Search

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with 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). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. Significance: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Neutrophil Extracellular Traps (NETs) are a key form of pro-inflammatory cell death of neutrophils characterized by the extrusion of extracellular webs of DNA containing bactericidal killing enzymes. NETosis is heavily implicated as a key driver of host damage in autoimmune diseases where injurious release of proinflammatory enzymes damage surrounding tissue and releases 70 known autoantigens. Recent evidence shows that both neutrophils and NETosis have a role to play in carcinogenesis, both indirectly through triggering DNA damage through inflammation, and directly contributing to a pro-tumorigenic tumor microenvironment. In this mini-review, we summarize the current knowledge of the various mechanisms of interaction and influence between neutrophils, with particular attention to NETosis, and cancer cells. We will also highlight the potential avenues thus far explored where we can intercept these processes, with the aim of identifying promising prospective targets in cancer treatment to be explored in further studies.

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

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.

All Supplementary Methods for the manuscript

Supplementary methods and figures

All supplementary tables for the manuscript

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with 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). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes.Significance:Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

All supplementary figures for the manuscript

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.

TP53 mutations are implicated in the progression of mucinous borderline tumors (MBOT) to mucinous ovarian carcinomas (MOC). Optimized immunohistochemistry (IHC) for TP53 has been established as a proxy for the TP53 mutation status in other ovarian tumor types. We aimed to confirm the ability of TP53 IHC to predict TP53 mutation status in ovarian mucinous tumors and to evaluate the association of TP53 mutation status with survival among patients with MBOT and MOC. Tumor tissue from an initial cohort of 113 women with MBOT/MOC was stained with optimized IHC for TP53 using tissue microarrays (75.2%) or full sections (24.8%) and interpreted using established criteria as normal or abnormal (overexpression, complete absence, or cytoplasmic). Cases were considered concordant if abnormal IHC staining predicted deleterious TP53 mutations. Discordant tissue microarray cases were re-evaluated on full sections and interpretational criteria were refined. The initial cohort was expanded to a total of 165 MBOT and 424 MOC for the examination of the association of survival with TP53 mutation status, assessed either by TP53 IHC and/or sequencing. Initially, 82/113 (72.6%) cases were concordant using the established criteria. Refined criteria for overexpression to account for intratumoral heterogeneity and terminal differentiation improved concordance to 93.8% (106/113). In the expanded cohort, 19.4% (32/165) of MBOT showed evidence for TP53 mutation and this was associated with a higher risk of recurrence, disease-specific death, and all-cause mortality (overall survival: HR = 4.6, 95% CI 1.5-14.3, p = 0.0087). Within MOC, 61.1% (259/424) harbored a TP53 mutation, but this was not associated with survival (overall survival, p = 0.77). TP53 IHC is an accurate proxy for TP53 mutation status with refined interpretation criteria accounting for intratumoral heterogeneity and terminal differentiation in ovarian mucinous tumors. TP53 mutation status is an important biomarker to identify MBOT with a higher risk of mortality.

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.

Majority of ovarian cancers are diagnosed at advanced stages with intra-peritoneal spread as the most common mode of disease metastasis. The formation of cancer spheroids is essential for the collective migration process, where shed tumour cells from the primary tumour form aggregates rather than disseminating as individual cells and seed within the peritoneal cavity. These cancer spheroids consist of leader cells (LC) and follower cells (FC), with the LC subset as key drivers of cellular movement and invasion. LCs have stem cell-like properties and are highly chemo-resistant with a specific survival addiction to several cell signalling pathways, such as the PI3K/AKT/mTOR pathway. We explore in this book chapter, the evidence supporting the role of LC in OC metastasis and the suppression of LC as an attractive therapeutic option for the treatment of advanced OC.

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.

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.

In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene

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.

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.

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.

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.

High-grade epithelial ovarian carcinomas containing mutated

Patient-derived xenograft (PDX) programs are crucial research tools in providing high-quality preclinical data for successful drug development, proof of principle of novel therapy efficacies, and the search for relevant biomarkers. These PDX programs are difficult to generate, costly to run and maintain, and must be able to change dynamically in line with technological and scientific advances. To date there are no specific guidelines for the development of a successful PDX program, particularly within the academic setting. The ultimate goal of most successful PDX programs is to generate high-quality research data that can be used to shape the landscape of novel oncologic therapies. The chance of success is increased by understanding the current PDX practices adopted by various research programs globally, and identifying key aspects of successful programs with a focus on areas for improvements.

The platinum analogues, cisplatin and carboplatin, are among the most widely used chemotherapeutic agents in oncology. Both agents have a broad spectrum of clinical activity in numerous malignancies including gynaecological cancers, germ cell tumours, head and neck cancer, thoracic cancers and bladder cancer. Although the final mechanism of inducing tumour cell apoptosis is similar for both compounds, cisplatin has been shown to be more effective in treating specific tumour types. Whilst more favourable toxicity profiles are often associated with carboplatin, this can frequently translate to inferior response in certain malignancies. This review succinctly collates the evidence for the preferential use of these platinum analogues in particular settings in addition to the long-standing dilemma surrounding the paucity of biomarkers predicting response to these agents.

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.

Introduction: High-grade serous ovarian cancer (HGSOC) can be divided into four subgroups based on molecular characteristics1,2. The proliferative/C5 subgroup is defined by MYCN-pathway activation and may be associated with stem cell-like behavior3. The majority of HGSOC originate from the secretory cells of the fallopian tube (FT), with both tumours and secretory cells characterised by PAX8 expression4. We have developed pre-malignant C5 sub-type Genetically Engineered Mouse Models (GEMM) to characterise key molecular events in the initiation of this sub-type and to provide an immune-competent model for testing therapeutics. Methods: We have generated independent GEMM with p53 dysfunction (p53lsl-R172H knock-in5) and over-expression of the MYCN-pathway, using either lsl-MYCN or lsl-lin28b transgenes6 directed by the doxycycline-inducible PAX8 promoter. Murine FT were harvested following two-weeks of transgene activation. One FT was harvested en bloc and paraffin embedded for analysis by H&E and immunohistochemistry for the presence of pre-malignant tubal lesions. The contralateral FT was macro-dissected into distal/mid-FT, which were snap frozen for analysis of gene expression by qRT-PCR and RNAseq. As mice with PAX8-directed expression of oncogenes (±MYCN/lin28b overexpression) developed enlarged kidneys due to renal tubular adenomata, for alternate mice, the mid/distal FT tissue was implanted into the bursae of wild-type CBA/nu mice for observation and later analysis. Results: FT were harvested from twelve p53lsl-R172H/lsl-lin28b mice and from ten p53lsl-R172H/lsl-MYCN mice (including relevant control mice). Dysplastic lesions were observed in a subset of p53lsl-R172H/lsl-MYCN or lsl-lin28b FT and not in controls. MYCN pathway activation was demonstrated by qRT-PCR analysis. Conclusion: Over-expression of the MYCN-pathway can drive FT epithelial dysplasia in the presence of p53 dysfunction. RNASeq analysis is being performed to elucidate early genetic events in dysplastic FT. Mice are now being harvested after eight-weeks of doxycycline activation to determine whether more advanced pre-malignant change occurs. CRISPR guides deleting relevant genes including p53, PTEN, Nf1 or Rb1 were generated to delete additional genes in MYCN pathway-transgenic FT, followed by implantation for observation of tumourigenesis. Note: This abstract was not presented at the meeting. Citation Format: Clare L. Scott, Gwo Y. Ho, Elizabeth E. Lieschke, Olga Kondrashova, Ronny Drapkin, David Bowtell, Matthew J. Wakefield. Genetically engineered mouse models of proliferative C5 high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4798. doi:10.1158/1538-7445.AM2017-4798

5583 Background: Elevated Glioma-associated Oncogene Homolog-1 (Gli1) protein expression is associated with Hedgehog (Hh) pathway activation in high-grade serous ovarian cancer (HGSOC). Inhibition of Hh signaling in Gli1-overexpressing HGSOC patient-derived xenograft (PDX) inhibited tumour growth, particularly in combination with chemotherapy. Early phase HGSOC clinical trials of vismodegib, a potent Hh inhibitor (SMO inhibitor), were disappointing. We identified a HGSOC PDX harboring both Indian Hh ligand-overexpression and bi-allelic deletion of TSC1, which latter event is reported to derepress the mTOR pathway, driving non-cannonical Gli1 expression. We explored the effect of vismodegib in combination with cisplatin or the mTOR inhibitor, everolimus, in this model. Methods: A cell-line was generated from the well-characterised PDX (identity confirmed by PDX-specific p53 mutation). In vitro response to vismodegib was assessed. qRT-PCR was performed to establish Hh-ligand and Gli1 expression with/without SMO inhibition. A PDX was generated from this cell-line and randomized to in vivo treatment with cisplatin, vismodegib, everolimus or vehicle alone, or vismodegib in combination with cisplatin or everolimus. Results: The HGSOC cell-line was sensitive to vismodegib in vitro (EC50 of 3.5µM) and qRT-PCR analysis revealed down-regulation of Hh-ligand and Gli1 expression following in vitro SMO inhibition, confirming on-target vismodegib activity. In vivo treatment with vismodegib or everolimus alone did not result in reproducible in vivo efficacy. The combination of vismodegib + everolimus caused short-lived responses in 3 of 6 mice. Strikingly, in vivo treatment with vismodegib in combination with cisplatin impaired median survival (19 days) when compared with cisplatin treatment alone (43 days; p = 0.039) due to rapid tumour progression. Conclusions: Combining chemotherapy with Hh inhibition in Hh ligand-overexpressing HGSOC PDX with mTOR pathway activation may be detrimental. These findings highlight the importance of an in-depth understanding of tumour biology in order to effectively combine therapeutic approaches.

e19598 Background: Cancer is highly prevalent in those above the age of 65 years. Elderly oncological patients are under-represented in clinical trials and there is a lack of clinical data to guide treatment decisions in this group of patients. We evaluated the cytotoxic treatment tolerability in this group of patients in a community hospital. Methods: Cytotoxic chemotherapies and tyrosine kinase inhibitors administered in Gold Coast Hospital to patients above 65 years of age between March 2010 and March 2011 were included. Data including number of comorbidities, concurrent medicines, ECOG status and treatment drug dose adjustment were obtained. The outcomes of treatment including treatment completion, early termination and its reason, toxicities and hospital admissions during treatment were also collected. Results: A total of 206 patients were identified. Majority had an ECOG score of 0 to 1 (89%) and number of co-morbidities between 0 and 3 (96%). Eighty percent of patients had initial or subsequent treatment dose reduction. Despite this, 53% of patients (n=109) did not complete the planned treatment; 68% had disease progression but 31% was due to treatment intolerance. There was a high number of reported grade 3 and 4 toxicities (n=84) and hospitalisations during treatment (n=154). Conclusions: There is a high rate of dose reduction and noncompletion of cytotoxic treatments even in a relatively healthy elderly population. We propose further prospective study incorporating in-depth geriatric assessment to evaluate elderly patients suitability for cytotoxic treatments. [Table: see text]