International Journal of Gynecological Pathology 00:1–9, Lippincott Williams & Wilkins, Baltimore Copyright © 2017 by the International Society of Gynecological Pathologists Original Article Clinical Targeted Next-Generation Sequencing Shows Increased Mutational Load in Endometrioid-type Endometrial Adenocarcinoma With Deficient DNA Mismatch Repair Paul J. Lee, M.D., Ph.D., Samantha McNulty, Ph.D., Eric J. Duncavage, M.D., Jonathan W. Heusel, M.D., Ph.D., and Ian S. Hagemann, M.D., Ph.D. Summary: A subset of endometrial adenocarcinomas (EACs) exhibit microsatellite instability and have deficient DNA mismatch repair (dMMR). The overall aim of the study was to compare the spectrum of mutations in endometrioid-type EAC with and without dMMR by using a clinically validated next-generation sequencing assay. We retrospectively identified 19 EACs with known mismatch repair status that had undergone targeted sequencing of a panel of cancer-related genes. The mismatch repair status was ascertained by immunohistochemistry against MLH1, PMS2, MSH2, and MSH6 mismatch proteins. Somatic mutations in EAC with dMMR were compared against those in cases with proficient MMR (pMMR). The dMMR EAC showed a normalized mean of 66.6 mutations/Mb per case compared with pMMR EAC with a mean of 26.2 (Po0.05). The most commonly mutated genes were PTEN (89% of dMMR, 50% of pMMR), PIK3CA (67% vs. 40%), ATM (89% vs. 40%), and FLT3 (67% vs. 50%). The transition/transversion ratio was 4.7 versus 2.8 for the dMMR and pMMR cohorts, respectively (Po0.05). Copy number variant analysis did not demonstrate significant differences between the dMMR and pMMR cohorts and was not correlated with histologic grade of EAC. In conclusion, tumorigenesis of EAC in the context of dMMR demonstrated heavier mutational burdens and higher transition/transversion ratio. The spectrum of genetic alterations can potentially help identify cases with microsatellite instability phenotype using next-generation sequencing data from a targeted cancer gene panel. Key Words: Endometrial adenocarcinoma—Endometrioid adenocarcinoma—DNA mismatch repair—Deep sequencing—DNA mutational anal- ysis—Copy number alterations—Single-nucleotide variants. Genetic deficiencies in DNA mismatch repair (dMMR) result in the microsatellite instability (MSI) phenotype, causing the accumulation of DNA replication errors (1). The loss of genomic proofreading causes increases in small insertions and/or deletions, which are most likely to occur in tandem repeat loci known as microsatellites (2). The exome is known to contain > 200,000 microsatellite loci, the vast majority of which are noncoding (3,4). The MSI phenotype is not associated with a specific mutational hot spot,” but direct detection is usually accomplished by polymerase From the Departments of Pathology and Immunology (P.J.L., S.M., E.J.D., J.W.H., I.S.H.); and Obstetrics and Gynecology (I.S.H.), Washington University School of Medicine, St Louis, Missouri. Supported by the Anatomic Pathology Fund, Department of Pathology and Immunology, Washington University School of Medicine. The authors declare no conflict of interest. Address correspondence and reprint requests to Ian S. Hagemann, MD, PhD, Campus Box 8118, 660 S. Euclid Avenue, St Louis, MO 63110. E-mail: hagemani@wustl.edu. Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website, www.intjgynpathology.com. 1 DOI: 10.1097/PGP.0000000000000459 Copyright r 2017 International Society of Gynecological Pathologists.