Delay to formalin fixation effect on breast biomarkers

Abstract

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 41C. 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 X6 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 (Po0.03). 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.

Preferential HER2 expression in liver metastases and EGFR expression in peritoneal metastases in patients with advanced gastric cancer

“…Gene amplification analysis of HER2 and EGFR by FISH could not be carried out in this study except for several recent autopsy cases because of the invalid results due to the delay in formalin fixation (up to 3-4 days) of the old archived specimens resected between 1995 and 2000 [22,23]. Nevertheless, we demonstrated the following interesting findings.…”

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

“…They concluded that delayed fixation of modified radical mastectomy tissues likely accounted for this finding and optimal selection of patients for hormonal therapies is dependent on tissue management strategies even before formal hormonal receptor protein testing procedures. 23 Khoury et al, 24 in their study of effects of delayed formalin fixation on 10 specimens that were kept at room temperature for 0, 10, 30 min, and 1, 2, 4, 8 h before fixation, concluded that ER expression began to decline after 2 h and PR expression after 1 h of cold ischemic time. Khoury et al 24 also reported that at 8 h of cold ischemic time, ER expression was completely negative, and they recommended that specimens be fixed ideally within 1 h of receipt, and not to be stored overnight at 4 1C.…”

“…23 Khoury et al, 24 in their study of effects of delayed formalin fixation on 10 specimens that were kept at room temperature for 0, 10, 30 min, and 1, 2, 4, 8 h before fixation, concluded that ER expression began to decline after 2 h and PR expression after 1 h of cold ischemic time. Khoury et al 24 also reported that at 8 h of cold ischemic time, ER expression was completely negative, and they recommended that specimens be fixed ideally within 1 h of receipt, and not to be stored overnight at 4 1C. Their finding was that overnight storage at 4 1C yielded similar results to leaving specimens without fixation at room temperature for 8 h. This finding is in contrast to our finding that non-refrigerated samples are affected more by prolonged cold ischemic time than refrigerated samples, whereas our finding of general decline in hormone receptor expression with increasing cold ischemic time periods is in keeping with their findings.…”

“…Therefore, the results are not applicable to most clinical specimens. Qiu et al 27 looked into the effect of delayed formalin fixation on three different clones of ER (1D5, 6F11, and SP1) and PR (PgR636, 16, and 1E2) on the same group of cases as that of Khoury et al 24 They found that for ER, delayed formalin fixation's effect on SP1 was less than 1D5 and 6F11 clones. The SP1 clone had more intense nuclear staining, low background, and no cytoplasmic staining when compared with 1D5 and 6F11 clones.…”

Do Clinical Features and Survival of Single Hormone Receptor Positive Breast Cancers Differ from Double Hormone Receptor Positive Breast Cancers?

“…(Ng et al, 2012) It only presents in a small number of cases (1-10%) and indeed re-testing of cases by some authors utilising more sensitive immunohistochemical detection systems and lower cut points suggests that at least some of these cases are double receptor positive using alternative assessment criteria (Hammond et al; Nadji et al, 2005;De Maeyer et al, 2008;Nadji, 2008). In addition hormone receptors are notoriously labile and unless tissues are fixed promptly and the cold ischaemia time between surgical removal and immersion in fixative is kept to a minimum, there is the confounding factor of receptor degradation and consequent false negative assay results (Khoury et al, 2009) All these factors need to be taken into consideration when considering the clinical relevance of the ER-PR+ result.…”