AbstractBackgroundAbout 70% of ovarian cancer (OC) patients relapse after initial chemotherapy, making it crucial to predict survival before second-line treatment. Our previous work discovered a blood-based DNA methylation prognostic signature (PLAT-M8) that uses 8 CpG sites related to chemoresistance. We aim to validate this biomarker and its correlation with clinicopathological features and treatment profiles in additional cohorts.MethodsExtracted DNA from whole blood was provided from the BriTROC 1 (n=47) and OV04 cohorts (n=57) upon the first relapse. Additional samples from Hammersmith Hospital (n=100) were collected during first-line chemotherapy (cycles 3-4 and 6). Bisulphite pyrosequencing was used to quantify DNA methylation at the previously identified 8 CpG sites. The methylation data obtained were combined with previous data from ScoTROC 1D and 1V (n=141) and OCTIPS (n=46). Cox regression was used to assess overall survival (OS) after relapse concerning clinicopathological characteristics. The DNA methylation Class (Class 1 vs 2) was determined by consensus clustering.FindingsBlood DNA methylation at relapse predicts better clinical outcomes. Methylation Class shows no association with outcome during first-line chemotherapy treatment. Methylation Class 1 is associated with shorter survival, as indicated by a meta-analysis of five cohorts (OS: HR 2.54, 1.67-3.85). Class 2 patients on carboplatin monotherapy have the best prognosis, while Class 1 patients on the same treatment have the poorest prognosis (OS: aHR 9.69, 2.38-39.47). Class 1 is linked to older patients (>75 years) with advanced-stage, platinum-resistant cases, correlating with residual disease, and shorter progression-free survival. In contrast, Class 2 of PLAT-M8 is linked to platinum-sensitive patients, and higher complete response rates by RECIST criteria, but shows no correlation with CA-125. These findings emphasise the potential of PLAT-M8 in guiding second-line chemotherapy decisions.InterpretationPLAT-M8 methylation biomarker is associated with survival in OC patients with relapse and hypothetically may predict platinum treatment response at second-line chemotherapy.FundingThis work was supported by funding from Ovarian Cancer Action (“Risk and Prevention” programme grant), Cancer Research UK programme grant (A13086) with support from the Cancer Research UK Imperial Centre, the National Institute for Health Research Imperial Biomedical Research Centre and the Ovarian Cancer Action Research Centre.Research in contextEvidence before this studyThere is a strong association between platinum-based chemotherapy and DNA methylation changes in blood DNA during ovarian cancer relapse. Previous findings identified eight specific CpG methylation changes (known as PLAT-M8) in blood at relapse following platinum-based chemotherapy that were associated with overall survival in patients enrolled in the ScoTROC 1 trial and the OCTIPS cohort. Using an ovarian cancer cell line model, the study also showed that functional DNA mismatch repair increased the frequency of platinum-induced methylation, providing insights into the observed epigenetic changes.Added values of this studyOur current study validates in five large relapsed ovarian cancer cohorts that: (1) PLAT-M8 is associated with various clinicopathological characteristics, such as age, stage, platinum sensitivity, RECIST response, and progression time; (2) PLAT-M8, particularly from blood samples taken at the time of the first relapse before second-line chemotherapy, can serve not only as prognostic indicators for overall survival but also time to death after relapse in ovarian cancer patients; (3) PLAT-M8 does not have prognostic value when blood samples are taken during first-line chemotherapy before relapse, after initial diagnosis; and (4) PLAT-M8 may stratify overall survival and time to death after relapse based on the second-line treatment received by patients. These findings pave the way for our ongoing research, showcasing the potential of this non-invasive approach in predicting second-line treatment response, guiding decisions, and enhancing outcomes for relapsed ovarian cancer patients.Implications of all the available evidenceThe lack of biomarkers guiding treatment decisions during second-line therapy highlights the need for more reliable biomarkers. As a prognostic biomarker, PLAT-M8 is considered simple yet impactful, as it only requires one blood sample taken before second-line treatment at the time of relapse. The advantages of this research include developing personalised treatment approaches, minimizing side effects and wasted time from ineffective medications, reducing the likelihood of subsequent relapse episodes, and improving clinical outcomes for patients. Ultimately, the use of biomarkers has the potential to reduce hospital stays and healthcare costs by optimizing treatment effectiveness and efficiency, while also enhancing the quality of life for patients.

Supplementary Methods, Tables, 2, 3, and 5, Figures 1-10 from HDAC4-Regulated STAT1 Activation Mediates Platinum Resistance in Ovarian Cancer

Supplementary Tables 1-7 from Assessment of Hepatocyte Growth Factor in Ovarian Cancer Mortality

Supplementary Table 1 from Ovarian Cancer Stem Cell–Like Side Populations Are Enriched Following Chemotherapy and Overexpress EZH2

Supplemental Figure 1. Flow of patients and samples through study Supplemental Figure 2. Receiver operating curve (ROC) for HRD as a predictor of CA125 response in the HGSOC subset. Supplementary Table 1. Clinical characteristics and patient outcome. Supplemental Table 2. Univariate logistic regression analyses of HRD status, tBRCA and CCNE1 as predictors of CA125 response. Supplemental Table 3. Multivariate logistic regression analysis of HRD and clinical factors as predictors of CA125 Complete Response. Supplemental Table 4: Univariate and multivariate logistic regression analyses of HRD status at the predefined threshold of {greater than or equal to}42 and an exploratory threshold of {greater than or equal to}33 as predictors of CA125 response in the HGSOC subset. Supplemental Table 5. Multivariate logistic regression analysis of tBRCA and clinical factors as predictors of CA125 complete response. Supplemental Table 6: Univariate and multivariate Cox PH analyses of PFS and OS for HRD status at the predefined threshold of {greater than or equal to}42 and an exploratory threshold of {greater than or equal to}33 in the HGSOC subset. Supplemental Table 7. Progression-free survival (PFS) and overall survival (OS) according HRD status, tBRCA mutation status, and CCNE1 amplification in the overall cohort (N=250). Supplemental Table 8. Progression-free survival (PFS) and overall survival (OS) according HRD status, tBRCA mutation status, and CCNE1 amplification in the HGSCO subgroup (N=179). Supplemental Table 9. Multivariate Cox PH analyses of HRD, CCNE1 amplification and clinical factors as predictors of PFS and OS.

Supplementary Figures 1-4 from Assessment of Hepatocyte Growth Factor in Ovarian Cancer Mortality

PDF file - 157K, Supplemental Table 1. Regulatory T cell genes included in this study (N=25). Supplemental Table 2. Regulatory T cell SNPs included in this study. Supplemental Table 3. Participating invasive epithelial ovarian cancer studies. Supplemental Table 4. Association between clinical variables and overall survival.

Background Data suggest that immunomodulation induced by DNA hypomethylating agents can sensitize tumors to immune checkpoint inhibitors. We conducted a phase 1 dose-escalation trial (NCT02998567) of guadecitabine and pembrolizumab in patients with advanced solid tumors. We hypothesized that guadecitabine will overcome pembrolizumab resistance. Methods Patients received guadecitabine (45 mg/m2 or 30 mg/m2, administered subcutaneously on days 1–4), with pembrolizumab (200 mg administered intravenously starting from cycle 2 onwards) every 3 weeks. Primary endpoints were safety, tolerability and maximum tolerated dose; secondary and exploratory endpoints included objective response rate (ORR), changes in methylome, transcriptome, immune contextures in pre-treatment and on-treatment tumor biopsies. Results Between January 2017 and January 2020, 34 patients were enrolled. The recommended phase II dose was guadecitabine 30 mg/m2, days 1–4, and pembrolizumab 200 mg on day 1 every 3 weeks. Two dose-limiting toxicities (neutropenia, febrile neutropenia) were reported at guadecitabine 45 mg/m2 with none reported at guadecitabine 30 mg/m2. The most common treatment-related adverse events (TRAEs) were neutropenia (58.8%), fatigue (17.6%), febrile neutropenia (11.8%) and nausea (11.8%). Common, grade 3+ TRAEs were neutropaenia (38.2%) and febrile neutropaenia (11.8%). There were no treatment-related deaths. Overall, 30 patients were evaluable for antitumor activity; ORR was 7% with 37% achieving disease control (progression-free survival) for ≥24 weeks. Of 12 evaluable patients with non-small cell lung cancer, 10 had been previously treated with immune checkpoint inhibitors with 5 (42%) having disease control ≥24 weeks (clinical benefit). Reduction in LINE-1 DNA methylation following treatment in blood (peripheral blood mononuclear cells) and tissue samples was demonstrated and methylation at transcriptional start site and 5’ untranslated region gene regions showed enriched negative correlation with gene expression. Increases in intra-tumoural effector T-cells were seen in some responding patients. Patients having clinical benefit had high baseline inflammatory signature on RNAseq analyses. Conclusions Guadecitabine in combination with pembrolizumab is tolerable with biological and anticancer activity. Reversal of previous resistance to immune checkpoint inhibitors is demonstrated.

Abstract Background: Methylation is reported to support cancer immune tolerance. We conducted a phase 1 dose-escalation trial [NCT02998567] of combination guadecitabine (G; DNA hypomethylating agent) and pembrolizumab (P) in patients (pts) with advanced cancers. We hypothesized that G can normalize the expression of epigenetically suppressed immune genes, increase interferon producing tumor-infiltrating lymphocytes (TILs), and enhance the anticancer activity of P. Methods: In dose escalation (Es), pts received G (45 mg/m2 or 30 mg/m2, administered SC on days 1-4) with P (200 mg, administered IV starting from cycle 2 onwards) as outpatient Q3W; in expansion (Ex), the RP2D of G (30 mg/m2) with P (200 mg) Q3W was administered. Pre-treatment and on-treatment tumor biopsies were evaluated for PD-L1 expression, tumor infiltrating lymphocytes, gene expression by RNAseq and methylome studies. Longitudinal analyses of peripheral blood CD3, CD4 and CD8 lymphocytes by flow cytometry were performed. Results: Overall, 34 pts (Es, n = 14; Ex, n = 20) were evaluable for safety. The most common treatment-related adverse events (TRAEs) were neutropenia (n = 21), fatigue (n = 6) and thrombocytopenia (n = 3), diarrhea (n = 2). G3+ TRAEs were neutropenia (n = 14), febrile neutropenia (n = 4), raised ALP (n = 1), raised AST (n=1), colitis (n = 1), diarrhoea (n = 1) and lung infection (n = 1). Two DLTs (neutropenia, febrile neutropenia) were reported at G 45mg/m2 with none reported at G 30mg/m2. There were no treatment-related deaths. In total, 28 pts (Es, n = 12; Ex, n = 16) were evaluable for antitumor activity studies (≥2 scans); ORR (CR+PR) and DCR (CR+PR+SD) were 3% and 57%; 10/15 pts with non-small cell lung cancer (13 pts resistant/refractory to PD-1/PD-L1 targeting agents) were evaluable, with a DCR of 80% and 5 pts having DCR > 6 months with 8 pts remaining on study treatment. Overall, 25 paired biopsies were obtained. Using LINE1 sequences to study global methylation, both tumor biopsies and peripheral blood showed reduced methylation post-G treatment. Preliminary data on tumor-infiltrating lymphocytes assessed by multicolor immunofluorescence in 9 paired biopsies showed a numerical increase in median values of T-helper (CD4+FOXP3-) (10.20 to 19.70, p = 0.5469), T-regulatory (CD4+FOXP3+) (5.1 to 6.7, p=0.8438), and T-cytotoxic (CD8+) cell densities (2.7 to 7.4, p=0.6523) . Comparing with matched pre-treatment, on treatment tumor had numerical increases in interferon alpha and gamma response pathway activation in serial biopsy RNAseq analyses but did not reach significance. Conclusions: G plus P resulted in no unexpected toxicities with evidence suggestive of biological and anti-cancer activity. Citation Format: Dionysis Papadatos-Pastos, Abhijit Pal, Melek Akay, Malaka Ameratunga, Sanjena Mithra, Joo-Ern Ang, Sofia Levva, Reece Caldwell, Ruth Riisnaes, Mateus Crespo, Wei Yuan, George Seed, Bora Gurel, Ines Figueiredo, Rita Pereira, Susana Miranda, Anna Ferreira, Suzanne Carreira, Claudia Bertan, Chloe Baker, Ricardo Morilla, Robert Brown, Nahal Masrour, Toby Prout, Anna Zachariou, Alison Turner, Mona Parmar, Mark Van de Velde, Ben Jenkins, Christina Yap, Nina Tunariu, Udai Banerji, Juanita Lopez, Anna Minchom, Johann De Bono. HyPeR: A phase 1, dose escalation and expansion trial of guadecitabine (SGI-110), a second-generation hypomethylating agent in combination with pembrolizumab (MK3475) in patients with refractory solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT129.

Despite increasing knowledge of tumorigenesis, the identity of the cancer cell-of-origin, i.e. the normal cell type that acquired the cancer-initiating event, remains largely unknown. Our approach of identifying the cell-of-origin is based on two observations: (1) the chromatin structure is cell-specific; and (2) the density of somatic mutations along the genome is associated with the regional profile of chromatin modifications. We have previously developed a method that quantifies the ability to predict the mutational distribution along the cancer genome from the profile of epigenetic modifications in different normal cell types. Here we present the largest application of our method using 2,550 whole genomes representing 32 distinct cancer types. To identify the cell-of-origin, we determined the correlation between the observed density of mutations along the genome and the predicted values based on chromatin modifications from 104 different normal tissue types. The normal cell type that showed the strongest correlation with a specific cancer mutational landscape was the candidate cell-of-origin. We found that in almost all cancer types the cell-of-origin can be characterized solely from DNA sequences. Interestingly, we found that the fallopian tube was the best match for high-grade serous ovarian cancer, providing independent evidence that this is the cancer’s site of origin. For breast cancer we found that the four distinct subtypes best-matched cells from the luminal cell lineage: basal-like breast cancer likely originates from luminal progenitors, whereas all other subtypes from luminal mature cells. This association holds true even when accounting for different alterations in the homologous recombination repair pathway, suggesting that subtypes are more determined by the cell-of-origin than the specific DNA repair defect. In addition, we found that we could identify the cell-of-origin using metastatic samples – a finding that may help in difficult clinical diagnoses. Moreover, we demonstrate that cancer drivers, both germline risk alleles and somatically mutated drivers, reside in active chromatin regions in the respective cell-of-origin. Taken together, our findings indicate that many of the somatic mutations accumulated while the cells maintained a chromatin structure similar to the cell-of-origin (likely occurring prior to transformation). Therefore, this historical record, captured in the DNA, can be used to identify, the often elusive, cancer cell-of-origin. Our approach can ultimately help better understand the potential of particular normal cell types to transform and initiate cancer, as well as the association of the cell-of-origin with tumor subtypes and sensitivity to treatment. Citation Format: Kirsten Kubler, Rosa Karlic, Nicholas J. Haradhvala, Kyungsik Ha, Jaegil Kim, Maja Kuzman, Wei Jiao, Sitanshu Gakkhar, Kent W. Mouw, Lior Z. Braunstein, Olivier Elemento, Andrew V. Biankin, Ilse Rooman, Mendy Miller, Christopher D. Nogiec, Edward Curry, Mari Mino-Kenudson, Leif W. Ellisen, Robert Brown, Alexander Gusev, Cristian Tomasetti, Hong-Gee Kim, Hwajin Lee, Kristian Vlahovicek, Charles Sawyers, Katherine A. Hoadley, Edwin Cuppen, Amnon Koren, Peter F. Arndt, David N. Louis, Lincoln Stein, William D. Foulkes, Paz Polak, Gad Getz. The premalignant state captured in the landscape of somatic mutations can reveal the cancer cell-of-origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2727.

Chromatin structure has a major influence on the cell-specific density of somatic mutations along the cancer genome. Here, we present a pan-cancer study in which we searched for the putative cancer cell-of-origin of 2,550 whole genomes, representing 32 cancer types by matching their mutational landscape to the regional patterns of chromatin modifications ascertained in 104 normal tissue types. We found that, in almost all cancer types, the cell-of-origin can be predicted solely from their DNA sequences. Our analysis validated the hypothesis that high-grade serous ovarian cancer originates in the fallopian tube and identified distinct origins of breast cancer subtypes. We also demonstrated that the technique is equally capable of identifying the cell-of-origin for a series of 2,044 metastatic samples from 22 of the tumor types available as primaries. Moreover, cancer drivers, whether inherited or acquired, reside in active chromatin regions in the respective cell-of-origin. Taken together, our findings highlight that many somatic mutations accumulate while the chromatin structure of the cell-of-origin is maintained and that this historical record, captured in the DNA, can be used to identify the often elusive cancer cell-of-origin.

The authors found that ESRRA when co-expressed at high levels with TGFβR3 may be prognostic for serous ovarian cancer overall survival in data from the Cancer Genome Atlas; however, this result was not validated in further datasets. A cell line was also identified in this study for future functional investigation of interactions between ESRRA and TGFβR3 in the context of oestrogen signaling in order to further elucidate their potential roles as prognostic biomarkers for serous ovarian cancer.FUNDING SUPPORT This work was supported by Cancer Research UK program A6689 (RB and CSWB) and the Medical Research Council (AP)CONFLICT OF INTEREST DISCLOSURES The authors have declared no conflicts of interest.AUTHOR CONTRIBUTIONS CSWB designed the study. AP, CSWB, and RB developed the methodology. AP and CSWB collected the data. AP, CSWB, and RB wrote the manuscript. CSWB is responsible for the overall content.

Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. 1 (chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU(VAR)3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by 1 and functionally related analogues. Our results reveal that only the structurally unique ETP core is required for inhibition, and such inhibition is time-dependent and irreversible (in the absence of DTT), ultimately resulting in protein denaturation. Mass spectrometric data provide a molecular basis for this effect, demonstrating covalent adduct formation between 1 and the protein. This provides a potential rationale for the selectivity observed in the inhibition of a variety of HKMTs by 1 in vitro and has implications for the activity of ETPs against these important epigenetic targets.

Abstract Background: AKT pathway activation is commonly described in ovarian cancer, and implicated in preclinical models of chemoresistance. Ascites occurs in a high proportion of patients at some stage in their illness. Purpose: To study the relationship between activation of the AKT pathway in human ovarian cancer ascites specimens (measured by the ratio of phosphorylated (P) to total (T) AKT pathway proteins) to patient survival and response to chemotherapy. In addition we studied the correlation of P/T AKT ratio to carboplatin sensitivity in a human ovarian cancer cell line panel. Method. 99 samples of ascites from 75 patients with ovarian cancer and a known treatment history were purified using anti-EpCam antibody coated magnetic beads (Dynal Biotech), stored at −80 degrees C, and then analysed with Mesoscale Discovery (MSD) ELISA for P/T AKT, GSK3β and p70S6K, and Pathscan ELISA for P/T 4EBP1 . Differences in survival in patient subgroups were analysed by logrank tests. Five cell lines (A2780, CH1, IGROV1, OVCAR3, SKOV3) were analysed in triplicate by sulphurhodamine B colorimetric assay to establish half maximal inhibitory concentration (IC50) for carboplatin and PI3K-AKT-mTOR pathway inhibitors (rapamycin, LY 294002, PI103, and Merck AKT inhibitor VIII), and the results correlated with baseline AKT pathway protein activation ratios. Results: Samples from patients who responded to chemotherapy (n=32) subsequent to sample collection had a lower median P/T S6K ratio (0.43) than those (n=28) who progressed (0.71, p=0.026, Mann-Whitney test; 39 samples were from patients who received no subsequent chemotherapy). Analysing the first sample for each patient, (n=75), those with a P/T AKT ratio above median (0.140) had a statistically significantly longer survival than those patients below median (28.0 months (m) vs 49.7 m, HR 1.74 (95% CI 1.01-3.01), p=0.048). A similar trend for P/T S6K ratio was also found, however this was not statistically significant (22.0 m vs 47.5 m, HR 1.56 (95%CI 0.90-2.71), p=0.112). In the ovarian cell line panel studied, a high P/T AKT ratio was found in the cells most resistant to carboplatin, whereas there was no relationship between AKT pathway activation and resistance to the AKT pathway inhibitors. Potential reasons for activation of the AKT pathway include amplification and mutation of PIK3CA and AKT2. Fluorescence in-situ hybridisation and sequencing of somatic DNA from these samples are ongoing. Conclusion: Patients who responded to subsequent chemotherapy had a significantly lower P/T S6K ratio than those who progressed. A P/T AKT ratio greater than 0.144 in malignant ascites predicts a statistically significant poorer prognosis. In vitro cell line models suggest a trend of P/T AKT ratio to correlate with carboplatin resistance. P/T AKT ratio should be studied prospectively as a prognostic and predictive biomarker in ovarian cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 788.

To the Editor: Green et al. ([1][1]) identified ABCB1 2677G>T/A genotype as a marker of response to paclitaxel therapy in ovarian cancer. Their study was done in 53 tumor samples from ovarian cancer patients treated with 175 or 135 mg/m2 paclitaxel for at least four cycles. Response was higher in

The tumour suppressor gene encoding p53 has been shown from experimental studies to have a crucial role in how cells respond to DNA damage. p53 has important functions in apoptosis, cell-cycle arrest and DNA repair, largely mediated by its activity on gene transcription. However, despite this wealth of in vitro data, its role in how tumours respond to DNA damage induced by chemotherapeutic drugs remains controversial. In this review, we highlight some of the problems surrounding design and analysis of studies of p53 as a prognostic marker of clinical outcome, using ovarian cancer as an example. We aim to build on the knowledge of the published literature in ovarian cancer to identify criteria for clinical studies that should give a more definitive estimate of the role of p53 in clinical drug resistance. A search of three public databases using keywords combined with Boolean operators identified 64 clinical publications investigating the relationship of p53 to clinical outcome following chemotherapy in ovarian cancer. Although 43% of 215 published analyses from the 64 papers reported a significant correlation between p53 status and a clinical endpoint relevant to chemoresistance, only six analyses fulfil minimum criteria and none of these finds a statistically significant correlation of p53 with chemotherapy-resistance endpoints. The results from published clinical studies suggest a more complex role of p53 mutation in the mechanism of resistance in ovarian cancer than is suggested by in vitro studies.