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Aims: The advent of specific ALK-targeting drugs has radically changed the outcome of patients with ALK translocated non-small-cell lung cancer (NSCLC). However, emerging resistance to treatment with ALK inhibitors in these patients remains a major concern. In previous studies, we analysed two ALK+ patient cohorts (TP53 wild-type/TP53 mutated) in terms of copy number alterations. All patients belonging to the TP53 wild-type group had mainly genetically stable genomes, with one exception showing chromosomal instability and amplifications of several gene loci, including TERT. Here, we aimed to determine the prevalence of TERT amplifications in these ALK+ lung cancer patients by analysing an independent cohort of 109 ALK translocated cases. We further analysed the copy numbers of numerous cancer-relevant genes and other genetic aberrations. Methods and Results: The prevalence of TERT amplifications was determined by means of FISH analyses. Copy numbers of 87 cancer-relevant genes were determined by NanoString nCounter technology, FoundationOne and lung-specific NGS panels in some of these TERT-amplified samples, and clinical data on patients with TERT-amplified tumours were collected. Our data revealed that five (4.6%) of all 109 analysed ALK+ patients harboured amplification of TERT and that these patients had genetically unstable genomes. Conclusion(s): Our preliminary study shows that ALK+ adenocarcinomas should be evaluated in the context of their genomic background in order to more clearly understand and predict patients' individual course of disease.Copyright © 2020 The Authors. Histopathology published by John Wiley & Sons Ltd

Identification of additional cancer-associated genes and secondary mutations driving the metastatic progression in pheochromocytoma and paraganglioma (PPGL) is important for subtyping, and may provide optimization of therapeutic regimens. We recently reported novel recurrent nonsynonymous mutations in the MYO5B gene in metastatic PPGL. Here, we explored the functional impact of these MYO5B mutations, and analyzed MYO5B expression in primary PPGL tumor cases in relation to mutation status. Immunohistochemistry and mRNA expression analysis in 30 PPGL tumors revealed an increased MYO5B expression in metastatic compared to non-metastatic cases. In addition, subcellular localization of MYO5B protein was altered from cytoplasmic to membranous in some metastatic tumors, and the strongest and most abnormal expression pattern was observed in a paraganglioma harboring a somatic MYO5B:p.G1611S mutation. In addition to five previously discovered MYO5B mutations, the present study of 30 PPGL (8 previous and 22 new samples) also revealed two, and hence recurrent, mutations in the gene paralog MYO5A. The three MYO5B missense mutations with the highest prediction scores (p.L587P, p.G1611S and p.R1641C) were selected and functionally validated using site directed mutagenesis and stable transfection into human neuroblastoma cells (SK-N-AS) and embryonic kidney cells (HEK293). In vitro analysis showed a significant increased proliferation rate in all three MYO5B mutated clones. The two somatically derived mutations, p.L587P and p.G1611S, were also found to increase the migration rate. Expression analysis of MYO5B mutants compared to wild type clones, demonstrated a significant enrichment of genes involved in migration, proliferation, cell adhesion, glucose metabolism, and cellular homeostasis. Our study validates the functional role of novel MYO5B mutations in proliferation and migration, and suggest the MYO5-pathway to be involved in the malignant progression in some PPGL tumors. © 2, Export Date: 6 August 2020; Article

Previous transcriptome-wide association studies (TWAS) have identified breast cancer risk genes by integrating data from expression quantitative loci and genome-wide association studies (GWAS), but analyses of breast cancer subtype-specific associations have been limited. In this study, we conducted a TWAS using gene expression data from GTEx and summary statistics from the hitherto largest GWAS meta-analysis conducted for breast cancer overall, and by estrogen receptor subtypes (ER+ and ER-). We further compared associations with ER+ and ER- subtypes, using a case-only TWAS approach. We also conducted multigene conditional analyses in regions with multiple TWAS associations. Two genes, STXBP4 and HIST2H2BA, were specifically associated with ER+ but not with ER- breast cancer. We further identified 30 TWAS-significant genes associated with overall breast cancer risk, including four that were not identified in previous studies. Conditional analyses identified single independent breast-cancer gene in three of six regions harboring multiple TWAS-significant genes. Our study provides new information on breast cancer genetics and biology, particularly about genomic differences between ER+ and ER- breast cancer.Copyright © 2020 The Authors. Genetic Epidemiology published by Wiley Periodicals, Inc.

Background: We examined the associations between germline variants and breast cancer mortality using a large meta-analysis of women of European ancestry. Method(s): Meta-analyses included summary estimates based on Cox models of twelve datasets using ~10.4 million variants for 96,661 women with breast cancer and 7697 events (breast cancer-specific deaths). Oestrogen receptor (ER)-specific analyses were based on 64,171 ER-positive (4116) and 16,172 ER-negative (2125) patients. We evaluated the probability of a signal to be a true positive using the Bayesian false discovery probability (BFDP). Result(s): We did not find any variant associated with breast cancer-specific mortality at P < 5 x 10-8. For ER-positive disease, the most significantly associated variant was chr7:rs4717568 (BFDP = 7%, P = 1.28 x 10-7, hazard ratio [HR] = 0.88, 95% confidence interval [CI] = 0.84-0.92); the closest gene is AUTS2. For ER-negative disease, the most significant variant was chr7:rs67918676 (BFDP = 11%, P = 1.38 x 10-7, HR = 1.27, 95% CI = 1.16-1.39); located within a long intergenic non-coding RNA gene (AC004009.3), close to the HOXA gene cluster. Conclusion(s): We uncovered germline variants on chromosome 7 at BFDP < 15% close to genes for which there is biological evidence related to breast cancer outcome. However, the paucity of variants associated with mortality at genome-wide significance underpins the challenge in providing genetic-based individualised prognostic information for breast cancer patients.Copyright © 2019, The Author(s).

Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD.Copyright © 2019, The Author(s).

Background: Plasma circulating tumour DNA (ctDNA) in prostate cancer has emerged as a promising minimally invasive tool with which to interrogate the cancer genome. However, detection of copy number variations (CNVs) in ctDNA has proved challenging, which poses a major problem in mCRPC, which commonly harbours pathogenic CNVs. Here, we use an ultra-sensitive next-generation sequencing (NGS) assay to identify CNVs in ctDNA from mCRPC patients and correlate findings with clinical outcomes in men commencing androgen receptor pathway inhibitors (ARPIs) or chemotherapy. Method(s): Plasma from mCRPC patients commencing therapy was collected and platelet poor plasma (PPP) fractions were processed uniformly and cell-free DNA (cfDNA) extracted. Plasma samples were analysed using the PredicineLITE NGS assay, which reports genomic alterations in 90 cancer genes. CNVs from this cohort were correlated with PSA response rate (Fisher's exact test), PSA progression-free survival (PSA-PFS), clinical/radiographic progression-free survival (clinical/ rPFS) and overall survival (OS). Result(s): Median follow-up was 19.85 months (mo) (IQR 12.5-23.0). In total, 32 pre-treatment samples were analysed (7 chemotherapy, 25 ARPI). The most common CNVs were PTEN loss (n = 12, 38% of cohort), RB1 loss (n = 5, 16%) and AR gain (n = 12, 38%). Notably, OS was decreased in patients with PTEN loss (median 9.7mo vs. not reached; P=.03) andRB1 loss (median 7.1movs. 17.1 mo; P=.1), while 48 POSTER ABSTRACTS PSA response rates were also lower in RB1 loss (1/5, 20% vs 19/27, 71%; P = .053). In addition, AR copy number gain was associated with decreased clinical/rPFS (median 3.4 mo vs 10.7 mo; P = .05) and inferior OS (median 9.7 mo vs not reached; P = .05). Conclusion(s): Using an ultra-sensitive NGS assay, we demonstrate the robust detection of CNVs in plasma ctDNA of patients with mCRPC. Copy number losses of PTEN and RB1, and gains ofARwere associated with worse clinical outcomes on chemotherapy and A

Background: Due to difficulties with routine metastatic tissue biopsy in mCRPC, the identification of prognostic and predictive biomarkers for treatment with androgen-receptor signalling inhibitors (ARSI) and chemotherapy remains an unmet clinical need. Plasma circulating tumor DNA (ctDNA) has emerged as a promising minimally-invasive tool with which to interrogate the cancer genome. However, detection of copy number variations (CNVs) in ctDNA has proved challenging. This poses a major problem in mCRPC, which commonly harbours pathogenic CNVs. Here we use an ultra-sensitive next-generation sequencing (NGS) assay to identify CNVs in ctDNA from mCRPC patients and correlate findings with clinical outcomes in men commencing ARSI (abiraterone or enzalutamide) or chemotherapy (docetaxel or cabazitaxel). Method(s): MCRPC patients commencing ARSI or chemotherapy were prospectively recruited at two Australian centers. Plasma was collected and platelet poor plasma (PPP) fractions were processed uniformly and cell-free DNA (cfDNA) extracted. Plasma samples were analysed using the PredicineLITE NGS assay, which reports genomic alterations in 90 cancer genes. CNVs from this cohort were correlated with PSA response rate (Fisher's exact test), PSA progression-free survival (PSA-PFS), clinical/radiographic progression-free survival (clinical/rPFS), and overall survival (OS). Result(s): Median follow-up was 19.85 months (mo) (IQR 12.5-23.0). In total, 32 pre-treatment samples were analyzed (7 chemotherapy, 25 ARSI). The most common CNVs were PTEN loss (n=12, 38% of cohort), RB1 loss (n=5, 16%) and AR gain (n=12, 38%). Notably, OS was decreased in patients with PTEN loss (median 9.7 mo vs. not reached; p=0.03) and RB1 loss (median 7.1 mo vs. 17.1 mo; p=0.1), while PSA response rates were also lower in RB1 loss (1/5, 20% vs. 19/27, 71%; p=0.053). In addition, AR copy number gain was associated with decreased clinical/rPFS (median 3.4 mo vs. 10.7 mo; p=0.05) and inferior OS (median 9.7

The anaplastic lymphoma kinase (ALK) rearrangement defines a distinct molecular subtype of non-small cell lung cancer (NSCLC). Despite the excellent initial efficacy of ALK inhibitors in patients with ALK+ lung cancer, resistance occurs almost inevitably. To date, there is no reliable biomarker allowing the identification of patients at higher risk of relapse. Here, we analysed a subset of 53 ALK+ tumours with and without TP53 mutation and ALK+ NSCLC cell lines by NanoString nCounter technology. We found that the co-occurrence of early TP53 mutations in ALK+ NSCLC can lead to chromosomal instability: 24% of TP53-mutated patients showed amplifications of known cancer genes such as MYC (14%), CCND1 (10%), TERT (5%), BIRC2 (5%), ORAOV1 (5%), and YAP1 (5%). MYC-overexpressing ALK+ TP53-mutated cells had a proliferative advantage compared to wild-type cells. ChIP-Seq data revealed MYC-binding sites within the promoter region of EML4, and MYC overexpression in ALK+ TP53-mutated cells resulted in an upregulation of EML4-ALK, indicating a potential MYC-dependent resistance mechanism in patients with increased MYC copy number. Our study reveals that ALK+ NSCLC represents a more heterogeneous subgroup of tumours than initially thought, and that TP53 mutations in that particular cancer type define a subset of tumours that harbour chromosomal instability, leading to the co-occurrence of pathogenic aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The Emu-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 Emu-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 Emu-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.Copyright © 2017 The Author(s).

Ovarian granulosa cell tumors (GCT) are hormonally-active neoplasms characterized, in the adult-subtype, by a mutation in the FOXL2 gene (C134W). They exhibit an indolent course with an unexplained propensity for late recurrence; ~80% of patients with aggressive, advanced stage tumors die from their disease; aside from surgery, therapeutic options are limited. To identify the molecular basis of advanced stage disease we have used whole transcriptome analysis of FOXL2 C134W mutation positive adult (a)GCT to identify genes that are differentially expressed between early (stage 1) and advanced (stage 3) aGCT. Transcriptome profiles for early (n=6) and stage 3 (n=6) aGCT, and for the aGCT-derived KGN, cell line identified 24 genes whose expression significantly differs between the early and stage 3 aGCT. Of these, 16 were more abundantly expressed in the stage 3 aGCT and 8 were higher in the stage 1 tumors. These changes were further examined for the genes which showed the greatest fold change: the cytokine CXCL14, microfibrillar-associated protein 5, insulin-like 3 and desmin. Gene Set Enrichment Analysis identified overexpression of genes on chromosome 7p15 which includes the homeobox A gene locus. The analysis therefore identifies a small number of genes with clearly discriminate patterns of expression arguing that the clinicopathological-derived distinction of the tumor stage is robust, whilst confirming the relative homogeneity of expression for many genes across the cohort and hence of aGCT. The expression profiles do however identify several overexpressed genes in both stage 1 and/or stage 3 aGCT which warrant further study as possible therapeutic targets.

Large genomic rearrangements (LGRs) account for at least 10 % of the mutations in BRCA1 and 5 % of BRCA2 mutations in outbred hereditary breast and ovarian cancer (HBOC) families. Data from some series suggest LGRs represent particularly penetrant mutations. 1,034 index cases from HBOC families underwent comprehensive BRCA1 and BRCA2 mutation testing, including screening for LGRs. The personal and family history of 254 identified mutation carriers were compared based on mutation type. Thirty-six LGRs were detected; 32/122 (26 %) BRCA1 and 4/132 (3 %) BRCA2 mutations. High risk features (bilateral breast cancer, diagnosis <40 years, ovarian cancer, male breast cancer) were more commonly associated with an LGR than a non-LGR mutation (p = 0.008), In families with a BRCA1 LGR the mean age of breast cancer diagnosis was younger than in families with a non-LGR BRCA1 mutation (42.5 vs. 46.1 years, p = 0.007). Across the entire group of mutation positive families the number of relatives affected by breast or ovarian cancer was increased [LGR 3.7 vs. non- LGR 2.8 per family, p value (adjusted for genotype) = 0.047]. Excluding index cases, the odds ratio for breast cancer in BRCA1 families with an LGR was 1.42 (95 % CI 1.24-1.63) and for ovarian cancer 1.66 (95 % CI 1.10-2.49). The increased cancer risk was reflected in significantly higher risk assessments by mutation prediction tools. LGRs are associated with higher cancer risks. If validated, LGRs could be included in cancer risk prediction tools to improve personalised cancer risk prediction estimates and may guide cost-minimising mutation screening strategies in some healthcare settings.Copyright © 2015, Springer Science+Business Media Dordrecht.

Background: The Familial Cancer Centre (FCC) at Peter Mac Callum Cancer Centre (PMCC) and Monash Health (MH) offers a long term annual review program (ARP) for carriers of hereditary gene mutations. This program can identity patients with unmet information or support needs related to managing their cancer risk and in communicating genetic risk information to at risk relatives. The Genetic Counselling Outcome Scale (GCOS) is a validated Patient Reported Outcome Measure. It captures the construct of Empowerment, which aligns with the ARP aims to facilitate patient adjustment and enhance confidence in the self-management of cancer risk. Using the GCOS we assessed mutation carriers' levels of Empowerment during different stages of participation in the ARP. Method(s): 1304 patients from PMCC and MH FCC's have been invited to complete three GCOS questionnaires in addition to their clinical ARP questionnaire over a period of five years (June 2013-June 2018). Data analysis: Preliminary quantitative analysis was undertaken on data collected from June 2013 to July 2014 using a multiple linear regression analysis predicting GCOS total scores. Result(s): Data from the first 439 (33%) patients, who completed an initial GCOS (at a median interval of 6 years post testing) has been analysed to date. Lower age (p < 0.001) and longer duration in the ARP (p < 0.05) were both significantly associated with higher GCOS scores (i.e., greater empowerment) (p < 0.05). We will present data from the updated set of responders by November 2015. Conclusion(s): The initial results provide preliminary evidence for the positive impact of the ARP in assisting patients gain a greater sense of control over their increased cancer risk. The final data will determine whether ARPs can impact on patient's level of empowerment. We also plan to analyse patients Empowerment scores associated with their pariticpation in an ARP and with additional data regarding use of cancer risk management strategies, to dete

Prostate cancer is worldwide the sixth leading cause of cancer related death in men thus early detection and successful treatment are still of major interest. The commonly performed screening of the prostate-specific antigen (PSA) is controversially discussed, as in many patients the prostate-specific antigen levels are chronically elevated in the absence of cancer. Due to the unsatisfying efficiency of available prostate cancer screening markers and the current treatment outcome of the aggressive hormone refractory prostate cancer, the evaluation of novel molecular markers and targets is considered an issue of high importance. MicroRNAs are relatively stable in body fluids orchestrating simultaneously the expression of many genes. These molecules are currently discussed to bear a greater diagnostic potential than protein-coding genes, being additionally promising therapeutic drugs and/or targets. Herein we review the potential impact of the microRNA let-7 family on prostate cancer and show how deregulation of several of its target genes could influence the cellular equilibrium in the prostate gland, promoting cancer development as they do in a variety of other human malignant neoplasias.

Sarcomatous transformation of a granulosa cell tumor (GCT) is a rare event. We describe the development of a rapidly progressive sarcomatous change in a woman who had initially presented with a classical GCT. A first recurrence occurred 23 months after the initial diagnosis when she was treated with external beam radiotherapy to her pelvis. A second recurrence 76 months following her initial surgery was consistent with a GCT. At 92 months, she presented with a further recurrence, outside of the radiotherapy field. This last recurrence had a different histologic appearance with features of sarcomatous change. Molecular analysis, using both reverse transcription- polymerase chain reaction and complementary DNA microarrays, has been used to analyze tissue obtained before and after the observed change in the tumor. The data show that GCT-specific genes, such as inhibin alpha, estrogen receptor, and follicle-stimulating hormone receptor, have been downregulated in the sarcomatous change. Significant upregulation of genes associated with an inflammatory response was also noted in the sarcoma, and this was consistent with the presence of a marked inflammatory infiltrate seen on histopathology. This study represents the novel application of microarray technology and demonstrates the unexpected finding of expression of the fibroblast activation protein gene in normal ovary. Although tumors such as this may be targets for the novel fibroblast activating protein-directed chemotherapeutic monoclonal antibody sibrotuzumab, the finding of expression in the normal ovary suggests the need for caution. © 2007, IGCS and ESGO.

Background: Our study focuses on identifying potential biomarkers for diagnosis and early detection of ovarian cancer (OC) through the study of transcription regulation of genes affected by estrogen hormone.Results: The results are based on a set of 323 experimentally validated OC-associated genes compiled from several databases, and their subset controlled by estrogen. For these two gene sets we computationally determined transcription factors (TFs) that putatively regulate transcription initiation. We ranked these TFs based on the number of genes they are likely to control. In this way, we selected 17 top-ranked TFs as potential key regulators and thus possible biomarkers for a set of 323 OC-associated genes. For 77 estrogen controlled genes from this set we identified three unique TFs as potential biomarkers.Conclusions: We introduced a new methodology to identify potential diagnostic biomarkers for OC. This report is the first bioinformatics study that explores multiple transcriptional regulators of OC-associated genes as potential diagnostic biomarkers in connection with estrogen responsiveness. We show that 64% of TF biomarkers identified in our study are validated based on real-time data from microarray expression studies. As an illustration, our method could identify CP2 that in combination with CA125 has been reported to be sensitive in diagnosing ovarian tumors. 2011 Kaur et al; licensee BioMed Central Ltd.

Background: Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples. Principal Findings: As a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs). Conclusions: The ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes.