Seth Sadis

Identification of targetable FGFR gene fusions in diverse cancers.

Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2, including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Because of the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts, which incorporate transcriptome analysis for gene fusions, are poised to identify rare, targetable FGFR fusions across diverse cancer types.

Phase IIb dose‐ranging study of the oral JAK inhibitor tofacitinib (CP‐690,550) or adalimumab monotherapy versus placebo in patients with active rheumatoid arthritis with an inadequate response to disease‐modifying antirheumatic drugs

OBJECTIVE To compare the efficacy, safety, and tolerability of 5 doses of oral tofacitinib (CP-690,550) or adalimumab monotherapy with placebo for the treatment of active rheumatoid arthritis (RA) in patients with an inadequate response to disease-modifying antirheumatic drugs. METHODS In this 24-week, double-blind, phase IIb study, patients with RA (n = 384) were randomized to receive placebo, tofacitinib at 1, 3, 5, 10, or 15 mg administered orally twice a day, or adalimumab at 40 mg injected subcutaneously every 2 weeks (total of 6 injections) followed by oral tofacitinib at 5 mg twice a day for 12 weeks. The primary end point was the responder rate according to the American College of Rheumatology 20% improvement criteria (ACR20) at week 12. RESULTS Treatment with tofacitinib at a dose of ≥3 mg twice a day resulted in a rapid response with significant efficacy when compared to placebo, as indicated by the primary end point (ACR20 response at week 12), achieved in 39.2% (3 mg; P ≤ 0.05), 59.2% (5 mg; P < 0.0001), 70.5% (10 mg; P < 0.0001), and 71.9% (15 mg; P < 0.0001) in the tofacitinib group and 35.9% of patients in the adalimumab group (P = 0.105), compared with 22.0% of patients receiving placebo. Improvements were sustained at week 24, according to the ACR20, ACR50, and ACR70 response rates as well as classifications of remission according to the 3-variable Disease Activity Score in 28 joints (DAS28) using C-reactive protein and the 4-variable DAS28 using the erythrocyte sedimentation rate. The most common treatment-emergent adverse events (AEs) in patients across all tofacitinib treatment arms (n = 272) were urinary tract infection (7.7%), diarrhea (4.8%), headache (4.8%), and bronchitis (4.8%). CONCLUSION Tofacitinib monotherapy at ≥3 mg twice a day was efficacious in the treatment of patients with active RA over 24 weeks and demonstrated a manageable safety profile.

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.

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

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. Mol Cancer Res; 13(4); 613–9. ©2015 AACR. Visual Overview

Monoclonal antibodies against macrophage colony-stimulating factor diminish the number of circulating intermediate and nonclassical (CD14++CD16+/CD14+CD16++) monocytes in rheumatoid arthritis patient

To the editor: Human blood monocytes are divided into 3 subsets: classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++).[1][1] The latter 2 subpopulations produce inflammatory cytokines in response to a wide variety of pattern receptor ligands and are constantly

Genomic Profiling of Penile Squamous Cell Carcinoma Reveals New Opportunities for Targeted Therapy

Penile squamous cell carcinoma (PeSCCA) is a rare malignancy for which there are limited treatment options due to a poor understanding of the molecular alterations underlying disease development and progression. Therefore, we performed comprehensive, targeted next-generation sequencing to identify relevant somatic genomic alterations in a retrospective cohort of 60 fixed tumor samples from 43 PeSCCA cases (including 14 matched primary/metastasis pairs). We identified a median of two relevant somatic mutations and one high-level copy-number alteration per sample (range, 0-5 and 0-6, respectively). Expression of HPV and p16 was detectable in 12% and 28% of patients, respectively. Furthermore, advanced clinical stage, lack of p16 expression, and MYC and CCND1 amplifications were significantly associated with shorter time to progression or PeSCCA-specific survival. Notably, four cases harbored EGFR amplifications and one demonstrated CDK4 amplification, genes for which approved and investigational targeted therapies are available. Importantly, although paired primary tumors and lymph node metastases were largely homogeneous for relevant somatic mutations, we identified heterogeneous EGFR amplification in primary tumor/lymph node metastases in 4 of 14 cases, despite uniform EGFR protein overexpression. Likewise, activating HRAS mutations occurred in 8 of 43 cases. Taken together, we provide the first comprehensive molecular PeSCCA analysis, which offers new insight into potential precision medicine approaches for this disease, including strategies targeting EGFR.

Abstract 2897: Discovery and characterization of driver MAPK and PI3K pathway mutations in tumors and association with drug response in cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The MAPK and PI3K pathways are frequently altered in human cancer and are targeted by dozens of agents in clinical trials. The successful application of these therapies, alone or in combination, may depend on the activation status of both pathways. Next-generation sequencing of cancer exomes provides a unique opportunity to systematically survey pathway alterations in cancer. Using somatic mutation data obtained from The Cancer Genome Atlas, we sought to catalog the members of the MAPK and PI3K pathways with driver mutations, the frequency of occurrence in common cancers and the frequency of co-occurrence. Furthermore, we sought to characterize the association of pathway mutation status with drug response in pre-clinical models. While the MAPK and PI3K pathways were frequently altered, the frequency of single and dual pathway alteration and the altered genes varied substantially across cancer types. The MAPK pathway was most frequently altered in rectal (62%), colon (59%), uterine (31%) and lung adenocarcinoma (45%) but infrequently altered in and breast cancer (4%). KRAS, BRAF and NRAS hotspot mutations were the most common pathway drivers, along with NF1 deleterious mutations in certain cancer types. The PI3K pathway was most frequently altered in uterine (84%), breast (40%) and glioblastoma (41%) but was rarely altered in lung adenocarcinoma (9%). Hotspot mutations in PIK3CA and hotspot and deleterious mutations in PTEN were the most common pathway alterations. In addition, predicted driver mutations occurred in PIK3R1, PIK3R3, MTOR, AKT1 and AKT3. Notably, MAPK and PI3K pathway alterations co-occurred in uterine (30%), colon (17%) and gastric (12%) cancers more so than would be expected by chance (p < 0.02). In contrast, other cancer types favored one pathway almost exclusively and thus had little co-occurrence. For example, breast cancer significantly favored PI3K pathway whereas lung adenocarcinoma favored MAPK pathway. To assess the effect of pathway mutation status on treatment response, we integrated hybrid-capture sequencing data from the Cancer Cell Line Encyclopedia with pharmacological data from over 150 compounds. We found that MAPK and PI3K pathway mutations most significantly associated with sensitivity to MEK and PI3K/AKT/mTOR inhibitors, respectively. Notably though, cell lines with co-occurring MAPK pathway and PIK3CA mutations were insensitive to MEK inhibitors and cell lines with co-occurring PI3K pathway and KRAS mutations were insensitive to PI3K inhibitors. Also, not all pathway mutations conferred equal sensitivity. For example, BRAF mutants were generally sensitive, KRAS mutants were mixed and NF1 mutants were generally insensitive to MEK inhibitors. Taken together, our work highlights the need to consider pathways and co-occurrence in the development of targeted therapies. Citation Format: Mark Tomilo, Paul D. Williams, Emma T. Bowden, Supra R. Gajjala, Santhoshi Bandla, Sean F. Eddy, Seth E. Sadis, Peter J. Wyngaard, Nickolay A. Khazanov, Daniel R. Rhodes. Discovery and characterization of driver MAPK and PI3K pathway mutations in tumors and association with drug response in cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2897. doi:10.1158/1538-7445.AM2013-2897

Abstract 3665: An EMT gene expression diagnostic predicts resistance to EGFR and MEK-targeted therapies in cell lines and patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The epithelial to mesenchymal transition (EMT) in cancer cells results in the acquisition of metastatic properties and may contribute to chemoresistance. Several studies have shown that transition to a mesenchymal phenotype leads to decreased dependence on EGFR-RAS signaling and insensitivity to EGFR inhibitors. To better understand the importance of EMT as a general predictor of drug response, we defined an EMT gene signature derived from a meta-analysis of differential gene expression signatures representing genes up-regulated following transfection of breast cell lines with various EMT regulators (Taube et al., 2010 Proc Natl Acad Sci USA 107:15449-54). We then determined the expression of the EMT signature across cell line panels and determined whether it predicted sensitivity or resistance to various targeted therapies. Consistent with previous results, expression of EMT signature was significantly associated with resistance to an EGFR inhibitor, lapatinib. Similarly, the EMT signature also predicted resistance to PQIP (IGF1R), GSK1120212 (MEK), GSK690693 (AKT), and perifosine (AKT/PI3K), suggesting that EMT may be a common resistance mechanism to a number of drugs that target growth factor signaling. As more of these targeted agents are entering clinical trials, the ability to characterize the signature may have important implications for drug development. To study the relevance of the EMT signature in clinical tumors, we compared the signature to a collection of tumor co-expression patterns, known as OncoScore modules, which were defined from 40,000+ tumor microarray experiments. Notably, the EMT signature was significantly associated with a major tumor co-expression pattern representing mesenchymal and/or stromal phenotype observed in almost all major solid tumor types. In retrospective microarray scoring analyses of key clinical datasets, the mesenchymal/stromal module predicted resistance to cetuximab. This finding was validated with an independent cohort of colorectal cancer patients treated with cetuximab using the Oncoscore Colon diagnostic. Oncoscore Colon is a qPCR test optimized for formalin-fixed paraffin-embedded tissue that measures the twelve key colon cancer transcriptional modules, including the mesenchymal module. Because the mesenchymal/stromal module monitors a fundamental phenotype of cancer cells important for drug response, this validated qPCR test has broad application to companion diagnostics development and personalized medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3665. doi:1538-7445.AM2012-3665

Targeted DNA and RNA Sequencing of Paired Urothelial and Squamous Bladder Cancers Reveals Discordant Genomic and Transcriptomic Events and Unique Therapeutic Implications

BACKGROUND Integrated molecular profiling has identified intrinsic expression-based bladder cancer molecular subtypes. Despite frequent histological diversity, robustness of subtypes in paired conventional (urothelial) and squamous components of the same bladder tumor has not been reported. OBJECTIVE To assess the impact of histological heterogeneity on expression-based bladder cancer subtypes. DESIGN, SETTING, AND PARTICIPANTS We performed clinically applicable, targeted DNA and/or RNA sequencing (multiplexed DNA and RNA sequencing [mxDNAseq and mxRNAseq, respectively]) on 112 formalin-fixed paraffin-embedded (FFPE) bladder cancer samples, including 12 cases with paired urothelial/squamous components and 21 bladder cancer cell lines. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Unsupervised hierarchical and consensus clustering of target gene expression enabled derivation of basal/luminal molecular subtyping. RESULTS AND LIMITATION Across 21 bladder cancer cell lines, our custom mxRNAseq panel was highly concordant with whole transcriptome sequencing, and assessed targets robustly determined expression-based basal/luminal subtypes from The Cancer Genome Atlas data (in silico) and internally sequenced FFPE tissues. Frequent deleterious TP53 (56%) and activating hotspot PIK3CA (30%) somatic mutations were seen across 69 high-quality tissue samples. Potentially targetable focal ERBB2 (6%) or EGFR (6%) amplifications were also identified, and a novel subgene copy-number detection approach is described. Combined DNA/RNA analysis showed that focally amplified samples exhibit outlier EGFR and ERBB2 expression distinct from subtype-intrinsic profiles. Critically, paired urothelial and squamous components showed divergent basal/luminal status in three of 12 cases (25%), despite identical putatively clonal prioritized somatic genomic alterations. Limitations include lack of profiled paired normal tissues for formal somatic alteration determination, and the need for formal analytical and clinical validation. CONCLUSIONS Our results support the feasibility of clinically relevant integrative bladder cancer profiling and challenge the intrinsic nature of expression subtypes in histologically diverse bladder cancers. PATIENT SUMMARY A targeted RNA sequencing assay is capable of assessing gene expression-based subtypes in individual components of clinical bladder cancer tissue specimens. Different histological components of the same tumor may yield divergent expression profiles, suggesting that expression-based subtypes should be interpreted with caution in heterogeneous cancers.

Abstract 5272: Cloud-based informatics enables the design and analysis of massively multiplex custom gene fusion panels for next-generation sequencing on FFPE RNA samples

Gene fusions, a combination of two genes, comprising their coding and/or regulatory sequences, are caused by structural rearrangements in DNA or in RNA transcripts. Many gene fusions are strong driver mutations in neoplasia, and are important in understanding basic biology, interaction with targeted therapy, and research into risk stratification and outcomes. Next-generation sequencing enables sensitive, specific and precise detection of particular fusion isoforms for defined gene pairs. Massively multiplex Ampliseq gene fusion assays enable enrichment of fusion transcripts using as little as 10 ng of RNA extracted from FFPE samples. Sequencing on Ion Torrent instruments reveals the full sequence of the gene fusion, for precise definition of the breakpoint and the expressed exons or promoter regions of both genes. We developed cloud-based software to support the design of a custom Ampliseq gene fusion panel, comprising 1 to 1,000 fusion isoform assays and any gene expression assays for normalization. We extensively mined the scientific literature on fusions and the COSMIC database to identify over 1000 fusion isoforms. We rigorously curated this data using automated and manual methods, including mapping, confirmation and correction of reported sequence to obtain genomic coordinates, identification of breakpoints, annotation of exon junctions, and selected wet lab testing. We created a database containing over 1000 high quality curated and annotated fusion isoforms, including 70 ALK, 60 RET, 26 ROS1, and 21 NTRK1 fusions. We designed Ampliseq primer pairs for each of these fusions using advanced assay design and pooling algorithms, such that all fusion and gene expression assays can be multiplexed into 1 or 2 compatible pools. Assays can be selected by gene or gene pair; detailed information about each assay selected includes isoform, genes, exon numbers, and links to COSMIC and to relevant publications. We developed cloud-based analysis software to analyze the BAM file resulting from amplification and sequencing of custom Ampliseq fusion panels on an Ion Torrent sequencer. This analysis leverages the rich annotation information from the assay design. The reads are mapped to a custom reference sequence tailored to the custom Ampliseq fusion assay, and applying an optimized algorithm to select confidently mapped reads based on read length and overlap with each gene of the gene pair based on the reference and annotated breakpoint. Gene fusions are detected based on the total number of fusion reads and optionally frequency, and on the properties of those reads. Software QC steps for total number of mapped reads, number of reads for gene expression controls, and elimination of cross-talk artifacts result in a highly sensitive and specific detection of fusions, with LOD below 1%. Fusion results for any or all samples can be viewed, annotated, filtered, and visualized, and exported. Citation Format: Fiona Hyland, Rajesh Gottimukkala, Efren Ballesteros, Heinz Breu, Yuandan Lou, Scott Myrand, Michael Hogan, Kelli Bramlett, Guoying Liu, Seth Sadis. Cloud-based informatics enables the design and analysis of massively multiplex custom gene fusion panels for next-generation sequencing on FFPE RNA samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5272.