Prospective study to validate the clinical utility of DNA diagnosis of peritoneal fluid cytology test in gastric cancer

Abstract

The clinical efficacy of DNA cytology test (CY) in gastric cancer (GC) has been retrospectively proposed using cancer-specific methylation of cysteine dioxygenase type 1 (CDO1). We confirmed the clinical utility of DNA CY in a prospective cohort. Four hundred GC samples were prospectively collected for washing cytology (UMIN000026191), and detection of the DNA methylation of CDO1 was assessed by quantitative methylation-specific PCR in the sediments. Endpoint was defined as the match rate between conventional CY1 and DNA CY1 (diagnostic sensitivity), and the DNA CY0 rate (diagnostic specificity) in pStage IA. DNA CY1 was detected in 45 cases (12.5%), while CY1 was seen in 31 cases (8.6%) of 361 chemotherapy-naïve samples, where the sensitivity and specificity of the DNA CY in the peritoneal solutions were 74.2% and 96.5%, respectively. The DNA CY was positive for 3.5/0/4.9/11.4/58.8% in pStage IA/IB/II/III/IV, respectively (P < .01). In the multivariate analysis, DNA CY1 was independently correlated with pathological tumor depth (pT) (P = .0012), female gender (P = .0099), CY1 (P = .0135), P1 (P = .019), and carcinoembryonic antigen (CEA) (P = .036). The combination of DNA CY1 and P factor nearly all covered the potential peritoneal dissemination (P1 and/or CY1 and/or DNA CY1) (58/61:95.1%). DNA CY1 had a significantly poorer prognosis than DNA CY0 in GC patients (P < .0001). DNA CY1 detected by CDO1 promoter DNA methylation has a great value to detect minimal residual disease of the peritoneum in GC clinics, representing poor prognosis as a novel single DNA marker.

Keywords: Cysteine dioxygenase type 1; gastric cancer; methylation-specific PCR; peritoneal dissemination; washing cytology test.

Conflict of interest statement

The authors have no competing interest to declare.

© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Figures

FIGURE 1

Bar graphs representing diagnostic sensitivity of the conventional CY1 (red bars) and the DNA CY1 (blue bars) according to pathological factor of gastric cancer (GC). A, Pathological T factor. B, Pathological N factor. C, Pathological stage

FIGURE 2

Quantitative methylation‐specific PCR (Q‐MSP) by using CDO1 DNA methylation. A, TaqMeth values of CDO1 in the peritoneal fluid samples of the 361 gastric cancers (GCs) (upper panel). The quantified outcomes of the DNA CY1 are magnified (lower panel). In the lower panel, red bar graphs represent conventional CY1, while blue graphs indicate conventional CY0. B, Q‐MSP curves of the representative cases. CDO1 TaqMeth values (Vs) were definitely represented as 39.8, 2.7, 1.2, and 0.3 in sample numbers 202, 156, 123, and 74, respectively (upper panel), while the minimum CDO1 TaqMeth V 0.003 among DNA CY1 cases was judged as positive in sample number 145

FIGURE 3

Prognosis analysis in DNA CY and CY. A, Overall survival of 346 gastric cancer (GC) patients associated with DNA CY. B, Overall survival of 165 advanced GC patients associated with DNA CY. C, Overall survival of 346 GC patients stratified according to the results of DNA CY and CY. D, Peritoneal dissemination–free survival of 346 GC patients stratified according to the results of DNA CY and CY. E, Overall survival of 51 GC patients with DNA CY and/or CY positive. F, Peritoneal dissemination–free survival of 51 GC patients with DNA CY and/or CY positive

FIGURE 4

Detailed assessment for the conventional CY1 cases (n = 27). In quantitative methylation‐specific PCR (Q‐MSP), CDO1 TaqMeth values (Vs) of the preoperative tumor biopsy samples (blue bars) were compared with those of the intraperitoneal fluid samples (red bars). Left scale represents CDO1 TaqMeth V in Q‐MSP. The red‐circled samples were judged to be negative in Q‐MSP because CDO1 TaqMeth V was 0