Method for diagnosing colorectal cancer and method for evaluating effect of adjuvant chemotheraphy applied to colorectal cancer patient

Abstract

The present invention provides a method for detecting colorectal cancer in a subject, said method comprising a step for determining the methylation state of at least one of the FGD5 gene and the GPC6 gene in the subject.

Description

Method for diagnosing colorectal cancer and method for evaluating the effect of adjuvant chemotherapy applied to colorectal cancer patients

[0001] The present invention relates to a method for diagnosing colorectal cancer and a method for evaluating the effect of adjuvant chemotherapy administered to colorectal cancer patients.

[0002] Morbidity and mortality from stage III colorectal cancer (CRC) remain high worldwide even after curative surgery. Adjuvant chemotherapy (ACT) is the standard treatment for improving clinical outcomes after curative surgery in stage III CRC. To improve clinical outcomes, accurate monitoring of minimal residual disease (MRD) is required to evaluate the efficacy of ACT and detect recurrence. Clinical findings by radiological means, i.e., computed tomography (CT) and magnetic resonance imaging (MRI), are recognized as definitive diagnoses of recurrence after surgery. However, accurate and readily available methods for tracking MRD using serum tumor markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are needed. Research is ongoing into the most potentially relevant approaches for liquid biopsy systems, such as transcripts in peripheral blood exosomes (Non-Patent Documents 1 and 2) and microRNAs (miRNAs) (Non-Patent Documents 3-5).

[0003] The 2017 State of the Union address launched cancer genomic medicine. Matched drugs addressing pathogenic targets in tumor tissues may improve clinical outcomes of malignant tumors (NPLs 6-8). Liquid biopsies have been used as biomarkers to detect genetic abnormalities in circulating tumor DNA (ctDNA) using plasma samples, including companion diagnostic (CDx) markers and comprehensive genomic profiling (CGP) tests, to enable drug targeting for patients with specific genomic abnormalities (NPL 9). Patients with non-small cell lung cancer (NSCLC) undergo liquid biopsies to detect epidermal growth factor receptor (EGFR) mutations R858L or T790M using CDx markers, and the results determine the optimal drug to be used, such as gefitinib or osimeritinib (NPLs 10, 11). The CGP, Guardant360 CDx, and FoundationOne® Liquid CDx tests were launched in August 2020 and applied following the current standard of care in the clinical treatment of severely advanced cases.

[0004] Liquid biopsy is the most effective approach for MRD monitoring, diagnosing recurrence earlier than conventional diagnostic tools such as high-resolution computed tomography (CT) and magnetic resonance imaging (MRI). To avoid misdiagnosis of recurrence, frequent postoperative blood testing is required to accurately track cancer-specific and robust events in peripheral blood that are invisible to the naked eye. Customized blood CGP testing is expected to play a central role in MRD monitoring in the future, facilitating the rapid selection of appropriate medications. However, current frequent MRD monitoring systems based on next-generation sequencing (NGS) are not practical due to the high sequencing costs. Given this background, there is a strong need for novel technologies that can predict recurrence after curative surgery for colorectal cancer at lower cost and with greater ease than conventional methods.

[0005] Iwaya T , Endo F , Takahashi F , Tokino T , Sasaki Y , Nishizuka SS . Frequent Tumor Burden Monitoring of Esophageal Squamous Cell Carcinoma With Circulating Tumor DNA Using Individually Designed Digital Polymerase Chain Reaction. Gastroenterology 2021;160(1): 463-465 e464.Mimori K, Fukagawa T, Kosaka Y, Kita Y, Ishikawa K, Etoh T, Iinuma H, Sasako M, Mori M. Hematogenous metastasis in gastric cancer requires isolated tumor cells and expression of vascular endothelial growth factor receptor-1. Clin Cancer Res 2008;14(9): 2609-2616.Matsumura T, Sugimachi K, Iinuma H, Takahashi Y, Kurashige J, Sawada G, Ueda M, Uchi R, Ueo H, Takano Y, Shinden Y, Eguchi H, Yamamoto H, Doki Y, Mori M, Ochiya T, Mimori K. Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer. Br J Cancer 2015;113(2): 275-281.Sugimachi K, Matsumura T, Hirata H, Uchi R, Ueda M, Ueo H, Shinden Y, Iguchi T, Eguchi H, Shirabe K, Ochiya T, Maehara Y,Mimori K. Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation. Br J Cancer 2015;112(3): 532-538.Takano Y, Masuda T, Iinuma H, Yamaguchi R, Sato K, Tobo T, Hirata H, Kuroda Y, Nambara S, Hayashi N, Iguchi T, Ito S, Eguchi H, Ochiya T, Yanaga K, Miyano S, Mimori K. Circulating exosomal microRNA-203 is associated with metastasis possibly via inducing tumor-associated macrophages in colorectal cancer. Oncotarget 2017;8(45): 78598-78613.Sicklick JK, Kato S, Okamura R, Schwaederle M, Hahn ME, Williams CB, De P, Krie A, Piccioni DE, Miller VA, Ross JS, Benson A, Webster J, Stephens PJ, Lee JJ, Fanta PT, Lippman SM, Leyland-Jones B, Kurzrock R. Molecular profiling of cancer patients enables personalized combination therapy: the I-PREDICT study. Nat Med 2019;25(5): 744-750.Tsimberidou AM, Hong DS, Ye Y, Cartwright C, Wheler JJ, Falchook GS, Naing A, Fu S, Piha-Paul S, Janku F, Meric-Bernstam F, Hwu P,Kee B, Kies MS, Broaddus R, Mendelsohn J, Hess KR, Kurzrock R. Initiative for Molecular Profiling and Advanced Cancer Therapy (IMPACT): An MD Anderson Precision Medicine Study. JCO Precis Oncol 2017;2017.Zehir A, Benayed R, Shah RH, Syed A, Middha S, Kim HR, Srinivasan P, Gao J, Chakravarty D, Devlin SM, Hellmann MD, Barron DA, Schram AM, Hameed M, Dogan S, Ross DS, DeLair JF, Mandel, Yao Mandel, DF. DL, Cheng DT, Chandramohan R, Mohanty AS, Ptashkin RN, Jayakumaran G, Prasad M, Syed MH, Rema AB, Liu ZY, Nafa K, Borsu L, Sadowska J, Casanova J, Bacares R, Kiecka IJ, Razumova A, Son JB, Stewart L, Baldi T, Mullaney, Al-Akima, H-Akima E, Abeshouse AA, Penson AV, Jonsson P, Camacho N, Chang MT, Won HH, Gross BE, Kundra R, Heins ZJ, Chen HW, Phillips S, Zhang H, Wang J, Ochoa A, Wills J, Eubank M, Thomas SB, Gardos SM, Reales DN, Galle J, R, Duran, C, Duran, C, Czech, Abida Feldman DR, Gounder MM, Hakimi AA, Harding JJ, Iyer G, Janjigian YY, Jordan EJ, Kelly CM, Lowery MA,Morris LGT, Omuro AM, Raj N, Razavi P, Shoushtari AN, Shukla N, Soumerai TE, Varghese AM, Yaeger R, Coleman J, Bochner B, Riely GJ, Saltz LB, Scher HI, Sabbatini PJ, Robson ME, Klimstra DS, Taylor BS, Baselga J, Schultz N, Hyman DM, Arcila ME, [ PubMed ] [ Cross Ref ] Solit DB, Ladanyi M, Berger MF. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med 2017;23(6): 703-713.Nakamura Y, Okamoto W, Kato T, Esaki T, Kato K, Komatsu Y, Yuki S, Masuishi T, Nishina T, Ebi H, Sawada K, Taniguchi H, Fuse N, Nomura S, Fukui M, Matsuda S, Sakamoto Y, Uchigata H, Kitajima K, Kuramoto N, Asakawa T, Olsen S, Odegaard JI, Sato A, Fujii S, Ohtsu A, Yoshino T. Circulating tumor DNA-guided treatment with pertuzumab plus trastuzumab for HER2-amplified metastatic colorectal cancer: a phase 2 trial. Nat Med 2021;27(11): 1899-1903.Wang C. EGFR T790M detection and osimertinib treatment response evaluation by liquid biopsy in lung adenocarcinoma patients with acquired resistance to first generation EGFR tyrosine kinase inhibitors. Diagn Pathol 2018;13(1): 49.Li M, Yang L, Hughes J, van den Hout A, Burns C, Woodhouse R, Dennis L, Hegde P, Oxnard GR, Vietz C. Driver Mutation Variant Allele Frequency in Circulating Tumor DNA and Association with Clinical Outcome in Patients with Non-Small Cell Lung Cancer and EGFR- and KRAS-Mutated Tumors. J Mol Diagn 2022;24(5): 543-553.,

[0006] An objective of the present invention is to provide a novel technique that can predict the onset and recurrence of colorectal cancer more easily than conventional methods.

[0007] As a result of extensive research into the above-mentioned problems, the present inventors have found that the methylation status of the FGD5 gene, the GPC6 gene, and the MSC gene in a subject is extremely useful as a marker for the presence of colorectal cancer cells, and have carried out further research based on this finding, thereby completing the present invention.

[0008] [1] A method for detecting colorectal cancer in a subject, comprising a step of determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject. [2] The method of [1], comprising a further step of determining the methylation status of an MSC gene in the subject. [3] The method of [1] or [2], wherein the subject has undergone radical surgery for colorectal cancer. [4] The method of any of [1] to [3], wherein the methylation status is determined based on a liquid sample derived from the subject. [5] The method of [4], wherein the liquid sample is a blood sample. [6] The method of any of [1] to [5], wherein the methylation status is determined by PCR. [7] A method for evaluating the effect of adjuvant chemotherapy for colorectal cancer in a subject, comprising a step of determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject. [8] The method of [7], wherein the methylation status of an MSC gene in the subject is further determined. [9] The method according to [7] or [8], wherein the subject has undergone radical surgery for colorectal cancer.

[10] The method according to any one of [7] to [9], wherein the determination of the methylation status is carried out based on a liquid sample derived from the subject.

[11] The method according to

[10] , wherein the liquid sample is a blood sample.

[12] The method according to any one of [7] to

[10] , wherein the determination of the methylation status is carried out by PCR.

[13] A diagnostic kit for colorectal cancer, comprising: (1) a PCR primer pair capable of amplifying a part or all of the base sequence of the FGD5 gene; and / or (2) a PCR primer pair capable of amplifying a part or all of the base sequence of the GPC6 gene.

[14] The kit of

[13] , wherein the PCR primer pair capable of amplifying part or all of the nucleotide sequence of the FGD5 gene is a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 4 and a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 5, and the PCR primer pair capable of amplifying part or all of the nucleotide sequence of the GPC6 gene is a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 6 and a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 7.

[15] (3) The kit of

[13] or

[14] , further comprising a PCR primer pair capable of amplifying part or all of the nucleotide sequence of the MSC gene.

[16] The kit of

[15] , wherein the PCR primer pair capable of amplifying part or all of the nucleotide sequence of the MSC gene is a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 8 and a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 9.

[0009] According to the present invention, it is possible to determine whether a subject has colorectal cancer or whether colorectal cancer has recurred in a subject after radical surgery for colorectal cancer more quickly than with conventional methods. Furthermore, according to the present invention, it is also possible to evaluate whether adjuvant chemotherapy for colorectal cancer is effective in a subject receiving the adjuvant chemotherapy.

[0010] Figure 1 shows an example of methylated ctDNA levels calculated using the AMUSE (amplicon of methylated sites using a specific enzyme) assay based on digital PCR findings (hereinafter, the methylated ctDNA level quantified using the AMUSE assay is referred to as the AMUSE score). HapII is a methylation site-specific enzyme. Because RNase P contains genomic DNA sequences that cannot be methylated, RNase P cfDNA was used as a control for HapII treatment. CFDNA of the target gene and cfDNA of the RNase P gene that was not treated with HapII were amplified, and the cfDNA (10) / RNase P (5) cfDNA ratio (2) was calculated. Next, cfDNA was amplified by dPCR using primers for the target gene and RNase P gene after HapII treatment, and the target cfDNA (8) / RNase P (6) cfDNA ratio (1.3) was calculated for the HapII-treated cfDNA. Finally, the methylation ratio of the target cfDNA compared to the RNase P cfDNA (0.65 = 2 / 1.3) was calculated. Figure 2 outlines the process used in the present invention to identify methylation biomarkers relevant to predicting colorectal cancer recurrence. From 473,864 probes from the Illumina Infinium Human Methylation 450K, 100 CpG island sites that were particularly highly methylated in colorectal cancer tissues were selected. Further selection criteria (specific to cancer cells, absent in blood cells, and absent in normal tissues) were applied, resulting in the identification of 50 probes. The identified locations were verified by amplifying the genomic region. These probes were further selected based on the following criteria: an inverse correlation between the methylated DNA level of the methylated site (methylation rate calculated from a bisulfite sequencing database, sometimes referred to as "beta value") and the expression of the target gene, and a significant correlation between the beta value and overall survival rate in stage III colorectal cancer cases. Abbreviations: CRC: colorectal cancer. Validation of the beta values ​​of the methylated sites of the FDG5, GPC6, and MSC genes.A) Colorectal cancer cases with high beta values ​​of promoter methylation sites of the FDG5, GPC6, and MSC genes had a poorer prognosis than cases with low beta values. B) A significant inverse correlation between gene expression and the beta value of each gene was statistically verified in plasma samples from TCGA CRC cases. Figure 4 shows the basic characteristics of the three biomarkers selected in this study. A) The methylation beta values ​​of CpG islands of FGD5, GPC6, and MSC were investigated from tumor / normal tissue pairs of 40 cases of colorectal cancer (stages I to IV). B) ROC curves drawn using three markers, two markers, and ctDNA amount. Abbreviations: ctDNA: circulating tumor DNA; ROC, receiver operating characteristic. Figure 5-1 shows the confirmation of postoperative recurrence suspected on CT images using the AMUSE score. The boxes (AC) below the graph indicate the ACT period (similar to Figures 5B-F and 6). The gray and black boxes indicate the AMUSE score and serum CEA protein level, respectively. The date of surgery (P), suspected recurrence on the image (Q), and recurrence diagnosis (R) are shown. These six cases have a higher Q than the 28 recurrence cases. Abbreviations: ACT: adjuvant chemotherapy; AMUSE, amplicon of methylated sites using a specific enzyme; CEA: carcinoembryonic antigen. Figure 5-2 is a continuation of Figure 5-1. Figure 6-1 shows the specificity of the AMUSE score in 19 cases without recurrence. The box (AC) below the graph indicates the duration of ACT. Two patients (5G and 5I) showed false-positive findings (arrows). Abbreviations: ACT: adjuvant chemotherapy; AMUSE: amplicon of methylated sites using a specific enzyme. Figure 6-2 is a continuation of Figure 6-1.Figure 7 shows the clinical benefit of the AMUSE assay. (A) Comparison of time to diagnosis of recurrence by CT (left bar) and AMUSE assay (right bar) in 22 cases with a positive AMUSE assay. (B) Comparison of recurrence-free survival rates between cases with AMUSE positive results at the second or third blood sampling time point (n=19) and cases with AMUSE positive results at both the second and third blood sampling time points (n=5) in recurrent cases. (C) Comparison of recurrence-free survival rates between cases with AMUSE positive results at the second or third blood sampling time point (n=42) and cases with AMUSE positive results at both the second and third blood sampling time points (n=5) in all cases. (D and E) Comparison of recurrent cases (D) and all cases (E) by serum CEA. Abbreviations: AMUSE: amplicon of methylated sites using a specific enzyme; CEA: carcinoembryonic antigen. Figure 8 shows stratification of postoperative changes in tumor burden affected by adjuvant chemotherapy. Four groups were stratified based on the pattern of changes in tumor burden affected by adjuvant chemotherapy. Group A) Tumor burden was reduced by surgery, and residual MRD was removed by ACT. Group B) Tumor burden was reduced by surgery but increased after ACT due to resistance or lack of sensitivity to the regimen. Group C) Tumor burden did not change due to a large number of remaining cancer cells that were insensitive to ACT. Group D) Tumor was completely removed by radical surgery alone. Abbreviations: ACT: adjuvant chemotherapy; MRD: minimal residual disease.

[0011] The present invention will be described in detail below.

[0012] 1. Method for detecting colorectal cancer in a subject The present invention provides a method for detecting colorectal cancer in a subject (hereinafter, sometimes referred to as the "detection method of the present invention"), which comprises a step of determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject.

[0013] In the detection method of the present invention, the methylation status of at least one of the FGD5 gene and the GPC6 gene is determined. In a preferred embodiment, the detection method of the present invention further determines the methylation status of the MSC gene. In a particularly preferred embodiment, the detection method of the present invention determines the methylation status of the FGD5 gene, the GPC6 gene, and the MSC gene.

[0014] The FGD5 gene (FYVE, RhoGEF and PH domain containing 5, Gene ID: 152273), also known as "ZFYVE23," is located at chromosomal location 8q13.3 in humans and is a gene with two exons. The FGD5 gene is known to be highly expressed in the spleen and lungs of normal humans. The nucleotide sequence of the human FGD5 gene is shown in SEQ ID NO: 1.

[0015] The GPC6 gene (glypican 6, Gene ID: 10082), also known as "OMIMD1," is located at chromosomal location 13q31.3-q32.1 in humans and is a gene with 12 exons. The GPC6 gene is known to be highly expressed in the gallbladder and urinary bladder in normal humans. The nucleotide sequence of the human GPC6 gene is shown in SEQ ID NO: 2.

[0016] The MSC gene (musculin, Gene ID: 9242), also known as "ABF1," "MYOR," "ABF-1," "bHLHa22," etc., is located at chromosome 8q13.3 in humans, and the MSC gene, which is a gene having two exons, is known to be highly expressed in the placenta and gallbladder of normal humans. The nucleotide sequence of the human MSC gene is shown in SEQ ID NO: 3.

[0017] In the detection method of the present invention, the methylation state of the above-mentioned gene is determined. However, methods for determining the methylation state of a gene are known to those skilled in the art, and any such method may be used. In the detection method of the present invention, "determining the methylation state of gene A" may also mean "determining the amount of methylation in gene A." The methylation state of a gene can be detected and its amount determined, for example, by methyl-DNA immunoprecipitation (MeDIP), methylation-sensitive restriction enzyme (MSRE) analysis, bisulfite sequencing, etc. In a preferred embodiment of the detection method of the present invention, the methylation state of a gene can be determined by combining methylation-sensitive restriction enzyme analysis with PCR. The PCR method combined with methylation-sensitive restriction enzyme analysis is preferably real-time PCR or digital PCR. Details of the method for determining the methylation state using a combination of methylation-sensitive restriction enzyme analysis and PCR are described in the Examples section of this specification.

[0018] As used herein, the term "determining the methylation status of gene A" refers to qualitatively analyzing the presence or absence of a methylated region in the entire length or a portion of gene A. The nucleotide length of the region for determining the methylation status is not particularly limited, but in one embodiment, it is preferable to set a length suitable for PCR amplification. The nucleotide length of the region for determining the methylation status may be, but is not limited to, typically 30 to 1000, preferably 50 to 300, more preferably 75 to 200, and particularly preferably 75 to 150 nucleotides. Note that when the region is amplified using a primer, this nucleotide length includes the primer binding site.

[0019] In the detection method of the present invention, when the determination of the methylation status is achieved by a method involving PCR such as digital PCR, the primer pairs that can be used in the PCR reaction may be appropriately designed using a method known per se to amplify part or all of the methylated regions of the FGD5 gene, GPC6 gene, and MSC gene. In a preferred embodiment of the detection method of the present invention, such primer pairs may be, but are not limited to, the following:

[0020] Primer pair for FGD5 gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 4 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 5. Primer pair for GPC6 gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 6 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 7. Primer pair for MSC gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 8 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9.

[0021] In one embodiment, probes for confirming amplification of target regions of the FGD5 gene, GPC6 gene, and MSC gene can be used in combination to determine the methylation status. Those skilled in the art can appropriately design such probes for confirming amplification, taking into account the nucleic acid sequence of the target region to be amplified. In one embodiment of the detection method of the present invention, the following probes can be used for confirming amplification of each gene, but are not limited to these.

[0022] Probe for confirming amplification of a target region of the FGD5 gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 10. Probe for confirming amplification of a target region of the GPC6 gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 11. Probe for confirming amplification of a target region of the MSC gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 12.

[0023] If the FGD5 gene, GPC6 gene, and / or MSC gene in a subject has a higher methylation status compared to a normal control, the subject is determined to have a high probability of having colorectal cancer. Furthermore, if the subject has previously had colorectal cancer, if the FGD5 gene, GPC6 gene, and / or MSC gene in the subject has a higher methylation status compared to a normal control, the subject is determined to have a high probability of having colorectal cancer recurrence. In one embodiment of the present invention, the methylation status of the FGD5 gene, GPC6 gene, and / or MSC gene in the subject can be scored and compared with the methylation status score in a normal subject. Note that a method known per se can be used to score the methylation status. If the methylation status score of the FGD5 gene, GPC6 gene, and / or MSC gene in the subject is (statistically significantly) higher than that of the normal control, the subject is determined to have a high probability of having colorectal cancer. Furthermore, if a subject has previously had colorectal cancer, and the methylation status score of the FGD5 gene, GPC6 gene, and / or MSC gene in the subject is (statistically significantly) higher than that of a normal control, it is determined that there is a high possibility that colorectal cancer has recurred in the subject. Alternatively, a cutoff value for the methylation status score can be set in advance, and if the subject's score is higher than the cutoff value, it can be determined that there is a high possibility that the subject has colorectal cancer or that there is a high possibility that colorectal cancer has recurred in the subject.

[0024] The "subject" in the detection method of the present invention is not particularly limited as long as it is a mammal suffering from colorectal cancer. Examples of the subject include, but are not limited to, livestock animals (such as cattle, sheep, cats, dogs, and horses), laboratory animals (such as rabbits, mice, and rats), and primates (such as humans and non-human primates). In a preferred embodiment, the subject is a human. In this specification, the term "subject" can also be used interchangeably with "individual," "patient," or "subject."

[0025] In one embodiment of the detection method of the present invention, the subject may have undergone radical surgery for colorectal cancer. Without wishing to be bound by theory, it is believed that the methylation status of the genes of interest in the present invention (i.e., the FGD5 gene, the GPC6 gene, and / or the MSC gene) can enable early detection of recurrence of colorectal cancer in subjects who have undergone radical surgery for colorectal cancer.

[0026] In the detection method of the present invention, "colorectal cancer" includes all types of colorectal cancer, such as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, etc. Note that "colorectal cancer" in the present invention may also include colon cancer and rectal cancer.

[0027] In the detection method of the present invention, the determination of the methylation status of the gene of interest in the present invention may be performed by any means as long as the methylation status can be determined; however, from the viewpoint of invasiveness, it is preferable to perform the determination based on a liquid sample derived from a subject. The liquid sample derived from a subject is not particularly limited as long as the methylation status of the gene of interest in the present invention can be determined by analyzing the liquid sample. Examples of liquid samples derived from a subject include blood, lymph, tissue fluid, and body cavity fluid, with blood being preferred, and plasma being more preferred. By determining the methylation status of the FDG5 gene, GPC6 gene, and / or MSC gene using cfDNA (cell-free DNA) and ctDNA (circulating tumor DNA) contained in plasma, early diagnosis of colorectal cancer in a subject can be achieved simply, at low cost, and minimally invasively.

[0028] 2. Method for Evaluating the Effect of Adjuvant Chemotherapy The present invention also provides a method for evaluating the effect of adjuvant chemotherapy being received by a subject, the method comprising a step of determining the methylation status of at least one of the FDG5 gene and the GPC6 gene in the subject receiving adjuvant chemotherapy for colorectal cancer (hereinafter, sometimes referred to as the "evaluation method of the present invention").

[0029] In the evaluation method of the present invention, the methylation status of at least one of the FGD5 gene and the GPC6 gene is determined. In a preferred embodiment, the evaluation method of the present invention further determines the methylation status of the MSC gene. In a particularly preferred embodiment, the evaluation method of the present invention determines the methylation status of the FGD5 gene, the GPC6 gene, and the MSC gene.

[0030] In the evaluation method of the present invention, the "FDG5 gene," the "GPC6 gene," the "MSC gene," the "subject," the "colon cancer," the "determination of gene methylation status," the "sample derived from the subject," etc. are the same as those described in the detection method of the present invention above.

[0031] In the evaluation method of the present invention, if the FGD5 gene, GPC6 gene, and / or MSC gene in a subject undergoing adjuvant chemotherapy for colorectal cancer have a high methylation status compared to a normal control, the subject is determined to have a high probability of having colorectal cancer, and therefore, the effect of the adjuvant chemotherapy currently being received by the subject is assessed to be low. Conversely, if the FGD5 gene, GPC6 gene, and / or MSC gene in a subject undergoing adjuvant chemotherapy for colorectal cancer have a similar methylation status compared to a normal control, the subject is determined to have a high probability of not having colorectal cancer, and therefore, the effect of the adjuvant chemotherapy currently being received by the subject is assessed to be high. Furthermore, if the subject has previously had colorectal cancer, if the FGD5 gene, GPC6 gene, and / or MSC gene in the subject have a high methylation status compared to a normal control, the subject is determined to have a high probability of having recurrence of colorectal cancer, and therefore, the effect of the adjuvant chemotherapy currently being received by the subject is assessed to be low. Conversely, if the FGD5 gene, GPC6 gene, and / or MSC gene in the subject have a methylation status comparable to that of the normal control, it is determined that the subject is likely to have no recurrence of colorectal cancer, and therefore, the adjuvant chemotherapy currently being received by the subject is evaluated as being highly effective. Furthermore, as explained in the detection method of the present invention above, the methylation status can be scored and used for evaluation.

[0032] 3. Diagnostic Kit for Colon Cancer The present invention also provides a diagnostic kit for colon cancer, comprising: (1) a PCR primer pair capable of amplifying a part or all of the nucleotide sequence of the FDG5 gene; and / or (2) a PCR primer pair capable of amplifying a part or all of the nucleotide sequence of the GPC6 gene (hereinafter, sometimes referred to as the "kit of the present invention").

[0033] The kit of the present invention comprises a PCR primer pair capable of amplifying part or all of the nucleotide sequence of at least one of the FGD5 gene and the GPC6 gene. In a preferred embodiment, the kit of the present invention further comprises a PCR primer pair capable of amplifying part or all of the nucleotide sequence of the MSC gene. In a particularly preferred embodiment, the kit of the present invention comprises a PCR primer pair capable of amplifying part or all of the nucleotide sequences of the FGD5 gene, the GPC6 gene, and the MSC gene.

[0034] The sequences of the primer pairs for amplifying the FDG5 gene, GPC6 gene, and MSC gene are not particularly limited as long as they can amplify part or all of these genes and amplify regions that can determine the methylation status of these genes. The nucleotide length of each primer is also not particularly limited, but can usually be 16 to 40, preferably 17 to 30, and more preferably 18 to 25.

[0035] In a preferred embodiment of the kit of the present invention, the primer pair may be, but is not limited to, the following:

[0036] Primer pair for FGD5 gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 4 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 5. Primer pair for GPC6 gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 6 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 7. Primer pair for MSC gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 8 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9.

[0037] In one embodiment, the kit of the present invention can include not only primer pairs but also probes for confirming amplification of the target regions of the FGD5 gene, GPC6 gene, and MSC gene. Such probes for confirming amplification can be appropriately designed by those skilled in the art, taking into account the nucleic acid sequence of the target region to be amplified. In one embodiment of the kit of the present invention, the following probes for confirming amplification of each gene can be included in the kit, but are not limited to these.

[0038] Probe for confirming amplification of a target region of the FGD5 gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 10. Probe for confirming amplification of a target region of the GPC6 gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 11. Probe for confirming amplification of a target region of the MSC gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 12.

[0039] The kit of the present invention may contain, in addition to the above-mentioned primer pairs and probes, reagents, positive / negative controls, instructions, and the like.

[0040] 4. Use of PCR primer pairs capable of amplifying part or all of the nucleotide sequence of the FGD5 gene and the GPC6 gene in in vitro / ex vivo diagnosis of colorectal cancer or evaluation of the effect of adjuvant chemotherapy for colorectal cancer The present invention also provides the use of: (1) a PCR primer pair capable of amplifying part or all of the nucleotide sequence of the FDG5 gene; and / or (2) a PCR primer pair capable of amplifying part or all of the nucleotide sequence of the GPC6 gene in in vitro / ex vivo diagnosis of colorectal cancer or evaluation of the effect of adjuvant chemotherapy for colorectal cancer (hereinafter sometimes referred to as "use of the present invention").

[0041] In the use of the present invention, a PCR primer pair capable of amplifying part or all of the base sequence of at least one of the FGD5 gene and the GPC6 gene is used for in vitro / ex vivo diagnosis of colorectal cancer. In a preferred embodiment, the use of the present invention further uses a PCR primer pair capable of amplifying part or all of the base sequence of the MSC gene. In a particularly preferred embodiment, the use of the present invention uses a PCR primer pair capable of amplifying part or all of the base sequences of the FGD5 gene, the GPC6 gene, and the MSC gene.

[0042] The sequences of the primer pairs for amplifying the FDG5 gene, GPC6 gene, and MSC gene are not particularly limited as long as they can amplify part or all of these genes and amplify regions that can determine the methylation status of these genes. The nucleotide length of each primer is also not particularly limited, but can usually be 16 to 40, preferably 17 to 30, and more preferably 18 to 25.

[0043] In a preferred embodiment of the use of the present invention, the primer pair may be, but is not limited to, the following:

[0044] Primer pair for FGD5 gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 4 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 5. Primer pair for GPC6 gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 6 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 7. Primer pair for MSC gene: a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 8 and a nucleic acid comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9.

[0045] In addition, in the use of the present invention, not only primer pairs but also probes for confirming amplification of the target regions of the FGD5 gene, GPC6 gene, and MSC gene can be used. Those skilled in the art can appropriately design such probes for confirming amplification taking into account the nucleic acid sequence of the target region to be amplified. In one embodiment, the following probes can be used to confirm amplification of each gene, but are not limited to these.

[0046] Probe for confirming amplification of a target region of the FGD5 gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 10. Probe for confirming amplification of a target region of the GPC6 gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 11. Probe for confirming amplification of a target region of the MSC gene: a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 12.

[0047] In using the present invention, the criteria for diagnosing a subject as having colorectal cancer and the criteria used to evaluate the effectiveness of adjuvant chemotherapy for colorectal cancer are the same as those described in "1. Method for detecting colorectal cancer in a subject" and "2. Method for evaluating the effectiveness of adjuvant chemotherapy."

[0048] 5. Diagnostic Method for Colorectal Cancer The present invention also provides a method for diagnosing colorectal cancer in a subject (hereinafter, sometimes referred to as the "diagnostic method of the present invention"), which comprises a step of determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject.

[0049] The method for determining the sequence and methylation status of each gene in the diagnostic method of the present invention is the same as that explained in "1. Method for detecting colorectal cancer in a subject."

[0050] In one embodiment, the diagnostic method of the present invention may comprise the step of administering a therapeutically effective amount of an anticancer agent to a subject diagnosed with colorectal cancer by using the diagnostic method of the present invention.

[0051] The anticancer drug administered to the subject is not particularly limited, and any anticancer drug can be used. Examples of anticancer drugs include, but are not limited to, alkylating agents (e.g., cyclophosphamide, cisplatin, etc.), antimetabolites (e.g., methotrexate, 5-FU, etc.), microtubule inhibitors (e.g., paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, etoposide, etc.), molecular targeted agents (e.g., imatinib, trastuzumab, etc.), immune checkpoint inhibitors (e.g., nivolumab, pembrolizumab, etc.), hormone therapy drugs (e.g., tamoxifen, flutamide, etc.), proteasome inhibitors (e.g., bortezomib, carfilzomib, etc.), and PARP inhibitors (e.g., olaparib, rucaparib, etc.).

[0052] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0053] This study was approved by the Research Ethics Review Board of the Cancer Institute Ariake Hospital. The IRB receipt number is 2010-1058 (March 7, 2019). Furthermore, the Clinical Research Ethics Review Board of the Kyushu University Medical School reviewed and approved the project (number 2020-321 (September 1, 2020)).

[0054] [method]

[0055] Clinical Cases: A total of 47 patients with stage IIIa or IIIb colon cancer (n = 30) and rectal cancer (n = 17) underwent curative surgery (Table 1). All patients received standard adjuvant capecitabine and oxaliplatin (eight courses over six months) followed by CAPOX therapy. Twenty-eight patients experienced recurrence between 170 and 1,070 days after surgery, and 19 patients were recurrence-free at least 1,090 days after surgery. A total of 180 blood samples were collected and extracted from recurrent patients before surgery, after surgery, during and after ACT, and at the time of recurrence. A total of 111 samples were collected from non-recurrent patients for analysis.

[0056]

[0057] Sample Storage and cfDNA Extraction: Patient-derived leaky plasma samples were centrifuged at 2,500 × G for 10 minutes at 4°C. The supernatant was dispensed into 2 mL tubes and stored in a -80°C freezer. After centrifugation at 16,000 × G for 10 minutes, the supernatant was dispensed. Using a King Fisher Duo Prime (Thermo Fisher Scientific), 2.5 mL and 30 μL of Magmax Extracellular DNA (cfDNA) Lysis / Binding Solution and magnetic beads were placed in the first row of Plate A, and all extracted samples were added. The second row was empty, and the third and fourth rows were filled with 1 mL of Magmax cfDNA Wash Solution. Plate B contained 50 μL of Magmax cfDNA Elution Solution in row 1, 500 μL of 80% ethanol in row 2, and 2 mL of 80% ethanol in row 3. cfDNA was extracted after 30 minutes using the cfDNA extraction protocol.

[0058] [Restriction enzyme treatment of eluted cfDNA] A total of 15 μL of cfDNA was dissolved in 5 μL of pure water as a control. For restriction enzyme treatment, 15 μL of cfDNA, 1 μL of HapII, 2 μL of 10x loading buffer, and 2 μL of pure water were added. The control and restriction enzyme treatment solutions were placed in a thermal cycler and incubated at 37°C for 1 hour to complete the restriction enzyme treatment.

[0059] [Sample Preparation for Digital PCR] Samples for digital PCR were prepared using the two completed solutions. First, a master solution for mixing the samples was prepared. Primers (F and R) and probes targeting FGD5, GPC6, and MSC were used. A total of 6 μL of RNase P, 6 μL of probe, 6 μL of each primer (F, R), 4 μL of pure water, and 60 μL of master mix were mixed. A total of 10.5 μL of the completed solution was dispensed, and 4 μL of each sample control and restriction enzyme treatment solution were mixed. 14.5 μL of the completed solution was subjected to digital PCR. The primer sequences are as follows:

[0060] FGD5 primer f (SEQ ID NO: 4) 5'-CCTTCTCAGCCTTGGCGAGFGGD5 primer r (SEQ ID NO: 5) 5'-GCGTCTTTCTTCGTCGTGGAA

[0061] GPC6 primer f (SEQ ID NO: 6) 5'-GCGGGCTTTCGGCTTGAGGPC6 primer r (SEQ ID NO: 7) 5'-CCAAGAGGGGAAGAATCACAGC

[0062] MSC primer f (SEQ ID NO: 8) 5'-AGCAGTCGCAGCGGAACGMSC primer r (SEQ ID NO: 9) 5'-CAGGGCAGGCTGGTCTTGA

[0063] FGD5 probe (SEQ ID NO: 10) 5'-CGCCAGTATCCCACTCGCACGGCGPC6 probe (SEQ ID NO: 11) 5'-CCGATCCAAGAAGGCATGGTGCAACATACAMSC probe (SEQ ID NO: 12) 5'-CCTGGAGAAGGCTTTGCTCAGCACGC

[0064] Methylation-sensitive restriction enzyme digestion: The eluted cfDNA was divided into two tubes: one for restriction enzyme digestion and the other for a no-enzyme control. A reaction mixture (20 μL) containing 15 μL of cfDNA, 1 μL of HapII (10 units / μL) (Takara Bio), and 1 μL of L buffer (10x) (Takara Bio) was prepared and incubated at 37°C for 1 hour. A control sample was prepared by diluting 15 μL of cfDNA to 20 μL with water and incubating at 37°C for 1 hour.

[0065] Methylation-specific digital PCR Digital PCR was performed using the QuantStudio 3D Digital PCR System (Thermo Fisher Scientific). A digital PCR reaction mixture (14.5 μL) was prepared containing 4 μL of cfDNA, 7.25 μL of QuantStudio 3D Digital PCR Master Mix v2 (ThermoFisher Scientific), 0.725 μL of forward primer (18 μM), 0.725 μL of reverse primer (18 μM), 0.725 μL of TaqMan probe (5 μM), and 0.725 μL of TaqMan Copy Number Reference Assay RNase P (ThermoFisher Scientific). The digital PCR mixture was loaded onto a QuantStudio 3D Digital PCR Chip v2 using the QuantStudio 3D Digital PCR Chip Loader. PCR was performed on a ProFlex 2xFlat PCR system using the following program: 39 cycles of 96°C for 10 minutes, 60°C for 2 minutes, 98°C for 30 seconds, 60°C for 2 minutes, and 10°C for 1 minute. Chip images were captured using the QuantStudio 3D Digital PCR Instrument and analyzed using the QuantStudio 3D Analysis Suite Software.

[0066] [Calculation of methylation ratio] Test samples digested with HapII and undigested control samples were prepared. The copy numbers of the target and RNase P in each sample were measured, and the methylation ratio was calculated as follows:

[0067]

[0068] Assay Design for Methylation Quantification Based on Methylation-Sensitive Restriction Enzymes: We designed a digital PCR assay to measure the methylation ratio of colorectal cancer-specific methylation sites using methylation-sensitive restriction enzymes. cfDNA was extracted from plasma and digested with HapII, a methylation-sensitive restriction enzyme. The HapII recognition site is cleaved when the amplicon is unmethylated. Therefore, when cfDNA was digested with HapII and PCR was performed using primers flanking the recognition site, amplification products were generated only when the HapII recognition site was methylated. After digesting cfDNA with HapII, digital PCR was performed using primers flanking the recognition site to measure the copy number of the methylation site. The copy number of the reference gene, RNase P, was also measured using a similar reaction. Because all cells contain the reference gene, the RNase P copies represent the total cfDNA released from normal and tumor cells. The copy number of colon cancer-specific methylation sites in HapII-digested samples was compared with that in samples without HapII, and the methylation ratio of the sites was calculated. Because pipetting bias can significantly affect the methylation ratio, the target site in each sample was divided by RNase P to calculate the accurate methylation ratio (Figure 1).

[0069] [result]

[0070] 1. Identification of optimal methylation sites In silico analysis was performed to select useful methylation sites. 473,864 probes from Illumina Infinium Human Methylation 450K were used for selection, and the number of methylation sites in colorectal cancer tissues (signal-to-noise ratio > 1.5) was narrowed down to 100 probes. Using data from The Cancer Genome Atlas (TCGA), probes with significant differences in methylation rates between tumor tissues and normal tissues, but low methylation rates between normal tissues and blood cells, were selected, narrowing the number of methylation sites to 50 probes. The specific selection criteria were as follows:

[0071] 1) Beta value (tumor / normal tissue) > 0.4 2) Beta value (normal blood) < 0.15 3) Beta value (normal tissue) < 0.2 (see Figure 2)

[0072] The 50 narrowed down methylation site beta loci were confirmed to be ubiquitously high, and the beta methylation of target sites was inversely correlated with mRNA expression. Furthermore, considering the inverse correlation between beta methylation of target sites and clinical outcome, the selection was narrowed down to three probes. The 59 patients with increased beta methylation of the FGD5 methylation site had a significantly worse prognosis than the 60 patients with decreased beta methylation of the FGD5 methylation site (p<0.05). Similar statistically significant results were also found for GPC6 and MSC (Figure 3). Consequently, the target methylation sites of the three genes, FGD5, GPC6, and MSC, were selected and used for further analysis of cfDNA in peripheral blood.

[0073] 2. Sensitivity and Specificity of Methylation Markers The methylation status of the three target sites was used to distinguish between colorectal cancer patients and those diagnosed by colonoscopy. The control group consisted of plasma samples from 10 patients who underwent colonoscopy at the Department of Surgery, Kyushu University Beppu Hospital between January 2019 and March 2020 without abnormal findings. The tumor group consisted of plasma samples from patients with colorectal cancer (stages 1-4; 40 patients, 10 per group) collected at the Cancer Research Institute Ariake Hospital. Of the three markers, FGD5 and GPC6 showed significantly increased methylation in the tumor group (p = 0.028 and p = 0.0231, respectively; Figure 4A). MSCs showed a tendency to have higher methylation levels in tumor tissue compared to normal tissue (p = 0.062) (Figure 4A). These results demonstrate that the three markers of the present invention can distinguish colorectal cancer patients, suggesting that these markers may be applicable to cancer screening. The receiver operating characteristic (ROC) curve for these markers showed a very high area under the curve (AUC) of 0.840 (Figure 4B), with a sensitivity of 65% and a specificity of 100%, both of which are very high compared to current practical methods.

[0074] 3. Comparison of the Sensitivity of MRD Monitoring by CT and the AMUSE Assay. Forty-seven patients with pathological stage IIIA / B and CurA colorectal cancer (28 recurrence-positive, 19 recurrence-negative) underwent surgery at the Cancer Institute Ariake Hospital between June 2012 and June 2017. Patients underwent CAPOX postoperative ACT. Pathological histological diagnoses of well-differentiated or poorly differentiated adenocarcinoma were included in this study. Methylation status was measured using plasma samples collected preoperatively, postoperatively, after ACT, and at the time of recurrence. Recurrence-free cases were defined as those without recurrence for a mean of 1,551 days (range, 1,093-1,897 days) after surgery. Patients with methylation levels of three genes in cfDNA above the cutoff value were diagnosed as recurrence-positive by liquid biopsy. Before clinical diagnosis of postoperative recurrence using CT imaging, 22 of 28 patients (78.5%) were diagnosed with recurrence (sensitivity, 78.5%).

[0075] 4. Potential for Rescuing False-Negative Cases of Postoperative Recurrence Using the AMUSE Assay Early detection of postoperative recurrence allows for complete removal and improves clinical outcomes in colorectal cancer. However, when serum tumor markers are within the normal range and CT findings are equivocal, a definitive diagnosis of recurrence may not be possible. In a case of rectal cancer, radiotherapy was administered to the recurrent lymph node (arrow) at time point R (Figure 5A). However, the site was noted 140 days earlier (suspicious; time point Q). On the other hand, the AMUSE assay of the present invention enabled the decision to perform immediate removal or definitive radiation therapy based on the AMUSE score at time point Q. Figure 3B shows a case in which ACT was administered to treat ascending colorectal cancer after right hemicolectomy. Small recurrent areas (arrows) were found in both lungs at time point Q. When the AMUSE assay of the present invention was used, recurrence could be diagnosed 500 days after surgery, but the level of the conventional serum marker (CEA) was low, and if conventional indicators were used, aggressive treatment would be hindered.

[0076] Follow-up was continued without extending adjuvant treatment or switching to other chemotherapy. Finally, at time point R, the increase in the size of the suspected area allowed a definitive diagnosis of lung recurrence. The opportunity for definitive treatment was lost, and only systemic chemotherapy was administered. However, in the case of sigmoid colon cancer (Figure 5C), the beta value of the AMUSE assay remained continuously positive from the first day of ACT to the day of diagnosis of recurrence, resulting in multiple missed opportunities for surgery at the liver metastasis site (time points Q1 and Q2). In this case, systemic chemotherapy was administered to control the metastatic site. Furthermore, a local recurrence of rectal cancer in a patient with ulcerative colitis who underwent total colectomy was misdiagnosed (time point Q) (Figure 5D). The beta value of the AMUSE assay increased immediately after ACT and remained positive until time point R, when the clinical diagnosis of recurrence was made. Therefore, in this case, the underlying MRD lesion was sensitive to chemotherapy. However, according to the initial protocol, the drug was discontinued. If the AMUSE assay had been applied to evaluate the efficacy of ACT, it would have been possible to extend patient survival by extending ACT. The AMUSE assay was performed on a patient who underwent radical anterior resection of rectal cancer and postoperative ACT (Figure 5E). Recurrent lymph nodes were found in the right pelvic region at time point R. However, they were also detected 392 days after surgery (time point Q), 652 days earlier than the date of recurrence diagnosis.

[0077] An example of a patient with rectal cancer who underwent radical surgery is shown in Figure 5F. After ACT, the patient was suspected of having a recurrence in the lungs, but was not treated based on imaging findings and serum tumor marker levels (time point Q). Ninety days later, recurrence was confirmed using the AMUSE assay and CT (time point R). The serum tumor marker CEA failed to detect recurrence at time point R.

[0078] 5. Clinical Benefits of the AMUSE Assay Nineteen patients with colorectal cancer were followed up for 1,093 to 1,897 days after each radical surgery and declared "non-recurrence." Seventeen of the 19 patients (89.5%) tested negative for the AMUSE assay, although two showed false-positive beta values ​​(Figures 6G and 6I, arrows).

[0079] Diagnosis by the AMUSE assay enabled diagnosis of recurrence an average of 208 days earlier than conventional clinical diagnosis (Figure 7A). Suspected recurrences were followed up because no clear findings were found for the diagnosis of recurrence. However, the use of CT confirmed the definitive diagnosis of recurrence. Some cases suggest that curative treatment of recurrent lesions would have been possible if the AMUSE assay approach had been implemented.

[0080] Of the 28 cases, the disease-free recurrence rate was compared between 23 cases in which recurrence was positive at the second blood sampling point at the start of postoperative ACT and the third blood sampling point during ACT, and 5 cases in which recurrence was positive at both the second and third blood sampling points (Table 2). Patients in the former group were significantly more likely to recur than those in the latter group (p = 0.013) (Figure 7B). Furthermore, when the disease-free recurrence rate was compared between the five patients who were positive in both tests and the other 42 patients, a significant recurrence was observed in the former group (p = 1.8E-05) (Figure 7C). Therefore, it was demonstrated that the AMUSE assay during ACT can predict recurrence.

[0081]

[0082] On the other hand, advanced CRC cases that were AMUSE positive throughout the ACT period and at the second and third time points simultaneously showed positive serum CEA levels at the same sampling points (Fig. 7E).However, the difference in survival rate between AMUSE-positive and AMUSE-negative patients was greater than that between CEA-positive and CEA-negative patients.

[0083] ACT, approved by the conference as the standard treatment for MRD after curative resection for postoperative stage III CRC, is administered. However, the ACT protocol prescribes a fixed dose and duration without considering each individual's tumor burden. Of the 47 cases, 39 were stratified, excluding 7 false-negative and 2 false-positive cases (Figure 8). Of note, only 6 of 39 cases (16.9%) in Group A received appropriate ACT treatment. ACT was ineffective in 7 cases (17.9%) in Group B and 15 cases (38.5%) in Group C, and ACT was not considered necessary in 11 cases (28.2%) in Group D.

[0084] According to the present invention, it is possible to determine whether a subject has colorectal cancer or whether colorectal cancer has recurred in a subject after radical surgery for colorectal cancer more quickly than conventional methods. Furthermore, according to the present invention, it is also possible to evaluate whether adjuvant chemotherapy for colorectal cancer is effective in a subject receiving the chemotherapy. Therefore, the present invention is extremely useful in the field of colorectal cancer treatment.

[0085] This application is based on patent application No. 2023-158487 filed in Japan (filing date: September 22, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for detecting colon cancer in a subject, comprising determining the methylation status of at least one of the FGD5 and GPC6 genes in the subject.

2. The method of claim 1, further comprising the step of determining the methylation status of the MSC gene in the subject.

3. The method according to claim 1 or 2, wherein the subject has undergone radical surgery for colorectal cancer.

4. The method according to claim 1 or 2, characterized in that the determination of the methylation status is carried out on the basis of a liquid sample derived from the subject.

5. The method of claim 4, wherein the liquid sample is a blood sample.

6. Method according to claim 1 or 2, characterized in that the determination of the methylation status is carried out by the PCR method.

7. A method for evaluating the effectiveness of adjuvant chemotherapy being received by a subject, comprising a step of determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject receiving adjuvant chemotherapy for colorectal cancer.

8. The method of claim 7, further comprising the step of determining the methylation status of the MSC gene in the subject.

9. The method according to claim 7 or 8, wherein the subject has undergone radical surgery for colon cancer.

10. Method according to claim 7 or 8, characterized in that the determination of the methylation status is carried out on the basis of a liquid sample derived from the subject.

11. The method of claim 10, wherein the liquid sample is a blood sample.

12. Method according to claim 7 or 8, characterized in that the determination of the methylation status is carried out by the PCR method.

13. A kit for diagnosing colon cancer, comprising: (1) a PCR primer pair capable of amplifying a part or all of the base sequence of the FGD5 gene; and / or (2) a PCR primer pair capable of amplifying a part or all of the base sequence of the GPC6 gene.

14. The kit according to claim 13, wherein the PCR primer pair capable of amplifying a part or all of the base sequence of the FGD5 gene is a nucleic acid comprising the base sequence shown in SEQ ID NO: 4 and a nucleic acid comprising the base sequence shown in SEQ ID NO: 5, and the PCR primer pair capable of amplifying a part or all of the base sequence of the GPC6 gene is a nucleic acid comprising the base sequence shown in SEQ ID NO: 6 and a nucleic acid comprising the base sequence shown in SEQ ID NO:

7.

15. The kit according to claim 13 or 14, further comprising (3) a PCR primer pair capable of amplifying a part or all of the base sequence of the MSC gene.

16. The kit according to claim 15, wherein the PCR primer pair capable of amplifying a part or the whole of the base sequence of the MSC gene is a nucleic acid comprising the base sequence shown in SEQ ID NO: 8 and a nucleic acid comprising the base sequence shown in SEQ ID NO: 9.

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