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Supplementary Data - PDF file 117K, Microarray analysis of differentially expressed genes in PHA-533533 resistant cell lines

Supplementary Table S1 - RT-PCR Primer sequences used in the study Supplementary Table S2 - CDK2 shRNA sequences used in the study Supplementary Table S3 - Cell lines and genes used in the analysis from Project Achilles Supplementary Table S4 - EC50 values for compounds from primary screen for OVCAR3 and SKOV3 cell lines Supplementary Table S5 - EC50 values for compounds from primary screen for OVCAR3 and OVCAR3-R1 cell lines Supplementary Table S7 - List of compounds and their primary targets tested in the secondary screen for OVCAR3 and OVCAR3-R1 cell lines Supplementary Table S8 - List of combination indexes and type of interaction between compounds tested in the matrix screen

IL-6 RTPCR primers

Supplementary Figures S1-S9 - PDF file 327K, Supplementary Figure S1: A) Baseline expression of CCNE1 and CDK2 in OVCAR-3 (CCNE1 amplified) and OVCAR-4 (CCNE1 gained) cell lines compared to SK-OV-3 (CCNE1 unamplified). *detects both isoforms of CDK2. B) CCNE1 and CDK2 gene expression by RT-PCR after gene knockdown normalized to no siRNA control cells within each cell line. Representative data shown. *detects both isoforms of CDK2. C) Cell viability normalized to no siRNA control cells after transfection with siRNAs against CCNE1 or CDK2. Statistical significance calculated by comparison to non-silencing (NS) siRNA in the same cell line using data from three independent MTS assays performed in triplicate. Average normalized absorbance at 490 nm and SEM plotted (n = 3), T-test *p-value 0.3 for gains and >0.8 for amplifications. Fraction of genome lost (log2 copy number ratio < -0.3) in ovarian tumor samples by B) number of predicted whole genome doublings and C) CCNE1 copy number status. T-test ***p-value < 0.001

Supplementary Methods - PDF file 70K, CDK2 sequencing methods and primer sequences

Selected compounds for secondary screen

Primary hits-JHOC-5

Low-grade serous ovarian carcinomas (LGSC) are associated with a poor response to chemotherapy and are molecularly characterized by RAS pathway activation. Using exome and whole genome sequencing, we identified recurrent mutations in the protein translational regulator EIF1AX and in NF1, USP9X, KRAS, BRAF, and NRAS. RAS pathway mutations were mutually exclusive; however, we found significant co-occurrence of mutations in NRAS and EIF1AX. Missense EIF1AX mutations were clustered at the N-terminus of the protein in a region associated with its role in ensuring translational initiation fidelity. Coexpression of mutant NRAS and EIF1AX proteins promoted proliferation and clonogenic survival in LGSC cells, providing the first example of co-occurring, growth-promoting mutational events in ovarian cancer. Cancer Res; 77(16); 4268–78. ©2017 AACR.

Purpose: Cyclin E1 (CCNE1) amplification is associated with primary treatment resistance and poor outcome in high-grade serous ovarian cancer (HGSC). Here, we explore approaches to target CCNE1-amplified cancers and potential strategies to overcome resistance to targeted agents.Experimental Design: To examine dependency on CDK2 in CCNE1-amplified HGSC, we utilized siRNA and conditional shRNA gene suppression, and chemical inhibition using dinaciclib, a small-molecule CDK2 inhibitor. High-throughput compound screening was used to identify selective synergistic drug combinations, as well as combinations that may overcome drug resistance. An observed relationship between CCNE1 and the AKT pathway was further explored in genomic data from primary tumors, and functional studies in fallopian tube secretory cells.Results: We validate CDK2 as a therapeutic target by demonstrating selective sensitivity to gene suppression. However, we found that dinaciclib did not trigger amplicon-dependent sensitivity in a panel of HGSC cell lines. A high-throughput compound screen identified synergistic combinations in CCNE1-amplified HGSC, including dinaciclib and AKT inhibitors. Analysis of genomic data from TCGA demonstrated coamplification of CCNE1 and AKT2. Overexpression of Cyclin E1 and AKT isoforms, in addition to mutant TP53, imparted malignant characteristics in untransformed fallopian tube secretory cells, the dominant site of origin of HGSC.Conclusions: These findings suggest a specific dependency of CCNE1-amplified tumors for AKT activity, and point to a novel combination of dinaciclib and AKT inhibitors that may selectively target patients with CCNE1-amplified HGSC. Clin Cancer Res; 23(7); 1862–74. ©2016 AACR.

Supplemental Material - Group Authorship from EIF1AX and NRAS Mutations Co-occur and Cooperate in Low-Grade Serous Ovarian Carcinomas

Primary hits-TOV21G

Supplementary Figure S1 - Validation of CCNE1 and CDK2 siRNA-mediated knockdown Supplementary Figure S2 - Validation of CDK2 knockdown mediated by shRNA in vitro and in vivo Supplementary Figure S3 - FACS plots validating incorporation and induction of shRNA into OVCAR3 and CAOV3 cells Supplementary Figure S4 - Characterisation of CDK resistant lines and supplementary data supporting drug treatment Supplementary Figure S5 - Extended Oncoprint figure from TCGA Supplementary Figure S6 - Validation of FT282 over-expression constructs

Supplemental materials and methods

RPPA Antibody List

Supplementary Table S1. Patient characteristics; Supplementary Table S2. Ki67 proliferation index; Supplementary Table S3. WES and WGS performance statistics; Supplementary Table S4. Amplicon sequencing primers; Supplementary Table S5. High confidence variants from exome and whole genome sequencing; Supplementary Table S6. Summary of SNVs and indels by sample; Supplementary Table S7. Significantly mutated genes (MuSiC output); Supplementary Table S8. Somatic variants in recurrently mutated genes; Supplementary Table S9. Validation cohort variants; Supplementary Table S10. TP53 variants in validation cohort; Supplementary Table S11. EIF1AX mutations reported in cancer

Ovarian clear cell adenocarcinoma (OCCA) is characterized by a particularly poor response to conventional chemotherapy and a short overall survival time in women with established disease. The development of targeted treatments for OCCA relies on a better understanding of its molecular characteristics. IL6 is strongly expressed in OCCA and may therefore provide a novel therapeutic target. Here we use CRISPR/Cas9 and conditional short hairpin interfering RNA to perform loss-of-function studies in human OCCA cell lines to explore the requirement for IL6 in vitro and in vivo. While reduction of IL6 expression exerted limited effects in vitro, its attenuation significantly impaired tumor growth and neovascularization in vivo. In contrast to typical signaling via STAT3, IL6 in OCCA signaled via a noncanonical pathway involving gp130, Src, and the Hippo pathway protein YAP. A high-throughput combination drug screen identified agents that enhanced cell killing following reduction of IL6 signaling. Intersection of screen hits obtained from two cell lines and orthogonal approaches to attenuation of IL6 yielded AKT and EGFR inhibitors as enhancers of the inhibitory monoclonal IL6 receptor antibody tocilizumab. This study defines for the first time the requirements for, and mechanisms of, signaling by IL6 in human OCCA cell lines and identifies potential combinatory therapeutic approaches. Given the molecular diversity of OCCA, further in vitro and in vivo studies are warranted to determine whether such approaches will overcome the limited efficacy of tocilizumab observed in ovarian cancer to date.Significance:This study defines the requirements for and mechanisms of noncanonical signaling by IL6 in human ovarian clear cell adenocarcinoma cell lines and identifies combinatory therapeutic approaches to be explored clinically.

Supplementary Figures

Additional description of methods for gene suppression studies, Western blot, immunohistochemistry, flow cytometry and cellular assays

Supplementary Figure S1. Representative hematoxylin and eosin stained sections from sequenced LGSC; Supplementary Figure S2. Age distribution analysis of patients from the Australian Ovarian Cancer Study. Includes patients with advanced stage, serous epithelial ovarian cancer (n = 684); Supplementary Figure S3. RAS pathway mutations in LGSC; Supplementary Figure S4. Verification of EIF1AX mutations; Supplementary Figure S5. Characterization of the AOCS2 cell line; Supplementary Figure S6. Functional effect of EIF1AX mutations and gene suppression in a LGSC cell line; Supplementary Figure S7. (a) EIF1AX and NRAS function in the mTOR and RAS/ERK signaling pathways to regulate protein translation, cell proliferation and cell survival

Supplementary Tables S1-5 - PDF filr 185K, Supplementary Table S1: Essentiality of CDKs in CCNE1 amplified cell lines; Supplementary Table S2: GeneGo gene expression pathways enriched in PHA-533533 resistant cell lines; Supplementary Table S3: Copy number changes identified by SNP microarray in PHA-533533 resistant cell lines; Supplementary Table S4: Gene Set Enrichment Analysis (GSEA) of positional gene sets enriched in amplified or deleted genes specific to PHA-533533 resistant cells; Supplementary Table S5: OVCAR-3 cell line and derivative resistant line karyotypes

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.

ObjectivesThe primary objective of this study was to describe treatment patterns after poly-ADP ribose polymerase (PARP) inhibitor in patients with epithelial ovarian cancer. Secondary objectives were to evaluate duration of response, time to first subsequent therapy, progression-free survival and overall survival.MethodsThis was a retrospective analysis of patients with epithelial ovarian cancer treated with PARP inhibitor therapy at six Australian gynecological oncology centers. Eligible patients were identified via clinics, trial databases and pharmacy dispensing logs between January 2005 and September 2019. Information regarding clinico-pathological characteristics and treatment outcomes were collated from medical records.ResultsA total of 85 patients with epithelial ovarian cancer were identified. Of these, 61% had germline BRCA1/2 mutations, 9% had somatic BRCA1/2 mutations, 5% had confirmed homologous recombination deficiency and 25% were BRCA1/2 wildtype mutations. A total of seventy-seven (91%) patients received chemotherapy after PARP inhibitor, with fifty-six (72.7%) of these patients receiving platinum-based chemotherapy. Four patients (5%) had a complete response, 15 (20%) a partial response, 15 (20%) stable disease and 41 (55%) progressive disease. Median duration of response to chemotherapy was 7.0 months (range 0.2–20.4). Median time to first subsequent therapy was 17.6 and 15.1 months in patients who received a PARP inhibitor as maintenance therapy and treatment, respectively. Median progression-free survival of first line treatment after PARP inhibitor was 9.6, 3.5 and 4.6 months for platinum doublet, single agent platinum and non-platinum chemotherapy, respectively. Adjusting for age and FIGO (Federation of Gynecological Oncologists classification) stage progression-free survival did not differ between treatment groups (p=0.14). Median overall survival for the cohort was 69 months, and patients with platinum sensitive ovarian cancer had improved survival compared with those with platinum refractory or resistant disease.ConclusionPlatinum doublet chemotherapy resulted in non-significant improved progression-free survival compared with other regimens, suggesting potential independent mechanisms of resistance between PARP inhibitor and platinum compounds.

Patients withIn this study, we characterized the treatment-naive immune context in patients withIn single-cell data,In conclusion, treatment-naive

Ovarian clear cell adenocarcinoma (OCCA) is characterized by a particularly poor response to conventional chemotherapy and a short overall survival time in women with established disease. The development of targeted treatments for OCCA relies on a better understanding of its molecular characteristics. IL6 is strongly expressed in OCCA and may therefore provide a novel therapeutic target. Here we use CRISPR/Cas9 and conditional short hairpin interfering RNA to perform loss-of-function studies in human OCCA cell lines to explore the requirement for IL6 in vitro and in vivo. While reduction of IL6 expression exerted limited effects in vitro, its attenuation significantly impaired tumor growth and neovascularization in vivo. In contrast to typical signaling via STAT3, IL6 in OCCA signaled via a noncanonical pathway involving gp130, Src, and the Hippo pathway protein YAP. A high-throughput combination drug screen identified agents that enhanced cell killing following reduction of IL6 signaling. Intersection of screen hits obtained from two cell lines and orthogonal approaches to attenuation of IL6 yielded AKT and EGFR inhibitors as enhancers of the inhibitory monoclonal IL6 receptor antibody tocilizumab. This study defines for the first time the requirements for, and mechanisms of, signaling by IL6 in human OCCA cell lines and identifies potential combinatory therapeutic approaches. Given the molecular diversity of OCCA, further in vitro and in vivo studies are warranted to determine whether such approaches will overcome the limited efficacy of tocilizumab observed in ovarian cancer to date. Significance: This study defines the requirements for and mechanisms of noncanonical signaling by IL6 in human ovarian clear cell adenocarcinoma cell lines and identifies combinatory therapeutic approaches to be explored clinically.

To determine the impact of chemotherapy dose reductions and dose delays on progression-free survival (PFS) in women with ovarian cancer receiving first line chemotherapy in a real world prospective cohort study.Patients with newly diagnosed epithelial ovarian (or peritoneal, fallopian tube) cancer enrolled in a national Australian prospective study, OPAL, who commenced three-weekly carboplatin (AUC 5 or 6) and paclitaxel 175 mg/m634 evaluable patients, 309 commenced CP and 325 DD-CP. Patient's age was similar in the two groups (median 62 years, range 21-79). All planned chemotherapy doses were completed by 66% vs 40% (p 0.001) in the CP and DD-CP groups respectively. There was at least one treatment delay in 28% vs 58% (p 0.001) in the CP and DD-CP groups, respectively, and 29% vs 49% (p 0.001), respectively, required at least a 15% dose reduction for either carboplatin or paclitaxel. Median PFS was 29.2 [22.9, 43.8] and 21.5 [19.4, 23.1] months in the CP and DD-CP groups respectively. Adjusting for age, histology and FIGO stage PFS did not differ between treatment groups. Median PFS was similar in patients irrespective of dose reduction or dose delay.Patients receiving DD-CP required more dose reductions and delays due to haematological toxicities and lower completion rates than CP without significant difference in median PFS between CP and DD-CP. Median PFS was similar in patients irrespective of dose reduction or dose delay.

We aimed to investigate the role of FDG-PET/CT in monitoring of response and immune-related adverse events (irAEs) following first-line combination-immune checkpoint inhibitor (combination-ICI) therapy for advanced melanoma. We retrospectively reviewed outcomes in patients who had (1) first-line nivolumab plus ipilimumab; (2) pre- and post-treatment FDG-PET/CT scans (pre-FDG-PET/CT and post-FDG-PET/CT) within 2 and 4 months of starting ICI, respectively; and (3) at least one lesion assessable by PET response criteria in solid tumors (PERCIST). Extracranial response was monitored by 3 monthly FDG-PET/CT. Whole-body metabolic tumor volume (wbMTV) was measured pre- and post-treatment and correlated with outcome. FDG-PET/CT manifestations of irAE were defined as new increased non-tumoral uptake on post-FDG-PET/CT and were correlated with clinical presentation. Thirty-one consecutive patients, median age 60 years (range, 30–78), were identified from 2016 to 2018. The median number of combination-ICI cycles to the first post-FDG-PET/CT response assessment was 3 (interquartile range (IQR), 2–4). The best-overall responses were complete metabolic response (CMR) in 25 (80%), partial metabolic response (PMR) in 3 (10%), and progressive metabolic disease (PMD) in 3 (10%) patients. Patients with PMD had significantly higher pre-treatment wbMTV (p = 0.009). At a median follow-up of 21.5 months, 26 (84%) patients were alive with median progression-free and overall survival not reached. Secondary progression occurred in 9/31 (29%) patients at a median of 8.2 months (IQR, 6.9–15.5), of those majority (78%) was detected by FDG-PET/CT. Of 36 findings on post-FDG-PET/CT suggestive of irAE, 29 (80%) had clinical confirmation. In 3 (7%), the FDG-PET/CT findings preceded clinical presentation. The most common FDG-PET/CT detectable irAEs were endocrinopathies (36%) and enterocolitis (35%). FDG-PET/CT response evaluation predicts the long-term outcome of patients treated with first-line combination-ICIs. Long-term treatment response monitoring for detection of extracranial secondary progression is feasible by FDG-PET/CT. Beyond response assessment, FDG-PET/CT frequently detects clinically relevant irAEs, which may involve multiple systems contemporaneously or at various time-points and may precede clinical diagnosis.

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.

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.

9503 Background: Combination anti-PD(L)1 and BRAF/MEK-targeted therapy (TT) improves PFS in stage IV melanoma vs TT. In stage IV melanoma recent data suggest immunotherapy 1st until progression, rather than BRAF-TT, improves OS, and induction TT upfront adds little benefit. NeoTrio explored the optimal combination of BRAF-TT and anti-PD1 using the NAT platform in pts with stage III melanoma (NCT02858921). Methods: 60 pts with resectable, RECIST measurable stage III (no in-transit) BRAFV600-mutant melanoma were randomized 1:1:1 to 3 arms of 6 wks of NAT followed by complete lymph node dissection (CLND): A) Pembro ALONE (200mg Q3W x 2); B) SEQ - D+T (150mg bd + 2mg od) for 1 wk followed by pembro (200mg x 2); C) CON – D+T+pembro (doses as SEQ). Pts had 46 wks pembro post-CLND. Primary endpoint was the pathological response rate (pRR) and pathological complete response (pCR) at wk 6. Secondary endpoints; RECIST RR at wk 6, event-free survival (EFS), RFS, OS, adverse events (AE) and translational endpoints. Results: At data cutoff 2 Jan 2022, 20 pts per arm had similar baseline characteristics; overall 42% female, med age 53 yrs, 82% BRAF V600E, 62% clinical N1b. Med f/u was 20 months (95% CI 17-31). The pCR rate and pRR were highest in CON arm, and similar in ALONE and SEQ arms (Table). Events (progression before surgery, recurrence after surgery or death) were highest in ALONE arm at this 1st analysis (Table). Assessment of the durability of path response subtypes in each arm is ongoing. Most common Rx related AE were fatigue (65%, 70%, 70%, ALONE, SEQ and CON respectively), pyrexia (0%, 25%, 85%) and rash (50%, 35%, 35%). Gd 3/4 AE occurred in 30%, 25% and 55%, respectively; pyrexia and hepatitis were common in CON during NAT. Rx interruptions during NAT occurred in 0, 3 and 19 pts, respectively; 1, 0 and 8 pts permanently discontinued. Post NAT surgical operability was the same or improved in 81%. Longitudinal analysis of melanoma tissue, microenvironment and microbiome is ongoing. Conclusions: CON D+T+pembro achieved the highest pRR, pCR rate, but with greater toxicity. Recurrences were seen in those with pCR/near pCR in BRAF-TT containing arms, but not in pembro ALONE, in keeping with previous data of NAT with checkpoint inhibitors vs BRAF-TT. Short course of D+T prior to PD1 did not improve path response, despite previous translational data showing increased tumour infiltrating T-cells early-during treatment with D+T. Follow up is ongoing. Clinical trial information: NCT02858921. [Table: see text]

5515 Background: Cyclin E1 gene amplification and protein over-expression is a marker of platinum resistance in high grade serous ovarian, fallopian tube or primary peritoneal cancer (HGSC), and may predict response to WEE1 inhibition. Adavosertib, a WEE1 inhibitor, has demonstrated activity in unselected women with recurrent ovarian and serous endometrial cancer. We aimed to evaluate the efficacy of adavosertib in women with recurrent platinum resistant HGSC with cyclin E1 over-expression, with and without gene amplification. Methods: IGNITE is a multicentre, phase 2 trial with 2 cohorts of women with recurrent platinum resistant HGSC. Tumors were assessed for cyclin E1 protein expression by IHC and CCNE1 copy number by FISH. Patients were assigned to Cohort 1 if tumors were cyclin E1 over-expressed (H-score>50) and amplified (≥8 copies), and Cohort 2 if tumors were overexpressed and nonamplified. Patients with evaluable disease by RECIST v1.1 or GCIG CA-125 criteria were included. Adavosertib 300mg PO was given daily on days 1-5 and 8-12 of a 21-day cycle. The primary endpoint was investigator assessed clinical benefit (CB) defined as absence of progression for ≥ 18 weeks. Here we present the 18-week response data for the first 32 patients treated from Cohort 2, with a data cut-off of August 2021. Results: Between Jan-2020 and May-2021, 32 patients were accrued to Cohort 2. Median age was 62 years (range 42-77) and 84% had received ≥2 prior lines of chemotherapy. Median cyclin E1 IHC H-score was 120 and 28 patients (88%) had measurable disease by RECIST. Median number of cycles commenced was 8 (range 1-19). Overall response rate (ORR) was 53% and CB rate was 61% for all evaluable patients. Seventeen patients (53%) required a dose reduction, most commonly for neutropenia or fatigue. Seventeen patients experienced ≥Grade 3 treatment related adverse event, and 4 patients (15%) discontinued due to toxicity. Conclusions: The efficacy results in a biomarker-selected cohort of patients are promising with a higher response rate than reported in previous studies of adavosertib in unselected women with recurrent HGSC. Duration of response and progression free survival data will be presented as data matures. Clinical trial information: ACTRN12619001185156P. [Table: see text]

BRCA1 and BRCA2 tumor suppressor genes are central components of the homologous recombination (HR) DNA repair pathway, a high-fidelity process required to maintain genome integrity in the presence of DNA damaging agents. Germline or somatic mutation in BRCA1 or BRCA2, and methylation of BRCA1 therefore strongly influence response of patients with high-grade serous ovarian cancer (HGSC) to chemotherapy or poly ADP ribose polymerase inhibitors (PARPi). The profound sensitivity of HR-defective HGSC to chemotherapy and PARPi provides a strong selective pressure for tumor cells that have partially or wholly restored HR repair function. Here we focus on mechanisms by which restoration of activity of mutant or methylated BRCA genes occurs, how it is detected, and some unresolved issues requiring attention before this important biomarker can be fully utilized to improve HGSC patient management.

BackgroundVaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8+Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate with longer survival in metastatic cutaneous melanoma. However, their capacity to respond to VM and their neoantigens is not known.MethodsUsing longitudinal samples, we explored the evolution of VM mutations by whole-exome sequencing and RNAseq, we also defined the immune context using multiplex immunohistochemistry and nanostring pan cancer immune profile. Then using fresh single cell suspensions of the metastatic samples, we explored VM T cells via mass cytometry and single cell RNAseq and T cell receptor sequencing (TCRseq). Finally, we investigated TRM, pre-TRM and exhausted T cell function against melanoma neo-antigens and melanoma differentiation antigens in vitro.ResultsPrimary VM was non-inflamed and devoid of CD8+ TRM cells. In contrast, both metastases showed proliferating CD8+ TRM were clustered at the tumor margin, with increased numbers in the second ICB-refractory metastasis. The first metastasis showed dense infiltration of CD8+ T cells, the second showed immune exclusion with loss of melanoma cell Major histocompatibility complex (MHC)-I expression associated with downregulation of antigen presentation pathway gene expression. CD8+ TRM from both metastases responded to autologous melanoma cells more robustly than all other CD8+ T cell subsets. In addition, CD8+ TRM shared TCR clones across metastases, suggesting a response to common antigens, which was supported by recognition of the same neoantigen by expanded tumor infiltrating lymphocytes.ConclusionsIn this study, we identified TRM clusters in VM metastases from a patient, but not primary disease. We showed TRM location at the tumor margin, and their superior functional response to autologous tumor cells, predicted neoantigens and melanoma differentiation antigens. These CD8+ TRM exhibited the highest tumor-responsive potential and shared their TCR with tumor-infiltrating effector memory T cells. This suggests VM metastases from this patient retain strong antitumor T cell functional responses; however, this response is suppressed in vivo. The loss of VG MHC-I expression is a common immune escape mechanism which was not addressed by anti-PD-1 monotherapy; rather an additional targeted approach to upregulate MHC-I expression is required.

Introduction PARP inhibitors (PARPi) have changed the management landscape for patients with epithelial ovarian cancer (EOC). However, as with many targeted therapies, treatment resistance is common. The response to treatment post-PARPi has not been well described in trials. Data is needed to better understand disease course, and guide treatment decisions. The primary aim is to describe the treatment patterns post-PARPi. Secondary aims are to describe patient characteristics who received chemotherapy post-PARPi and DoR to chemotherapy. Methods Retrospective analysis of women with EOC treated with PARPi either in the maintenance or treatment setting and via government-funded or clinical trial access at six gynaecological oncology centres. Between 2007–2019 eligible women were identified via clinics, trial databases and pharmacy dispensing logs. Information regarding clinico-pathological characteristics and treatment outcomes were collated from medical records. Results Eighty-five women with EOC were identified. 90.6% received chemotherapy post-PARPi, with 72.7% receiving platinum-based chemotherapy. Clinicopathological characteristics in table 1. Best responses observed were 5.2% CR, 19.5% PR, 19.5% SD, and 55.8% PD. Median DOR was 7.0 months (range, 0.2 – 20.4 months) in 77 patients receiving chemotherapy post-PARPi. Eight patients did not receive further systemic therapy due to poor performance status. Women who received platinum doublet chemotherapy post-PARPi had a longer mPFS than those receiving platinum single agent or non-platinum chemotherapy (9.1 months vs 3.3 months vs 5.0 months, respectively p=0.0004). Conclusion Most patients received-platinum based chemotherapy post-PARPi with a modest response rate. Potential overlapping mechanisms of resistance to PARPi and platinum require further study to improve patient outcomes.

BackgroundMucosal melanoma is a rare subtype of melanoma originating from mucosal tissues (1), metastases are very aggressive and respond poorly to therapy, including immune checkpoint inhibitors (ICI) such as anti-CTLA4 and anti-PD1 antibodies (2–5). CD8+ T cells constitute the most abundant immune infiltrate in metastatic melanoma, of which the Tissue Resident Memory subset (TRM) is of particular interest (6). CD8+ TRM cells express the highest levels of immune checkpoint receptors, proliferate in response to ICI and correlate with longer disease-free and overall survival (6–8). The immune landscape in mucosal melanoma remains poorly characterized. We aimed to: 1) phenotype CD8+ T cells and TRM infiltrating metastatic mucosal melanoma, 2) characterize the clonality of TRM in relation to other CD8+ T cell subsets and 3) define the capacity of CD8+ T cells and TRM to respond to melanoma cells and to in vivo and in vitro anti-PD1 treatment.MethodsWe investigated the CD8+ T and TRM cells infiltrating two temporally- and spatially-distant subcutaneous metastases, these originated from a primary vaginal mucosal melanoma. One metastasis was excised prior to anti-PD1 treatment and one was anti-PD1 refractory, having progressed on treatment. We used mass cytometry and single-cell RNA and TCR sequencing to characterise the phenotype and clonality of the T cells, multiplex immunohistochemistry to define their spatial relationship with tumour cells and other T cells, and functional assays to determine TRM response to tumour cells (figure 1).ResultsCD8+ TRM frequency increased with time and anti-PD1 treatment, forming clusters at the tumour margin. T cells in the anti-PD1 refractory lesion were more activated than T cells in the first tumour and were bound by anti-PD1 antibody in vivo. T cells could not be stimulated by anti-PD1 directly ex vivo. Both metastatic lesions shared common T cell clusters including TRM. Furthermore, TRM in each tumour shared T cell clones, suggesting the presence of common antigens between metastatic sites. Indeed, the two metastases had a similar mutational profile. In vitro expanded tumour infiltrating lymphocytes from both lesions recognized tumour cells from both lesions and the same neoantigen generated from a single point mutation in the gene CDKN1C. Finally, tumour cells stimulated TRM cells more robustly than other T cells subsets.Abstract 548 Figure 1Graphical depiction of the methods used to characterise T cells in mucosal metastatic melanomaConclusionsIn this patient with vaginal mucosal melanoma, subsequent melanoma metastases of clonal origin attracted CD8+ T cells of similar specificity, among which TRM cells responded more vigorously to tumour cells than other T cells subsets.AcknowledgementsThe authors would like to acknowledge imCORE La Hoffmann- Roche Ltd. for funding.Ethics ApprovalPatients diagnosed with stage 3 or 4 metastatic melanoma and undergoing clinically indicated surgery were enrolled in prospective studies approved by the Peter MacCallum Cancer Centre human ethics research committee (13/141). All experimental protocols have been approved and clinical data has been collected prospectively.ReferencesCarvajal RD, Hamid O, Ariyan C. Mucosal Melanoma. [cited 2020 Apr 1]; Available from: https://www.uptodate.com/contents/mucosal-melanomaDel Vecchio M, Di Guardo L, Ascierto PA, Grimaldi AM, Sileni VC, Pigozzo J, et al. Efficacy and safety of ipilimumab 3 mg/kg in patients with pretreated, metastatic, mucosal melanoma. Eur J Cancer Oxf Engl 1990; 2014 Jan;50(1):121–7.Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for patients with advanced mucosal melanoma. The Oncologist 2013 Jun;18(6):726–32.D’Angelo SP, Larkin J, Sosman JA, Lebbe C, Brady B, Neyns B, et al. Efficacy and safety of nivolumab alone or in combination with ipilimumab in patients with mucosal melanoma: a pooled analysis. J Clin Oncol Off J Am Soc Clin Oncol. 2017 Jan 10;35(2):226–35.Hamid O, Robert C, Ribas A, Hodi FS, Walpole E, Daud A, et al. 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BackgroundMetastatic Merkel cell carcinoma (mMCC) is an aggressive neuroendocrine malignancy of the skin with a poor prognosis. Immune checkpoint inhibitors (ICIs) have shown substantial efficacy and favorable safety in clinical trials.MethodsMedical records of patients (pts) with mMCC treated with ICIs from August 2015 to December 2018 at Peter MacCallum Cancer Centre in Australia were analyzed. Response was assessed with serial imaging, the majority with FDG-PET/CT scans. RNA sequencing and immunohistochemistry for PD-L1, CD3 and Merkel cell polyomavirus (MCPyV) on tumor samples was performed.Results23 pts with mMCC were treated with ICIs. A median of 8 cycles (range 1 to 47) were administered, with treatment ongoing in 6 pts. Objective responses (OR) were observed in 14 pts (61%): 10 (44%) complete responses (CR) and 4 (17%) partial responses (PR). Median time to response was 8 weeks (range 6 to 12) and 12-month progression-free survival rate was 39%. Increased OR were seen in pts aged less than 75 (OR 80% vs 46%), no prior history of chemotherapy (OR 64% vs 50%), patients with an immune-related adverse event (OR 100% vs 43%) and in MCPyV-negative tumors (OR 69% vs 43%). Pts with a CR had lower mean metabolic tumor volume on baseline FDG-PET/CT scan (CR: 35.7 mL, no CR: 187.8 mL, p=0.05). There was no correlation between PD-L1 positivity and MCPyV status (p=0.764) or OR (p=0.245). 10 pts received radiation therapy (RT) during ICI: 4 pts started RT concurrently (OR 75%, CR 50%), 3 pts had isolated ICI-resistant lesions successfully treated with RT and 3 pts with multisite progression continued to progress despite RT. Overall, 6 pts (26%) had grade 1–2 immune-related adverse events.ConclusionICIs showed efficacy and safety in mMCC consistent with trial data. Clinical and imaging predictors of response were identified.

Background: Immune checkpoint blockade is now established as standard of care in several malignancies. Trials involving combined cytotoxic T lymphocyte associated protein 4 (CTLA4) and programmed cell death protein 1 (PD1) blockade demonstrate improved tumour responses in melanoma but at the cost of severe grade 3-4 immune related adverse events (irAEs) in 55%, and endocrine irAEs in up to 10% [1]. Immune-mediated damage to endocrine glands can be a diagnostic and management challenge. We aimed to review the incidence, biochemical evolution and imaging findings of endocrine toxicity related to combined anti CTLA-4 and anti-PD-1 therapy. Methods: We undertook a retrospective chart review of patients who received combined ipilimumab and nivolumab for metastatic melanoma at a tertiary referral centre between 2016-2019. We recorded onset and duration of abnormal biochemistry in endocrine irAEs, reviewed all available MRI images for pituitary size (mm) and appearance and 18-F FDG PET images for features of hypophysitis, thyroiditis and pancreatitis. Results: 162 patients received combination therapy. At least one irAE was recorded in 135 patients (83%), 100 (62%) required glucocorticoids, and 84 (52%) had an unplanned hospital presentation due to irAEs. Thyroiditis occurred in 50 (30.9%), with median time to onset of 30.9 days (range 1-234 days). 35 cases were identified with routine biochemistry performed every 4-6 weeks. TSH receptor antibody was measured in 13 patients and all were negative. 29 (58%) developed permanent hypothyroidism. Central cortisol deficiency was documented in 31 (19%) with a median time to diagnosis of 67.5 days (range 5-286). 4 cases were diagnosed on routine biochemistry and 14 presented with symptoms prompting investigation. 13 were diagnosed after routine neuroimaging demonstrated a pituitary abnormality, and a further 27 patients without the clinical syndrome had features of hypophysitis on neuroimaging. New onset diabetes occurred in 3 people, in which pancreatic inflammation on imaging was found in 2. A further 3/5 patients with an asymptomatic elevated lipase were found to have abnormal pancreatic imaging. In one patient with no features of endocrine or exocrine failure, there was a significant increase in FDG uptake and a subsequent loss of pancreatic volume. Conclusion: We report real world incidence of endocrine irAEs with combination immunotherapy. Routine biochemistry leads to the detection of some but not all cases. Early recognition and avoidance of unplanned presentations remains a challenge. Opportunistic assessment of endocrine gland appearance on routine imaging studies may provide useful early diagnostic information. Reference: Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. (2015) 1:23-34. 10.1056/NEJMoa1504030