BACKGROUND Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas. METHODS We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes. RESULTS Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma. CONCLUSIONS The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma. (Funded by the National Institutes of Health.)

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In recent years, there has been increased interest in carcinomas of the urologic tract, that demonstrate association with the polyoma virus BK arising in immunosuppressed individuals, though the nature of this association is uncertain. To begin to understand this phenomenon, we reviewed the clinical, morphological, and immunohistochemical features of 11 carcinomas of the urologic tract, mainly urothelial (N = 9) and collecting duct carcinomas (N = 2), occurring during immunosuppression, and expressing polyoma virus T-antigen by immunohistochemistry. These were compared to a control group of carcinomas (N = 8), also arising during immunosuppression, but without T-antigen expression. A subset of both groups were also studied by hybrid capture-based DNA sequencing, probing not only for 479 cancer-related human genes, but also for polyoma and other viral sequences. Polyoma T-antigen-expressing tumors arose in 7 males and 4 females, at a median age of 66, and were aggressive, high-grade tumors with more than 1 variant morphologic pattern identified in 81% of cases, and a majority (73%) presenting at high stage category (>pT3). Diffuse polyoma T-antigen staining was seen in 91% of cases, with co-localization of aberrant p53 staining in 89%. Sequencing detected a lower number of deleterious mutations among T-antigen-expressing cases (average 1.62; 1/8 with TP53 mutation) compared to control cases (average 3.5, 2/4 with TP53 mutation). Only BK virus was detected with clonal integration and breakpoints randomly distributed across the human and viral genomes in 5/5 of the polyoma T-antigen-expressing carcinomas, and in none of the controls (0/4). In summary, these findings identify aggressive clinicopathologic features of polyoma T-antigen-expressing carcinomas, document BK as the strain involved, and associate BK viral integration with T-antigen expression and p53 aberrancy. While the apparent randomness of viral insertion sites is functionally unclear, the differing rates of mutations between T-antigen-expressing and control cases is intriguing.

572 Background: Comprehensive molecular profiling of CRC can inform treatment decisions by identifying patient subgroups at varying risks of death. Microsatellite instability (MSI) is prognostic in CRC and is used to select patients for immunotherapy. High tumor mutational burden (TMB) is associated with genomic instability and is prognostic in melanoma. Expression of p16 protein is prognostic in many tumor types. We used proteomic and genomic profiling to measure MSI, TMB and p16 in CRC tumors and to assess associations with patient survival. Methods: In archived clinical samples of CRC, 76 proteins were quantitated with mass spectrometry-based proteomics. MSI was measured by WGS and RNA-seq; unstable loci were quantified in tumor and normal samples. Cutoffs were derived via ROC analysis: high TMB was defined as > 4.5 somatic mutations per megabase; p16 as > 108 amol/ug. Patients were grouped by microsatellite status (MSI vs. microsatellite stable [MSS]), TMB (high vs. low), and p16 protein expression level. Survival curves were compared with the Mantel-Cox log-rank test. Results: Of 145 samples, 39 (27%) had high TMB and 29 (20%) had MSI. Patients with MSI tumors had longer OS than patients with MSS tumors (HR: 0.096; p = 0.003). Similarly, patients with high TMB had longer OS than those with low TMB (HR: 0.076; p < 0.001). High p16 expression was prognostic of poor survival (HR: 2.874; p = 0.019). Among patients with MSS tumors or low TMB, those with low p16 levels had longer OS than patients with high p16 (HR: 0.257; p = 0.002 and HR: 0.249; p = 0.002, for MSS and low TMB, respectively). A combination of MSS, low TMB, and low p16 also differentiated between long and short survivors (HR: 0.249; p = 0.002). These associations remained after adjustment for tumor sidedness. Further analyses of clinical correlates will be presented. Conclusions: A combination of MSS, low TMB and low p16 expression characterized a subset of patients with longer survival. This is important because patients with MSS tumors have limited treatment options but may respond to CDK4/6 inhibitors due to low p16 expression. Molecular profiling of CRC may identify patient subgroups with a relatively poor prognosis who could benefit from personalized therapy.

Desmoplastic melanomas (DMs) comprise 4% of the overall melanoma burden and have a 5-year survival rate of 85%. DMs are dermal tumors characterized by spindled melanocytes situated within abundant desomplastic stroma. These unusual histological features commonly lead to misdiagnosis. Currently, there are no known genetic drivers. A better understanding of the underlying biology of desmoplastic melanoma would provide biomarkers and therapeutic opportunities. Towards this goal, we performed low-coverage genome and high-coverage exome sequencing of 20 DMs in a discovery cohort, followed by targeted sequencing of 293 candidate genes on a validation cohort of 42 cases. Additionally, high-resolution aCGH was performed on samples from both cohorts. A high mutation burden (median 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers sequenced to date. Mutation patterns strongly indicate that UV-radiation is the dominant mutagen and implicate a superficially located cell of origin despite their predominantly intradermal presentation. Novel alterations included recurrent promoter mutations and amplification of NF-kappa B inhibitor epsilon, NFKBIE (IkBϵ) in 14.5% of samples. The promoter mutations typically affect both alleles and occur over a highly conserved DNA region. The mutations are predicted to disrupt a canonical Ets Like Factor 1 (ELF1) binding site. In total, these data imply aberrant NF-kappa B signaling as a pathogenic feature of desmoplastic melanoma. Commonly mutated oncogenes in melanomas, in particular BRAF V600E and NRAS Q61K/R, were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS, and PIK3CA. Rb and p53 pathway alterations occurred respectively in 71% and 66% of tumors, affecting RB1, FBXW7, CDK4, PPP6C, CCND1, CDKN2A, TP53, and MDM2. Finally, TERT promoter mutations or amplifications occurred in 90% of tumors. The consequences of the mutations on protein expression levels was confirmed by immunostaining for NF1, EGFR, Rb, CDK4, CCND1, p16, p53, and Mdm2. Collectively, many of these oncogenic mutations are potentially druggable. In conclusion, desmoplastic melanomas harbor distinct genetic alterations that explain their unique biology, and this study illuminates genetic biomarkers and nominates targets for therapeutic intervention. Citation Format: Alan H. Shain, Maria Garrido, Thomas Botton, Eric Talevich, Iweh Yeh, Zack Sanborn, Jongsuk Chung, Nicholas Wang, Hojabr Kakavand, Graham Mann, John Thompson, Thomas Wiesner, Ritu Roy, Adam Olshen, Alexander Gagnon, Joe Gray, Nam Huh, Joe Hur, Klaus Busam, Richard Scolyer, Raymond Cho, Rajmohan Murali, Boris Bastian. Exome sequencing of desmoplastic melanoma reveals recurrent NFKBIE promoter mutations and diverse MAPK/PI3K pathway activating mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2968. doi:10.1158/1538-7445.AM2015-2968

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Background: Esophageal squamous cell carcinoma (ESCC) comprises 90% of all esophageal cancer cases globally and is the most common histology in low-resource settings. Eastern Africa has a disproportionately high incidence of ESCC. Methods: We describe the genomic profiles of 61 ESCC cases from Tanzania and compare them to profiles from an existing cohort of ESCC cases from Malawi. We also provide a comparison to ESCC tumors in The Cancer Genome Atlas. Results: We observed substantial transcriptional overlap with other squamous histologies via comparison with The Cancer Genome Atlas (TCGA) PanCan dataset. DNA analysis revealed known mutational patterns, both genome-wide as well as in genes known to be commonly mutated in ESCC. TP53 mutations were the most common somatic mutation in tumors from both Tanzania and Malawi but were detected at lower frequencies than previously reported in ESCC cases from other settings. In a combined analysis, two unique transcriptional clusters were identified: a proliferative/epithelial cluster and an invasive/migrative/mesenchymal cluster. Mutational signature analysis of the Tanzanian cohort revealed common signatures associated with aging and cytidine deaminase activity (APOBEC) and an absence of signature 29, which was previously reported in the Malawi cohort. Conclusion: This study defines the molecular characteristics of ESCC in Tanzania, and enriches the Eastern African dataset, with findings of overall similarities but also some heterogeneity across two unique sites. Impact: Despite a high burden of ESCC in Eastern Africa, genomic investigations in this region are nascent. This represents the largest comprehensive genomic analysis ESCC from sub-Saharan Africa to date.

Background: We evaluate the feasibility, quality, and analytical potential of whole-transcriptome RNA-seq on over one thousand clinical FFPE tumor samples. Transcriptional profiling of RNA is used for clinical decision making in many tumor types. Clinical analysis of RNA is complicated by the common use of formalin-fixed paraffin-embedded (FFPE) tissue storage, which can cause low yield and RNA degradation. Methods: RNA was extracted from FFPE material using commercially available kits using an RNAseH based ribodeplete. Multiple libraries per sample are sequenced using standard Illumina sequencing. Bowtie2, RSEM, and custom software are used for alignment, transcript quantification, fusion detection, and variant expression analysis. Results: We observe a &gt;85% success rate on whole transcriptome RNA-seq on our cohort of more than 1000 samples. We find reliable transcript quantification upon successful sequencing, and on a subset of samples quantify differences between FFPE and fresh-frozen material when using ribodeplete. We also compare transcriptional profiles of clinical FFPE samples to an independent set of fresh frozen, poly-A capture samples from The Cancer Genome Atlas (TCGA) and show differences between poly-A capture and ribodeplete RNA isolation methods as well as FFPE vs. FF effects. We present a robust mapping methodology for comparison of public FF poly-A dataset to our FFPE ribodeplete samples. We demonstrate that we are able to utilize this joint gene expression space to perform site of origin prediction on FFPE samples. This is especially important for clinical application in analyzing Cancer of Unknown Primary (CUP) samples as well as detecting outlier samples whose molecular features may suggest additional therapeutic avenues. Finally, we show reliable detection of fusion transcripts from FFPE RNA-seq material from whole-transcriptome analysis and expression of somatic tumor variants detected from DNA sequencing. Conclusions: Large scale sequencing of RNA from clinical FFPE materials provides reliable transcriptomic results comparable to existing public databases of RNA, enabling research on cohorts of tumors that are FFPE-banked and unavailable as frozen tissue. Citation Format: Yulia Newton, Justin Golovato, Mark Johnson, Shahrooz Rabizadeh, Zack Sanborn, Steve Benz, Charles J. Vaske. Analysis of whole transcriptome RNA-seq of large numbers of clinical FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3398.