Effects of Tissue Fixatives on Immunohistochemical Expression of MSI Makers in Colon Adenocarcinoma.
Patrick A Adegboyega. LSU Health Sciences Center, Shreveport, LA
Background: Colorectal adenocarcinomas with microsatellite instability (MSI) do not respond well to Fluorouracil-based chemotherapy; and do have treatment outcome that differs from that seen in microsatellite stable tumors. In clinical settings, immunohistochemical staining for makers of MSI [Mismatch repair (MMR) gene products] is used to screen for the presence of MSI; and has been shown to have comparable sensitivity and specificity with MSI detection by PCR. However, the effects of tissue fixatives on the immunohistochemical expression of MMR gene products are not known. This study explores the effects of two routinely used tissue fixatives [Dissect Aid and 10% Neutral Buffered Formalin (NBF)] on the immunohistochemical expression of three MMR gene products (MLH1, MSH2 and MSH6).
Design: Study materials consisted of 7 colectomy specimens received for tumor diagnosis and staging. Samples of normal colon and tumor from each specimen were fixed in NBF and Dissect Aid solutions. Matched samples from each fixative were submitted for routine processing and paraffin embedding after fixation for the following day(s): 1, 7, 14, 28, 42, 56, 84 and 112. Immunohistochemistry for MLH1, MSH2 and MSH6 was performed on representative sections of each block. Immunoreactivity scoring was done in a blinded fashion using a semi-quantitative score of 0, 1, 2, 3, and 4. The code for the fixatives was broken after scoring was done.
Results: MLH1 immunoreactivity scores for samples processed in fixative A was at least one score less than that observed for corresponding samples in fixative B; and almost undetectable by the end of week 4 for samples processed in fixative A but remaining strong throughout the study for samples processed in fixative B. Both benign and tumor samples stained strongly positive for MSH2 and MSH6 in all tissue samples processed in fixative B and the strong staining reaction was maintained throughout the study. In comparison, negative staining reaction was observed in both tumor and benign mucosa for all samples processed with fixative A – with as early as 24 hours fixation.
Conclusions: For all three MSI markers investigated, fixative B (10% Neutral Buffered Formalin solution) is the preferred fixative for immunohistochemical assay. Fixative A (Dissect Aid) may produce erroneous negative immunostaining results for MSI markers, even with as little as 24 hours fixation. These findings highlight the need for protocol modifications to separate the mesentery from the colon for fixation of colon in NBF and mesentery in Dissect Aid for optimal processing of both specimen components.
Wednesday, March 2, 2011 9:30 AM
Poster Session V # 33, Wednesday Morning