Effect of Ischemic Time, Fixation Time, and Fixative Type on HER2/neu Immunohistochemical and Fluorescence In Situ Hybridization Results in Breast Cancer

Abstract

Accurate determination of HER2/neu status in breast carcinoma is essential. Alteration of preanalytic variables is known to affect HER2/neu results. American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) issued guidelines to standardize fixation for increased HER2/neu accuracy. We studied the effects of changing preanalytic variables on HER2/neu immunohistochemical and fluorescence in situ hybridization (FISH) results in a known HER2/neu+ invasive carcinoma. The clinical specimen was processed according to ASCO/CAP guidelines, with remaining tumor stored fresh without any fixatives for 4 days at 4°C and cut into core biopsy-sized pieces. Each was fixed in 10% formalin, 15% formalin, Pen-Fix (Richard-Allan Scientific, Kalamazoo, MI), Bouin solution, Sakura molecular fixative (Sakura Tissue-Tek Xpress, Torrance, CA), or zinc formalin for 0 to 168 hours. Immunohistochemical studies and FISH were performed. Compared with the clinical specimen, the samples showed no tumor degradation or marked difference by immunohistochemical studies, except the 1-hour 10% formalin and Bouin samples, or FISH, except the Bouin-fixed samples. Our study demonstrates that HER2/neu results remain accurate beyond ASCO/CAP-recommended preanalytic variables, with the exception of Bouin solution for FISH analysis.

The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma

“…The same group of authors published another similar study to analyze the HER2 IHC and fluorescence in-situ hybridization results, and found that cold ischemic time of 4 days at 4 1C did not alter the HER2 results. 25,26 It is important to note, however, that these two studies were performed on a single case of strongly hormone receptor-positive ER/PR/HER2 and cold ischemic time tumor and on a single case of unequivocally HER2-positive tumor. Therefore, the results are not applicable to most clinical specimens.…”

High concordance between HercepTest immunohistochemistry and ERBB2 fluorescence in situ hybridization before and after implementation of American Society of Clinical Oncology/College of American Pathology 2007 guidelines

“…We observed a similar lack of discordance in immunohistochemical accuracy when we changed fixative types, fixation times, and ischemia times for HER2 and other hormonal receptor studies for breast cancer. 18,19 On the basis of the change in guidelines for scoring 3 þ immunohistochemical positivity in 10-30% of tumor cells, one would expect an increase in equivocal (2 þ ) cases as well as a decrease in positive (3 þ ) cases given the more stringent cutoff. Middleton et al 20 reported a 64% reduction of inconclusive cases, from 10.8-3.4%, and an increase in their concordance rate from 98-98.5% after implementation of ASCO/CAP guidelines, supporting the change in ERBB2 testing guidelines.…”

Assessment of HER-2 status in invasive breast cancer in Brazil

“…24,25 A low concordance between local and reference laboratories has also been reported in Brazil, and the authors have argued that it may be related to inexperience with HER-2 scoring, a low-volume load of HER-2 assays, and technical issues related to IHC in local laboratories. 26 Given the impact of preanalytic variables IHC and FISH results, 27 and aiming at improving the accuracy of HER-2 testing, the ASCO/CAP guidelines include recommendations regarding type of fixative, time between sample collection and placement into fixative, and fixation duration. According to the 2007 guideline, time from tissue acquisition to fixation should be as short as possible, with samples for HER-2 testing being fixed in 10% neutral buffered formalin for a minimum of 6 and a maximum of 48 hours.…”