Aurélie Auguste

The PI3K/Akt/mTOR pathway in ovarian cancer: therapeutic opportunities and challenges.

The phosphatidylinositol 3 kinase (PI3K) pathway is frequently altered in cancer, including ovarian cancer (OC). Unfortunately, despite a sound biological rationale and encouraging activity in preclinical models, trials of first-generation inhibitors of mammalian target of rapamycin (mTOR) in OC have demonstrated negative results. The lack of patient selection as well as resistance to selective mTOR complex-1 (mTORC1) inhibitors could explain the disappointing results thus far. Nonetheless, a number of novel agents are being investigated, including dual mTORC1/mTORC2, Akt, and PI3K inhibitors. Although it is likely that inhibition of the PI3K/Akt/mTOR pathway may have little effect in unselected OC patients, certain histological types, such as clear cell or endometrioid OC with frequent phosphatidylinositol-4,5-biphosphate 3-kinase, catalytic subunit alpha (PIK3CA) and/or phosphatase and tensin homolog (PTEN) alterations, may be particularly suited to this approach. Given the complexity and redundancy of the PI3K signaling network, PI3K pathway inhibition may be most useful in combination with either chemotherapy or other targeted therapies, such as MEK inhibitors, anti-angiogenic therapy, and hormonal therapy, in appropriately selected OC patients. Here, we discuss the relevance of the PI3K pathway in OC and provide an up-to-date review of clinical trials of novel PI3K inhibitors alone or in combination with cytotoxics and novel therapies in OC. In addition, the challenges of drug resistance and predictive biomarkers are addressed.

DNA damage response as a therapeutic target in gynecological cancers.

Purpose of review The proven activity of poly ADP ribose polymerase (PARP) inhibitors in BRCA-mutated homologous recombination deficient (HRD) ovarian cancer has led to the availability to patients with ovarian cancer of the first targeted therapy with an associated predictive biomarker. Our focus has recently turned towards expanding the clinical utility of PARP inhibitors beyond BRCA mutated ovarian cancer, and to a search for novel targets within DNA damage response (DDR). Recent findings Early trials in unselected patients with ovarian cancer showed responses to PARP inhibition in BRCA-wildtype ovarian cancer, and recent genomic studies have demonstrated that germline or somatic aberrations in other homologous recombination genes are present in a significant proportion of ovarian cancers. In addition, PARP inhibition may be of value in molecularly defined subsets of endometrial or cervical cancers. Novel DDR inhibitors such as ATR, ATM, WEE1 or DNA-PK inhibitors are also being tested in patients. Finally, combinatorial strategies of DDR inhibitors with antiangiogenic agents, phosphoinositide 3-kinase inhibitors or immunotherapies may further increase therapeutic efficacy. Summary In the future, patients with gynaecological malignancies may be rationally selected for PARP inhibition on the basis of comprehensive evaluation of homologous recombination genomic alterations, or HRD assays. Furthermore, novel DDR inhibitors have the potential to expand the repertoire of therapeutic options available to these patients.

Markers of the p53 pathway further refine molecular profiling in high-risk endometrial cancer: A TransPORTEC initiative

BACKGROUND The morphological classification of high-risk endometrial cancer is of limited prognostic value. Recent attempts to stratify tumours according to molecular signatures have shown considerable promise. Here we attempted to further refine molecular classifications using markers of the p53 pathway. METHODS We analysed the expression of p53 as well as three downstream markers of the p53 pathway, p21, mdm2 and phospho-p63 (pp63), by immunohistochemistry in a series of 114 endometrial cancers (86 endometrioid, 28 non-endometrioid subtype) with high-risk features (such as high tumour grade and deep myometrial invasion) and correlated results with clinical outcome. The Cancer Genome Atlas (TCGA) data were used to analyse TP63 mutations and copy-number alterations using cBioPortal. TP53 was silenced in two endometrial cancer cell lines to study its effect on p21 and p63. RESULTS About half of the tumours showed a p53 mutant phenotype and there was a strong negative correlation with p21 expression. Being marker positive for pp63 or mdm2 was associated with a significantly increased likelihood of dying, [hazard ratios 5.93 (95% CI 2.37-7.27) and 7.48 (95% CI 3.04-9.39), respectively]. These findings were seen in both p53 wildtype and p53 mutant tumours. Only 11% of TCGA endometrial cancers had a functional TP63 alteration. Upon silencing of TP53, p21 expression was decreased in one cell line, but no effects on p63 were observed. CONCLUSION Markers of the p53 pathway improve stratification of endometrial cancers and provide novel insights into the role of this pathway in the disease.

Impact of neoadjuvant chemotherapy on the immune microenvironment in advanced epithelial ovarian cancer: Prognostic and therapeutic implications: Prognostic and therapeutic implications

Over the last decade, increasing evidence highlights the role of the host immune system in the control of tumor growth and the prognostic implications of tumor infiltrating lymphocytes (TILs) in ovarian cancer. Most data support a better prognosis with accumulation of CD3+ and CD8 + TILs and a poor outcome associated with increased regulatory T cells. However, only a small number of studies have focused on the effect of neoadjuvant chemotherapy (NACT) on the tumor immune microenvironment. This review will provide an update on the prognostic value of TIL subpopulations at diagnosis and a comprehensive overview of the recent studies evaluating the impact of neoadjuvant chemotherapy on TILs and their relationship to clinical outcome in advanced ovarian cancer. This information could help in future investigations of immunotherapy as maintenance following primary treatment.

Anomalies de la réparation de l’ADN et cancers gynécologiques

The demonstration of frequent defects in the DNA damage response in high grade ovarian cancer has paved the way for a new therapeutic approach aimed at exploiting this unique vulnerability. The efficacy of poly (ADP) ribose polymerase inhibitors (PARPi) in patients with homologous recombination (HR) DNA repair deficient ovarian cancer (OC) resulting from a BRCA1/2 mutation has provided the proof of concept for synthetic lethality. Thus, olaparib is now approved by the EMA as maintenance therapy after response to a platinum regimen for patients with recurrent, platinum-sensitive, high-grade serous, BRCA1/2-mutated ovarian cancer. Furthermore, several recent trials in OC have demonstrated that the benefit of PARPi may not be limited to patients with BRCA mutations. These data, combined with genomic studies suggesting that a significant proportion of OC may harbor somatic and germline alterations in other HR genes open huge perspectives for exploiting DNA repair as a therapeutic strategy. The current priorities are to (i) determine whether new biomarkers of homologous recombination deficiency may identify the BRCA wild-type subset likely to derive benefit from PARPi; (ii) to determine whether the efficacy of PARPi can be improved by combinatorial strategies (with chemotherapy, radiotherapy, immunotherapy, anti-angiogenesis or DNA repair inhibitors) and (iii) to develop new approaches exploiting DNA repair deficiencies in ovarian and other gynecological tumors.

Refinement of high-risk endometrial cancer classification using DNA damage response biomarkers: a TransPORTEC initiative

The TransPORTEC consortium previouslclassified high-risk endometrial cancer including poor-risk histologies such as clear cells, into four molecular subtypes “POLE mutated,” “microsatellite unstable,” “TP53 mutated,” and “no specific molecular profile.” We evaluated whether DNA damage response biomarkers could further refine this high-risk tumors classification, in particular the heterogeneous “no specific molecular profile” and “TP53 mutated” subsets recently qualified as poor prognosis in high-risk endometrial cancer. DNA damage response biomarkers including proteins involved in DNA damage (δ-H2AX), homologous recombination (RAD51), regulators of error-prone Non Homologous End-Joining (DNA-pk, FANCD2), and PARP-1 were evaluated in 116 high-risk tumors by immunohistochemistry. CD8 and PD-1 expression by immunochemistry and mutation analyses were performed previously. Survival outcome were calculated using Kaplan-Meier and Log-rank test. None of the DNA damage response biomarkers alone were prognostic. However markers were informative within molecular subsets. Among the “no specific molecular profile” subset, δ-H2AX+ was significantly predictive of poor disease free survival (Hazard Ratio = 2.56; p = 0.026), and among “TP53 mutated,” a DNA-pk+/FANCD2- profile (favouring error-prone Non Homologous End-Joining) predicted worst disease free survival (Hazard Ratio = 4.95; p = 0.009) resulting in five distinct prognostic subgroups from best to worst prognosis: group1 “POLE mutated/Microsatellite unstable” > group2 “no specific molecular profile with no DNA damage” > group3 “TP53 mutated/Non Homologous End-Joining negative” > group4 “no specific molecular profile with high DNA damage” > group5 “TP53 mutated/Non Homologous End-Joining positive”; p = 0.0002). Actionable targets were also different among subsets. Group3 had significantly higher infiltration of PD-1+ immune cells (p = 0.003), segregating with group1. Group2 had frequent PI3K pathway mutations and ER positivity. While group5, with the worst prognosis, had high DNA damage and PARP-1 expression providing a rationale for PARP inhibition. Our findings have refined the TransPORTEC prognostic classification of high-risk endometrial cancer into five distinct subgroups by integrating DNA damage response biomarkers and identified molecular subtype-specific therapeutic strategies.

Small Cell Carcinoma of the Ovary

Small cell carcinoma of the ovary (SCCO) is a rare tumor in a very young population with a bad prognosis, even when diagnosed as stage 1A. Recent data suggest a possible implication of SMARCA4 gene in SCCOHT oncogenesis and suggest they may simply be an ovarian variant of malignant rhabdoid tumors. Multimodal approach including intensive chemotherapy, radical surgery and possibly radiotherapy is actually often proposed.

Abstract B02: DNA repair landscape in High Grade Ovarian Cancer (HGOC) and evolution with neo-adjuvant chemotherapy

Background: HGOC is frequently diagnosed at an advanced stage where NeoAdjuvant Chemotherapy (NACT) provides an essential component of the treatment strategy. HGOC are initially very chemosensitive, putatively due to DNA repair defects. While BRCA mutations explain impaired homologous recombination in 12% to 15% of HGOC, loss of other DNA repair proteins may also be relevant. Unfortunately despite initial response rates of 80%, most invariably relapse. Chemo-resistance mechanisms are poorly understood and previous studies have only focused on the characterization at diagnosis. However given their chemosensitivity, profiling the post-NACT tumor (cleared of sensitive cells and enriched for chemoresistant clones) may be more relevant to uncover mechanisms of platinum resistance. We aimed to evaluate the evaluate the expression of key DNA repair biomarkers in a large series of HGOC tumors obtained at baseline, Post-NACT and at relapse. Methods: TMA was constructed using sequential FFPE tumor samples of high-grade ovarian cancer collected at diagnosis (pre-NACT) (N=143), post-NACT (N=139) and at relapse (N=36) in 170 patients, encompassing 115 patients with sequential tumor samples. Expression of 53BP1, PAR, PARP-1 and ATM were scored by immunohistochemistry using an H-score (0-300) with biomarker negative defined as H-score = Results: At diagnosis, a significant proportion of tumors showed loss of DNA repair proteins: 61% were PAR-negative, and 24%, 15% and 3% negative for ATM, 53BP1 and PARP-1 respectively and a fifth of tumors showed concomitant loss in 2 or more DNA proteins. There was an overall decrease in DNA repair protein expression with NACT, which was significant for PAR (pre-NACT vs post-NACT H-score= 45 vs 19, p=0.0025, N=201) and PARP-1(H-score=208 vs 190; p=0.0149, N=205). In addition paired samples analysis of matched pre- and post-NACT samples showed complete loss of biomarker expression after treatment in a subset (24%, 16%, 6% and 3% for PAR, ATM, TP53BP1 and PARP-1). When comparing post-NACT to relapsed samples, PAR expression increased significantly (mean H-score=19 vs 54, p=0,025). The prognostic value of loss of each DNA repair at diagnosis and after NACT was evaluated. At diagnosis, 53BP1-negative tumors were associated with a significantly worst PFS (HR=3.484; p: 0.0025). Post-NACT, ATM-negative tumors had a significantly worst PFS (HR=1.690; p: 0.0374). As PAR, TP53BP1 and ATM were shown not to be correlated in individual tumors (Pearson correlation), the impact of combined biomarker loss on PFS was evaluated. At diagnosis, double TP53BP1/PAR-negative tumors predicted shorter PFS (PFS: median=17.65 vs 23.23 mths, p:0.0372, HR=0.2237, N=58), while post-NACT double ATM/PAR-negativity was significantly predictive of poor outcome in terms of PFS and OS (PFS: median=17.35 vs 23.77 mths, p:0.0198, HR=0.4759; OS: median=35 vs 50 mths, p:0.0120, HR=0.3602; N=80). Conclusions: We present the first study of change in DNA repair protein expression with NACT. At diagnosis, HGOC is associated with loss of key DNA repair proteins in a significant proportion of patients and NACT can induce significant loss of DNA repair proteins. Combined loss of DNA repair proteins was significantly predictive of survival. Work in ongoing to extend this analysis to a greater panel of DNA repair biomarkers. Citation Format: Aurelie Auguste, Soizick Mesnage, Audrey Le Formal, Elena Cojocaru, Francoise Drusch, Julien Adam, Sebastien Gouy, Enrica Bentivegna, Catherine Lhomme, Patricia Pautier, Catherine Genestie, Alexandra Leary. DNA repair landscape in High Grade Ovarian Cancer (HGOC) and evolution with neo-adjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B02.

Abstract A1-27: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) beyond SMARCA4 mutations: A comprehensive genomic analysis

Background: Small cell ovarian carcinoma of the hypercalcemic type (SCCOHT) is an extremely rare and aggressive tumor that affects mainly young women (median age = 24 years). Prognosis is poor as most patients die within 2 years of diagnosis. Until recently, the literature describing the genomic profile of SCCOHT was scarce. In the last few months, four groups have now confirmed that SMARCA4 mutations are a frequent event in SCCOHT. Methods: We performed an integrated genomic analysis using WES, RNA-seq and CGH approaches to identify other recurrent genomic alteration and potential therapeutic targets. Candidate SNVs identified by WES were validated by Sanger and RNA-Seq. 8 frozen tumors were available, including 6 with matching normal control DNA. In addition 33 tumor samples were available as FFPE. Results: Despite their high grade and aggressive clinical course, SCCOHT tumors display genomic stability, low mutation load (0.53mut.Mb) and few SCNAs. However when present, genomic alterations were recurrent. Integrated WES and CGH analysis revealed a frequent and unusual 19p CN LOH (in 5 of 6 tumors). Although overall mutation rate was low, the mutations that did occur were novel and recurrent in 50%-80% of SCCOHT. In addition to SMARCA4 mutation (M+), we validated 10 novel recurrent M+ (including ABCA7, ANKRD24, CACTIN, EMR1, FBN3, FUT5, GRIN3B, KANK3, KRI1 and PLK5) in 50%-80% of tumors at high allelic frequencies (mean=0.87). Similarly, when present, amplifications were recurrent, common to a third of tumors: such as the mitochondrial redox enzymes (SHMT2, NDUFA4L2) or the transmembrane transporter LRP1 were amplified (Log2>2.3). No common fusion transcripts were identified. Since SCCOHT often display initial chemosensitivity, but rapid progressions, we investigated the differential genomic profiles of treatment naive versus chemotherapy treated tumors to uncover candidate resistance genes. The only alteration significantly enriched in post treatment tumors were M+ in the putative efflux pump, ABCA7. These M+ could not be identified even at low allele fractions in treatment naive tumors, but were identified in all 3 treated tumors with a mean allelic frequency of 0.83. In terms of therapeutic implications, modulating mitochondrial metabolism or targeting the membrane transporter LRP1 or the ABCA7 efflux pump could provide therapeutic venues in the future. However the most promising approaches for now may be targeting cell cycle checkpoint regulators or chromatin remodelling. M+ in the tumor suppressor PLK5 were confirmed in 79% of a cohort of 33 SCCOHT and many were predicted as damaging. Inactivation of PLK5 is associated with a loss of G2/M checkpoint regulation and cell harbouring PLK5 mutations were sensitive to PLK1 inhibitors. More interestingly, SMARCA4-M+ SCCOHT demonstrated complete loss of SMARCA4 expression as well as loss of the only other SWI/SNF catalytic subunit SMARCA2 suggesting that double SMARCA4/SMARCA2 negative SCCOHTs are characterized by a catalytically inactive SWI/SNF complex. This hypothesis was supported by changes in RNA expression levels in the main transcriptional targets for SWI/SNF (RHOA, RB and the E2F family). Importantly, the double SMARCA2/4 negative BIN-67 SCCOHT cell line displaying both a SMARCA4 M+ and loss of SMARCA2 expression was exquisitely sensitive to HDAC and MT inhibitors, while cell lines with either a SMARCB1-M+ or SMARCA4-M+ with retained SMARCA2 expression were not. These data make double SMARCA2/4 negative by IHC a potential predictive biomarker for histone modifying inhibitors in SCCOHT or potentially in other double SMARCA2/4 negative malignancies. Conclusions: We present the 1st integrated genomic analysis of SCCOHT. SCCOHT demonstrate a remarkably homogeneous genomic profile and potentially actionable alterations. In particular, dual null SMARCA2/SMARCA4 SCCOHTs could be selected for clinical trials with EZH2, HDAC or MT inhibitors. Citation Format: Aurelie Auguste, Marc Deloger, Joanna Cyrta, Audrey Le Formal, Catherine Richon, Nathalie Droin, Beatrice Brunn, Olivier Caron, Mojgan Devouassoux, Catherine Lhomme, Patricia Pautier, Catherine Genestie, Alexandra Leary. Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) beyond SMARCA4 mutations: A comprehensive genomic analysis. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-27.