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Cancer of unknown primary (CUP) is a syndrome defined by clinical absence of a primary cancer after standardised investigations. Gene expression profiling (GEP) and DNA sequencing have been used to predict primary tissue of origin (TOO) in CUP and find molecularly guided treatments; however, a detailed comparison of the diagnostic yield from these two tests has not been described. Here, we compared the diagnostic utility of RNA and DNA tests in 215 CUP patients (82% received both tests) in a prospective Australian study. Based on retrospective assessment of clinicopathological data, 77% (166/215) of CUPs had insufficient evidence to support TOO diagnosis (clinicopathology unresolved). The remainder had either a latent primary diagnosis (10%) or clinicopathological evidence to support a likely TOO diagnosis (13%) (clinicopathology resolved). We applied a microarray (CUPGuide) or custom NanoString 18-class GEP test to 191 CUPs with an accuracy of 91.5% in known metastatic cancers for high-medium confidence predictions. Classification performance was similar in clinicopathology-resolved CUPs - 80% had high-medium predictions and 94% were concordant with pathology. Notably, only 56% of the clinicopathology-unresolved CUPs had high-medium confidence GEP predictions. Diagnostic DNA features were interrogated in 201 CUP tumours guided by the cancer type specificity of mutations observed across 22 cancer types from the AACR Project GENIE database (77,058 tumours) as well as mutational signatures (e.g. smoking). Among the clinicopathology-unresolved CUPs, mutations and mutational signatures provided additional diagnostic evidence in 31% of cases. GEP classification was useful in only 13% of cases and oncoviral detection in 4%. Among CUPs where genomics informed TOO, lung and biliary cancers were the most frequently identified types, while kidney tumours were another identifiable subset. In conclusion, DNA and RNA profiling supported an unconfirmed TOO diagnosis in one-third of CUPs otherwise unresolved by clinicopathology assessment alone. DNA mutation profiling was the more diagnostically informative assay. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Objective This study aimed to determine the healthcare experiences, quality of life, and psychosocial needs of patients with cancer of unknown primary (CUP) early after diagnosis; comparing their experiences to patients with advanced cancer of a known primary (non-CUP control patients) and published general population reference data where available. Methods This study was a cross-sectional, multi-site study comparing CUP patients (n = 139) compared to non-CUP controls (n = 45). Demographic, clinical information and patient-reported outcome questionnaire data were collected at baseline. Results Differences in healthcare experienced were found between CUP and non-CUP controls with CUP patients reporting higher scores for unmet medical communication/information needs compared with non-CUP control patients (p = 0.013) as well as greater uncertainty in illness (p = 0.042). Whilst no differences were found between CUP and non-CUP controls on the EORTC and PROMIS measures, of those that ‘received written information about your cancer…’ and asked ‘…how useful was it?’ fewer CUP patients reported finding the information useful 40% vs 61%, and more were likely to not have received written information at all 59% vs 32%; (p = 0.002). Additionally, of those that found information about their cancer online, fewer patients with CUP reported finding it useful 32% vs 48% control patients (p = 0.005). Conclusions CUP patients have unmet medical communication/information needs and greater uncertainty in illness but do not differ in health-related quality of life domains compared to patients with advanced cancer of a known primary.

Patients diagnosed with Cancer of Unknown Primary (CUP) experience high levels of psychological distress and report poor understanding of their cancer. We aimed to investigate: (1) if CUP patients with poorer understanding of their cancer diagnosis and testing experience more symptoms of psychological distress than those with better understanding; (2) if the relationship between patients' understanding of their cancer and psychological distress is mediated by illness uncertainty; and (3) explore whether patients' degree of understanding of their cancer can be predicted by clinical and socio-demographic factors.209 CUP patients completed a questionnaire measuring anxiety, depression, illness uncertainty, fatigue, pain, sleep and understanding of their cancer. Using an apriori theoretical framework, we employed structural equation modelling to investigate predictors of patient's understanding of their cancer and psychological distress and the relationships between understanding, illness uncertainty and distress.The structural equation model displayed good fit indices and supported the hypothesised relationship of patient's understanding of their cancer and the extent of psychological distress, which was mediated via illness uncertainty. Physical symptoms were positively associated with psychological distress and illness uncertainty. Younger age was predictive of lower patient's understanding of their cancer and higher levels of psychological distress.Patients with CUP, particularly those who are younger and experiencing more physical symptoms, report higher levels of psychological distress and may require additional mental health support. Our findings highlight a need to improve CUP patient's understanding about their illness, which could help reduce their illness uncertainty and alleviate psychological distress.

Background Diagnosis and management of cancers of unknown primary (CUP) remain challenging. This study examines the referral patterns, management and outcomes of patients referred to Australia’s first dedicated CUP clinic. Methods Retrospective medical record review was conducted for patients seen at the Peter MacCallum Cancer Centre CUP clinic between July 2014 and August 2020. Overall survival (OS) was analysed for patients with a CUP diagnosis where treatment information was available. Results Of 361 patients referred, fewer than half had completed diagnostic work-up at the time of referral. A diagnosis of CUP was established in 137 (38%), malignancy other than CUP in 177 (49%) and benign pathology in 36 (10%) patients. Genomic testing was successfully completed in 62% of patients with initial provisional CUP and impacted management in 32% by identifying a tissue of origin or actionable genomic alteration. The use of site-specific, targeted therapy or immunotherapy was independently associated with longer OS compared to empirical chemotherapy. Conclusion Our specialised CUP clinic facilitated diagnostic work-up among patients with suspected malignancy and provided access to genomic testing and clinical trials for patients with a CUP diagnosis, all of which are important to improve outcomes in this patient population.

A list of copy number alterations, rearrangements and short variants detected by Foundation Medicine NGS

Supplementary Tables, Figures and Video legends, Tables 2,3,5 and all Supplementary Figures. Supplementary Table 2. Confirmation of cis configuration of BRCA1 primary and secondary mutations in case 4 by colony PCR. Supplementary Table 3. IC50 (mircoM) values of the PARPi and platinum drugs in parental OVCAR8 cell line and OVCAR8 RAD51C KO clone, and the fold change in IC50 values. Supplementary Table 5. Sequences of primers used for site-directed mutagenesis. Supplementary Figure 1. Foundation Medicine NGS analysis of the 12 cases with archival tissue and/or pre-treatment and post-progression biopsies. Supplementary Figure 2. Sanger sequencing trace of the primary and secondary BRCA1 mutations in cis configuration in case 4 post-progression biopsy sample. Supplementary Figure 3. In vitro response to PARP inhibitor therapy and platinum agents in RAD51C deficient cell lines, with primary or secondary mutations in RAD51C. Supplementary Figure 4. RAD51 foci formation in geminin positive cells deficient for RAD51C, complemented with primary or secondary mutations in RAD51C. Supplementary Figure 5. Diagram of HR reporter assay. Supplementary Figure 6. RAD51C expression in MCF10A cells and in yeast. Supplementary Figure 7. Analysis of serial sections by direct PCR sequencing approach of a post-progression biopsy containing multiple secondary mutations in RAD51C. Supplementary Figure 8. Molecular Dynamics Modeling of WT RAD51D protein and RAD51D protein with secondary mutation c.770_776delinsA, p.S257_R259delinsK. Supplementary Figure 9. In vitro response to PARP inhibitor therapy and cisplatin in RAD51D deficient CHO cell line, with primary or secondary mutation in RAD51D. Supplementary Figure 10. Examination of the parental PEO4 cell line, PEO4 cells with the homozygous frameshift RAD51D mutation (c.762_763del, D254E*fs72) in the same exon as the primary mutation and PEO4 cells with the homozygous secondary RAD51D mutation (c.770_776delinsA, S257_R259delinsK). Supplementary Figure 11. Modeling of tumor clonal fractions in the post-progression biopsy sample with germline RAD51C mutation and multiple secondary mutations.

Molecular Dynamics Modeling of WT RAD51D monofilament interaction with dsDNA.

Copy number estimation by SNP array in the archival tumor tissue of the patient identified to have a germline RAD51C mutation (c.577C

Purpose: Maintenance therapy with olaparib has improved progression-free survival in women with high-grade serous ovarian cancer (HGSOC), particularly those harboring BRCA1/2 mutations. The objective of this study was to characterize long-term (LT) versus short-term (ST) responders to olaparib.Experimental Design: A comparative molecular analysis of Study 19 (NCT00753545), a randomized phase II trial assessing olaparib maintenance after response to platinum-based chemotherapy in HGSOC, was conducted. LT response was defined as response to olaparib/placebo >2 years, ST as BRCA1/2 status, three-biomarker homologous recombination deficiency (HRD) score, BRCA1 methylation, and mutational profiling. Another olaparib maintenance study (Study 41; NCT01081951) was used as an additional cohort.Results: Thirty-seven LT (32 olaparib) and 61 ST (21 olaparib) patients were identified. Treatment was significantly associated with outcome (P < 0.0001), with more LT patients on olaparib (60.4%) than placebo (11.1%). LT sensitivity to olaparib correlated with complete response to chemotherapy (P < 0.05). In the olaparib LT group, 244 genetic alterations were detected, with TP53, BRCA1, and BRCA2 mutations being most common (90%, 25%, and 35%, respectively). BRCA2 mutations were enriched among the LT responders. BRCA methylation was not associated with response duration. High myriad HRD score (>42) and/or BRCA1/2 mutation was associated with LT response to olaparib. Study 41 confirmed the correlation of LT response with olaparib and BRCA1/2 mutation.Conclusions: Findings show that LT response to olaparib may be multifactorial and related to homologous recombination repair deficiency, particularly BRCA1/2 defects. The type of BRCA1/2 mutation warrants further investigation. Clin Cancer Res; 23(15); 4086–94. ©2017 AACR.

Purpose: A significant number of women with serous ovarian cancer are intrinsically refractory to platinum-based treatment. We analyzed somatic DNA copy number variation and gene expression data to identify key mechanisms associated with primary resistance in advanced-stage serous cancers.Experimental Design: Genome-wide copy number variation was measured in 118 ovarian tumors using high-resolution oligonucleotide microarrays. A well-defined subset of 85 advanced-stage serous tumors was then used to relate copy number variation to primary resistance to treatment. The discovery-based approach was complemented by quantitative-PCR copy number analysis of 12 candidate genes as independent validation of previously reported associations with clinical outcome. Likely copy number variation targets and tumor molecular subtypes were further characterized by gene expression profiling.Results: Amplification of 19q12, containing cyclin E (CCNE1), and 20q11.22-q13.12, mapping immediately adjacent to the steroid receptor coactivator NCOA3, was significantly associated with poor response to primary treatment. Other genes previously associated with copy number variation and clinical outcome in ovarian cancer were not associated with primary treatment resistance. Chemoresistant tumors with high CCNE1 copy number and protein expression were associated with increased cellular proliferation but so too was a subset of treatment-responsive patients, suggesting a cell-cycle independent role for CCNE1 in modulating chemoresponse. Patients with a poor clinical outcome without CCNE1 amplification overexpressed genes involved in extracellular matrix deposition.Conclusions: We have identified two distinct mechanisms of primary treatment failure in serous ovarian cancer, involving CCNE1 amplification and enhanced extracellular matrix deposition. CCNE1 copy number is validated as a dominant marker of patient outcome in ovarian cancer.

Supplementary figures, tables and materials & methods Supplementary Figure 1. Schematic presentation of steps in immunoassay development, validation and studies for screening TP53 autoantibody in human serum samples. Supplementary Figure 2. Identifying a common cut-off value for TP53 autoantibody immunoassay. Supplementary Table 1. Characteristics of patients with invasive epithelial ovarian/tubal/peritoneal cancer in MDACC-NROSS set Supplementary Table 2. Characteristics of patients with invasive epithelial ovarian/tubal/peritoneal cancer in AOCS set Supplementary Table 3. Characteristics of patients with invasive epithelial ovarian/tubal/peritoneal cancer by stage and primary site in the UKCTOCS study Supplementary Figure 3. Examples of longitudinal analysis of CA125 and TP53 autoantibody titers in pre-diagnostic serial serum samples from ovarian cancer patients in the UKCTOCS Study. Supplementary Figure 4. ROC curve analysis for TP53 autoantibody and CA125 biomarkers in UKCTOCS trial. Supplementary Figure 5. Comparison of TP53 autoantibody titers between cancer cases with TP53 wild-type and mutant genes in the AOCS biobanking study. Supplementary Figure 6. The list of TP53 mutant protein candidates for preliminary screening of autoantibody against specific TP53 mutant proteins. Supplementary Table 4. Summary of specific TP53 mutant autoantibody titers of individual cancer patients with corresponding TP53 protein mutations in the AOCS biobanking study Supplementary Table 5. Summary of multiplex immunoassay results for screening TP53 mutant-specific autoantibody using samples from the AOCS biobanking study.

Supplemental Figures 1-6, Supplemental Tables 1-2, Supplemental Materials & Methods, Supplemental References Figure S1. Increased tumour growth is not a result of decreased apoptosis. Figure S2. Pericytes increase OVCAR-5 cell proliferation in vitro. Figure S3. Pericytes promote metastasis in vivo. Figure S4. Pericytes promote growth and initiate metastasis in the non-metastatic OVCAR-8 cells in vivo. Figure S5. Pericytes recruit αSMAcells expressing BM-MSC markers to OVCAR-5 xenografts. Figure S6. CD34 and PDGFRβ expression do not correlate with early relapse in serous ovarian cancer patients. Table S1: List of probe sets for the 146 overlapping genes up-regulated in both the pericytes and ovarian stroma. Table S2A-D: Top GO cellular component, molecular function & biological processes terms; and KEGG pathways enriched in early versus late relapse patients. Supplemental Methods: Details of methods used for in silico analyses, GFP transduction of cells, immunostaining, invasion assays and morphometric analyses.

Supplementary Tables S1-S6, Supplementary References. Supplementary Table S1. Characteristics of included studies; S2. Complete-case analysis of ten years estimated hazard ratios (HR) for all cause death in patients with ovarian cancer. S3. Complete-case analysis of ten years estimated subhazard ratios (SHR) for ovarian cancer death in patients with ovarian cancer. S4. Ten years estimated subhazard ratios (SHR) for ovarian cancer death in patients with ovarian cancer (with multiple imputation for missing grade and stage). S5. Complete-case analysis of ten years estimated hazard ratios (HR) for all cause death in patients with high grade serous ovarian cancer. S6. Ten years estimated hazard ratios (HR) for all cause death in patients with high grade serous ovarian cancer (with multiple imputation for missing stage).

Supplementary Data from Integrated Genome-Wide DNA Copy Number and Expression Analysis Identifies Distinct Mechanisms of Primary Chemoresistance in Ovarian Carcinomas

Supplementary Figure 1. Mutational analysis of TP53 in the olaparib arm. (A) Analysis of TP53 mutation type. (B) BRCA1 and BRCA2 mutation type Supplementary Table 1. Platinum sensitivity and outcome on olaparib maintenance treatment. Supplementary Table 2. Mutational analysis of the FoundationOne® panel from the LT and ST responders in the olaparib arm. Supplementary Table 3. Mutational analysis of PTEN in the olaparib and placebo arms. Supplementary Table 4. Analysis of BRCA1, BRCA2, and TP53 mutation frequency in olaparib patients in the LT and ST responder group.

Purpose: The TP53 tumor-suppressor gene is mutated in >95% of high-grade serous ovarian cancers. Detecting an autologous antibody response to TP53 that might improve early detection.Experimental Design: An immunoassay was developed to measure TP53 autoantibody in sera from 378 cases of invasive epithelial ovarian cancer and 944 age-matched healthy controls from the United States, Australia, and the United Kingdom. Serial preclinical samples from cases and controls were also assayed from the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS).Results: Using a cutoff value of 78 U/mL to achieve a specificity of 97.4%, TP53 autoantibody was elevated in 30% of 50 cases from MD Anderson, 21.3% of 108 cases from the Australian Ovarian Cancer Study, and 21% of 220 cases from the UKCTOCS. Among 164 cases with rising CA125 detected with the UKCTOCS risk of ovarian cancer algorithm (ROCA), 20.7% had elevated TP53 autoantibody. In cases missed by the ROCA, 16% of cases had elevated TP53 autoantibody. Of the 34 ovarian cancer cases detected with the ROCA, TP53 autoantibody titers were elevated 11.0 months before CA125. In the 9 cases missed by the ROCA, TP53 autoantibody was elevated 22.9 months before cancer diagnosis. Similar sensitivity was obtained using assays with specific mutant and wild-type TP53.Conclusions: TP53 autoantibody levels provide a biomarker with clinically significant lead time over elevation of CA125 or an elevated ROCA value. Quantitative assessment of autoantibodies in combination with CA125 holds promise for earlier detection of invasive epithelial ovarian cancer. Clin Cancer Res; 23(19); 5912–22. ©2017 AACR.

Methods, figures and tables

Gastric cancer is a leading cause of global cancer mortality, but comparatively little is known about the cellular pathways regulating different aspects of the gastric cancer phenotype. To achieve a better understanding of gastric cancer at the levels of systems topology, functional modules, and constituent genes, we assembled and systematically analyzed a consensus gene coexpression meta-network of gastric cancer incorporating >300 tissue samples from four independent patient populations (the “gastrome”). We find that the gastrome exhibits a hierarchical scale-free architecture, with an internal structure comprising multiple deeply embedded modules associated with diverse cellular functions. Individual modules display distinct subtopologies, with some (cellular proliferation) being integrated within the primary network, and others (ribosomal biosynthesis) being relatively isolated. One module associated with intestinal differentiation exhibited a remarkably high degree of autonomy, raising the possibility that its specific topological features may contribute towards the frequent occurrence of intestinal metaplasia in gastric cancer. At the single-gene level, we discovered a novel conserved interaction between the PLA2G2A prognostic marker and the EphB2 receptor, and used tissue microarrays to validate the PLA2G2A/EphB2 association. Finally, because EphB2 is a known target of the Wnt signaling pathway, we tested and provide evidence that the Wnt pathway may also similarly regulate PLA2G2A. Many of these findings were not discernible by studying the single patient populations in isolation. Thus, besides enhancing our knowledge of gastric cancer, our results show the broad utility of applying meta-analytic approaches to genome-wide data for the purposes of biological discovery. (Cancer Res 2006; 66(1): 232-41)

BackgroundCancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers where a primary tissue of origin (TOO) is uncertain. Most patients with CUP have limited treatment options and poor survival outcomes. Immune checkpoint inhibitors (ICIs) can be efficacious in some patients with CUP, but the optimal predictive biomarkers are unknown. We therefore assessed immune and genomic biomarkers as well as predicted TOO in patients with CUP, including a subset treated with ICIs.MethodsPatients with CUP were subject to gene-expression profiling (GEP) and DNA panel sequencing. Immune and stromal-related gene expression was explored by NanoString, including genes associated with immunotherapy response (IR) in other solid malignancies. ICI responsive cancer types were assigned based on Food and Drug Administration-approved indications, and either detection of a latent primary tumor or the TOO was suspected based on genomics informed pathology review. Tumor mutation burden (TMB) and gene mutations were also assessed.ResultsA total of 219 patients with CUP were included, 215 assessed for TOO in a previous study, with the majority (163) receiving both RNA and DNA tests. Of GEP profiled cases, 33% (59/175) had a high IR gene-expression score. Of the DNA sequenced cases, 16% (32/203) had high TMB (>10 mutations/Mb), including two with mismatch repair deficiency. Low correlation was observed between TMB and an IR score (R=0.26, pConclusionsA significant fraction of CUP tumors had genomic features previously associated with ICI response. High IR score was the most sensitive predictive feature of ICI response, warranting evaluation in a larger patient series.

SummaryUnderstanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.

Low-grade serous ovarian cancer (LGSC) responds poorly to platinum based chemotherapy and is characterized by activating RAS-MAPK pathway mutations, including oncogenic BRAF. Drugs that target this pathway are effective in BRAF-mutant melanoma but other cancer types, such as colorectal cancers, are less sensitive. Early phase trials report a 15% response rate to MEK inhibitors in LGSC patients, however it is not known which features may predict response. We aimed to determine clinical characteristics and treatment response in women with LGSC and to determine whether response to targeted pathway inhibition is associated with specific mutation profiles in LGSC cell lines. Tumor samples from a cohort of grade 1 and 2 serous ovarian cancer patients were analyzed using targeted, exome or whole genome sequencing. Patient characteristics, treatment and clinical outcome were assessed. Cell lines derived from patients with LGSC with known mutation profiles, AOCS2 (KRAS/BRAF/NRAS wild-type), MPSC1 (BRAFV600L, NRASQ16R), VOA1056 (NRASQ16R) and HCC5075 (KRASG12V), were treated with BRAF (dabrafenib) or MEK inhibitors (trametinib, pimasertib and binimetinib) and response was compared to cell lines derived from high-grade serous cancer (HGSC) and BRAF-mutant melanoma. Women diagnosed with grade 1 or 2 serous carcinoma between 1994-2015 were identified from 1654 invasive ovarian serous cancer cases in the Australian Ovarian Cancer Study and the GynBiobank. HGSC cases were excluded following histopathology review and TP53 mutation screening. Amongst 65 confirmed LGSC patients, 18 (27.7%) had a KRAS mutation, 9 (13.8%) had a BRAF mutation and 7 (10.8%) had a NRAS mutation. Women with advanced stage LGSC and residual disease following debulking surgery had a similarly poor progression-free and overall-survival compared with HGSC patients. We saw a dramatic response to BRAF inhibition in a patient with BRAFV600E-positive LGSC, however, the LGSC cell lines did not respond to dabrafenib. This is not surprising as the cell lines did not harbour the hot-spot BRAFV600E mutation. MPSC1 has a BRAFV600L and a NRASQ16R mutation, but dabrafenib did not inhibit growth. HCC5075 (KRASG12V) was sensitive to all three MEK inhibitors, but response to MEK inhibition in the other LGSC cell lines was modest. In conclusion, LGSC are generally chemotherapy resistant and molecular analyses can identify targetable mutations. However, LGSC are heterogenous with respect to underlying mutations and response to pathway inhibitors is likely to depend on which mutations and pathways are activated. BRAF mutations are not uncommon in patients with LGSC and should be routinely tested as BRAF inhibitors can be an effective treatment for these patients. MEK inhibitors may also be effective in a subset of cases. The results highlight the need for novel clinical trial design, as traditional clinical trials are unlikely to be effective in rare ovarian cancer sub-types. Citation Format: Tania Moujaber, Dariush Etemadmoghadam, Cristina Mapagu, Catherine Kennedy, Yoke-Eng Chiew, Casina Kan, Nikilyn Nevins, Sivatharsny Srirangan, Sian Fereday, Nadia Traficante, Australian Ovarian Cancer Study group, David Bowtell, Rosemary Balleine, Paul Harnett, Anna deFazio. Mutations in low-grade serous ovarian cancer and response to BRAF and MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2584.

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

ABCB1 (adenosine triphosphate-binding cassette transporter B1) mediates cellular elimination of many chemotherapeutic agents including paclitaxel, which is commonly used to treat ovarian cancer. A significant association between common single nucleotide polymorphisms (SNPs) in ABCB1 and progression-free survival has been reported in patients with ovarian cancer. Variable paclitaxel clearance due to genotype specific differences in ABCB1 activity in cancer cells and/or normal tissues may underlie the association. Using cell-based models, we evaluated the correlations between ABCB1 expression, polymorphisms, transporter activity and paclitaxel sensitivity in ovarian cancer (n = 10) and lymphoblastoid (n = 19) cell lines. Close associations between ABCB1 expression, transporter function and paclitaxel sensitivity were found in lymphoblastoid cell lines, although we could not demonstrate an association with common SNPs. In ovarian cancer cell lines, ABCB1 expression was low and the association between expression and function was lost. These results suggest that ABCB1 related survival difference in ovarian cancer patients is more likely to be due to differential whole body paclitaxel clearance mediated by normal cells rather than a direct effect on cancer cells.

Background: The E3 ubiquitin ligases SIah1 and 2 mediate a cellular response to hypoxia through its control of prolyl-hydroxylases and consequently HIF1α stability. Given the impact of hypoxia on heart function under stress, we have evaluated the effect of genetic deletion of Siah1 and 2 on cardiac function in response to myocardial infarction. Objective: To determine the physiological significance of the ubiquitin ligase Siah genes under in vivo ischemia condition. Materials and methods: To substantiate the role of Siah2 in ischemia-induced cell death, we tested difference in the response of myocardial tissue to ischemia using Siah1a +/− /Siah2 −/− mutant mice and their wildtype littermates. Analysis of heart tissue from Siah1a +/− /Siah2 −/− mice subjected to a model of 24 hours anterior descending coronary artery occlusion revealed their resistance to ischemic injury when compared with their littermates. Cardiac function, measured as EF %, was significantly less impaired in the mutant mice. Heart tissue obtained from Siah1a +/− /Siah2 −/− mice subjected to myocardial ischemia, exhibited a significantly lower degree of apoptosis (Tunel Staining), and myocardial infarct size (Evans blue, TTC). Conclusions: Collectively, these data establish that reduced expression of Siah1/2 exerts a cardioprotective function under ischemic conditions. The underlying molecular mechanisms linking cardioprotection to ischemic challenge will be discussed.

Purpose: A significant number of women with serous ovarian cancer are intrinsically refractory to platinum-based treatment. We analyzed somatic DNA copy number variation and gene expression data to identify key mechanisms associated with primary resistance in advanced-stage serous cancers. Experimental Design: Genome-wide copy number variation was measured in 118 ovarian tumors using high-resolution oligonucleotide microarrays. A well-defined subset of 85 advanced-stage serous tumors was then used to relate copy number variation to primary resistance to treatment. The discovery-based approach was complemented by quantitative-PCR copy number analysis of 12 candidate genes as independent validation of previously reported associations with clinical outcome. Likely copy number variation targets and tumor molecular subtypes were further characterized by gene expression profiling. Results: Amplification of 19q12, containing cyclin E (CCNE1), and 20q11.22-q13.12, mapping immediately adjacent to the steroid receptor coactivator NCOA3, was significantly associated with poor response to primary treatment. Other genes previously associated with copy number variation and clinical outcome in ovarian cancer were not associated with primary treatment resistance. Chemoresistant tumors with high CCNE1 copy number and protein expression were associated with increased cellular proliferation but so too was a subset of treatment-responsive patients, suggesting a cell-cycle independent role for CCNE1 in modulating chemoresponse. Patients with a poor clinical outcome without CCNE1 amplification overexpressed genes involved in extracellular matrix deposition. Conclusions: We have identified two distinct mechanisms of primary treatment failure in serous ovarian cancer, involving CCNE1 amplification and enhanced extracellular matrix deposition. CCNE1 copy number is validated as a dominant marker of patient outcome in ovarian cancer.

Background: X chromosome inactivation, which silences gene expression from one of the two X chromosomes in females, is usually random. Skewed X inactivation has been implicated in both the expression and the suppression of X-linked disease phenotypes and has been reported to occur more frequently in breast and ovarian cancer patients, including BRCA1 or BRCA2 mutation carriers, than in control subjects. Methods: We assessed the pattern of X chromosome inactivation using methylation-specific polymerase chain reaction amplification of the exon 1 microsatellite region of the X-linked androgen receptor (AR) gene in DNA from blood samples obtained from control subjects without a personal history of breast or ovarian cancer (n = 735), ovarian cancer patients (n = 313), familial breast cancer patients who did not carry mutations in BRCA1 or BRCA2 (n = 235), and affected and unaffected carriers of mutations in BRCA1 (n = 260) or BRCA2 (n = 63). We defined the pattern of X chromosome inactivation as skewed when the same X chromosome was active in at least 90% of cells. The association between skewed X inactivation and disease and/or BRCA mutation status was assessed by logistic regression analysis. The association between skewed X inactivation and age at cancer diagnosis was assessed by Cox proportional hazards regression analysis. All statistical tests were two-sided. Results: The age-adjusted frequency of skewed X inactivation was not statistically significantly higher in ovarian cancer or familial breast cancer case subjects compared with control subjects. Skewed X inactivation was higher in BRCA1 mutation carriers than in control subjects (odds ratio [OR] = 2.7, 95% confidence interval [CI] = 1.1 to 6.2; P = .02), particularly among unaffected women (OR = 6.1, 95% CI = 1.5 to 31.8; P = .005). Among BRCA1 mutation carriers, those with skewed X inactivation were older at diagnosis of breast or ovarian cancer than those without skewed X inactivation (hazard ratio [HR] of breast or ovarian cancer = 0.37, 95% CI = 0.14 to 0.95; P = .04). Among BRCA2 mutation carriers, skewed X inactivation also occurred more frequently in unaffected carriers than in those diagnosed with breast or ovarian cancer (OR = 5.2, 95% CI = 0.5 to 28.9; P = .08) and was associated with delayed age at onset (HR = 0.59, 95% CI = 0.37 to 0.94; P = .03). Conclusions: Skewed X inactivation occurs at an increased frequency in BRCA1 (and possibly BRCA2) mutation carriers compared with control subjects and is associated with a statistically significant increase in age at diagnosis of breast and ovarian cancer. © The Author 2008

Transforming growth factor-beta (TGF-beta) is thought to regulate ductal and lobuloalveolar development as well as involution in the mammary gland. In an attempt to understand the role TGF-beta plays during normal mammary gland development, and ultimately cancer, we previously generated transgenic mice that express a dominant-negative TGF-beta type II receptor under control of the metallothionine promoter (MT-DNIIR). Upon stimulation with zinc sulfate, the transgene was expressed in the mammary stroma and resulted in an increase in ductal side branching. In this study, mammary gland transplantation experiments confirm that the increase in side branching observed was due to DNIIR activity in the stroma. Development during puberty through the end buds was also accelerated. Cbl is a multifunctional intracellular adaptor protein that regulates receptor tyrosine kinase ubiquitination and downregulation. Mice with a targeted disruption of the c-Cbl gene displayed increased side branching similar to that observed in MT-DNIIR mice; however, end bud development during puberty was normal. Transplantation experiments showed that the mammary stroma was responsible for the increased side branching observed in Cbl-null mice. Cbl expression was reduced in mammary glands from DNIIR mice compared to controls and TGF-beta stimulated expression of Cbl in cultures of primary mammary fibroblasts. In addition, both TGF-beta and Cbl regulated platelet-derived growth factor receptor-alpha (PDGFR alpha) expression in vivo and in isolated mammary fibroblasts. The hypothesis that TGF-beta mediates the levels of PDGFR alpha protein via regulation of c-Cbl was tested. We conclude that TGF-beta regulates PDGFR alpha in the mammary stroma via a c-Cbl-independent mechanism. Finally, the effects of PDGF-AA on branching were determined. Treatment in vivo with PDGF-AA did not affect branching making a functional interaction between TGF-beta and PDGF unlikely.

Current epidemiological evidence supports a pathogenetic model of gastric cancer involving intermediate stages that include chronic gastritis and intestinal metaplasia. This study explores the molecular features of gastric cancer and premalignant stages using DNA microarray-based gene expression profiling and relates these findings to clinical, pathological, and ethnic parameters. A total of 124 tumor and adjacent mucosa samples were analyzed using spotted cDNA microarrays containing 9381 nonredundant gene elements. Tumor specimens were diffuse, intestinal, or mixed gastric cancer and adjacent mucosa, which generally displayed signs of chronic gastritis or intestinal metaplasia. Expression patterns could be discerned that readily defined premalignant and tumor subtypes. Chronic gastritis exhibits a pronounced mitochondrial gene expression signature, which may be linked to Helicobacter pylori pathogenesis. Intestinal metaplasia was associated with increased expression of many intestinal differentiation genes, many of which were not overexpressed in tumors. Samples were obtained from 91 Australian and 33 Chinese patients to explore potential variation in gene expression between these populations. Despite differences in the incidence, and potentially the etiology, of gastric cancer between these ethnic groups, we found the tumors to be molecularly similar. The identification of molecular signatures that are characteristic of subtypes of gastric cancer and associated premalignant changes should enable further analysis of the steps involved in the initiation and progression of this disease.

Background: The five year survival rate for women with ovarian cancer (OC) is less than 40%. Crucial to improving the outcomes for ovarian cancer patients is the development of accurate pre-clinical models of human epithelial OC, which can be used to unravel the mechanisms underlying its evolution and behaviour. Methods: We have generated a novel xenograft model of human high-grade serous OC (HG-SOC) in order to enable preclinical molecular and drug response assessment of individual HG-SOC: this includes a comparison of standard OC xeno-transplantion approaches (intra-peritoneal, subcutaneous and sub renal capsular), with the novel use of the rodent ovarian bursa, the orthotopic site. Utilising the rodent bursa may better replicate the relevant microenvironment and increase the success of EOC xeno-transplantion. Other aspects designed to optimize the model include the use of NOD-SCID-IL-2rg recipient mice, systemic estrogen supplementation and transplantation of fresh human HG-SOC fragments which have had no prior in vitro culture. Histological, functional and molecular analysis of the novel xenograft cohort (at baseline and following xenotransplantation) includes histological review; documentation of in vitro Homologous Recombination (HR) DNA repair and drug response capabilities (using novel α-irradiation and explant drug assays); classification according to molecular subtype (Tothill classfier); documentation of NHEJ pathway status, BRCA1/2 status and other DNA repair gene status. In vivo drug treatment studies are being performed, with the choice of treatment targeted to the specific molecular characteristics of the HG-SOC in question. Results: Fourteen consecutive chemotherapy naive potentially HG-SOC samples have been collected. Corresponding clinical data has been collected. Data concerning DNA repair capability and response to DNA damaging agents will be presented, including IHC for markers of DNA damage (γH2AX), DNA repair (RAD51) and apoptosis (capsase 3 cleavage). HG-SOC in this cohort have been classified according to Tothilll (Tothill et al 2008). Eight appropriate HG-SOC have been transplanted and 5 of the first 6 have successfully xenografted, with phenotyping of xenografts underway. In vivo analysis of response to cisplatin treatment and other relevant therapeutics will be presented. Conclusions: A novel xenograft model has been developed of human HG-SOC, which includes comparative characterization of important prognostic features both in the baseline panel of fresh human HG-SOC and in subsequent xenografts. This clinically, functionally and molecularly annotated consecutive xenograft cohort of HG-SOC will provide outstanding utility for the development of improved therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3276. doi:1538-7445.AM2012-3276