- Letter to the Editor
- Open Access
Personalized analysis of minimal residual cancer cells in peritoneal lavage fluid predicts peritoneal dissemination of gastric cancer
Journal of Hematology & Oncology volume 14, Article number: 164 (2021)
Peritoneal dissemination (PD) is a major type of gastric cancer (GC) recurrence and leads to rapid death. Current approaches cannot precisely determine which patients are at high risk of PD to provide early intervention. In this study, we developed a technology to detect minimal residual cancer cells in peritoneal lavage fluid (PLF) samples with a personalized assay profiling tumor-specific mutations. In a prospective cohort of 104 GC patients, the technology detected all the cases that developed PD with 100% sensitivity and 85% specificity. The minimal residual cancer cells in PLF were associated with a significantly increased risk of PD (HR = 145.13; 95% CI 20.20–18,435.79; p < 0.001), which was the strongest independent predictor over pathologic diagnosis and cytological diagnosis. In pathologically high-risk (pT4) patients, the PLF mutation profiling model exhibited a greater specificity of 91% and a positive predictive value of 88% while retaining a sensitivity of 100%. This approach may help in the postsurgical management of GC patients by detecting PD far before metastatic lesions grow to a significant size detectable by conventional methods such as MRI and CT scanning.
To the Editor,
Peritoneal dissemination (PD) is a major type of gastric cancer (GC) recurrence and a strong indicator of poor prognosis [1, 2]. Although multiple therapeutic solutions such as hyperthermic intraperitoneal chemotherapy (HIPEC) have been developed to prevent PD [3,4,5], current diagnostic approaches cannot precisely determine which patients will develop PD. When detectable by CT/MRI or causing symptoms, PD lesions are often of significant size, with no effective treatments available. Minimal residual disease (MRD) detection based on tumor-specific mutations in plasma cell-free DNA (cfDNA) has shown promising performance in prognostic prediction and disease monitoring in several tumor types, including breast, colorectal and lung cancers [6,7,8,9,10].
Here, we developed customized mutation profiling technology to detect minimal residual cancer cells from peritoneal lavage fluid (PLF). For each case, 20 tumor-specific mutations were selected from exome sequencing of the tumor tissue, and a personalized assay based on Mutation Capsule, a mutation profiling technology, was developed to detect the mutations. The assay was applied to the genomic DNA from cell pellets in the matched PLF samples, which were collected after abdominal exploration and before any manipulation of the stomach in the surgery. A model was developed to determine the fraction of cancer cells among normal cells in PLF based on the number and fraction of the mutations detected (Fig. 1a). The materials and methods are shown in detail in Additional file 1.
To validate the accuracy of the assay, we made a standard reference with serial dilutions of PLC/PRF/5 cells into A549 cells (Additional file 2: Table S1). We selected 20 SNPs unique to PLC/PRF/5 to profile in the genomic DNA of the cell dilutions (Additional file 2: Table S2). We found a strong linear correlation between the theoretical and estimated dilution ratios up to a dilution of 1:100,000 (R2 = 0.9998) (Fig. 1b). Background noise observed at 0% PLC/PRF/5 cell input was < 0.0007% among 20 independent replicates, and the assay confidently detected a 0.001% fraction (Fig. 1c, d). To evaluate the biological noise of mutations from nontumor cells in the PLF sample, we profiled 20 mutations that were not detected in the matched tumor sample. We found background noise in all PLF clinical samples to be < 0.01%, which was used as the cutoff for MRD detection in the subsequent analysis (Fig. 1e).
We applied MRD analysis to a cohort of 104 GC patients with radical resection (Additional file 3: Figure S1; Additional file 2: Table S3). Based on exome sequencing of tumor tissue, 17 (median, range 3–23) mutations were selected and profiled on matched PLF samples (Additional file 2: Table S4, S5). The cancer cell fraction ranged from 0 to 23.41%, and 42 cases (40%) were MRD-positive. At the 41-month follow-up, 27 out of the 104 patients experienced PD, and all (100%) of them were MRD-positive. Six patients developed lymphatic metastasis, and 4 (67%) were MRD-positive (Fig. 2a, b; Additional file 2: Table S6). In addition, 15% (11/71) of the nonrecurrence patients were MRD-positive. The MRD analysis achieved 100% sensitivity, 85% specificity and 71% positive predictive value (PPV), and the MRD-positive patients showed a high risk of PD (HR = 145.13; 95% CI 20.20–18,435.79; p < 0.001) (Fig. 2c, d). In addition, MRD-positive patients were associated with decreased recurrence-free survival (RFS) and overall survival, and all 10 patients who died during the follow-up were MRD-positive patients with disease recurrence (Fig. 2d, e; Additional file 3: Figure S2A).
We further compared the predictive value of the MRD analysis with clinical risk factors (Additional file 3: Figure S2B, C). The MRD analysis showed the best performance (Fig. 2c) and was the strongest independent predictor of RFS for PD based on the Cox proportional hazard model (Additional file 2: Table S7). Interestingly, the MRD analysis exhibited a lower false-positive rate among stage pT4 patients (n = 56), delivering 100% (23/23) sensitivity, 91% (30/33) specificity and 88% (23/26) PPV (Fig. 2c, f).
In conclusion, our mutation-based MRD analysis exhibited highly improved performance compared with cytology or other PLF-based assays, making early detection and early intervention far before PDs become clinically detectable possible. This approach may help improve postoperative treatment to prevent PD and improve the overall survival of GC.
Availability of data and materials
The dataset supporting the conclusions of this article is available in the Genome Sequence Archive for Human repository [HRA000528 in https://bigd.big.ac.cn/gsa-human/].
Peritoneal lavage fluid
Hyperthermic intraperitoneal chemotherapy
Minimal residual disease
Positive predictive value
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This study was supported by the National Key R&D Program of China (2018YFC1312100) and the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-001, 2019-I2M-1-003 and 2017-I2M-4-002).
Ethics approval and consent to participate
All patients provided written informed consent, and the study was approved by the Ethics Committee of National Cancer Center, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (Approval Number: 17-093/1349).
Consent for publication
Yuchen Jiao, Dongbing Zhao, Pei Wang, Qianqian Song and Pinli Yue have filed patents/patent applications based on the technology and data generated from this work. Yuchen Jiao is one of the cofounders, has owner interest in Genetron Holdings, and receives royalties from Genetron. The remaining authors disclose no conflicts.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional file 1.
Materials and Methods.
Additional file 2. Table S1
.Theoretical and estimated dilution ratio of cancer cells. Table S2. Detected mutations in standard curve. Table S3. Clinicopathological characteristics. Table S4. Summary of whole-exome sequencing and traced mutations. Table S5. Personalized somatic mutations in tumor tissue and paired PLF. Table S6. Raw data on the cancer cell fraction and clinical risk factors for the prediction of PD. Table S7. Recurrence-free survival analysis by clinicopathological variables and MRD analysis.
Additional file 3. Fig. S1
.Patient enrollment, sample collection workflow and the prognosis prediction of patients. Fig. S2. Kaplan–Meier estimates of recurrence-free survival (RFS) based on clinical risk factors and MRD analysis. (A) Kaplan–Meier estimates of RFS based on MRD analysis in 104 gastric cancer patients. Peritoneal dissemination and lymphatic metastasis patients were combined as a recurrence group (n = 33). The Kaplan–Meier survival analysis shows the probability of RFS by (B) cytological diagnosis with peritoneal lavage fluid (n = 98) and (C) pT stage according to the 8th edition of the Union for International Cancer Control (n = 98) for peritoneal dissemination. A patient was classified as test-positive if the cytological diagnosis of peritoneal lavage fluid was positive (B) and the pathologic diagnosis was pT4 stage gastric cancer (C). CCF: cancer cell fraction. Shaded areas in the Kaplan–Meier plots indicate 95% CIs. HR: hazard ratio.
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Zhao, D., Yue, P., Wang, T. et al. Personalized analysis of minimal residual cancer cells in peritoneal lavage fluid predicts peritoneal dissemination of gastric cancer. J Hematol Oncol 14, 164 (2021). https://doi.org/10.1186/s13045-021-01175-2
- Gastric cancer
- Peritoneal lavage fluid
- Peritoneal dissemination
- Personalized mutation assay
- Minimal residual disease