Gene of the month: VSTM2A.

The V-set and transmembrane domain containing 2A (VSTM2A) gene is located on chromosome 7. In the physiological state, VSTM2A regulates preadipocyte cell differentiation. VSTM2A is highly expressed in normal human brain tissue and minimally expressed in other normal tissues. Mucinous tubular and spindle cell carcinoma (MTSCC) of the kidney is a distinct renal tumour subtype with signature chromosomal copy number alterations and an indolent outcome in the majority of cases. VSTM2A overexpression is highly enriched in this renal cancer subtype and has been shown to have potential diagnostic value in distinguishing MTSCC from renal tumours with overlapping histological appearances.

VSTM2A Overexpression Is a Sensitive and Specific Biomarker for Mucinous Tubular and Spindle Cell Carcinoma (MTSCC) of the Kidney.

Our recent study revealed recurrent chromosomal losses and somatic mutations of genes in the Hippo pathway in mucinous tubular and spindle cell carcinoma (MTSCC). Here, we performed an integrative analysis of 907 renal cell carcinoma (RCC) samples (combined from The Cancer Genome Atlas and in-house studies) and the Knepper data set of microdissected rat nephrons. We identified VSTM2A and IRX5 as novel cancer-specific and lineage-specific biomarkers in MTSCC. We then assessed their expression by RNA in situ hybridization (ISH) in 113 tumors, including 33 MTSCC, 40 type 1 papillary RCC, 8 type 2 papillary RCC, 2 unclassified RCC, 15 clear cell RCC, and 15 chromophobe RCC. Sensitivity and specificity were calculated as the area under the receiver operating characteristics curve (AUC). All MTSCC tumors demonstrated moderate to high expression of VSTM2A (mean ISH score=255). VSTM2A gene expression assessed by RNA sequencing strongly correlated with VSTM2A ISH score (r(2)=0.81, P=0.00016). The majority of non-MTSCC tumors demonstrated negative or low expression of VSTM2A. IRX5, nominated as a lineage-specific biomarker, showed moderate to high expression in MTSCC tumors (mean ISH score=140). IRX5 gene expression assessed by RNA sequencing strongly correlated with IRX5 ISH score (r(2)=0.69, P=0.00291). VSTM2A (AUC: 99.2%) demonstrated better diagnostic efficacy than IRX5 (AUC: 87.5%), and may thus serve as a potential diagnostic marker to distinguish tumors with overlapping histology. Furthermore, our results suggest MTSCC displays an overlapping phenotypic expression pattern with the loop of Henle region of normal nephrons.

Ipilimumab plus nivolumab and DNA-repair defects in AR-V7-expressing metastatic prostate cancer.

AR-V7-expressing metastatic prostate cancer is an aggressive phenotype with poor progression-free survival (PFS) and overall survival (OS). Preliminary evidence suggests that AR-V7-positive tumors may be enriched for DNA-repair defects, perhaps rendering them more sensitive to immune-checkpoint blockade. We enrolled 15 metastatic prostate cancer patients with AR-V7-expressing circulating tumor cells into a prospective phase-2 trial. Patients received nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses, then maintenance nivolumab 3 mg/kg every 2 weeks. Targeted next-generation sequencing was performed to determine DNA-repair deficiency (DRD) status. Outcomes included PSA response rates, objective response rates (ORR), PSA progression-free survival (PSA-PFS), clinical/radiographic PFS and OS. Median age of participants was 65, median PSA was 115 ng/mL, 67% had visceral metastases, and 60% had ≥4 prior systemic therapies. Six of 15 men (40%) had DRD mutations (three in BRCA2, two in ATM, one in ERCC4; none had microsatellite instability). Overall, the PSA response rate was 2/15 (13%), ORR was 2/8 (25%) in those with measurable disease, median PSA-PFS was 3.0 (95%CI 2.1-NR) months, PFS was 3.7 (95%CI 2.8-7.5) months, and OS was 8.2 (95%CI 5.5-10.4) months. Outcomes appeared generally better in DRD+ vs. DRD- tumors with respect to PSA responses (33% vs. 0%; P=0.14, nonsignificant), ORR (40% vs. 0%; P=0.46, nonsignificant), PSA-PFS (HR 0.19; P<0.01, significant), PFS (HR 0.31; P=0.01, significant), and OS (HR 0.41; P=0.11, nonsignificant). There were no new safety concerns. Ipilimumab plus nivolumab demonstrated encouraging efficacy in AR-V7-positive prostate cancers with DRD mutations, but not in the overall study population.

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.

Molecular Correlates of In Vitro Responses to Dacomitinib and Afatinib in Bladder Cancer.

BACKGROUND: The HER family of proteins (EGFR, HER2, HER3 and HER4) have long been thought to be therapeutic targets for bladder cancer, but previous clinical trials targeting these proteins have been disappointing. Second generation agents may be more effective. OBJECTIVE: The aim of this study was to evaluate responses to two second-generation irreversible tyrosine kinase inhibitors, dacomitinib and afatinib, in bladder cancer cell lines. METHODS: Cell lines were characterized by targeted next generation DNA sequencing, RNA sequencing, western blotting and flow cytometry. Cell survival responses to dacomitinib or afatinib were determined using (3-[4,5-dimethylthioazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) or [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosylfate (PMS) cell survival assays. RESULTS: Only two cell lines of 12 tested were sensitive to afatinib. Sensitivity to afatinib was significantly associated with mutation in either HER2 or HER3 (p < 0.05). The two cell lines sensitive to afatinib were also responsive to dacomitinib ralong with an additional 4 other cell lines out of 16 tested. No characteristic was associated with dacomitinib sensitivity. Molecular profiling demonstrated that only two genes were high in both afatinib and dacomitinib sensitive cells. Further rhigher expression of RAS pathway genes was noted for dacomitinib responsive cells. CONCLUSIONS: This study confirms that cell line screening can be useful in pre-clinical evaluation of targeted small molecule inhibitors and suggests that compounds with similar structure(s) and target(s) may have distinct sensitivity profiles. Further rcombinational targeting of additional molecularly relevant pathways may be important in enhancing responses to HER targeted agents in bladder cancer.

Inflammation-Induced Oxidative Stress Mediates Gene Fusion Formation in Prostate Cancer.

Approximately 50% of prostate cancers are associated with gene fusions of the androgen-regulated gene TMPRSS2 to the oncogenic erythroblast transformation-specific (ETS) transcription factor ERG. The three-dimensional proximity of TMPRSS2 and ERG genes, in combination with DNA breaks, facilitates the formation of TMPRSS2-ERG gene fusions. However, the origins of DNA breaks that underlie gene fusion formation in prostate cancers are far from clear. We demonstrate a role for inflammation-induced oxidative stress in the formation of DNA breaks leading to recurrent TMPRSS2-ERG gene fusions. The transcriptional status and epigenetic features of the target genes influence this effect. Importantly, inflammation-induced de novo genomic rearrangements are blocked by homologous recombination (HR) and promoted by non-homologous end-joining (NHEJ) pathways. In conjunction with the association of proliferative inflammatory atrophy (PIA) with human prostate cancer, our results support a working model in which recurrent genomic rearrangements induced by inflammatory stimuli lead to the development of prostate cancer.

Two-pass alignment improves novel splice junction quantification.

MOTIVATION: Discovery of novel splicing from RNA sequence data remains a critical and exciting focus of transcriptomics, but reduced alignment power impedes expression quantification of novel splice junctions. RESULTS: Here, we profile performance characteristics of two-pass alignment, which separates splice junction discovery from quantification. Per sample, across a variety of transcriptome sequencing datasets, two-pass alignment improved quantification of at least 94% of simulated novel splice junctions, and provided as much as 1.7-fold deeper median read depth over those splice junctions. We further demonstrate that two-pass alignment works by increasing alignment of reads to splice junctions by short lengths, and that potential alignment errors are readily identifiable by simple classification. Taken together, two-pass alignment promises to advance quantification and discovery of novel splicing events. CONTACT: arul@med.umich.edu, nesvi@med.umich.edu AVAILABILITY AND IMPLEMENTATION: Two-pass alignment was implemented here as sequential alignment, genome indexing, and re-alignment steps with STAR. Full parameters are provided in Supplementary Table 2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptome meta-analysis of lung cancer reveals recurrent aberrations in NRG1 and Hippo pathway genes.

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyse 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumours without known driver mutations. In addition, we observe exon-skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations.