Search

Liquid biopsy components from blood, such as cell free DNA (cfDNA) and circulating tumor cells (CTCs), are prognostic for overall survival in advanced prostate cancer patients and allow the study of clonal evolution. cfDNA is easily obtained and has been widely used for molecular characterization and reflects pooled genomic profiles in a patient, but has limitations regarding gene copy number calls. CTC single-cell genomic studies generate precise gene copy number calls and elucidate intrapatient intercellular genomic heterogeneity. The main limitation of CTC analyses has been the low CTC count found in many cancer patients. We elected to study whether liquid biopsy by apheresis in advanced prostate cancer patients increases the yield of CTC to study tumor genomics, intrapatient heterogeneity, and ex vivo organotypic 3D models. Advanced metastatic prostate cancer patients being considered for clinical trials were invited to consent to apheresis. Apheresis CTC counts using CellSearchTM (Menarini) were acquired from 16 patients. The contents of the CellSearch cartridges were sorted into pure single cells by fluorescence-activated cell sorting and subsequently assessed by array comparative genomic hybridization (aCGH, Agilent Technology) for copy number aberrations (CNA). Exome and aCGH from tissue biopsies were compared to the single cell aCGH results. We generated patient-derived organoid (PDOs) cultures from apheresis products by preenrichment using density gradient (Lymphoprep) and subsequent CTC enrichment by EpCAM positive selection (EasySep StemCell Technologies). PDOs were characterized by immunofluorescence (IF) as DAPI+/CK+/EpCAM+ and CD45- cells and subsequently by aCGH for CNA. All sixteen patients (median age of 70 years; range 60-77 years) tolerated apheresis without any adverse effects. CTC counts from peripheral blood (PB) prior to apheresis ranged from 13 to 711 (median = 96), and did not significantly change post apheresis. The estimated CTC yield per apheresis ranged from 660-35473 per apheresis product (median = 3351). This constitutes an increase of 102-fold when compared to median CTC capture from 7.5mL of PB. A total of 170 single CTCs from 15 apheresis patients were genomically profiled and the copy number aberration profiles confirmed prostate cancer with multiple genomic hallmarks including CNAs such as AR amplification, chromosome 8q gain (MYC locus), and PTEN, RB1, BRCA2, TP53, CHD1 loss. CNA profiles of PDOs showed similar genomic aberrations to same patient CTCs and also reflected intrapatient heterogeneity detected by single CTC analysis. In conclusion, apheresis from advanced prostate cancer patients is a well-tolerated procedure and in our study increased the CTC yield by 102-fold when compared to PB. CTC and PDOs from apheresis products shared similar CNA profile compared with tissues biopsies and furthermore gave us an insight of the tumor heterogeneity and clonal evolution. Citation Format: Maryou B.K. Lambros, Veronica Gil, Mateus Crespo, Mariane S. Fontes, Alan Mackay, Gemma Folwer, Gemma Folwer, Berni Ebbs, Rui Neves, Penny Flohr, Susana Miranda, Semini Sumanasuriya, Daniel N. Rodrigues, Rita Pereira, Geroge Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Niven Mehra, Jane Goodall, Claudia Bertan, Suzanne Carreira, Nikolas H. Stoecklein, Leon W.M.M. Terstappen, Gunther Boysen, Joahnn S. De Bono. Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A051.

Introduction Frequently the number of 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; doi: 10.1073/pnas.1313594110) approach to screen liters of blood and thereby substantially increasing the number of CTC available for further characterization. Here we present the results obtained from 32 metastatic cancer patients subjected to DLA in the participating institutions. Methods Before the DLA procedure, whole blood was drawn in a CellSave blood collection tube and a 7.5 ml aliquot was processed with the ‘gold standard’ reference CellSearch® (Janssen Diagnostics, USA). DLAs from metastatic cancer patients were performed for ≈90 minutes to obtain 40 mL of product containing ≈4x109 mononuclear cells (MNC) representing ≈1 liter of blood. The obtained DLA samples were then divided, fixed with CellSave preservative, prepared and processed with each of the analysis techniques as described in the Standard Operating Procedures developed for DLA in the CTCTrap consortium (https://www.utwente.nl/tnw/mcbp/protocolsandtools/). Results DLAs were obtained from 20 metastatic prostate cancer patients and 12 metastatic breast cancer patients at four different European academic medical institutions. Using a SOP for the DLA procedure, similar DLA products (MNC concentration: 64x106/mL, SD = 38x106) could be generated without any noticeable side effects. CTC in 7.5 mL of blood ranged from 0 to 324 (mean = 61, median = 18). DLA processed with CellSearch represented 7 to 212 mL of blood (mean = 100, median = 97), CTC ranged from 0 to 2913 (mean = 330, median = 105). Resulting in a significant increase in CTC yield (p = 0.004) ranging from 0x to 40x (mean = 13, median = 9) when comparing 1mL of whole blood to 1mL of DLA. Filtration of 50x106 WBC of DLA, through 5um microsieves yielded only 0 to 12 CTC (mean = 2, median = 0, n = 16). Leukocyte depletion of 18 mL of DLA followed by filtration yielded 0 to 178 CTC (mean = 37, median = 4, n = 22) not yielding a relative increase versus CellSearch DLA. Leukocyte depletion followed by CellSearch yielded 271 to 1620 CTC (mean = 792, median = 484, n = 3) also not yielding a relative increase versus CellSearch DLA. In 7 patients 0 CTC were detected in 7.5mL of blood, in 4 out of these 7 patients CTC were detected in DLA. Conclusion The yield of CTC can be significantly increased by the use of DLA in patients with CTC detected in 7.5 mL of blood. Technology to select CTC from DLAs will need to be further improved before one can make optimal use of the large processed blood volumes. Citation Format: Kiki C. Andree, Anouk Mentink, Joost F. Swennenhuis, Leon W. Terstappen, Nikolas H. Stoecklein, Rui P. Neves, Rita Lampignano, Hans Neubauer, Tanja Fehm, Johannes C. Fischer, Elisabetta Rossi, Mariangela Manicone, Umberto Basso, Piero Marson, Rita Zamarchi, Yohann Loriot, Valérie Lapierre, Vincent Faugeroux, Marianne Oulhen, Francoise Farace, Gemma Fowler, Mariane Sousa Fontes, Berni Ebbs, Maryou Lambros, Mateus Crespo, Penelope Flohr, Johann S. de Bono. Diagnostic leukapheresis results in a significant increase in CTC yield in metastatic breast and 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 1723. doi:10.1158/1538-7445.AM2017-1723

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

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.