Influence Of Two Different Decalcification Agents On Antigen Expression And Molecular Profiles In Bone Metastatic Carcinomas Tissue

Purpose Biomarker analysis to establish the mutation assay of actionable targets on bone metastatic carcinomas(BMCs)is a routinely recommended clinical screen.We investigated the influence of different decalcification procedures on cell antigenicity and DNA quality to obtain a reasonable process,which could provide reliable evidence-based medicine evidence for the correct diagnosis and treatment of bone metastasis.Method Tumor tissues of breast cancer(BC),non-small cell lung cancer(NSCLC)and colorectal cancer(CRC)were collected to mimic bone biopsies as clinical samples and decalcified by hydrochloric acid(HCL)or ethylenediaminetetraacetic acid(EDTA).We performed immunohistochemistry,DNA integrity measurements and molecular analysis to evaluate influences of decalcification.In order to verify reliability in mutational profile in HCL decalcification,we also enrolled another real clinical cohort of 71 bone metastases of NSCLC patients and detected their EGFR mutation status,in which 18 cases matched corresponding primary tumors.Result We found that compared with controls(without decalcification),the percentage of immune-positive cells was on average lower in both decalcified cases,and the decrease after HCL exposure was much more obvious.Among them,C-MET for NSCLC was the most sensitive to decalcification agent,with a decrease of 77.5%(P=0.041)and 82.5%(P=0.038)after 1 hours and 2 hours HCL-treatment and 22.5%(P=0.180)and 57.5%(P=0.039)after 2 hours and 4 hours EDTA-treatment respectively.The expression of prospective biomarkers of BC and CRC were observed dramatic loss after 2 hours HCL-treatment(26.6%-41.4%),but slight decrease after decalcification with EDTA(5%-34.8%).As to HER2-FISH,the signal ratio decreased by 57.4% after 2 hours HCL exposure,and there is no significant difference between EDTA group and control group in signal ratio.In addition,after decalcification of HCL,results of the size ladder PCR indicated degraded DNA,most of which were small chunks of 100 bp.The concentration also dramatically decreased.But these results of EDTA group remained consistent with control group.Moreover,the positive mutation of oncogenic driver such as EGFR,KRAS,and PIK3 CA lost in half HCL-treated cases but only in few(9%)EDTA-treated samples.Validation on paired primary NSCLC specimens and HCL-treated bone metastases demonstrated that the concordance rate on EGFR mutations(mutant or wild-type)was 61.1% and 7cases were discordant.Conclusion We conclude that EDTA-based decalcification could provide better conservation on immunoreactivity and nuclear integrity.Therefore we suggest that EDTA-based decalcification should be preferred for bone biopsies tissue and the influence of decalcification should be taken into consideration when making diagnosis for bone metastasis patients...