Estrogen Receptor (ER) and Progesterone Receptor (PR) Immunohistochemistry (IHC) Results in Breast Carcinoma Using Varying Fixation Times in Different Fixatives
AC Lowe, N Moatamed, R Pucci, IP Shintaku, S Shapourifar-Tehrani, B Shackley, SK Apple. UCLA, Los Angeles, CA
Background: Accurate determination of ER and PR status in breast carcinoma is essential because their presence warrants hormonal therapy. The high levels of discordance in Her-2/neu and ER test results reported in the literature (up to 20% and 30%, respectively) are thought to be partly due to variation in pre-analytic factors. To address this, ASCO/CAP made recommendations to normalize fixation for breast biomarker IHC, stating that breast core needle biopsies (CNB) should be fixed >6 and <48 hours (hrs) and excisional biopsies >8 and <72 hrs in 10% formalin.
Design: A mastectomy specimen with a 4 cm known invasive lobular carcinoma (ER+, PR+ and Her-2/neu-) underwent routine clinical sampling and breast biomarker IHC as per ASCO/CAP guidelines: 10 hr fixation in 10% formalin. The remaining tumor was stored fresh at 4o C for 4 days and subsequently cut into 92 CNB-sized pieces (0.5-1.5 cm in length and 0.2 cm in diameter). Each piece was fixed in 20 mL of 10% formalin for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, 72, or 168 hrs or 20 mL of Pen-Fix, Bouin's solution, Sakura molecular fixative, zinc formalin, or 15% formalin at similar intervals (only Pen-Fix for 168 hrs). Samples were processed on the Sakura Tissue-Tek VIP tissue processor with no additional fixation. One H&E slide was made for each block. IHC was performed with the 6F11 ER and 636 PR antibody clones with appropriate controls. Two pathologists independently interpreted the IHC blindly, documenting the intensity and percentage of tumor ER and PR staining.
Results: The patient's tumor showed >95% of tumor cells with 3+ ER and PR IHC staining in the clinical sample. All 92 blocks had invasive tumor, except one, which had lobular carcinoma in situ (LCIS), on which IHC was interpreted. After 4 days at 4o C, the tumor showed no degradation and no differences in ER and PR staining for all samples fixed in 10% formalin. In fact, there was no significant difference in ER and PR staining for 15% formalin, zinc formalin, Pen-Fix, Sakura molecular fixative, and most of the Bouin's-fixed samples. Only two Bouin's-fixed samples (48, 72 hrs) had a significant decrease in ER staining, which was still interpreted as positive. No effect was seen with PR.
Conclusions: In our study, the pre-analytical variables of fixative type and fixation time, ranging from 1 hr to 1 week, did not affect the accuracy of ER and PR IHC results in CNB-sized tumor sections.
Tuesday, March 23, 2010 1:00 PM
Poster Session IV # 21, Tuesday Afternoon