Effect of Ischemic Time, Fixation Time and Fixatives on Her-2/Neu IHC and FISH Results in Breast Cancer.
Alarice C Lowe, Gouri Nanjangud, I Peter Shintaku, Saeedeh Shapourifar-Tehrani, Nagesh Rao, Neda Moatamed, David Lu, Sophia Apple. UCLA, Los Angeles, CA
Background: Accurate determination of Her-2/neu status in breast carcinoma is essential. Variation in pre-analytic factors may be a source of discordant results. Recently, ASCO/CAP recommended a <1 hour (hr) ischemic time and fixation for 6-72 hrs in 10% formalin to normalize pre-analytic variables. We studied the effects of an ischemic time of 4 days at 4o C and the pre-analytical variables of fixative type and fixation time, ranging from 0 to 168 hrs, on Her-2/neu IHC and FISH.
Design: We studied a 10 cm invasive ductal carcinoma with known Her-2/neu overexpression by IHC and FISH on the previous biopsy. The clinical sample was fixed in 10% formalin for 12 hours within 1 hr of receipt. The remaining tumor was stored fresh at 4o C for 4 days and subsequently cut into 97 core needle biopsy-sized pieces. 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 15% formalin, Pen-Fix, Bouin's solution, Sakura molecular fixative or zinc formalin at similar intervals. One sample was not fixed at all. All were processed on the Sakura Tissue-Tek VIP tissue processor. IHC for Her-2/neu (DAKO) were done. Two pathologists independently reviewed the H&E's and interpreted the IHC blindly. All blocks had FISH Her-2/neu analysis using FDA approved PathVysion.
Results: 89 of 97 blocks had invasive tumor, which showed no histologic degradation after 4 days of ischemic time. The clinical sample showed 3+ HER-2/neu staining by IHC and FISH amplification. IHC: The 10% formalin samples fixed for 1 and 3 hrs show weaker, variable IHC staining (1+ score). The 1 hr Bouin and 1 hr molecular-fixed samples also showed 1+ IHC staining. Almost all the remaining samples of all fixative types and durations, including the unfixed sample, showed IHC scores of 3+. FISH: Of the 89 blocks with invasive tumor, 57 (64%) showed amplification and 32 (36%) resulted in no hybridization. All 10% formalin-fixed samples showed amplification. The Bouin-fixed samples show amplification at 1 hr but failed to hybridize at all other times. The unfixed sample did not hybridize. No samples hybridized without amplification.
Conclusions: For IHC, the 1 and 3 hr 10% formalin, 1 hr Bouin and 1 hr molecular-fixed samples showed weaker IHC Her-2/neu staining; all other samples retained IHC staining quality. For FISH, 10% formalin fixation worked best, followed by 15% formalin, zinc formalin, Pen-Fix and molecular fixatives. Bouin fixation resulted in no hybridization. IHC and FISH for Her-2/neu were accurate beyond the ASCO/CAP guidelines for fixation even with 4 days of ischemic time.
Wednesday, March 2, 2011 9:30 AM
Poster Session V # 7, Wednesday Morning