Molecular comparison of pure ovarian fibroma with serous benign ovarian tumours.

OBJECTIVE: Ovarian fibromas and adenofibromas are rare ovarian tumours. They are benign tumours composed of spindle-like stromal cells (pure fibroma) or a mixture of fibroblast and epithelial components (adenofibroma). We have previously shown that 40% of benign serous ovarian tumours are likely primary fibromas due to the neoplastic alterations being restricted to the stromal compartment of these tumours. We further explore this finding by comparing benign serous tumours to pure fibromas. RESULTS: Performing copy number aberration (CNA) analysis on the stromal component of 45 benign serous tumours and 8 pure fibromas, we have again shown that trisomy of chromosome 12 is the most common aberration in ovarian fibromas. CNAs were more frequent in the pure fibromas than the benign serous tumours (88% vs 33%), however pure fibromas more frequently harboured more than one CNA event compared with benign serous tumours. As these extra CNA events observed in the pure fibromas were unique to this subset our data indicates a unique tumour evolution. Gene expression analysis on the two cohorts was unable to show gene expression changes that differed based on tumour subtype. Exome analysis did not reveal any recurrently mutated genes.

Germline whole exome sequencing of a family with appendiceal mucinous tumours presenting with pseudomyxoma peritonei.

BACKGROUND: Familial cases of appendiceal mucinous tumours (AMTs) are extremely rare and the underlying genetic aetiology uncertain. We identified potential predisposing germline genetic variants in a father and daughter with AMTs presenting with pseudomyxoma peritonei (PMP) and correlated these with regions of loss of heterozygosity (LOH) in the tumours. METHODS: Through germline whole exome sequencing, we identified novel heterozygous loss-of-function (LoF) (i.e. nonsense, frameshift and essential splice site mutations) and missense variants shared between father and daughter, and validated all LoF variants, and missense variants with a Combined Annotation Dependent Depletion (CADD) scaled score of ≥10. Genome-wide copy number analysis was performed on tumour tissue from both individuals to identify regions of LOH. RESULTS: Fifteen novel variants in 15 genes were shared by the father and daughter, including a nonsense mutation in REEP5. None of these germline variants were located in tumour regions of LOH shared by the father and daughter. Four genes (EXOG, RANBP2, RANBP6 and TNFRSF1B) harboured missense variants that fell in a region of LOH in the tumour from the father only, but none showed somatic loss of the wild type allele in the tumour. The REEP5 gene was sequenced in 23 individuals with presumed sporadic AMTs or PMP; no LoF or rare missense germline variants were identified. CONCLUSION: Germline exome sequencing of a father and daughter with AMTs identified novel candidate predisposing genes. Further studies are required to clarify the role of these genes in familial AMTs.

PathOS: a decision support system for reporting high throughput sequencing of cancers in clinical diagnostic laboratories.

BACKGROUND: The increasing affordability of DNA sequencing has allowed it to be widely deployed in pathology laboratories. However, this has exposed many issues with the analysis and reporting of variants for clinical diagnostic use. Implementing a high-throughput sequencing (NGS) clinical reporting system requires a diverse combination of capabilities, statistical methods to identify variants, global variant databases, a validated bioinformatics pipeline, an auditable laboratory workflow, reproducible clinical assays and quality control monitoring throughout. These capabilities must be packaged in software that integrates the disparate components into a useable system. RESULTS: To meet these needs, we developed a web-based application, PathOS, which takes variant data from a patient sample through to a clinical report. PathOS has been used operationally in the Peter MacCallum Cancer Centre for two years for the analysis, curation and reporting of genetic tests for cancer patients, as well as the curation of large-scale research studies. PathOS has also been deployed in cloud environments allowing multiple institutions to use separate, secure and customisable instances of the system. Increasingly, the bottleneck of variant curation is limiting the adoption of clinical sequencing for molecular diagnostics. PathOS is focused on providing clinical variant curators and pathology laboratories with a decision support system needed for personalised medicine. While the genesis of PathOS has been within cancer molecular diagnostics, the system is applicable to NGS clinical reporting generally. CONCLUSIONS: The widespread availability of genomic sequencers has highlighted the limited availability of software to support clinical decision-making in molecular pathology. PathOS is a system that has been developed and refined in a hospital laboratory context to meet the needs of clinical diagnostics. The software is available as a set of Docker images and source code at https://github.com/PapenfussLab/PathOS .

Genomic characterisation of Eµ-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene.

The Eµ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eµ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eµ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.

Id2 and E Proteins Orchestrate the Initiation and Maintenance of MLL-Rearranged Acute Myeloid Leukemia.

E proteins and their antagonists, the Id proteins, are transcriptional regulators important for normal hematopoiesis. We found that Id2 acts as a key regulator of leukemia stem cell (LSC) potential in MLL-rearranged acute myeloid leukemia (AML). Low endogenous Id2 expression is associated with LSC enrichment while Id2 overexpression impairs MLL-AF9-leukemia initiation and growth. Importantly, MLL-AF9 itself controls the E-protein pathway by suppressing Id2 while directly activating E2-2 expression, and E2-2 depletion phenocopies Id2 overexpression in MLL-AF9-AML cells. Remarkably, Id2 tumor-suppressive function is conserved in t(8;21) AML. Low expression of Id2 and its associated gene signature are associated with poor prognosis in MLL-rearranged and t(8;21) AML patients, identifying the Id2/E-protein axis as a promising new therapeutic target in AML.

Molecular profiling of low grade serous ovarian tumours identifies novel candidate driver genes.

Low grade serous ovarian tumours are a rare and under-characterised histological subtype of epithelial ovarian tumours, with little known of the molecular drivers and facilitators of tumorigenesis beyond classic oncogenic RAS/RAF mutations. With a move towards targeted therapies due to the chemoresistant nature of this subtype, it is pertinent to more fully characterise the genetic events driving this tumour type, some of which may influence response to therapy and/or development of drug resistance. We performed genome-wide high-resolution genomic copy number analysis (Affymetrix SNP6.0) and mutation hotspot screening (KRAS, BRAF, NRAS, HRAS, ERBB2 and TP53) to compare a large cohort of ovarian serous borderline tumours (SBTs, n = 57) with low grade serous carcinomas (LGSCs, n = 19). Whole exome sequencing was performed for 13 SBTs, nine LGSCs and one mixed low/high grade carcinoma. Copy number aberrations were detected in 61% (35/57) of SBTs, compared to 100% (19/19) of LGSCs. Oncogenic RAS/RAF/ERBB2 mutations were detected in 82.5% (47/57) of SBTs compared to 63% (12/19) of LGSCs, with NRAS mutations detected only in LGSC. Some copy number aberrations appeared to be enriched in LGSC, most significantly loss of 9p and homozygous deletions of the CDKN2A/2B locus. Exome sequencing identified BRAF, KRAS, NRAS, USP9X and EIF1AX as the most frequently mutated genes. We have identified markers of progression from borderline to LGSC and novel drivers of LGSC. USP9X and EIF1AX have both been linked to regulation of mTOR, suggesting that mTOR inhibitors may be a key companion treatment for targeted therapy trials of MEK and RAF inhibitors.

Prevalence of PALB2 mutations in Australian familial breast cancer cases and controls.

INTRODUCTION: PALB2 is emerging as a high-penetrance breast cancer predisposition gene in the order of BRCA1 and BRCA2. However, large studies that have evaluated the full gene rather than just the most common variants in both cases and controls are required before all truncating variants can be included in familial breast cancer variant testing. METHODS: In this study we analyse almost 2000 breast cancer cases sourced from individuals referred to familial cancer clinics, thus representing typical cases presenting in clinical practice. These cases were compared to a similar number of population-based cancer-free controls. RESULTS: We identified a significant excess of truncating variants in cases (1.3 %) versus controls (0.2 %), including six novel variants (p = 0.0001; odds ratio (OR) 6.58, 95 % confidence interval (CI) 2.3-18.9). Three of the four control individuals carrying truncating variants had at least one relative with breast cancer. There was no excess of missense variants in cases overall, but the common c.1676A > G variant (rs152451) was significantly enriched in cases and may represent a low-penetrance polymorphism (p = 0.002; OR 1.24 (95 % CI 1.09-1.47). CONCLUSIONS: Our findings support truncating variants in PALB2 as high-penetrance breast cancer susceptibility alleles, and suggest that a common missense variant may also lead to a low level of increased breast cancer risk.

Loss of heterozygosity: what is it good for?

BACKGROUND: Loss of heterozygosity (LOH) is a common genetic event in cancer development, and is known to be involved in the somatic loss of wild-type alleles in many inherited cancer syndromes. The wider involvement of LOH in cancer is assumed to relate to unmasking a somatically mutated tumour suppressor gene through loss of the wild type allele. METHODS: We analysed 86 ovarian carcinomas for mutations in 980 genes selected on the basis of their location in common regions of LOH. RESULTS: We identified 36 significantly mutated genes, but these could only partly account for the quanta of LOH in the samples. Using our own and TCGA data we then evaluated five possible models to explain the selection for non-random accumulation of LOH in ovarian cancer genomes: 1. Classic two-hit hypothesis: high frequency biallelic genetic inactivation of tumour suppressor genes. 2. Epigenetic two-hit hypothesis: biallelic inactivation through methylation and LOH. 3. Multiple alternate-gene biallelic inactivation: low frequency gene disruption. 4. Haplo-insufficiency: Single copy gene disruption. 5. Modified two-hit hypothesis: reduction to homozygosity of low penetrance germline predisposition alleles. We determined that while high-frequency biallelic gene inactivation under model 1 is rare, regions of LOH (particularly copy-number neutral LOH) are enriched for deleterious mutations and increased promoter methylation, while copy-number loss LOH regions are likely to contain under-expressed genes suggestive of haploinsufficiency. Reduction to homozygosity of cancer predisposition SNPs may also play a minor role. CONCLUSION: It is likely that selection for regions of LOH depends on its effect on multiple genes. Selection for copy number neutral LOH may better fit the classic two-hit model whereas selection for copy number loss may be attributed to its effect on multi-gene haploinsufficiency. LOH mapping alone is unlikely to be successful in identifying novel tumour suppressor genes; a combined approach may be more effective.

Mutational landscape of mucinous ovarian carcinoma and its neoplastic precursors.

BACKGROUND: Mucinous ovarian tumors are an unusual group of rare neoplasms with an apparently clear progression from benign to borderline to carcinoma, yet with a controversial cell of origin in the ovarian surface epithelium. They are thought to be molecularly distinct from other ovarian tumors but there have been no exome-level sequencing studies performed to date. METHODS: To understand the genetic etiology of mucinous ovarian tumors and assess the presence of novel therapeutic targets or pathways, we undertook exome sequencing of 24 tumors encompassing benign (5), borderline (8) and carcinoma (11) histologies and also assessed a validation cohort of 58 tumors for specific gene regions including exons 4-9 of TP53. RESULTS: The predominant mutational signature was of C>T transitions in a NpCpG context, indicative of deamination of methyl-cytosines. As well as mutations in known drivers (KRAS, BRAF and CDKN2A), we identified a high percentage of carcinomas with TP53 mutations (52 %), and recurrent mutations in RNF43, ELF3, GNAS, ERBB3 and KLF5. CONCLUSIONS: The diversity of mutational targets suggests multiple routes to tumorigenesis in this heterogeneous group of tumors that is generally distinct from other ovarian subtypes.

Studying cancer genomics through next-generation DNA sequencing and bioinformatics.

Cancer is a complex disease driven by multiple mutations acquired over the lifetime of the cancer cells. These alterations, termed somatic mutations to distinguish them from inherited germline mutations, can include single-nucleotide substitutions, insertions, deletions, copy number alterations, and structural rearrangements. A patient's cancer can contain a combination of these aberrations, and the ability to generate a comprehensive genetic profile should greatly improve patient diagnosis and treatment. Next-generation sequencing has become the tool of choice to uncover multiple cancer mutations from a single tumor source, and the falling costs of this rapid high-throughput technology are encouraging its transition from basic research into a clinical setting. However, the detection of mutations in sequencing data is still an evolving area and cancer genomic data requires some special considerations. This chapter discusses these aspects and gives an overview of current bioinformatics methods for the detection of somatic mutations in cancer sequencing data.

The drug vehicle and solvent N-methylpyrrolidone is an immunomodulator and antimyeloma compound.

N-methyl-2-pyrrolidone (NMP) is a common solvent and drug vehicle. We discovered unexpected antineoplastic and immunomodulatory activity of NMP in a cMYC-driven myeloma model. Coincident to this, NMP was identified as an acetyllysine mimetic and candidate bromodomain ligand. Accordingly, NMP-treated cells demonstrated transcriptional overlap with BET-bromodomain inhibition, including downregulation of cMYC and IRF4. NMP's immunomodulatory activity occurred at sub-BET inhibitory concentrations, and, despite phenotypic similarities to lenalidomide, its antimyeloma activity was independent of the IMiD targets cereblon and Ikaros-1/3. Thus, low-affinity yet broad-spectrum bromodomain inhibition by NMP mediates biologically potent, cereblon-independent immunomodulation and at higher doses targets malignant cells directly via BET antagonism. These data reveal that NMP is a functional acetyllysine mimetic with pleotropic antimyeloma and immunomodulatory activities. Our studies highlight the potential therapeutic benefits of NMP, the consequences of current human NMP exposures, and the need for reassessment of scientific literature where NMP was used as an "inert" drug-delivery vehicle.

Bioinformatics pipelines for targeted resequencing and whole-exome sequencing of human and mouse genomes: a virtual appliance approach for instant deployment.

Targeted resequencing by massively parallel sequencing has become an effective and affordable way to survey small to large portions of the genome for genetic variation. Despite the rapid development in open source software for analysis of such data, the practical implementation of these tools through construction of sequencing analysis pipelines still remains a challenging and laborious activity, and a major hurdle for many small research and clinical laboratories. We developed TREVA (Targeted REsequencing Virtual Appliance), making pre-built pipelines immediately available as a virtual appliance. Based on virtual machine technologies, TREVA is a solution for rapid and efficient deployment of complex bioinformatics pipelines to laboratories of all sizes, enabling reproducible results. The analyses that are supported in TREVA include: somatic and germline single-nucleotide and insertion/deletion variant calling, copy number analysis, and cohort-based analyses such as pathway and significantly mutated genes analyses. TREVA is flexible and easy to use, and can be customised by Linux-based extensions if required. TREVA can also be deployed on the cloud (cloud computing), enabling instant access without investment overheads for additional hardware. TREVA is available at http://bioinformatics.petermac.org/treva/.

RNF43 is a tumour suppressor gene mutated in mucinous tumours of the ovary.

Mucinous carcinomas represent a distinct morphological subtype which can arise from several organ sites, including the ovary, and their genetic characteristics are largely under-described. Exome sequencing of 12 primary mucinous ovarian tumours identified RNF43 as the most frequently somatically mutated novel gene, secondary to KRAS and mutated at a frequency equal to that of TP53 and BRAF. Further screening of RNF43 in a larger cohort of ovarian tumours identified additional mutations, with a total frequency of 2/22 (9%) in mucinous ovarian borderline tumours and 6/29 (21%) in mucinous ovarian carcinomas. Seven mutations were predicted to truncate the protein and one missense mutation was predicted to be deleterious by in silico analysis. Six tumours had allelic imbalance at the RNF43 locus, with loss of the wild-type allele. The mutation spectrum strongly suggests that RNF43 is an important tumour suppressor gene in mucinous ovarian tumours, similar to its reported role in mucinous pancreatic precancerous cysts.

Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

MicroRNA genes and their target 3'-untranslated regions are infrequently somatically mutated in ovarian cancers.

MicroRNAs are key regulators of gene expression and have been shown to have altered expression in a variety of cancer types, including epithelial ovarian cancer. MiRNA function is most often achieved through binding to the 3'-untranslated region of the target protein coding gene. Mutation screening using massively-parallel sequencing of 712 miRNA genes in 86 ovarian cancer cases identified only 5 mutated miRNA genes, each in a different case. One mutation was located in the mature miRNA, and three mutations were predicted to alter the secondary structure of the miRNA transcript. Screening of the 3'-untranslated region of 18 candidate cancer genes identified one mutation in each of AKT2, EGFR, ERRB2 and CTNNB1. The functional effect of these mutations is unclear, as expression data available for AKT2 and EGFR showed no increase in gene transcript. Mutations in miRNA genes and 3'-untranslated regions are thus uncommon in ovarian cancer.

Next-generation sequencing for cancer diagnostics: a practical perspective.

Next-generation sequencing (NGS) is arguably one of the most significant technological advances in the biological sciences of the last 30 years. The second generation sequencing platforms have advanced rapidly to the point that several genomes can now be sequenced simultaneously in a single instrument run in under two weeks. Targeted DNA enrichment methods allow even higher genome throughput at a reduced cost per sample. Medical research has embraced the technology and the cancer field is at the forefront of these efforts given the genetic aspects of the disease. World-wide efforts to catalogue mutations in multiple cancer types are underway and this is likely to lead to new discoveries that will be translated to new diagnostic, prognostic and therapeutic targets. NGS is now maturing to the point where it is being considered by many laboratories for routine diagnostic use. The sensitivity, speed and reduced cost per sample make it a highly attractive platform compared to other sequencing modalities. Moreover, as we identify more genetic determinants of cancer there is a greater need to adopt multi-gene assays that can quickly and reliably sequence complete genes from individual patient samples. Whilst widespread and routine use of whole genome sequencing is likely to be a few years away, there are immediate opportunities to implement NGS for clinical use. Here we review the technology, methods and applications that can be immediately considered and some of the challenges that lie ahead.