Preservation of biomolecules in breast cancer tissue by a formalin-free histology system

Abstract

BackgroundThe potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer – estrogen receptor and HER2 – at the RNA and protein levels.MethodsParallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR.ResultsThe immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p < 0.05). Also HER2 result was similar to that of formalin-fixed counterparts after elimination of antigen retrieval step (Spearman Rank R = 0.84, p < 0.05). The result of HER2 amplification by FISH and CISH was identical in the molecular fixative and formalin-fixed samples; although a shorter digestion step was required when using the former fixative. Real-time PCR for both estrogen receptor and HER2 were successful in all of the molecular fixative specimens.ConclusionThe formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

Evaluation of Reliability of FISH Versus Brightfield Dual-probe In Situ Hybridization (BDISH) for Frontline Assessment of HER2 Status in Breast Cancer Samples in a Community Setting

“…It has been determined that delay in fixation of >2 hours at ambient temperature and underfixation ( < 6 h of fixation) generally result in apparent loss of nuclear material that could lead to weakened signal and damaged morphology. 6,9 FISH is considered the gold standard to evaluate HER2 status in breast cancer. 10,11 As this method is not readily available in Pathology laboratories, IHC remain the most widespread technique used for this evaluation.…”

“…Probes were directed to the HER2 gene and to the centromere of chromosome. 6 Nonfluorescence ISH techniques were performed manually using the kit Dako DuoCISH (Dako, Carpinteria, CA), and ZytoVision CISH (ZytoDot 2C SPEC HER2/ CEN 17 Probe kit; Zytovision GmbH, Bremerhaven, Germany). The silver-based dual-color in situ hybridization, Dual ISH (Ventana), was automatically performed in the BenchMark XT instrument (Ventana).…”

Tissue fixation and the effect of molecular fixatives on downstream staining procedures

“…Some, for example UMFIX in combination with rapid tissue processing, have been adopted in some centres and have been reported to increase throughput when adopted [40], but most have not been widely adopted, partly because of the expense of transferring to a more costly fixation regime and purchase of equipment and partly because of the inevitable re-validation of antibody panels in the clinical setting [24,41].…”

Fast Flex® Validation Study: Comparison of Two Tissue Processors

“…For tissue processing, there are options available for laboratories to process tissues the same day they arrive. These same-day tissue processors use microwave technology, and/or optimized fixatives, and/or reagent systems to accomplish the goals (3,4). The same-day tissue processing format used in Fast Flex ® processing involves optimized reagents along with improved agitation.…”