The RAD51-FFPE Test; Calibration of a Functional Homologous Recombination Deficiency Test on Diagnostic Endometrial and Ovarian Tumor Blocks

PARP inhibitor (PARPi) sensitivity is related to tumor-specific defects in homologous recombination (HR). Therefore, there is great clinical interest in tests that can rapidly and reliably identify HR deficiency (HRD). Functional HRD tests determine the actual HR status by using the (dis)ability to accumulate RAD51 protein at sites of DNA damage as read-out. In this study, we further improved and calibrated a previously described RAD51-based functional HRD test on 74 diagnostic formalin-fixed paraffin-embedded (FFPE) specimens (RAD51-FFPE test) from endometrial cancer (EC n = 25) and epithelial ovarian cancer (OC n = 49) patients. We established optimal parameters with regard to RAD51 foci cut-off (≥ 2) and HRD threshold (15%) using matched endometrial and ovarian carcinoma specimens for which HR status had been established using a RAD51-based test that required ex vivo irradiation of fresh tissue (RECAP test). The RAD51-FFPE test detected BRCA deficient tumors with 90% sensitivity and RECAP-HRD tumors with 87% sensitivity, indicating that it is an attractive alternative to DNA-based tests with the potential to be applied in routine diagnostic pathology.

Download Full-text

Abstract 5070: Validation of a fresh-tissue based prognostic gene signature in formalin-fixed, paraffin-embedded melanomas

10.1158/1538-7445.am2011-5070 ◽

2011 ◽

Author(s):

Georg Brunner ◽

Ludwig Suter ◽

Hans-Joachim Schulze ◽

Carola Berking ◽

Achim Heinecke ◽

...

Keyword(s):

Gene Signature ◽

Fresh Tissue ◽

Prognostic Gene ◽

Prognostic Gene Signature ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Download Full-text

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.7.1607637 ◽

1992 ◽

Vol 40 (7) ◽

pp. 1047-1051 ◽

Cited By ~ 61

Author(s):

B J Kerns ◽

P A Jordan ◽

M B Moore ◽

P A Humphrey ◽

A Berchuck ◽

...

Keyword(s):

Breast Cancers ◽

Fresh Tissue ◽

P53 Overexpression ◽

P53 Expression ◽

Ovarian Cancers ◽

Formalin Fixed Paraffin ◽

Wide Range ◽

Formalin Fixed Paraffin Embedded ◽

Fresh Frozen ◽

Formalin Fixed

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.

Download Full-text

OR19-01 IHC-Guided Capture of Adrenocortical Zones from Archival Formalin-Fixed, Paraffin-Embedded Adrenal Tissue Allows the Study Of RNA Expression Profiles

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.939 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Samuel Plaska ◽

Jessica Baker ◽

Amy Blinder ◽

Jung Soo Lim ◽

William E Rainey

Keyword(s):

Expression Profiles ◽

Tumor Biology ◽

Zona Glomerulosa ◽

Ffpe Tissue ◽

Zona Fasciculata ◽

Fresh Tissue ◽

Adrenal Tissue ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Abstract Introduction: Characterization of human adrenal RNA has relied on fresh tissue, or laser capture from OCT-embedded frozen tissue. These approaches have allowed identification of differences between zones of the adrenal cortex, but the need for fresh or frozen material has limited access for most researchers and has limited the number of samples that can be studied. In order to expand tissue availability and researcher access, we examined the use of archival formalin-fixed, paraffin-embedded (FFPE) adrenals for the capture and analysis of RNA from each adrenal zone. Methods: 13 normal adrenals were obtained from deceased renal donors. Following FFPE, tissue serial sections were examined for zonal histology. Immunohistochemistry (IHC) was used to guide the capture of specific adrenal zones from FFPE tissue. CYP11B2, CYP17A1, and CYB5A IHC was used to visualize the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR) respectively. Serial unstained sections were then dissected under a dissection microscope based on IHC-guided visualization of the ZG, ZF, and ZR. Following capture of zones and isolation of RNA, quantitative PCR (qPCR) was used to examine zonal expression of transcripts for the IHC markers. Using housekeeping genes for normalization, specific zonal markers identified successfully isolated zones. Results: Adrenals with clear zonal expression of CYP11B2 (ZG), HSD3B2 (ZG/ZF), and CYB5A (ZR) were included for study. Based on marker inclusion criteria, 3 ZG, 3 ZF, 3 ZR, and 3 medulla samples were studied. Using these samples, confirmatory markers of ZG (VSNL1), ZF (CYP17A1), ZR (SLC27A2), and medulla (TH) were analyzed by qPCR. Levels of these transcripts segregated as expected in the adrenal zones. Discussion: The use of archival normal and pathologic adrenal tissue for RNA analysis would increase the number of samples available to many research groups. Our data demonstrates the ability to capture and use FFPE tissue for the capture and analysis of RNA. Future comparison of normal adrenal zones to adrenal tumor phenotypes will improve our understanding of tumor biology and provide potential genetic markers for targeted therapeutics.

Download Full-text