WO2025063294A1 - Method for diagnosing colorectal cancer and method for evaluating effect of adjuvant chemotheraphy applied to colorectal cancer patient - Google Patents

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

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

Morbidity and mortality from stage III colorectal cancer (CRC) remain high worldwide even after curative surgery. Adjuvant chemotherapy (ACT) is the standard treatment to improve 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, there is a need for accurate and easily accessible methods to track MRD using serum tumor markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). The most promising and suitable approaches in liquid biopsy systems, such as transcripts in peripheral blood exosomes (Non-Patent Documents 1 and 2) and microRNAs (miRNAs) (Non-Patent Documents 3 to 5), are currently under investigation.

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 (Non-Patent Documents 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 (Non-Patent Document 9), to enable drug adaptation to patients with specific genomic abnormalities. Patients with non-small cell lung cancer (NSCLC) undergo liquid biopsy to detect epidermal growth factor receptor (EGFR) mutations with R858L or T790M using CDx markers, and the results determine the optimal drug to be used, such as Gefitinib or Osimertinib (Non-Patent Documents 10, 11). The CGP, Guardant360 CDx, and FoundationOne® Liquid CDx tests were launched in August 2020 and were applied following the current standard of care in the clinical treatment of severely advanced cases.

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 tests are required to accurately track cancer-specific and robust events in peripheral blood that are not detectable by the naked eye. Customized blood CGP tests are expected to play a central role in MRD monitoring in the future and facilitate the rapid selection of appropriate drugs. However, the current frequent MRD monitoring system based on next-generation sequencing (NGS) is not realistic due to the high sequencing costs. In this context, there is a strong demand for the development of new technologies that can predict recurrence after curative surgery for colorectal cancer at a lower cost and more simply than conventional methods.

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 equires 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, Mo ri 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 bon a 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, Nambar a S, Hayashi N, Iguchi T, Ito S, Eguchi H, Ochiya T, Yanaga K, Miyano S, Mimori K. Ci rculating 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, L ippman 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, Mendelso hn 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, Doga n S, Ross DS, Hechtman JF, DeLair DF, Yao J, Mandelker 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 KA, Al-Ahmadie H, Vakiani 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, Durany R, Cambria R, Abida W, Cercek A, Feldman DR, Gounder MM, Hakimi AA, Harding JJ, Iyer G, Janj igian 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, Solit DB, Ladany i 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 pl us trastuzumab for HER2-amplified metastatic colorectal cancer: a phase 2 trial. Nat Med 2021;27(11): 1899-1903. Li C, Jia R, Liu H, Zhang B, 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 T umor 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.

The objective of the present invention is to provide a new technology that can predict the onset and recurrence of colorectal cancer more easily than conventional methods.

As a result of intensive research into the above-mentioned problems, the inventors discovered that the methylation status of the FGD5 gene, GPC6 gene and MSC gene in a subject is extremely useful as a marker for the presence of colorectal cancer cells, and by conducting further research based on this finding, they have completed the present invention.
That is, the present invention is as follows.

[1]
A method for detecting colon cancer in a subject, comprising determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject.
[2]
The method described in [1], further comprising a step of determining the methylation status of the MSC gene in the subject.
[3]
The method according to [1] or [2], wherein the subject has undergone radical surgery for colorectal cancer.
[4]
The method according to any one of [1] to [3], wherein the determination of the methylation status is carried out based on a liquid sample derived from the subject.
[5]
The method according to [4], wherein the liquid sample is a blood sample.
[6]
The method according to any one of [1] to [5], wherein the methylation status is determined by a PCR method.
[7]
A method for evaluating the effect of adjuvant chemotherapy being received by a subject, comprising 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 described in [7], further comprising a step of determining the methylation status of the MSC gene in the subject.
[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 methylation status is determined by a PCR method.
[13]
A diagnostic kit for colon cancer, including:
(1) a PCR primer pair capable of amplifying a part or the whole of the nucleotide sequence of the FGD5 gene; and/or
(2) A PCR primer pair capable of amplifying a part or the whole of the nucleotide sequence of the GPC6 gene.
[14]
A PCR primer pair capable of amplifying a part or the whole of the base sequence of the FGD5 gene is a nucleic acid having a base sequence shown in SEQ ID NO: 4 and a nucleic acid having a base sequence shown in SEQ ID NO: 5;
A PCR primer pair capable of amplifying a part or the whole 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.
[13] The kit described in.
[15]
(3) The kit according to [13] or [14], further comprising a PCR primer pair capable of amplifying a part or all of the base sequence of the MSC gene.
[16]
The kit according to [15], wherein the PCR primer pair capable of amplifying a part or all of the base sequence of the MSC gene is a nucleic acid having a base sequence shown in SEQ ID NO: 8 and a nucleic acid having a base sequence shown in SEQ ID NO: 9.

The present invention makes it possible to determine whether a subject is suffering from colorectal cancer and whether colorectal cancer has recurred in a subject after radical surgery for colorectal cancer more quickly than with conventional methods. Furthermore, the present invention also makes it possible to evaluate whether adjuvant chemotherapy for colorectal cancer is effective in a subject receiving the chemotherapy.

Figure 1 shows an example of the calculation of methylated ctDNA levels by 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 group for HapII treatment. The cfDNA of the target gene and the 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 as HapII-treated cfDNA. Finally, the methylation ratio of the target cfDNA compared to the RNase P cfDNA (0.65 = 2/1.3) was calculated. FIG. 2 is a diagram outlining the flow of the search for methylation biomarkers related to the prediction of colorectal cancer recurrence employed in the present invention. Among 473,864 probes of Illumina Infinium Human Methylation 450K, 100 sites of CpG islands that were particularly highly methylated in colorectal cancer tissues were selected. Then, further selection was performed by applying additional selection criteria (specific to cancer cells, not present in blood cells, and not present in normal tissues), and 50 probes were identified. The identified positions were verified by amplifying the genomic region. These probes were further selected under the following conditions: an inverse correlation between the methylation DNA level of the methylated site (methylation rate calculated from a database of bisulfite sequences, sometimes referred to as "beta value") and the expression of the target gene, and a significant correlation between the overall survival rate and beta value in stage III colorectal cancer cases. Abbreviations: CRC: colorectal cancer Verification of beta values of methylation sites of FDG5, GPC6, and MSC genes. A) Colon cancer cases with high beta values of promoter methylation sites of FDG5, GPC6, and MSC genes had poorer prognosis than cases with low beta values. B) A significant inverse correlation between gene expression and beta values of each gene was statistically verified in plasma samples of TCGA CRC cases. Figure 4 shows the basic characteristics of the three biomarkers selected in the present invention. A) The methylation beta values of CpG islands of FGD5, GPC6, and MSC were investigated from 40 cases of colorectal cancer (stage I to IV) tumor/normal tissue pairs. B) ROC curves drawn by 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) under the graphs indicate the duration of ACT (similar to Figures 5B-F and 6). The boxes in gray and black indicate the AMUSE score and serum CEA protein level, respectively. The date of surgery (P), the suspected recurrence on the images (Q), and the recurrence diagnosis (R) are shown. These six cases have a higher Q than the 28 recurrent 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) under the graph indicates the period 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. 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 positive AMUSE assay. (B) Comparison of recurrence-free survival rates between cases with positive AMUSE at the second or third blood sampling time point (n=19) and cases with positive AMUSE at both the second and third blood sampling time points (n=5) in recurrence cases. (C) Comparison of recurrence-free survival rates between cases with positive AMUSE at the second or third blood sampling time point (n=42) and cases with positive AMUSE at both the second and third blood sampling time points (n=5) in all cases. (D and E) Consideration of recurrence 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 the stratification of postoperative changes in tumor burden affected by adjuvant chemotherapy. Four groups were stratified according to 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 and lack of sensitivity to ACT. Group D) Tumor was completely removed by definitive surgery alone. Abbreviations: ACT: adjuvant chemotherapy; MRD: minimal residual disease.

The present invention is explained in detail below.

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"), comprising a step of determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject.

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.

The FGD5 gene (FYVE, RhoGEF and PH domain containing 5, Gene ID: 152273), also known as "ZFYVE23", is located at chromosomal position 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 in normal human individuals. The base sequence of the human FGD5 gene is shown in SEQ ID NO:1.

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 human individuals. The base sequence of the human GPC6 gene is shown in SEQ ID NO: 2.

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

In the detection method of the present invention, the methylation state of the above-mentioned gene is determined, but the method of determining the methylation state of a gene is known to those skilled in the art, and any 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 the amount can be 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 a PCR method. The PCR method combined with methylation-sensitive restriction enzyme analysis is more preferably real-time PCR or digital PCR. Details of the determination of the methylation state by combining methylation-sensitive restriction enzyme analysis with a PCR method are described in the examples of this specification.

In this specification, the term "determining the methylation status of gene A" means qualitatively analyzing the presence or absence of a methylated region in the entire length or a part 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, usually 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.

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

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

In one embodiment, probes for confirming amplification of the target regions of the FGD5 gene, GPC6 gene, and MSC gene can be used in combination to determine the methylation status. A person skilled in the art can appropriately design such probes for confirming amplification, taking into consideration 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.

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.

If the FGD5 gene, GPC6 gene and/or MSC gene in the subject has a high methylation state compared to the normal control, the subject is determined to have a high possibility of having colorectal cancer. In addition, if the subject has previously had colorectal cancer, if the FGD5 gene, GPC6 gene and/or MSC gene in the subject has a high methylation state compared to the normal control, the subject is determined to have a high possibility of having recurrent colorectal cancer. In one embodiment of the present invention, the methylation state of the FGD5 gene, GPC6 gene and/or MSC gene in the subject can be scored and the score can be compared with the score of the methylation state in a normal subject. Note that the method for scoring the methylation state may be a method known per se. If the score of the methylation state 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 possibility of having colorectal cancer. In addition, if the subject previously had colorectal cancer, 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, it is determined that the subject is likely to have colorectal cancer recurrence. 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 the subject is likely to have colorectal cancer or that the subject is likely to have colorectal cancer recurrence.

The "subject" in the detection method of the present invention is not particularly limited as long as it is a mammalian animal suffering from colorectal cancer. Examples of subjects include, but are not limited to, livestock animals (such as cows, 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 referred to as "individual," "patient," "subject," etc.

In one embodiment of the detection method of the present invention, the subject may be a subject who has undergone curative surgery for colorectal cancer. Without wishing to be bound by theory, it is believed that the methylation state 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 curative surgery.

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

In the detection method of the present invention, the determination of the methylation state of the gene of interest in the present invention may be performed by any means as long as the methylation state can be determined, but 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 the subject is not particularly limited as long as the methylation state of the gene of interest in the present invention can be determined by analyzing the liquid sample. Examples of the liquid sample derived from the subject include blood, lymph, tissue fluid, and body cavity fluid, and preferably blood, and more preferably plasma. By determining the methylation state 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 colon cancer in the subject can be made simply, at low cost, and in a minimally invasive manner.

2. Method for Evaluating the Effect of Adjuvant Chemotherapy The present invention also provides a method for evaluating the effect of adjuvant chemotherapy received by a subject, comprising a step of determining the methylation state 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").

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.

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

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 state compared to a normal control, the subject is determined to have a high possibility of having colorectal cancer, and therefore, the effect of the adjuvant chemotherapy currently being received by the subject is evaluated 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 degree of methylation state compared to a normal control, the subject is determined to have a high possibility of not having colorectal cancer, and therefore, the effect of the adjuvant chemotherapy currently being received by the subject is evaluated to be high. In addition, if a subject has previously suffered from colorectal cancer, if the FGD5 gene, GPC6 gene and/or MSC gene in the subject have a high methylation state compared to a normal control, the subject is determined to have a high possibility of having recurrence of colorectal cancer, and therefore, the effect of the adjuvant chemotherapy currently being received by the subject is evaluated to be low. Conversely, if the FGD5 gene, GPC6 gene and/or MSC gene in the subject have a methylation state comparable to that of the normal control, it is determined that the subject is highly 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. Also, as explained in the detection method of the present invention described above, the methylation state can be scored and used for evaluation.

3. Colon cancer diagnostic kit The present invention also relates to a colon cancer diagnostic kit comprising:
(1) a PCR primer pair capable of amplifying a part or the whole of the base sequence of the FDG5 gene; and/or
(2) To provide a PCR primer pair capable of amplifying a part or the whole of the base sequence of the GPC6 gene (hereinafter, sometimes referred to as the "kit of the present invention").

The kit of the present invention includes a PCR primer pair capable of amplifying a part or all of the base sequence of at least one of the FGD5 gene and the GPC6 gene. In a preferred embodiment, the kit of the present invention further includes a PCR primer pair capable of amplifying a part or all of the base sequence of the MSC gene. In a particularly preferred embodiment, the kit of the present invention includes a PCR primer pair capable of amplifying a part or all of the base sequence of the FGD5 gene, the GPC6 gene, and the MSC gene.

The sequences of the primer pairs for amplifying the FDG5 gene, the GPC6 gene, and the MSC gene are not particularly limited, so long as they can amplify part or all of these genes and can amplify the regions in these genes that can determine the methylation status. There are also no particular limitations on the nucleotide length of each primer, but it can usually be 16 to 40, preferably 17 to 30, and more preferably 18 to 25.

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

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

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, the GPC6 gene, and the MSC gene. A person skilled in the art can appropriately design such probes for confirming amplification, taking into consideration 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.

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.

The kit of the present invention may include, in addition to the primer pairs and probes described above, reagents, positive/negative controls, instructions, etc.

4. Use of a PCR primer pair capable of amplifying a part or the whole of the base sequence of the FGD5 gene and the GPC6 gene in in vitro/ex vivo diagnosis of colon cancer or evaluation of the effect of adjuvant chemotherapy for colon cancer The present invention also relates to the use of a PCR primer pair capable of amplifying a part or the whole of the base sequence of the FGD5 gene and the GPC6 gene in in vitro/ex vivo diagnosis of colon cancer or evaluation of the effect of adjuvant chemotherapy for colon cancer.
(1) a PCR primer pair capable of amplifying a part or the whole of the base sequence of the FDG5 gene; and/or
(2) The present invention provides a use of a PCR primer pair capable of amplifying a part or the whole of the base sequence of the GPC6 gene (hereinafter, sometimes referred to as "use of the present invention").

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, in the use of the present invention, a PCR primer pair capable of amplifying part or all of the base sequence of the MSC gene is further used. In a particularly preferred embodiment, in the use of the present invention, a PCR primer pair capable of amplifying part or all of the base sequence of the FGD5 gene, the GPC6 gene, and the MSC gene is used.

The sequences of the primer pairs for amplifying the FDG5 gene, the GPC6 gene, and the MSC gene are not particularly limited, so long as they can amplify part or all of these genes and can amplify the regions in these genes that can determine the methylation status. There are also no particular limitations on the nucleotide length of each primer, but it can usually be 16 to 40, preferably 17 to 30, and more preferably 18 to 25.

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

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

In addition, when using 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. A person skilled in the art can appropriately design such probes for confirming amplification, taking into consideration the nucleic acid sequence of the target region to be amplified. In one embodiment, the following probes can be used for confirming amplification of each gene, but are not limited to these.

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.

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 explained in "1. Method for detecting colorectal cancer in a subject" and "2. Method for evaluating the effectiveness of adjuvant chemotherapy."

5. Diagnosis Method for Colorectal Cancer The present invention also provides a method for diagnosing 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 (hereinafter, sometimes referred to as the "diagnostic method of the present invention").

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 described in "1. Method for detecting colorectal cancer in a subject."

In one embodiment, the diagnostic method of the present invention may include a step of administering a therapeutically effective amount of an anticancer agent to a subject diagnosed as having colorectal cancer by using the diagnostic method of the present invention.

The anticancer drug administered to the subject is not particularly limited, and any drug can be used. Examples of anticancer drugs include, but are not limited to, alkylating agents (e.g., cyclophosphamide, cisplatin, etc.), antimetabolic agents (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.).

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

This study was approved by the Research Ethics Committee of the Cancer Institute Ariake Hospital. The IRB receipt number is 2010-1058 (March 7, 2019). In addition, the Clinical Research Ethics Committee of the Kyushu University Medical Department reviewed and approved the project (number 2020-321 (September 1, 2020)).

[Method]

[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 capecitabine and oxaliplatin (8 courses over 6 months) as adjuvant therapy and CAPOX treatment. Twenty-eight patients relapsed between 170 and 1070 days after surgery, and 19 were non-relapsed at least 1090 days after surgery. A total of 180 blood samples were collected and extracted from relapsed patients before surgery, after surgery, during ACT, after ACT, and at the time of relapse. A total of 111 samples were collected from non-relapsed patients for analysis.

[Sample storage and cfDNA extraction]
The patient-derived leaky plasma samples were centrifuged at 2500×G for 10 min 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 min, 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 the 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.

[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 10× 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.

[Sample preparation for digital PCR]
The two completed solutions were used to prepare samples for digital PCR. First, a master solution for mixing 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's 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:

FGD5 primer f (SEQ ID NO: 4)
5'-CCTTCTCAGCCTTGGCGAG
FGD5 primer r (SEQ ID NO: 5)
5'-GCGTCTTTCTTCGTCGTGGAA

GPC6 primer f (SEQ ID NO: 6)
5'-GCGGGCTTTCGGCTTGAG
GPC6 primer r (SEQ ID NO: 7)
5'-CCAAGAGGGGAAGAATCACAGC

MSC primer f (SEQ ID NO:8)
5'-AGCAGTCGCAGCGGAACG
MSC primer r (SEQ ID NO:9)
5'-CAGGGCAGGCTGGTCTTGA

FGD5 probe (SEQ ID NO: 10)
5'-CGCCAGTATCCCACTCGCACGGC
GPC6 probe (SEQ ID NO: 11)
5'-CCGATCCAAGAAGGCATGGTGCAACATACA
MSC probe (SEQ ID NO: 12)
5'-CCTGGAGAAGGCTTTGCTCAGCACGC

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

[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 cfDNA, 7.25 μL QuantStudio 3D Digital PCR Master Mix v2 (ThermoFisher Scientific), 0.725 μL forward primer (18 μM), 0.725 μL reverse primer (18 μM), 0.725 μL TaqMan probe (5 μM), and 0.725 μL TaqMan Copy Number Reference Assay RNase P (ThermoFisher Scientific). Digital PCR mixtures were loaded onto a QuantStudio 3D Digital PCR Chip v2 using the QuantStudio 3D Digital PCR Chip Loader. PCR was run on a ProFlex 2xFlat PCR system using the following program: 96°C for 10 min, 60°C for 2 min, 39 cycles of 98°C for 30 s, 60°C for 2 min, 10°C. Chip images were captured using the QuantStudio 3D Digital PCR Instrument and analyzed using the QuantStudio 3D Analysis Suite Software.

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

[Assay design for methylation quantification based on methylation-sensitive restriction enzymes]
A digital PCR assay was designed to measure the methylation ratio of colon 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 not methylated. Therefore, if cfDNA is digested with HapII and then PCR is performed using primers that flank the recognition site, amplification products will only occur if the HapII recognition site is methylated. After cfDNA is digested with HapII, digital PCR was performed using primers that flank the recognition site to measure the copy number of the methylation site. The copy number of the reference gene, RNase P, was also measured by a similar reaction. Since all cells contain the reference gene, the copies of RNase P represent the total cfDNA released from normal and tumor cells. The copy number of colon cancer-specific methylation sites in samples digested with HapII was compared to those without HapII, and the methylation ratio of the site was calculated. Because pipetting bias can have a significant effect on methylation ratios, the target site in each sample was divided by RNase P to calculate the exact methylation ratio (Figure 1).

[Results]

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 (signal-to-noise ratio > 1.5) in colorectal cancer tissues was narrowed down to 100 probes. Using data from The Cancer Genome Atlas (TCGA), the number of methylation sites was narrowed down to 50 probes by selecting probes with a significant difference in methylation rate between tumor tissues and normal tissues, but low methylation rates in normal tissues and blood cells. The specific selection criteria are as follows:

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)

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

2. Sensitivity and specificity of methylation markers The methylation status of the three target sites was used to distinguish patients with colorectal cancer from 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 and had no abnormal findings. The tumor group used plasma from patients with colorectal cancer (stages 1-4; 40 patients, 10 in each group) collected at the Cancer Research Institute Ariake Hospital. Of the three markers, FGD5 and GPC6 had significantly increased methylation in the tumor group (p = 0.028, p = 0.0231, respectively; Figure 4A). MSCs showed a tendency to have higher methylation values in tumor tissues compared to normal tissues (p = 0.062) (Figure 4A). These results indicate that the three markers of the present invention can distinguish patients with colorectal cancer, suggesting that these markers may be applicable for cancer screening. The receiver operating characteristic (ROC) curve of 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.

3. Comparison of the sensitivity of MRD monitoring by CT and AMUSE assay Forty-seven patients with pathological stage III A/B and CurA colon cancer (28 recurrence-positive, 19 recurrence-negative) underwent surgery at the Cancer Institute Ariake Hospital between June 2012 and June 2017. Patients were treated with CAPOX for postoperative ACT. Pathological histological diagnoses of well-differentiated-poorly differentiated adenocarcinoma were included in the study. Methylation status was measured using plasma samples collected preoperatively and postoperatively, after ACT, and at the time of recurrence. For patients without recurrence, cases without recurrence were defined as those without recurrence for a mean of 1551 days (1093-1897 days) after surgery. Patients with methylation values of the three genes in cfDNA above the cutoff value were diagnosed as positive for recurrence by liquid biopsy. Twenty-two of 28 patients (78.5%) were diagnosed with recurrence before clinical diagnosis of postoperative recurrence using CT images (sensitivity, 78.5%).

4. Possibility of Rescuing False-Negative Cases of Postoperative Recurrence Using the AMUSE Assay Early detection of postoperative recurrence allows complete removal and improves clinical outcomes of colorectal cancer. However, when serum tumor markers are within the normal range and CT findings are unclear, 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 than time point R (suspicious; time point Q). On the other hand, when the AMUSE assay of the present invention was used, the decision to immediately remove or to radically irradiate was made based on the AMUSE score at time point Q. Also, Figure 3B shows a case in which ACT was administered to 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 aggressive treatment would be hindered if the conventional indicator was used.

The follow-up was continued without extending the adjuvant treatment or switching to another chemotherapy. Finally, at time point R, the increase in the size of the suspicious area allowed a definitive diagnosis of lung recurrence. The opportunity to perform a definitive treatment was missed, and only systemic chemotherapy was administered. However, in the case of sigmoid colon cancer (Figure 5C), the beta value of the AMUSE assay was continuously positive from the first day of ACT to the day of diagnosing the recurrence, and the opportunity to perform surgery at the site of liver metastasis was missed multiple times (time points Q1 and Q2). In this case, systemic chemotherapy was administered to control the metastatic site. Furthermore, the local recurrence of rectal cancer in a patient with ulcerative colitis with malignant tumor 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. Thus, 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 the patient's survival time 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.

An example of a patient with rectal cancer who underwent curative surgery is shown in Figure 5F. After ACT, the patient had suspected pulmonary recurrence 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.

5. Clinical Benefits of the AMUSE Assay Nineteen patients with colorectal cancer were followed up for 1093-1897 days after each radical operation and declared “non-recurrence.” Seventeen of the 19 patients (89.5%) were negative in the AMUSE assay, but two points showed false-positive beta values (Figures 6G and 6I, arrows).

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

Among 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 at 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). In addition, when the disease-free recurrence rate was compared between the 5 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.

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 (Figure 7E). However, the difference in survival rates between AMUSE positive and AMUSE negative patients was greater than the difference in survival rates between CEA positive and CEA negative patients.

ACT, approved by the conference as the standard treatment for MRD after curative resection for postoperative stage III CRC, is performed, but the ACT protocol prescribes a fixed dose and duration without considering each individual's tumor burden. Of the 47 cases, 39 cases were stratified, excluding 7 false negatives and 2 false positives (Figure 8). It is noteworthy that the proportion of cases that received appropriate ACT treatment was only 6 out of 39 cases (16.9%) in group A. 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.

The present invention makes it possible to determine whether a subject is suffering from colorectal cancer, and whether colorectal cancer has recurred in a subject after radical surgery for colorectal cancer, more quickly than with conventional methods. Furthermore, the present invention also makes it 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.

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 into this specification.

Claims (16)

A method for detecting colorectal cancer in a subject, comprising determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject. The method of claim 1, further comprising a step of determining the methylation status of the MSC gene in the subject. The method according to claim 1 or 2, wherein the subject has undergone radical surgery for colorectal cancer. The method according to claim 1 or 2, characterized in that the determination of the methylation status is carried out based on a liquid sample derived from the subject. The method of claim 4, wherein the liquid sample is a blood sample. The method according to claim 1 or 2, characterized in that the methylation status is determined by PCR. A method for evaluating the effect of adjuvant chemotherapy being received by a subject, the method comprising 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. The method of claim 7, further comprising a step of determining the methylation status of the MSC gene in the subject. The method according to claim 7 or 8, wherein the subject has undergone radical surgery for colorectal cancer. The method according to claim 7 or 8, characterized in that the determination of the methylation status is carried out based on a liquid sample derived from the subject. The method of claim 10, wherein the liquid sample is a blood sample. The method according to claim 7 or 8, characterized in that the methylation status is determined by PCR. A diagnostic kit for colon cancer, including:
(1) a PCR primer pair capable of amplifying a part or the whole of the nucleotide sequence of the FGD5 gene; and/or
(2) A PCR primer pair capable of amplifying a part or the whole of the nucleotide sequence of the GPC6 gene. A PCR primer pair capable of amplifying a part or the whole of the base sequence of the FGD5 gene is a nucleic acid having a base sequence shown in SEQ ID NO: 4 and a nucleic acid having a base sequence shown in SEQ ID NO: 5;
A PCR primer pair capable of amplifying a part or the whole 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.
The kit of claim 13. (3) The kit according to claim 13 or 14, further comprising a PCR primer pair capable of amplifying a part or all of the base sequence of the MSC gene. The kit according to claim 15, wherein the PCR primer pair capable of amplifying a part or all of the base sequence of the MSC gene is a nucleic acid containing the base sequence shown in SEQ ID NO: 8 and a nucleic acid containing the base sequence shown in SEQ ID NO: 9.