Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles.

Aberrant expression of ETS transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS::FLI1 and EWS::ERG fusions in Ewing sarcoma (EwS) and TMPRSS2::ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS::FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs) and are highly elevated in plasma of EwS patients with metastatic disease. High HSAT2,3 levels in EWS::FLI1 or ERG expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs which transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

Neuropathological Applications of Microscopy with Ultraviolet Surface Excitation (MUSE): A Concordance Study of Human Primary and Metastatic Brain Tumors.

Whereas traditional histology and light microscopy require multiple steps of formalin fixation, paraffin embedding, and sectioning to generate images for pathologic diagnosis, Microscopy using Ultraviolet Surface Excitation (MUSE) operates through UV excitation on the cut surface of tissue, generating images of high resolution without the need to fix or section tissue and allowing for potential use for downstream molecular tests. Here, we present the first study of the use and suitability of MUSE microscopy for neuropathological samples. MUSE images were generated from surgical biopsy samples of primary and metastatic brain tumor biopsy samples (n = 27), and blinded assessments of diagnoses, tumor grades, and cellular features were compared to corresponding hematoxylin and eosin (H&E) images. A set of MUSE-treated samples subsequently underwent exome and targeted sequencing, and quality metrics were compared to those from fresh frozen specimens. Diagnostic accuracy was relatively high, and DNA and RNA integrity appeared to be preserved for this cohort. This suggests that MUSE may be a reliable method of generating high-quality diagnostic-grade histologic images for neuropathology on a rapid and sample-sparing basis and for subsequent molecular analysis of DNA and RNA.

Transcriptomic classes of BCR-ABL1 lymphoblastic leukemia.

In BCR-ABL1 lymphoblastic leukemia, treatment heterogeneity to tyrosine kinase inhibitors (TKIs), especially in the absence of kinase domain mutations in BCR-ABL1, is poorly understood. Through deep molecular profiling, we uncovered three transcriptomic subtypes of BCR-ABL1 lymphoblastic leukemia, each representing a maturation arrest at a stage of B-cell progenitor differentiation. An earlier arrest was associated with lineage promiscuity, treatment refractoriness and poor patient outcomes. A later arrest was associated with lineage fidelity, durable leukemia remissions and improved patient outcomes. Each maturation arrest was marked by specific genomic events that control different transition points in B-cell development. Interestingly, these events were absent in BCR-ABL1+ preleukemic stem cells isolated from patients regardless of subtype, which supports that transcriptomic phenotypes are determined downstream of the leukemia-initialing event. Overall, our data indicate that treatment response and TKI efficacy are unexpected outcomes of the differentiation stage at which this leukemia transforms.

Dr.Nod: computational framework for discovery of regulatory non-coding drivers in tissue-matched distal regulatory elements.

The discovery of cancer driver mutations is a fundamental goal in cancer research. While many cancer driver mutations have been discovered in the protein-coding genome, research into potential cancer drivers in the non-coding regions showed limited success so far. Here, we present a novel comprehensive framework Dr.Nod for detection of non-coding cis-regulatory candidate driver mutations that are associated with dysregulated gene expression using tissue-matched enhancer-gene annotations. Applying the framework to data from over 1500 tumours across eight tissues revealed a 4.4-fold enrichment of candidate driver mutations in regulatory regions of known cancer driver genes. An overarching conclusion that emerges is that the non-coding driver mutations contribute to cancer by significantly altering transcription factor binding sites, leading to upregulation of tissue-matched oncogenes and down-regulation of tumour-suppressor genes. Interestingly, more than half of the detected cancer-promoting non-coding regulatory driver mutations are over 20 kb distant from the cancer-associated genes they regulate. Our results show the importance of tissue-matched enhancer-gene maps, functional impact of mutations, and complex background mutagenesis model for the prediction of non-coding regulatory drivers. In conclusion, our study demonstrates that non-coding mutations in enhancers play a previously underappreciated role in cancer and dysregulation of clinically relevant target genes.

SCALP syndrome with a germline heterozygous DOCK6 mutation and somatic mosaic NRAS Q61R mutation.

We present a case of SCALP syndrome, which was diagnosed in a male infant with the characteristic findings of sebaceous nevi, central nervous system malformations, aplasia cutis congenita, limbal dermoid, and giant congenital melanocytic nevi, or pigmented nevi. We identified a germline compound heterozygous DOCK6 mutation and a somatic mosaic NRAS Q61R mutation in the giant congenital melanocytic nevus. This report will increase clinician awareness of SCALP syndrome and augment the literature in characterizing this rare syndrome, including its genetic background.

A distinct subpopulation of leukemia initiating cells in acute precursor B lymphoblastic leukemia: quiescent phenotype and unique transcriptomic profile.

In leukemia, a distinct subpopulation of cancer-initiating cells called leukemia stem cells (LSCs) is believed to drive population expansion and tumor growth. Failing to eliminate LSCs may result in disease relapse regardless of the amount of non-LSCs destroyed. The first step in targeting and eliminating LSCs is to identify and characterize them. Acute precursor B lymphoblastic leukemia (B-ALL) cells derived from patients were incubated with fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (NBDG) and sorted based on NBDG uptake. Cell subpopulations defined by glucose uptake were then serially transplanted into mice and evaluated for leukemia initiating capacity. Gene expression profiles of these cells were characterized using RNA-Sequencing (RNA-Seq). A distinct population of NBDG-low cells was identified in patient B-ALL samples. These cells are a small population (1.92% of the entire leukemia population), have lower HLA expression, and are smaller in size (4.0 to 7.0 µm) than the rest of the leukemia population. All mice transplanted with NBDG-low cells developed leukemia between 5 and 14 weeks, while those transplanted with NBDG-high cells did not develop leukemia (p ≤ 0.0001-0.002). Serial transplantation of the NBDG-low mouse model resulted in successful leukemia development. NBDG-medium (NBDG-med) populations also developed leukemia. Interestingly, comprehensive molecular characterization of NBDG-low and NBDG-med cells from patient-derived xenograft (PDX) models using RNA-Seq revealed a distinct profile of 2,162 differentially-expressed transcripts (DETs) (p<0.05) with 70.6% down-regulated in NBDG-low cells. Hierarchical clustering of DETs showed distinct segregation of NBDG-low from NBDG-med and NBDG-high groups with marked transcription expression alterations in the NBDG-low group consistent with cancer survival. In conclusion, A unique subpopulation of cells with low glucose uptake (NBDG-low) in B-ALL was discovered. These cells, despite their quiescence characteristics, once transplanted in mice, showed potent leukemia initiating capacity. Although NBDG-med cells also initiated leukemia, gene expression profiling revealed a distinct signature that clearly distinguishes NBDG-low cells from NBDG-med and the rest of the leukemia populations. These results suggest that NBDG-low cells may represent quiescent LSCs. These cells can be activated in the appropriate environment in vivo, showing leukemia initiating capacity. Our study provides insight into the biologic mechanisms of B-ALL initiation and survival.

Environmental pesticide exposure and non-Hodgkin lymphoma survival: a population-based study.

BACKGROUND: There is evidence indicating that pesticide exposure is a risk factor for non-Hodgkin lymphoma (NHL) development. However, the association between pesticide exposure and NHL survival is not well-established. METHODS: Using the California Cancer Registry, we identified patients with a first primary diagnosis of NHL from 2010 to 2016 and linked these patients with CalEnviroScreen 3.0 to obtain production agriculture pesticide exposure to 70 chemicals from the state-mandated Pesticide Use Reporting (PUR) by census tract from 2012 to 2014. In addition, data from PUR was integrated into a geographic information system that employs land-use data to estimate cumulative exposure to specific pesticides previously associated with NHL (glyphosate, organophosphorus, carbamate, phenoxyherbicide, and 2,4-dimethylamine salt) between 10 years prior up to 1 year after NHL diagnosis. Multivariable Cox proportional hazards regression models were used to evaluate the association between total pesticide exposure from CalEnviroScreen 3.0 and individual pesticide exposure from geographic land use data and lymphoma-specific and overall survival. RESULTS: Among 35,808 NHL patients identified, 44.2% were exposed to pesticide in their census tract of residence. Glyphosate, organophosphorus, carbamate, phenoxyherbicide, and 2,4-dimethylamine salt exposure was observed in 34.1%, 26.0%, 10.6%, 14.0%, and 12.8% of NHL patients, respectively. Total pesticide exposure at the time of diagnosis was not associated with lymphoma-specific or overall survival. In addition, no association was consistently found between glyphosate, organophosphorus, carbamate, phenoxyherbicide, and 2,4 dimethylamine salt exposure and lymphoma-specific or overall survival. CONCLUSIONS: Although we found no consistent associations between agricultural pesticide exposure at the neighborhood level and worse survival, these results provide a platform for designing future studies to determine the association between pesticide and NHL.

High-Plex Spatial RNA Profiling Reveals Cell Type‒Specific Biomarker Expression during Melanoma Development.

Early diagnosis of melanoma is critical for improved survival. However, the biomarkers of early melanoma evolution and their origin within the tumor and its microenvironment, including the keratinocytes, are poorly defined. To address this, we used spatial transcript profiling that maintains the morphological tumor context to measure the expression of >1,000 RNAs in situ in patient-derived formalin-fixed, paraffin-embedded tissue sections in primary melanoma and melanocytic nevi. We profiled 134 regions of interest (each 200 µm in diameter) enriched in melanocytes, neighboring keratinocytes, or immune cells. This approach captured distinct expression patterns across cell types and tumor types during melanoma development. Unexpectedly, we discovered that S100A8 is expressed by keratinocytes within the tumor microenvironment during melanoma growth. Immunohistochemistry of 252 tumors showed prominent keratinocyte-derived S100A8 expression in melanoma but not in benign tumors and confirmed the same pattern for S100A8's binding partner S100A9, suggesting that injury to the epidermis may be an early and readily detectable indicator of melanoma development. Together, our results establish a framework for high-plex, spatial, and cell type‒specific resolution of gene expression in archival tissue applicable to the development of biomarkers and characterization of tumor microenvironment interactions in tumor evolution.

Whole exome sequencing of a gut-associated lymphoid tissue neoplasm points to precursor or early form of sporadic colon carcinoma.

Gut-associated lymphoid tissue (GALT) carcinoma is a colorectal neoplasm characterized by cystically dilated neoplastic glands that extend into prominent, well-circumscribed submucosal lymphoid tissue. Although often subtle, lamina propria between and around the neoplastic glands (identified by plasma cells, scattered eosinophils, etc.) is frequent in cases with classic morphology, arguing (at least in such cases) in favor of adenoma extending into lymphoglandular complexes rather than true invasive carcinoma. Some have postulated that the tumor arises from M-cells, specialized epithelial cells overlying GALT, and others have suggested it represents a unique pathway to carcinoma, specific to the environmental conditions of epithelium overlying lymphoid tissue. Although both hypotheses are intriguing, definitive phenotypic and genetic support is currently lacking. To address these possibilities, we undertook whole exome sequencing and immunohistochemical characterization of a GALT neoplasm recently identified on our clinical service. We discovered well-known mutations in both APC and KRAS, as well as mutations in several Wnt pathway components (MED12, BCL9L, RFX4, DACT3). No immunohistochemical expression of GP2, a marker of M-cell differentiation, was identified. Expression of CDX2, SATB2, and the DNA mismatch repair proteins was observed, while expression of both CK7 and CK20 was absent. No PD-L1 expression was present on tumor cells, but PD-L1 expression was noted in a subset of tumor-adjacent mononuclear cells. Overall, the findings suggest that GALT neoplasms, although morphologically distinct, may be a precursor or early form of typical sporadic colon carcinoma.

Landscape of somatic single nucleotide variants and indels in colorectal cancer and impact on survival.

Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR = 0.42, 95% CI: 0.21-0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR = 1.53, 95% CI: 1.21-1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features.

Germline and somatic DNA repair gene alterations in prostate cancer.

BACKGROUND: Emerging evidence has suggested that DNA repair gene alterations may be important in prostate cancer pathogenesis. In the current study, the authors sought to characterize alterations in DNA repair pathway genes in both primary and metastatic prostate tumors with attention to tissue distribution as well as specific genomic alterations. METHODS: The authors studied the distribution and type of alterations in 24 genes that are considered important for DNA repair in 944 prostate cancers harvested from localized and metastatic tumors. Tumor DNA underwent hybrid capture for all coding exons of 287 or 395 cancer-related genes plus select introns from 19 or 31 genes frequently rearranged in cancer. Captured libraries were sequenced to a median exon coverage depth of >×500. Specific genomic alterations were characterized and the frequencies of mutations by tissue site (prostate vs metastases) were compared using logistic regression. RESULTS: A total of 152 patients from the cohort of 944 men (16%) harbored a germline or somatic mutation in ≥1 DNA repair genes. The most frequently mutated genes were BRCA2 (11.4%) and ATM (5.8%), followed by MSH6 (2.5%) and MSH2 (2.1%). Mutations were identified in approximately 20.1% of primary prostate tumors compared with 18.8% of bone metastases. When stratified by tissue site, the highest rates of DNA repair mutations were found in solid organ metastases, including brain and visceral metastases, compared with prostate. CONCLUSIONS: DNA repair gene mutations are more common in metastatic than localized prostate tumors. Visceral and other solid organ metastases appear enriched for these mutations compared with localized tumors or bone and lymph node metastases.

Genome-wide germline correlates of the epigenetic landscape of prostate cancer.

Oncogenesis is driven by germline, environmental and stochastic factors. It is unknown how these interact to produce the molecular phenotypes of tumors. We therefore quantified the influence of germline polymorphisms on the somatic epigenome of 589 localized prostate tumors. Predisposition risk loci influence a tumor's epigenome, uncovering a mechanism for cancer susceptibility. We identified and validated 1,178 loci associated with altered methylation in tumoral but not nonmalignant tissue. These tumor methylation quantitative trait loci influence chromatin structure, as well as RNA and protein abundance. One prominent tumor methylation quantitative trait locus is associated with AKT1 expression and is predictive of relapse after definitive local therapy in both discovery and validation cohorts. These data reveal intricate crosstalk between the germ line and the epigenome of primary tumors, which may help identify germline biomarkers of aggressive disease to aid patient triage and optimize the use of more invasive or expensive diagnostic assays.

Comparative toxicoproteogenomics of mouse and rat liver identifies TCDD-resistance genes.

The aryl hydrocarbon receptor (AHR) mediates many toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone does not explain the widely different outcomes among organisms. To identify the other factors involved, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS) providing widely different resistance to TCDD toxicity, as well as C57BL/6 and DBA/2 mice which exhibit a ~ tenfold divergence in TCDD sensitivity (exposures of 5-1000 µg/kg TCDD). We supplement these with whole-genome sequencing, together with transcriptomic and proteomic analyses of the corresponding rat models, Long-Evans (L-E) and Han/Wistar (H/W) rats (having a ~ 1000-fold difference in their TCDD sensitivities; 100 µg/kg TCDD), to identify genes associated with TCDD-response phenotypes. Overall, we identified up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (33.8%, 11.7%, 5.2% and 0.3% of 3076 genes altered unique to rWT, DEL, C57BL/6 and INS respectively following 1000 µg/kg TCDD). Hepatic Pxdc1 was significantly repressed in all three TCDD-sensitive animal models (C57BL/6 and rWT mice, and L-E rat) after TCDD exposure. Three genes, including Cxxc5, Sugp1 and Hgfac, demonstrated different AHRE-1 (full) motif occurrences within their promoter regions between rat strains, as well as different patterns of mRNA abundance. Several hepatic proteins showed parallel up- or downward alterations with their RNAs, with three genes (SNRK, IGTP and IMPA2) showing consistent, strain-dependent changes. These data show the value of integrating genomic, transcriptomic and proteomic evidence across multi-species models in toxicologic studies.

Regional perturbation of gene transcription is associated with intrachromosomal rearrangements and gene fusion transcripts in high grade ovarian cancer.

Genomic rearrangements are a hallmark of cancer biology and progression, allowing cells to rapidly transform through alterations in regulatory structures, changes in expression patterns, reprogramming of signaling pathways, and creation of novel transcripts via gene fusion events. Though functional gene fusions encoding oncogenic proteins are the most dramatic outcomes of genomic rearrangements, we investigated the relationship between rearrangements evidenced by fusion transcripts and local expression changes in cancer using transcriptome data alone. 9,953 gene fusion predictions from 418 primary serious ovarian cancer tumors were analyzed, identifying depletions of gene fusion breakpoints within coding regions of fused genes as well as an N-terminal enrichment of breakpoints within fused genes. We identified 48 genes with significant fusion-associated upregulation and furthermore demonstrate that significant regional overexpression of intact genes in patient transcriptomes occurs within 1 megabase of 78 novel gene fusions that function as central markers of these regions. We reveal that cancer transcriptomes select for gene fusions that preserve protein and protein domain coding potential. The association of gene fusion transcripts with neighboring gene overexpression supports rearrangements as mechanism through which cancer cells remodel their transcriptomes and identifies a new way to utilize gene fusions as indicators of regional expression changes in diseased cells with only transcriptomic data.

Activation of hedgehog signaling associates with early disease progression in chronic lymphocytic leukemia.

Targeted sequencing of 103 leukemia-associated genes in leukemia cells from 841 treatment-naive patients with chronic lymphocytic leukemia (CLL) identified 89 (11%) patients as having CLL cells with mutations in genes encoding proteins that putatively are involved in hedgehog (Hh) signaling. Consistent with this finding, there was a significant association between the presence of these mutations and the expression of GLI1 (χ2 test, P < .0001), reflecting activation of the Hh pathway. However, we discovered that 38% of cases without identified mutations also were GLI1+ Patients with GLI1+ CLL cells had a shorter median treatment-free survival than patients with CLL cells lacking expression of GLI1 independent of IGHV mutation status. We found that GANT61, a small molecule that can inhibit GLI1, was highly cytotoxic for GLI1+ CLL cells relative to that of CLL cells without GLI1. Collectively, this study shows that a large proportion of patients have CLL cells with activated Hh signaling, which is associated with early disease progression and enhanced sensitivity to inhibition of GLI1.

Future Promises and Concerns of Ubiquitous Next-Generation Sequencing.

Since the first draft of the human genome was completed, next-generation DNA sequencing technology has dramatically reduced the cost of sequencing a genome. Computational analysis has not advanced as fast as the instruments that generate the data, and storing all the data remains a challenge. Nevertheless, personal genomics has arrived and is already being used in the clinic. Significant privacy issues remain, however, and these are not widely understood. The Genetic Information Non-Discrimination Act (GINA) needs to be extended and the probabilistic nature of genetic predisposition must be better explained to both the public and physicians. We must also be wary that this promising new technology and its applications do not amplify existing healthcare disparities.