Next-Gen Sequencing Exposes Frequent MED12 Mutations and Actionable Therapeutic Targets in Phyllodes Tumors.

UNLABELLED: Phyllodes tumors are rare fibroepithelial tumors with variable clinical behavior accounting for a small subset of all breast neoplasms, yet little is known about the genetic alterations that drive tumor initiation and/or progression. Here, targeted next-generation sequencing (NGS) was used to identify somatic alterations in formalin-fixed paraffin-embedded (FFPE) patient specimens from malignant, borderline, and benign cases. NGS revealed mutations in mediator complex subunit 12 (MED12) affecting the G44 hotspot residue in the majority (67%) of cases spanning all three histologic grades. In addition, loss-of-function mutations in p53 (TP53) as well as deleterious mutations in the tumor suppressors retinoblastoma (RB1) and neurofibromin 1 (NF1) were identified exclusively in malignant tumors. High-level copy-number alterations (CNA) were nearly exclusively confined to malignant tumors, including potentially clinically actionable gene amplifications in IGF1R and EGFR. Taken together, this study defines the genomic landscape underlying phyllodes tumor development, suggests potential molecular correlates to histologic grade, expands the spectrum of human tumors with frequent recurrent MED12 mutations, and identifies IGF1R and EGFR as potential therapeutic targets in malignant cases. IMPLICATIONS: Integrated genomic sequencing and mutational profiling provides insight into the molecular origin of phyllodes tumors and indicates potential druggable targets in malignant disease. Visual Overview: http://mcr.aacrjournals.org/content/early/2015/04/02/1541-7786.MCR-14-0578/F1.large.jpg.

Tumor evolution and progression in multifocal and paired non-invasive/invasive urothelial carcinoma.

Although multifocal tumors and non-invasive/invasive components are commonly encountered in surgical pathology, their genetic relationship is often poorly characterized. We used next-generation sequencing (NGS) to characterize somatic alterations in a patient with five spatially distinct, high-grade papillary urothelial carcinomas (UCs), with one tumor harboring an underlying invasive component. NGS of 409 cancer-related genes was performed on DNA isolated from formalin-fixed paraffin-embedded (FFPE) blocks representing each papillary tumor (n = 5), the invasive component of one tumor, and matched normal tissue. We identified nine unique non-synonymous somatic mutations across the six UC samples, including five present in each carcinoma sample, consistent with clonal origin and limited intertumoral heterogeneity. Copy number and loss of heterogeneity (LOH) profiles were similar in all six carcinomas; however, the invasive carcinoma component uniquely showed focal CDKN2A loss and chromosome 9 LOH and did not harbor gains of chromosomes 5p or X that were present in the other tumor samples. Phylogenetic analysis supported the invasive component arising from a shared progenitor prior to the outgrowth of cells in the non-invasive tumors. Results were extended to three additional cases of upper tract UC with paired non-invasive/invasive components, which identified driving alterations exclusive to both non-invasive and invasive components. Lastly, we performed targeted RNA sequencing (RNAseq) using a custom bladder cancer panel, which confirmed gene expression signature differences between paired non-invasive/invasive components. The results and approaches presented here may be useful in understanding the clonal relationships in multifocal cancers or paired non-invasive/invasive components from routine FFPE specimens.

Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors.

Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with <20 ng of DNA/RNA from formalin-fixed paraffin-embedded (FFPE) tissues], coupled with an informatics pipeline to specifically identify relevant predefined variants and created a knowledge base of related potential treatments, current practice guidelines, and open clinical trials. We validated OCP using molecular standards and more than 300 FFPE tumor samples, achieving >95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.