To more thoroughly evaluate the mutation-specific IHC on tumors bearing a variety of exon 19 deletions, 24 tumors harboring less common exon 19 deletions (9, 12, 18, 24 bp) were identified, and unstained slides were prepared using 4-um-thick tissue sections cut directly from the FFPE tumor blocks

To more thoroughly evaluate the mutation-specific IHC on tumors bearing a variety of exon 19 deletions, 24 tumors harboring less common exon 19 deletions (9, 12, 18, 24 bp) were identified, and unstained slides were prepared using 4-um-thick tissue sections cut directly from the FFPE tumor blocks. DNA Extraction Hematoxylin and eosinCstained sections of FFPE tissue were reviewed for each sample to identify areas of tumor. as a screen to identify most candidates for EGFR inhibitors. Somatic mutations within the tyrosine kinase domain name of EGFR are found in approximately 20% of lung adenocarcinomas and are the most reliable predictors of response to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib (Sharma et al, 2007).1 Multiple studies support that, in addition to their predictive value in treatment selection, mutations are also prognostic for survival benefit.2,3 Specifically, patients with these tumors survive significantly longer on EGFR TKIs than with conventional cytotoxic chemotherapy. 4 EGFR-mutant lung adenocarcinomas also form a distinct clinically favorable biological subset, regardless of EGFR TKI therapy.2 Mutated Ionomycin EGFR is more often found in better differentiated adenocarcinomas with or without a bronchioloalveolar component.5,6 It is virtually absent in other lung cancer subtypes except for adenosquamous carcinoma.7,8 In-frame deletions in exon 19 and the exon 21 L858R substitution are the most common mutations and, combined, symbolize approximately 90% of all mutants.9 Analysis for common mutations is now performed in many institutions to help direct treatment decisions. Direct DNA sequencing is usually a common detection method but has well-known sensitivity limitations depending on the proportion of tumor cells present in the material available for DNA extraction. Other DNA-based methods have been developed to address issues of sensitivity and turnaround time associated with direct sequencing.10 However, the Ionomycin cost and Rabbit polyclonal to ASH1 complexity of molecular methods has slowed their widespread implementation outside of major academic centers and commercial laboratories and drives the continued desire for less robust predictors of response such copy number and conventional immunohistochemistry (IHC) for total EGFR. IHC for total EGFR is an especially poor substitute as it correlates poorly or not at all with the presence of mutations.11,12 Another more challenging IHC strategy is to develop antibodies that react only with the mutant form of a given oncoprotein. Desire for this approach is usually driven by the fact that IHC is usually a technology available to essentially all pathology departments, can be automated, and can be performed on samples where the number or proportion of tumor cells poses difficulties for molecular assessments based on bulk DNA extraction from tissue. Cell Signaling Technology has recently developed two mutant-specific antibodies for IHC directed against the most common mutant forms of mutation status. We provide a careful assessment of putative false-positive and false-negative results, including a detailed analysis of how they relate to the molecular heterogeneity in exon 19 deletions and we propose an algorithm for their possible clinical implementation. Materials and Methods Tumor Samples Two hundred eighteen lung adenocarcinoma samples, procured at Memorial Sloan-Kettering Malignancy Center under Ionomycin IRB-approved protocols, between the years 1999 and 2008 were used for this study. The vast majority of cases were classified as adenocarcinoma, mixed subtype. A total of 194 formalin-fixed paraffin-embedded (FFPE) lung adenocarcinoma samples with available molecular data were selected for tissue microarray (TMA) construction. These included 18 L858R mutants, 31 cases with exon 19 deletions (deletion sizes: 9 bp Ionomycin [= 4], 12 bp [= 1], 15 bp [= 20], 18 bp [= 3], 24 bp [= 3]), and 145 cases without either mutation. The TMAs were constructed using triplicate 0.6-mm tissue cores. Three cores from different areas were selected from each tumor. Serial 4-um-thick tissue sections were freshly slice from your TMAs.