Delay to formalin fixation effect on breast biomarkers

Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Modern Pathology (Impact Factor: 6.19). 10/2009; 22(11):1457-67. DOI: 10.1038/modpathol.2009.117
Source: PubMed


Delay to formalin fixation may invalidate hormone receptors and HER2 analyses. Invalid results of tumor markers could significantly alter the type of adjuvant therapy a patient receives and potentially impact outcome. The purpose of this study was to determine the effects of progressive delay to formalin fixation on breast cancer biomarkers. Ten palpable invasive breast cancers were resected and underwent immediate gross evaluation. For each case, the procured tumor was divided into eight parts and consecutively fixed after 0, 10, 30 min, 1, 2, 4, and 8 h; one section was kept in saline and stored overnight at 4 degrees C. Two tissue microarray blocks were constructed. Estrogen and progesterone receptors and HER2 immunohistochemistry and fluorescence in situ hybridization were carried out. Statistical analyses including non-parametric sign test, exact McNemar's test and Page's L test were used. All 10 cases were invasive ductal carcinomas. Q score > or =6 was identified in five cases for estrogen receptor and four for progesterone receptor. Mean Q score started to decline at the 2 h mark for estrogen receptor and 1 h mark for progesterone receptor. Lowest score was at 8 h mark for estrogen receptor and overnight for progesterone receptor. HER2 fluorescence in situ hybridization started to be compromised for interpretation at the 1 h mark and became statistically significant at the 2 h mark (P<0.03). To avoid delay to formalin fixation as a factor negatively affecting on breast biomarkers, we recommend not to delay formalin fixation for more than 1 h and not to store specimens overnight.


Available from: Sheila N J Sait, Jun 04, 2014
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    • "Storage of slides at 4°C is shown in several studies to help prevent antigen degradation [5,10-12] but may also be sub-optimal due to the humid environment and thus hydrolysis occurring from exogenous water [8]. Although not related to storage per se, it should be mentioned that inappropriate and prolonged fixation may damage antigenicity or cause diffusion artefacts, and that these effects may differ from one antigen to another [13]. Of course, the type of fixative also has an important impact on antigenicity [14]. "
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    ABSTRACT: The use of paraffin slides and tissue microarrays (TMA) is indispensable for translational research. However, storage of paraffin slides over time has a substantial detrimental effect on the quality and reliability of immunohistochemistry stains. Particularly affected by this issue may be any collaborative efforts where paraffin slides or TMAs are shipped to central laboratories and then 'biobanked' for some time until use. This article summarizes some of the key issues affecting loss of antigenicity on paraffin slides and some simple storage solutions to help maintain high quality immunohistochemistry results when paraffin slides must be stored for a certain time prior to use.
    Clinical and Translational Medicine 03/2014; 3(1):4. DOI:10.1186/2001-1326-3-4
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    • "However, a decrease in HER2 IHC score was observed only in SCH specimens with a 24-h delay to fixation, whereas the HER2/CEP17 ratio of FISH in SCH was decreased from 2.3 to 1.3 if the tumor sample was left at room temperature for only 6 h before fixation. In breast cancer, it has been reported that a delay to formalin fixation affects FISH results but not IHC scores [16]. Therefore, we would expect that FISH results would be more vulnerable to delayed fixation than IHC in gastric cancer specimens. "
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    ABSTRACT: Accurate and reliable assessment of human epidermal growth factor receptor type 2 (HER2) status is important for selecting patients with gastric cancer who may benefit from trastuzumab treatment. Here we examined the impact of formalin fixing conditions on HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in xenografted tumor tissues. Xenografted tumor tissues of the human gastric cancer cell lines NCI-N87, SCH, and SNU-16 were collected and kept at room temperature for 0, 6, or 24 h before being fixed with 10 % neutral buffered formalin (NBF) for 24 h or 5, 7, or 10 days and embedded in paraffin. Use of 10 % NBF, 20 % NBF, or nonbuffered formalin as fixative was investigated. The HER2 IHC scores for NCI-N87, SCH, and SNU-16 tumors were 3+, 2+, and 1+, respectively, when specimens were fixed with 10 % NBF for 24 h immediately after resection of the tumors. Specimens left for longer than 6 h before fixation had shrinkage of the tumor periphery and decreased immunostaining intensity in this region in all specimens. In SCH and SNU-16 specimens, starting fixation 24 h after tumor tissue collection induced autolysis and reduction of the number of stained cells, and 10-day-fixation lowered the HER2 score. Prolongation of fixation time did not affect FISH results, but if samples were left for more than 6 h before fixation, the FISH score was strongly reduced in SCH specimens (2.3 to 1.3). Reduced IHC staining intensity was observed with 20 % NBF and nonbuffered formalin compared to 10 % NBF. The time to and length of fixation of tumor specimens can affect HER2 IHC and FISH scores. The fixative used can affect IHC results.
    Gastric Cancer 01/2014; 17(4). DOI:10.1007/s10120-013-0329-8 · 3.72 Impact Factor
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    • "Our earlier work suggests that the HER2 epitope is not affected when delay is less than 2–3 hours [3]. However, others have suggested substantial loss of epitope when assessing longer time points [4], [5]. "
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    ABSTRACT: HER2/Neu (ErbB-2) overexpression, which occurs in 15-20% of breast cancer cases, is associated with better response to treatment with the drug trastuzumab. PhosphoHER2 (pHER2) has been evaluated for prediction of response to trastuzumab. Both markers are heterogeneously detected and are potentially subject to loss as a consequence of delayed time to fixation. Here, we quantitatively assess both markers in core needle biopsies (CNBs) and matched tumor resections to assess concordance between the core and the resection and between HER2 and pHER2. A selected retrospective collection of archival breast cancer cases yielded 67 cases with both core and resection specimens. Both HER2 and pTyr(1248)HER2 were analyzed by the AQUA® method of quantitative immunofluorescence on each specimen pair. Both HER2 immunoreactivity (P<0.0001) and pTyr(1248)HER2 immunoreactivity (P<0.0001) were lower in resections relative to CNB specimens. However, clinical implications of this change may not be evident since no case changed from 3+ (CNB) to negative (resection). Assessment of pTyr(1248)HER2 showed no direct correlation with HER2 in either CNB or resection specimens. The data suggest that measurement of both HER2 and phospho- Tyr(1248)HER2, in formalin-fixed tissue by immunological methods is significantly affected by pre-analytic variables. The current study warrants the adequate handling of resected specimens for the reproducible evaluation of HER2 and pHER2. The level of pTyr(1248)HER2, was not correlated to total HER2 protein. Further studies are required to determine the significance of these observations with respect to response to HER2 directed therapies.
    PLoS ONE 11/2013; 8(11):e79901. DOI:10.1371/journal.pone.0079901 · 3.23 Impact Factor
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