JP7747459B2 - Method for determining sensitivity to FGFR inhibitors - Google Patents

Description

The present disclosure includes a method for determining the sensitivity of cancer patients to FGFR inhibitors.

Fibroblast growth factor receptor (FGFR) inhibitors have been undergoing clinical trials for a variety of cancers, and their efficacy has been demonstrated in urothelial carcinoma and intrahepatic cholangiocarcinoma. Several compounds have been approved by the FDA based on the results of preclinical and clinical trials. These clinical trials use genetic alterations in the FGFR gene, such as amplification, translocations that cause gene fusions, and point mutations, as criteria for patient selection and enrollment (eligibility criteria). However, there are a significant number of patients who do not respond to FGFR inhibitors despite having genetic alterations that meet the eligibility criteria, and genetic alterations in the FGFR gene determined by genomic analysis are not necessarily an appropriate criterion.

International Publication No. 2019/111998

T Yamamoto et al., Cancers 2020, 12(8), 2010

One object of the present disclosure is to provide a method for determining the sensitivity of a cancer patient to an FGFR inhibitor. A further object of the present disclosure is to provide compositions and methods for treating cancer. A further object of the present disclosure is to provide a method for determining the sensitivity of cancer cells to an FGFR inhibitor. A further object of the present disclosure is to provide a method for selecting cells. A further object of the present disclosure is to provide a method for screening for FGFR inhibitors.

In one aspect, the present disclosure provides a method for determining sensitivity of a cancer patient to an FGFR inhibitor, comprising:
The present invention provides a method for determining whether a patient is susceptible to an FGFR inhibitor, comprising: determining an FGFR/EGFR expression ratio in the cancer of the patient; and comparing the FGFR/EGFR expression ratio with a reference value, wherein the patient is determined to be sensitive to an FGFR inhibitor if the FGFR/EGFR expression ratio is higher than the reference value.

In one aspect, the present disclosure provides a composition containing an FGFR inhibitor for treating cancer, the composition being for administration to a cancer patient determined to be sensitive to an FGFR inhibitor by the determination method described above.

In one aspect, the present disclosure provides a composition comprising an FGFR inhibitor for treating cancer, the composition being to be administered to a patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value.

In one aspect, the present disclosure provides a method for treating cancer, the method comprising administering an FGFR inhibitor to a cancer patient determined to be sensitive to an FGFR inhibitor by the determination method.

In one aspect, the present disclosure provides a method for treating cancer, the method comprising administering an FGFR inhibitor to a patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value.

In one aspect, the present disclosure provides a method for determining the sensitivity of a cancer cell to an FGFR inhibitor, comprising:
The present invention provides a method for determining that a cancer cell is sensitive to an FGFR inhibitor, comprising: examining an FGFR/EGFR expression ratio in the cancer cell; and comparing the FGFR/EGFR expression ratio with a standard value, wherein the cancer cell is determined to be sensitive to an FGFR inhibitor if the FGFR/EGFR expression ratio is higher than the standard value.

The present disclosure provides, in one aspect, a method for selecting cells, comprising:
The present invention provides a method for selecting cancer cells, comprising: determining an FGFR/EGFR expression ratio in cancer cells; and comparing the FGFR/EGFR expression ratio with a standard value, wherein the cancer cells are selected when the FGFR/EGFR expression ratio is higher than the standard value.

In one aspect, the present disclosure provides a method for screening for FGFR inhibitors, the method comprising contacting cancer cells having an FGFR/EGFR expression ratio higher than a reference value with a candidate substance for an FGFR inhibitor.

The present disclosure provides methods for determining the sensitivity of cancer patients to FGFR inhibitors, compositions and methods for treating cancer, methods for determining the sensitivity of cancer cells to FGFR inhibitors, methods for selecting cells, and methods for screening FGFR inhibitors.

FIG. 1 shows the results of a correlation test between the sumFGFR/EGFR ratio (X axis) and GEI (Y axis). FIG. 2 shows the results of a correlation test between the (FGFR3+FGFR4)/EGFR ratio (X axis) and GEI (Y axis). FIG. 3 shows the results of a correlation test between the FGFR3/EGFR ratio (X-axis) and GEI (Y-axis). FIG. 4 shows the results of a correlation test between the FGFR4/EGFR ratio (X-axis) and GEI (Y-axis). FIG. 5 shows the results of a correlation test between FGFR3 expression (X-axis) and GEI (Y-axis). FIG. 6 shows the results of a correlation test between FGFR4 expression (X-axis) and GEI (Y-axis). FIG. 7 shows the results of a correlation test between the sumFGFR/ERBB2 ratio (X axis) and GEI (Y axis). FIG. 8 shows the results of a correlation test between the sumFGFR/MET ratio (X axis) and GEI (Y axis). FIG. 9 shows the results of a comparison between the responsive group and the non-responsive group using the sumFGFR/EGFR ratio (Y axis) as an index. FIG. 10 shows the ROC curve and AUC plotted based on FIG.

Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings.

In one aspect, the present disclosure provides a method for determining sensitivity of a cancer patient to an FGFR inhibitor, comprising:
The present invention relates to a method for determining whether a patient is susceptible to an FGFR inhibitor, comprising: determining an FGFR/EGFR expression ratio in the cancer of the patient; and comparing the FGFR/EGFR expression ratio with a reference value, wherein the patient is determined to be sensitive to an FGFR inhibitor if the FGFR/EGFR expression ratio is higher than the reference value.

As used herein, a fibroblast growth factor receptor (FGFR) inhibitor refers to a substance that inhibits signal transduction mediated by FGFR. There are four paralogs of FGFR: FGFR1, FGFR2, FGFR3, and FGFR4. An FGFR inhibitor inhibits one or more of these four FGFRs. In one embodiment, an FGFR inhibitor inhibits FGFR3 and/or FGFR4. In a further embodiment, an FGFR inhibitor inhibits FGFR1, FGFR2, and FGFR3. In a further embodiment, an FGFR inhibitor inhibits FGFR1, FGFR2, FGFR3, and FGFR4. An FGFR inhibitor that inhibits FGFR1, FGFR2, FGFR3, and FGFR4 is also referred to as a pan-FGFR inhibitor. An FGFR inhibitor may be a small molecule compound, a protein, an antibody, a peptide, or a nucleic acid. FGFR inhibitors include erdafitinib (JNJ42756493, pan-FGFR inhibitor), rogaratinib (BAY1163877, FGFR1-3 inhibitor), AZD4547 (FGFR1-3 inhibitor), infigratinib (BGJ398, FGFR1-3 inhibitor), fisogatinib (BLU554, FGFR4 inhibitor), LY2874455 (pan-FGFR inhibitor), futibatinib (TAS-120, pan-FGFR inhibitor), ASP5878 (pan-FGFR inhibitor), and delazantinib (ARQ087, FGFR1-3 inhibitor). In one embodiment, the FGFR inhibitor is erdafitinib.

Examples of cancer include colorectal cancer, stomach cancer, prostate cancer, breast cancer, cervical cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, kidney cancer, pancreatic cancer, urothelial cancer (including bladder cancer), biliary tract cancer (including intrahepatic bile duct cancer), brain tumor, leukemia, myelodysplastic syndrome, multiple myeloma, and malignant lymphoma. Preferably, the cancer is colorectal cancer, intrahepatic bile duct cancer, or urothelial cancer (including bladder cancer). The large intestine is divided into the colon and the rectum, and colorectal cancer includes colon cancer and rectal cancer.

Cancers include those with genetic alterations that enhance signal transduction from FGFR, those that enhance signal transduction from EGFR, or both, as well as those without such genetic alterations. Genetic alterations that enhance signal transduction from FGFR include FGFR amplification, translocations that cause gene fusion, and point mutations. Genetic alterations that enhance signal transduction from EGFR include EGFR exon deletion mutations and point mutations, RAS (KRAS/NRAS) mutations, and BRAF ^V600E mutations. In one embodiment, cancers do not have genetic alterations that enhance signal transduction from FGFR or those that enhance signal transduction from EGFR. In a further embodiment, cancers do not have genetic alterations that enhance signal transduction from FGFR or those that enhance signal transduction from EGFR.

The FGFR/EGFR expression ratio in a patient's cancer is typically determined in vitro. The FGFR/EGFR expression ratio in a patient's cancer can be determined using a patient sample. Examples of such samples include cancer cells, cancer tissues, cancer spheroids, and blood samples (e.g., whole blood, plasma, and serum). Cancer cells or cancer tissues may be obtained from a primary tumor or a metastatic tumor. In one embodiment, the cancer cells or cancer tissues are cells or tissues obtained from a primary tumor. Cancer cells or cancer tissues can be obtained by surgery or biopsy. A cancer spheroid refers to a cell mass containing tumor-initiating cells, and tumor-initiating cells refer to cells that form tumors when transplanted into an immunocompromised animal. When the sample is a cancer spheroid, a cancer patient sample refers to a cancer spheroid prepared from the patient's cancer tissue (also referred to as a patient-derived cancer spheroid). Cancer spheroids can be prepared by known methods, such as the method described in International Publication No. WO 2019/111998. It is known that cancer cells (circulating tumor cells, CTCs) released from cancer tissue circulate in the blood of cancer patients, and gene expression has been examined using these cells (Pantel K. et al., Nat Rev Gastroenterol Hepatol, 14:73-74, 2017). Therefore, blood samples can also be used as samples in the present disclosure. In one embodiment, the sample is a cancer spheroid. In the present disclosure, the term "cancer patient sample" is used to refer to samples that have undergone the necessary processing for measuring FGFR and EGFR, such as protein or nucleic acid isolation or concentration, freezing, or fixation.

The sample may be obtained before treatment with an FGFR inhibitor, at one or more time points after initiation of treatment, or both. In one embodiment, the FGFR/EGFR expression ratio in the patient's cancer comprises the FGFR/EGFR expression ratio before treatment with an FGFR inhibitor, in which case the sample comprises a sample from the cancer patient before treatment with an FGFR inhibitor. In a further embodiment, the FGFR/EGFR expression ratio in the patient's cancer comprises the FGFR/EGFR expression ratio before treatment with an FGFR inhibitor and the FGFR/EGFR expression ratio at one or more time points after initiation of treatment with an FGFR inhibitor, in which case the sample comprises a sample from the cancer patient before treatment with an FGFR inhibitor and a sample at one or more time points after initiation of treatment with an FGFR inhibitor. Changes in sensitivity to FGFR inhibitors can be monitored by examining the FGFR/EGFR expression ratio in samples after initiation of treatment.

The expression of FGFR and EGFR may be measured by any method capable of measuring their mRNA expression or protein expression. The amino acid and nucleic acid (mRNA) sequences of human FGFR1, FGFR2, FGFR3, FGFR4, and EGFR are known, and representative sequences are disclosed below: FGFR1 (Genbank No. FJ809917 and Genbank No. BC018128), FGFR2 (Genbank No. AB030073 and Genbank No. M87770), FGFR3 (Genbank No. AB209441 and Genbank No. AF245114), FGFR4 (Genbank No. BC011847 and Genbank No. AK301169), and EGFR (Genbank No. BC094761 and Genbank No. HQ912715). Measurement methods include RNA sequencing (RNA-Seq), PCR (polymerase chain reaction), microarrays, and immunological methods such as immunostaining, ELISA, Western blot, and dot blot. As used herein, the term "PCR" encompasses any method that utilizes PCR, such as reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), and real-time PCR. In one embodiment, FGFR and EGFR expression is measured by PCR. Those skilled in the art can appropriately process cancer patient samples according to the measurement method, and can appropriately prepare or select primers or probes suitable for measuring FGFR and EGFR, and reagents such as anti-FGFR antibodies and anti-EGFR antibodies for use in immunological methods, based on the amino acid or nucleic acid sequences of FGFR and EGFR.

FGFR expression may be the sum of the expression of four types of FGFR1, FGFR2, FGFR3, and FGFR4, or the sum of the expression of two or three types, or the expression of any one type. FGFR expression may be measured for FGFR1, FGFR2, FGFR3, and FGFR4 that is expressed in the target cancer. For example, the FGFR/EGFR expression ratio may be the ratio of the sum of the expression of FGFR1, FGFR2, FGFR3, and FGFR4 to the expression of EGFR (referred to as sumFGFR/EGFR ratio), the ratio of the sum of the expression of FGFR3 and FGFR4 to the expression of EGFR (referred to as (FGFR3+FGFR4)/EGFR ratio), the ratio of the expression of FGFR3 to the expression of EGFR (referred to as FGFR3/EGFR ratio), or the ratio of the expression of FGFR4 to the expression of EGFR (referred to as FGFR4/EGFR ratio). For example, since expression of FGFR3 and FGFR4 is predominant in colorectal cancer, the sumFGFR/EGFR ratio, (FGFR3+FGFR4)/EGFR ratio, FGFR3/EGFR ratio, or FGFR4/EGFR ratio may be examined.

Sensitivity to FGFR inhibitors is determined by comparing the FGFR/EGFR expression ratio with a reference value. If the FGFR/EGFR expression ratio is higher than the reference value, the cancer patient can be determined to be sensitive to FGFR inhibitors. The reference value can be a value determined from the FGFR/EGFR expression ratios in the cancers of multiple patients whose sensitivity to FGFR inhibitors is known. Those skilled in the art can determine an appropriate reference value taking into account factors such as the type of sample used for the determination, the method for measuring FGFR and EGFR expression, and the type of cancer.

The reference value can be an FGFR/EGFR expression ratio that can statistically significantly separate multiple cancer patients known to be sensitive to FGFR inhibitors into a sensitive group and an insensitive group. This value can be calculated, for example, using receiver operating characteristic (ROC) analysis. Alternatively, the reference value can be a value that can separate a certain percentage (e.g., 10%, 20%, 30%, 40%, or 50%) of the top FGFR/EGFR expression ratios of multiple cancer patients known to be sensitive to FGFR inhibitors. The statistical significance of the difference between the two groups can be analyzed using any method. Examples of statistical analysis methods include the chi-square test and Fisher's exact test.

The FGFR/EGFR expression ratio may be compared with multiple reference values. For example, in multiple cancer patients whose sensitivity to FGFR inhibitors is known, the sensitivity of each patient may be ranked into three or more levels, and two or more FGFR/EGFR expression ratios that can separate each rank group with a statistically significant difference may be used as reference values. This allows the rank of cancer patients' sensitivity to FGFR inhibitors to be determined. Statistical significance may be analyzed by any method. Examples of statistical analysis methods include the chi-square test and Fisher's exact test. Alternatively, the reference values may be multiple values that can separate a certain percentage (e.g., 10%, 20%, 30%, 40%, or 50%) of the top FGFR/EGFR expression ratios of each patient in multiple cancer patients whose sensitivity to FGFR inhibitors is known.

Comparing the FGFR/EGFR expression ratio with the reference value may or may not involve statistical analysis. Statistical analysis methods include one-way ANOVA, two-way ANOVA, Dunnett's test, and Tukey's test.

It is known that patients selected based on genetic alterations in FGFR genes include a significant number of patients who are insensitive to FGFR inhibitors. Furthermore, under these selection criteria, cancer patients without genetic alterations in FGFR genes would not be selected as candidates for treatment with FGFR inhibitors. The method disclosed herein enables highly accurate selection of cancer patients, increasing the treatment options for cancer patients without genetic alterations and expanding the application of FGFR inhibitors.

Cancer patients determined to be sensitive to FGFR inhibitors can be treated with FGFR inhibitors. Therefore, in one aspect, the present disclosure provides a composition comprising an FGFR inhibitor for treating cancer, the composition being for administration to a cancer patient determined to be sensitive to an FGFR inhibitor by the determination method of the present disclosure. In a further aspect, the present disclosure provides a composition comprising an FGFR inhibitor for treating cancer, the composition being for administration to a cancer patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value. In another aspect, the present disclosure provides a method for treating cancer, the method comprising administering an FGFR inhibitor to a cancer patient determined to be sensitive to an FGFR inhibitor by the determination method of the present disclosure. The treatment method may further comprise determining sensitivity to an FGFR inhibitor by the determination method of the present disclosure prior to administration of the FGFR inhibitor. In a further aspect, the present disclosure provides a method for treating cancer, the method comprising administering an FGFR inhibitor to a cancer patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value. The treatment method may include, prior to administration of an FGFR inhibitor, determining the FGFR/EGFR expression ratio in the patient's cancer and comparing the FGFR/EGFR expression ratio with a reference value.

For the treatment of cancer, an effective amount of an FGFR inhibitor is administered to a cancer patient. As used herein, an effective amount refers to an amount that can produce the desired effect in cancer treatment, such as tumor disappearance, tumor shrinkage, or inhibition of tumor growth, or prevention of recurrence. The FGFR inhibitor is administered at a dose determined appropriately depending on the patient's age, weight, disease severity, concomitant medications, etc., and may be administered at, for example, 0.01 mg/kg to 100 mg/kg body weight, 0.1 mg/kg to 100 mg/kg body weight, 0.1 mg/kg to 10 mg/kg body weight, or 0.1 mg/kg to 1 mg/kg body weight per day. In one embodiment, the FGFR inhibitor is erdafitinib, and is administered at 1 mg/day to 30 mg/day, 1 mg/day to 15 mg/day, 1 mg/day to 12 mg/day, or 1 mg/day to 10 mg/day. FGFR inhibitors can be administered orally or parenterally (e.g., intravenously, intramuscularly, or subcutaneously). FGFR inhibitors may be administered once, twice, three times, four times, or more times daily, and may be administered daily or at regular intervals.

FGFR inhibitors may be administered before, during, or after endoscopic resection, surgical resection, radiation therapy, or chemotherapy for the treatment of cancer. That is, cancer treatment includes treatment before and after resection of cancerous tissue. For example, treatment of colorectal cancer may be treatment of unresectable colorectal cancer or treatment to prevent recurrence after resection of cancerous tissue.

In one embodiment, the FGFR inhibitor is used in combination with one or more other drugs. The one or more drugs can be selected from anticancer drugs such as 5-fluorouracil (5-FU), tegafur, tegafur-uracil (UFT), doxifluridine (5'-DFUR), carmofur, tegafur-gimeracil-oteracil potassium (S-1), capecitabine (Cape), regorafenib, trifluridine-tipiracil hydrochloride (FTD/TPI), mitomycin C, irinotecan (IRI), and oxaliplatin (OX), as well as molecularly targeted drugs such as vascular endothelial growth factor (VEGF) inhibitors (e.g., bevacizumab, aflibercept), vascular endothelial growth factor receptor (VEGFR) inhibitors (e.g., ramucirumab), and EGFR inhibitors (e.g., cetuximab, panitumumab, erlotinib).

When two or more drugs are used in combination, some or all of these drugs may be contained in a single composition, or all of the drugs may be contained in separate compositions. In this disclosure, the use of two or more drugs in combination means that the two or more drugs are used to treat the same subject at the same time, and the administration schedules of these drugs may be the same or different. Furthermore, when described herein as the use of two or more drugs in combination, this also includes the use of these drugs in combination with yet another drug.

In one embodiment, an FGFR inhibitor is used in combination with an EGFR inhibitor. EGFR inhibitors include cetuximab, panitumumab, and erlotinib.

FGFR inhibitors can also be administered in combination with chemotherapy, such as FOLFOX (intravenous 5-FU + levofolinate + OX), FOLFIRI (5-FU + levofolinate + IRI), FOLFOXIRI (5-FU + levofolinate + OX + IRI), CapeOX (Cape + OX), SOX (S-1 + OX), or IRIS (S-1 + IRI), or a combination of these with one or more molecularly targeted drugs. Alternatively, FGFR inhibitors can be administered after treatment with chemotherapy, such as FOLFOX, FOLFIRI, FOLFOXIRI, CapeOX, SOX, or IRIS, or a combination of these with one or more molecularly targeted drugs. In one embodiment, the one or more molecularly targeted drugs include an EGFR inhibitor. In one embodiment, the EGFR inhibitor is cetuximab, panitumumab, or erlotinib.

Pharmaceutical compositions containing FGFR inhibitors can be formulated using conventional methods. Pharmaceutical compositions may be in the form of tablets, capsules, powders, granules, suspensions, syrups, elixirs, injections, etc. Depending on the dosage form, in addition to the FGFR inhibitor, the pharmaceutical composition may contain pharmaceutically acceptable additives such as excipients, disintegrants, binders, lubricants, stabilizers, buffers, surfactants, preservatives, sweeteners, etc., and/or pharmaceutically acceptable carriers such as lactose, starch, dextrin, water, saline, oil, alcohol, etc.

Based on the FGFR/EGFR expression ratio, the sensitivity of cancer cells to FGFR inhibitors can be determined, and cancer cells sensitive to FGFR inhibitors can be selected. Therefore, based on the FGFR/EGFR expression ratio, cancer cells that can be used for screening FGFR inhibitors can be selected.

In one aspect, the present disclosure provides a method for determining the sensitivity of a cancer cell to an FGFR inhibitor, comprising:
The present invention relates to a method for determining whether a cancer cell is sensitive to an FGFR inhibitor, the method comprising: determining an FGFR/EGFR expression ratio in the cancer cell; and comparing the FGFR/EGFR expression ratio with a standard value, wherein the cancer cell is determined to be sensitive to an FGFR inhibitor if the FGFR/EGFR expression ratio is higher than the standard value.

In a further aspect, the present disclosure provides a method for selecting cells, comprising:
The present invention relates to a method for selecting cancer cells, comprising: determining an FGFR/EGFR expression ratio in cancer cells; and comparing the FGFR/EGFR expression ratio with a standard value, wherein the cancer cells are selected when the FGFR/EGFR expression ratio is higher than the standard value.

In a further aspect, the present disclosure relates to a method for screening for FGFR inhibitors, the method comprising contacting cancer cells having an FGFR/EGFR expression ratio higher than a reference value with a candidate substance for an FGFR inhibitor. To select cancer cells having an FGFR/EGFR expression ratio higher than the reference value, the method may further comprise examining the FGFR/EGFR expression ratio in the cancer cells and comparing the FGFR/EGFR expression ratio with the reference value prior to contacting the cancer cells with the candidate substance.

In the above-described methods for assessing cancer cell sensitivity, cell selection, and FGFR inhibitor screening, the measurement of FGFR and EGFR expression, calculation of the FGFR/EGFR expression ratio, determination of a reference value, and comparison with the reference value can be performed as described for the cancer patient sensitivity assessment method of the present disclosure. For example, the reference value can be determined from the FGFR/EGFR expression ratio in multiple cancer cells known to be sensitive to FGFR inhibitors. Those skilled in the art can determine an appropriate reference value taking into account factors such as the type of cancer cell and the method for measuring FGFR and EGFR expression. Various cancer cell lines, particularly cancer cell lines whose sensitivity to FGFR inhibitors is unknown, can be used as cancer cells for which the FGFR/EGFR expression ratio is examined in the above-described sensitivity assessment and selection methods. Cancer cells selected by the above-described selection method can be used in the above-described screening method. Candidate substances can include, but are not limited to, single compounds such as small molecular weight compounds, proteins, antibodies, peptides, and nucleic acids; libraries of compounds, antibodies, nucleic acids, and the like; cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, and the like.

In one aspect, the present disclosure provides use of an FGFR inhibitor for the manufacture of a medicament for treating cancer, wherein the medicament is administered to a cancer patient determined to be sensitive to an FGFR inhibitor by the determination method of the present disclosure.
In one aspect, the present disclosure provides use of an FGFR inhibitor for the manufacture of a medicament for treating cancer, wherein the medicament is administered to a patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value.
In one aspect, the present disclosure provides an FGFR inhibitor for treating cancer, which is administered to a cancer patient determined to be sensitive to an FGFR inhibitor by the determination method of the present disclosure.
In one aspect, the present disclosure provides an FGFR inhibitor for treating cancer, wherein the FGFR inhibitor is administered to a patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value.

An exemplary embodiment of the present disclosure is shown below.

[1]
A method for determining the sensitivity of a cancer patient to an FGFR inhibitor, comprising:
A method comprising: determining an FGFR/EGFR expression ratio in a patient's cancer; and comparing the FGFR/EGFR expression ratio with a reference value, wherein the patient is determined to be sensitive to an FGFR inhibitor if the FGFR/EGFR expression ratio is higher than the reference value.
[2]
[3] The method according to 1, wherein the cancer is colorectal cancer, urothelial cancer, or intrahepatic bile duct cancer.
3. The method according to claim 1 or 2, wherein the cancer is colon cancer.
[4]
4. The method according to any one of 1 to 3 above, wherein the FGFR inhibitor is erdafitinib.
[5]
5. The method according to any one of 1 to 4 above, wherein the FGFR/EGFR expression ratio is examined using cancer cells, cancer tissues, or cancer spheroids of the patient.
[6]
6. The method according to any one of 1 to 5, wherein the FGFR/EGFR expression ratio is a sumFGFR/EGFR ratio, a (FGFR3+FGFR4)/EGFR ratio, an FGFR3/EGFR ratio, or an FGFR4/EGFR ratio.

[7]
A composition comprising an FGFR inhibitor for treating cancer, the composition being intended for administration to a cancer patient determined to be sensitive to an FGFR inhibitor by the method described in any one of 1 to 6 above.
[8]
A composition comprising an FGFR inhibitor for treating cancer, the composition being to be administered to a patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value.
[9]
9. The composition according to claim 7 or 8, wherein the cancer is colorectal cancer, urothelial cancer, or intrahepatic bile duct cancer.
[10]
10. The composition according to any one of 7 to 9, wherein the cancer is colon cancer.
[11]
11. The composition according to any one of 7 to 10, wherein the FGFR inhibitor is erdafitinib.

[12]
A method for treating cancer, comprising administering an FGFR inhibitor to a cancer patient determined to be sensitive to an FGFR inhibitor by the method described in any one of 1 to 6 above.
[13]
13. The method according to 12 above, further comprising determining sensitivity to an FGFR inhibitor by any of the methods according to 1 to 6 above, prior to administration of the FGFR inhibitor.
[14]
A method for treating cancer, comprising administering an FGFR inhibitor to a patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value.
[15]
Prior to administration of FGFR inhibitors,
15. The method of 14, further comprising determining the FGFR/EGFR expression ratio in the patient's cancer, and comparing the FGFR/EGFR expression ratio with a reference value.
[16]
16. The method according to any one of 12 to 15, wherein the cancer is colorectal cancer, urothelial cancer, or intrahepatic bile duct cancer.
[17]
17. The method according to any one of 12 to 16, wherein the cancer is colorectal cancer.
[18]
18. The method according to any one of 12 to 17, wherein the FGFR inhibitor is erdafitinib.

[19]
A method for determining the sensitivity of a cancer cell to an FGFR inhibitor, comprising:
A method comprising: determining an FGFR/EGFR expression ratio in cancer cells; and comparing the FGFR/EGFR expression ratio with a standard value, wherein the cancer cells are determined to be sensitive to an FGFR inhibitor if the FGFR/EGFR expression ratio is higher than the standard value.
[20]
A method for selecting cells, comprising:
A method comprising: determining an FGFR/EGFR expression ratio in cancer cells; and comparing the FGFR/EGFR expression ratio with a standard value, wherein the cancer cells are selected if the FGFR/EGFR expression ratio is higher than the standard value.
[21]
21. The method according to 19 or 20, wherein the FGFR/EGFR expression ratio is a sumFGFR/EGFR ratio, a (FGFR3+FGFR4)/EGFR ratio, a FGFR3/EGFR ratio, or a FGFR4/EGFR ratio.

[22]
A method for screening for an FGFR inhibitor, comprising contacting a cancer cell having an FGFR/EGFR expression ratio higher than a reference value with a candidate substance for an FGFR inhibitor.
[23]
23. The method according to claim 22, wherein the cancer cells are cells selected by the method according to claim 20 or 21.
[24]
Prior to contact with the candidate substance,
24. The method according to 22 or 23, further comprising: examining the FGFR/EGFR expression ratio in cancer cells; and comparing the FGFR/EGFR expression ratio with a standard value, and selecting cancer cells having an FGFR/EGFR expression ratio higher than the standard value.

Specific examples are described below, but these examples do not in any way limit the invention described in the claims.

1. Method
1.1. Patient-Derived Cancer Spheroids Patient-derived cancer spheroids were prepared using tumor samples obtained during surgery from colorectal cancer patients, as described in WO 2019/111998. The harvested tumor fragments were minced and digested with type I collagenase (Thermo Fisher Scientific, Waltham, MA, USA). Epithelial cells were then harvested, suspended in Matrigel (Corning, Corning, NY, USA), and cultured in tumor medium with or without 50 ng/mL EGF (Epidermal Growth Factor) (PeproTech, Cranbury, NJ, USA) and 100 ng/mL bFGF (Basic Fibroblast Growth Factor) (PeproTech).

1.2. Drug Sensitivity Testing. Luciferase-expressing spheroids were generated by infecting cancer spheroids with a lentivirus containing a luciferase-encoding sequence, as described in WO 2019/111998. Luciferase-expressing spheroids were cultured in 96-well cell culture plates (Corning; 3 μL per well). Luminescence measurements were performed on days 1 and 4, and the effect of drug treatment was evaluated by calculating the growth effect index (GEI). The GEI was defined as the growth rate of the drug-treated group relative to the growth rate in the vehicle-only group. Erdafitinib (JNJ42756493, a pan-FGFR1-4 inhibitor; Active Biochem, Kowloon, Hong Kong) and erlotinib (an EGFR inhibitor; ChemScene, Monmouth Junction, NJ, USA) were added at 100 nM ( ^10-7 M) and 1 μM ( ^10-6 M), respectively. The cutoff GEI value between responsive and non-responsive was set at 0.7 (70%).

1.3. Preparation of DNA and RNA samples
DNA: Matrigel spheroids were suspended in Cell Recovery Solution (Corning) and collected in a 1.5 mL tube. Matrigel was disintegrated by rotation at 4°C for 30 minutes. Spheroids were centrifuged for 5 minutes and washed twice with PBS at 4°C. DNA was purified using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany).
After aspirating the medium, lysis buffer (Takara Bio, Kusatsu, Japan) was added directly to each well of the spheroid culture. RNA was purified using the NucleoSpin RNA II kit (Takara Bio).

1.4. Mutation Analysis Next-generation sequencing of cancer-related genes was performed by Macrogen. Amplicon libraries were prepared using the Ion AmpliSeq Comprehensive Cancer Panel (Thermo Fisher). DNA sequences were sequenced using an Ion Proton sequencer (Thermo Fisher). Sequencing data were processed using Ion Torrent Suite software v5.0.4 (Thermo Fisher). Gene mutations were extracted using Torrent Variant Caller v5.0.4 (Thermo Fisher) against the hg19 human genome.

1.5. Mutation filtration. The extracted gene mutations were annotated using ANNOVAR software, and only those occurring at a frequency of 20% or higher were further extracted for nonsynonymous substitutions (gene mutations resulting in amino acid changes primarily due to single base substitutions), frameshift mutations (gene mutations resulting in significant changes in amino acid sequence due to base deletion or insertion, resulting in a shift in the translational frame), and splicing mutations (gene mutations occurring at splice sites). Polymorphic alleles were then filtered out by referencing two databases: the Human Genetic Variation Database (HGVD) and 1KJPN. Incorrect mutations (testing errors) were filtered out using Integrative Genomics Viewer software v2.3 (Broad Institute, Cambridge, MA, USA).

1.6. RNA-Seq analysis
RNA-Seq analysis was performed by Macrogen. Prepared RNA libraries were sequenced to a depth of approximately 40 million reads per sample using an Illumina HighSeq 2000 sequencer. Sequence reads were mapped to the hg19 human genome using the HISAT2 program, and transcripts were collected using the String Tie program. Read counts were scored using the feature Counts program.

2. Results The responsiveness of patient-derived cancer spheroids is known to reflect the drug sensitivity of cancer patients (WO 2019/111998; T Yamamoto et al., Cancers 2020, 12(8), 2010). To obtain a more accurate molecular marker for FGFR inhibitor sensitivity, we calculated the mRNA expression ratio of FGFR to EGFR based on RNA-Seq data from cancer spheroids derived from colorectal cancer patients. FGFR exists as four paralogs: FGFR1, FGFR2, FGFR3, and FGFR4. However, FGFR3 and FGFR4 are predominantly expressed in colorectal epithelial cells. First, we used the RNA-Seq data to sum the expression levels of FGFR1 to FGFR4 by adding the number of reads per million base pairs for each FGFR paralog (sumFGFR). We then divided this value by the expression level of EGFR, which was calculated in the same way. Hereinafter, this value will be referred to as the sumFGFR/EGFR ratio or the F/E ratio.

The sumFGFR/EGFR ratio was relatively high (14.8-207) in six of the seven colon cancer spheroid lines that responded to erdafitinib alone, whereas it was 14 or less in 15 of the lines that did not respond to erdafitinib (Table 1). HC28T was the only exception, responding to erdafitinib despite an F/E ratio of 11.3 (<14). On the other hand, three lines (HC129T, HC22T, and HC40T) had F/E ratios of 26-36 (higher than 14) but had GEIs of 0.80-0.95, and were resistant to erdafitinib. Two of these three lines (HC129T and HC40T) also failed to respond to combination therapy with erdafitinib and erlotinib (GEIs of 0.74 and 0.86, respectively). HC129T had an FGFR1 R820H mutation, and HC40T had an activating PIK3CA E545K mutation. For HC22T, the combination of erdafitinib and erlotinib was effective, with a GEI of 0.59.
R: Responsive, NR: Non-responsive. A GEI < 0.7 was defined as responsive.

Next, we examined the F/E ratios of cholangiocarcinoma and urothelial carcinoma cell lines whose preclinical sensitivity to FGFR inhibitors has been published using the Broad Institute Cancer Cell Line Encyclopedia (https://portals.broadinstitute.org/ccle). These in vitro sensitivity data were obtained from classical cell lines, not patient-derived cancer spheroids. Among the cholangiocarcinoma cell lines tested for sensitivity, two showed FGFR inhibitor-responsive F/E ratios of 9.5 and 9.0, respectively, while the four non-responsive cell lines had significantly lower F/E ratios ranging from 0.1 to 1.8 (Table 2). Among urothelial carcinoma cell lines, four showed F/E ratios ranging from 2.81 to 4.9, while the 17 non-responsive cell lines had F/E ratios ranging from 0.01 to 2.75 (Table 3). SW780 was the only exception, responding to FGFR inhibitors but with an F/E ratio of 1.02. SW780 had both an FGFR3 gene fusion and an S771F mutation.

R: Responsive, NR: Non-responsive.

R: Responsive, NR: Non-responsive.

Furthermore, correlation tests were performed between various expression ratios and GEI. Pearson's correlation coefficients (r) were calculated between the ratios of sumFGFR, FGFR3 + FGFR4, FGFR3, or FGFR4 expression levels to EGFR expression levels (sumFGFR4/EGFR ratio, (FGFR3 + FGFR4)/EGFR ratio, FGFR3/EGFR ratio, or FGFR4/EGFR ratio) and GEI, and between FGFR3 or FGFR4 expression levels and GEI. Pearson's correlation coefficients (r) were also calculated between the ratios of sumFGFR expression levels to ERBB2 expression levels (sumFGFR/ERBB2 ratio) or sumFGFR expression levels to MET expression levels (sumFGFR/MET ratio) and GEI. The results are shown in Figures 1 to 8. The sumFGFR4/EGFR ratio, (FGFR3 + FGFR4)/EGFR ratio, FGFR3/EGFR ratio, and FGFR4/EGFR ratio were r=-0.52, -0.52, -0.61, and -0.49, respectively, confirming a correlation with GEI. On the other hand, the expression levels of FGFR3 and FGFR4 were r=-0.20 and -0.21, respectively, showing no correlation. The sumFGFR/ERBB2 ratio and sumFGFR/MET ratio were also r=-0.02 and -0.22, respectively, showing no correlation.

A comparison of the responder and non-responder groups was performed using the sumFGFR/EGFR ratio as an index, and ROC analysis yielded an AUC of 0.841, 95% Cl of 0.684-0.999, and p of 0.0093 (Figures 9 and 10), confirming a correlation between the sumFGFR/EGFR ratio and sensitivity to FGFR inhibitors.

HC6T, HC20T, and HC9T, which are shown to respond to erdafitinib in Table 1, are also known to respond to AZD4547 (FGFR1-3 inhibitor), infigratinib (BGJ398, FGFR1-3 inhibitor), fisogatinib (BLU554, FGFR4-selective inhibitor), rogaratinib (BAY1163877, FGFR1-3 inhibitor), LY2874455 (pan-FGFR1-4 inhibitor), and futibatinib (TAS-120, pan-FGFR1-4 inhibitor), and the gene copy number and mRNA expression levels of FGFR1-4 and their ligands do not match the responsiveness of patient-derived cancer spheroids (WO 2019/111998; T Yamamoto et al., Cancers 2020, 12(8), 2010). These results indicate that the FGFR/EGFR ratio can be used as a biomarker for determining sensitivity to FGFR inhibitors, including erdafitinib, and that the FGFR/EGFR ratio is a better predictor of FGFR inhibitor sensitivity than genetic alterations or expression levels of the FGFR gene alone. FGFR and EGFR share a common downstream signaling pathway, and it is speculated that FGFR inhibitors may be effective in cells with low EGFR expression levels and little signal influx from EGFR.

Claims (13)

A method for determining sensitivity of a cancer patient to an FGFR inhibitor, comprising:
a method comprising: determining an FGFR/EGFR expression ratio in a patient's cancer; and comparing the FGFR/EGFR expression ratio with a reference value, wherein the patient is determined to be sensitive to an FGFR inhibitor when the FGFR/EGFR expression ratio is higher than the reference value ;
where:
The FGFR/EGFR expression ratio is the expression ratio of FGFR and EGFR mRNA; and
the FGFR/EGFR expression ratio is a sumFGFR/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4;
the FGFR/EGFR expression ratio is a (FGFR3+FGFR4)/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3 and/or FGFR4;
the FGFR/EGFR expression ratio is an FGFR3/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3; or
The FGFR/EGFR expression ratio is an FGFR4/EGFR ratio, and the FGFR inhibitor inhibits one or more types of FGFR selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more types of FGFR include FGFR4 . The method of claim 1, wherein the cancer is colorectal cancer, urothelial cancer, or intrahepatic bile duct cancer. The method of claim 1 or 2, wherein the cancer is colorectal cancer. The method of any one of claims 1 to 3, wherein the FGFR inhibitor is erdafitinib. The method of any one of claims 1 to 4, wherein the FGFR/EGFR expression ratio is examined using cancer cells, cancer tissue, or cancer spheroids from the patient. A composition comprising an FGFR inhibitor for treating cancer, the composition being intended for administration to a cancer patient determined to be sensitive to an FGFR inhibitor by the method of any one of claims 1 to 5 . a composition for treating cancer, comprising an FGFR inhibitor, the composition being to be administered to a patient whose cancer has an FGFR/EGFR expression ratio higher than a reference value ;
where:
The FGFR/EGFR expression ratio is the expression ratio of FGFR and EGFR mRNA; and
the FGFR/EGFR expression ratio is a sumFGFR/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4;
the FGFR/EGFR expression ratio is a (FGFR3+FGFR4)/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3 and/or FGFR4;
the FGFR/EGFR expression ratio is an FGFR3/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3; or
The FGFR/EGFR expression ratio is an FGFR4/EGFR ratio, and the FGFR inhibitor inhibits one or more types of FGFR selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more types of FGFR include FGFR4 . The composition of claim 6 or 7 , wherein the cancer is colorectal cancer, urothelial cancer, or intrahepatic bile duct cancer. The composition according to any one of claims 6 to 8 , wherein the cancer is colon cancer. The composition according to any one of claims 6 to 9 , wherein the FGFR inhibitor is erdafitinib. A method for determining the sensitivity of a cancer cell to an FGFR inhibitor, comprising:
a method comprising: determining an FGFR/EGFR expression ratio in cancer cells; and comparing the FGFR/EGFR expression ratio with a reference value, wherein the cancer cells are determined to be sensitive to an FGFR inhibitor when the FGFR/EGFR expression ratio is higher than the reference value ;
where:
The FGFR/EGFR expression ratio is the expression ratio of FGFR and EGFR mRNA; and
the FGFR/EGFR expression ratio is a sumFGFR/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4;
the FGFR/EGFR expression ratio is a (FGFR3+FGFR4)/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3 and/or FGFR4;
the FGFR/EGFR expression ratio is an FGFR3/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3; or
The FGFR/EGFR expression ratio is an FGFR4/EGFR ratio, and the FGFR inhibitor inhibits one or more types of FGFR selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more types of FGFR include FGFR4 . A method for selecting cells sensitive to an FGFR inhibitor , comprising:
a method comprising: determining an FGFR/EGFR expression ratio in a cancer cell; and comparing the FGFR/EGFR expression ratio with a reference value, wherein the cancer cell is selected when the FGFR/EGFR expression ratio is higher than the reference value ;
where:
The FGFR/EGFR expression ratio is the expression ratio of FGFR and EGFR mRNA; and
the FGFR/EGFR expression ratio is a sumFGFR/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4;
the FGFR/EGFR expression ratio is a (FGFR3+FGFR4)/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3 and/or FGFR4;
the FGFR/EGFR expression ratio is an FGFR3/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3; or
The FGFR/EGFR expression ratio is an FGFR4/EGFR ratio, and the FGFR inhibitor inhibits one or more types of FGFR selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more types of FGFR include FGFR4 . A method for screening for an FGFR inhibitor, comprising contacting a cancer cell having an FGFR/EGFR expression ratio higher than a reference value with a candidate substance for an FGFR inhibitor in vitro ;
where:
The FGFR/EGFR expression ratio is the expression ratio of FGFR and EGFR mRNA; and
the FGFR/EGFR expression ratio is a sumFGFR/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4;
the FGFR/EGFR expression ratio is a (FGFR3+FGFR4)/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3 and/or FGFR4;
the FGFR/EGFR expression ratio is an FGFR3/EGFR ratio, and the FGFR inhibitor inhibits one or more FGFRs selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more FGFRs include FGFR3; or
The FGFR/EGFR expression ratio is an FGFR4/EGFR ratio, and the FGFR inhibitor inhibits one or more types of FGFR selected from FGFR1, FGFR2, FGFR3, and FGFR4, and the one or more types of FGFR include FGFR4 .