Mariane Sousa Fontes

Circulating tumor cells, tumor-derived extracellular vesicles and plasma cytokeratins in castration-resistant prostate cancer patients

Purpose The presence of Circulating Tumor Cells (CTCs) in Castration-Resistant Prostate Cancer (CRPC) patients is associated with poor prognosis. In this study, we evaluated the association of clinical outcome in 129 CRPC patients with CTCs, tumor-derived Extracellular Vesicles (tdEVs) and plasma levels of total (CK18) and caspase-cleaved cytokeratin 18 (ccCK18). Experimental Design CTCs and tdEVs were isolated with the CellSearch system and automatically enumerated. Cut-off values dichotomizing patients into favorable and unfavorable groups of overall survival were set on a retrospective data set of 84 patients and validated on a prospective data set of 45 patients. Plasma levels of CK18 and ccCK18 were assessed by ELISAs. Results CTCs, tdEVs and both cytokeratin plasma levels were significantly increased in CRPC patients compared to healthy donors (HDs). All biomarkers except for ccCK18 were prognostic showing a decreased median overall survival for the unfavorable groups of 9.2 vs 21.1, 8.1 vs 23.0 and 10.0 vs 21.5 months respectively. In multivariable Cox regression analysis, tdEVs remained significant. Conclusions Automated CTC and tdEV enumeration allows fast and reliable scoring eliminating inter- and intra- operator variability. tdEVs provide similar prognostic information to CTC counts.

Effect on Overall Survival of Locoregional Treatment in a Cohort of De Novo Metastatic Prostate Cancer Patients: A Single Institution Retrospective Analysis From the Royal Marsden Hospital

Micro‐Abstract We retrospectively evaluated the effect of locoregional treatment (LRT) on overall survival (OS) in 300 metastatic at diagnosis (M1) prostate cancer patients. LRT was associated in univariate and multivariate analysis with longer OS, which remained significant for radiotherapy but not for transurethral prostatectomy. These data support further prospective evaluation of the benefit of local control in this patient population. Background: The optimal management of the primary tumor in metastatic at diagnosis (M1) prostate cancer (PCa) patients is not yet established. We retrospectively evaluated the effect of locoregional treatment (LRT) on overall survival (OS) hypothesizing that this could improve outcome through better local disease control and the induction of an antitumor immune response (abscopal effect). Patients and Methods: M1 at diagnosis PCa patients referred to the Prostate Targeted Therapy Group at the Royal Marsden between June 2003 and December 2013 were identified. LRT was defined as either surgery, radiotherapy (RT) or transurethral prostatectomy (TURP) administered to the primary tumor at any time point from diagnosis to death. Kaplan–Meier analyses generated OS data. The association between LRT and OS was evaluated in univariate (UV) and multivariate (MV) Cox regression models. Results: Overall 300 patients were identified; 192 patients (64%) experienced local symptoms at some point during their disease course; 72 patients received LRT (56.9% TURP, 52.7% RT). None of the patients were treated with prostatectomy. LRT was more frequently performed in patients with low volume disease (35.4% vs. 16.2%; P < .001), lower prostate‐specific antigen (PSA) level at diagnosis (median PSA: 75 vs. 184 ng/mL; P = .005) and local symptoms (34.2% vs. 4.8%; P < .001). LRT was associated in UV and MV analysis with longer OS (62.1 vs. 55.8 months; hazard ratio [HR], 0.74; P = .044), which remained significant for RT (69.4 vs. 55.1 months; HR, 0.54; P = .002) but not for TURP. RT was associated with better OS independent of disease volume at diagnosis. Conclusion: These data support the conduct of randomized phase III trials to evaluate the benefit of local control in patients with M1 disease at diagnosis.

Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap): Toward a real liquid biopsy in metastatic breast and prostate cancer

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a “liquid biopsy”. In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as “gold standard” reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0–32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1–4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Single-Cell Analyses of Prostate Cancer Liquid Biopsies Acquired by Apheresis

Purpose: Circulating tumor cells (CTCs) have clinical relevance, but their study has been limited by their low frequency. Experimental Design: We evaluated liquid biopsies by apheresis to increase CTC yield from patients suffering from metastatic prostate cancer, allow precise gene copy-number calls, and study disease heterogeneity. Results: Apheresis was well tolerated and allowed the separation of large numbers of CTCs; the average CTC yield from 7.5 mL of peripheral blood was 167 CTCs, whereas the average CTC yield per apheresis (mean volume: 59.5 mL) was 12,546 CTCs. Purified single CTCs could be isolated from apheresis product by FACS sorting; copy-number aberration (CNA) profiles of 185 single CTCs from 14 patients revealed the genomic landscape of lethal prostate cancer and identified complex intrapatient, intercell, genomic heterogeneity missed on bulk biopsy analyses. Conclusions: Apheresis facilitated the capture of large numbers of CTCs noninvasively with minimal morbidity and allowed the deconvolution of intrapatient heterogeneity and clonal evolution. Clin Cancer Res; 24(22); 5635–44. ©2018 AACR.

Abstract 993: Diagnostic leukapheresis (DLA): Molecular characterisation and organoid culture of circulating tumor cells (CTC) from metastatic castration resistant prostate cancer (mCRPC)

Introduction: CTC count is an independent predictor of overall survival in mCRPC. Isolation of CTC from peripheral blood (PB) for genomic and functional analysis is challenging, especially in patients (pts) with low CTC count. It has been shown that DLA increases CTC yield. However, it has yet to be proven whether CTC isolation from DLA can be used in complementary studies such as molecular characterization and growth of organoid culture for drug sensitivity studies. Here we present preliminary data of an on-going study, which evaluates DLA in mCRPC pts, focusing on safety, CTC enrichment, molecular characterization and feasibility for organoid culture. Methods: mCRPC pts considered for clinical trials were selected according to performance status (ECOG 0-1) and number of CTC found in 7.5ml PB (>20 cells/7.5mL). DLA products (200x106 cells) were processed using the CellSearch CTC kit (Janssen Diagnostics, LLC) according to manufacturer procedures. The contents of CellSearch cartridges were sorted into single cell by fluorescence activated cell sorting (FACS) and subsequently assessed by array comparative genomic hybridization (aCGH) for copy number aberrations (CNA). Enrichment of CTC for organoid culture was performed by density gradient of mononuclear cells followed by positive selection using magnetic beads. Results: Overall 12 mCRPC patients underwent DLA without any complication or toxicity. The mean CTC count was 90 CTC/7.5 ml peripheral blood (median = 31) and ranged from 20 to 324. CellSearch CTC count in the DLA yielded a mean of 466 (median=203) and ranged from 60 to 2496 with an up to 40-fold increase (mean = 13, median = 6) in CTC count separation when comparing 1mL of PB to 1mL of DLA. Molecular analyses of FACS single CTC from the DLA by aCGH showed that these CTC genomic profiles had the typical hallmarks of mCRPC with CNAs including AR and MYC locus (8q) amplification, and PTEN, RB1, TP53, CHD1 loss. Additionally, ex vivo culture of CTC-derived organoids was successfully achieved. aCGH of these organoids matched the genomic profile that of the CTC from the same patient. Conclusion: DLA from mCRPC pts was well tolerated and yields higher CTC capture than PB and may provide an alternative to tissue biopsy and routine blood volumes. Our strategy allowed us to isolate genomic DNA with good quality for molecular characterization and viable CTC for organoid culture and functional studies. Citation Format: Maryou B. Lambros, Veronica S. Gil, Mateus Crespo, Mariane S. Fontes, Rui N. Neves, Niven Mahra, Gemma Fowler, Berni Ebbs, Penny Flohr, George Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Joost F. Swennenhuis, Kiki C. Andree, Semini Sumanasuriya, Matthew Clarke, Pasquale Rescigno, Zafeiris Zafeiriou, Joaquin Mateo, Diletta Bianchini, Nikolas H. Stoecklein, Leon W. Terstappen, Gunther Boysen, Johann S. De Bono. Diagnostic leukapheresis (DLA): Molecular characterisation and organoid culture of circulating tumor cells (CTC) from metastatic castration resistant prostate cancer (mCRPC) [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 993. doi:10.1158/1538-7445.AM2017-993

Abstract 4679: Mismatch repair defects in lethal prostate cancer

INTRODUCTION: A subset of prostate cancers (PCs) has a hypermutated phenotype. We hypothesized that lethal PCs with a high mutational load (ML) have DNA repair defects including mismatch-repair (MMR) aberrations and that such aberrations may impact outcome. METHODS: Diagnostic and metastatic castration resistant prostate cancer (mCRPC) tissues were tested by A) immunohistochemistry (IHC) for MSH2, MSH6, MLH1, and PMS2; B) microsatellite instability (MSI) (Promega v1.2 assay); C) targeted next generation sequencing (NGS) of the coding regions of 113 genes, a panel enriched for DNA-repair genes that includes MMR genes. Utilizing NGS data we also evaluated ML and MSI by NGS (MSINGS; Pritchard et al, 2014). IHC was used as reference for sensitivity (SE) and specificity (SP) of methods B) and C).Youden’s index was used to determine the optimal cut off for the ROC for IHC in relation to MSINGS and ML. Demographics and clinical data were retrospectively collected from electronic records. Relationship between ML in diagnostic and mCRPC samples was evaluated by paired t-test. The relationship between ML and MMR deficient tumors on IHC (MMRd) and/or MSI tumors by PCR (MMRd/MSI) was analyzed by negative binomial regression model. The relationship between MMRd/MSI tumors and overall survival (OS) was analyzed using univariate and multivariate Cox regression, adjusting for radical treatment (prostatectomy or radiotherapy), Gleason score, age, PSA and stage and presence of metastatic disease at diagnosis. RESULTS: We analyzed 306 PC biopsies (180 hormone-sensitive [HS] and 126 castration resistant [CR]) from 208 patients. Overall 16 patients (7.7%) had either MMRd or MSI. Matched, same-patient, HSPC and mCRPC tumor biopsies were available for 82 patients. Only one case with MMRd status in HS was not confirmed in matched mCRPC. This patient was considered MMR proficient for the survival analysis. MSI had a SE of 80% and a SP of 97%. An MSI by NGS cut-off of 0.096 had SE of 60% and SP of 99%, with an area under ROC curve of 0.83. A ML cut-off of 11 mutations per panel had SE of 60% and SP of 99%, with an area under ROC curve of 0.79. Although ML in diagnostic samples correlates with ML in mCRPC, ML was significantly higher in mCRPC MMRd/MSI cases (p CONCLUSION: We provide evidence that MMRd/MSI status represents a negative prognostic factor for survival for metastatic CRPC. MMRd/MSI status in diagnostic biopsies accurately identifies MMRd/MSI in mCRPC. MSINGS and ML can detect MMRd/MSI tumors. Citation Format: Pasquale Rescigno, Daniel Nava Rodrigues, Wei Yuan, Suzanne Carreira, Maryou Lambros, George Seed, Ruth Riisnaes, Susana Miranda, David Dolling, Matthew Clarke, Mateus Crespo, Claudia Bertan, Gunther Boysen, Joaquin Mateo, Ana Ferreira, Adam Sharp, Ines Figueiredo, Semini Sumanasuriya, Mariane Sousa Fontes, Diletta Bianchini, Zafeiris Zafeiriou, Johann Sebastian de Bono. Mismatch repair defects in lethal prostate 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 4679. doi:10.1158/1538-7445.AM2017-4679

Abstract CT067: 2-hydroxyoleic acid (2-OHOA), a novel activator of sphingomyelin synthase with antitumor activity in refractory glioblastoma: results of the first-in-human dose-escalation (DE) study in patients with advanced solid tumors (AST) and refractory gliomas/glioblastomas

Background: 2-OHOA, is a synthetic hydroxylated lipid that activates sphingomyelin synthase and regulates the lipid content of cell membranes resulting in translocation of Ras to the cytoplasm and inactivation of Ras/MAPK, PI3K/Akt and PKC/cyclin/CDK signaling pathways. 2-OHOA causes autophagic cell death of glioma cell lines in vitro, and reduces tumor growth in numerous xenograft mice models including glioblastoma (GBM), prostate, leukemia, breast and colon cancer. It also crosses the blood brain barrier. This first-in-human trial was designed to determine the safety, tolerability, and recommended phase 2 dose (RP2D), alongside the pharmacokinetic (PK), pharmacodynamic (PD) and anti-tumor profile of 2-OHOA. Methods: Eligible pts with AST or GIII/IV gliomas/glioblastomas received 2-OHOA as a daily PO suspension in 21 day cycles using a 3+3 DE design. Adverse events (AE) were assessed by CTCAEv4; Tumor response was assessed every 2 cycles using RECIST 1.1/RANO criteria. Results: 32 pts were enrolled on the DE stage. 28 pts were evaluable for safety (13 gliomas and 15 AST). Pts were treated with doses from 500mg-16g/day in 7 cohorts. Drug-related AEs >Grade 3 of nausea, vomiting and diarrhea (n = 6) were seen at high doses. DLTs were diarrhea (n = 3) and vomiting (n = 1) at 12 and 16g/day. Extent of exposure (AUC) was generally proportional to the dose. The PK profile showed dose accumulation from 8g/day. Average t1/2 ranged from 1-2h to 8-12h with longer half-lives seen at higher doses Steady state was reached with 12 and 16g/day. 4 pts had clinical benefit for >6mo, 25% of refractory GBM patients treated (3/12) had objective responses by RANO criteria : 1 partial response (PR) and 2 stable disease (SD). The GBM pt with confirmed PR was treated at 1g/daily for 41 cycles and continues on treatment; the other 2 GBM pts were treated at 12g/day and had SD for 9 cycles. Conclusions: The MTD and RP2D of 2-OHOA is 12g/daily. Clinical benefit was observed in 4 pts including durable responses in 25% of advanced treatment-refractory GBM pts treated. Recruitment is ongoing including an expansion cohort for advanced gliomas/glioblastomas. Clinical trial information: NCT01792310 Citation Format: Juanita S. Lopez, Mariane Fontes, Niamh Coleman, Analia Azaro, Yvette Drew, Pablo Escriba, Xavier Busquets, Gareth Veal, Vicenc Tur, Antoine Perier, Elisabet Sicart, Rhoda Molife, Ruth Plummer, Jordi Rodon. 2-hydroxyoleic acid (2-OHOA), a novel activator of sphingomyelin synthase with antitumor activity in refractory glioblastoma: results of the first-in-human dose-escalation (DE) study in patients with advanced solid tumors (AST) and refractory gliomas/glioblastomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT067.