Samantha E. Boyle

Identification of Candidate Growth Promoting Genes in Ovarian Cancer through Integrated Copy Number and Expression Analysis

Ovarian cancer is a disease characterised by complex genomic rearrangements but the majority of the genes that are the target of these alterations remain unidentified. Cataloguing these target genes will provide useful insights into the disease etiology and may provide an opportunity to develop novel diagnostic and therapeutic interventions. High resolution genome wide copy number and matching expression data from 68 primary epithelial ovarian carcinomas of various histotypes was integrated to identify genes in regions of most frequent amplification with the strongest correlation with expression and copy number. Regions on chromosomes 3, 7, 8, and 20 were most frequently increased in copy number (>40% of samples). Within these regions, 703/1370 (51%) unique gene expression probesets were differentially expressed when samples with gain were compared to samples without gain. 30% of these differentially expressed probesets also showed a strong positive correlation (r≥0.6) between expression and copy number. We also identified 21 regions of high amplitude copy number gain, in which 32 known protein coding genes showed a strong positive correlation between expression and copy number. Overall, our data validates previously known ovarian cancer genes, such as ERBB2, and also identified novel potential drivers such as MYNN, PUF60 and TPX2.

Copy Number Analysis Identifies Novel Interactions Between Genomic Loci in Ovarian Cancer

Ovarian cancer is a heterogeneous disease displaying complex genomic alterations, and consequently, it has been difficult to determine the most relevant copy number alterations with the scale of studies to date. We obtained genome-wide copy number alteration (CNA) data from four different SNP array platforms, with a final data set of 398 ovarian tumours, mostly of the serous histological subtype. Frequent CNA aberrations targeted many thousands of genes. However, high-level amplicons and homozygous deletions enabled filtering of this list to the most relevant. The large data set enabled refinement of minimal regions and identification of rare amplicons such as at 1p34 and 20q11. We performed a novel co-occurrence analysis to assess cooperation and exclusivity of CNAs and analysed their relationship to patient outcome. Positive associations were identified between gains on 19 and 20q, gain of 20q and loss of X, and between several regions of loss, particularly 17q. We found weak correlations of CNA at genomic loci such as 19q12 with clinical outcome. We also assessed genomic instability measures and found a correlation of the number of higher amplitude gains with poorer overall survival. By assembling the largest collection of ovarian copy number data to date, we have been able to identify the most frequent aberrations and their interactions.

Analysis of RAD51C germline mutations in high‐risk breast and ovarian cancer families and ovarian cancer patients

There is strong evidence that overtly inactivating mutations in RAD51C predispose to hereditary breast and ovarian cancer but the prevalence of such mutations, and whether they are associated with a particular clinical phenotype, remains unclear. Resolving these questions has important implications for the implementation of RAD51C into routine clinical genetic testing. Consequently, we have performed a large RAD51C mutation screen of hereditary breast and ovarian cancer families, and the first study of unselected patients diagnosed with ovarian cancer. Our data confirm a consistent but low frequency (2/335 families) of inactivating RAD51C mutations among families with a history of both breast and ovarian cancer and an absence of mutations among breast cancer only families (0/1,053 families). Our data also provide support for the designation of the missense variant p.Gly264Ser as a moderate penetrance allele. Hum Mutat 33:95–99, 2012.

Are there any more ovarian tumor suppressor genes? A new perspective using ultra high-resolution copy number and loss of heterozygosity analysis.

Ovarian cancer is characterized by complex genetic alterations, including copy number loss and copy number‐neutral loss of heterozygosity (LOH). These alterations are assumed to represent the “second hit” of the underlying tumor suppressor gene (TSG), however, relative to the number of LOH hotspots reported, few ovarian TSGs have been identified. We conducted a high‐resolution LOH analysis using SNP arrays (500K and SNP6.0) of 106 primary ovarian tumors of various histological subtypes together with matching normal DNA. LOH was detected in at least 35% of samples on chromosomes 17, 19p, 22q, Xp, 13q, 8p, 6q, 4q, 5q, 1p, 16q, and 9q with a median minimal region of overlap of only 300 kb. Subtype‐specific differences in LOH frequency were noted, particularly for mucinous cases. We also identified 192 somatic homozygous deletions (HDs). Recurrent HDs targeted known TSGs such as CDKN2A (eight samples), RB1 (five samples), and PTEN (three samples). Additional recurrent HDs targeted 16 candidate TSGs near minimal regions of LOH on chromosomes 17, 13, 8p, 5q, and X. Given the importance of HDs in inactivating known genes, these candidates are highly likely to be ovarian TSGs. Our data suggest that the poor success of previous LOH studies was due to the inability of previous technology to resolve complex genomic alterations and distinguish true LOH from allelic imbalance. This study shows that recurrent regions of LOH and HD frequently align with known TSGs suggesting that LOH analysis remains a valid approach to discovering new candidates.

The transcription cofactor c-JUN mediates phenotype switching and BRAF inhibitor resistance in melanoma

The transcriptional regulator c-JUN is a key mediator of the metastatic potential and drug resistance in melanoma. Inhibiting two MAPKs is better than one Mitogen-activated protein kinase (MAPK) pathways, such as the ERK and JNK pathways, mediate critical cellular processes, such as survival, stress responses, and proliferation. These pathways can be hijacked by cancer cells, leading to uncontrolled cell division and metastasis. Many melanoma patients have activating mutations in an upstream kinase, BRAF, in the ERK pathway, but inhibitors of BRAF only produce short-term improvement. Using panels of melanoma cell lines and BRAF inhibitor–treated patient samples, Ramsdale et al. found that increased abundance of the transcription cofactor c-JUN, which is activated by the JNK pathway, mediated both inherent and adaptive resistance to BRAF inhibitors and contributed to metastatic potential. Blocking c-JUN abundance or its activation by the kinase JNK enhanced the efficacy of BRAF inhibitors against melanoma cells. Thus, targeting both MAPK pathways may overcome resistance to treatment with only inhibitors of the ERK pathway. Most patients with BRAF-mutant metastatic melanoma display remarkable but incomplete and short-lived responses to inhibitors of the BRAF kinase or the mitogen-activated protein kinase kinase (MEK), collectively BRAF/MEK inhibitors. We found that inherent resistance to these agents in BRAFV600-mutant melanoma cell lines was associated with high abundance of c-JUN and characteristics of a mesenchymal-like phenotype. Early drug adaptation in drug-sensitive cell lines grown in culture or as xenografts, and in patient samples during therapy, was consistently characterized by down-regulation of SPROUTY4 (a negative feedback regulator of receptor tyrosine kinases and the BRAF-MEK signaling pathway), increased expression of JUN and reduced expression of LEF1. This coincided with a switch in phenotype that resembled an epithelial-mesenchymal transition (EMT). In cultured cells, these BRAF inhibitor-induced changes were reversed upon removal of the drug. Knockdown of SPROUTY4 was sufficient to increase the abundance of c-JUN in the absence of drug treatment. Overexpressing c-JUN in drug-naïve melanoma cells induced similar EMT-like phenotypic changes to BRAF inhibitor treatment, whereas knocking down JUN abrogated the BRAF inhibitor-induced early adaptive changes associated with resistance and enhanced cell death. Combining the BRAF inhibitor with an inhibitor of c-JUN amino-terminal kinase (JNK) reduced c-JUN phosphorylation, decreased cell migration, and increased cell death in melanoma cells. Gene expression data from a panel of melanoma cell lines and a patient cohort showed that JUN expression correlated with a mesenchymal gene signature, implicating c-JUN as a key mediator of the mesenchymal-like phenotype associated with drug resistance.

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.

Loss of heterozygosity: what is it good for?

BackgroundLoss 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.MethodsWe analysed 86 ovarian carcinomas for mutations in 980 genes selected on the basis of their location in common regions of LOH.ResultsWe 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.ConclusionIt 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.

A community-based model of rapid autopsy in end-stage cancer patients

To the Editor: Systematic genomic studies, including the Cancer Genome Atlas (TCGA)1 and the International Cancer Genome Consortium (ICGC)2, have provided an unprecedented catalog of driver mutations in human cancer. However, these studies use mainly primary, pre-treatment tumor material obtained at surgery with curative intent. There is an urgent need to identify and characterize resistance mechanisms to understand how cancers can evade even the best medical efforts and kill patients; therefore, access to end-stage disease is important. Solid cancers show considerable spatial3, temporal4,5 and genomic heterogeneity at diagnosis. Selective pressure and mutagenic impact of treatment6 drives intra-patient evolution of cancer cell populations4,7. Understanding acquired resistance requires access to paired preand post-treatment samples4,7; however, curative surgery is typically confined to patients with locoregional disease, and opportunities for tumor sampling in advanced disseminated disease are limited. Here, we describe Cancer Tissue Collection After Death (CASCADE), an autopsy program that overcomes logistical challenges to enable collection of samples at end stage for research in melanoma and breast, ovarian and prostate cancers. For the CASCADE study, we aimed to recruit cancer patients close to the end of life, including those outside the minority of patients who die in hospitals. To preserve tissue integrity, autopsies must commence within a few hours of death, requiring access to around-the-clock services. Intervention in the emotionally charged end-of-life environment must be managed in an ethical manner and to a high standard. Finally, we aimed for the study to be highly cost-effective. We believe our approach to meeting these challenges is applicable to researchers in other large urban centers. Here we summarize the main steps in CASCADE’s operating protocol and our experiences from the initial 3 years and 30 autopsies performed (Fig. 1). Information about institutional review board approvals (including a detailed patient informationand-consent form), the autopsy procedure and certain laboratory processes is given in Supplementary Methods and Supplementary Figure 1. Recruitment of participants was led by the clinicians. Such discussions require careful consideration, in timing and in language, and were initiated only if there was a perception that tissue donation would be acceptable to the patients and their families. Factors suggesting acceptability include the emotional stability of the participant and family members and their clarity about and acceptance of the terminal nature of the disease. On occasion, participants prompted discussion by asking about organ or body donation. Consent discussions typically involved oncologists and/or palliative care physicians employed at recruiting hospitals who had established a care relationship with the participant and their family during the patient’s cancer journey. Frequently, the study was introduced at one meeting and discussed over several subsequent clinic visits, allowing patients and their families time to consider participation. We view the involvement of family members in the consent process as essential to support the participant and facilitate decisionmaking. Involvement of family members also ensures that they are fully aware of the autopsy process and helps to clarify funeral arrangements for the study team. After obtaining consent, study investigators collated clinical information, including that related to past and current treatment and diagnostic procedures such as imaging, on an ongoing basis. Between September 2012 and August 2015, 40 patients were approached, and 37 (92.5%) expressed interest in participating. Of those 32 patients (80%) consented; the other 5 had rapid clinical deterioration precluding

CD271 Expression on Patient Melanoma Cells Is Unstable and Unlinked to Tumorigenicity.

The stability of markers that identify cancer cells that propagate disease is important to the outcomes of targeted therapy strategies. In human melanoma, conflicting data exist as to whether hierarchical expression of CD271/p75/NGFR (nerve growth factor receptor) marks cells with enriched tumorigenicity, which would compel their specific targeting in therapy. To test whether these discrepancies relate to differences among groups in assay approaches, we undertook side-by-side testing of published methods of patient-derived melanoma xenografting (PDX), including comparisons of tissue digestion procedures or coinjected Matrigel formulations. We found that CD271(-) and CD271(+) melanoma cells from each of seven patients were similarly tumorigenic, regardless of assay variations. Surprisingly variable CD271 expression patterns were observed in the analyses of sibling PDX tumors (n = 68) grown in the same experiments from either CD271(-) or CD271(+) cells obtained from patients. This indicates unstable intratumoral lineage relationships between CD271(-) and CD271(+) melanoma cells that are inconsistent with classical, epigenetically based theories of disease progression, such as the cancer stem cell and plasticity models. SNP genotyping of pairs of sibling PDX tumors grown from phenotypically identical CD271(-) or CD271(+) cells showed large pairwise differences in copy number (28%-48%). Differences were also apparent in the copy number profiles of CD271(-) and CD271(+) cells purified directly from each of the four melanomas (1.4%-23%). Thus, CD271 expression in patient melanomas is unstable, not consistently linked to increased tumorigenicity and associated with genetic heterogeneity, undermining its use as a marker in clinical studies. Cancer Res; 76(13); 3965-77. ©2016 AACR.

A non-canonical role for desmoglein-2 in endothelial cells: implications for neoangiogenesis

Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by non-desmosome-forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34+CD45dim hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34+CD45dimDSG2+ progenitor cells are multi-potent and pro-angiogenic in vitro. Using a ‘knockout-first’ approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2lo/lo) and observed that, in response to reduced levels of Dsg2: (i) CD31+ ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrow-derived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell–cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.