MAST BOLOGNA, 25-26 OCTOBER, 2016 DEPArray User Meeting Molecular - PowerPoint PPT Presentation

MAST • BOLOGNA, 25-26 OCTOBER, 2016

DEPArray™ User Meeting Molecular profile of single CTCs: an opportunity for patients with cholangiocarcinoma? cholangiocarcinoma? Carolina Reduzzi PhD student Biomarker Unit, Dept Experimental Oncology and Molecular Medicine Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy MAST • BOLOGNA, 25-26 OCTOBER, 2016

CHOLANGIOCARCINOMA Patel, T. (2011) Cholangiocarcinoma—controversies and challenges Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2011.20 • Arises from the transformation of cholangiocytes forming the bile ducts • CCAs are divided in intra-hepatic (ICC) and extra-hepatic(ECC) • It is a rare disease with a very poor prognosis • Surgical resection is the only potentially curative therapy, but most cases are inoperable MAST • BOLOGNA, 25-26 OCTOBER, 2016

CHOLANGIOCARCINOMA • 127 CCAs (70 intra-hepatic, 57 extra-hepatic) • Panel of 56 genes - the majority of tumors harbours at - the majority of tumors harbours at least 1 mutation ( KRAS and TP53 ) - potentially actionable mutations are present in a high percentage of tumors Simbolo et al. Oncotarget, 2014 MAST • BOLOGNA, 25-26 OCTOBER, 2016

CCA patients could benefit from targeted therapies Sia et al. Oncogene, 2013 Clinical trials with targeted therapies have failed to produce significant benefit, but patients were grouped together irrespective of their genetic alterations. The administration of therapies should be based on genetic alteration, but it is often impossible to obtain biopsies � A possible solution is the use of CTCs MAST • BOLOGNA, 25-26 OCTOBER, 2016

Is it possible to identify CTCs in CCA patients? 1) 16 patients, positivity rate= 25% 2) 88 patients, positivity rate= 17% 2) 88 patients, positivity rate= 17% CTC detection: CellSearch Positivity threshold: 2 CTC/ 7.5 ml MAST • BOLOGNA, 25-26 OCTOBER, 2016

Is it possible to identify CTCs in CCA patients? • ScreenCell: CTC CTM • size-based entichment of CTCs and CTMs • identification by morphological criteria (nucleo-cytoplasmic ratio ≥0.75, large nuclear size (≥20 µm), irregular nuclear contour and nuclear hyperchromatism) • 31 blood samples • 31 blood samples Baseline Baseline During therapy During therapy (baseline/ during therapy, 9 ml) n=17 n=14 • 17 patients CTC CTM CTC CTM (intra/extra-hepatic CCA) positivity rate 100% 53% 100% 43% median 10 1 16 0 range 1-66 0-8 3-71 0-64 No correlations between CTC/CTM number and clinical outcome MAST • BOLOGNA, 25-26 OCTOBER, 2016

Protocol for CTC characterization Blood draw Identification K2EDTA tubes � Positive selection markers (PE-channel): Unbiased CTC enrichment EpCAM, CK, EGFR � Negative selection marker (APC-channel): CD45 � DAPI Fixation & Labeling � Vimentin (FITC-channel) Dielectrophoretic single cell Enrichment method sorting by DEPArray Whole genome amplification (WGA) by Ampli1 kit Molecular analysis � Unbiased: based on density � High volume of blood (15-30 ml) MAST • BOLOGNA, 25-26 OCTOBER, 2016

Protocol for CTC characterization: Enrichment methods 8 spiking experiments with 50, 25, 10 MCF7 1) ScreenCell: N# MCF7 50 50 50 50 50 50 25 10 MCF7 expected* 36 36 36 36 36 36 18 7 EpCAM+/CK+/CD45- 11 13 8 13 14 11 4 4 (recovery rate) (31) (37) (22) (37) (39) (31) (22) (56) 34% Mean recovery 2) OncoQuick: 9 spiking experiments with 50, 25, 10 MCF7 N# MCF7 50 50 50 25 25 25 10 10 10 MCF7 expected* 36 36 36 18 18 18 7 7 7 EpCAM+/CK+/CD45- 26 30 27 11 14 10 7 8 7 (recovery rate) (72) (83) (75) (61) (78) (56) (100) (100) (100) 81% Mean recovery *corrected for the cartridge dead volume MAST • BOLOGNA, 25-26 OCTOBER, 2016

Protocol for CTC characterization: Enrichment methods 17 blood samples from 11 CCA patients (baseline and during therapy) Enrichment: OncoQuick High leukocyte contamination! 19 CTCs CTC+ve 8 CTC-ve 9 9 • 33 cartridges for 17 samples (time-consuming and expensive) • 8/19 CTCs lost during parking • Most of the recovered CTCs were not single MAST • BOLOGNA, 25-26 OCTOBER, 2016

Protocol for CTC characterization: Enrichment methods 3) Parsortix: Enrichment based on size and deformability Recovery rate 3 CCA cell lines � Staining with CellTracker � Spike of tumor cells in 5ml di sangue � Parsortix � Harvest in 96wells plate � Count of the fluorescent recovered cells Spiked Recovered Spiked Recovered Mean Mean Cell line Cell line Recovery rate Recovery rate cells cells recovery rate 50 37 74 EGI 25 20 80 75 10 7 70 50 43 86 84% vs 81% HuH28 25 24 96 87 (OncoQuick) 10 8 80 50 39 78 HuCCT 25 23 92 90 10 10 100 MAST • BOLOGNA, 25-26 OCTOBER, 2016

Protocol for CTC characterization: Enrichment methods ...what about leukocyte contamination? � 9 cartridges 9 blood samples from 9 CCA patients � 8 CTCs (baseline and during therapy) Enrichment: Parsortix 7 CTCs recovered � Ampli1 WGA kit + Ampli1 QC kit � mutational profile (Cancer Hotspot Panel v2 - Thermo Fisher) MAST • BOLOGNA, 25-26 OCTOBER, 2016

Beyond standard identification: negative selection of CTCs Blood draw K2EDTA tubes Identification � Positive selection markers (PE-channel): CTC enrichment: Parsortix EpCAM, CK, EGFR � Negative selection marker (APC-channel): Fixation & Labeling CD45 � DAPI � DAPI � Vimentin (FITC-channel) Dielectrophoretic single cell sorting by DEPArray CD14 and CD16 Whole genome amplification (WGA) by Ampli1 kit Monocyte Natural killer cells Molecular analysis MAST • BOLOGNA, 25-26 OCTOBER, 2016

Beyond standard identification: negative selection of CTCs 23 blood samples from 14 CCA 154 “double-negative” cells patients (baseline and during therapy) CTCs or WBCs? MAST • BOLOGNA, 25-26 OCTOBER, 2016

Beyond standard identification: negative selection of CTCs 32 double-negative cells Ampli1 Low pass kit CNA profiles + 4 pools of WBCs from 8 patients Patient A Patient B Patient C Patient D Patient E Cell ID A1 A2 A3 A4 B1 B2 B3 C1 C2 D1 D2 D3 E1 E2 E3 E4 Cell type CTC WBC WBC CTC WBC WBC WBC WBC CTC WBC CTC WBC CTC CTC WBC CTC Ploidy Ploidy 3 3 2 2 2 2 4 4 2 2 2 2 2 2 2 2 5 5 2 2 2 2 2 2 4 4 3 3 2 2 6 6 Patient F Patient G Patient H Cell ID F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 G1 G2 H1 H2 H3 H4 Cell type CTC WBC WBC WBC WBC CTC WBC CTC WBC CTC WBC WBC WBC WBC N.E. WBC Ploidy 3 2 2 2 2 4 2 4 2 3 2 2 2 2 2 2 • All WBC pools had normal Copy Number profiles • 11 double-negative cells (34%) showed Copy Number Alterations MAST • BOLOGNA, 25-26 OCTOBER, 2016

Beyond standard identification: negative selection of CTCs Patient A A1 A2 A3 A4 CTC CTC WBC WBC CTC CTC A4 3 2 2 4 WBC A2 A3 WBC CTC CTC WBC WBC MAST • BOLOGNA, 25-26 OCTOBER, 2016

Beyond standard identification: negative selection of CTCs Patient E E1 E2 E3 E4 CTC CTC CTC WBC CTC CTC 4 3 2 6 CTC WBC CTC CTC CTC WBC MAST • BOLOGNA, 25-26 OCTOBER, 2016

Beyond standard identification: negative selection of CTCs 8 patients Standard CTC negative: CTC positive: identification: 4 patients 4 patients 3 3 1 1 4 4 Unbiased Unbiased identification: Epithelial+ Epithelial Non-epithelial Non-epithelial CTCs CTCs CTCs By using standard identification 4 patients would have been incorrectly considered as CTC-negative The majority of patients had only either epithelial or non-epithelial CTCs MAST • BOLOGNA, 25-26 OCTOBER, 2016

Beyond standard identification: negative selection of CTCs Vimentin expression in “Double-negative” CTCs is heterogeneous heterogeneous “Triple-negative” CTCs? MAST • BOLOGNA, 25-26 OCTOBER, 2016

CONCLUSIONS MAST • BOLOGNA, 25-26 OCTOBER, 2016

Thanks to: Maria Grazia Daidone Vera Cappelletti Antonia Martinetti Filippo De Braud Rosita Motta Elisa Sottotetti Luigi Celio Patrizia Miodini Filippo Cascone Katia Dotti all the patients who recognized the importance of research in the cancer field by accepting to donate their blood for these studies Grant from the Italian Health Ministry to MGD MAST • BOLOGNA, 25-26 OCTOBER, 2016