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?

MAST • BOLOGNA, 25-26 OCTOBER, 2016

cholangiocarcinoma?

Carolina Reduzzi PhD student Biomarker Unit, Dept Experimental Oncology and Molecular Medicine Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy

CHOLANGIOCARCINOMA

MAST • BOLOGNA, 25-26 OCTOBER, 2016

• 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

Patel, T. (2011) Cholangiocarcinoma—controversies and challenges

• Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/nrgastro.2011.20

• the majority of tumors harbours at
• Panel of 56 genes
• 127 CCAs (70 intra-hepatic, 57 extra-hepatic)

CHOLANGIOCARCINOMA

MAST • BOLOGNA, 25-26 OCTOBER, 2016

• 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

CCA patients could benefit from targeted therapies

MAST • BOLOGNA, 25-26 OCTOBER, 2016

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

• ften impossible to obtain biopsies A possible solution is the use of CTCs

Is it possible to identify CTCs in CCA patients?

1) 16 patients, positivity rate= 25% 2) 88 patients, positivity rate= 17%

MAST • BOLOGNA, 25-26 OCTOBER, 2016

2) 88 patients, positivity rate= 17% CTC detection: CellSearch Positivity threshold: 2 CTC/ 7.5 ml

Is it possible to identify CTCs in CCA patients?

• ScreenCell:
• 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)

CTC CTM Baseline During therapy

• 31 blood samples

MAST • BOLOGNA, 25-26 OCTOBER, 2016

Baseline n=17 During therapy n=14 CTC CTM CTC CTM positivity rate 100% 53% 100% 43% median 10 1 16 range 1-66 0-8 3-71 0-64

No correlations between CTC/CTM number and clinical outcome

• 31 blood samples

(baseline/ during therapy, 9 ml)

• 17 patients

(intra/extra-hepatic CCA)

Protocol for CTC characterization

Fixation & Labeling Unbiased CTC enrichment Blood draw K2EDTA tubes

Identification Positive selection markers (PE-channel): EpCAM, CK, EGFR DAPI Vimentin (FITC-channel) Negative selection marker (APC-channel): CD45

MAST • BOLOGNA, 25-26 OCTOBER, 2016

Dielectrophoretic single cell sorting by DEPArray Molecular analysis Whole genome amplification (WGA) by Ampli1 kit

Unbiased: based on density High volume of blood (15-30 ml) Enrichment method

Protocol for CTC characterization: Enrichment methods

8 spiking experiments with 50, 25, 10 MCF7

N# MCF7 50 50 50 50 50 50 25 10 MCF7 expected* 36 36 36 36 36 36 18 7 EpCAM+/CK+/CD45- (recovery rate) 11 (31) 13 (37) 8 (22) 13 (37) 14 (39) 11 (31) 4 (22) 4 (56) Mean recovery

34%

1) ScreenCell:

MAST • BOLOGNA, 25-26 OCTOBER, 2016

*corrected for the cartridge dead volume

2) OncoQuick:

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- (recovery rate) 26 (72) 30 (83) 27 (75) 11 (61) 14 (78) 10 (56) 7 (100) 8 (100) 7 (100) Mean recovery

81%

9 spiking experiments with 50, 25, 10 MCF7

CTC+ve 8 CTC-ve 9

Protocol for CTC characterization: Enrichment methods

17 blood samples from 11 CCA patients (baseline and during therapy) Enrichment: OncoQuick 19 CTCs

High leukocyte contamination!

MAST • BOLOGNA, 25-26 OCTOBER, 2016

9

• 33 cartridges for 17 samples (time-consuming

and expensive)

• 8/19 CTCs lost during parking
• Most of the recovered CTCs were not single

3) Parsortix:

Enrichment based on size and deformability

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

Recovery rate

Cell line Spiked Recovered Recovery rate Mean

Protocol for CTC characterization: Enrichment methods

MAST • BOLOGNA, 25-26 OCTOBER, 2016

Cell line Spiked cells Recovered cells Recovery rate Mean recovery rate EGI 50 37 74 75 25 20 80 10 7 70 HuH28 50 43 86 87 25 24 96 10 8 80 HuCCT 50 39 78 90 25 23 92 10 10 100

84% vs 81% (OncoQuick)

...what about leukocyte contamination?

9 blood samples from 9 CCA patients (baseline and during therapy) Enrichment: Parsortix 9 cartridges 8 CTCs

Protocol for CTC characterization: Enrichment methods

MAST • BOLOGNA, 25-26 OCTOBER, 2016

7 CTCs recovered Ampli1 WGA kit + Ampli1 QC kit mutational profile (Cancer Hotspot Panel v2 - Thermo Fisher)

Beyond standard identification: negative selection of CTCs

Fixation & Labeling CTC enrichment: Parsortix Blood draw K2EDTA tubes

Identification Positive selection markers (PE-channel): EpCAM, CK, EGFR DAPI Negative selection marker (APC-channel): CD45

MAST • BOLOGNA, 25-26 OCTOBER, 2016

Dielectrophoretic single cell sorting by DEPArray Molecular analysis Whole genome amplification (WGA) by Ampli1 kit

DAPI Vimentin (FITC-channel)

CD14 and CD16

Monocyte Natural killer cells

23 blood samples from 14 CCA patients (baseline and during therapy)

154 “double-negative” cells

Beyond standard identification: negative selection of CTCs

MAST • BOLOGNA, 25-26 OCTOBER, 2016

CTCs or WBCs?

Ampli1 Low pass kit CNA profiles 32 double-negative cells + 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 3 2 2 4 2 2 2 2 5 2 2 2 4 3 2 6

Beyond standard identification: negative selection of CTCs

MAST • BOLOGNA, 25-26 OCTOBER, 2016

Ploidy 3 2 2 4 2 2 2 2 5 2 2 2 4 3 2 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

Patient A A1 A2 A3 A4 CTC WBC WBC CTC 3 2 2 4

CTC

A4 A2 A3

CTC WBC WBC

Beyond standard identification: negative selection of CTCs

MAST • BOLOGNA, 25-26 OCTOBER, 2016

CTC CTC WBC WBC

Beyond standard identification: negative selection of CTCs

CTC CTC CTC WBC Patient E E1 E2 E3 E4 CTC CTC WBC CTC 4 3 2 6

MAST • BOLOGNA, 25-26 OCTOBER, 2016

CTC CTC CTC WBC

Beyond standard identification: negative selection of CTCs

8 patients

Standard identification: CTC positive: 4 patients CTC negative: 4 patients Unbiased

1 3 4

MAST • BOLOGNA, 25-26 OCTOBER, 2016

Unbiased identification:

Epithelial CTCs Epithelial+ Non-epithelial CTCs Non-epithelial CTCs

1 3 4

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

Beyond standard identification: negative selection of CTCs

Vimentin expression in “Double-negative” CTCs is heterogeneous

MAST • BOLOGNA, 25-26 OCTOBER, 2016

heterogeneous “Triple-negative” CTCs?

CONCLUSIONS

MAST • BOLOGNA, 25-26 OCTOBER, 2016

Thanks to:

Maria Grazia Daidone Vera Cappelletti

MAST • BOLOGNA, 25-26 OCTOBER, 2016

Rosita Motta Patrizia Miodini Antonia Martinetti Elisa Sottotetti Filippo Cascone all the patients who recognized the importance of research in the cancer field by accepting to donate their blood for these studies Filippo De Braud Luigi Celio Katia Dotti Grant from the Italian Health Ministry to MGD