Patient information

The exposed group included 120 CRC patients (84 males, 36 females), aged 28 to 80 years, with a mean age of 59.3 years. Tumor locations included 25 in the left colon, 17 in the right colon, and 78 in the rectum. A total of 87 patients were administered chemotherapy, while 30 patients received concurrent chemoradiotherapy. Additionally, 3 patients exclusively received immunotherapy. Detailed demographics and treatment regimens are presented in Table S2 (Supplemental Digital Content 2).

Changes in MMR expression after PT

MMR expression loss was observed in 6 pre-treatment biopsy samples (1 MSH6, 1 MSH2, 4 MLH1 and PMS2). Post-treatment surgical samples revealed loss in 9 cases (4 MSH6, 2 PMS2, 2 MSH6 and MSH2, 4 MLH1 and PMS2) (Fig. 1A). Three cases showed inconsistent MMR expression post-treatment, all transitioning from pMMR to dMMR (Fig. 1B; Table 1).

Fig. 1

MMR expression in colorectal cancer before and after preoperative treatment. (A) Heatmap of MMR proteins expression scores pre- and post-treatment; (B) Representative cases of MMR expression conversion after preoperative treatment (↑ positive staining of normal cells, ↑ negative staining of tumor cells); (C) Comparison of MMR scores between pre- and post- treatment and the control group. *P < 0.05, ** P < 0.01, ***P < 0.001

Table 1 Clinicopathological characteristics of patients with colorectal cancer stratified by MMR expression conversion

Pre-treatment samples demonstrated significantly higher scores for MSH6 (P < 0.001), MSH2 (P < 0.01), and total MMR score (P < 0.01) compared to post-treatment samples. However, no significant differences were noted for MLH1 (P = 0.655) and PMS2 (P = 0.324) (Fig. 1C).

When compared to the control group’s surgical samples, post-treatment surgical samples showed significantly lower MSH2 scores and higher MLH1 scores, with no significant differences in MSH6, PMS2, and total MMR scores (Fig. 1C). Heterogeneous staining for MSH6 was common in surgical samples, regardless of treatment, primarily manifesting as varied staining within the same gland rather than between different regions (Figure S2, Supplemental Digital Content).

Analysis of MSI status in cases with deficient or weak MMR expression

22 samples from 14 patients who with a score less than 6 underwent MSI status analysis. Among the 5 pre-treatment dMMR cases, both pre- and post-treatment samples were classified as MSI-H (Table 2). For the remaining dMMR and weakly MMR expressing cases, both pre- and post-treatment samples were classified as MSS.

Table 2 Analysis of MSI status in cases with deficient or weakly expressed MMR proteinsCorrelation between changes in MMR expression and clinicopathologic features

We attempted to compare the changes in protein expression with various clinicopathological features and found that, PMS2 was found to be associated with gender. However, changes in MSH6, MSH2, and MLH1 did not correlate with any clinicopathological features (Table S3, Supplemental Digital Content 2). The change in total MMR score was significantly associated with fluorouracil (5-FU) and capecitabine treatment. Specifically, cases with reduced MMR expression treated with 5-FU were significantly fewer compared to untreated cases (Fig. 2A). In contrast, cases with reduced MMR expression treated with capecitabine were significantly more frequent compared to untreated cases (Fig. 2B).

Fig. 2

Relationship between fluorouracil (A) and capecitabine (B) and changes in total MMR scores after preoperative treatment. *P < 0.05, ** P < 0.01

Changes in HER2 expression after PT

A total of 99 cases underwent HER2 testing before and after PT (Fig. 3A). Post-treatment samples showed significantly higher HER2 scores compared to pre-treatment samples (P < 0.001; Fig. 3B), while control group scores were not significantly different. No clinicopathological features were identified as associated with HER2 expression changes except cTNM stage (Table S3, Supplemental Digital Content 2).

Fig. 3

HER2 expression in colorectal cancer before and after preoperative treatment. (A) Heatmap of HER2 expression scores pre- and post-treatment; (B) Comparison of HER2 scores between pre- and post- treatment and the control group. ***P < 0.001

Prognostic analysis results

Prognostic analysis results indicated that within the exposed group, seven patients had incomplete data regarding disease progression or overall survival. Specifically, three lacked progression information, three were missing survival data, and one patient was lost to follow-up. As of the final follow-up date, four patients had deceased due to cancer-related causes. The average PFS and OS were 791.3 days and 1823.3 days, respectively (Fig. 4A-B, Table S4, Supplemental Digital Content 2).

Fig. 4

Prognostic analysis in patients with CRC following preoperative treatment. Panel A represents the PFS curve, while Panel B represents the OS curve. Panels C-I represent the comparisons of PFS across various subgroups

Supporting information

Univariate analysis revealed significant associations between PFS and cTNM stage, ypTNM stage, treatment modalities, KRAS mutation and the use of oxaliplatin, bevacizumab, or cetuximab (Fig. 4C-H, Table S4, Supplemental Digital Content 2). However, neither the baseline expression of MMR proteins and HER2, nor the changes in their expression following treatment, showed significantly associated with PFS (Table S4, Supplemental Digital Content 2). Although univariate analysis did not identify an association between TRG scores and PFS, multivariate analysis demonstrated TRG as an independent predictor of PFS (Fig. 4I, Table S4, Supplemental Digital Content 2). Additionally, ypTNM stage was also identified as an independent factor influencing PFS in the multivariate assessment (Table S4, Supplemental Digital Content 2).

We further analyzed the relationship between MSH6, MSH2, total MMR, HER2, and their expression changes post-treatment with PFS across different clinical stages. The results indicated that in metastatic colorectal cancer, the changes in HER2 expression were significantly associated with PFS. In contrast, MMR and its expression changes did not exhibit an association with PFS, regardless of the disease being in a localized or metastatic stage (Table S5, Supplemental Digital Content 2).

It is noteworthy that no statistically significant associations were identified between any of the evaluated factors and OS (data not shown), potentially due to the limited sample size and the short follow-up duration.