Methylated Reprimo Cell-Free DNA as a Non-Invasive Biomarker for Gastric Cancer

Journal
International Journal of Molecular Sciences
ISSN
1422-0067
Date Issued
2025-04-03
Author(s)
María José Maturana
Oslando Padilla
Pablo M. Santoro
Maria Alejandra Alarcón
Wilda Olivares
Alejandro Blanco
Marcelo Garrido
Edmundo Aravena
Carlos Barrientos
Alfonso Calvo-Belmar
Alejandro H. Corvalán
Type
journal-article
DOI
URL
Abstract
Restrictions resulting from the COVID-19 pandemic abruptly reversed the slow decline of the diagnosis and mortality rates of gastric cancer (GC). This scenario highlights the importance of developing cost-effective methods for mass screening and evaluation of treatment response. In this study, we evaluated a non-invasive method based on the circulating methylated cell-free DNA (cfDNA) of Reprimo (RPRM), a tumor suppressor gene associated with the development of GC. Methylated RPRM cfDNA was analyzed in three de-identified cohorts: Cohort 1 comprised 81 participants with GC and 137 healthy donors (HDs); Cohort 2 comprised 27 participants with GC undergoing gastrectomy and/or chemotherapy analyzed at the beginning and after three months of treatment; and Cohort 3 comprised 1105 population-based participants in a secondary prevention program who underwent esophagogastroduodenal (EGD) endoscopy. This cohort includes 180 normal participants, 845 participants with premalignant conditions (692 with chronic atrophic gastritis [AG] and 153 with gastric intestinal metaplasia/low-grade dysplasia [GIM/LGD]), 21 with high-grade dysplasia/early GC [HGD/eGC], and 59 with advanced GC [aGC]). A nested case-control substudy was performed using a combination of methylated RPRM cfDNA and pepsinogens (PG)-I/II ratio. The dense CpG island of the promoter region of the RPRM gene was bisulfite sequenced and analyzed to develop a fluorescence-based real-time PCR assay (MethyLight). This assay allows the determination of the absolute number of copies of methylated RPRM cfDNA. A targeted sequence of PCR amplicon products confirmed the gastric origin of the plasma-isolated samples. In Cohort 1, the mean value of GCs (32,240.00 copies/mL) was higher than that of the HD controls (139.00 copies/mL) (p < 0.0001). After dividing this cohort into training–validation subcohorts, we identified an area under the curve of 0.764 (95% confidence interval (CI) = 0.683–0.845) in the training group. This resulted in a cut-off value of 87.37 copies/mL (sensitivity 70.0% and specificity 80.2%). The validation subcohort predicted a sensitivity of 66.67% and a specificity of 83.33%. In Cohort 2 (monitoring treatment response), RPRM levels significantly decreased in responders (p = 0.0042) compared to non-responders. In Cohort 3 (population-based participants), 18.9% %, 24.1%, 30.7%, 47.0%, and 71.2% of normal, AG, GIM/LGD, HGD/eGC, and aGC participants tested positive for methylated RPRM cfDNA, respectively. Overall sensitivity and specificity in distinguishing normal/premalignant conditions vs. GC were 65.0% (95% CI 53.52% to 75.33%) and 75.9% (95% CI 73.16% to 78.49%), respectively, with an accuracy of 75.11% (95% CI 72.45% to 77.64%). Logistic regression analyses revealed an OR of 1.85 (95% CI 1.11–3.07, p = 0.02) and an odds ratio (OR) of 3.9 (95% CI 1.53–9.93, p = 0.004) for the risk of developing GIM/LGD and HGD/eGC, respectively. The combined methylated RPRM cfDNA and PG-I/II ratio reached a sensitivity of 78.9% (95% CI 54.43% to 93.95%) and specificity of 63.04% (95% CI 52.34% to 72.88%) for detecting HGD/eGC vs. three to six age- and sex-matched participants with premalignant conditions. Our results demonstrate that methylated RPRM cfDNA should be considered a direct biomarker for the non-invasive detection of GC and a predictive biomarker for treatment response.
Subjects
adult

; 

aged

; 

biomarkers

; 

tumor

; 

case-control studies

; 

cell-free nucleic acids

; 

covid-19

; 

dna methylation

; 

female

; 

humans

; 

male

; 

middle aged

; 

stomach neoplasms

; 

antineoplastic agent

; 

circulating free dna

; 

eosin

; 

hematoxylin

; 

pepsinogen i

; 

pepsinogen ii

; 

protein

; 

reprimo

; 

tumor marker

; 

unclassified drug

; 

cell free nucleic acid

; 

tumor marker

; 

adult

; 

advanced cancer

; 

aged

; 

amplicon

; 

area under the curve

; 

article

; 

atrophic gastritis

; 

bisulfite sequencing

; 

cancer surgery

; 

case control study

; 

cohort analysis

; 

controlled study

; 

cpg island

; 

diagnostic accuracy

; 

dna methylation

; 

enzyme linked immunosorbent assay

; 

esophagogastroduodenoscopy

; 

false positive result

; 

female

; 

fluorescence quantitative polymerase chain reaction

; 

fluorescent polymerase chain reaction

; 

histopathology

; 

human

; 

human tissue

; 

intestinal metaplasia of the stomach

; 

major clinical study

; 

male

; 

non invasive measurement

; 

palliative chemotherapy

; 

precancer

; 

predictive value

; 

promoter region

; 

prospective study

; 

response evaluation criteria in solid tumors

; 

secondary prevention

; 

sensitivity and specificity

; 

stomach cancer

; 

stomach tissue

; 

total gastrectomy

; 

transcription initiation site

; 

treatment response

; 

blood

; 

coronavirus disease 2019

; 

diagnosis

; 

genetics

; 

middle aged

; 

pathology

; 

stomach tumor

; 

biomarkers

; 

cancer prevention

; 

cancer screening

; 

cfdna

; 

gastric cancer

; 

liquid biopsy

; 

methylated rprm

; 

non-invasive diagnosis