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EP-4735641-A1 - DETECTION OF HYPERMETHYLATED GENES FOR DIAGNOSING COLORECTAL CANCER

EP4735641A1EP 4735641 A1EP4735641 A1EP 4735641A1EP-4735641-A1

Abstract

The present invention relates to a method for diagnosing or identifying a colorectal cancer in a subject, through the detection of the abnormal hypermethylation levels of specific genes in a biological sample of said subject. The inventors indeed identified DNA methylation biomarkers that, alone or in combination, can help diagnosing or following-up colorectal cancer patients. Further, it can be used for determining, and/or adapting a suitable therapeutic regimen for a subject diagnosed for colorectal cancer. The present invention also relates to kits comprising primers or probes to detect, diagnose, or identify hypermethylated genes.

Inventors

  • WANG-RENAULT, Shufang
  • ABDELLI, Justine
  • TALY, Valérie
  • LAURENT-PUIG, PIERRE
  • BEINSE, Guillaume
  • NAYAGOM, Benjamin
  • DJAMAI, Hanane
  • BAURES, Aurélia
  • GOMIS, Barbara
  • TALY, Jean-François

Assignees

  • Methys DX
  • Université Paris Cité
  • Centre National de la Recherche Scientifique
  • Assistance Publique - Hôpitaux de Paris
  • SORBONNE UNIVERSITE
  • Institut National de la Santé et de la Recherche Médicale

Dates

Publication Date
20260506
Application Date
20240701

Claims (17)

  1. 1. An in vitro method for detecting or monitoring a colorectal cancer (CRC) in a subject, preferably a human subject, which method comprises detecting, or determining the level of methylation of at least one of AQP5-AS1, ZSCAN23, or COL4A1 genes, in a biological sample from the subject, wherein the biological sample contains genomic DNA.
  2. 2. The method of claim 1, comprising detecting, or determining the level of methylation of AQP5-AS1 and ZSCAN23 genes.
  3. 3. The method of claim 1, comprising detecting, or determining the level of methylation of AQP5-AS1 and COL4A1 genes.
  4. 4. The method of claim 1, comprising detecting, or determining the level of methylation of ZSCAN23 and COL4A1 genes.
  5. 5. The method of any one of claims 1 to 4, comprising detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23, and COL4A1 genes.
  6. 6. The method of any one of claims 1 to 5, which method further comprises detecting, or determining the level of methylation of at least one of NR5A2, C9orf50, WIFI and NPY genes.
  7. 7. The method of any of claims 1 to 6, which method further comprises detecting, or determining the level of methylation of at least one of ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, or LRRC4 (SND1) genes.
  8. 8. The method of any of claims 1 to 7, comprising detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23, and COL4A1 genes, and at least two, preferably three, preferably four genes selected in the group consisting of : NR5A2, C9orf50, WIFI, NPY, ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1) genes.
  9. 9. The method of any of claims 1 to 7, comprising detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23, and COL4A1 genes, and at least two, preferably three, preferably four genes selected in the group consisting of : NR5A2, C9orf50, WIFI, and NPY.
  10. 10. The method of any of claims 1 to 9, wherein said level or amount of methylation is determined in the nucleotide region(s) of : - SEQ ID NO:40 in the ZSCAN23 gene, - SEQ ID NO:41 in the C9orf50 gene, - SEQ ID NO:42 in the AQP5-AS1 gene, - SEQ ID NO:43 in the NR5A2 gene, - SEQ ID NO:44 in the ADARB2 gene, - SEQ ID NO:45 in the COL4A1 gene, - SEQ ID NO:46 in the NPY gene, - SEQ ID NO:47 in the WIFI gene, - SEQ ID NO:48 in the CPNE8 gene, - SEQ ID NO:49 in the LINC00693 (RBMS3) gene, - SEQ ID NO:50 in the LINC00900 gene, - SEQ ID NO:51 in the DYDC2 gene, and/or - SEQ ID NO: 52 in the LRRC4 (SND1) gene, wherein said level or amount of methylation is preferably determined in the nucleotide region(s) of : - SEQ ID NO 53 in the ZSCAN23 gene, - SEQ ID NO:54 in the C9orf50 gene, - SEQ ID NO 55 in the AQP5-AS1 gene, - SEQ ID NO:56 in the NR5A2 gene, - SEQ ID NO:57 in the ADARB2 gene, - SEQ ID NO:58 in the COL4A1 gene, - SEQ ID NO:59 in the NPY gene, - SEQ ID NO: 60 in the WIFI gene, - SEQ ID NO:61 in the CPNE8 gene, - SEQ ID NO:62 in the LINC00693 (RBMS3) gene, - SEQ ID NO: 63 in the LINC00900 gene, - SEQ ID NO:64 in the DYDC2 gene, and/or - SEQ ID NO: 65 in the LRRC4 (SND1) gene.
  11. 11. The method of any of claims 1 to 10, wherein said level or amount of methylation is determined by using : - the primers of SEQ ID NO: 1 and 2, and the probe of SEQ ID NO:27 for the ZSCAN23 gene, - the primers of SEQ ID NO:3 and 4, and the probe of SEQ ID NO:28 for the C9orf50 gene, - the primers of SEQ ID NO:5 and 6, and the probe of SEQ ID NO:29 for the AQP5-AS1 gene, - the primers of SEQ ID NO: 7 and 8, and the probe of SEQ ID NO:30 for the NR5A2 gene, - the primers of SEQ ID NO: 9 and 10, and the probe of SEQ ID NO: 31 for the ADARB2 gene, - the primers of SEQ ID NO: 11 and 12, and the probe of SEQ ID NO:32 for the COL4A1 gene, - the primers of SEQ ID NO: 13 and 14, and the probe of SEQ ID NO:33 for the NPY gene, - the primers of SEQ ID NO: 15 and 16, and the probe of SEQ ID NO:34 for the WIFI gene, - the primers of SEQ ID NO: 17 and 18, and the probe of SEQ ID NO:35 for the CPNE8 gene - the primers of SEQ ID NO: 19 and 20, and the probe of SEQ ID NO:36 for the LINC00693 (RBMS3) gene, - the primers of SEQ ID NO:21 and 22, and the probe of SEQ ID NO:37 for the LINC00900 gene, - the primers of SEQ ID NO:23 and 24, and the probe of SEQ ID NO:38 for the DYDC2 gene, and/or - the primers of SEQ ID NO:25 and 26, and the probe of SEQ ID NO:39 for the LRRC4 (SND1) gene.
  12. 12. The method according to any of claims 1 to 11, wherein the sample is a body fluid, preferably selected from the group consisting of plasma, serum, blood, urine and feces, still preferably wherein the sample is a plasma or serum sample and the DNA is circulating cell-free DNA (ccfDNA), preferably circulating tumor DNA (ctDNA).
  13. 13. The method according to any of claims 1 to 12, wherein if said gene(s) are hypermethylated as compared with reference value(s), then said subject is diagnosed or identified as suffering from a colorectal cancer.
  14. 14. The method according to any of claims 1 to 12, for an early diagnosis in a subject who is at risk of developing a colorectal cancer.
  15. 15. The method according to any of claims 1 to 12, for an early diagnosis of a cancerous lesion in the colon in a subject who has been developing metastasis from a primary tumor of unknown origin.
  16. 16. The method according to any of claims 1 to 12, for assessing the risk of relapse.
  17. 17. The method according to any of claims 1 to 12, for monitoring the progress of colorectal cancer in a subject diagnosed with colorectal cancer, which method preferably comprises a) determining the level of methylation of any of said genes, in a biological sample of said subject, at a first time point, b) determining the level of methylation of said genes selected previously in the step a), in a biological sample of said subject, at a second time point, and c) comparing the level of methylation determined in step a) to the level or amount determined in step b) or to reference values.

Description

DETECTION OF HYPERMETHYLATED GENES FOR DIAGNOSING COLORECTAL CANCER FIELD OF THE INVENTION The present invention relates to the field of oncology. More particularly, the present invention relates to a method for detecting or monitoring a colorectal cancer (CRC) in a subject, through the detection of the abnormal hypermethylation levels of specific genes in a biological sample of said subject. The inventors indeed identified a panel of DNA methylation biomarkers that, alone or in combination, can help diagnosing or following-up CRC patients. BACKGROUND OF THE INVENTION The colorectal cancer (CRC) represents an important challenge in terms of public health. In fact, the CRC is the third cancer in terms of incidence with nearly 2 million of new cases in 2020. There were also more than 916,000 deaths, representing more than 9% of the world's mortality in 2020. Surgery is the most common treatment for all stages of colon cancer. Most of the time, surgery is followed by adjuvant chemotherapy (ACT) in patients with histological high-risk stage II and III. ACT can decrease the risk of recurrence, but the survival is contrasted. The high mortality rate can be explained by a late diagnosis, limited monitoring tools and a low adaptability of treatments. This illustrates the real need for a new method, noninvasive, cost- effective and sensitive for the diagnosis and monitoring of CRC patients. Thus, an improvement in diagnostic and tracking methods could allow the reduction of its mortality by allowing its detection at an early stage. The diagnostic methods now available have important limits, such as their invasiveness, cost or low sensitivity, especially at early stages. There is still a need for methods for early diagnosis and accurate monitoring of colorectal cancer. SUMMARY OF THE INVENTION A first aspect of the invention relates to an in vitro method for detecting or monitoring a colorectal cancer (CRC) in a subject, preferably a human subject, which method comprises detecting, or determining the level of methylation of at least one of AQP5-AS1, ZSCAN23, or COL4A1 genes, in a biological sample from the subject, wherein the biological sample contains genomic DNA. In a particular embodiment, the method comprises detecting, or determining the level of methylation of AQP5-AS1 and ZSCAN23 genes. In another particular embodiment, the method comprises detecting, or determining the level of methylation of AQP5-AS1 and COL4A1 genes. In another particular embodiment, the method comprises detecting, or determining the level of methylation of ZSCAN23 and COL4A1 genes. In a particular embodiment, the method comprises detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23, and COL4A1 genes. In a particular embodiment, the method further comprises detecting, or determining the level of methylation of NR5A2, C9orf50, WIFI or NPY gene. In a particular embodiment, the method further comprises detecting, or determining the level of methylation of at least one of ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, or LRRC4 (SND1) genes. In a particular embodiment, the method comprises detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23 and COL4Algenes, and at least two, preferably three, preferably four genes selected in the group consisting of : NR5A2, C9orf50, WIFI, NPY, ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1) genes. In a particular embodiment, the method comprises detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23, and COL4A1 genes, and at least two, preferably three, preferably four genes selected in the group consisting of : NR5A2, C9orf50, WIFI, and NPY. In a particular embodiment, said level or amount of methylation is determined in the nucleotide region(s) of : - SEQ ID NO:40 in the ZSCAN23 gene, - SEQ ID NO:41 in the C9orf50 gene, - SEQ ID NO:42 in the AQP5-AS1 gene, - SEQ ID NO:43 in the NR5A2 gene, - SEQ ID NO:44 in the ADARB2 gene, - SEQ ID NO:45 in the COL4A1 gene, - SEQ ID NO:46 in the NPY gene, - SEQ ID NO:47 in the WIFI gene, - SEQ ID NO:48 in the CPNE8 gene, - SEQ ID NO:49 in the LINC00693 (RBMS3) gene, - SEQ ID NO:50 in the LINC00900 gene, - SEQ ID NO:51 in the DYDC2 gene, and/or - SEQ ID NO: 52 in the LRRC4 (SND1) gene. In a particular embodiment, said level or amount of methylation is determined in the nucleotide region(s) of : - SEQ ID NO 53 in the ZSCAN23 gene, - SEQ ID NO:54 in the C9orf50 gene, - SEQ ID NO 55 in the AQP5-AS1 gene, - SEQ ID NO:56 in the NR5A2 gene, - SEQ ID NO:57 in the ADARB2 gene, - SEQ ID NO:58 in the COL4A1 gene, - SEQ ID NO:59 in the NPY gene, - SEQ ID NO: 60 in the WIFI gene, - SEQ ID NO:61 in the CPNE8 gene, - SEQ ID NO:62 in the LINC00693 (RBMS3) gene, - SEQ ID NO: 63 in the LINC00900 gene, - SEQ ID NO:64 in the DYDC2 gene, and/or - SEQ ID NO: 65 in the LRRC4 (SND1) gene. In a particular embodiment, said level or amount of methylation is de