Objective:To investigate the expression pattern of pan-TRK protein in colorectal cancers with NTRK gene fusion and mismatch repair deficient (dMMR) and to analyze its molecular pathological characteristics.Methods:A total of 117 dMMR colorectal cancers diagnosed in the Department of Pathology of Henan Provincial People′s Hospital, Zhengzhou, China from 2020 to 2023 were collected. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and DNA/RNA-based next-generation sequencing (NGS) were used to detect pan-TRK protein expression and fusion partner genes in tumors, and to further explore the correlation between pan-TRK staining patterns and partner genes.Results:IHC and FISH were performed successfully in formalin-fixed paraffin-embedded tissues from 117 dMMR colorectal cancer patients. There were 15 (15/117, 12.8%) cases with positive pan-TRK, including 6 cases with strong staining in tumor cell membrane and cytoplasm, 2 cases with weakly granular staining in tumor cytoplasm, 2 cases with moderate dot-like staining in near 5% tumor cell nuclei, 1 case with moderately to strongly granular staining in the cytoplasm and membrane of tumor cells, 1 case with moderately to weakly granular staining in about 60% of the tumor cells, 1 case with strongly staining in about 1% of the tumor cells, 1 case with moderately to strongly staining in about 3% of the tumor cells and 1 case with diffuse, moderate para-nuclear dot-like and weakly perinuclear granular staining. NTRK1 gene disruption was detected in 6 cases (6/117, 5.1%) and consistent with diffusely strong expression of pan-TRK. Based on DNA/RNA NGS, it was further confirmed that the 6 cases with NTRK1 gene disruption all carried TPM3-NTRK1 fusion gene, and all had high microsatellite instability and high tumor mutation burden. No KRAS, NRAS, BRAF V600E or TP53 gene mutations were detected. Four patients carried frame shift mutations in RNF43. Other molecular changes included 3 cases with ROS1 gene mutation, 2 cases with BRAC, ALK, and EGFR gene mutations, 2 cases with ATM gene mutation, and 2 cases with KIT gene mutation. These were missense/frame shift mutations that were associated with no clinical significance. The nine pan-TRK-positive cases without NTRK gene fusion detected with DNA-based NGS were further confirmed with RNA-based NGS, and no NTRK gene fusion was found. The sensitivity and specificity of NTRK gene fusion detected using IHC were 100.0% and 92.5%, respectively. The sensitivity and specificity of diffusely strong membranous/cytoplasmic staining were both 100.0%.Conclusions:Pan-TRK protein has various expression patterns in dMMR colorectal cancer. Its diffusely strong expression is highly suggestive of NTRK1 gene fusion. TPM3-NTRK1 gene fusion is a common form of NTRK gene fusion in dMMR colorectal cancer.

Objective:To investigate the expression pattern of pan-TRK protein in colorectal cancers with NTRK gene fusion and mismatch repair deficient (dMMR) and to analyze its molecular pathological characteristics.Methods:A total of 117 dMMR colorectal cancers diagnosed in the Department of Pathology of Henan Provincial People′s Hospital, Zhengzhou, China from 2020 to 2023 were collected. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and DNA/RNA-based next-generation sequencing (NGS) were used to detect pan-TRK protein expression and fusion partner genes in tumors, and to further explore the correlation between pan-TRK staining patterns and partner genes.Results:IHC and FISH were performed successfully in formalin-fixed paraffin-embedded tissues from 117 dMMR colorectal cancer patients. There were 15 (15/117, 12.8%) cases with positive pan-TRK, including 6 cases with strong staining in tumor cell membrane and cytoplasm, 2 cases with weakly granular staining in tumor cytoplasm, 2 cases with moderate dot-like staining in near 5% tumor cell nuclei, 1 case with moderately to strongly granular staining in the cytoplasm and membrane of tumor cells, 1 case with moderately to weakly granular staining in about 60% of the tumor cells, 1 case with strongly staining in about 1% of the tumor cells, 1 case with moderately to strongly staining in about 3% of the tumor cells and 1 case with diffuse, moderate para-nuclear dot-like and weakly perinuclear granular staining. NTRK1 gene disruption was detected in 6 cases (6/117, 5.1%) and consistent with diffusely strong expression of pan-TRK. Based on DNA/RNA NGS, it was further confirmed that the 6 cases with NTRK1 gene disruption all carried TPM3-NTRK1 fusion gene, and all had high microsatellite instability and high tumor mutation burden. No KRAS, NRAS, BRAF V600E or TP53 gene mutations were detected. Four patients carried frame shift mutations in RNF43. Other molecular changes included 3 cases with ROS1 gene mutation, 2 cases with BRAC, ALK, and EGFR gene mutations, 2 cases with ATM gene mutation, and 2 cases with KIT gene mutation. These were missense/frame shift mutations that were associated with no clinical significance. The nine pan-TRK-positive cases without NTRK gene fusion detected with DNA-based NGS were further confirmed with RNA-based NGS, and no NTRK gene fusion was found. The sensitivity and specificity of NTRK gene fusion detected using IHC were 100.0% and 92.5%, respectively. The sensitivity and specificity of diffusely strong membranous/cytoplasmic staining were both 100.0%.Conclusions:Pan-TRK protein has various expression patterns in dMMR colorectal cancer. Its diffusely strong expression is highly suggestive of NTRK1 gene fusion. TPM3-NTRK1 gene fusion is a common form of NTRK gene fusion in dMMR colorectal cancer.