LIQUID BIOPSY

Miguel Abal Investigador I3SNS

Oncoloxía Médica Traslacional Instituto de Investigación Sanitaria de Santiago (IDIS)

Complexo Hospitalario Universitario de Santiago/SERGAS

www.idisantiago.es

Recent technological advances have enabled the detection and detailed characterization of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) in blood samples from patients with cancer. Often referred to as a "liquid biopsy," CTCs and ctDNA are expected to provide real-time monitoring of tumor evolution and therapeutic efficacy, with the potential for improved cancer diagnosis and treatment.

A major reason for treatment failures is our inability to monitor tumor evolution and adapt treatment accordingly.

Liquid biopsy

Solid biopsy

Pantel K , and Alix-Panabières C Cancer Res 2013;73:6384-6388

Circulating Tumor Cell (CTC)

• liquid biopsy (clinical tool) • new therapeutic target to prevent and/or eradicate metastasis

We need high sensitive and specific techniques to isolate and quantify CTC

Schematic view of CTC enrichment methods

Pantel & Alix-Panabières. Circulating tumour cells in cancer patients: challenges and perspectives. Trends Mol Med. 2010 Sep;16(9):398-406

The EPISPOT assay procedure.

The microfluidic circulating tumor cell (CTC) chip

Efficient and selective separation of viable CTCs from peripheral whole blood samples, mediated by the interaction of target CTCs with antibody (EpCAM)-coated microposts under precisely controlled laminar flow conditions.

A circulating tumor cell (CTC) selection microfluidic device integrated to an electrokinetic enrichment device for preconcentrating CTCs directly from whole blood to allow for molecular profiling.

Microfluidic devices for CTC capture and characterisation

Tools:

CellTracks® AutoPrep® System

CellTracks Analyzer II®

7.5mL

CK-/CD45 +

CK+/CD45 -

Ferrofluid-nanoparticules/EpCAM CYTOKERATINE CD45 DAPI

The CellSearch™Circulating Tumor Cell Kit is intended for the enumeration of circulating tumor cells (CTC) of epithelial origin (CD45-, EpCAM+, and cytokeratins8, 18+, and/or 19+) in whole blood.

FDA APPROVAL FOR CLINICAL USE

(Cristofanilli et al., N Engl J Med

2004) (Cohen et al., J Clin Oncol

2008) (de Bono et al., Clin Cancer Res

2008)

(Cristofanilli et al., N Engl J Med 2004)

(Cohen et al., J Clin Oncol 2008)

(de Bono et al., Clin Cancer Res 2008)

Progression-free survival (PFS) and overall survival (OS) of metastatic colorectal cancer patients with < three and ≥ three circulating tumor cells (CTCs) in 7.5 mL of blood (A, B) before therapy, (C, D) 1 to 2, 3 to 5, 6 to 12, and 13 to 20 weeks after initiation of therapy, and (E, F) by circulating tumor cell status at baseline and 3 to 5 weeks.

Cohen S J et al. JCO 2008;26:3213-3221

12-weeks

CT

Prediction of therapy response based on CTC-biomarker analysis

Staging

CT

1 month

Cycle 1 Cycle 2 Cycle 3 Cycle 4

Baseline 4-weeks

Anti EpCAM Preamplification

mC

RC

(n=

50

)

RNA extraction qPCR for selected transcripts

vs

21

CTC-markers can predict therapy response more accurately

and earlier than CT imaging

For any technology to be used in the clinic, demonstration of analytic validity (the accuracy of the test to measure the target of interest), clinical validity (the value of the test to predict the clinical outcome), and ultimately clinical utility (ability of the test to lead to improved clinical outcome when treatment choice is informed by test results) is required. A recent study assessed the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data (20 studies; approx. 2000 patients). The data confirmed the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improved the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not. (Bidard et al., Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol. 2014;15(4):406-14).

The value of CTC enumeration for treatment decision making in metastatic breast cancer was prospectively tested in the Southwest Oncology Group (SWOG) S0500 clinical trial. The SWOG trial evaluated the benefit of an early change in chemotherapy for patients with persistently increased CTCs at first follow-up after starting first-line chemotherapy. Of 595 evaluable patients, 123 patients with persistently elevated CTCs on day 21 of therapy were randomized to either continue the same treatment or to switch to an alternative chemotherapy of physician's choice. In this trial, an early switch to an alternative chemotherapy did not increase overall survival (OS). Although CTCs were strongly prognostic, the absence of a survival benefit from changing treatment based on elevated CTC counts suggests that earlier detection of relapse can only be important when a more effective treatment is available: Switching from one ineffective therapy to another ineffective therapy does not change outcome. Instead, changing treatment based on CTC molecular characterization might be a more promising approach to test.

DEP Array semi-conductor chip

Silicon Biosystems Patented Technology:

Moving DEP Cages

+ + + + - -

-

nDEP Cage nDEP Cage

+ + + + - -

Non-uniform electric field

generated by the chip electrodes (cross section)

Cell trapping by DEP cages

cage-move

+ + +

+ - +

+ + +

+ + +

+ - +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + -

+ + +

+ + +

+ - +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ - +

+ + +

+ + +

- + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ - +

+ + +

+ + +

+ - +

+ + +

+ + +

+ + +

+ + +

+ + +

+ - +

+ + +

+ - +

+ + +

+ + +

Moving DEP Cages Enables Outstanding Performance in Single-Cell Sorting

Main

chamber

Parking Recovery

1. Inject, trap and image all cells

2. Move all cells of interest

into Parking chamber

3. Move separately

to Recovery chamber

and flush

Homogeneous pools of cells from FFPE

300 pure cells tumor vs. stromal from FFPE tissue

Direct multiplex PCR

Deep Sequencing

data analysis

100% purity

Digitalization improves resolution

Sequencing

Quantitation

T GG TA CA G T T

10

T NA TA G G AC T

20

AA TG GG AA AA

30

TTTA AA G TGC

40

AAC CA G T CT G

50

AG T CAA CA G A

60

T TT CT T CCAA

70

T TA NGT T GAC

80

AG GT GTAGG T

90

C C TAC TA A TA

100

C T GT AC C TA T

110

A GC TT TAT GT

120

C CACA

AG AT T T

130

C TAT GA GTA T

140

C T GAT CA TA C

150

T GT C TTAC T T

160

TG ATAA A AC C

170

T CCAANT C CC

180

NC T AT CA T T T

190

T T GGT T TC CA

200

T C T T CC T GGC

210

AA AC T CA T T T

220

C TT C TA A TA C

230

T GTA T CA T C T

240

GC TC C T GT A

AT

250

C TA AT A GA GC

260

T TC C T T TA GT

270

T GC C CCC C T A

280

T C TT TA T T GT

290

GA C GA GG GGT

300

C GT T GC CA A A

310

GA GT GA TC T G

320

A GGGA A GT TA

330

A A GG ATAC A G

340

T T C C T TGT CT

350

AT C GGC TC C T

360

GCT T C TGAG A

370

GG GA

AGT T GT T

380

GTC TC TA C C C

390

C AGA CCT GAA

400

GC T CT C T T C T

410

GGT GGG GC T G

420

T TG GC T CT GG

430

T C T GC TC T GA

440

A GAA A AT T CC

450

C T GGCC T TC C

460

CTT GT AGGAA

470

GGC CA G AT CT

480

T CCC TAA A AA

490

AT TA GC C

CT GT

500

CAC TCAGT AC

510

A A T C T T T CAT

520

T TG GT GT CCT

530

TCCT TT C CAC

540

AT T T C CA AC A

550

GC C CT T TT TC

560

CT AGGGGC CC

570

T GC AA T T T CT

580

GGC TAT GT GC

590

C C T TC TT T GC

600

CACAA T TGAA

610

AC AC TT AACA

620

AT CT

Model 3100

BC 1.2.d.4

50_50F_E03_09.ab1

50_50F

Lane 9

Signal G:515 A:390 T:445 C:290

DT3100POP6{BD}v2.mob

Points 592 to 11557 Base 1: 592

Page 1 of 2

Fri, Apr 28, 2000 7:36 PM

Tue, Apr 4, 2000 6:31 AM

Spacing: 13.65

TTT CT CT

630

GGT T CCTAAA

640

AT T GC CT CT C

650

T GCAT CA TT A

660

T GGTAG CT GA

670

AT TT G TT AC T

680

T GGC TCA TT G

690

CT TCAGC C AA

700

AAC T CTT G CC

710

TT AT GG C C GG

720

GTC C TCC CAC

730

T CCCT GAC AT

740

GC TGTCAT CA

750

TT T

T CTT CTA G

760

TGT AG CT GCT

770

GGTC CCAATG

780

C TTT T AAAA T

790

A GT C TTAC AA

800

T CT GGG TTCG

810

C AT TCTGGAC

820

CAA C A NGGTT

830

CT GNCA TC C A

840

AT T TT T A CCT

850

ATA GNGAGTC

860

GNATTA CC GG

870

GA T CCTT TAN

880

A GT C

CGA CCTG

890

CA GGCAT GC A

900

A CCTT GGCGT

910

A TT AT GGGN

Model 3100

BC 1.2.d.4

50_50F_E03_09.ab1

50_50F

Lane 9

Signal G:515 A:390 T:445 C:290

DT3100POP6{BD}v2.mob

Points 592 to 11557 Base 1: 592

Page 2 of 2

Fri, Apr 28, 2000 7:36 PM

Tue, Apr 4, 2000 6:31 AM

Spacing: 13.65

Cancer Mutation Panel for CTCs

Ampli1- WGA

Gene CTC WBCpool

MU27 PIK3CA Ex 9 mut wt wt wt wt wt wt wt DO wt wt wt wt wt wt wt

Ex 20 mut mut mut mut mut mut mut mut mut mut DO wt DO wt wt wt

Her2 CNV+ NA n n n n n n DO n n n n NA n n

pool

MU22 PIK3CA Ex 9 mut wt wt wt wt wt wt wt wt wt wt DO wt wt wt

Ex 20 mut mut mut mut mut mut mut mut mut mut mut mut wt wt wt

Her2 CNV+ NA n n n n n n n n DO DO NA n n

pool

MU28 PIK3CA Ex 9 mut wt wt wt DO DO wt wt wt wt wt wt

Ex 20 mut wt wt wt wt wt wt wt wt wt wt wt

Her2 CNV+ NA n n DO n n n DO NA n NA

MU09 PIK3CA Ex 9 mut wt wt wt wt wt wt wt wt wt

Ex 20 mut mut mut mut mut wt wt wt wt wt

Her2 CNV+ a a a a a a NA NA NA

pool

MU18 PIK3CA Ex 9 mut wt mut wt wt wt wt wt

Ex 20 mut mut wt wt wt wt wt wt

Her2 CNV+ DO NA n n DO NA n

MU37 PIK3CA Ex 9 mut mut mut wt wt wt wt

Ex 20 mut wt wt wt wt wt wt

Her2 CNV+ a a a a NA n

MU23 PIK3CA Ex 9 mut mut mut wt DO wt wt wt

Ex 20 mut wt wt wt DO wt wt wt

Her2 CNV+ n DO n n NA n n

MU21 PIK3CA Ex 9 mut wt wt wt wt wt wt

Ex 20 mut wt wt wt wt wt wt

Her2 CNV+ a a a NA n n

MU29 PIK3CA Ex 9 mut wt wt wt DO DO

Ex 20 mut wt wt wt wt wt

Her2 CNV+ n n n n NA

MU05 PIK3CA Ex 9 mut wt wt wt

Ex 20 mut wt wt wt

Her2 CNV+ a NA NA

CTC Characterization by DEPArray™ and Ampli1™ yield Actionable Information

Source: Division of Oncogenomics, Pathology Dept., Uni Regensburg

PI3K-inhibitor

Lapatinib Herceptin

Gene CTC WBCpool

MU27 PIK3CA Ex 9 mut wt wt wt wt wt wt wt DO wt wt wt wt wt wt wt

Ex 20 mut mut mut mut mut mut mut mut mut mut DO wt DO wt wt wt

Her2 CNV+ NA n n n n n n DO n n n n NA n n

pool

MU22 PIK3CA Ex 9 mut wt wt wt wt wt wt wt wt wt wt DO wt wt wt

Ex 20 mut mut mut mut mut mut mut mut mut mut mut mut wt wt wt

Her2 CNV+ NA n n n n n n n n DO DO NA n n

pool

MU28 PIK3CA Ex 9 mut wt wt wt DO DO wt wt wt wt wt wt

Ex 20 mut wt wt wt wt wt wt wt wt wt wt wt

Her2 CNV+ NA n n DO n n n DO NA n NA

MU09 PIK3CA Ex 9 mut wt wt wt wt wt wt wt wt wt

Ex 20 mut mut mut mut mut wt wt wt wt wt

Her2 CNV+ a a a a a a NA NA NA

pool

MU18 PIK3CA Ex 9 mut wt mut wt wt wt wt wt

Ex 20 mut mut wt wt wt wt wt wt

Her2 CNV+ DO NA n n DO NA n

MU37 PIK3CA Ex 9 mut mut mut wt wt wt wt

Ex 20 mut wt wt wt wt wt wt

Her2 CNV+ a a a a NA n

MU23 PIK3CA Ex 9 mut mut mut wt DO wt wt wt

Ex 20 mut wt wt wt DO wt wt wt

Her2 CNV+ n DO n n NA n n

MU21 PIK3CA Ex 9 mut wt wt wt wt wt wt

Ex 20 mut wt wt wt wt wt wt

Her2 CNV+ a a a NA n n

MU29 PIK3CA Ex 9 mut wt wt wt DO DO

Ex 20 mut wt wt wt wt wt

Her2 CNV+ n n n n NA

MU05 PIK3CA Ex 9 mut wt wt wt

Ex 20 mut wt wt wt

Her2 CNV+ a NA NA

“if CTCs can be isolated from cancer patients as viable cells that can be genotyped and functionally characterized over the course of therapy, they have the potential to identify treatments that most effectively target the evolving mutational profile of the primary tumor “ (Yu et al., Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility. Science. 2014 July 11; 345(6193): 216–220).

The isolation of viable CTCs is technically challenging, success associated with unmanipulated CTCs. CTCs proliferated best as tumor spheres when cultured in serum-free media supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) under hypoxic conditions (4% O2). Nonadherent culture conditions were critical, because CTCs senesced after a few cell divisions in adherent monolayer culture

CONCLUSION single-cell analyses have provided higher-resolution evidence of intratumor heterogeneity with the finding of substantial clonal diversity and subclonal heterogeneity, such that no two individual tumor cells are genetically identical. Beyond spatial heterogeneity, solid tumors also exhibit temporal heterogeneity, evolving over time under selection pressure from treatment. Thus, there is an increased appreciation that the management of metastatic disease should rely on analysis of contemporary tumor tissue rather than on the primary tumor diagnosed years ago. However, obtaining serial samples of metastatic tissue is impractical and complicated by spatial heterogeneity and sampling bias. Analysis of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) thus holds appeal and promise for noninvasive real-time assessment of tumor molecular profiles during the course of disease. Evaluation of CTCs and ctDNA may enable more sensitive monitoring of treatment efficacy and thereby guide drug selection, even potentially in the adjuvant setting where no such tools exist today.

Miguel Abal Translational Medical Oncology (IDIS) Complexo Hospitalario Universitario de Santiago de Compostela (SERGAS) Trav. Choupana s/n 15706 Santiago de Compostela (Spain) phone. +34 981955073 e-mail: miguel.abal.posada@sergas.es

Fragments of DNA are shed into the bloodstream from dying cells during cellular turnover or other forms of cell death.

More than 90% of healthy individuals having less than 25 ng cfDNA per mL.

cfDNA in the circulation is typically fragmented to 160 to 180 bp in length, corresponding to nucleosome-protected DNA observed in apoptotic cells.

In certain conditions, including inflammation, exercise, or tissue injury, cfDNA levels can be substantially higher.

ctDNA

ctDNA may be derived from primary tumors, metastatic lesions, or CTCs. The fraction of ctDNA that is tumor derived in patients with cancer has a

variable contribution ranging from <0.1% to >10% of the DNA molecules. The variability in levels of ctDNA is not well understood and is thought to be

affected by tumor burden, stage, cellular turnover, accessibility to the circulation, and factors affecting blood volume.

Although patients with similar tumor types may have varying absolute levels of ctDNA at the time of diagnosis, the relative levels of ctDNA within an individual have been shown to correlate with tumor burden and response to therapy.

Subject 11 had a sigmoid adenocarcinoma and two liver metastases that were treated with systemic chemotherapy before surgery (Chemotherapy 1). The subject underwent a sigmoid colectomy, left hepatic lobectomy and RFA of a solitary right hepatic lesion (Surgery 1). Imaging studies at 2 months showed recurrence in the liver, and the subject underwent a right hepatectomy (Surgery 2). Given the high risk of recurrence, chemotherapy was reinitiated (Chemotherapy 2). At 8 months, imaging showed three recurrent liver lesions and a suspicious celiac lymph node. The subject underwent RFA of these lesions and resection of the celiac node (Surgery 3). After surgery, the subject received additional chemotherapy (Chemotherapy 3); however, later imaging revealed multiple pulmonary metastases.

Technologies for ctDNA analysis

BEAMing Up Personalized Medicine: Detecting Cancer-Driving Mutations in Circulating DNA

BEAMing (Beads, Emulsification, Amplification, and Magnetics)