Disentangling oncogenic amplicons in esophageal adenocarcinoma.

Esophageal adenocarcinoma is a prominent example of cancer characterized by frequent amplifications in oncogenes. However, the mechanisms leading to amplicons that involve breakage-fusion-bridge cycles and extrachromosomal DNA are poorly understood. Here, we use 710 esophageal adenocarcinoma cases with matched samples and patient-derived organoids to disentangle complex amplicons and their associated mechanisms. Short-read sequencing identifies ERBB2, MYC, MDM2, and HMGA2 as the most frequent oncogenes amplified in extrachromosomal DNAs. We resolve complex extrachromosomal DNA and breakage-fusion-bridge cycles amplicons by integrating of de-novo assemblies and DNA methylation in nine long-read sequenced cases. Complex amplicons shared between precancerous biopsy and late-stage tumor, an enrichment of putative enhancer elements and mobile element insertions are potential drivers of complex amplicons' origin. We find that patient-derived organoids recapitulate extrachromosomal DNA observed in the primary tumors and single-cell DNA sequencing capture extrachromosomal DNA-driven clonal dynamics across passages. Prospectively, long-read and single-cell DNA sequencing technologies can lead to better prediction of clonal evolution in esophageal adenocarcinoma.

Understanding the malignant potential of gastric metaplasia of the oesophagus and its relevance to Barrett's oesophagus surveillance: individual-level data analysis.

OBJECTIVE: Whether gastric metaplasia (GM) of the oesophagus should be considered as Barrett's oesophagus (BO) is controversial. Given concern intestinal metaplasia (IM) may be missed due to sampling, the UK guidelines include GM as a type of BO. Here, we investigated whether the risk of misdiagnosis and the malignant potential of GM warrant its place in the UK surveillance. DESIGN: We performed a thorough pathology and endoscopy review to follow clinical outcomes in a novel UK cohort of 244 patients, covering 1854 person years of follow-up. We complemented this with a comparative genomic analysis of 160 GM and IM specimens, focused on early molecular hallmarks of BO and oesophageal adenocarcinoma (OAC). RESULTS: We found that 58 of 77 short-segment (<3 cm) GM (SS-GM) cases (75%) continued to be observed as GM-only across a median of 4.4 years of follow-up. We observed that disease progression in GM-only cases and GM+IM cases (cases with reported GM on some occasions, IM on others) was significantly lower than in the IM-only cases (Kaplan-Meier, p=0.03). Genomic analysis revealed that the mutation burden in GM is significantly lower than in IM (p<0.01). Moreover, GM does not bear the mutational hallmarks of OAC, with an absence of associated signatures and driver gene mutations. Finally, we established that GM found adjacent to OAC is evolutionarily distant from cancer. CONCLUSION: SS-GM is a distinct entity from SS-IM and the malignant potential of GM is lower than IM. It is questionable whether SS-GM warrants inclusion in BO surveillance.

Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1-6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

Global Epidemiology and Genetics of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading cancers worldwide. Classically, HCC develops in genetically susceptible individuals who are exposed to risk factors, especially in the presence of liver cirrhosis. Significant temporal and geographic variations exist for HCC and its etiologies. Over time, the burden of HCC has shifted from the low-moderate to the high sociodemographic index regions, reflecting the transition from viral to nonviral causes. Geographically, the hepatitis viruses predominate as the causes of HCC in Asia and Africa. Although there are genetic conditions that confer increased risk for HCC, these diagnoses are rarely recognized outside North America and Europe. In this review, we will evaluate the epidemiologic trends and risk factors of HCC, and discuss the genetics of HCC, including monogenic diseases, single-nucleotide polymorphisms, gut microbiome, and somatic mutations.

Rearrangement processes and structural variations show evidence of selection in oesophageal adenocarcinomas.

Oesophageal adenocarcinoma (OAC) provides an ideal case study to characterize large-scale rearrangements. Using whole genome short-read sequencing of 383 cases, for which 214 had matched whole transcriptomes, we observed structural variations (SV) with a predominance of deletions, tandem duplications and inter-chromosome junctions that could be identified as LINE-1 mobile element (ME) insertions. Complex clusters of rearrangements resembling breakage-fusion-bridge cycles or extrachromosomal circular DNA accounted for 22% of complex SVs affecting known oncogenes. Counting SV events affecting known driver genes substantially increased the recurrence rates of these drivers. After excluding fragile sites, we identified 51 candidate new drivers in genomic regions disrupted by SVs, including ETV5, KAT6B and CLTC. RUNX1 was the most recurrently altered gene (24%), with many deletions inactivating the RUNT domain but preserved the reading frame, suggesting an altered protein product. These findings underscore the importance of identification of SV events in OAC with implications for targeted therapies.

Accuracy of mutational signature software on correlated signatures.

Mutational signatures are characteristic patterns of mutations generated by exogenous mutagens or by endogenous mutational processes. Mutational signatures are important for research into DNA damage and repair, aging, cancer biology, genetic toxicology, and epidemiology. Unsupervised learning can infer mutational signatures from the somatic mutations in large numbers of tumors, and separating correlated signatures is a notable challenge for this task. To investigate which methods can best meet this challenge, we assessed 18 computational methods for inferring mutational signatures on 20 synthetic data sets that incorporated varying degrees of correlated activity of two common mutational signatures. Performance varied widely, and four methods noticeably outperformed the others: hdp (based on hierarchical Dirichlet processes), SigProExtractor (based on multiple non-negative matrix factorizations over resampled data), TCSM (based on an approach used in document topic analysis), and mutSpec.NMF (also based on non-negative matrix factorization). The results underscored the complexities of mutational signature extraction, including the importance and difficulty of determining the correct number of signatures and the importance of hyperparameters. Our findings indicate directions for improvement of the software and show a need for care when interpreting results from any of these methods, including the need for assessing sensitivity of the results to input parameters.

Functional genomics identifies specific vulnerabilities in PTEN-deficient breast cancer.

BACKGROUND: Phosphatase and tensin homolog (PTEN) is one of the most frequently inactivated tumor suppressors in breast cancer. While PTEN itself is not considered a druggable target, PTEN synthetic-sick or synthetic-lethal (PTEN-SSL) genes are potential drug targets in PTEN-deficient breast cancers. Therefore, with the aim of identifying potential targets for precision breast cancer therapy, we sought to discover PTEN-SSL genes present in a broad spectrum of breast cancers. METHODS: To discover broad-spectrum PTEN-SSL genes in breast cancer, we used a multi-step approach that started with (1) a genome-wide short interfering RNA (siRNA) screen of ~ 21,000 genes in a pair of isogenic human mammary epithelial cell lines, followed by (2) a short hairpin RNA (shRNA) screen of ~ 1200 genes focused on hits from the first screen in a panel of 11 breast cancer cell lines; we then determined reproducibility of hits by (3) identification of overlaps between our results and reanalyzed data from 3 independent gene-essentiality screens, and finally, for selected candidate PTEN-SSL genes we (4) confirmed PTEN-SSL activity using either drug sensitivity experiments in a panel of 19 cell lines or mutual exclusivity analysis of publicly available pan-cancer somatic mutation data. RESULTS: The screens (steps 1 and 2) and the reproducibility analysis (step 3) identified six candidate broad-spectrum PTEN-SSL genes (PIK3CB, ADAMTS20, AP1M2, HMMR, STK11, and NUAK1). PIK3CB was previously identified as PTEN-SSL, while the other five genes represent novel PTEN-SSL candidates. Confirmation studies (step 4) provided additional evidence that NUAK1 and STK11 have PTEN-SSL patterns of activity. Consistent with PTEN-SSL status, inhibition of the NUAK1 protein kinase by the small molecule drug HTH-01-015 selectively impaired viability in multiple PTEN-deficient breast cancer cell lines, while mutations affecting STK11 and PTEN were largely mutually exclusive across large pan-cancer data sets. CONCLUSIONS: Six genes showed PTEN-SSL patterns of activity in a large proportion of PTEN-deficient breast cancer cell lines and are potential specific vulnerabilities in PTEN-deficient breast cancer. Furthermore, the NUAK1 PTEN-SSL vulnerability identified by RNA interference techniques can be recapitulated and exploited using the small molecule kinase inhibitor HTH-01-015. Thus, NUAK1 inhibition may be an effective strategy for precision treatment of PTEN-deficient breast tumors.

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analyzed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations; conversely, fluke-negative CCAs (clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3' untranslated region deletion as a mechanism of FGFR2 upregulation. Integration of noncoding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores-mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics, and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.Significance: Integrated whole-genome and epigenomic analysis of CCA on an international scale identifies new CCA driver genes, noncoding promoter mutations, and structural variants. CCA molecular landscapes differ radically by etiology, underscoring how distinct cancer subtypes in the same organ may arise through different extrinsic and intrinsic carcinogenic processes. Cancer Discov; 7(10); 1116-35. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Genome-scale mutational signatures of aflatoxin in cells, mice, and human tumors.

Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene-this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence.

The Drosophila Dicer-1 Partner Loquacious Enhances miRNA Processing from Hairpins with Unstable Structures at the Dicing Site.

In Drosophila, Dicer-1 binds Loquacious-PB (Loqs-PB) as its major co-factor. Previous analyses indicated that loqs mutants only partially impede miRNA processing, but the activity of minor isoforms or maternally deposited Loqs was not eliminated in these studies. We addressed this by generating a cell line from loqs-null embryos and found that only ∼40% of miRNAs showed clear Loqs dependence. Genome-wide comparison of the hairpin structure and Loqs dependence suggested that Loqs substrates are influenced by base-pairing status at the dicing site. Artificial alteration of base-pairing stability at this position in model miRNA hairpins resulted in predicted changes in Loqs dependence, providing evidence for this hypothesis. Finally, we found that evolutionarily young miRNA genes tended to be Loqs dependent. We propose that Loqs may have roles in assisting the de novo emergence of miRNA genes by facilitating dicing of suboptimal hairpin substrates.