Schistosoma infection, KRAS mutation status, and prognosis of colorectal cancer

To the Editor: Colorectal cancer (CRC) is a major cause of cancer-related death worldwide, and several pathogens with carcinogenic potential, such as Schistosoma, have been associated with an increased risk of CRC. As a frequently mutated gene in CRC patients, kirsten rat sarcoma viral oncogene homolog (KRAS) helps to determine the selection of CRC therapies. Therefore, we aimed to elucidate the association among Schistosoma infection, KRAS mutation status, and the prognosis of CRC in this study. We enrolled 489 CRC patients (schistosomal CRC [SCRC]: n = 30, non-schistosomal CRC [NSCRC]: n = 459) who were pathologically diagnosed at Union Hospital, Tongji Medical College of Huazhong University of Science and Technology from January 1, 2010 to June 31, 2019 in this corhort study. Each patient was pathologically diagnosed with primary CRC [Figure 1A], and the degree of differentiation and histological type were described according to the American Joint Committee on Cancer criteria. The criteria for positive infection included the presence of intact or calcified eggs, active granulomas, or adult worms [Figure 1A]. Those with metastatic disease and/or unconfirmed tumor information were excluded. The clinical features, including sex, age, year of diagnosis, stage, histological type, tumor differentiation, primary site, primary tumor size, chemotherapy, radiotherapy, KRAS mutation status (verified by quantitative polymerase chain reaction [qPCR]), survival time, and survival status, were collected. The study was approved by the Medical Ethics Committee of Union Hospital Affiliated with Tongji Medical College of Huazhong University of Science and Technology (No. 2018-S377).Figure 1: Association between Schistosoma infection and prognosis of colorectal cancer. (A) Hematoxylin–eosin staining of CRC tissues, scale bar: 500 μm. Left: NSCRC; Right: SCRC. The arrows indicated deposited Schistosoma eggs. (B) Overall survival of SCRC and NSCRC patients. (C) Disease-free survival of SCRC and NSCRC patients. CRC: Colorectal cancer; MST: Mean survival time; NSCRC: Non-schistosomal colorectal cancer; SCRC: Schistosomal colorectal cancer.All statistical analyses were performed using SPSS statistics software (version 26.0, IBM Corporation, Armonk, NY, USA). The statistical significance of baseline clinicopathological characteristics was assessed with the Chi-squared test or Fisher's exact test as appropriate. Univariate and multivariate Cox analyses were conducted to estimate the relationship between Schistosoma infection and the overall survival (OS) of patients with CRC. The Kaplan–Meier test was performed for survival analysis. Propensity score matching (PSM) was applied to balance the clinicopathological variables of the patients at a 1:4 ratio with the Schistosoma infection (yes or no) as the dependent variable and confounding factors (sex, age, histology, and primary site) as the covariates. A two-sided P value < 0.05 was considered statistically significant. Baseline characteristics of the CRC patients were shown in Supplementary Table 1, https://links.lww.com/CM9/B784. Kaplan–Meier survival analysis revealed that patients with SCRC exhibited statistically significantly worse OS (mean survival time [MST] of SCRC: 51.9 months [95% confidence interval [CI]: 43.0–60.7] vs. MST of NSCRC: 65.3 months [95% CI: 62.9–67.6], χ2 = 9.908, P = 0.002) and disease-free survival (DFS, MST of SCRC: 51.5 months [95% CI: 42.4–60.6] vs. MST of NSCRC: 63.1 months [95% CI: 60.7–65.6], χ2 = 5.298, P = 0.021) compared to those with NSCRC [Figures 1B and 1C]. Moreover, univariate and multivariate Cox regression analyses showed that Schistosoma infection was an independent risk factor for CRC in terms of OS. Specifically, compared with NSCRC patients, worse OS was associated with SCRC patients (univariate Cox analysis: hazard ratio [HR], 2.689; 95% CI, 1.416–5.108; P = 0.003. Multivariate Cox analysis: HR, 2.507; 95% CI, 1.275–4.932; P=0.008) [Supplementary Table 2, https://links.lww.com/CM9/B784]. For DFS, the univariate Cox regression analysis identified Schistosoma infection as a risk factor for CRC (HR, 2.062; 95% CI, 1.098–3.873; P = 0.024). However, after controlling for potential confounding factors in the multivariate Cox regression analysis, Schistosoma infection was no longer an independent risk factor for CRC (HR, 1.849; 95% CI, 0.956–3.575; P = 0.068) [Supplementary Table 3, https://links.lww.com/CM9/B784]. There was no statistically significant difference in the mutation rate of overall KRAS (verified through qPCR) between the SCRC and NSCRC groups (63.3% [19/30] vs. 48.8% [224/459], χ2 = 2.379, P = 0.123). However, a detailed analysis of specific mutation sites revealed an interesting distinction. Patients with SCRC were more likely to have KRASG12S/D mutations than patients with NSCRC (43.3% [12/30] vs. 18.1% [83/459], χ2 = 11.379, P = 0.001) [Supplementary Table 4, https://links.lww.com/CM9/B784]. Due to limitations of the KRAS mutation qPCR detection kit (where detection primers for the G12S and G12D sites are combined in one reaction tube), mutations at G12S or G12D were indistinguishable. To address this limitation, we expanded our analysis by performing next-generation sequencing on an additional 230 colorectal cancer patients. After applying 1:4 PSM, a well-balanced study population was obtained, consisting of a SCRC group (n = 8) and a matched NSCRC group (n = 32). It is worth noting that in the SCRC group, the mutation rate of KRASG12D was statistically significantly higher compared to the NSCRC group (4/8 vs. 3/32, χ2 = 4.773, P=0.029) [Supplementary Table 5, https://links.lww.com/CM9/B784]. Notably, the KRASG12S mutation was absent in both groups [Supplementary Table 5, https://links.lww.com/CM9/B784]. Finally, The Kaplan–Meier survival analysis showed that KRAS mutations were not associated with OS (MST of SCRC: 64.1 months [95% CI, 60.9–67.4] vs. MST of NSCRC: 64.9 months [95% CI: 61.7–68.0], χ2 = 0.063, P = 0.802) or DFS (MST of SCRC: 62.4 months [95% CI: 59.2–65.7] vs. MST of NSCRC: 62.4 months [95% CI: 59.0–65.8], χ2 = 0.025, P = 0.874) among CRC patients. Similarly, KRASG12S/D mutations were not associated with OS (MST of SCRC: 63.4 months [95% CI: 58.6–68.1] vs. MST of NSCRC: 64.7 months [95% CI: 62.2–67.2], χ2 = 0.016, P = 0.899) or DFS (MST of SCRC: 61.9 months [95% CI: 57.0–66.8] vs. MST of NSCRC: 62.3 months [95% CI: 59.7–65.0], χ2 = 0.005, P = 0.944) in this patient population [Supplementary Figure 1, https://links.lww.com/CM9/B784]. Increasing evidence indicates that schistosomiasis is associated with the development of certain malignancies (such as bladder cancer, liver cancer, and other urogenital tumors), but the effects of Schistosoma infection on the prognosis of CRC have rarely been studied. A report from the National Cooperative Group on Pathology and Prognosis of CRC indicated that CRC patients with schistosomiasis had a 5-year survival rate of 45.6% [196/430], significantly lower than that of patients without schistosomiasis (50.9% [1383/2717]).[1] The study by Wang et al[2] on 30 patients with Schistosoma japonicum-associated rectal cancer revealed that schistosomiasis was an independent prognostic factor for worse DFS and OS in CRC. In this study, the Kaplan–Meier survival analysis showed that SCRC patients had worse OS and DFS than NSCRC patients, which further supported the association between Schistosoma infection and worse prognosis in CRC. A previous study conducted by Soliman et al[3], including 59 Egyptian colorectal carcinoma patients, found that schistosomiasis was linked to KRAS mutations. This finding prompted us to investigate the correlation between Schistosoma infection and KRAS mutations in a Chinese population. Although we did not find an association between overall KRAS mutations and Schistosoma infection, upon further analysis of mutation sites, we observed a statistically significantly higher mutation rate of KRASG12D in SCRC patients compared to NSCRC patients. While we did not replicate the exact results found by Soliman et al[3], we consider that this discrepancy may be attributed to differences in ethnic backgrounds, Schistosoma species, or potentially limited sample sizes. Additionally, it is worth noting that KRASG12D is the most common mutation in KRAS-mutant tumors (5894/17,741, 33.2%).[4] Nevertheless, the development of KRASG12D inhibitors has been notably hampered because of the structural characteristics of the KRASG12D site. The high KRASG12D mutation rate in SCRC means that there may be fewer treatments for SCRC than for NSCRC. However, we did not discover any associations between KRAS mutations and OS or DFS in CRC patients in this study. This is consistent with a previous study from our group, in which we revealed that the KRAS mutation was not found to be an independent predictive factor for OS in CRC patients.[5] Therefore, it is possible that the prognosis of schistosomiasis is not directly driven by KRAS mutations. Strengths of our study include a large sample size, comprehensive clinical and pathological information, and the use of PSM to address confounding factors. We also ensured diagnostic accuracy through a rigorous review by experienced pathologists. This study investigates the association between Schistosoma infection and the KRAS mutation sites, providing valuable insights into the underlying mechanisms. In summary, this study highlights the association of Schistosoma infection with poor prognosis and a higher KRASG12D mutation rate in CRC patients. KRAS mutations lead to resistance to anti-epithelial growth factor receptor treatment, and no effective inhibitor exists for KRASG12D, making SCRC patients more likely to encounter inadequate treatment options. The prevention and treatment of schistosomiasis require further investigation. Future research should focus on elucidating the relationship between Schistosoma infection and KRASG12D mutation in CRC patients. Funding This work was supported by grants from the National Key Research and Development Program of China (Nos. 2022YFF1203300 and 2018YFC1313300), the National Natural Science Foundation of China (Nos. 81702392 and 81472707), the Chinese South Western Oncology Group (No. CSWOG-CCET005), and the Beijing Bethune Public Welfare Foundation (No. BJ-GYQZHX2021006). Conflicts of interest None.