BACKGROUNDHistopathologically stained archived tissue slides are stored in hospital archives for years to decades. They are the largest available source of biological materials and are a potentially useful resource that can be used for retrospective epidemiological studies. DNA recovered from the slides can be used for several downstream molecular processes including polymerase chain reaction, single nucleotide polymorphism analysis, and whole genome sequencing. The DNA from these slides can be utilized to compare gene signatures of normal and diseased tissues. However, extraction of high-quality DNA from archived stained hematoxylin and eosin (H&E) slides remains challenging.AIMTo standardize a new protocol for extracting DNA from archived H&E-stained tissue slides for further molecular assays.METHODSA total of 100 archived H&E-stained cancer slides were subjected to a total of five methods of DNA extraction. Methods were varied in the deparaffinization step, tissue rehydration, duration of lysis, and presence or absence of proteinase K. The extracted DNA was quantified using a NanoDrop spectrophometer and the quality was analyzed by agarose gel electrophoresis. Then each sample was subjected to polymerase chain reaction (PCR) to amplify the internal control gene GAPDH, thereby confirming the DNA intactness, which could be further utilized for other downstream applications.RESULTSOf the five different methods tested, the third method wherein xylene was used for tissue deparaffinization followed by 72 h of digestion and without proteinase K inactivation yielded the highest amount of DNA with good purity. The yield was significantly higher when compared to other methods. In addition, 90% of the extracted DNA showed amplifiable GAPDH gene.CONCLUSIONHere we present a step-by-step, cost-effective, and reproducible protocol for the extraction of PCR-friendly DNA from archived H&E-stained cancer tissue slides that can be used for further downstream molecular applications.

Purpose To investigate the role of NRF2 signaling in conferring superior prognosis in patients with HPV positive (HPV+ ve) head & neck squamous cell carcinomas (HNSCC) compared to HPV negative (HPV− ve) HNSCC and develop molecular markers for selection of HPV+ ve HNSCC patients for treatment de-escalation trials. Methods We compared NRF2 activity (NRF2, KEAP1, and NRF2-transcriptional targets), p16, and p53 levels between HPV+ ve HNSCC and HPV− ve HNSCC in prospective and retrospective tumor samples. Cancer cells were transfected with HPV E6/E7 plasmid to elucidate if HPV infection repress NRF2 activity and sensitize to an anticancer drug. Results Prospective analysis revealed a marked reduction in expression of NRF2, and its target antioxidant genes (NQO1, HMOX-1, GCLC, and GCLM) in HPV+ ve tumors compared to HPV− ve tumors. A retrospective analysis by IHC revealed significantly lower NQO1 in p16high tumors compared to p16low tumors and the NQO1 expression correlated negatively with p16 and positively with p53. Analysis of the TCGA database confirmed low constitutive NRF2 activity in HPV+ ve HNSCC compared to HPV− ve HNSCC and revealed that HPV+ ve HNSCC patients with ‘low NQO1’ expression showed better overall survival compared to HPV+ ve HNSCC patients with ‘high NQO1’ expression. Ectopic expression of HPV E6/E7 plasmid in cancer cells repressed constitutive NRF2 activity, increased KEAP1, reduced total GSH levels, and enhanced cytotoxic effects of cisplatin. Conclusion Low constitutive NRF2 activity contributes to better prognosis in patients with HPV+ ve HNSCC compared to HPV− ve HNSCC. Co-expression of p16high, NQO1low, and p53low could serve as a predictive biomarker for the selection of HPV+ ve HNSCC patients for de-escalation trials.

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