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Treatment of locally advanced rectal cancer includes chemotherapy, radiation, and surgery but patient responses to neoadjuvant treatment are variable. We have shown that rectal tumors are comprised of multiple genetically distinct sub-clones. Unique sub-clones within tumors may harbor mutations which contribute to inter-patient variation in response to neoadjuvant chemoradiotherapy (nCRT). Analysis of the influence of nCRT on the extent and nature of intra-tumoral genetic heterogeneity in rectal cancer may provide insights into mechanisms of resistance.Locally advanced rectal cancer patients underwent pre-treatment biopsies. At the time of surgery, tissue from the treated tumor was obtained and analyzed. Pre- and post-treatment specimens were subjected to whole exome and confirmatory deep sequencing for somatic mutations. Copy number variation was assessed using OncoScan SNP arrays. Genomic data were analyzed using PyClone to identify sub-clonal tumor population following nCRT. Alterations that persisted or were enriched in the post-treatment tumor specimen following nCRT were defined for each patient.Thirty-two samples were obtained from ten patients. PyClone identified 2 to 10 genetic sub-clones per tumor. Substantial changes in the proportions of individual sub-clones in pre- versus post-treatment tumor material were found in all patients. Resistant sub-clones recurrently contained mutations in TP53, APC, ABCA13, MUC16, and THSD4. Recurrent copy number variation was observed across multiple chromosome regions after nCRT. Pathway analysis including variant alleles and copy number changes associated with resistant sub-clones revealed significantly altered pathways, especially those linked to the APC and TP53 genes, which were the two most frequently mutated genes.Intra-tumoral heterogeneity is evident in pre-treatment rectal cancer. Following treatment, sub-clonal populations are selectively modified and enrichment of a subset of pre-treatment sub-clones is seen. Further studies are needed to define recurrent alterations at diagnosis that may contribute to resistance to nCRT.

Background: Half of muscle-invasive bladder cancer patients will relapse with metastatic disease and molecular tests to predict relapse are needed. TP63 has been proposed as a prognostic biomarker in bladder cancer, but reports associating it with clinical outcomes are conflicting. Since TP63 is expressed as multiple isoforms, we hypothesized that these conflicting associations with clinical outcome may be explained by distinct opposing effects of differential TP63 isoform expression. Methods: Using RNA-Seq data from The Cancer Genome Atlas (TCGA), TP63 isoform-level expression was quantified and associated with clinical covariates (e.g. survival, stage) across 8,519 patients from 29 diseases. A comprehensive catalog of TP63 isoforms was assembled using gene annotation databases and de novo discovery in bladder cancer patients. Quantifications and un-annotated TP63 isoforms were validated using quantitative RT-PCR and a separate bladder cancer cohort. Findings: DNp63 isoform expression was associated with improved bladder cancer patient survival in patients with a luminal subtype (HR = 0.89, CI 0.80–0.99, Cox p = 0.034). Conversely, TAp63 isoform expression was associated with reduced bladder cancer patient survival in patients with a basal subtype (HR = 2.35, CI 1.64–3.37, Cox p < 0.0001). These associations were observed in multiple TCGA disease cohorts and correlated with epidermal differentiation (DNp63) and immune-related (TAp63) gene signatures. Interpretation: These results comprehensively define TP63 isoform expression in human cancer and suggest that TP63 isoforms are involved in distinct transcriptional programs with opposing effects on clinical outcome. Keywords: TP63, Isoforms, Cancer, Bladder Cancer, Biomarker