A Multicenter Collaborative Survey of Peritoneal Lavage Cytology in Gastric Cancer Surgery: Interinstitutional Methodological Differences and Positive Rate

Abstract

Objective:

This study aimed to clarify the current status of cytological methodologies in high-volume gastric cancer centers and explore the relationship between methodology and positive rates.

Background:

International standards for peritoneal lavage cytological methods for collection, handling, and cytopreparation in gastric cancer have not been established yet.

Methods:

A questionnaire survey on cytological methodology was conducted in 61 institutions within the Japan Clinical Oncology Group in 2024. Aggregated data from patients with clinical T3 to T4 gastric cancer with cytology from 2017 to 2022 were collected to calculate positivity rates in each institution for the comparison of methodologies between institutions with high- and low-positivity rates.

Results:

Thirty-three institutions (64%) collected samples from 2 sites, primarily from the Douglas pouch and left subphrenic area. Fifty-eight institutions (95%) used ≤100 mL of normal saline for injection, and 51 (87%) performed intraoperative rapid cytology. Twenty-five institutions (41%) used additives in samples. Scraping glass slides and centrifugal direct smears were predominant cytopreparation methods in 31 (51%) and 22 (36%) institutions, respectively, and ethanol fixation was employed in 53 (87%). In 61 institutions (11,367 patients), the median cytological positivity rate for clinical T3 to T4 gastric cancer was 8.5% (2.1%–28.3%). Institutions with higher positivity rates more often employed ethanol fixation (97% vs. 77%, P = 0.026) and used ≤50 mL of normal saline for injection (61% vs. 37%, P = 0.074).

Conclusions:

Even among Japanese high-volume centers, cytological methodologies for gastric cancer lack uniformity, thereby leading to substantial variability in the proportion of positive cytology.

INTRODUCTION

Peritoneal lavage cytology is incorporated into the staging system of Japanese Gastric Cancer Association and Union for International Cancer Control, and positive cytology is defined as distant metastasis.^1,2 Patients with positive lavage cytology have a dismal long-term prognosis, even if it is an only incurable factor and macroscopic curative resection is achieved, and are at a higher risk of peritoneal recurrence within 2 years after surgery.³ Postoperative chemotherapy with S-1 has improved the survival outcomes, with a reported 5-year survival rate of 26% to 27.1%^4,5; however,^3–5 positive cytology remains a strong poor prognostic factor.⁶

No cytological methodologies have been standardized yet, and the positivity rates of peritoneal cytology in patients with gastric cancer vary from 4% to 41%.⁷ The Union for International Cancer Control does not specify the methods for cytological sampling; however, these methods are clearly outlined in the Japanese Classification of Gastric Carcinoma. Therefore, less variability would be expected between institutions in Japan than in the West. However, the handling of cytological samples and pathological evaluation methods have not been standardized, even in Japan. Furthermore, no studies have reported interinstitutional concordance across different countries. Given that various cytological techniques were employed, international standards for gastric cancer cytology must be established to eliminate disparities among medical institutions and countries and establish methods that achieve high interinstitutional concordance.

This study employing a questionnaire-based survey aimed to clarify the current status of cytological sample collection, handling, and diagnostic procedures in high-volume institutions attending to patients with gastric cancer in Japan and provide fundamental data as basis for future discussions on unifying cytological methods. In addition, it seeks to explore appropriate methods with less interinstitutional variability.

PATIENTS AND METHODS

A questionnaire was distributed through the mailing list of the Stomach Cancer Study Group within the Japan Clinical Oncology Group (JCOG). This survey covered the period from March to April 2024 across 66 institutions participating in the JCOG Stomach Cancer Study Group.

The survey respondents were patients with clinical T3 to T4 gastric cancer in whom peritoneal lavage cytology was performed between 2017 and 2022. Patients with peritoneal metastasis confined to the perigastric region, classified as P1a in the 15th edition of the Japanese Classification of Gastric Carcinoma, were included; however, those with distant metastasis other than positive cytology were excluded. The questionnaire (Supplemental Table 1, see https://links.lww.com/AOSO/A524) included indications for cytology, site of collecting cytological specimens according to the surgical approach, volume of normal saline injected to the abdominal cavity, additives used in the harvested samples, cytopreparation methods, fixation methods, time from sample collection to fixation, staining methods, number of cytotechnologists, and time to diagnosis (intraoperative rapid diagnosis or routine diagnosis). Intraoperative rapid cytology involved fixation, staining, and procedures designed to provide a quick report, typically within 1 h intraoperatively. Although individual patient data were not collected, aggregated data about the number of cytology-positive patients were compiled by institution. The cytology-positive rate of clinical T3 to T4 gastric cancer in each institution was calculated. Subsequently, the institutions were divided into 2 groups based on the median cytology-positive rate, and questionnaire items were compared between these 2 groups. In most institutions, endoscopic ultrasonography is not routinely performed for advanced gastric cancer. Staging is performed through endoscopy, upper gastrointestinal series, and computed tomography.

The questionnaire was sent to researchers through a mailing list that included a Microsoft Forms link, allowing for response collection. As this survey did not involve access to individual patient data, institutional review board approval was waived.

STATISTICAL ANALYSIS

Fisher’s exact test was employed to compare categorical variables. A 2-sided P-value of <0.05 was considered significant. All statistical analyses were performed using R Statistics version 4.3.1 (R Foundation, Vienna, Austria).

RESULTS

Responses were obtained from 65 institutions. After excluding 3 institutions that provided insufficient data and one that does not routinely perform cytology for gastric cancer, data from 61 institutions were analyzed.

Distribution of Cytological Methods Across Institutions

Figure 1 illustrates the cytological methods across all 61 participating institutions. The most frequent method involved collecting samples from 2 sites, using ≤50 mL of saline without additives, maintaining a fixation time <1 h, preparing cytological specimens by scraping glass slides, using ethanol for fixation, and excluding immunocytochemistry (ICC). In most institutions, fewer than 2 cytotechnologists were involved in screening each cytological specimen.

FIGURE 1.

Differences in cytology methods among 61 institutions.

Figure 2 shows the institutional policies for determining the final diagnosis through intraoperative rapid and/or routine methods. Among the 39 institutions utilizing both intraoperative rapid and routine cytology, the results from routine cytology were adopted as the final diagnosis. Among these, 8 institutions rarely revised the intraoperative rapid cytological results based on routine cytological findings.

FIGURE 2.

Institutional policies for intraoperative rapid and routine cytology.

Figure 3 presents the site of collecting cytological specimens according to the surgical approach. Specimens were primarily collected from the Douglas pouch (80%–92%) and the left subphrenic space (57%–67%) regardless of the surgical approach.

FIGURE 3.

Site of cytological specimen harvest in (A) staging laparoscopy, (B) laparoscopic or robotic gastrectomy, and (C) open surgery.

Staining Methods Used in Cytology

Figure 4 presents the staining methods used in intraoperative rapid and routine cytology. In intraoperative rapid cytology, Papanicolaou staining alone was the most common (45%), followed by Papanicolaou staining plus one (34%) and plus 2 or more stains (15%). In routine cytology, Papanicolaou plus Giemsa staining was the most frequent (34%), followed by Papanicolaou staining alone (19%) and Papanicolaou plus Giemsa plus Periodic Acid-Schiff staining (17%). In addition, one institution used ICC with carcinoembryonic antigen (CEA) and Ber-EP4, whereas another used CEA and MOC 31; only 1 institution used Ber-EP4, and the cytology-positive rates in all 3 institutions were 16.7%, 10.2%, and 9.1%, respectively.

FIGURE 4.

Proportion of staining methods in (A) intraoperative rapid cytology and (B) routine cytology, including duplications.

Differences in CY1-Positive Rates by Institution

Among 11,367 patients with clinical T3 to T4 gastric cancer, 1038 showed cytology positivity (CY1), resulting in a CY1 rate of 9.1% (95% confidence interval, 8.6%–9.7%).

The number of patients with clinical T3 to T4 gastric cancer per institution ranged from 76 to 504, with a median of 167. Figure 5 presents the distribution of CY1 by medical institution. The median and average CY1 rates across institutions were 8.5% and 9.8%, respectively, ranging from 2.1% to 28.3%. The institutions with the lowest and highest positivity rates had 195 and 198 patients, respectively. The positivity rates and hospital type or geographical location showed a weak relationship.

FIGURE 5.

Distribution of positive cytology rates by medical institution. The error bars indicate the 95% confidence interval, and the dotted line indicates the median CY1 rates across institutions.

Comparison Between Institutions With High- and Low-Positive Cytological Rates

Table 1 presents a comparison between institutions divided into high (n = 31) and low (n = 30) groups based on the median CY1 rates (8.5%). The utilization of ethanol for fixation was significantly more prevalent in the high group (97% vs 77%, P = 0.026). Although not reaching significance, the high group tended to use <50 mL of normal saline for injection (61% vs. 37%, P = 0.074). The proportion of using routine cytology for the final diagnosis did not correlate with the positivity rates (84% vs. 70%, P = 0.235). In the 47 institutions using routine cytology, the time from harvest to fixation did not affect the positivity rate (65% vs. 43%, P = 0.149). In a subgroup analysis based on institutional case volume (>170 or ≤170 patients), the use of ≤50 mL of saline and ethanol fixation were associated with higher rates of cytology positivity in both groups, although the difference did not reach statistical significance.

TABLE 1.

Comparison Between Institutions With High- and low-Positive Cytology Rate

	Positivity Rate		P
	Low (n = 30)	High (n = 31)
Number of collection sites
1	12 (40)	9 (29)	0.426
2	18 (60)	22 (71)
Saline volume (mL)
≤50	11 (37)	19 (61)	0.074
>50	19 (63)	12 (39)
Final diagnostic method
Routine	21 (70)	26 (84)	0.235
Intraoperative rapid	9 (30)	5 (16)
Time from harvest to fixation in routine cytology (n = 47)
<1 h	9 (43)	17 (65)	0.149
≥1 h	12 (57)	9 (35)
Additive
No	16 (53)	20 (65)	0.440
Yes	14 (47)	11 (35)
Cytopreparation
Centrifugal direct smear	11 (37)	12 (39)	1.000
Noncentrifugal direct smear	19 (63)	19 (61)
Fixation
Ethanol	23 (77)	30 (97)	0.026
Nonethanol	7 (23)	1 (3)
Immunocytochemistry
Yes	0	3 (10)	0.238
No	30 (100)	28 (90)
Number of cytotechnologist
1	5 (17)	6 (19)	1.000
≥2	25 (83)	25 (81)

DISCUSSION

This study based on questionnaire survey clarified the current status of cytology, encompassing sample collection, specimen preparation, and diagnosis in high-volume institutions dealing with gastric cancer in Japan. To date, no comprehensive reports have summarized cytological methodologies, and this study is expected to provide essential baseline data on cytological methodologies. Based on the methods used by institutions with high positivity rates, the following approach is suggested to be preferable: injecting ≤50 mL of normal saline, processing samples within 1 h, and fixing specimens in ethanol.

To obtain samples, the most common procedure involved the injection of up to 100 mL of normal saline into the Douglas pouch and left subphrenic space, followed by ethanol fixation and Papanicolaou staining. Despite minimal interinstitutional variations in the collection methods, substantial differences were noted in sample handling, including time to fixation, use and type of additives, and specimen preparation techniques such as centrifugation. These discrepancies highlight challenges for the standardization of peritoneal lavage cytology across institutions.

The minimal between-institution variations in cytological specimen collection may be due to adherence to the gastric cancer treatment guidelines in Japan. For gastric cancers of clinical T2 or deeper, ascites is collected if present; otherwise, 100 mL of saline is injected, and lavage fluid is retrieved from the Douglas pouch. Conversely, the National Comprehensive Cancer Network, European Society for Medical Oncology, and the English version of Japanese guidelines recommend cytology for potentially resectable gastric cancer; however, they do not provide detailed methodologies for specimen collection and handling, resulting in the lack of international standards.^8,9 A systematic review of staging laparoscopy noted that the most common lavage volume is 200 (range, 200–1000) mL, with sampling from the upper quadrants and pelvis in approximately half of the patients.¹⁰ In Western countries, the rate of cytology positivity in resectable gastric cancer showed considerable variation, ranging from 7.8% to 36%,^11,12 potentially indicating methodological differences. The standardization of cytological methodologies may minimize variability and facilitate the formulation of uniform treatment algorithms. Moreover, because positive peritoneal cytology is regarded as a metastatic disease even in the absence of visible peritoneal implants, such patients can be included in palliative chemotherapy trials or excluded from adjuvant chemotherapy trials.^13–15 This highlights the need for methodological standardization in future clinical studies.

Sample preparation methods exhibited notable interinstitutional variability, employing various techniques. Most institutions fixed routine cytological samples within 1 h, though approximately 30% required over 4 h. Heparin and ethylenediaminetetraacetic acid were used to prevent coagulation caused by blood components; however, they may alter cell morphology, potentially complicating the diagnosis. Therefore, their use depends on institutional policies. Although cytospin smears can improve diagnostic accuracy by increasing cell density,¹⁶ conventional scraping glass slide smears remained more common owing to the lower costs and workload. Liquid-based cytology offers benefits such as simplified fixation, removal of blood components using hemolytic agents, and preparation of unstained slides¹⁷; however, its adoption is still limited.

Most institutions have used Papanicolaou and Giemsa staining without ICC. Minimal free cancer cells detected by CEA reverse transcription polymerase chain reaction are associated with high peritoneal recurrence and poor prognosis,¹⁸ highlighting the significance of cytological detection. Although ICC complements Papanicolaou staining in pancreatic cancer,¹⁹ no significant effect was observed on gastric cancer cytology positivity. The low number of institutions using ICC may have influenced this outcome, warranting further investigations into its potential to improve detection rates in low-positivity institutions.

Institutions with higher cytology positivity rates often used lower saline volumes and more frequently employed ethanol fixation. Ethanol fixation may improve the accuracy of positive cell detection by effectively preserving the morphology of cells. In addition, it is a simple and reliable method that minimizes variations in specimen quality, regardless of the preparer or institution. Conversely, spray fixation, which is a common alternative, requires a shorter fixation time and is thus advantageous for intraoperative rapid cytology. However, directly spraying the fixative onto the smear surface can result in uneven fixation, potentially affecting diagnostic accuracy. When fewer cancer cells are deposited in the Douglas pouch and subphrenic area, the use of large saline volumes may lead to the recovery of only a small amount of supernatant. However, smaller saline volumes are expected to more effectively collect fluid from deeper areas with even fewer cancer cells.

The concordance between pathologists may have affected the difference in positivity rates among institutions. In general cytology, the concordance between pathologists is high in benign compared with that in malignant samples but decreases in atypical or suspicious samples.^20,21 However, in gastric cancer cytology, relatively fewer samples were classified as atypical or suspicious malignancy. Given that experienced pathologists maintain a consistently high diagnostic accuracy,²² the interinstitutional differences in positivity rates observed in this study are not likely due to interobserver difference rather than methodological variations in sample collection and preparation. This point should be elucidated in future trials using individual patient data and pathological central reviews.

This study has several limitations. First, the interinstitutional variability in cytology-positive rates may be attributed to differences in patient backgrounds. Second, differences in the expertise of cytotechnologists and cytopathologists, a key factor in cytological interpretation, were not evaluated. Finally, individual patient data were not analyzed given the institution-based setting.

CONCLUSION

This study based on a questionnaire survey demonstrated the current status of cytological methodologies on gastric cancer in high-volume centers in Japan. The results suggested that ethanol fixation contributes to the higher positive rates in cytological diagnosis.

ACKNOWLEDGMENTS

The authors sincerely appreciate all investigators from the 66 institutions of the JCOG Stomach Cancer Study Group who participated in this survey.

K.F. designed the study, analyzed the data, and wrote the original draft. M.T. and K.T. edited the questionnaire survey and reviewed and revised the paper. K.N., R.T., S.S., J.K., Y. Kakiuchi, I.N., S.H., S.N., K.F., Y. Kurokawa, N.B., and T.Y. reviewed and edited the paper. All authors approved the final version.

Supplementary Material

as9-6-e607-s001.pdf ^{(353.1KB, pdf)}