Clinicopathologic Features and Prognostic Value of DNA Mismatch Repair Statues for Stage (cid:0)/(cid:0) Colorectal Cancer in Northeast China.

Background MSI CRCs were associated with better prognosis and limited predictive value for adjuvant chemotherapy. However, whether the same is true in Northeastern China is still unclear. The aim of the present study was to evaluate the association of clinicopathologic features and MMR/MSI status determined with immunohistochemistry analysis in Northeast China patients with stage (cid:0)/(cid:0) CRCs. Particularly, we sought to detect the relationship between MMR/MSI status and ecacy of oxaliplatin and uoropyrimidine based adjuvant chemotherapy. adjuvant OS,


Abstract
Background MSI CRCs were associated with better prognosis and limited predictive value for adjuvant chemotherapy.
However, whether the same is true in Northeastern China is still unclear. The aim of the present study was to evaluate the association of clinicopathologic features and MMR/MSI status determined with immunohistochemistry analysis in Northeast China patients with stage / CRCs. Particularly, we sought to detect the relationship between MMR/MSI status and e cacy of oxaliplatin and uoropyrimidine based adjuvant chemotherapy.

Methods
In total, 476 pathological specimens from eligible stage / CRCs were analyzed with IHC between 2016 and 2018, of which 63 CRCs were diagnosed with MMR protein de ciency. Clinicopathological features and overall survival (OS) were compared between these above two groups.

Conclusions
MMR protein appeared distinct associations with tumor staging, serum CEA level and tumor size. And MMR protein was an independent prognostic marker in patients with stage CRC, whereas dMMR CRC patients did not seem to bene t from oxaliplatin combined with uorouracil-based adjuvant chemotherapy.
Take Home Message 1. In this work, IHC was used to analyze the MMR protein status of surgical specimens obtained from CRC patients, and the clinicopathological characteristics and prognosis were compared between dMMR and pMMR CRCs.
2. In addition, our results suggest that the de ciency of MMR protein in tissue of color rectal cancer was associated with a better prognosis in stage patients, but not to bene t from oxaliplatin/5-FU based adjuvant chemotherapy.
3. This study is of reference for the chemotherapy treatment of dMMR CRCs in the northeast of China.

Background
Following lung cancer and prostate cancer in males and lung cancer and breast cancer in females, colorectal cancer (CRC), the third most commonly diagnosed malignant and the third leading cause of carcinoma-related mortality, continues a major international health problem [1]. There were more than 1.8 million new colorectal carcinoma cases and approximately 900,000 deaths in 2017 [2].
Currently, it was wildly accepted that chromosomal instability (CIN) and microsatellite instability (MSI) were the major pathways for CRC development [3]. CIN carcinomas, the most commonly pathway, were characteristic by mutations in genes resulting in chromosomal aneuploidy, loss of heterozygosity, and structural chromosomal rearrangements. However, MSI tumors were usually caused by the de ciency of mismatch repair (MMR) genes including MSH1, PMS2, MSH2 and MSH6 with the morbidity of approximately 15% of all sporadic CRCs [4]. Compared with CIN CRCs, dMMR/MSI colorectal malignancy tend to be proximal and histologically exhibit poor differentiation, a mucinous cell type and a better prognosis [5 6]. Therefore, it's crucial to identify the MMR/MSI status of CRCs.
Although there were currently three approaches for detection including immunohistochemistry (IHC), polymerase chain reaction (PCR) based methods and next generation sequencing (NGS), the sensitivity and speci city among the three methods were with high concordance rate (92-97%) [7].

Patients and materials
The ethics committee of Second A liated Hospital of Jilin University approved the present retrospective study, and the research was performed in accordance with the Helsinki Declaration of World Medicine Association. Since December 2016, our institution has routinely performed immunohistochemistry staining for the four MMR proteins in all newly diagnosed colorectal carcinoma resection specimens. After rigorous screening, eventually, 476 patients who went through CRC radical resection were considered eligible from December 1st, 2016 to December 1st, 2018 in our institution. The clinical data of the eligible CRCs (such as gender, age, BMI, tumor location, tumor size, Carcinoma Embryonic Antigen (CEA) and postoperative chemotherapy (POC) etc.) was obtained from medical records. The pathologic data (such as T category, pathologic N category, differentiation, tumor pathologic type and vascular invasion) was collected from pathological examination result, and IHC was used to evaluated MMR status in CRC patients. The overall survival (OS) of the CRCs was de ned as the time from radical surgery to death. The Follow-up information was obtained at outpatient clinic in our institution or using a telephone questionnaire directly with the nal follow-up date of September 1st, 2021. Proximal colon carcinomas were de ned as cecum, ascending colon or transverse colon. Pathological stage (TNM) depended on the tumor invasion depth, lymph nodes and metastases status according to the American Joint Committee on Cancer (AJCC) cancer staging manual. Adjuvant chemotherapy was performed postoperatively was determined by the TNM stage and the patient's willingness with the regimen of oxaliplatin combined with capecitabine.

IHC
The immunohistochemistry analyses for surgical specimens obtained from CRC patients who underwent radical resection were performed in Department of Pathology, Second A liated Hospital of Jilin University. IHC was performed on formalin-xed para n-embedded tissues using a standard avidin-biotin complex peroxidase procedure with an autostainer (YZB/USA 2016-2012). Here are the brief steps. 2-µmthinck formalin-xed and para n-embedded tumor tissue sections were heating for two hours at 70 •C. The heat-induced epitope retrieval was performed using a PT link machine(California, America) after the sections were depara nized and rehydrated. The slides were incubated with primary antibodies for PMS2 (clone: ZA-0542; 1:1; Wuxi), MLH1(clone: ZM-0154; 1:1; Wuxi), MSH2(clone: ZA-0622; 1:1; Wuxi), MSH6(clone: G24072; 1:1; Ventana). The slides were counterstained with hematoxylin and then sealed with neutral balsam. In all the runs, positive and negative controls were included. The tissue sections omitting the primary antibodies were regarded as negative controls, and the tissues known to express the proteins were positive controls.

Evaluation Of IHC
All the IHC stains of the CRC samples were reviewed by two pathologists (Z.Y. Wang and D.W. Huang). The expression of four MMR proteins (PMS2, MLH1, MSH2 and MSH6) was de ned as abnormal (loss) when nuclear straining of carcinoma cells was absent whereas the surrounding stromal cells performed positive nuclear straining. Pro cient mismatch repair (pMMR) was de ned as expression of all the four MMR proteins in tumor tissues, while nuclear staining absence at least one MMR proteins was regarded as de cient mismatch repair (dMMR) (Figure 1).

The Inclusion And Exclusion Criteria
The inclusion criteria were showed as follows: 1) Age between 18-75 y. 2) Pathologically diagnosed as CRC. 3) TNM / . 4) colorectal R0 resection was applied. The following exclusion criteria applied to patients in the present research were: 1) History of malignant carcinoma. 2) Severe respiratory tract, liver, kidney or cardiovascular disease. 3) Underwent preoperative neoadjuvant therapy, which was considered likely to affect MMR protein expression [14]. 4) Clinicopathological data cannot be collected accurately.

Statistical analysis
Statistical analyses were performed using SPSS for MAC, version 26.0 (IBM Corporation). Mann-Whitney U test or t test was used for continuous variables, while χ2 test or Fisher exact test was used for comparing categorical data. Multivariate analyses were evaluated with Cox proportional hazards models and survival curves were created using the Kaplan-Meier method. All P values calculated in the analysis were 2-sided, and P values less than 0.05 were considered statistically signi cant. Table 1 Patients' clinicopathologic characteristics with respect to mismatch repair protein expression.   The clinicopathological features of 29 type 1 and 24 type 2 dMMR CRCs are presented in Table 2. A loss of MSH2/MSH6 protein expression was associated with gender, where the MMR-protein negative carcinomas occurred more frequently in male (P=0.011).

Result
However, there was no signi cant differences between the two groups among the other clinicopathological features (P>0.05).
To further analyze the association of MMR status and prognosis in patients with CRC, Kaplan-Meier analyses were performed ( Figure 3). The survivorship analysis (Kaplan-Meier) showed 86% OS rate in dMMR group and a 68% OS rate in pMMR group after 5 years (P=.004, Kaplan-Meier log-rank). In stage CRC patients, the estimated OS rate for patients with loss of MMR protein was 89% and patients without de ciency was 74% after 5 years, which demonstrated that dMMR was associated with a favorable prognosis in stage patients (P=.014, Kaplan-Meier log-rank). However, OS did not differ from the two groups in patients with stage colorectal carcinomas (P=.353, Kaplan-Meier log-rank). Abbreviation: pMMR, mismatch repair pro cient. dMMR, mismatch repair de cient. OS, overall survival.
To determine whether dMMR was independent prognostic factor associated with CRC clinical outcomes, a univariate and multivariate analysis was performed using the Cox proportional hazard model (

Declarations
Availability of data and materials The datasets generated and analyzed during the current study available from the corresponding author on reasonable request.
This study was not funded by any outside source.
contributions AS and MW conceived the study design. ZZ and DL acquired the data for the study. YG, YY, and RQ analyzed and interpreted the data. ZW read the pathological section. AS drafted the manuscript. YY and ZZ revised the manuscript critically. The authors read and approved the nal manuscript.

Ethics declarations
Ethics approval and consent to participate The study protocol was approved by the institutional review board of The Second Hospital of Jilin University. And the ethics approval number was 2021111. Due to the retrospective design of the study, the local ethic committee con rmed that informed consent was not necessary from participants. The demand of patient informed consent was deserted because of the retrospective nature of this study.

Consent for publication
Not applicable.    prognosis value for MMR protein status. Figure 4