The Effect of 96-Hour Formalin Fixation on the Immunohistochemical Evaluation of Estrogen Receptor, Progesterone Receptor, and HER2 Expression in Invasive Breast Carcinoma

Department of Pathology, Magee-Womens Hospital, University of Pittsburgh Medical Center, PA 15213, USA.
American Journal of Clinical Pathology (Impact Factor: 2.51). 05/2012; 137(5):691-8. DOI: 10.1309/AJCPQRAG67GJRPMT
Source: PubMed


We studied the impact of 96 hours of formalin fixation on estrogen receptor (ER), progesterone receptor (PR), and HER2 testing by comparing immunohistochemical results from core biopsy specimens fixed under current American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines with results for corresponding resection samples fixed for 96 hours. Samples enriched with cases showing weak to moderate receptor expression on core biopsy were included in the study. Cases were scored using ASCO/CAP guidelines. Of the 47 cases, only 1 case (2%) showed a qualitative change in result. However, this change was a positive ER result (H score, 1) on the 96-hour fixed resected sample compared with a negative ER result (H score, 0) for the core biopsy. Minimal changes in semiquantitative H scoring were noted for ER and PR that were likely due to tumor heterogeneity and/or intraobserver variability as the variation occurred in both directions. ER, PR, and HER2 immunohistochemical results should be considered valid for cases fixed up to 96 hours.

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    • "Overfixation was shown to be less important as tissue blocks fixed up to 72 hrs and 96 hrs did not show a reduction in ER, PR, and HER2 status [13, 14]. Fixation for more than 57 days could reduce immunostaining [15, 16]. As such long fixation times are not clinically relevant, overfixation would not be a concern in routine pathology labs. "
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    ABSTRACT: Although immunohistochemistry (IHC) is a widely used technique to classify tumors in ER-positive versus ER-negative ones, interlab variabilities can occur. This study aims to investigate the influences of preanalytical and analytical factors on IHC results. For this purpose, the different steps of the preparation of IHC sections and scoring procedures were compared between two participating laboratories and a central lab. There was a significant positive correlation between the IHC results of the participating laboratories and those of the central lab (correlation coefficient > 0.600; P<0.05 ). Nevertheless, some discordant cases for immunostaining (5.3% for ER and 5.6% for PR) and for scoring (10.5% for PR) occur at site 1. Comparing IHC results with ESR1 gene expression results revealed a significant positive correlation (correlation coefficients > 0.769; P<0.05 ). PCR results of ER target genes showed some heterogeneity in the ER-signalling pathway. These results suggest that differences in the IHC procedure between these laboratories did not have a big influence on the end result. Nevertheless, discordant cases caused by preanalytical and analytical lab-specific procedures have been identified.
    Full-text · Article · Mar 2014
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    • "The cold ischemia time (which in our study corresponds to the VPAC time) starts when the specimen is excised and ends with incision of tissue and placement in a suitable tissue fixative [18], [19]. Appreciation of the scientific importance of primary tissue handling procedures is growing, particularly for its impact on preservation of nucleic acids and proteins [20], [21], [22], [23], [24]. When dealing with cell cultures and xenograft implantations researchers may ideally wish to collect the sample for experiments directly in the surgery room in order to keep the cold ischemia time as short as possible. "
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    ABSTRACT: Primary cultures represent an invaluable tool to set up functional experimental conditions; however, creation of tissue cultures from solid tumors is troublesome and often unproductive. Several features can affect the success rate of primary cultures, including technical issues from pre-analytical procedures employed in surgical theaters and pathology laboratories. We have recently introduced a new method of collection, transfer, and preservation of surgical specimens that requires immediate vacuum sealing of excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory. Here we investigate the feasibility and performance of short-term primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues. Tissue fragments were sampled from 52 surgical specimens of tumors larger than 2 cm for which surgical and VPAC times (the latter corresponding to cold ischemia time) were recorded. Cell viability was determined by trypan blue dye-exclusion assay and hematoxylin and eosin and immunohistochemical stainings were performed to appreciate morphological and immunophenotypical features of cultured cells. Cell viability showed a range of 84-100% in 44 out of 52 (85%) VPAC preserved tissues. Length of both surgical and VPAC times affected cell viability: the critical surgical time was set around 1 hour and 30 minutes, while cells preserved a good viability when kept for about 24 hours of vacuum at 4°C. Cells were maintained in culture for at least three passages. Immunocytochemistry confirmed the phenotype of distinct populations, that is, expression of cytokeratins in epithelioid cells and of vimentin in spindle cells. Our results suggest that VPAC preserved tissues may represent a reliable source for creation of primary cell cultures and that a careful monitoring of surgical and cold ischemia times fosters a good performance of primary tissue cultures.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "As an example, for the pre-analytical phase bold claims have been recently made about the impact cold ischemia time (i.e., time to fixation) may have on HER2 testing (Pekmezci et al., 2012; Yildiz-Aktas et al., 2012a,b). The shorter the cold ischemia time the better is the quality of HER2 staining (Pekmezci et al., 2012; Yildiz-Aktas et al., 2012a,b), and results are poorer for non-refrigerated samples (Yildiz-Aktas et al., 2012a). "
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    ABSTRACT: HER2 overexpression and anti-HER2 agents represent probably the best story of success of individualized therapy in breast cancer. Due to the important therapeutic implications, the issue under the spotlight has been, since ever, the correct identification of true HER2 positivity on tissue specimens. Eligibility to anti-HER2 agents is strictly dependent on the demonstration of HER2 overexpression (by immunohistochemistry) or of HER2 gene amplification by in situ techniques (fluorescence in situ hybridization, FISH), however there are controversial issues involving cases with "equivocal" HER2 status based on conventional techniques (about 20% of specimens). In terms of HER2 expression a major debate is the presence of full-length and truncated forms of the protein and controversial clinical data have been reported on the therapeutic implications of these HER2 fragments. In terms of HER2 gene assessment, the occurrence of amplification of the chromosome 17 centromeric region (CEP17) has been proven responsible for misleading HER2 FISH results, precluding anti-HER2 based therapy to some patients. Finally HER2 activating mutations have been recently described as a biological mechanisms alternative to HER2 gene amplification. In this review we will focus on the controversies that pathologists and oncologists routinely face in the attempt to design the most tailored treatment for breast cancer patients. We will focus on the HER2 gene and on the protein, both at technical and interpretational levels.
    Full-text · Article · May 2013 · Frontiers in Oncology
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