Clinicopathological and molecular profiling of sporadic synchronous multiple primary colorectal cancers: focus on microsatellite instability status

Introduction

Colorectal cancer (CRC) remains a major cause of cancer-related morbidity and mortality, and its burden continues to increase in many regions (1), with the incidence of synchronous multiple primary colorectal cancer (SCRC) also increasing. SCRCs—defined as two or more primary colorectal carcinomas diagnosed simultaneously within the colon and/or rectum—are being recognized more frequently in routine practice. SCRC has been reported to account for approximately 1.1% to 8.1% of all CRCs (2-7). SCRC is subdivided by etiology into hereditary and sporadic, with some multiple primary CRCs occurring in individuals with hereditary syndromes, such as SCRC due to familial adenomatous polyposis (FAP) and Lynch syndrome, while others are associated with hereditary genetic syndromes without a clear relationship. Currently, there are more extensive studies on FAP and Lynch syndrome, but few studies have analyzed sporadic synchronous multiple primary colorectal cancer (SSCRC) in depth. Most studies on SSCRC have focused on clinical characterization and epidemiology (8), and only a few have conducted comprehensive studies of clinical phenotypes and molecular characterization (9).

In addition, molecular biology studies of synchronous colon cancer have focused on the analysis of microsatellite instability (MSI) (6,10,11). MSI status is a key molecular phenotype for CRC prognostic assessment and therapeutic strategy development, and its role has become even more prominent with advances in the treatment of microsatellite instability-high (MSI-H) CRC, which plays a crucial role in guiding the application of immunotherapies such as programmed cell death protein 1 (PD-1) inhibitors in particular (12). However, it is not clear whether MSI status is consistent across lesions in the same SSCRC patient. To scientifically and rigorously investigate the impact of MSI status on the clinicopathological features and prognosis of SSCRC patients, this study performed MSI status testing on all tumor lesions in SSCRC cases. The results were systematically organized and subsequently analyzed to provide comprehensive insights. We present this article in accordance with the STROBE reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0027/rc).

Methods

Patients

Retrospective inclusion criteria for SSCRC were applied as follows: (I) histopathological confirmation of adenocarcinoma; (II) presence of at least two primary colorectal tumors separated by normal mucosa; (III) absence of distant metastasis from the primary CRC; and (IV) diagnosis together or within 6 months of each other (V). Exclusion criteria for the cases are as follows: (I) metastatic or recurrent cancer; (II) patients with FAP or Lynch syndrome; (III) patients with cancer arising from ulcerative colitis; (IV) incomplete medical records. The inclusion criteria for patients with solitary primary colorectal cancer (SPCRC) were as follows: (I) histologically confirmed colorectal adenocarcinoma; (II) presence of a single primary tumor without synchronous or metachronous colorectal lesions; (III) no evidence of hereditary CRC syndromes; and (IV) availability of complete clinicopathological and follow-up data. We included patients with SSCRC who underwent surgical treatment at The First Affiliated Hospital of Shandong First Medical University from January 2020 to December 2023, all of whom had not received neoadjuvant therapy. To facilitate a better statistical analysis of patient prognosis, we also included 202 patients with single primary CRC who underwent surgical treatment throughout the year 2020. All cancer lesions in the SSCRC and SPCRC patients included in the study underwent pathological examination. All cancer lesions in the SSCRC patients included in the study were tested for MSI status.

Tumors were staged according to the TNM classification (13). For anatomical analyses, lesions were categorized by location into right-sided colon (including the appendix, cecum, ascending colon, hepatic flexure, and transverse colon), left-sided colon (including the splenic flexure, descending colon, sigmoid colon, and rectosigmoid junction), and rectum.

Clinical and pathological information for each patient was collected through electronic medical records, and follow-up was conducted using methods including phone calls, outpatient visits, and re-admissions. Initially, patients were followed up every 3 months after surgery, and if no recurrence occurred by one year post-surgery, follow-up was reduced to every 6 months. Regular check-ups included serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) testing, with a computed tomography (CT) scan every 6 months postoperatively. The follow-up ended on August 31, 2024, or at the time of patient death. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Board of The First Affiliated Hospital of Shandong First Medical University (No. YXLL-KY-2024(116)). Informed consent was obtained from all individual participants.

MSI analysis

DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissues using standard protocols. MSI status was assessed using a polymerase chain reaction (PCR)-based assay targeting five mononucleotide markers (BAT25, BAT26, NR21, NR24, and NR27). PCR amplification was performed according to the manufacturer’s instructions, and fragment analysis was conducted using a capillary electrophoresis system. Tumors were classified as MSI-H if ≥2 markers showed instability, microsatellite instability-low (MSI-L) if only one marker was unstable, and microsatellite stable (MSS) if no markers showed instability (14,15).

MSI status was classified as MSI-H, MSI-L, and MSS according to standard criteria.For analytical purposes, MSI-H tumors were compared with MSI-L/MSS tumors based on their similar clinicopathological behavior reported in previous studies. Based on inter-lesional MSI concordance, SSCRC patients were categorized into the following groups: Group I, patients with concordant MSI-H tumors across all lesions; Group II, patients with concordant MSI-L/MSS tumors across all lesions; and Group III, patients with discordant MSI status across lesions.

To evaluate lesion-level MSI concordance in synchronous tumors, PCR-based MSI testing was performed on all available tumor lesions in SSCRC cases. In contrast, immunohistochemical (IHC) assessment of mismatch repair (MMR) proteins and BRAF mutation analysis were conducted on index lesions. This approach was mainly due to the retrospective design of the study and practical constraints, including limited tissue availability and variability in archived formalin-fixed paraffin-embedded specimens.

BRAF mutations and mismatch-repair gene expression analysis

In SSCRC, the most pathologically advanced cancer was designated as the index tumor. When more than one tumor were diagnosed with identical pathological stage, the largest one was considered the index tumor and the other lesions were considered the companion tumors. Due to workload and some objective factors, we used immunohistochemistry to assess the MMR status and BRAF status of the index tumor in each SSCRC patient. We analyzed the expression of MMR genes and BRAF genes in the index tumor.

IHC staining for MMR proteins was performed on formalin-fixed paraffin-embedded tissue sections. The following primary antibodies were used: MLH1 (clone ES05), MSH2 (clone FE11), MSH6 (clone EP49), and PMS2 (clone EP51) (all from Zhongshan Golden Bridge Biotechnology, Beijing, China). Staining was performed using the EnVision two-step method according to the manufacturer’s instructions. Normal tissue with known expression of the respective proteins was used as a positive control, and phosphate-buffered saline (PBS) was used as a negative control in place of the primary antibody. Nuclear staining in tumor cells was evaluated, and loss of expression of any MMR protein was defined as deficient mismatch repair (dMMR), while retained nuclear staining was considered proficient mismatch repair (pMMR) (16).

Statistical analysis

Continuous variables are presented as mean ± SD (or median with interquartile range when appropriate) and compared using Student’s t-test for normally distributed data; otherwise, the Mann-Whitney U test was applied. Categorical variables were analyzed using the χ² test or Fisher’s exact test as appropriate. Survival outcomes were estimated with Kaplan-Meier curves and compared by the log-rank test. Overall survival (OS) was defined as the time from the date of surgery to death from any cause or last follow-up. Progression-free survival (PFS) was defined as the time from the date of surgery to disease progression, recurrence, or death from any cause, whichever occurred first. Patients without events were censored at the date of last follow-up. Cox proportional hazards models were used for univariable and multivariable analyses, with hazard ratios (HRs) reported alongside 95% confidence intervals. Variables with P<0.10 in univariable analysis or those considered clinically relevant (including tumor stage and N category) were included in the multivariable Cox regression model. A two-sided P value <0.05 was considered statistically significant. No missing data were observed in the analyzed variables. In addition, information on postoperative adjuvant therapy, including chemotherapy and immunotherapy, was collected from medical records when available and descriptively analyzed.

Results

Clinicopathologic characteristics of patients with SSCRC versus SPCRC

We obtained 106 primary tumors from 46 patients with SSCRC who underwent surgical resection. Among them, 36 patients had two tumors, 8 had three tumors, 1 had four tumors, and 1 had six tumors. Table 1 summarizes the clinical and pathological characteristics between SSCRC and SPCRC patients. Seven factors showed statistically significant differences: tumor location (P<0.001), tumor size (P=0.048), tumor differentiation (P<0.001), T category (P=0.04), N category (P=0.04), overall cancer stage (P=0.01), and perineural invasion (P<0.001). Other clinical and pathological features, such as age, sex, histological type, and vascular invasion, did not show significant differences between SSCRC and SPCRC patients.

Molecular characterization of patients with SSCRC versus SPCRC BRAF mutations

As shown in Table 2, BRAF mutations were detected in 4.0% of SPCRC patients and 4.3% of SSCRC patients. There was no significant difference in the BRAF mutation rate between SSCRC and SPCRC patients (P>0.99).

MMR

In the 46 SSCRC patients, according to the IHC results of each index tumor, 9 cases (19.6%) were classified as dMMR, while 37 cases (80.4%) were classified as pMMR. Among the 202 SPCRC patients, 10 cases (5.0%) were dMMR, and 192 cases (95%) were pMMR. As shown in Table 2, dMMR was more common in SSCRC than in SPCRC (19.6% vs. 5.0%; P=0.003). Furthermore, we further analyzed the expression loss of MMR proteins leading to dMMR status between SSCRC and SPCRC patients (Table 2), and the results showed no significant difference (P=0.84).

MSI

Among the 106 synchronous lesions analyzed, 24 (22.6%) were classified as MSI-H, 3 (2.8%) as MSI-L, and 79 (74.5%) as MSS. When applying the two-tier MSI classification system, 24 tumors were categorized as MSI-H, whereas 82 were considered MSI-L/MSS. At the patient level, MSI status among the 46 SSCRC cases was stratified into MSI-L/MSS (n=37, 80.4%) and MSI-H (n=9, 19.6%).

Survival analysis of patients with SSCRC versus SPCRC

Postoperative adjuvant chemotherapy was administered in a subset of patients, most commonly using fluoropyrimidine- and oxaliplatin-based regimens. A small proportion of patients received immune checkpoint inhibitors during follow-up. The distribution of treatment patterns is summarized in Table S1. The median follow-up time was 46 months (range, 1–55 months). Survival outcomes were evaluated in 202 patients with SPCRC and 46 patients with SSCRC. Over the follow-up period, death occurred in 27 individuals (13.4%) in the SPCRC cohort and in 9 individuals (19.6%) in the SSCRC cohort. OS and PFS data were complete for all patients in both groups. Median OS was not reached, as the Kaplan-Meier curves did not decline below 50% in either cohort. Therefore, mean survival time was reported as an alternative descriptive measure. The mean OS for SPCRC patients was 49.8 months, while for SSCRC patients, it was 41.5 months. The mean PFS time for SPCRC patients was 48.4 months, and for SSCRC patients, it was 40.2 months. As shown in Figure 1A, in Kaplan-Meier analysis, SSCRC patients experienced worse OS than SPCRC patients (log-rank, P=0.03). However, there was no significant difference in PFS between the two groups.

Figure 1 Kaplan-Meier survival analyses were performed for SPCRC patients and SSCRC patients, including their subgroups. (A) Survival analyses were performed to compare overall survival and progression-free survival between patients with synchronous colorectal carcinomas and those with solitary colorectal carcinomas. (B) Survival analyses were performed to compare overall survival and progression-free survival in subgroups of patients with synchronous colorectal cancers based on MSI concordance, versus patients with solitary colorectal carcinomas. I, all individual tumors MSI-H; II, all individual tumors MSI-L/MSS. MSI-H, microsatellite instability-high; MSI-L, microsatellite instability-low; MSS, microsatellite stable; SSCRC, sporadic synchronous multiple primary colorectal cancer; SPCRC, solitary primary colorectal cancer.

Analysis of SSCRC patients in each subgroup based on MSI status consistency

Assessment of intertumoral MSI concordance across lesions within each of the 46 patients with synchronous disease revealed the following distribution: group I (concordant MSI-H tumors), 9 patients (19.6%); group II (concordant MSI-L/MSS tumors), 37 patients (80.4%); and no patients with discordant MSI status were identified in this cohort. Table 3 summarizes the clinicopathological and molecular features of groups I and II. Four factors showed statistically significant differences: gender (P=0.005), tumor location (P=0.006), N category (P=0.02), and overall cancer stage (P=0.01). Other factors, such as age, tumor size, number of tumors, tumor differentiation, histological type, T category, vascular invasion, perineural invasion, and BRAF mutation status, did not show significant differences between Group I and Group II patients. Figure 1B shows the survival curves for the two subgroups of SSCRC patients and SPCRC patients. Although the differences in PFS and OS between the groups were not statistically significant, Figure 1B suggests a trend of poorer prognosis in Group II (concordant MSI-L/MSS tumors).

Univariable and multivariable analyses of possible prognostic factors

To explore variables associated with unfavorable outcomes, univariable and multivariable analyses were performed. For PFS, the results showed that advanced N category (P<0.001), vascular invasion (P=0.02), perineural invasion (P=0.004), and BRAF mutation (P<0.001) were risk factors for poor PFS (Table 4). When these factors were further subjected to multivariable analysis, advanced N category (HR =4.918, 95% CI: 2.058–11.752, P<0.001), perineural invasion (HR =2.157, 95% CI: 1.012–4.601, P=0.047), and BRAF mutation (HR =8.454, 95% CI: 3.112–22.967, P<0.001) were identified as independent risk factors for poor PFS (Table 4). For OS, the results showed that advanced N category (P<0.001), vascular invasion (P=0.01), perineural invasion (P=0.004), and BRAF mutation (P<0.001) were risk factors for poor OS (Table 5). When these factors were further subjected to multivariable analysis, advanced N category (HR =4.265, 95% CI: 1.794–10.140, P=0.001) and BRAF mutation (HR =7.252, 95% CI: 2.714–19.378, P<0.001) were identified as independent risk factors for poor OS (Table 5). MSI status was evaluated in univariable analysis but was not included in the multivariable model due to the limited number of MSI-H cases and lack of statistical significance. Importantly, tumor stage-related variables (including N category) were included in the multivariable models to account for potential confounding effects.

Discussion

Over the last decade, immunotherapy—particularly PD-1 inhibition—has substantially reshaped the management of multiple malignancies, including CRC (17). It is estimated that approximately 12% of CRC cases harbor sporadic MSI/dMMR (18). Clinical evidence indicates that PD-1 blockade can produce favorable responses in dMMR/MSI-H CRC and is currently recommended as a first-line option for metastatic dMMR/MSI-H CRC (19). Accordingly, MMR/MSI status has become increasingly influential in therapeutic decision-making. Nevertheless, in patients with sporadic synchronous multiple primary CRC (SSCRC), whether MMR/MSI profiles remain concordant across different lesions within the same individual is still uncertain. In light of ongoing advances in CRC immunotherapy (20-24) and the treatment response observed in our case(s), we suggest that molecular subtype-guided strategies are warranted for SSCRC. For the fully MSI-H group, immune therapies such as PD-1 inhibitors are recommended as the first-line treatment. For patients with both MSI-H and MSI-L/MSS lesions, a combination of PD-1 inhibitors and chemotherapy may be more appropriate. For patients with fully MSI-L/MSS tumors, neoadjuvant chemotherapy, with or without targeted therapy, can be considered. Due to the molecular heterogeneity of different lesions, we recommend performing MSI status testing on each lesion in newly diagnosed SSCRC patients to optimize treatment strategies. Therefore, this study examined the MSI status of all lesions in 46 SSCRC patients over the past 4 years of treatment. Our results showed that the MSI status was consistent across different SSCRC lesions. Based on this, we classified SSCRC into two groups: the fully MSI-H group and the fully MSI-L/MSS group. Among these two groups, the fully MSI-L/MSS group had a higher incidence, which is consistent with the higher frequency of pMMR status in SPCRC. However, dMMR was observed at a significantly higher frequency in the SSCRC group than in the SPCRC group. No patients with both MSI-H and MSI-L/MSS lesions were observed in this study, which may be attributed to the relatively small sample size. This complete concordance may be influenced by the relatively small sample size and may not fully reflect the potential intertumoral heterogeneity reported in larger cohorts. However, the relatively small number of MSI-H cases in this study may limit the statistical robustness of subgroup comparisons and survival analyses; therefore, these findings should be interpreted with caution.

This study compared the clinicopathological characteristics, molecular features, and prognostic outcomes between SSCRC and SPCRC patients. A recent study by Nosho et al. (10)reported that patients with synchronous CRCs experienced poorer OS and disease-free survival compared with those with solitary CRC. This is consistent with our findings. Our study revealed that, compared to SPCRC cases, the SSCRC index lesions (which are larger and more advanced among SSCRCs) had poorer tumor differentiation, larger average size, later staging, and a higher incidence of perineural invasion. Additionally, the proportion of dMMR was higher (Tables 1,2). These factors contributed to a higher tumor burden, increased tumor aggressiveness, larger surgical resection areas, and greater surgical complexity in SSCRC patients compared to SPCRC patients. As a result, SSCRC patients are at a higher risk of local recurrence and metastasis, leading to a higher postoperative morbidity. We believe this may explain the lower OS rate in SSCRC patients compared to SPCRC patients, as shown in Figure 1A (P=0.03). In terms of PFS, although no statistical differences were observed between the SSCRC and SPCRC groups, Figure 1A shows a trend of poorer disease-free survival in SSCRC patients. In addition, postoperative treatment heterogeneity, including differences in adjuvant chemotherapy and the use of immune checkpoint inhibitors, may have influenced the survival outcomes observed in this study. Notably, BRAF mutation emerged as the strongest independent prognostic factor in this study, with hazard ratios exceeding 7, highlighting its critical role in risk stratification. In addition, its interaction with MSI status may further influence prognosis and warrants further investigation. In addition, key clinicopathological variables, including tumor stage, were accounted for in the multivariable analysis to minimize potential confounding.

In cases of multiple primary tumors, our study found a high proportion (20%) of tumors with MSI-H in synchronous CRC patients. In this study, MSI-L tumors were not analyzed as a separate subgroup due to the limited sample size. Consistent with previous reports, MSI-L tumors were grouped together with MSS tumors, given their similar biological and clinical characteristics. MSI-H is typically associated with improved OS and disease-free survival (25,26). The increased presence of MSI-H in SSCRC may contribute to an improved OS rate, which is consistent with our findings (Figure 1B). As shown in the Figure 1B, group I (all individual tumors MSI-H) had a better prognosis than group II (all individual tumors MSI-L/MSS). This suggests that patients in group II may require more aggressive or personalized treatment strategies. Although no statistically significant survival difference was observed, likely due to the limited number of MSI-H cases, MSI status remains an important predictive biomarker for immunotherapy response and may guide treatment decisions in SSCRC. Based on these findings, we speculate that SSCRC patients may derive greater therapeutic benefit from immunotherapy than SPCRC patients, further emphasizing the importance of MSI status assessment for each lesion in SSCRC patients.

We also observed a distinct distribution pattern of SSCRC within the colon. Our findings revealed significant differences in tumor location between isolated and synchronous CRCs. Contrary to previous studies that reported SSCRC occurring more frequently in the right colon compared to SPCRC (27,28), our data suggest that SSCRC has a higher incidence in the left colon (comprising the splenic flexure, descending colon, sigmoid colon, and rectosigmoid junction) and a lower incidence in the rectum. In contrast, SPCRC tends to have a higher incidence in the rectum and a lower incidence in the left colon (Table 1). This discrepancy may be related to differences in study populations, sample size, or regional characteristics.

It should be noted that, in this study, comprehensive molecular profiling was not performed uniformly across all lesions. While PCR-based MSI testing was conducted for all synchronous tumors to assess inter-lesional concordance, MMR immunohistochemistry and BRAF mutation analysis were limited to index lesions. This approach was primarily adopted because index lesions are generally considered to represent the most clinically relevant and biologically advanced tumors in SSCRC.

However, given the recognized intertumoral heterogeneity in synchronous CRC, this limitation may reduce the ability to fully characterize molecular concordance beyond MSI status. Therefore, our findings regarding concordance primarily apply to MSI status rather than the full spectrum of molecular alterations. Future studies with uniform multi-platform molecular testing across all lesions are warranted to better characterize intrapatient molecular heterogeneity.

Despite the clinical significance of these findings, this study has several limitations. First, the patients in this study were selected from hospital-based samples, which may introduce selection bias compared to population-based samples. Second, due to the retrospective nature of this study, treatment information was not uniformly available for all patients. Differences in adjuvant chemotherapy and immunotherapy could not be fully accounted for in the survival analysis, which may have introduced potential confounding. Additionally, molecular analysis in this study was performed only on cancerous lesions, which may not fully capture the broader molecular context, including potential information from non-cancerous tissue, such as background alterations or hereditary predisposition. MMR immunohistochemistry and BRAF mutation analyses were only performed on index lesions, which may limit a comprehensive assessment of molecular heterogeneity across all synchronous tumors. Further studies involving larger, population-based cohorts and comprehensive molecular analysis of both cancerous and non-cancerous tissues are needed to validate these findings.

Conclusions

This study demonstrates that MSI status is highly concordant across lesions in SSCRC, with 19.6% of patients classified as MSI-H and 80.4% as MSI-L/MSS. Although no statistically significant association with survival was observed, this may be related to the limited number of MSI-H cases. Importantly, MSI status remains clinically relevant as a predictive biomarker for immunotherapy eligibility. Notably, BRAF mutation emerged as the strongest independent prognostic factor for both PFS and OS, with hazard ratios exceeding 7, identifying a high-risk subgroup requiring closer monitoring and more aggressive management. These findings support the importance of comprehensive molecular profiling in SSCRC and suggest that MSI status and BRAF mutation may help guide individualized treatment strategies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0027/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0027/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0027/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-1-0027/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Institutional Review Board of The First Affiliated Hospital of Shandong First Medical University (No. YXLL-KY-2024(116)). Informed consent was obtained from all individual participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Han B, Zheng R, Zeng H, et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 2024;4:47-53. [Crossref] [PubMed]
2. Arriba M, Sánchez R, Rueda D, et al. Toward a Molecular Classification of Synchronous Colorectal Cancer: Clinical and Molecular Characterization. Clin Colorectal Cancer 2017;16:31-7. [Crossref] [PubMed]
3. Kato T, Alonso S, Muto Y, et al. Clinical characteristics of synchronous colorectal cancers in Japan. World J Surg Oncol 2016;14:272. [Crossref] [PubMed]
4. van Leersum NJ, Aalbers AG, Snijders HS, et al. Synchronous colorectal carcinoma: a risk factor in colorectal cancer surgery. Dis Colon Rectum 2014;57:460-6. [Crossref] [PubMed]
5. Lam AK, Chan SS, Leung M. Synchronous colorectal cancer: clinical, pathological and molecular implications. World J Gastroenterol 2014;20:6815-20. [Crossref] [PubMed]
6. Hu H, Chang DT, Nikiforova MN, et al. Clinicopathologic features of synchronous colorectal carcinoma: A distinct subset arising from multiple sessile serrated adenomas and associated with high levels of microsatellite instability and favorable prognosis. Am J Surg Pathol 2013;37:1660-70. [Crossref] [PubMed]
7. Lam AK, Carmichael R, Gertraud Buettner P, et al. Clinicopathological significance of synchronous carcinoma in colorectal cancer. Am J Surg 2011;202:39-44. [Crossref] [PubMed]
8. Bae JM, Cho NY, Kim TY, et al. Clinicopathologic and molecular characteristics of synchronous colorectal cancers: heterogeneity of clinical outcome depending on microsatellite instability status of individual tumors. Dis Colon Rectum 2012;55:181-90. [Crossref] [PubMed]
9. Zhao Y, Wu J, Pei F, et al. Molecular Typing and Clinical Characteristics of Synchronous Multiple Primary Colorectal Cancer. JAMA Netw Open 2022;5:e2243457. [Crossref] [PubMed]
10. Nosho K, Kure S, Irahara N, et al. A prospective cohort study shows unique epigenetic, genetic, and prognostic features of synchronous colorectal cancers. Gastroenterology 2009;137:1609-20.e1-3.
11. Aslanian HR, Burgart LJ, Harrington JJ, et al. Altered DNA mismatch repair expression in synchronous and metachronous colorectal cancers. Clin Gastroenterol Hepatol 2008;6:1385-8. [Crossref] [PubMed]
12. Beamer LC, Grant ML, Espenschied CR, et al. Reflex immunohistochemistry and microsatellite instability testing of colorectal tumors for Lynch syndrome among US cancer programs and follow-up of abnormal results. J Clin Oncol 2012;30:1058-63. [Crossref] [PubMed]
13. American Joint Committee on Cancer. AJCC Cancer Staging Manual. 8th ed. New York: Springer; 2017.
14. Xicola RM, Llor X, Pons E, et al. Performance of different microsatellite marker panels for detection of mismatch repair-deficient colorectal tumors. J Natl Cancer Inst 2007;99:244-52. [Crossref] [PubMed]
15. Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998;58:5248-57.
16. Reis-Filho JS. Detecting MSI and TRK fusion using NGS: ESMO recommendations. Presented at: ESMO 2018 Congress; October 19–23, 2018; Munich, Germany. Available online: https://www.retesarcoma.it/pdf/final_programme_esmo2018.pdf
17. Egen JG, Ouyang W, Wu LC. Human Anti-tumor Immunity: Insights from Immunotherapy Clinical Trials. Immunity 2020;52:36-54. [Crossref] [PubMed]
18. Echle A, Ghaffari Laleh N, Quirke P, et al. Artificial intelligence for detection of microsatellite instability in colorectal cancer-a multicentric analysis of a pre-screening tool for clinical application. ESMO Open 2022;7:100400. [Crossref] [PubMed]
19. Benson AB, Venook AP, Al-Hawary MM, et al. Colon Cancer, Version 2.2021, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw 2021;19:329-59. [Crossref] [PubMed]
20. Llosa NJ, Luber B, Siegel N, et al. Immunopathologic Stratification of Colorectal Cancer for Checkpoint Blockade Immunotherapy. Cancer Immunol Res 2019;7:1574-9. [Crossref] [PubMed]
21. Ganesh K, Stadler ZK, Cercek A, et al. Immunotherapy in colorectal cancer: rationale, challenges and potential. Nat Rev Gastroenterol Hepatol 2019;16:361-75. [Crossref] [PubMed]
22. Overman MJ, McDermott R, Leach JL, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol 2017;18:1182-91. [Crossref] [PubMed]
23. Le DT, Durham JN, Smith KN, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017;357:409-13. [Crossref] [PubMed]
24. Le DT, Uram JN, Wang H, et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med 2015;372:2509-20. [Crossref] [PubMed]
25. Ricco G, Gallio C, Benhima N, et al. Mismatch repair deficiency/microsatellite instability (dMMR/MSI-H) in rectal cancer patients treated with standard neoadjuvant therapy: a systematic review and meta-analysis. Cancer Treat Rev 2025;141:103039. [Crossref] [PubMed]
26. Petrelli F, Dottorini L, Zaniboni A, et al. Immune checkpoint inhibitors for dMMR/MSI localized rectal cancer: A systematic review of treatment strategies. Crit Rev Oncol Hematol 2025;215:104921. [Crossref] [PubMed]
27. Tanegashima T, Shiota M, Toyosaki K, et al. Biology and evolving management of resectable dMMR/MSI-H cancers: current status and future perspectives. Cancer Immunol Immunother 2025;74:364. [Crossref] [PubMed]
28. Ambrosini M, Manca P, Nasca V, et al. Epidemiology, pathogenesis, biology and evolving management of MSI-H/dMMR cancers. Nat Rev Clin Oncol 2025;22:385-407. [Crossref] [PubMed]