WO2015181804A2 - Procédé et test de détection non invasive d'un cancer gastrique à un stade précoce par l'utilisation combinée d'adn sans cellule de reprimo méthylé et de pepsinogène i/ii - Google Patents

Procédé et test de détection non invasive d'un cancer gastrique à un stade précoce par l'utilisation combinée d'adn sans cellule de reprimo méthylé et de pepsinogène i/ii Download PDF

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WO2015181804A2
WO2015181804A2 PCT/IB2015/054104 IB2015054104W WO2015181804A2 WO 2015181804 A2 WO2015181804 A2 WO 2015181804A2 IB 2015054104 W IB2015054104 W IB 2015054104W WO 2015181804 A2 WO2015181804 A2 WO 2015181804A2
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methylated
gastric cancer
reprimo
rprm
cell
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PCT/IB2015/054104
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WO2015181804A3 (fr
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Alejandro CORVALÁN
Eudocia SANTIBÁÑEZ
María José MATURANA
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Pontificia Universidad Católica De Chile
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to the field of the diagnostic of cancer in human subjects.
  • the present invention provides a method and an assay for the noninvasive detection of early gastric cancer by the combined use of methylated reprimo cell-free dna and pepsinogen l/ll
  • Cancer is defined as unregulated growth of abnormal cells, which form masses or tumors. This overgrowth of some tissues can occur for various reasons, such as mutations, flaws in the body monitoring system, etc.
  • the signs of cancer vary based on the type and location of the tumor, thus common tests include biopsy of the tumor, blood tests (which look for chemicals such as tumor markers), bone marrow biopsy, chest x-ray, complete blood count, CT scan, liver function tests, MRI scan, etc. Cancer causes about 15% of all human deaths.
  • the most common types of cancer in men are lung cancer, prostate cancer, colorectal cancer, and stomach cancer, and in women, the most common types are breast cancer, colorectal cancer, lung cancer, and cervical cancer.
  • the risk of cancer increases significantly with age and rates are increasing as more people live to an old age and as lifestyle changes occur in the developing world.
  • Some cancers are more common in certain parts of the world. For example, in Japan, there are many cases of stomach cancer, but in the United States, this type of cancer is unusual, differences in diet or environmental factors may play a role.
  • gastric cancer accounts for 8% and 10% of new cases and deaths by cancer, respectively. Although over 70% of these events occur in developing countries, a rise in incidence in young white cohorts might explain why gastric cancer has been postulated to be among the top ten leading causes of death by 2030.
  • Mass screening programs have been able to identify a high proportion of tumors limited to the mucosa and submucosa of the stomach, so called early gastric cancer. Thus, these programs should have an impact in reducing the mortality rate of gastric cancer. Indeed, in Japan and Korea, case-control as well as prospective longitudinal studies based on large-scale population-based surveys, showed a significant reduction in gastric cancer mortality. However, mass-screening programs may not be the most practical approach because of acceptance, availability, and cost.
  • GC Gastric cancer
  • Gastric cancer has been described as the final step of a dynamic sequence of precursor lesions which progresses from atrophic gastritis (AG), to intestinal metaplasia (IM), to dysplasia and, finally, gastric cancer.
  • This sequence is initiated by Helicobacter pylori infection, which can remain as a chronic infection or establish atrophic gastritis.
  • Long-term follow-up studies have shown an increasing annual incidence from 0.1 % in the case of atrophic gastritis and intestinal metaplasia, to 6% in dysplasia for the development of gastric cancer.
  • Pepsinogen (PG) l/ll ratio in peripheral blood samples is the most extensively studied biomarker.
  • studies have shown it to have poor sensitivity (36.8-62.3%) and to be a better marker for the assessment of atrophic gastritis than gastric cancer.
  • the PG tests reflect the degree of multifocal atrophic gastritis, a well-characterized entity defined by atrophy and intestinal metaplasia of the corpus mucosa. Because gastric precancerous process arises and may be limited to the antrum rather than the corpus mucosa, longitudinal studies have shown normal PG l/ll ratio in almost half of incidental gastric cancer cases. Therefore, serum PG tests are insufficiently sensitive for the direct detection of gastric cancer.
  • document WO2010131984 describes the early detection of gastric tumors.
  • the documents describe members of the GTM gene family that can be differentially expressed in gastric tumor tissue, and thus can be used as markers for the detection of gastric and other types of cancer.
  • the document provides GTMs for the detection of tumors, including gastric tumors, and in particular human zymogen granule protein 16 (ZG16).
  • the GTMs can be used in isolation or together with other known GTMs to provide for novel signatures to be used in the detection of tumors, including gastric tumors.
  • GTM is not an specific marker for gastric cancer, and declares that the most frequently used markers for gastric cancer, CA19-9, CA72-4 and carcino-embryonic antigen (CEA), where those markers allow the detection of only 15-50% of gastric tumors of any stage, declining to approximately 2-1 1 % for early stage disease.
  • GTM gastric cancer
  • CEA carcino-embryonic antigen
  • FIG. 1 Immunohistochemical staining for RPRM protein expression in gastric cancer cases.
  • A Representative example of tumor and non-tumor adjacent mucosa showing negative and positive cytoplasmic staining of RPRM in more than 10% of cells, respectively.
  • Original magnification 100X
  • B Higher magnification of normal area showing positive staining of RPRM.
  • C Higher magnification of tumor area showing negative RPRM staining.
  • D Higher magnification of tumor area showing positive RPRM staining.
  • FIG. 3 (A) Next-generation sequencing of paired tumor and plasma gastric cancer cases. Each color represents one clone per case in tumor and plasma samples, respectively. Three, four and six identical clones were found (similar color) in tumor and plasma from three cases, respectively.
  • Figure 4. Receiver operating characteristic (ROC) curve for cfDNA of RPRM promoter methylation, H. pylori assay and serum PG tests to determine the best cutoff values in the training cohort (60 gastric cancer patients and 101 healthy control subjects) for noninvasive detection of gastric cancer. The jagged line represents the ROC curve. The dashed line represents the line of no discrimination between good and bad classification.
  • ROC curve for cfDNA of RPRM methylation demonstrate an AUC of 0.789 (95% CI 0.718 to 0.85) to determine an optimal cut-off value of 82 copies/mL (Sensitivity 68.3%, 95% CI 55% to 79.7% and Specificity 86.1 %, 95% CI 77.8% to 92.2%).
  • (B) ROC curve for H. pylori demonstrate an AUC of 0.765 (95% CI 0.676-0.84) to determine an optimal cut-off value of 86.8 EIU (Sensitivity 85.7%, 95% CI 63.6% to 96.8% and Specificity 63%, 95% CI 52.3% to 72.9%).
  • (C) ROC curve for serum PG tests demonstrate an AUC of 0.771 (95% CI 0.681 to 0.845) to determine an optimal cut-off value of 4.6 (Sensitivity 68.4%, 95% CI 43.5% to 87.3% and Specificity 76.1 %, 95% CI 66.1 % to 84.4%).
  • Figure 5. Age differences between healthy control donors and gastric cancer patients.
  • FIG. 1 Reprimo (RPRM) promoter structure and bisulfite sequence of the promoter region in PBMCs and gastric carcinoma cell lines.
  • the present invention provides the combined use of methylated reprimo (RPRM) cell-free DNA (cfDNA) and Pepsinogen l/ll (PG l/ll) for early gastric cancer non- invasive detection.
  • RPRM methylated reprimo
  • cfDNA cell-free DNA
  • PG l/ll Pepsinogen l/ll
  • the invention addressed the evaluation of the role of cfDNA of RPRM methylation along with H. pylori (Hpy) and serum pepsinogen (PG) tests for noninvasive detection of gastric cancer, where gastric cancer, chronic gastritis and healthy control subjects were prospectively assessed for cfDNA of RPRM, Hpy and PG by MethyLight and ELISA assays, respectively. Data analysis was performed by statistical software, such as R, SPSS and GraphPad packages.
  • Gastric cancer tissues displayed higher levels of RPRM methylation than chronic gastritis where cfDNA of RPRM methylation is a direct marker of GC.
  • the combination of RPRM methylation with serum PG tests substantially improved sensitivity 87.5%, specificity 64.4% and negative predictive value 99.6%, allowing a reliable noninvasive detection of gastric cancer.
  • cfDNA tumor-derived cell-free DNA
  • RPRM methylated Reprimo
  • methylated RPRM cfDNA detected 56.3% of early gastric cancer cases, similar to the test for PG l/ll ratio.
  • the combination of methylated RPRM cfDNA and PG l/ll showed a substantial increase in sensitivity for detection of early gastric cancer to 87.5% (95% CI 61 .62 to 98.08).
  • both the odds ratio and the accuracy of the combined approach tended to decrease with the progression of the cascade.
  • this is the first combined biomarker approach that reaches the highest sensitivity for the non-invasive assessment of early gastric cancer maintaining a good performance for the assessment of gastric cancer.
  • Fig.3A three, four and six identical clones were identified for each case, respectively.
  • RPRM methylation cfDNA The gastric origin of RPRM methylation in cfDNA along with differences before and after treatment suggested RPRM promoter methylation as a sensitive and specific biomarker for the noninvasive detection of gastric cancer.
  • RPRM methylation cfDNA and well established markers such as H. pylori and serum PG tests were evaluated in 81 gastric cancer patients and 137 healthy donors.
  • Table 1 provides demographic information regarding gastric cancer and control subjects. Although age of the cancer group was older than control group (p ⁇ 0.0001 ), proportion of male to female participants was similar (ratio 1 .9:1 vs. 2:1 , respectively). The ratio of intestinal to diffuse gastric cancer cases was 2.5:1 and the ratio of early to advanced stages was 1 :4.2.
  • RPRM Methylated Reprimo
  • Quantitative asessment of CpG-island methylation was carried out using the fluorescence-based real-time PCR, MethyLight assay, a high- throughput methylation assay that utilizes highly sensitive and accurate fluorescence- based Real-Time PCR (Taqman) and is ideal for small amount of genomic DNA such gastric biopsies or cfDNA in plasma.
  • Two sets of primers and hydrolysis probes (TaqMan) where designed specifically for bisulfite converted DNA sequences, one spanning 12 CpG dinucleotides in the proximal promoter of RPRM gene, and the other set overlapping methylation-independent sequences on MYOD1 promoter as a control for input genomic DNA.
  • MethyLight was performed on a PCR amplification and detection instrument (for example, in a LightCycler 480 instrument, Roche®) with 4 ⁇ _ of bisulfite modified DNA, 1 X of hot start reaction mix for PCR such as LightCycler FastStart DNA Master Hybprobe, Roche®, 0.6 ⁇ of each primer, and 0.2 ⁇ of single-stranded oligonucleotide hybridization probe.
  • the PCR profile consisted of an initial denaturation for 10 minutes at 95°C; followed by 50 cycles of 10 seconds at 95°C and 55 seconds at 60 °C.
  • Cut-off values > 0 were considered a positive result.
  • Serum blood sample was obtained during an upper endoscopic procedure and levels of PG l/ll ratio were performed through an enzyme-linked immunosorbent assay (ELISA) test according to the assay manufacturers' instructions (such as Biohit Pic®).
  • ELISA enzyme-linked immunosorbent assay
  • H. pylori immunoglobulin (Ig) G assays can be also performed similarly. PGI/II ratios ⁇ 3.0 were considered a positive result.
  • the promoter region of RPRM gene (National Center for Biotechnology Information [NCBI] accession number NM _019845) contains a dense CpG island located from base 154.335.529 to 154.335.021 (Homo sapiens chromosome 2, GRCh37.p13 Primary Assembly) relative to the chromosome localization (Fig. 6A, Primers and probe sequences are listed in Table 5).
  • NCBI National Center for Biotechnology Information
  • the promoter region was cloned into pUC57 vector using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA) and the number of copies of RPRM was calculated using concentration and size of linearized plasmid.
  • a 10-fold serial dilution of the plasmid (10e-1 to 10e-8 copies/mL) was prepared and run in triplicates.
  • ROC Receiver Operating Characteristic
  • Fisher's Exact Test p values less than 0.05 were considered statistically significant.
  • the positive and negative predictive values for the entire cohort were estimated using a nested case-control design based on the prevalence of EGC (1 .7%) from the entire Population-Based Gastric Cancer Screening Program described in the Examples.
  • methylated RPRM cfDNA was considered when more than 0 copies/mL were detected in plasma.
  • positive result was considered when serum PG l/ll rate was inferior to 3.
  • a positive result occurs when either of the biomarkers is positive.
  • methylated RPRM cfDNA combined with PG l/ll showed an overall sensitivity of 87.5% (95% Cl :61 .6-98.1 ) and specificity of 64.4% (95% CI :54.6-73.5) for early gastric cancer detection.
  • Positive- and negative- predictive values estimated for the cohort were 4.1 % (95% CI :2.3-6.7) and 99.6% (95%CI :98.8-99.9), respectively.
  • methylated RPRM cfDNA and PG l/ll in methods and/or assay for the non-invasive assessment of early gastric cancer may reduce gastric cancer incidence and mortality in the population.
  • Example 1 Clinical samples Three hundred and twenty subjects were enrolled in the study. Forty chronic gastritis and gastric cancer patients who underwent an upper endoscopic procedure were prospectively enrolled. In all cases tissue and plasma samples were obtained during the upper endoscopic procedure. In gastric cancer patients, tumor and matched noncancerous gastric normal mucosa were obtained.
  • One hundred and thirty-seven healthy control subjects were recruited from Blood Banks as control subjects. Endoscopic examination was performed in 1 1 of these healthy controls to rule out the presence of gastric cancer.
  • 19 gastric cancer cases (8 total gastrectomy and 1 1 palliative chemotherapy) plasma samples were obtained before and three months after gastric cancer treatment.
  • Example 2 A nested case-control study
  • EGC stage I GC
  • 107 matched controls 49 atrophic gastritis, 44 intestinal metaplasia and 14 dysplasia. Cases were individually matched by sex and age ( ⁇ 4 years) to 1 -3 controls from each lesion of the precancerous cascade.
  • Cases and controls were selected from a population-based GC Screening Program that included a cohort of 1344 enrolled symptomatic patients, from whom serum and plasma samples were taken during upper gastrointestinal endoscopy (UGE).
  • UGE upper gastrointestinal endoscopy
  • Inclusion criteria were men and women aged 40 years and older, with upper gastrointestinal symptoms for more than 15 days and no usage of H2 blockers, nonsteroidal anti-inflammatory drugs (NSAIDs) or corticosteroids.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • corticosteroids corticosteroids
  • Methylated RPRM cfDNA was assessed by MethyLight (Roche) and PG l/l I by Enzyme-linked immunosorbent assay (ELISA) test (Biohit).

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Abstract

La présente invention concerne un procédé de détection non invasive d'un cancer gastrique à un stade précoce, comprenant les étapes consistant à mesurer l'ADN sans cellule de Reprimo méthylé; à déterminer le rapport de pepsinogène I/II; et à déterminer si le résultat est positif ou négatif.
PCT/IB2015/054104 2014-05-30 2015-05-29 Procédé et test de détection non invasive d'un cancer gastrique à un stade précoce par l'utilisation combinée d'adn sans cellule de reprimo méthylé et de pepsinogène i/ii WO2015181804A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016205971A1 (fr) * 2015-06-26 2016-12-29 Pontificia Universidad Catolica De Chile Méthode ultrasensible de détection de biomarqueur du cancer de l'estomac
WO2018209361A3 (fr) * 2017-05-12 2019-01-10 President And Fellows Of Harvard College Diagnostic de cancer précoce universel
CN114107213A (zh) * 2021-11-12 2022-03-01 苏州大学 Rprm基因在调控组织电离辐射耐受性上的应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6465177B1 (en) * 1998-10-26 2002-10-15 John Wayne Cancer Institute Detection of loss of heterozygosity in tumor and serum of melanoma patients
US6331393B1 (en) * 1999-05-14 2001-12-18 University Of Southern California Process for high-throughput DNA methylation analysis
EP1829978B1 (fr) * 2004-12-13 2011-09-21 Bio-Dixam LLC Procede consistant a detecter la methylation d'un gene et procede consistant a rechercher un neoplasme en detectant la methylation
EP2557425A3 (fr) * 2007-10-26 2013-07-10 Biohit Oyj Procédés et produits permettant de diagnostiquer des maladies auto-immunes et le cancer gastrique lié à une gastrite atrophique
CL2009001907A1 (es) * 2009-09-28 2010-05-28 Univ Pontificia Catolica Chile Metodo de deteccion precoz de cancer gastrico que comprende detectar la presencia de marcadores especificos de cancer gastrico en muestras de plasma.
CN102782500B (zh) * 2010-03-03 2015-03-18 东丽株式会社 胃癌检测用的标志物和胃癌检测方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016205971A1 (fr) * 2015-06-26 2016-12-29 Pontificia Universidad Catolica De Chile Méthode ultrasensible de détection de biomarqueur du cancer de l'estomac
WO2018209361A3 (fr) * 2017-05-12 2019-01-10 President And Fellows Of Harvard College Diagnostic de cancer précoce universel
CN111417731A (zh) * 2017-05-12 2020-07-14 哈佛学院校长同事会 通用早期癌症诊断
CN114107213A (zh) * 2021-11-12 2022-03-01 苏州大学 Rprm基因在调控组织电离辐射耐受性上的应用

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