Study On Reuse Of Formalin-fixed Paraffin-embedded Sections After Immunohistochemical Staining For Molecular Detection

Objective: The purpose of this study was to evaluate the quality of DNA from the formalin-fixed paraffin-embedded(FFPE)specimens after immunohistochemical(IHC)staining and investigate DNA extraction by IHC staining of the specimens in small in number or difficult to obtain and the feasibility of molecular detection and fluorescence in situ hybridization detection.Methods: We collected 50 FFPE biopsy specimens of non-small cell lung cancer(NSCLC),30 FFPE biopsy specimens of colorectal cancer(CRC)and75 FFPE biopsy specimens of breast cancer(BC)form the Department of Pathology of Jinling Hospital from June 2017 to December 2017 and sliced each into 12 sections,of which,6 to immunohistochemical Envision two-step staining for DNA extraction(the experimental group)and the remaining 6 were directly subjected to DNA extraction(the control group).NSCLC FFPE samples were used to detect the mutations of epidermal growth factor receptor(EGFR),mouse Sarcoma virus carcinogenic gene(KRAS)and mouse sarcomas filter virus carcinogen B1(BRAF)gene.Humanβ-catenin(CTNNB1)gene and E-cadherin(CDH1)were detected in CRC FFPE samples.BC FFPE sliced each into 3 sections for HE,IHC and FISH detection,respectively.One of the sections was randomly selected for HE staining,and the remaining 2 sections were taken as one experimental group,IHC staining was performed before FISH detection,and the remaining section was included in the control group for direct FISH detection.Results: The DNA concentration and purity of the experimental group of NSCLC and CRC specimens were not significantly different from those of the control group(P>0.05).Of the NSCLC FFPE specimens of the experimental group,26(52%)showed the mutation of EGFR,4(8%)exhibited that of KRAS and 1(2%)manifested that of BRAF.Of the CRC FFPE specimens of the experimental group,2(6.7%)showed the mutation of CTNNB1 gene.Of the CRC FFPE specimens of the experimental group,14(46.7%)showed the incidence of CDH1 promoter methylation.The results of EGFR,KRAS,BRAF,CTNNB1 and CDH1 gene test results in the NSCLC and CRC specimens were highly consistent with the control group,and the coincidence rate were 100%(P=0.000).Of the BC FFPE specimens of the experimental group,19(25.3%)showed the positive rate of HER2 and 56(74.7%)showed the negative rate of HER2.The coincidence rate of FISH in the experimental group and the control group was the same(P< 0.001).The expression of C-erb B-2 protein in BC FFPE specimens was(3+)in 15 cases(20%),(2+)in 23 cases(30.7%),(1+)in 7 cases(9.3%)and(0)in 30cases(40%).Except for the cases in which the expression of C-erb B-2 protein in BC FFPE specimens was(2+),the positive coincidence rate between IHC and FISH in BC experimental group was 14(93.3%)and the negative coincidence rate between IHC and FISH in BC experimental group was 36(97.3%).The total coincidence rate between IHC and FISH in BC experimental group was 50(96.2%).Conclusion: High-quality DNA can be extracted after immunohistochemical staining from FFPE specimens,especially those small in number or difficult to obtain,and can be used for downstream molecular analysis of target genes,which is a good method for specimen recycling and provides a solution for subsequent molecular test of scarce or difficult-to-obtain clinical samples.