Multiple Immunostainings with Different Epitope Retrievals-The FOLGAS Protocol.

We describe a sequential multistaining protocol for immunohistochemistry, immunofluorescence and CyTOF imaging for formalin-fixed, paraffin-embedded specimens (FFPE) in the formalin gas-phase (FOLGAS), enabling sequential multistaining, independent from the primary and secondary antibodies and retrieval. Histomorphologic details are preserved, and crossreactivity and loss of signal intensity are not detectable. Combined with a DAB-based hydrophobic masking of metal-labeled primary antibodies, FOLGAS allows the extended use of CyTOF imaging in FFPE sections.

Integration of p16/HPV DNA Status with a 24-miRNA-Defined Molecular Phenotype Improves Clinically Relevant Stratification of Head and Neck Cancer Patients.

Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD-L1 expression/PD-1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.

Alteration of Tissue Marking Dyes Depends on Used Chromogen during Immunohistochemistry.

Pathological biopsy protocols require tissue marking dye (TMD) for orientation. In some cases (e.g., close margin), additional immunohistochemical analyses can be necessary. Therefore, the correlation between the applied TMD during macroscopy and the examined TMD during microscopy is crucial for the correct orientation, the residual tumour status and the subsequent therapeutic regime. In this context, our group observed colour changes during routine immunohistochemistry. Tissue specimens were marked with various TMD and processed by two different methods. TMD (blue, red, black, yellow and green) obtained from three different providers (A, B and C, and Whiteout/Tipp-Ex®) were used. Immunohistochemistry was performed manually via stepwise omission of reagents to identify the colour changing mechanism. Blue colour from provider A changed during immunohistochemistry into black, when 3,3'-Diaminobenzidine-tetrahydrochloride-dihydrate (DAB) and H2O2 was applied as an immunoperoxidase-based terminal colour signal. No other applied reagents, nor tissue texture or processing showed any influence on the colour. The remaining colours from provider A and the other colours did not show any changes during immunohistochemistry. Our results demonstrate an interesting and important pitfall in routine immunohistochemistry-based diagnostics that pathologists should be aware of. Furthermore, the chemical rationale behind the observed misleading colour change is discussed.