Research of ntrk fusion by immunhistochemical andmolecular methods in non-small cell lung cancers

Non-small cell lung carcinomas are the most common type of lung cancer. Squamous cell carcinoma, adenocarcinoma and large cell carcinoma constitute the majority of non-small cell lung carcinomas. Other types are seen less frequently. The most important parameter in prognosis and treatment in nonsmall cell lung carcinomas is the stage of the disease. While surgical resection is recommended for early-stage tumors, immunotherapy and treatments targeting the driver oncogene have come to the fore in recent years in addition to chemoradiotherapy for advanced- stage tumors. The discovery of specific targetable genetic changes suitable for therapeutic agents in non- small cell lung carcinomas has increased the importance of molecular diagnostic tests that detect these genetic changes by routine practice. Tyrosine kinase inhibitors targeting genetic changes in EGFR, ALK, ROS1, RET, BRAF V600E, MET Exon 14, and NTRK genes have been approved for certain subtypes of non-small cell carcinomas. The NTRK genes are a family of genes consisting of NTRK1, NTRK2, and NTRK3, which encode the TrkA, TrkB, and TrkC proteins, respectively. Trk proteins are physiologically involved in the regulation of neuronal development and some cognitive functions. Moreover, NTRK gene fusions have been identified as the main genetic change in some tumors. NTRK fusion is frequently encountered in rare tumors such as secretory carcinoma of the breast, breast analogue secretory carcinoma of the salivary gland, infantile fibrosarcoma, and congenital mesoblastic nephroma. Furthermore, NTRK fusions have been reported rarely in more common tumors such as non-small cell lung carcinomas, colorectal carcinomas, and papillary thyroid carcinomas. The distinct response of recently approved tyrosine kinase inhibitors such as entrectinib and larotectinib in NTRK fusion-positive tumors has increased the importance of detecting NTRK fusion. In addition to immunohistochemical methods, molecular fluorescence in situ hybridization, reverse transcriptase polymerase chain reaction and new generation sequencing tests are used for NTRK fusion detection. In this study, 295 non-small cell lung carcinoma cases diagnosed in our institution were included. Out of the cases, 148 (50,2%) had biopsy samples available, while 147 (49,8%) had resection material. Of all cases, 188 (63,7%) were diagnosed with squamous cell carcinoma, 85 (28,8%) with adenocarcinom, 9 (3,1%) with non-small cell lung carcinoma, 10 (3,4%) with sarcomatoid carcinoma, 3 (1,0%) large cell carcinoma. All cases were examined in terms of Pan- TRK immunohistochemical expression, clinicopathological parameters and prognosis. Immunohistochemical examination was applied to all cases for NTRK fusion detection. Immunohistochemical Pan-TRK positivity was confirmed by RT-PCR. NTRK fusion was reconfirmed by fluorescence in situ hybridization method in cases with positive NTRK fusion detected by reverse transcriptase polymerase chain reaction. A statistically significant difference was found between clinicopathological data in terms of tumor diameter, tumor stage, lymph node metastasis stage and distant metastasis stage, and total survival time of the cases (p<0,05). As the tumor size increased, tumor stage, lymph node metastasis stage and distant metastasis stage progressed, total survival time decreased. Immunohistochemically, Pan-TRK (clone EPR17341) expression was observed in 24 (8,1%) cases. Cytoplasmic expression was found in four (1,4%) cases, focal cytoplasmic expression in 10 (3,4%) cases, focal nuclear expression in 10 (3,4%) cases. Pan-TRK expression was detected in 18 (9,6%) cases with squamous cell carcinoma, 3 (3,5%) with adenocarcinoma, 2 (22,2%) cases with non-small cell lung carcinoma, NOS, 1 (10,0%) with sarcomatoid carcinoma, although/however no expression was observed with Pan-TRK in 3 (100%) cases of large cell carcinoma. In terms of Pan-TRK expression statistically significant difference was not observed between the groups according to gender, age, number of tumor foci, tumor diameter, tumor types, tumor subtypes, angiolymphatic invasion status, perineural invasion status, tumor stroma, tumor stage, lymph node metastasis, distant metastasis and recurrence status (p>0,05). There was no statistically significant difference between Pan-TRK positive and negative cases in terms of disease-free survival and total life expectancy (p>0,05). Reverse transcriptase polymerase chain reaction test was applied to the cases who was found to be positive immunohistochemically. NTRK3 fusion was detected in 3 (12,5%) of 24 cases. Among the fusion positive cases 2 (67%) were squamous cell carcinoma, one (33%) was adenocarcinoma. In two cases, the test could not be performed because the sample was insufficient. In conclusion, in this study, we identified Pan-TRK expression through immunohistochemistry in 8,1% of non-small cell lung carcinomas, consistent with the literature. Among the immunohistochemically positive cases, we detected NTRK3 fusion by reverse transcriptase polymerase chain reaction in 12,5% of the cases, and we confirmed our findings using FISH. Our results indicate that immunohistochemistry can be used as a screening test in settings where molecular testing is not available, but it always requires molecular validation, as demonstrated by meticulous examination by experienced pathologists. Our finding of NTRK3 fusion in squamous cell carcinomas with limited treatment options, which constitutes the majority of our cases, suggested that the driver function of this gene may be more effective in this group. We believe that our findings can be supported by larger series, which would enhance the generalizability and reliability of our results.