemical staining in the cytoplasm of tumor cells was counted as phosphorylated RB protein positive. To evaluate phosphorylated RB protein expression, the expression rate (%) of phosphorylated RB (Ser780) protein in the whole cell population was evaluated in each case. The labeling index (LI) of Ki-67 in immunohistochemistry was determined by the positive rate of cancer cells in the most strongly stained areas. In addition, the region which was severely keratinized was excluded.

[9. Statistical analysis)
Data are shown as the mean of three measurements ± SD. Significance was assessed by Student's t-test or Mann-Whitney U test comparing between the two groups. Fisher's exact test was performed to examine the correlation between clinicopathological parameters and phosphorylated RB protein expression levels. Differences were considered significant when the p-value was less than 0.05 or less than 0.01.

<Result>
[1. Sensitivity to SN38 is inversely related to cell doubling time. ]

[Example 1]
It was examined whether human colon cancer cells express Top I (topoisomerase-I). As a result, human colon cancer cells HCT116, LIM1215, SW480, SW620, CCK-81, CoCM-1, SW837 and Caco-2 all expressed Top I (topoisomerase-I). (Fig. 1).

[Example 2]
Therefore, it was examined whether Top I expression in colorectal cancer cells had a correlation with sensitivity to SN38. The above eight types of human colon cancer cells were treated with each concentration of SN38, and IC50 values were calculated. Table 1 shows the results.

  As shown in Table 1 above, human colon cancer cells were found to have cell lines sensitive to SN38 and cell lines not sensitive to SN38.

  Further, it was revealed that HCT116 cells, LIM1215 cells, SW480 cells, and SW620 cells of human colon cancer cells have sensitivity to SN38. In addition, the IC50 value of SN38 in each cell was less than 4 nM.

  On the other hand, it was revealed that CCK-81 cells, CoCM-1 cells, SW837 cells, and Caco-2 cells of human colon cancer cells have resistance to SN38. In addition, the IC50 value of SN38 in each cell was greater than 80 nM.

  As a result of the above test, it was newly found that no correlation was found between the sensitivity to SN38 and the expression level of Top I in colorectal cancer cell lines (FIG. 1, Table 1).

[Example 3]
Therefore, the doubling time was measured for each of the above colon cancer cell lines. Then, the relationship between the doubling time and the IC50 value of SN38 was shown in a graph (FIG. 2).

As shown in FIG. 2, the doubling time in each of the above cell lines and the IC38 value of SN38 clearly showed a positive correlation (R ² = 0.9315).

  From these results, it has now become clear that colon cancer cells having high proliferative properties have higher sensitivity to SN38.

[2. The phosphorylation status of the RB protein correlates with the sensitivity of the cells to SN38. ]
[Example 4]
Next, in each of the above colon cancer cell lines, a test by Western blot analysis was performed to detect a phosphorylated RB protein that could not bind to the transcription factor E2F due to cell proliferation.

  As a result, in the cell lines (HCT116 cells, LIM1215 cells, SW480 cells and SW620 cells) that were found to be sensitive to SN38 in the above test, all of these RB proteins were excessively phosphorylated. This was newly confirmed this time (Fig. 3). The total amount of RB protein in each cell line was constant.

  On the other hand, the cell lines (CCK-81 cells, CoCM-1 cells and SW837 cells) that were found to be resistant to SN38 in the above test show a low degree of phosphorylation of RB protein. It was confirmed (FIG. 3). The total amount of RB protein in each cell line was constant.

  From the above test results, it has now become clear that the sensitivity to SN38 can be evaluated in colon cancer cell lines by confirming whether Ser780 or Ser807 / 811 is excessively phosphorylated in the RB protein.

  Since it was confirmed that Ser807 / 811 of the RB protein was phosphorylated only in Caco-2 cells that are resistant to SN38 (FIG. 3), further investigation was performed.

[Example 5]
Caco-2 cells are known to express BCRP (breast cancer resistance protein) that transports SN38. Therefore, it was examined whether or not each of the above colon cancer cell lines, in which the presence or absence of excessive phosphorylation of Ser780 and Ser807 / 811 in the RB protein, expressed BCRP.

  As a result, it was newly confirmed that only the Caco-2 cells expressed BCRP, and that the other SN38-resistant cell lines did not express BCRP (FIG. 4). This suggests that only Caco-2 cells have resistance to SN38 due to extracellular secretion of SN38 by the expressed BCRP, despite the phosphorylation of Ser807 / 811 of the RB protein.

  The above results revealed that the efficacy of irinotecan for cancer can be predicted based on the degree of phosphorylation of RB protein. And, a cancer cell having excessive phosphorylation in the RB protein has sensitivity to irinotecan, that is, it can be determined that irinotecan has therapeutic efficacy, a cancer cell having no excessive phosphorylation Has resistance to irinotecan, that is, it can be determined that irinotecan has no therapeutic efficacy. Further, even if the cancer cells have excessive phosphorylation in the RB protein, those that further express BCRP have resistance to irinotecan, that is, it can be determined that irinotecan has no therapeutic efficacy But this time, it is newly revealed.

  From this, it can be understood that the efficacy of irinotecan can also be predicted by examining the degree of phosphorylation of RB protein even in cancer tissues of cancer patients. Therefore, by further examining the degree of phosphorylation of RB protein using a tissue sample obtained from a cancer patient, it was verified whether the efficacy of irinotecan can be reasonably predicted.

[3. The response to irinotecan in patient samples can be predicted by the phosphorylation status of RB protein detected by immunohistochemical staining. ]

[Example 6]
Next, it was examined whether the responsiveness to irinotecan in a patient sample could be predicted by using the expression level of phosphorylated RB protein in the sample as an index.

  Twenty-three patients with colorectal cancer were classified into treatment-responsive and treatment-non-responsive groups based on their response to treatment with irinotecan, as described in the Materials and Methods section (Table 2). .

  Table 3 shows the characteristics of patients in the irinotecan treatment response group and the treatment non-response group.

  There was no significant difference between the treatment response group and the non-treatment response group in overall survival (data not shown).

[Example 7]
Next, we analyzed the correlation between various clinicopathological factors and phosphorylated RB protein expression levels determined by immunohistochemical staining. Representative results of immunohistochemical staining confirming the phosphorylated RB protein expression level in the tumor are shown in FIG.

  Phosphorylated RB protein expression levels in 23 samples from 23 patients with colorectal cancer were then assessed as described in Materials and Methods, and appropriate for immunohistochemically stained tumor cells. Defined cut-off values.

  As a result, when the phosphorylated RB protein (Ser780) positivity was set to a cutoff value of 25% (P value = 0.0006, Table 4), a significantly higher positive rate was observed in colon cancer tissues in the group responding to treatment with irinotecan. This is the first time I have found that.

Example 8
On the other hand, the labeling index (LI) of Ki-67 (MIB-1), which has been conventionally used clinically as a diagnostic marker for tumor growth, shows a statistically significant difference between the treatment-responsive group and the non-treatment-responsive group. It was found for the first time that this was not the case (FIGS. 6 and 7).

  Based on these results, the use of patient tissue samples to predict response to irinotecan is more than using the conventionally used Ki-67 labeling index. It was newly found that it would be more useful to confirm by immunohistochemical staining using the phosphorylated RB protein as an indicator.

[4. Knockdown of both CDK4 and CDK6 reduces SN38-induced G2 / M phase accumulation, along with dephosphorylation of RB proteins. ]

[Example 9]
Next, further studies were performed to confirm that phosphorylation of the RB protein did play an important role in the efficacy of irinotecan in cancer.

  Therefore, we investigated whether suppressing the expression of phosphorylated RB protein would impair the efficacy of irinotecan in cancer. Cyclin-dependent kinases such as CDK2, CDK4, CDK6 and CDK4 / 6 are well known to phosphorylate RB proteins. Therefore, knockdown of cyclin-dependent kinase was performed in an SN38-sensitive colon cancer cell line. The colon cancer cell line was treated with siRNA to suppress the expression of CDK2, CDK4, CDK6 and CDK4 / 6 (FIG. 8).

  As a result, in SW620 and SW480 cells, the RB protein was most significantly converted to the non-phosphorylated form after the simultaneous knockdown of CDK4 and CDK6. On the other hand, in those cells, knocking down CDK2 did not affect the degree of phosphorylation of RB protein (FIG. 9).

[Example 10]
In addition, in SW620 cells and SW480 cells, treatment with SN38 for 72 hours induced G2 / M phase arrest in a dose-dependent manner (FIG. 10).

[Example 11]
However, G2 / M arrest induced by treatment with 5 nM SN38 for 72 h was suppressed by siCDK4 / 6 (Figure 9), which most significantly reduced the level of phosphorylation of RB protein (Figure 9). ).

[5. Susceptibility to SN38 is attenuated by knocking down both CDK4 and CDK6. ]

[Example 12]
Therefore, it was further examined whether CDK4 / 6 activity affected the sensitivity to SN38.

  As a result, SW620 cells showed resistance to SN38 after both CDK4 and CDK6 were suppressed (FIG. 12). Also, transfection of siCDK4 / 6 increased the IC50 value in SN38 to 58.4 nM, whereas the control had an IC50 value of 1.4 nM (FIG. 13). Therefore, sensitivity to irinotecan may be affected by the activity of CDK4 / 6 in cancer.

[6. Summary)
We have shown for the first time that the efficacy of irinotecan in cancer can be reasonably predicted by the expression of phosphorylated RB protein.

  First, they show that irinotecan targets proliferating cells and acts to inhibit Top I during S phase, but Top I expression is not correlated with irinotecan efficacy in cancer. . And in in vitro studies using new cancer cell lines, a positive correlation was found between the phosphorylation status of RB protein in cancer cells and the efficacy of SN38 in various cancer cell lines. Was proved. We also showed that further examination of BCRP expression eliminated false positives and that the efficacy of irinotecan in patients with cancer could further enhance the predictive effect.

  Furthermore, a retrospective study using cancer patient tissue samples and clinical data revealed that the cancer tissue in the irinotecan treatment response group had a significantly higher positive rate of phosphorylation at the serine site of the RB protein. By preliminarily examining the positive rate of phosphorylation of the serine site of RB protein in the tissues of patients suffering from, it was demonstrated that the efficacy of irinotecan in patients suffering from cancer can be predicted and monitored.

  Furthermore, compared to the index of the positive rate of Ki-67, which has been used as a proliferative marker in cancer patients in the past in clinical practice, the phosphorylation at the serine site of RB protein, a new proliferative marker found in our clinical study, It has now been newly confirmed that the positive rate of oxidation is significantly higher. The expression of Ki-67 increases during S phase, but is reported to reach a maximum in M phase, suggesting that it has efficacy as an M phase marker. Given that irinotecan attacks cancer cells in the S phase, it is more likely that the newly discovered phosphorylated RB protein will have a greater effect on irinotecan sensitivity than Ki-67. It is reasonable.

  In addition, RB protein is known to be phosphorylated by CDK, leading to cell cycle progression, and there are reports suggesting a relationship between CDK2 activity and prognosis. As a result of knocking down and examining CDK2, CDK4, CDK6 and CDK4 / 6 that convert to a state, it is very interesting that in irinotecan-sensitive cancer, not CDK2 but rather both CDK4 and CDK6 are suppressed by RB protein Became the lowest phosphorylation state, and the cells newly found to be resistant to SN38.

  The present invention relates to irinotecan in various cancers such as colon cancer (colorectal cancer), gastric cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, squamous cell carcinoma, non-Hodgkin's lymphoma, and pediatric malignant solid tumor of the patient. The use of the methods and kits for predicting or monitoring a response has the effect of reasonably predicting the efficacy of irinotecan in a patient suffering from the cancer. Then, by using the method and the kit of the present invention, it becomes possible to examine in advance the positive rate of phosphorylation of the serine site of the RB protein in the tissue of the patient suffering from the above-mentioned cancer, and to become susceptible to a cancer sensitive to irinotecan. Provides a chance to prescribe irinotecan early and appropriately for patients who have had cancer, and avoids prescribing irinotecan for patients with cancer that is not susceptible to irinotecan, as well as providing alternative treatment options. Providing the opportunity to make early choices has made it possible to greatly increase the therapeutic potential of cancer.

Claims (8)

A first step of measuring the amount of phosphorylated RB protein in an excised specimen sample obtained from a region containing a cancer tissue of a patient and a control sample, and a phosphorylated RB protein measured in the excised specimen sample and a control sample methods for the and a second step of comparing the amount to predict the response to irinotecan in cancer patients, or monitors. The method according to claim 1, wherein the phosphorylation site of the phosphorylated RB protein is Ser780 and / or Ser807 / 811 of the RB protein. The amount of the phosphorylated RB protein is measured using a measurement method selected from the group consisting of Western blotting, immunohistochemical staining, immunofluorescence, immunoassay, ELISA or mass spectrometry. Item 3. The method according to Item 1 or 2. The amount of the phosphorylated RB protein is measured using a phosphorylation site-specific anti-Rb antibody or a phosphorylation site-specific anti-Rb antibody fragment, according to any one of claims 1 to 3, The described method. The method according to claim 4, wherein the phosphorylation site-specific anti-RB antibody detects phosphorylation of Ser780 and / or Ser807 / 811 of RB protein. Furthermore, the expression of BCRP (breast cancer resistance protein) was determined from a group consisting of Western blotting, immunohistochemical staining, immunofluorescence, immunoassay, ELISA or mass spectrometry in an extirpated sample obtained from a region containing the cancer tissue of the patient. The method according to any one of claims 1 to 5, comprising a step of measuring using a selected measurement method. Predicting or monitoring the response to irinotecan in a cancer of a patient by the method according to any one of claims 1 to 6, comprising a phosphorylation site-specific anti-RB antibody or a phosphorylation site-specific anti-RB antibody fragment. Kit to do. The phosphorylation site-specific anti-RB antibody or the phosphorylation site-specific anti-RB antibody fragment is characterized in that the phosphorylation of Ser780 and / or Ser807 / 811 of RB protein is detected. The kit as described.