John A. Puskas Ph.D. 2/3/2017

Advances in Liquid Biopsy for Solid Tumors

NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 10 AUGUST 2013

Liquid Biopsy Origin Other Fluid Sources: CSF Urine

Liquid Biopsy Clinical Applications

Adapted from Alix-Panabieres et. al. (2016) Clinical Appilications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy . Cancer discovery

Real-time monitoring

of therapy CTC counts (BC)

KRAS mt on ctDNA (CRC)

AR mt on ctDNA (PC)

Screening and early detection of cancer EGFR mt ctDNA and

CTC counts in (NSCLC)

Stratification and

therapeutic intervention HER2 or ER

expression on CTCs (BC)

CTC counts (BC)-Metabreast trial

Therapeutic targets and resistance mechanisms

KRAS mt (CRC) EGFR my (NSCLC)

Lack of ER expression (BC)

AR mt or ARv7 expression (PC)

Risk for metastatic relapse (prognosis) CTC counts in solid tumors (e.g. breast, prostate, colorectal,

lung and bladder cancers

KRAS mt in ctDNA (CRC)

Clinical Applications

Real time liquid biopsy

Non-invasive blood sampling

Personalized Treatment

CTCs and cfDNA

Ann Transl Med. 2014 Nov;2(11):107. doi: 10.3978/j.issn.2305-5839.2014.08.11.

CTCs and Cell-Free DNA Pros and Cons

Liquid Biopsy Platforms Available at/or Coming to Moffitt Cancer Center

Janssen Diagnostics CellSearch®

ANGLE Parsortix

ddPCR

CellTracks AutoPrep Liquid Biopsy CTCs: The CellSearch® System

CellTracks Analyzer II MagNest®

FDA approved for Metastatic Breast, Colorectal and Prostate Cancers

The CellSearch® System Kit Comparison Epithelial (CTC) cells Epcam capture CK+ PE DAPI+ CD45- APC

FITC channel open MCC 50100 Breast Cancer

Melanoma (CMC) cells CD146 capture MEL+ PE CD45- APC CD34- APC MCC 50140 Melanoma

Epithelial (CXC) cells Epcam capture

CK+ FITC DAPI+ CD45- APC

PE channel open MCC 17924 Bladder Cancer

Endothelial (CEC) and Melanoma cells CD146 capture CD105+ PE DAPI+ CD45- APC

The CellSearch® Antibody Characterization

MDA-MB-231 CELLS

Composite CK-PE DAPI CD45-APC HER-2/neu FITC

SK-BR-3 CELLS

Her2/neu

Other Fully Characterized Antibodies: ER, AR, Ki67, PD-L1

The CellSearch® System: Pros and Cons

Pros Cons

FDA approved Strict CTC definition: Epcam+, DAPI+ CK8, 18, 19+ and CD45-

Automated Limited Available Channels (4) RUO Applications Relatively Expensive

Liquid Biopsy CTCs: ANGLE Parsortix PR1 Separates rare circulating cell populations based on size and deformability and is an epitope-independent enrichment method.

ANGLE Parsortix PR1: How it Works.

Int J Cancer. 2016 Jun 15; 138(12): 2894–2904. Published online 2016 Feb 26. doi: 10.1002/ijc.30007

6.5, 8 and 10 microns sizes available

Harvest cells by reversing flow

ANGLE Parsortix PR1: Pros and Cons

Pros Cons No strict phenotypic definition Cell enrichment only Can detect EMT CTCs Not FDA approved, RUO Cells are viable Cell recovery rates ~75% Color channels defined by user Visualization not included Can isolate CTCs and ctDNA from the same sample

Leukocyte contamination still present

NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 10 AUGUST 2013

Liquid Biopsy: ctDNA Other potential sources: CSF Urine Cystic Pleural

Applications of Digital PCR

• Circulating Mutations – BRAF V600E – EGFR T790M

• Sub clones in tissue • Mutational screening, minimal residual disease,

tumor evolution • Other fluids-

– Urine, CSF, Pleural

Liquid Biopsy: ctDNA, Why is it Important? Fr

eque

ncy

of c

ases

with

det

ecta

ble

ctD

NA

(%)

Mut

ant f

ragm

ents

per

5m

l

ddPCR Principle: Limiting Dilution PCR

Traditional PCR: One fluorescence

measurement

Digital PCR: Thousands of distinct

fluorescence measurements

VS.

https://en.wikipedia.org/wiki/Digital_polymerase_chain_reaction

How the assay works

Liquid Biopsy: ddPCR EGFR T790M assay Process Overview

EGFR

mut

ant

EGFR Wild Type

T790M mutant droplets

Negative EGFR T790M WT

False positives, droplets with

mix of WT and mutant

ddPCR EGFR T790M Assay Data Analysis

ddPCR EGFR T790M Assay: Sensitivity

0.012 0.42

13.7

7.1

2.23 1.39

0

2

4

6

8

10

12

14

16

NTC WT CTR sample 41%

sample 40.5%

sample 40.1%

sample 40.01%

Conc

0.012 0.42

16.6

8.2

2.8 1.44

0

2

4

6

8

10

12

14

16

18

NTC WT CTR sample 51%

sample 50.5%

sample 50.1%

sample 50.01%

conc

0.012 0.42

34.3

17.5

4.7 0.96

0

5

10

15

20

25

30

35

40

NTC WT CTR sample 61%

sample 60.5%

sample 60.1%

sample 60.01%

Conc

Liquid Biopsy: ddPCR Pros and Cons

Pros Cons

Quantitative 50ng sample/well (150ng)

Fast Little or No Multiplexing

Inexpensive Need to know mutation

Sensitivity

The Future of Liquid Biopsy

+ + + + - -

Thanks!

Morsani Molecular Diagnostic Lab Anthony Magliocco MD Carolyn Loret DeMola Gisela Caceres Ph.D. Mike Gruidl Ph.D. Elena Ryzhova Ph.D. Ravi Kothapalli Ph.D. Liang Nong MD

Moffitt Cancer Center Jhanelle Gray MD