Preliminary Study On The Identification Of BRAF^V600E Mutant Gene In Colorectal Cancer By Near Infrared Spectroscopy

Colorectal cancer（CRC）was a common malignant tumor,with an increasing trend in morbidity and mortality.BRAF^V600Egene mutation was suggested to play important roles in the occurrence and development of CRC,and the determination of BRAF^V600Egene mutation was of great significance to predict the prognosis and to guide the treatment of CRC.At present,the commonly used methods for the detection of BRAF gene mutation were immunohistochemistry（IHC）and gene sequencing,but with some limitations.Therefore,we tried to explore and find a new and rapid identification method for the detection of BRAF^V600Egene in CRC for assisting clinicopathological diagnosis.Near-infrared（NIR）light was a kind of electromagnetic wave between visible light（VIS）and mid-infrared（MIR）light.According to American Society for Testing Materials（ASTM）,the NIR spectral region was defined as a spectral region with a wavelength of 780-2526 nm（wave number,1200-4000cm-1）.In the study,the near-infrared diffuse reflectance spectroscopy characterized by high specificity and rapid identification was used to scan paraffin-embedded CRC tissue sections,and the measured spectrum was processed by chemometrics technology.According to the processed spectrum data and with the detected BRAF gene mutation types of the tissue sections as the reference value,a discriminant model of CRC tissue BRAF gene mutation was established,and the classification accuracy for BRAF gene mutation detection was compared between the NIRS and the IHC（clinically used at present）,in order to explore a new and rapid identification method of BRAF gene mutation in CRC tissues.Methods1.58 CRC tissues that had been analyzed by second-generation sequencing to determine BRAF mutation status were selected,of which 8 were mutant type and 50 were wild-type.2.12 paraffin-embedded CRC tissue sections（6 wild type and 6 mutant type）were selected,The NIRS diffuse reflection based on integrating sphere was used to record the signal and the discriminant analysis method was set up to establish a NIRS-discriminant analysis（DA）model.3.Four paraffin-embedded CRC tissue sections（2 wild type and 2 mutant type）with detected BRAF^V600Egene mutation by second generation sequencing were selected in order to verify the BRAF^V600Emutant gene NIRS-DA model.4.BRAF^V600Emutant gene NIRS-DA model was used to scan the remaining42 paraffin-embedded CRC tissue sections to predict the BRAF^V600Egene.5.The expression levels of BRAF^V600Egene mutation-associated protein in42 CRC tissue specimens were detected by IHC,and the IHC results were compared with the prediction of BRAF^V600Emutant gene NIRS and second-generation sequencing.ResultsThe model was established by using paraffin-embedded CRC tissue sections,including 6 cases of wild type and 6 cases of mutant type,and the classification accuracy of calibration（CAC）was 100%,showing that the BRAF^V600Emutant gene NIRS-DA model was established.The paraffin-embedded CRC tissue sections（2 cases of wild type and 2 cases of mutant type）in order to verify the BRAF^V600Emutant gene NIRS-DA model for the identification of the BRAF^V600Egene mutation,and the result is consistent with the second-generation sequencing.The NIRS-DA model was used to predict the gene mutation of the 42 paraffin-embedded CRC tissue sections detected by the second generation sequencing,7 cases were positive（+）,35 cases were negative（-）,and the classification accuracy of prediction（CAP）was 83.3%。As for the expression of the BRAF^V600Egene mutation associated IHC protein in CRC tissues,the results showed that VE1 expression was mainly located in the cytoplasm of tumor cells,and the detection results showed that there were 13 cases of weakly positive（+）,1 case of moderately positive（++）and 28 cases of negative（-）,and the classification accuracy was 66.7%.Among the NIRS prediction of 42 cases of CRC tissues BRAF wild type,the results showed that NIRS recognition to the 7 cases of BRAF mutation type,using IHC BRAF^V600E（VE1）antibody sign for BRAF^V600Emutation detected in 14 cases.Comparion of the results with second-generation sequencing,the positive rate of NIRS is 83.3%（35/42）,and the positiverate of IHC is 66.7%（28/42）.There was no statistical significance between these two methods（P>0.05）.ConclusionThe successfully established NIRS-DA model was used to predict the gene mutation of the CRC tissue specimens,and the results showed that the NIRS-DA model results were highly consistent with the detection results of the second-generation sequencing,no significant difference with the results of IHC.However,the experiment,a preliminary study of methodology,had the limitation of small sample size and many variable factors,thus the role of NIRS in the identification of BRAF^V600Egene mutation as a new detection method for clinical application needed to be further studied.