Socrates: identification of genomic rearrangements in tumour genomes by re-aligning soft clipped reads.

MOTIVATION: Methods for detecting somatic genome rearrangements in tumours using next-generation sequencing are vital in cancer genomics. Available algorithms use one or more sources of evidence, such as read depth, paired-end reads or split reads to predict structural variants. However, the problem remains challenging due to the significant computational burden and high false-positive or false-negative rates. RESULTS: In this article, we present Socrates (SOft Clip re-alignment To idEntify Structural variants), a highly efficient and effective method for detecting genomic rearrangements in tumours that uses only split-read data. Socrates has single-nucleotide resolution, identifies micro-homologies and untemplated sequence at break points, has high sensitivity and high specificity and takes advantage of parallelism for efficient use of resources. We demonstrate using simulated and real data that Socrates performs well compared with a number of existing structural variant detection tools. AVAILABILITY AND IMPLEMENTATION: Socrates is released as open source and available from http://bioinf.wehi.edu.au/socrates CONTACT: papenfuss@wehi.edu.au Supplementary information: Supplementary data are available at Bioinformatics online.

Assessment of DNA methylation profiling and copy number variation as indications of clonal relationship in ipsilateral and contralateral breast cancers to distinguish recurrent breast cancer from a second primary tumour.

BACKGROUND: Patients with breast cancer have an increased risk of developing subsequent breast cancers. It is important to distinguish whether these tumours are de novo or recurrences of the primary tumour in order to guide the appropriate therapy. Our aim was to investigate the use of DNA methylation profiling and array comparative genomic hybridization (aCGH) to determine whether the second tumour is clonally related to the first tumour. METHODS: Methylation-sensitive high-resolution melting was used to screen promoter methylation in a panel of 13 genes reported as methylated in breast cancer (RASSF1A, TWIST1, APC, WIF1, MGMT, MAL, CDH13, RARß, BRCA1, CDH1, CDKN2A, TP73, and GSTP1) in 29 tumour pairs (16 ipsilateral and 13 contralateral). Using the methylation profile of these genes, we employed a Bayesian and an empirical statistical approach to estimate clonal relationship. Copy number alterations were analysed using aCGH on the same set of tumour pairs. RESULTS: There is a higher probability of the second tumour being recurrent in ipsilateral tumours compared with contralateral tumours (38 % versus 8 %; p <0.05) based on the methylation profile. Using previously reported recurrence rates as Bayesian prior probabilities, we classified 69 % of ipsilateral and 15 % of contralateral tumours as recurrent. The inferred clonal relationship results of the tumour pairs were generally concordant between methylation profiling and aCGH. CONCLUSION: Our results show that DNA methylation profiling as well as aCGH have potential as diagnostic tools in improving the clinical decisions to differentiate recurrences from a second de novo tumour.

CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

PURPOSE: CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. EXPERIMENTAL DESIGN: We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. RESULTS: CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CONCLUSIONS: CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

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.

Identification of copy number alterations associated with the progression of DCIS to invasive ductal carcinoma.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive ductal carcinoma (IDC). Annotation of the genetic differences between the two lesions may assist in the identification of genes that promote the invasive phenotype. Synchronous DCIS and IDC cells were microdissected from FFPE tissue and analysed by molecular inversion probe (MIP) copy number arrays. Matched IDC and DCIS showed highly similar copy number profiles (average of 83% of the genome shared) indicating a common clonal origin although there is evidence that the DCIS continues to evolve in parallel with the co-existing IDC. Four chromosomal regions of loss (3q, 6q, 8p and 11q) and four regions of gain (5q, 16p, 19q and 20) were recurrently affected in IDC but not in DCIS. CCND1 and MYC showed increased amplitude of gain in IDC. One region of loss (17p11.2) was specific to DCIS. IDC-specific regions include genes with previous links to breast cancer progression and potential therapeutic targets such as AXL, SPHK1 and PLAUR.

Longitudinal monitoring of disease burden and response using ctDNA from dried blood spots in xenograft models.

Whole-genome sequencing (WGS) of circulating tumour DNA (ctDNA) is now a clinically important biomarker for predicting therapy response, disease burden and disease progression. However, the translation of ctDNA monitoring into vital preclinical PDX models has not been possible owing to low circulating blood volumes in small rodents. Here, we describe the longitudinal detection and monitoring of ctDNA from minute volumes of blood in PDX mice. We developed a xenograft Tumour Fraction (xTF) metric using shallow WGS of dried blood spots (DBS), and demonstrate its application to quantify disease burden, monitor treatment response and predict disease outcome in a preclinical study of PDX mice. Further, we show how our DBS-based ctDNA assay can be used to detect gene-specific copy number changes and examine the copy number landscape over time. Use of sequential DBS ctDNA assays could transform future trial designs in both mice and patients by enabling increased sampling and molecular monitoring.

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.

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.

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.

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

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 BRAF(V600)-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.

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 > or =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.