Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer.

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival. mRNA expression clustering refined prior clustering analyses and identified a poor-survival "neuronal" subtype in which the majority of tumors lacked small cell or neuroendocrine histology. Clustering by mRNA, long non-coding RNA (lncRNA), and miRNA expression converged to identify subsets with differential epithelial-mesenchymal transition status, carcinoma in situ scores, histologic features, and survival. Our analyses identified 5 expression subtypes that may stratify response to different treatments.

Identification of an early survival prognostic gene signature for localized osteosarcoma patients.

Osteosarcoma is the most prevalent bone tumor in pediatric patients. Neoadjuvant chemotherapy has improved osteosarcoma patient survival, however the 5-year survival rate for localized osteosarcoma is 75% with a 30-50% recurrence rate. We, therefore, sought to identify a prognostic gene signature which could predict poor prognosis in localized osteosarcoma patients. Using the TARGET osteosarcoma transcriptomic dataset, we identified a 13-hub gene signature associated with overall survival and time to death of localized osteosarcoma patients, with the high-risk group showing a 22% and the low-risk group showing 100% overall survival. Furthermore, network analysis identified five modules of co-expressed genes that significantly correlated with survival, and identified 65 pathways enriched across 3 modules, including Hedgehog signaling, which includes 2 of the 13 genes, IHH and GLI1. Subsequently, we demonstrated that GLI antagonists inhibited growth of a recurrent localized PDX-derived cell line with elevated IHH and GLI1 expression, but not a non-relapsed cell line with low pathway activation. Finally, we show that our signature outperforms previously reported signatures in predicting poor prognosis and death within 3 years in patients with localized osteosarcoma.

Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis.

Analysis of lung alveolar type 2 (AT2) progenitor stem cells has highlighted fundamental mechanisms that direct their differentiation into alveolar type 1 cells (AT1s) in lung repair and disease. However, microRNA (miRNA) mediated post-transcriptional mechanisms which govern this nexus remain understudied. We show here that the let-7 miRNA family serves a homeostatic role in governance of AT2 quiescence, specifically by preventing the uncontrolled accumulation of AT2 transitional cells and by promoting AT1 differentiation to safeguard the lung from spontaneous alveolar destruction and fibrosis. Using mice and organoid models with genetic ablation of let-7a1/let-7f1/let-7d cluster (let-7afd) in AT2 cells, we demonstrate prevents AT1 differentiation and results in aberrant accumulation of AT2 transitional cells in progressive pulmonary fibrosis. Integration of enhanced AGO2 UV-crosslinking and immunoprecipitation sequencing (AGO2-eCLIP) with RNA-sequencing from AT2 cells uncovered the induction of direct targets of let-7 in an oncogene feed-forward regulatory network including BACH1/EZH2 which drives an aberrant fibrotic cascade. Additional analyses by CUT&RUN-sequencing revealed loss of let-7afd hampers AT1 differentiation by eliciting aberrant histone EZH2 methylation which prevents the exit of AT2 transitional cells into terminal AT1s. This study identifies let-7 as a key gatekeeper of post-transcriptional and epigenetic chromatin signals to prevent AT2-driven pulmonary fibrosis.

Plasma protein biomarkers for primary graft dysfunction after lung transplantation: a single-center cohort analysis.

The clinical use of circulating biomarkers for primary graft dysfunction (PGD) after lung transplantation has been limited. In a prospective single-center cohort, we examined the use of plasma protein biomarkers as indicators of PGD severity and duration after lung transplantation. The study comprised 40 consecutive lung transplant patients who consented to blood sample collection immediately pretransplant and at 6, 24, 48, and 72 h after lung transplant. An expert grader determined the severity and duration of PGD and scored PGD at T0 (6 h after reperfusion), T24, T48, and T72 h post-reperfusion using the 2016 ISHLT consensus guidelines. A bead-based multiplex assay was used to measure 27 plasma proteins including cytokines, growth factors, and chemokines. Enzyme-linked immunoassay was used to measure cell injury markers including M30, M65, soluble receptor of advanced glycation end-products (sRAGE), and plasminogen activator inhibitor-1 (PAI-1). A pairwise comparisons analysis was used to assess differences in protein levels between PGD severity scores (1, 2, and 3) at T0, T24, T48, and T72 h. Sensitivity and temporal analyses were used to explore the association of protein expression patterns and PGD3 at T48-72 h (the most severe, persistent form of PGD). We used the Benjamini-Hochberg method to adjust for multiple testing. Of the 40 patients, 22 (55%) had PGD3 at some point post-transplant from T0 to T72 h; 12 (30%) had PGD3 at T48-72 h. In the pairwise comparison, we identified a robust plasma protein expression signature for PGD severity. In the sensitivity analysis, using a linear model for microarray data, we found that differential perioperative expression of IP-10, MIP1B, RANTES, IL-8, IL-1Ra, G-CSF, and PDGF-BB correlated with PGD3 development at T48-72 h (FDR < 0.1 and p < 0.05). In the temporal analysis, using linear mixed modeling with overlap weighting, we identified unique protein patterns in patients who did or did not develop PGD3 at T48-72 h. Our findings suggest that unique inflammatory protein expression patterns may be informative of PGD severity and duration. PGD biomarker panels may improve early detection of PGD, predict its clinical course, and help monitor treatment efficacy in the current era of lung transplantation.

Interchromosomal template-switching as a novel molecular mechanism for imprinting perturbations associated with Temple syndrome.

BACKGROUND: Intrachromosomal triplications (TRP) can contribute to disease etiology via gene dosage effects, gene disruption, position effects, or fusion gene formation. Recently, post-zygotic de novo triplications adjacent to copy-number neutral genomic intervals with runs of homozygosity (ROH) have been shown to result in uniparental isodisomy (UPD). The genomic structure of these complex genomic rearrangements (CGRs) shows a consistent pattern of an inverted triplication flanked by duplications (DUP-TRP/INV-DUP) formed by an iterative DNA replisome template-switching mechanism during replicative repair of a single-ended, double-stranded DNA (seDNA), the ROH results from an interhomolog or nonsister chromatid template switch. It has been postulated that these CGRs may lead to genetic abnormalities in carriers due to dosage-sensitive genes mapping within the copy-number variant regions, homozygosity for alleles at a locus causing an autosomal recessive (AR) disease trait within the ROH region, or imprinting-associated diseases. METHODS: Here, we report a family wherein the affected subject carries a de novo 2.2-Mb TRP followed by 42.2 Mb of ROH and manifests clinical features overlapping with those observed in association with chromosome 14 maternal UPD (UPD(14)mat). UPD(14)mat can cause clinical phenotypic features enabling a diagnosis of Temple syndrome. This CGR was then molecularly characterized by high-density custom aCGH, genome-wide single-nucleotide polymorphism (SNP) and methylation arrays, exome sequencing (ES), and the Oxford Nanopore long-read sequencing technology. RESULTS: We confirmed the postulated DUP-TRP/INV-DUP structure by multiple orthogonal genomic technologies in the proband. The methylation status of known differentially methylated regions (DMRs) on chromosome 14 revealed that the subject shows the typical methylation pattern of UPD(14)mat. Consistent with these molecular findings, the clinical features overlap with those observed in Temple syndrome, including speech delay. CONCLUSIONS: These data provide experimental evidence that, in humans, triplication can lead to segmental UPD and imprinting disease. Importantly, genotype/phenotype analyses further reveal how a post-zygotically generated complex structural variant, resulting from a replication-based mutational mechanism, contributes to expanding the clinical phenotype of known genetic syndromes. Mechanistically, such events can distort transmission genetics resulting in homozygosity at a locus for which only one parent is a carrier as well as cause imprinting diseases.

Comparative Molecular Analysis of Gastrointestinal Adenocarcinomas.

We analyzed 921 adenocarcinomas of the esophagus, stomach, colon, and rectum to examine shared and distinguishing molecular characteristics of gastrointestinal tract adenocarcinomas (GIACs). Hypermutated tumors were distinct regardless of cancer type and comprised those enriched for insertions/deletions, representing microsatellite instability cases with epigenetic silencing of MLH1 in the context of CpG island methylator phenotype, plus tumors with elevated single-nucleotide variants associated with mutations in POLE. Tumors with chromosomal instability were diverse, with gastroesophageal adenocarcinomas harboring fragmented genomes associated with genomic doubling and distinct mutational signatures. We identified a group of tumors in the colon and rectum lacking hypermutation and aneuploidy termed genome stable and enriched in DNA hypermethylation and mutations in KRAS, SOX9, and PCBP1.

A Pan-Cancer Analysis Reveals High-Frequency Genetic Alterations in Mediators of Signaling by the TGF-ß Superfamily.

We present an integromic analysis of gene alterations that modulate transforming growth factor ß (TGF-ß)-Smad-mediated signaling in 9,125 tumor samples across 33 cancer types in The Cancer Genome Atlas (TCGA). Focusing on genes that encode mediators and regulators of TGF-ß signaling, we found at least one genomic alteration (mutation, homozygous deletion, or amplification) in 39% of samples, with highest frequencies in gastrointestinal cancers. We identified mutation hotspots in genes that encode TGF-ß ligands (BMP5), receptors (TGFBR2, AVCR2A, and BMPR2), and Smads (SMAD2 and SMAD4). Alterations in the TGF-ß superfamily correlated positively with expression of metastasis-associated genes and with decreased survival. Correlation analyses showed the contributions of mutation, amplification, deletion, DNA methylation, and miRNA expression to transcriptional activity of TGF-ß signaling in each cancer type. This study provides a broad molecular perspective relevant for future functional and therapeutic studies of the diverse cancer pathways mediated by the TGF-ß superfamily.

A Comprehensive Pan-Cancer Molecular Study of Gynecologic and Breast Cancers.

We analyzed molecular data on 2,579 tumors from The Cancer Genome Atlas (TCGA) of four gynecological types plus breast. Our aims were to identify shared and unique molecular features, clinically significant subtypes, and potential therapeutic targets. We found 61 somatic copy-number alterations (SCNAs) and 46 significantly mutated genes (SMGs). Eleven SCNAs and 11 SMGs had not been identified in previous TCGA studies of the individual tumor types. We found functionally significant estrogen receptor-regulated long non-coding RNAs (lncRNAs) and gene/lncRNA interaction networks. Pathway analysis identified subtypes with high leukocyte infiltration, raising potential implications for immunotherapy. Using 16 key molecular features, we identified five prognostic subtypes and developed a decision tree that classified patients into the subtypes based on just six features that are assessable in clinical laboratories.

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas.

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smoking and/or human papillomavirus (HPV). SCCs harbor 3q, 5p, and other recurrent chromosomal copy-number alterations (CNAs), DNA mutations, and/or aberrant methylation of genes and microRNAs, which are correlated with the expression of multi-gene programs linked to squamous cell stemness, epithelial-to-mesenchymal differentiation, growth, genomic integrity, oxidative damage, death, and inflammation. Low-CNA SCCs tended to be HPV(+) and display hypermethylation with repression of TET1 demethylase and FANCF, previously linked to predisposition to SCC, or harbor mutations affecting CASP8, RAS-MAPK pathways, chromatin modifiers, and immunoregulatory molecules. We uncovered hypomethylation of the alternative promoter that drives expression of the ΔNp63 oncogene and embedded miR944. Co-expression of immune checkpoint, T-regulatory, and Myeloid suppressor cells signatures may explain reduced efficacy of immune therapy. These findings support possibilities for molecular classification and therapeutic approaches.

The Cancer Genome Atlas Comprehensive Molecular Characterization of Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is not a single disease, but several histologically defined cancers with different genetic drivers, clinical courses, and therapeutic responses. The current study evaluated 843 RCC from the three major histologic subtypes, including 488 clear cell RCC, 274 papillary RCC, and 81 chromophobe RCC. Comprehensive genomic and phenotypic analysis of the RCC subtypes reveals distinctive features of each subtype that provide the foundation for the development of subtype-specific therapeutic and management strategies for patients affected with these cancers. Somatic alteration of BAP1, PBRM1, and PTEN and altered metabolic pathways correlated with subtype-specific decreased survival, while CDKN2A alteration, increased DNA hypermethylation, and increases in the immune-related Th2 gene expression signature correlated with decreased survival within all major histologic subtypes. CIMP-RCC demonstrated an increased immune signature, and a uniform and distinct metabolic expression pattern identified a subset of metabolically divergent (MD) ChRCC that associated with extremely poor survival.