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Evaluating utility and feasibility of mismatch repair testing of colorectal cancer patients in a low-middle-income country


Study design

This was a joint project between the Centre for Colorectal Disease at St Vincent’s University Hospital, Dublin, Ireland & a number of hospitals in Sudan. The twin aims were a) to determine the feasibility of introducing universal MSI testing in CRC to a LMIC and b) to perform a retrospective cohort study to explore the clinicopathological features, MMR/BRAF status, and overall survival (OS) among Sudanese patients diagnosed with CRC at three major medical centres namely Soba University Hospital, Ibn-Sina Hospital, and Gezira Medical Laboratory in the time period from Jan. 1st 2016 to Dec. 31st 2018.

Case selection and sample inclusion

Sample inclusion methodology

Pathological tumour material of all patients diagnosed with CRC at three major medical settings in Sudan from January 1st, 2016 to December 31st, 2018 were filtered. These included cases with biopsy or resection colorectal samples reported to fulfil criteria for CRC in the routine histopathology review. We identified a total number of 68, 60, & 61 cases diagnosed as CRC in Soba University Hospital, Gezira Medical Laboratory, and Ibn Sina Hospital in the specified period respectively. 50 patients were included based on the availability and quality of thematerial, specifically the overall appearance of the paraffin block, the amount of tumour and normal tissueavailable, and most importantly the quality of fixation and embedding of the tissue. After filtering, the selected cases included 29 cases from Soba University Hospital, 11 cases from Gezira Medical Laboratory, and 10 cases from Ibn Sina Hospital. 112 paraffin-embedded tissue blocks of CRC biopsies/resections from 50 CRC cases fulfilling the histopathological criteria of CRC were obtained. Blocks were selected to provide samples representing CRC and normal colon tissue for each case. Ultimately only samples with adenocarcinoma histopathological type (48 samples) that contains adequately processed primary tumor (44 out of the 48 samples) were processed for MMR expression and BRAF expression/mutation assessment.

Demographic and clinical data

Data including age, gender, ethnic/sub-ethnic group, prior 10 years residence, history of malignancy, tumour anatomical site and clinical cancer stage (if available) were collected for all cases.

The diagnostic and therapeutic pathway for each patient is summarized (Table 1). Patient’s follow-up was conducted through review of medical records after follow up visits (Table 1) and personal contact (physical meetings or phone calls) if feasible. Data on neoadjuvant/adjuvant chemotherapy, CRC disease-free survival/recurrence (DFS), CRC recurrence, and CRC overall survival (OS) were collected if available. We evaluated CRC disease-free survival and recurrence if proven by either colonoscopy, imaging, or serum CEA elevation. We assessed CRC OS on confirmation of CRC related mortality when other causes of death were excluded.

Table 1 Protocol for diagnosis and treatment of CRC at the Sudanese Medical Centres.

Histopathological review

Histopathological characteristics were evaluated in the local hospitals; namely histopathological type, tumour grade, and tumour stage (Dukes and TNM stage) using standardised criteria14.

Immunohistochemical analysis of MMR status and BRAF gene mutation expression

Immunohistochemistry

Immunohistochemistry (IHC) was performed at the Centre for Colorectal Disease, SVUH (RG). 112 blocks from 50 cases were examined. All paraffin blocks were cut on an automated Leica RM255 microtome and stained on a Leica ST5020 automated staining machine with an integrated Leica CV5030 glass coverslipper. All slides for immunohistochemical staining were baked in a 60 degree centigrade oven for two hours. H&E slides were microscopically re-examined by a Pathologist (SA) to confirm the presence of invasive CRC. 6 cases did not contain invasive carcinoma in any of the blocks received. No further testing was performed, and MMR IHC was performed on 44 cases.

Assessing MMR status

MMR status was assessed using IHC for MMR proteins, hMLH1 (BD Bioscience, clone G168-728), hPMS2 (BD Biosciences, clone A16-4, hMSH2 (Calbiochem, clone FE11) and hMSH6 (BD Biosciences, clone 44). Automated IHC was performed on the BOND instrument (Leica). The protocol involved heat-induced antigen retrieval with Bond Epitope Retrieval 2 solution for 30 min. Slides were incubated with antibody diluted 1 in 200 for 15 min at room temperature. Visualisation of the antibody antigen reaction was via the Bond Polymer Refine Detection kit (Leica).

Nuclear staining in any area of the tumour was classified as showing no loss of the mismatch repair proteins. Tumours showing complete loss of nuclear staining of the mismatch repair proteins in the entire tumour with concurrent positive staining of nuclei of non-neoplastic cells were classified as having loss of expression of that mismatch repair protein. Samples demonstrating MLH1 + PMS2 loss (n = 3) were examined by BRAF IHC and /or mutation testing if the staining was equivocal.

BRAF immunohistochemistry

Automated immunostaining was performed on the Ventana Ultra (Roche). The protocol involved dewaxing with Ezprep solution, followed by heat-induced epitope retrieval with CC1buffer for 64 min and then endogenous peroxidase inhibition. Slides were then incubated with the ready to use BRAF V600E mutation-specific monoclonal antibody, (Ventana, clone VE1 (CE-IVD)) for 16 min at 37 °C. Chromogenic detection was carried out using the OptiView DAB IHC kit (Roche) along with an Optiview amplification kit (Roche). Four minutes Optivew amplification H2O2 and four minutes Optiview amplification multimer incubation times were used. Slides were counterstained with haematoxylin II (Roche) for 4 min followed by bluing reagent (Roche) for 4 min.

Positive staining was seen as the presence of unequivocal cytoplasmic granular staining of any intensity in the tumour cells. Negative staining showed the absence of cytoplasmic staining in the tumour cells.

Molecular assessment of BRAF gene mutations

DNA was isolated from the tumour blocks using the Cobas DNA Sample Preparation kit (Roche). Real time PCR analysis of BRAF V600 mutation status with the BRAF/NRAS Mutation Test Roche Oncology Life Science Research (LSR) kit (Roche) was performed on the automated Cobas z480 analyser (Roche). A mutant control and negative control were included to confirm the validity of the analysis.

Statistical analysis

All statistical analyses were performed using R (versions: 4.0.2 and 4.0.3) and STATA (version: 419.12.0.870). The Chi-square test was used to estimate correlation for categorical variables, while the Student t-test was applied for continuous variables. The Fisher exact test was used for small numbers where the conditions for the Chi-square test were not fulfilled. Kaplan–Meier survival analysis with log-rank test was used to estimate overall survival (OS) and its correlation with categorical variables while Cox regression was used for continuous variables. OS and DFS were calculated starting from the date of first diagnosis. Censor was death for OS and disease relapse and/or metastasis for DFS. All tests were two-tailed and a P-value of < 0.05 was considered statistically significant.

Ethics declaration

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committees of Soba University Hospital, and National Cancer Institute, University of Gezira, Sudan.

Consent to participate

Informed consent was obtained from all individuals included in the study.



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