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Purpose: Advanced-stage mucinous ovarian carcinoma (MOC) has poor chemotherapy response and prognosis and lacks biomarkers to aid stage I adjuvant treatment. Differentiating primary MOC from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathologic and gene-expression data were analyzed to identify prognostic and diagnostic features. Experimental Design: Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n = 333), mucinous borderline ovarian tumors (MBOT, n = 151), and upper GI (n = 65) and lower GI tumors (n = 55). Results: Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2 years from diagnosis, compared with expansile pattern in stage I MOC [hazard ratio (HR), 2.77; 95% confidence interval (CI), 1.04–7.41, P = 0.042]. Increased expression of THBS2 and TAGLN was associated with shorter OS in MOC patients (HR, 1.25; 95% CI, 1.04–1.51, P = 0.016) and (HR, 1.21; 95% CI, 1.01–1.45, P = 0.043), respectively. ERBB2 (HER2) amplification or high mRNA expression was evident in 64 of 243 (26%) of MOCs, but only 8 of 243 (3%) were also infiltrative (4/39, 10%) or stage III/IV (4/31, 13%). Conclusions: An infiltrative growth pattern infers poor prognosis within 2 years from diagnosis and may help select stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confers an adverse prognosis and is upregulated in the infiltrative subtype, which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies.

Supplementary Methods

Supplementary Figures S1-S13

Purpose:Advanced-stage mucinous ovarian carcinoma (MOC) has poor chemotherapy response and prognosis and lacks biomarkers to aid stage I adjuvant treatment. Differentiating primary MOC from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathologic and gene-expression data were analyzed to identify prognostic and diagnostic features.Experimental Design:Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n = 333), mucinous borderline ovarian tumors (MBOT, n = 151), and upper GI (n = 65) and lower GI tumors (n = 55).Results:Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2 years from diagnosis, compared with expansile pattern in stage I MOC [hazard ratio (HR), 2.77; 95% confidence interval (CI), 1.04–7.41, P = 0.042]. Increased expression of THBS2 and TAGLN was associated with shorter OS in MOC patients (HR, 1.25; 95% CI, 1.04–1.51, P = 0.016) and (HR, 1.21; 95% CI, 1.01–1.45, P = 0.043), respectively. ERBB2 (HER2) amplification or high mRNA expression was evident in 64 of 243 (26%) of MOCs, but only 8 of 243 (3%) were also infiltrative (4/39, 10%) or stage III/IV (4/31, 13%).Conclusions:An infiltrative growth pattern infers poor prognosis within 2 years from diagnosis and may help select stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confers an adverse prognosis and is upregulated in the infiltrative subtype, which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies.

Supplementary Tables S1-S11

In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene RAD51C are established drivers of defective homologous recombination and are emerging biomarkers of PARP inhibitor (PARPi) sensitivity. RAD51C promoter methylation (meRAD51C) is detected at similar frequencies to mutations, yet its effects on PARPi responses remain unresolved. In this study, three HGSC patient-derived xenograft (PDX) models with methylation at most or all examined CpG sites in the RAD51C promoter show responses to PARPi. Both complete and heterogeneous methylation patterns were associated with RAD51C gene silencing and homologous recombination deficiency (HRD). PDX models lost meRAD51C following treatment with PARPi rucaparib or niraparib, where a single unmethylated copy of RAD51C was sufficient to drive PARPi resistance. Genomic copy number profiling of one of the PDX models using SNP arrays revealed that this resistance was acquired independently in two genetically distinct lineages. In a cohort of 12 patients with RAD51C-methylated HGSC, various patterns of meRAD51C were associated with genomic “scarring,” indicative of HRD history, but exhibited no clear correlations with clinical outcome. Differences in methylation stability under treatment pressure were also observed between patients, where one HGSC was found to maintain meRAD51C after six lines of therapy (four platinum-based), whereas another HGSC sample was found to have heterozygous meRAD51C and elevated RAD51C gene expression (relative to homozygous meRAD51C controls) after only neoadjuvant chemotherapy. As meRAD51C loss in a single gene copy was sufficient to cause PARPi resistance in PDX, methylation zygosity should be carefully assessed in previously treated patients when considering PARPi therapy. Significance: Homozygous RAD51C methylation is a positive predictive biomarker for sensitivity to PARP inhibitors, whereas a single unmethylated gene copy is sufficient to confer resistance.

We look at how strongly shocks to wealth affect labour supply, using Italian data. We use asset price shocks to provide a measure of wealth changes that is exogenous to the household's saving and labour supply. Results point to significant effects of wealth on: hours of work; whether agents leave their jobs; and, labour earnings. The magnitude of these effects can be substantial, for example for individuals who suffered larger wealth losses during the financial crisis. Responses are similar for men and women on average, but older working‐age individuals have relatively strong responses that drive the population results. Short‐run effects are somewhat persistent.

Female fertility and offspring health are critically dependent on an adequate supply of high-quality oocytes, the majority of which are maintained in the ovaries in a unique state of meiotic prophase arrest. While mechanisms of DNA repair during meiotic recombination are well characterized, the same is not true for prophase-arrested oocytes. Here we show that prophase-arrested oocytes rapidly respond to γ-irradiation–induced DNA double-strand breaks by activating Ataxia Telangiectasia Mutated, phosphorylating histone H2AX, and localizing RAD51 to the sites of DNA damage. Despite mobilizing the DNA repair response, even very low levels of DNA damage result in the apoptosis of prophase-arrested oocytes. However, we show that, when apoptosis is inhibited, severe DNA damage is corrected via homologous recombination repair. The repair is sufficient to support fertility and maintain health and genetic fidelity in offspring. Thus, despite the preferential induction of apoptosis following exogenously induced genotoxic stress, prophase-arrested oocytes are highly capable of functionally efficient DNA repair. These data implicate DNA repair as a key quality control mechanism in the female germ line and a critical determinant of fertility and genetic integrity.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease., Acquired resistance limits the efficacy of PARP inhibitors (PARPi) in high grade serous ovarian cancer (HGSOC). Here, the authors show that inhibition of RNA polymerase I transcription using CX-5461 increases the therapeutic efficacy of PARPi and overcomes PARPi resistance in PDX models of HGSOC.

Objective Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. Methods We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117–166). Results Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). Conclusions Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

PURPOSE The ALLOCATE study was designed as a pilot to demonstrate the feasibility and clinical utility of real-time targeted molecular profiling of patients with recurrent or advanced ovarian cancer for identification of potential targeted therapies. PATIENTS AND METHODS A total of 113 patients with ovarian cancer of varying histologies were recruited from two tertiary hospitals, with 99 patient cases suitable for prospective analysis. Targeted molecular and methylation profiling of fresh biopsy and archived tumor samples were performed by screening for mutations or copy-number variations in 44 genes and for promoter methylation of BRCA1 and RAD51C. RESULTS Somatic genomic or methylation events were identified in 85% of all patient cases, with potentially actionable events with defined targeted therapies (including four resistance events) detected in 60% of all patient cases. On the basis of these findings, six patients received molecularly guided therapy, three patients had unsuspected germline cancer–associated BRCA1/ 2 mutations and were referred for genetic counseling, and two intermediate differentiated (grade 2) serous ovarian carcinomas were reclassified as low grade, leading to changes in clinical management. Additionally, secondary reversion mutations in BRCA1/ 2 were identified in fresh biopsy samples of two patients, consistent with clinical platinum/poly (ADP-ribose) polymerase inhibitor resistance. Timely reporting of results if molecular testing is done at disease recurrence, as well as early referral for patients with platinum-resistant cancers, were identified as factors that could improve the clinical utility of molecular profiling. CONCLUSION ALLOCATE molecular profiling identified known genomic and methylation alterations of the different ovarian cancer subtypes and was deemed feasible and useful in routine clinical practice. Better patient selection and access to a wider range of targeted therapies or clinical trials will further enhance the clinical utility of molecular profiling.

We use individual life history data from 12 European countries, and duration analysis that controls for unobserved heterogeneity, to investigate whether observed patterns of investment in illiquid assets and housing are consistent with predictions based on "temptation preferences". A motivating model takes into account the standard motives for saving, but also recognises that illiquid financial assets and housing may be used by individuals who find it hard to control the temptation linked to abundant cash on hand. Even controlling for many possible motives for purchasing housing, and for unobserved heterogeneity, a large fraction of individuals become significantly more likely to buy housing after investing in illiquid assets; this finding is consistent with the predictions of the model.

Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

Mucinous ovarian carcinoma (MOC) is a unique subtype of ovarian cancer with an uncertain etiology, including whether it genuinely arises at the ovary or is metastatic disease from other organs. In addition, the molecular drivers of invasive progression, high-grade and metastatic disease are poorly defined. We perform genetic analysis of MOC across all histological grades, including benign and borderline mucinous ovarian tumors, and compare these to tumors from other potential extra-ovarian sites of origin. Here we show that MOC is distinct from tumors from other sites and supports a progressive model of evolution from borderline precursors to high-grade invasive MOC. Key drivers of progression identified are TP53 mutation and copy number aberrations, including a notable amplicon on 9p13. High copy number aberration burden is associated with worse prognosis in MOC. Our data conclusively demonstrate that MOC arise from benign and borderline precursors at the ovary and are not extra-ovarian metastases., Whether mucinous ovarian carcinoma (MOC) arises from cells at the ovary or from metastases from other primary sites is an unanswered question. Here, Cheasley et al perform a genetic analysis of the disease, showing that MOC arises at the ovary.

Devil facial tumour disease (DFTD) comprises two genetically distinct transmissible cancers (DFT1 and DFT2) endangering the survival of the Tasmanian devil (Sarcophilus harrisii) in the wild. DFT1 first arose from a cell of the Schwann cell lineage; however, the tissue-of-origin of the recently discovered DFT2 cancer is unknown. In this study, we compared the transcriptome and proteome of DFT2 tumours to DFT1 and normal Tasmanian devil tissues to determine the tissue-of-origin of the DFT2 cancer. Our findings demonstrate that DFT2 expresses a range of Schwann cell markers and exhibits expression patterns consistent with a similar origin to the DFT1 cancer. Furthermore, DFT2 cells express genes associated with the repair response to peripheral nerve damage. These findings suggest that devils may be predisposed to transmissible cancers of Schwann cell origin. The combined effect of factors such as frequent nerve damage from biting, Schwann cell plasticity and low genetic diversity may allow these cancers to develop on rare occasions. The emergence of two independent transmissible cancers from the same tissue in the Tasmanian devil presents an unprecedented opportunity to gain insight into cancer development, evolution and immune evasion in mammalian species.

Adrenal chromaffin cells and sympathetic neurons synthesize and release catecholamines, and both cell types are derived from neural crest precursors. However, they have different developmental histories, with sympathetic neurons derived directly from neural crest precursors while adrenal chromaffin cells arise from neural crest-derived cells that express Schwann cell markers. We have sought to identify the genes, including imprinted genes, which regulate the development of the two cell types in mice. We developed a method of separating the two cell types as early as E12.5, using differences in expression of enhanced yellow fluorescent protein driven from the tyrosine hydroxylase gene, and then used RNA sequencing to confirm the characteristic molecular signatures of the two cell types. We identified genes differentially expressed by adrenal chromaffin cells and sympathetic neurons. Deletion of a gene highly expressed by adrenal chromaffin cells, NIK-related kinase, a gene on the X-chromosome, results in reduced expression of adrenaline-synthesizing enzyme, phenyl-N-methyl transferase, by adrenal chromaffin cells and changes in cell cycle dynamics. Finally, many imprinted genes are up-regulated in chromaffin cells and may play key roles in their development.

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.

Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated in the tumor, leading to homologous recombination deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived xenograft PH039, which lacks demonstrable repair gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by repeated 21-day niraparib treatments in vivo. PH039 acquired PARPi resistance by the third cycle of treatment and demonstrated unimpeded growth during subsequent exposure to either niraparib or rucaparib. Transcriptional profiling throughout the time course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of RAD51C methylation in patient tumor samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated that loss of RAD51C methylation prior to on-study biopsy was associated with limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide an important model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

While loss of BRCA1 promoter methylation has been shown to cause PARP inhibitor (PARPi) resistance in high-grade serous ovarian carcinoma (HGSC), the impacts of RAD51C methylation (meRAD51C) remain unresolved. In this study, three PARPi-responsive HGSC patient-derived xenografts (PDX) with RAD51C gene silencing and homologous recombination deficiency were found to have either homogeneous or heterogeneous patterns of meRAD51C. PDX could lose meRAD51C following PARPi treatment (rucaparib/niraparib), where a single unmethylated RAD51C copy was sufficient to drive PARPi-resistance. Genomic profiling revealed this resistance was acquired independently in two distinct PDX lineages. Furthermore, we describe a patient sample where 1/3 RAD51C gene copies lost methylation following neoadjuvant chemotherapy. We show meRAD51C is a positive predictive biomarker for PARPi response and should be screened for routinely in patients. However, methylation loss in a single gene copy is sufficient to cause PARPi resistance and should be carefully assessed in previously treated patients considering PARPi therapy.

We look at how strongly shocks to wealth affect labour supply, using Italian data. We use asset price shocks to provide a measure of wealth changes that is exogenous to the household’s saving and labour supply. Results point to significant effects of wealth on: hours of work; whether agents leave their jobs; and, labour earnings. The magnitude of these effects can be substantial, for example for individuals who suffered larger wealth losses during the financial crisis. Responses are similar for men and women on average, but older working‐age individuals have relatively strong responses that drive the population results. Short‐run effects are somewhat persistent.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumour type with limited treatment options. OCS is hypothesised to develop via the combination theory from a single progenitor, resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). We show OCS from 18 women to be monoclonal through analysis of DNA variants from isolated carcinoma and sarcoma components. RNA sequencing indicated the carcinoma components were more mesenchymal when compared with pure ovarian carcinomas, supporting the conversion theory. We used pre-clinical OCS models to test the efficacy of microtubule-targeting drugs, including eribulin, which has been shown to reverse EMT characteristics. We demonstrated that microtubule inhibitors, vinorelbine and eribulin, were more effective than standard-of-care platinum-based chemotherapy. Eribulin reduced mesenchymal characteristics, N-MYC expression and cholesterol biosynthesis. Finally, eribulin induced a strong immune response, supporting immunotherapy combinations in the clinic.

Recently, genetic analyses of primary mucinous ovarian tumours (MOT) have considerably enhanced our understanding of the biology of such neoplasms, supporting a progressive model of carcinogenesis from benign/borderline tumours to carcinomas. Nevertheless, the histogenesis of these neoplasms remains a subject of discussion and several cell types of origin have been proposed; a proportion of these tumours are associated with Brenner (transitional cell) tumours, therefore some are believed to be derived from metaplastic mucinous epithelium lining cystic transitional cell nests (Walthard rests).

High-grade mucinous ovarian cancer (HGMOC) is often a misnomer as the majority of cases are metastatic disease with a gastro-intestinal origin. The standard platinum-based ovarian cancer (OC) chemotherapy regimens are often ineffective, and there are insufficient data to support the use of colorectal cancer (CRC) chemotherapy regimens due to the rarity of HGMOC. We described a cohort of four consecutive suspected HGMOC cases treated at the Royal Women’s Hospital, Melbourne in 2012. Two cases were treated as primary MOC, whereas the other two were considered to be metastatic CRC based on histopathological and clinical evidence. From the RNAseq analysis, we identified two cases of HGMOC whose gene expression profiles were consistent with mucinous epithelial OC, one case that was treated as metastatic CRC with gene expression profile correlated with CRC and one case with neuroendocrine (NET) gene expression features. Interestingly, glucagon was over-expressed in this tumor that was subsequently confirmed by immunohistochemistry. These findings suggest a rare glucagonoma-like NET appendiceal tumor that had metastasized to the surface of ovary and were unresponsive to CRC chemotherapy regimens. In summary, a carefully curated panel of expression markers and selected functional genomics could provide diagnosis and treatment guidance for patients with possible HGMOC.

Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence1,2. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function3-5. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.

The high mortality (>80%) seen in high-grade serous ovarian cancer (HGSOC) highlights the unmet clinical need for treatments capable of producing long-term sustained responses. The related cancer, ovarian carcinosarcoma (OCS), has even poorer survival rates, which is due in part to being a rare cancer but also because of its unique phenotype. OCS are composed of both epithelial (carcinoma) and mesenchymal (sarcoma) components and molecular analysis has shown that most OCS are monoclonal and are derived from a common progenitor with sarcomatous transformation occurring by a stable epithelial-to-mesenchymal transition (EMT) process involving reprogramming of gene expression. Paclitaxel is an effective microtubule-stabilizing drug that is used in the treatment of HGSOC and OCS, however, resistance to platinum-taxane treatment usually occurs within 2-3 years and earlier in many OCS. We have been investigating the microtubule inhibitors, vinorelbine and eribulin, which each bind to distinct regions of microtubules and have a different mechanism of inhibition compared to paclitaxel. Eribulin has also been shown to reverse EMT, inducing vasculature remodeling and changes to the tumor-immune microenvironment. This dual activity of eribulin provides greater potential for improved efficacy in the clinic. Focusing on platinum resistant/refractory patient-derived xenografts (PDX) of HGSOC and OCS we assessed response to paclitaxel, vinorelbine and eribulin treatment and observed that around 50% of PDX were sensitive (HGSOC 8/13, 8/13 and 4/8 respectively; OCS 3/6 for all treatments). To deliver higher doses of drug to tumors but also limit toxicity, antibody-drug conjugates (ADCs) are being developed. MORAb-202 is an eribulin-anti-folate receptor alpha (FRα) ADC. It has been reported that 80-90% of HGSOC express FRα and in our HGSOC PDX cohort 14/15 (93%) were positive. However, only 1/5 (20%) OCS model showed any FRα and this was restricted to epithelial glandular structures, Citation Format: Cassandra J. Vandenberg, Gwo-Yaw Ho, Ksenija Nesic, Elizabeth M. Swisher, Sean M. Grimmond, Matthew J. Wakefield, Holly E. Barker, David D. Bowtell, Clare L. Scott. Improving efficacy of microtubule inhibitors in the treatment of high-grade serous ovarian cancer and ovarian carcinosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1074.

Background: Acquired PARP inhibitor (PARPi) resistance in High Grade Serous Ovarian Carcinoma (HGSOC) commonly occurs via restored homologous recombination DNA repair due to secondary mutations in BRCA1/2. However, overexpression of certain BRCA1 splice isoforms can also contribute to PARPi resistance in HGSOC. This includes the D11q isoform of BRCA1, where deleterious mutations in the 11q region of BRCA1 (exon 10) can be spliced out resulting in a truncated but partially functional BRCA1 protein. Here we describe four HGSOC Patient Derived Xenografts (PDX) with primary BRCA1 11q mutations, where D11q isoform expression has been shown to correlate with platinum and PARPi responses in vivo. Methods: PDX were derived from chemo-naive and pre-treated patients (Table 1). PDX were treated with PARPi rucaparib (300mg/kg) and cisplatin (4mg/kg). D11q isoform expression was assessed using two-step RT-qPCR. DNA sequencing was performed using the BROCA Cancer Risk Panel. Results: PARPi resistant PDX #1032 and #1049 had high D11q expression relative to D11q-high cell line UWB1.289 and the PARPi-responsive models (Table 1). Interestingly, PDX #1049 harboured a second BRCA1 exon 11 donor splice site mutation, shown previously to enhance splicing and increase abundance of the D11q isoform. Indeed, other proximal splice site mutations have been found following relapse in patients with BRCA1 11q mutations post-PARPi. Conclusions: Our work in PDX provides additional functional evidence that BRCA1 D11q expression impacts on PARPi and platinum responses, and is a clinically relevant mechanism of PARPi resistance in a subset of patients with BRCA1 mutations. Whilst high D11q expression can be associated with secondary BRCA1 splice site mutations (PDX #1049), it can also occur in their absence (PDX #1032). Thus, BRCA1 D11q isoform expression should be considered a potential mechanism of PARP-resistance in patients with BRCA1 11q mutations. Table 1.Summary of results.PDX modelPrimary BRCA1 mutationBRCA1 D11q isoform expressionSecondary BRCA1 mutationsOther mutations (BROCA)PDX platinum responsePDX PARPi responsePatient treatments prior to PDX generation#1049Germline BRCA1: c.2475delCVery highBRCA1: c.4096+3A>C (33%) - splice site mutation known to cause increased D11q expressionTP53: c.428_429insCAStable diseaseNon-responsive - progressive disease3 lines of platinum chemotherapy and PARPi#1032Somatic BRCA1: c.1251delTHighNone detectedTP53: c.97-1_97-6del; FANCA: c.3788_3790delResistant - progressive diseaseNon-responsive - progressive disease3 lines of platinum chemotherapy and PARPi#56Germline BRCA1: c.894_895delTGModerateNone detectedNone detectedSensitive - complete responseMixed responsesNo prior treatment#206Germline BRCA1: c.3817C>TLowNone in PDX, one found in post-PARPi patient biopsy: BRCA1:c.3746_4081delTP53:c.451C>T; BLM rearrangementSensitive - complete responseResponsive - complete response1 line of platinum chemotherapy Citation Format: Ksenija Nesic, Cassandra J. Vandenberg, Olga Kondrashova, John J. Krais, Neil Johnson, Elizabeth M. Swisher, Matthew J. Wakefield, Clare L. Scott. BRCA1 D11q isoform expression impacts PARP inhibitor responses in high grade serous ovarian carcinoma patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2057.

Objectives Mucinous ovarian carcinoma (MOC) is a rare ovarian cancer subtype that responds poorly to conventional chemotherapy. Recurrent and advanced disease have poor survival and there are no specific guidelines for their treatment. We used a large cohort of genomic and immunohistochemical data to evaluate the likelihood of success of possible therapeutic interventions. Methods We used DNA sequencing data (n=185) and genome-wide copy number (n=199) from primary MOC to identify key genetic events, homologous recombination deficiency scores and mismatch repair deficiency. Immunohistochemistry data was obtained for CK7, CK20, PAX8, p16, CDX2, HER2 and ER (n=162–256) and tumour infiltrating lymphocytes were counted on H&E stained slides (n=40). Results Therapies exploiting homologous recombination deficiency are unlikely to be effective in MOC, as only 1.5% had a homologous recombination deficiency score of more than 50. Mismatch repair deficiency was very rare ( Conclusions MOC is genetically diverse but with a number of potential targets. Importantly, the clinically observed lack of response to cisplatin is supported by a corresponding lack of a genomic signature, and MOC are unlikely to respond to PARP inhibitors. The role of immunotherapy is unclear. Testing novel therapeutic options in appropriate patient-derived models will be crucial and we are currently developing organoid cultures from this disease.

Gene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off-target mutations in gene-edited pigs remains largely undefined. We have previously generated GGTA1 knock-in pigs for xenotransplantation using FokI-dCas9, a variant of Cas9 that is reported to reduce the frequency of off-target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI-dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant-calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock-in and wild-type pigs. Platypus variant-calling software was used to detect single-nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock-in construct in the gene-edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off-target sites, and were not detected in a second line of knock-in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off-target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off-target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI-dCas9-edited cells. Our results demonstrate that FokI-dCas9 is capable of high-fidelity gene editing with negligible off-target or undesired on-target mutagenesis.

Introduction:Lens management in phakic patients with rhegmatogenous retinal detachments undergoing para plana vitrectomy surgery remains controversial among vitreoretinal surgeons. When combined phacovitrectomy is performed, the biometry decisions can be challenging both in the face of macula-off rhegmatogenous retinal detachments and previous refractive surgery. This study analyses current trends in practise.Methods:A scenario-related survey was sent to all members of the British and Eire Association of Vitreoretinal Surgeons and to vitreoretinal surgeons in the northern Indian state of Punjab.Results:In post-laser-assisted in situ keratomileusis patients with a visually significant cataract and a macula-on rhegmatogenous retinal detachment, the majority of British and Eire Association of Vitreoretinal Surgeons members (67.3%) and surgeons in Punjab (91.4%) would perform a stand-alone para plana vitrectomy and defer cataract surgery. When a combined phacovitrectomy is performed in this scenario, the majority of British and Eire Association of Vitreoretinal Surgeons (68%) would implant an intraocular lens (using either Haigis-L or European Society of Cataract and Refractive Surgeons/American Society of Cataract and Refractive Surgeons calculators), whereas the majority of Punjab surgeons (79.3%) would leave the patient aphakic. In a patient with a cataract and macula-off rhegmatogenous retinal detachment undergoing combined phacovitrectomy surgery, without any previous refractive surgery, the majority of British and Eire Association of Vitreoretinal Surgeons members (47.3%) would use the opposite eye biometry, whereas most Punjab surgeons (62.7%) would leave the patient aphakic.Discussion:Vitreoretinal surgeons in both the United Kingdom and Punjab predominantly avoid combined surgery in rhegmatogenous retinal detachment cases, even when faced with a visually significant cataract. When combined phacovitrectomy is performed, most vitreoretinal surgeons preference using the opposite eye biometry for macula-off cases and Haigis-L (myope) or online calculators for post-laser-assisted in situ keratomileusis cases.

High-grade serous ovarian cancer (HGSOC) accounts for the majority of ovarian cancer and has a dismal prognosis. PARP inhibitors (PARPi) have revolutionized disease management of patients with homologous recombination (HR) DNA repair-deficient HGSOC. However, acquired resistance to PARPi by complex mechanisms including HR restoration and stabilisation of replication forks is a major challenge in the clinic. Here, we demonstrate CX-5461, an inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress at rDNA leading to activation of DNA damage response and DNA damage involving MRE11-dependent degradation of replication forks. CX-5461 cooperates with PARPi in exacerbating DNA damage and enhances synthetic lethal interactions of PARPi with HR deficiency in HGSOC-patient-derived xenograft (PDX)in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi and destabilises replication forks irrespective of HR pathway status, overcoming two well-known mechanisms of resistance to PARPi. Importantly, CX-5461 exhibits single agent efficacy in PARPi-resistant HGSOC-PDX. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Therefore, CX-5461 is a promising therapy alone and in combination therapy with PARPi in HR-deficient HGSOC. CX-5461 is also an exciting treatment option for patients with relapsed HGSOC tumors that have poor clinical outcome.

High-grade serous endometrial carcinoma (HGSEC) accounts for just 10% of endometrial cancer (EC) cases but is responsible for at least 40% of all EC-related deaths. It typically arises in post-menopausal women, with 70% of patients presenting with stage III or IV disease, does not respond to hormone therapy unlike the less aggressive forms of EC, and has a lower overall survival rate of just 18-27%, which has not improved over the past two decades. The primary treatment for HGSEC is surgery, followed by a combination of standard chemotherapies (platinum and taxane) with or without localised radiotherapy. However, recurrent HGSEC is less responsive to chemotherapy than are other subtypes of EC and even initial responses to chemotherapy are poor. Therefore, there is a great unmet clinical need to find better treatment options for women with this aggressive cancer. Apart from TP53 (mutated in up to 90% of cases), the other most frequently mutated genes in HGSEC are PPP2R1A (31%), PIK3CA (22%), FBXW7 (28%), CHD4 (17%) and BRCA2 (12%). Focal amplifications of the genes MYC, ERBB2, CCNE1, FGFR3 and SOX17 are also common. The presence of ERBB2 amplification and/or HER2 over-expression in around 30% of HGSEC suggests these patients may respond to HER2-targeting drugs, such as trastuzumab. However, only modest benefit has so far been seen for single-agent HER2-targeted therapies (ie trastuzumab or lapatinib) against HGSEC, suggesting resistance mechanisms are present. Another feature of HGSEC that could be exploited therapeutically is homologous recombination deficiency (HRD), which may be targeted with PARP inhibitors (PARPi). It is not clear what proportion of HGSEC are HRD and neither HER2-targeting drugs or PARPi have been approved for the treatment of HGSEC. Due to its rarity and a lack of pre-clinical models, HGSEC has so far been understudied, resulting in a lack of effective treatment options. We currently have 33 HGSEC patients consented to the WEHI-Stafford Fox Rare Cancer Program and have developed pre-clinical models from fresh patient tumour samples received (4 patient-derived xenograft (PDX) models validated, with 3 pending). Preliminary molecular analysis of whole-genome sequencing (5 samples, one of which gave rise to a PDX model), whole-exome sequencing (4 samples), and cancer panel sequencing (3 samples, 2 of which gave rise to PDX models; one harbouring ERBB2 amplification and one harbouring an AKT mutation) data from our HGSEC cohort has been performed. This has identified potential treatment targets, including ERBB2 amplifications and mutations in HR genes. I am using the PDX models for initial in vivo therapeutic characterization studies and to develop organoid models for use in high-throughput drug assays in vitro. This will guide subsequent novel drug combination testing in our PDX models. By combining specific targeted drugs I hope to overcome de novo resistance mechanisms and prevent acquired resistance. Results from this study will guide future decisions about therapeutic strategies to improve survival of women with HGSEC. Citation Format: Holly E. Barker, Ratana Lim, Amandine Carmagnac, Cassandra Vandenberg, Gayanie Ratnayake, Genevieve Dall, Briony Milesi, Angela Komiti, Emily O'Grady, Joshua Tram, Kym Pham Stewart, Justin Bedo, Jocelyn Penington, Joep Vissers, Sean Grimmond, Matthew Wakefield, Tony Papenfuss, Clare Scott. Identifying effective combinations of targeted therapies, using novel pre-clinical models, to improve treatment options for high-grade serous endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO037.

Whilst the majority of Endometrial Cancers (ECs) are common Type 1 endometrioid cancers, accounting for 75-80% of cases, with a good prognosis, Type 2 ECs include high-grade, clinically aggressive histologies, with poor response rates to hormonal therapies. Serous endometrial carcinoma is the second most common type, accounting for ~10 percent of cases, most with a p53 abnormality and a lesser proportion with HER2 overexpression/amplification/mutation. Clear cell endometrial carcinoma accounts for Citation Format: Clare L. Scott, Ratana Lim, Amandine Carmagnac, Cassandra Vandenberg, Gayanie Ratnayake, Genevieve Dall, Joshua Tram, Justin Bedo, Jocelyn Penington, Joep Vissers, Sean Grimmond, Matthew Wakefield, Anthony Papenfuss, Holly Barker. Building the infrastructure required to research novel therapies for 35% of women with endometrial cancer who have rare subtypes [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO026.

PARP inhibitor (PARPi) resistance in high-grade serous ovarian carcinoma (HGSOC) can be acquired as a result of restored homologous recombination (HR) due to secondary or reversion mutations in HR genes, such as BRCA1, BRCA2, and RAD51C, or due to loss of BRCA1 promoter methylation (meBRCA1). We have demonstrated that homozygous meBRCA1 can be lost or reverted to heterozygous methylation following treatment with platinum-based chemotherapy, resulting in HR-competent PARPi-resistant tumors. RAD51C promoter methylation (meRAD51C) is detected in approximately 2% of HGSOC cases and, as with meBRCA1, is associated with gene silencing and HR deficiency. We are exploring PARPi response in meRAD51C preclinical models to determine clinical relevance. Two patient-derived xenograft (PDX) models of HGSOC with RAD51C gene silencing caused by meRAD51C have distinct meRAD51C profiles (measured by methylation-specific high-resolution melt analysis and targeted bisulfite next-generation sequencing), and different responses to PARPi treatment pressure. PDX PH039 loses methylation and regains RAD51C expression after only 2 cycles of PARPi retreatment (niraparib), resulting in PARPi-refractory tumors by cycle 3-4. Illumina EPIC methylation array analysis of PH039 revealed increasing global methylation losses following each round of PARPi treatment. Lack of meRAD51C stability and rapid development of PARPi resistance in PH039 may be due to the high degree of meRAD51C heterogeneity within the tumor favoring selection of pre-existing HR-competent clones under PARPi pressure. In contrast, PDX 183 has a relatively homogeneous and stable meRAD51C profile. We have multiple examples of using unique PDX models to demonstrate various important features of PARPi resistance, including loss of promoter methylation for BRCA1 vs. RAD51C. Thus, meRAD51C confers response to PARPi in HGSOC, but PARPi treatment pressure can cause loss of methylation and drug resistance in some tumors. The contrasting PARPi responses of these PDX provide a platform for the study of meRAD51C stability in vivo and may present therapeutic opportunities to improve meRAD51C durability and PARPi responses in patients. Citation Format: Kasenija Nesic, Rachel M Hurley, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Giada V. Zapparoli, Ashan Musafer, Ming E. Wong, John Weroha, Xiaonan Hou, Hu Li, Vivian Negron, Kevin Peterson, Paula Schneider, Elizabeth M. Swisher, Melissa Southey, Alexander Dobrovic, Matthew Wakefield, Scott H. Kaufmann, Clare L. Scott. DNA repair gene promoter methylation patterns adapt and influence PARP inhibitor response [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA02.

Introduction: PARP inhibitors (PARPi) have revolutionized disease management of patients with homologous recombination (HR) DNA repair-deficient high-grade serous ovarian cancer (HGSOC). However, acquired resistance to PARPi is a major challenge in the clinic. The specific inhibitor of RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) has demonstrated single-agent antitumor activity in p53 wild-type and p53-mutant hematologic malignancies (first-in-human trial, dose escalation study of CX-5461 at Peter MacCallum Cancer Centre) (Khot et al., Cancer Discov 2019). CX-5461 has also been reported to exhibit synthetic lethality with BRCA1/2 deficiency through stabilization of G-quadruplex DNA (GQ) structures. Here, we investigate the efficacy of CX-5461 in treating HGSOC. Experimental Design: The mechanisms by which CX-5461 induces DNA damage response (DDR) and displays synthetic lethality in HR-deficient HGSOC cells are explored. We present in vivo data of mice bearing two functionally and genomically profiled HGSOC-patient-derived xenograft (PDX)s treated with CX-5461 and olaparib, alone and in combination. We also investigate CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Results: Utilizing ovarian cancer cell lines, we demonstrate that sensitivity to CX-5461 is associated with “BRCA1 mutation” and “MYC targets” gene expression signatures. In addition, sensitivity to CX-5461 is associated with high basal rates of Pol I transcription. Importantly, we demonstrate a novel mechanism for CX-5461 synthetic lethal interaction with HR deficiency mediated through the induction of replication stress at rDNA repeats. Our data reveal CX-5461-mediated DDR in HR-deficient cells does not involve stabilization of GQ structures as previously proposed. On the contrary, we show definitively that CX-5461 inhibits Pol I recruitment leading to rDNA chromatin defects including stabilization of R-loops, single-stranded DNA, and replication stress at the rDNA. Mechanistically, we demonstrate CX-5461 leads to replication-dependent DNA damage involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 has a different sensitivity spectrum to olaparib and cooperates with PARPi in exacerbating replication stress, leading to enhanced therapeutic efficacy in HR-deficient HGSOC-PDX in vivo compared to single-agent treatment of both drugs. Further, CX-5461 exhibits single-agent efficacy in olaparib-resistant HGSOC-PDX overcoming PARPi-resistance mechanisms involving fork protection. Importantly, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Conclusions: CX-5461 is a promising therapy alone and in combination therapy with PARPi in HR-deficient HGSOC. CX-5461 also has exciting potential as a treatment option for patients with relapsed HGSOC tumors that have high MYC activity and poor clinical outcome; these patients currently have very limited effective treatment options. This abstract is also being presented as Poster A71. Citation Format: Elaine Sanij, Katherine Hannan, Jiachen Xuan, Shunfei Yan, Jessica A. Ahern, Anna S. Trigos, Natalie Brajanovski, Jinbae Son, Keefe T. Chan, Olga Kondrashova, Elizabeth Lieschke, Matthew J. Wakefield, Sarah Ellis, Carleen Cullinane, Gretchen Poortinga, Kum Kum Khanna, Linda Mileshkin, Grant A. McArthur, John Soong, Els M. Berns, Ross D. Hannan, Clare L. Scott, Karen E. Sheppard, Richard B. Pearson. Inhibition of RNA polymerase I transcription activates targeted DNA damage response and enhances the efficacy of PARP inhibitors in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR13.

We investigate whether different ages of first-time house buying lead to persistent differences in homeownership between cohorts. Our data span nearly 40 years and multiple cycles of England's volatile house prices. Ownership rates at thirty have differed substantially, with a significant negative association with prices. The persistence of differences is assessed using synthetic cohort techniques. Two methods of dealing with measurement error problems both indicate that cohorts with low ownership rates at thirty catch up almost all of the ownership gap by forty. Earlier access to homeownership may result in the ownership of slightly larger homes at around forty.

Conventional means of identifying variants in high-throughput sequencing align each read against a reference sequence, and then call variants at each position. Here, we demonstrate an orthogonal means of identifying sequence variation by grouping the reads as amplicons prior to any alignment. We used AmpliVar to make key-value hashes of sequence reads and group reads as individual amplicons using a table of flanking sequences. Low-abundance reads were removed according to a selectable threshold, and reads above this threshold were aligned as groups, rather than as individual reads, permitting the use of sensitive alignment tools. We show that this approach is more sensitive, more specific, and more computationally efficient than comparable methods for the analysis of amplicon-based high-throughput sequencing data. The method can be extended to enable alignment-free confirmation of variants seen in hybridization capture target-enrichment data.

Genomic instability and mutations are fundamental aspects of human malignancies, leading to progressive accumulation of the hallmarks of cancer. For some time, it has been clear that key mutations may be used as both prognostic and predictive biomarkers, the best-known examples being the presence of germline BRCA1 or BRCA2 mutations, which are not only associated with improved prognosis in ovarian cancer, but are also predictive of response to poly(ADP-ribose) polymerase (PARP) inhibitors. Although biomarkers as specific and powerful as these are rare in human malignancies, next-generation sequencing and improved bioinformatic analyses are revealing mutational signatures, i.e. broader patterns of alterations in the cancer genome that have the power to reveal information about underlying driver mutational processes. Thus, the cancer genome can act as a stratification factor in clinical trials and, ultimately, will be used to drive personalized treatment decisions. In this review, we use ovarian high-grade serous carcinoma (HGSC) as an example of a disease of extreme genomic complexity that is marked by widespread copy number alterations, but that lacks powerful driver oncogene mutations. Understanding of the genomics of HGSC has led to the routine introduction of germline and somatic BRCA1/2 testing, as well as testing of mutations in other homologous recombination genes, widening the range of patients who may benefit from PARP inhibitors. We will discuss how whole genome-wide analyses, including loss of heterozygosity quantification and whole genome sequencing, may extend this paradigm to allow all patients to benefit from effective targeted therapies. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

Atypical enteropathogenic Escherichia coli (aEPEC) causes endemic diarrhea, diarrheal outbreaks, and persistent diarrhea in humans, but the mechanism by which aEPEC causes disease is incompletely understood. Virulence regulators and their associated regulons, which often include adhesins, play key roles in the expression of virulence factors in enteric pathogenic bacteria. In this study we identified a transcriptional regulator, RalR, in the rabbit-specific aEPEC strain, E22 (O103:H2) and examined its involvement in the regulation of virulence. Microarray analysis and quantitative real-time reverse transcription-PCR demonstrated that RalR enhances the expression of a number of genes encoding virulence-associated factors, including the Ral fimbria, the Aap dispersin, and its associated transport system, and downregulates several housekeeping genes, including fliC . These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins and by adherence and motility assays. To investigate the mechanism of RalR-mediated activation, we focused on its most highly upregulated target operons, ralCDEFGHI and aap . By using primer extension, electrophoretic mobility shift assay, and mutational analysis, we identified the promoter and operator sequences for these two operons. By employing promoter- lacZ reporter systems, we demonstrated that RalR activates the expression of its target genes by binding to one or more 8-bp palindromic sequences (with the consensus of TGTGCACA) located immediately upstream of the promoter core regions. Importantly, we also demonstrated that RalR is essential for virulence since infection of rabbits with E22 carrying a knockout mutation in the ralR gene completely abolished its ability to cause disease.

AraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli , enteroaggregative E. coli , and Citrobacter rodentium . Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, of C. rodentium . Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target, sefA . Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression of sefA by binding to a region upstream of the sefA promoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a lethal human intestinal pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. EHEC is transmitted by the fecal-oral route and has a lower infectious dose than most other enteric bacterial pathogens in that fewer than 100 CFU are able to cause disease. This low infectious dose has been attributed to the ability of EHEC to survive in the acidic environment of the human stomach. In silico analysis of the genome of EHEC O157:H7 strain EDL933 revealed a gene, patE , for a putative AraC-like regulatory protein within the prophage island, CP-933H. Transcriptional analysis in E. coli showed that the expression of patE is induced during stationary phase. Data from microarray assays demonstrated that PatE activates the transcription of genes encoding proteins of acid resistance pathways. In addition, PatE downregulated the expression of a number of genes encoding heat shock proteins and the type III secretion pathway of EDL933. Transcriptional analysis and electrophoretic mobility shift assays suggested that PatE also activates the transcription of the gene for the acid stress chaperone hdeA by binding to its promoter region. Finally, assays of acid tolerance showed that increasing the expression of PatE in EHEC greatly enhanced the ability of the bacteria to survive in different acidic environments. Together, these findings indicate that EHEC strain EDL933 carries a prophage-encoded regulatory system that contributes to acid resistance.

Transcriptional control is dependent on a vast network of epigenetic modifications. One epigenetic mark of particular interest is tri-methylation of lysine 27 on histone H3 (H3K27me3), which is catalysed and maintained by Polycomb Repressive Complex 2 (PRC2). Although this histone mark is studied widely, the precise relationship between its local pattern of enrichment and regulation of gene expression is currently unclear. We have used ChIP-seq to generate genome-wide maps of H3K27me3 enrichment, and have identified three enrichment profiles with distinct regulatory consequences. First, a broad domain of H3K27me3 enrichment across the body of genes corresponds to the canonical view of H3K27me3 as inhibitory to transcription. Second, a peak of enrichment around the transcription start site (TSS) is commonly associated with 'bivalent' genes, where H3K4me3 also marks the TSS. Finally and most surprisingly, we identified an enrichment profile with a peak in the promoter of genes that is associated with active transcription. Genes with each of these three profiles were found in different proportions in each of the cell types studied. The data analysis techniques developed here will be useful for the identification of common enrichment profiles for other histone modifications that have important consequences for transcriptional regulation.

This paper uses a realistic structural lifecycle model of consumption and housing decisions to understand how data might distinguish different mechanisms that explain the correlation between house prices and consumption. The model includes price and earnings shocks estimated from data (the latter including aggregate and idiosyncratic components), and incorporates realistic features of the UK mortgage market. We simulate the model using more than 30 years of realized shocks and under counterfactual scenarios. Our results confirm the intuition of earlier studies: house price shocks should have a larger effect on the consumption of older households and earnings shocks on young households.

Background: The Australian Ovarian Cancer Assortment Trial (ALLOCATE) was designed as a pilot study to demonstrate feasibility of molecularly profiling patients with recurrent ovarian cancer with the aim of allocating patients to targeted therapies based on the genomic profile of their tumors. Materials and Methods: Two next-generation sequencing (NGS) panels, as well as a BRCA1 methylation assay, were used for molecular profiling of most common subtypes of ovarian cancer. A custom Illumina TruSeq Amplicon Low Input (v2) panel with dual-strand coverage was designed to target 38 genes commonly mutated and clinically important in ovarian cancer. The second assay was a NGS modification of the Multiplex Ligation-dependent Probe Amplification (MLPA) assay that was designed to target 11 genes with common copy number alterations (CNA) in ovarian cancer, including extensive BRCA1/2 coverage for large exonic deletions (Kondrashova et al., 2015). A thorough analytic validation was performed to ensure that both tests were fit for diagnostic use. Patients with recurrent epithelial ovarian cancer were eligible for the study. Where feasible, patients underwent biopsies of recurrent tumor that were snap frozen. Otherwise, archival FFPE tumor blocks were retrieved. Sequencing was performed using Illumina Miseq and HiSeq 2500 with target median coverage of 2000x (amplicon panel) and 800x (MLPA-Seq). Data were analyzed using an internally built pipeline, an upgraded version of AmpliVar (Hsu et al 2015), with Variant Effect Predictor (Mclaren et al., 2016) used for variant annotation. Results: Between December 2013 and October 2016, 113 patients with recurrent ovarian cancer were recruited from two tertiary hospitals, with 15 cases (13%) excluded due to insufficient tumor material or poor-quality DNA. Ninety-eight cases (87%) were analyzed and reports issued back to the referring clinician. Fifty-six patients (61%) in the study had recurrent high-grade serous ovarian cancer (HGSC). Of these, TP53 mutations were identified in 91%. Events in genes other than TP53 were detected in 44 cases, most commonly MYC and CCNE1 amplifications and BRCA1/2 mutations. BRCA1/2 reversions were identified in two cases, explaining their lack of response to platinum/PARPi. Fifteen patients (16%) had recurrent low-grade serous ovarian cancer (LGSC), with KRAS or BRAF mutations identified in four cases. Two HGSC tumors were reclassified as LGSC on the basis of a lack of TP53 mutation, presence of KRAS mutation, and subsequent pathology review. Other cases in the study included mucinous, clear cell, and mixed-histology carcinomas and a metastatic carcinosarcoma. In terms of clinical utility, 6 patients (7%) received a matched therapy. Three HGSC patients with somatic BRCA1/2 mutations were treated with PARP inhibitors. Another HGSC patient with ERBB2 amplification was treated with trastuzumab. One LGSC patient with a BRAF mutation was enrolled on a BRAF inhibitor clinical trial. A second LGSC patient was enrolled in a trial of anastrazole. Furthermore, 7 patients (14%) with HGSC who were previously untested were found to have a germline BRCA1/2 mutation and were subsequently referred to a familial cancer clinic for further management and cascade testing. The limitations in the study included the turnaround time and advanced stage of disease at enrolment, which significantly affected the clinical utility of the test. Conclusion: We demonstrated that molecular profiling of recurrent ovarian cancer using the ALLOCATE panel was feasible and reflected the known genomic characteristics of the different subtypes. However, challenges remain, including appropriate patient selection and efficient turnaround time for reporting. Furthermore, improved access to targeted therapies or clinical trials will also enhance the clinical utility of the ALLOCATE panel. Citation Format: Olga Kondrashova, George Au-Yeung, Leakhena Leas, Gwo-Yaw Ho, Sebastian Lunke, Kathryn Alsop, Clare Scott, Anne Hamilton, Sumitra Ananda, Alison Freimund, Michael Quinn, Orla McNally, Nadia Traficante, Tiffany Cowie, Matthew Wakefield, Arthur Hsu, Alex Dobrovic, Michael Christie, Graham Taylor, David Bowtell, Linda Mileshkin, Paul Waring. Australian Ovarian Cancer Assortment Trial–Allocating ovarian cancer patients into clinical trials based on molecular profiling. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B35.

Background: Acquired PARP inhibitor (PARPi) resistance in high-grade serous ovarian cancer (HGSOC) as a result of restored homologous recombination has been observed following secondary mutations that restore full-length protein in BRCA1, BRCA2, RAD51C, and RAD51D. Additionally, loss of BRCA1 methylation has also been shown to confer resistance. However, little is known about the role of RAD51C methylation in acquired PARPi resistance. In ARIEL2 Part 1, a phase 2 study of the PARPi rucaparib in ovarian carcinoma, four (2%) tumors demonstrated RAD51C methylation. The present study utilizes HGSOC patient derived xenografts (PDXs) and recurrent samples from ARIEL2 to assess the role of RAD51C methylation in the development of PARPi resistance. Methods: To drive PARPi resistance, PDX039, an extremely PARPi-sensitive model lacking demonstrable mutations in DNA repair genes, was treated cyclically with niraparib (100 mg/kg) for 21 days, after which the tumor was allowed to regrow and re-established in new mice for the next treatment round. To evaluate the frequency of methylation change, RAD51C methylation was analyzed in 12 rucaparib-treated mice (300 or 450 mg/kg) harboring PDX183, a PARPi-sensitive model without mutations in DNA repair genes. Global changes in gene expression following development of PARPi resistance were assessed by RNA sequencing. RAD51C promoter methylation was evaluated by bisulfite sequencing. Subsequent functional analysis included qRT-PCR, IHC, and western blot. DNA damage response pathways are being evaluated by immunofluorescence ex vivo following niraparib, rucaparib, or IR. Results: PDX039 grew through PARPi treatment by the third and fourth cycle of therapy. RAD51C was the only DNA repair gene to show significant change in RNAseq analysis (log2 fold-change=8.43; p=2e-192), corresponding with a loss of RAD51C methylation. Moreover, after just one round of PARPi treatment, RAD51C methylation was lost in 1 of 12 PARPi-treated PDX183 xenografts. RAD51C methylation loss ultimately resulted in restoration of expression, for which functional analysis is ongoing. Analysis of patient samples is currently underway. Conclusions: In HGSOC PDX models, RAD51C methylation affords PARPi sensitivity in the absence of DNA repair gene mutations. Treatment pressure with PARPi can reverse RAD51C methylation and restore RAD51C expression. Isolated changes in methylation of the RAD51C locus are sufficient to restore HR and convey PARPi resistance. Citation Format: Rachel M. Hurley, Ksenija Nesic, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Paula A. Schneider, Xiaonan Hou, Cristina Correia, Karen S. Flatten, Giada V. Zapparoli, Alexander Dobrovic, Kevin K. Lin, Thomas C. Harding, Andrea E. Wahner Hendrickson, Elizabeth M. Swisher, Matthew Wakefield, S. John Weroha, Clare L. Scott, Scott H. Kaufmann. Loss of RAD51C promoter hypermethylation confers PARP inhibitor resistance [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 5885.

In April 2001, the UK government introduced Stakeholder Pensions – a new private pension arrangement. The reform also changed the structure of tax-relieved pension contribution ceilings, increasing their generosity for lower-earners. We examine the impact of these changes on private pension coverage using individual level data. We use a difference-in-differences strategy with an estimator that is modified to allow for dichotomous outcomes. Contrary to the conventional wisdom that the Stakeholder Pension reforms had little or no impact on saving behaviour, our results indicate that the change to the contribution ceilings affected private pension coverage rates among lower-earners, especially among women.

The major histocompatibility complex (MHC) contain genes which play a key role in immune response and mate choice, and are therefore of functional importance to molecular ecologists. Here we describe the design of 10 MHC Class I-associated microsatellite loci from the tammar wallaby. All 10 loci are highly polymorphic, with the expected heterozygosity ranging from 0.547 to 0.919. Six loci successfully cross-amplify in other macropodid species. These microsatellites will serve as useful tools for studying the level of MHC diversity, the impact of selection on genetic variation and the unique structure of the tammar wallaby MHC.