Abstract
Background: Tumor biopsies obtained from patients are often limited in size and availability, and the ability to perform multiple diagnostic assays depends on the quantity and quality of the tissue. Here we describe and evaluate a method for performing DNA-based mutational analyses after immunohistochemistry analysis has been performed, using a single tissue section. Method: Immunohistochemistry analysis was performed on 4-5 μm formalin-fixed paraffin-embedded tumor tissue sections and immunohistochemistry-stained sections were stored for subsequent genomic analysis. DNA was isolated from these immunohistochemistry-stained sections and DNA quality was assessed using a multiplexpolymerase chain reaction method as well as real time quantitative polymerase chain reaction of commonly used reference genes. Subsequently, genomic DNA was pre-amplified and mutations in KRAS, BRAF, NRAS and PIK3CA were detected by validated Taqman assays. Comparisons were made with results from unstained formalinfixed paraffin-embedded sections obtained from the same paraffin block. Results: Our results demonstrate that genomic DNA isolated from immunohistochemistry-stained and unstained formalin-fixed paraffin-embedded tissue sections are comparable in quality and are suitable for down-stream analysis using polymerase chain reaction based assays. We also found that the sensitivity and specificity in detecting hotspot mutations are comparable in both sources of genomic DNA. This study reports 100% concordance in detecting hotspot mutations in KRAS, BRAF, NRAS and PIK3CA using quantitative real-time polymerase chain reaction between stained and unstained formalin-fixed paraffin-embedded sections. Conclusion: We conclude that by using our novel approach, it is possible to perform immunohistochemistry staining followed by genomic analysis using a single 4-5 μm section of formalin-fixed paraffin-embedded tissue.
Highlights
Histopathological and immunohistochemical (IHC) analysis have been the predominant methods used to diagnose cancer
We conclude that by using our novel approach, it is possible to perform immunohistochemistry staining followed by genomic analysis using a single 4-5 μm section of formalin-fixed paraffin-embedded tissue
We evaluated the ability to isolate genomic DNA from tissue sections that have previously been used for IHC
Summary
Histopathological and immunohistochemical (IHC) analysis have been the predominant methods used to diagnose cancer. A large number of genomic alterations such as gene amplifications, point mutations, translocations, deletions, or insertions have been extensively documented in various types of cancers [1,2]. Identifying relationships between genomic alterations and cancer has provided a number of valuable targets for targeted therapies, such as BRAF mutations in melanoma [6,7] and ALK translocations in lung cancer [8,9]. Identifying genomic alterations along with histopathological and IHC analysis would enable clinicians to stratify patients based on the molecular characteristics of the tumor to deliver targeted therapies. Some well-known examples of such alterations and related therapies are vemurafenib for BRAF-mutant melanoma and crizotinib for lung cancers with EML4-ALK translocation. We describe and evaluate a method for performing DNA-based mutational analyses after immunohistochemistry analysis has been performed, using a single tissue section
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