Gemma Fowler

DNA-Repair Defects and Olaparib in Metastatic Prostate Cancer

BACKGROUND Prostate cancer is a heterogeneous disease, but current treatments are not based on molecular stratification. We hypothesized that metastatic, castration-resistant prostate cancers with DNA-repair defects would respond to poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibition with olaparib. METHODS We conducted a phase 2 trial in which patients with metastatic, castration-resistant prostate cancer were treated with olaparib tablets at a dose of 400 mg twice a day. The primary end point was the response rate, defined either as an objective response according to Response Evaluation Criteria in Solid Tumors, version 1.1, or as a reduction of at least 50% in the prostate-specific antigen level or a confirmed reduction in the circulating tumor-cell count from 5 or more cells per 7.5 ml of blood to less than 5 cells per 7.5 ml. Targeted next-generation sequencing, exome and transcriptome analysis, and digital polymerase-chain-reaction testing were performed on samples from mandated tumor biopsies. RESULTS Overall, 50 patients were enrolled; all had received prior treatment with docetaxel, 49 (98%) had received abiraterone or enzalutamide, and 29 (58%) had received cabazitaxel. Sixteen of 49 patients who could be evaluated had a response (33%; 95% confidence interval, 20 to 48), with 12 patients receiving the study treatment for more than 6 months. Next-generation sequencing identified homozygous deletions, deleterious mutations, or both in DNA-repair genes--including BRCA1/2, ATM, Fanconis anemia genes, and CHEK2--in 16 of 49 patients who could be evaluated (33%). Of these 16 patients, 14 (88%) had a response to olaparib, including all 7 patients with BRCA2 loss (4 with biallelic somatic loss, and 3 with germline mutations) and 4 of 5 with ATM aberrations. The specificity of the biomarker suite was 94%. Anemia (in 10 of the 50 patients [20%]) and fatigue (in 6 [12%]) were the most common grade 3 or 4 adverse events, findings that are consistent with previous studies of olaparib. CONCLUSIONS Treatment with the PARP inhibitor olaparib in patients whose prostate cancers were no longer responding to standard treatments and who had defects in DNA-repair genes led to a high response rate. (Funded by Cancer Research UK and others; ClinicalTrials.gov number, NCT01682772; Cancer Research UK number, CRUK/11/029.).

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.

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 3973: Diffusion-weighted imaging of bone metastases as treatment response biomarker in prostate cancer

INTRODUCTION: Response assessment of bone metastases (BM) remains a challenge for drug development in metastatic castration resistant prostate cancer (mCRPC) as standard imaging techniques, computed tomography and bone scintigraphy, fail to characterize BM. Diffusion-weighted imaging (DWI) is a functional MRI technique that studies the motion of water molecules within a tissue informing on cellularity. We hypothesized that changes in whole body (WB) DWI informs on BM response to treatment in mCRPC. METHODS: We conducted a phase II trial of the PARP inhibitor olaparib in mCRPC (TOPARP-A; CRUK/11/029); the primary endpoint was response rate defined using RECIST 1.1, PSA falls of ≥50% and conversion of circulating tumour cell (CTC) counts from ≥5 to RESULTS: Overall, 33/42 pt consented to the WB-DWI substudy of whom 21 had WB-DWI at baseline and at 12w. Of these 29% (6/21) were classified as responders to olaparib as per the primary endpoint definition and had not progressed prior to 12w. At baseline, median CTC count was 46 CTC/7.5ml blood and median PSA was 411 ng/ml for this cohort. When assessing all the areas of DWI signal abnormality, median volume of BM per patient was 445ml and mADC was 782 x10-6 mm2/s. Baseline CTC counts and PSA were significantly associated with volume of BM (ρ = 0.59, p = 0.005; ρ = 0.64, p = 0.002 respectively). All pts who responded to olaparib showed a decrease in volume of BM (median -41.1%, range -58.8%, -6.3%), whilst in non-responders a decrease was not observed in any pt (median 20.7%, range 0.0%, 76.9%); the difference between responders and non-responders was statistically significant (p = 0.001). Increases in mADC after 12 weeks of treatment were associated with increased odds of response (Odds Ratio: 1.08, 95% CI 1.00, 1.15, p = 0.04). Additionally, we detected a significant positive association between changes in volume of BM estimated by DWI and best percentage change in PSA and CTC (ρ = 0.63, p = 0.002 and ρ = 0.77, p CONCLUSION: Decrease in volume and increase in mADC of BM assessed by WB-DWI are indicators of response to olaparib in BM from mCRPC. These data require validation but support the use of WB-DWI for assessing BM during treatment. Citation Format: Raquel Perez-Lopez, Matthew D. Blackledge, Helen Mossop, Joaquin Mateo, David Collins, Veronica A. Morgan, Alison McDonald, Shahneen Sandhu, Aurelius Omlin, Diletta Bianchini, Zafeiris Zafeiriou, Pasquale Rescigno, Michael Kolinsky, Daniel Nava Rodrigues, Penny Flohr, Berni Ebbs, Gemma Fowler, Nuria Porta, Emma Hall, Martin Leach, Johann S. de Bono, Dow-Mu Koh, Nina Tunariu. Diffusion-weighted imaging of bone metastases as treatment response biomarker in prostate cancer. [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 3973.

Abstract 2243: Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts)

Background: NSCLC exhibits intratumor heterogeneity, with subpopulations of cells undergoing epithelial-mesenchymal transition. Such CTCs from NSCLC pts may be missed by the EpCAM-based CELLSEARCH® system (CS). The label-free ClearCell® FX inertial microfluidic system (FX) isolates CTCs based on size and inertia and may lead to more accurate CTC capture. PD-1/PD-L1 inhibitors have shown benefit in PD-L1+ NSCLC pts, but responses are still seen in PD-L1- pts, suggesting limitations in tumor PD-L1 testing. Also, PD-L1 testing on CTCs may be more practical than tumor rebiopsies and may provide insights into cancer heterogeneity. Methods: FX was validated with EpCAM-high and EpCAM-low cancer cell lines labeled with CellTracker dyes spiked into healthy volunteer (HV) blood for repeatability and reliability. Enriched cells were detected with the automated Bioview Duet imaging system for recovery (%). Identical spiking studies were conducted on CS for comparison. Antibodies for 5-color immunofluorescence (IF) (CK, CD45, DAPI, TTF1 [to detect lung adenocarcinoma cells], PD-L1) were optimized on EpCAM-high and EpCAM-low cell lines. 8ml of blood from NSCLC pts were enriched with FX for 5-color IF characterization. 7.5ml of blood from the same pts taken at identical timepoints were enumerated with CS for comparison. Blood was obtained from HV for CTC enumeration on FX and CS as controls. Results: Cell recovery of EpCAM-high cancer cell lines using FX produced similar counts to CS, e.g. lung NCI-H2066 cells: FX 64.9%±4.5 vs CS 74.4%±9.2 (p = 0.05); lung H1975 cells: FX 74.1%±11.9 vs CS 74.7%±10.7 (p = 0.91). In contrast, for EpCAM-low cells, a significant difference in cell recovery between FX and CS was seen, e.g. lung A549 cells: FX 59.3%±5.8 vs CS 26.1%±7.6 (p Conclusions: FX resulted in consistently high cell recovery rates regardless of EpCAM status. Higher CTC counts were isolated with FX vs CS in 90% of NSCLC pts. 40% of NSCLC adenocarcinoma pts had PD-L1+ TTF1+ CTCs, but PD-L1 CTC heterogeneity was seen in other pts, which may in part explain differences in responses to PD-1/PD-L1 inhibitors in NSCLC. Citation Format: Zai Ahmad, Jen Fraser-Fish, Rajiv Kumar, Bernadette Ebbs, Gemma Fowler, Penny Flohr, Mateus Crespo, Sanjay Popat, Jaishree Bhosle, Udai Banerji, Mary O’Brien, Johann S. de Bono, Timothy A. Yap. Characterization of PD-L1 expression on circulating tumor cells (CTCs) isolated with a label-free inertial microfluidic system from advanced non-small cell lung cancer patients (NSCLC pts). [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 2243.