AGA/ASCO/ASTRO/SSOGastrointestinal Cancers Symposium

Orlando, FLJanuary 26, 2008

Circulating tumor cells: are they predictive markers?

Neal J. Meropol, M.D.Fox Chase Cancer Center

“Predictive” vs. “Prognostic”

• Predictive: explains variability in response to treatment

– Traditional application before treatment

• Prognostic: explains variability irrespective of treatment

Natural History of Circulating Tumor Cells

Paterlini-Brechot and Benali, Cancer Letters, 2007

Potential advantages of circulating tumor cells (CTCs) compared to

other blood markers

• Representative of tumor access to circulation• Permits multiple simultaneous analyses

– Enumerate, phenotype, gene expression• Cytopathology• in vivo pharmacodynamic assessment, gene

expression profiling• Sensitivity/Specificity

Why Study CTCs?

• Prognosis• Monitor disease course

– Minimal residual disease– Early relapse– Response to therapy

• Treatment selection• Drug development (pharmacodynamics)

– Target acquisition– Down stream effects

Methods for CTC Detection

• Density gradient• Immunomagnetic separation (beads, ferrofluid)• Size-based filtration

Count

Cytopathology

RT-PCR

Genotyping20%-70% of patients with colorectal cancer have

detectable CTCs

Immunomagnetic Separation

Magnetic Incubations

-

7.5 ml Blood + 6.5mL Buffer

PlasmaAspiration & Addition of

EPCAM Ferrofluid

Station 1 Stations2 & 3

Primary Magnetic

Separation & Resuspension

Station 4

-

Addition of Cytokeratin-PECD45-APC, &

DAPI

Station 5

Centrifuge

Place on Instrument

Described in: WJ Allard et al, Clin Cancer Res 10: 6897-6904, 2004

Immunomagnetic Separation

Stations 6,7, & 8 Station 9b

Staining Incubation, Magnetic Wash &

Free Particle Removal

Final Resuspension

Station 9a Image Gallery

Automatic Transfer of Sample for Fluorescent

Image Analysis

Characterization of CTCs by Flow Cytometry

SJ Cohen et al. Clin Colorectal Cancer, 2006

CD45-,CK+

CD45-,CK+,EGFR+

CD45+

beads

beads

CD45 PerCP

CD45 PerCPEGFR APC

Forward Light ScatterC

ytok

era

tinP

EO

rtho

gona

l Lig

ht S

catte

r

Cyt

oker

atin

PE

Nuc

leic

aci

d dy

e

CD45-,CK+

CD45-,CK+,EGFR+

CD45+

beads

beads

CD45 PerCP

CD45 PerCPEGFR APC

Forward Light ScatterC

ytok

era

tinP

EO

rtho

gona

l Lig

ht S

catte

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Cyt

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atin

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Nuc

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CTC, green and redEGFR+, redWBC, blueBeads, yellow

N. J. Meropol, S. J. Cohen, N. Iannotti, B. H. Saidman, K. D. Sabbath, M. C. Miller, G. V. Doyle, H. Tissing, L. W.M.M. Terstappen, C.J.A. Punt

Fox Chase Cancer Center, Philadelphia, PA; Hematology Oncology Associates, Port St. Lucie, FL; Medical Oncology Associates, Kingston, PA;

Medical Oncology and Hematology, PC, Waterbury, CT; Immunicon Corporation, Huntingdon Valley, PA; Radboud University Medical Center,

Nijmegen, The Netherlands

Circulating tumor cells (CTC) predict progression free (PFS) and overall

survival (OS) in patients with metastatic colorectal cancer

ASCO 2007

Objectives and Eligibility

Overall Objective

To determine the association between circulating tumor cell number and clinical outcomes in patients with metastatic colorectal cancer

EligibilityAdults with measurable metastatic colorectal cancer, initiating first-, second-, or third-line therapy

Methods

• Multicenter international study• Radiographic tumor measurement at baseline

and every 6-12 weeks after treatment initiation (RECIST criteria)

• Peripheral blood was collected for CTC enumeration at baseline and subsequently at 1-2, 3-5, 6-12, and 13-20 weeks after treatment initiation

• Blood mailed overnight at room temperature, and processed at 1 of 4 laboratories within 96 hours

11%12%13%14%16%18%21%26%33%47%53%0

2

4

6

8

10

12

14

16

18

20

22

24

26

0 >= 1 >= 2 >= 3 >= 4 >= 5 >= 6 >= 7 >= 8 >= 9 >= 10

CTC / 7.5mL of Blood (Baseline Draw)

Med

ian

OS

fro

m B

asel

ine

(Mo

nth

s) Median OS for Patients with Metastatic Colorectal Cancer Based Upon number of CTC

Prior to the Initiation of Therapy (N=413)

ASCO 2007

Baseline CTC: Progression Free Survival%

Pro

gre

ssio

n F

ree

Time from Baseline Blood Draw (Months)0 2 4 6 8 10 12 14 16 18 22 24 26 28 30

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20

P = 0.0002

CTC/7.5 mL Median in Months(95% CI)

<3 CTCs 7.9 (7.0 - 8.6)≥3 CTCs 4.5 (3.7 - 6.3)

<3 CTCs≥3 CTCs

305108

269 84

229 60

187 42

138 28

8816

44 8

32 3

20 2

15 2

81

60

30

00

00

00

ASCO 2007

Baseline CTC: Overall Survival%

Su

rviv

al

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Time from Baseline Blood Draw (Months)0 2 4 6 8 10 12 14 16 18 22 24 26 28 3020

<3 CTC≥3 CTC

305108

289102

276 86

252 66

227 49

180 36

134 24

107 13

7812

6011

43 7

32 4

22 2

11 1

41

20

CTC/7.5 mL Median in Months (95% CI)

<3 CTC 18.5 (15.5 - 21.2) ≥3 CTC 9.4 (7.5 - 11.6)

P < 0.0001

ASCO 2007

CTC During Treatment: PFS%

Pro

bab

ility

of

Pro

gre

ssio

n F

ree

Su

rviv

al

Time from Blood Draw (Months)0 2 4 6 8 10 12 14 16 18 22 24 26 28 30

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20

N Median PFS in Months (95% CI)

<3 CTC ≥3 CTC1-2 wks 315 7.3 (6.5 - 8.1) 3.8 (1.9 - 5.1)3-5 wks 329 6.8 (6.1 - 7.6) 1.9 (1.2 - 4.4)6-12 wks 284 6.5 (5.8 - 7.7) 2.0 (0.5 - 2.5)

P < 0.0001 at each timepoint

ASCO 2007

CTC During Treatment: Overall Survival%

Pro

bab

ilit

y o

f S

urv

ival

Time from Blood Draw (Months)0 2 4 6 8 10 12 14 16 18 22 24 26 28 30

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20

Median OS in Months (95% CI)

N <3 CTC ≥3 CTC1-2 wks 357 15.7 (14.3 - 18.4) 6.1 (4.9 - 8.9)3-5 wks 333 16.4 (14.1 - 18.3) 4.4 (2.6 - 8.7)6-12 wks 310 15.8 (13.8 - 19.2) 3.3 (1.8 - 5.6)

P < 0.0001 at each timepoint

ASCO 2007

Predictors of PFS and OS:Multivariable Model – Baseline (N=373)

Variable PFS OS

HR P HR P

CTC number

<3 vs. >3 1.8 0.000 2.4 0.000

Line of therapy

1st vs. 2nd vs. 3rd

1.6 0.000 1.4 0.007

Age <65 vs. >65

1.5 0.001 1.9 0.000

PS 0 vs. 1 vs. 2

1.2 0.084 1.5 0.001

ASCO 2007

Predictors of PFS and OS:Multivariable Model – 3-5 Weeks (N=302)

Variable PFS OS

HR P HR P

CTC number

<3 vs. >3 2.2 0.000 4.5 0.000

Line of therapy

1st vs. 2nd vs. 3rd

1.7 0.000 1.7 0.001

Age <65 vs. >65

1.6 0.000 2.1 0.000

PS 0 vs. 1 vs. 2

1.2 0.109 1.3 0.032

ASCO 2007

Circulating Tumor Cells at Time of 1st Followup Image Add Prognostic Information

%P

rob

abil

ity

of

Su

rviv

al

Time from Baseline Blood Draw (Months)0 2 4 6 8 10 12 14 16 18 22 24 26 28 30

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20

CTC Response N OS in Months(95% CI)

<3 CTC S/PR/CR 271 18.8 (17.0 - 25.1)<3 CTC PD/Death 64 8.3 (5.8 - 11.2)>3 CTC S/PR/CR 13 7.1 (5.4 - 10.8)>3 CTC PD/Death 16 3.1 (2.0 - 4.4)

vs. P < 0.0001

ASCO 2007

Circulating Tumor Cells at Time of 1st Followup Image Add Prognostic Information

%P

rob

abil

ity

of

Su

rviv

al

Time from Baseline Blood Draw (Months)0 2 4 6 8 10 12 14 16 18 22 24 26 28 30

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20

CTC Response N OS in Months(95% CI)

<3 CTC S/PR/CR 271 18.8 (17.0 - 25.1)<3 CTC PD/Death 64 8.3 (5.8 - 11.2)>3 CTC S/PR/CR 13 7.1 (5.4 - 10.8)>3 CTC PD/Death 16 3.1 (2.0 - 4.4)

vs. P = 0.0001

ASCO 2007

Circulating Tumor Cells at Time of 1st Followup Image Add Prognostic Information

%P

rob

abil

ity

of

Su

rviv

al

Time from Baseline Blood Draw (Months)0 2 4 6 8 10 12 14 16 18 22 24 26 28 30

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20

CTC Response N OS in Months(95% CI)

<3 CTC S/PR/CR 271 18.8 (17.0 - 25.1)<3 CTC PD/Death 64 8.3 (5.8 - 11.2)>3 CTC S/PR/CR 13 7.1 (5.4 - 10.8)>3 CTC PD/Death 16 3.1 (2.0 - 4.4)

ASCO 2007

Decrease in CTC at 3-5 Weeks is Associated with Improved PFS in Patients with > 3 CTC at Baseline

% P

rob

abil

ity

of

Pro

gre

ssio

n F

ree

Su

rviv

al

Time from 3-5 Week Blood Draw (Months)0 2 4 6 8 10 12 14 16 18 22 24 26 28 30

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20

Baseline 3-5 wks N Med PFS(95% CI)

≥3 CTC <3 CTC 52 6.2 (4.6 - 7.0)≥3 CTC ≥3 CTC 28 1.6 (1.2 - 2.7)

P = 0.02

ASCO 2007

CTC Association with PFS and OS by Lines of Therapy

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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time from Baseline Blood Draw in Months

<3 CTC

>=3 CTC

1st Line Therapy mCRC Patients (N=296)

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<3 CTC

>=3 CTC

2nd & 3rd Line Therapy mCRC Patients (N=117)

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>=3 CTC

1st Line Therapy mCRC Patients (N=296)

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<3 CTC

>=3 CTC

2nd & 3rd Line Therapy mCRC Patients (N=117)

Cohen et al. AGA/ASCO/ASTRO/SSO GI Cancers Symposium, 2008

1st line

2nd line

PFS OS

HR=1.44 (1.04-1.98)

HR=2.22 (1.49-3.30)

HR=1.90 (1.26-2.85)

HR=2.98 (1.85-4.77)

Conclusions

• In patients with metastatic colorectal cancer, CTC number before and after initiation of treatment is a significant independent predictor of progression free survival and overall survival

• CTC enumeration is complementary to imaging, and may provide early evidence of treatment success or failure

• Further research is required to determine whether change in therapy based upon elevated CTC number at early followup will improve patient outcomes

Are CTCs Predictive Markers?

Maybe

• Traditional application before treatment– no data yet regarding role of

phenotyping/genotyping CTCs and response to therapy

• Alternative application early after treatment initiation– suggestive evidence that CTCs indicate

resistance to treatment

Clinical validation study design requirements

• Standardized assay platform for clinical decision making

• Prospective randomized trial– archival well-annotated clinical specimens

do not exist• Randomized population of adequate size to

answer clinical question– Requires previous validation with assay

platform

Metastatic disease study design:early change in therapy

Begin Treatment A

Assess at 1-3 weeks

Favorable CTCs?

Continue Treatment A

Yes*

Continue Treatment A

Change to Treatment B

R

No

*assumes that continuation of Treatment

A is best in Favorable CTC group

Metastatic disease study design:selection of initial therapy

Favorable CTCs?

Standard initial therapy

Yes*

Standard initial therapy

More aggressive initial therapy

R

No

*assumes that standard therapy is “best” in

Favorable CTC group

Surveillance study design 1: randomize early

Resected patients at risk for

recurrence

Routine surveillance

Routine surveillance +

CTC evaluation, with aggressive intervention if

CTC “recurrence”

R

Surveillance study design 2: randomize later

Resected patients at risk for recurrence

Routine surveillance

plus CTC evaluation

CTC “Recurrence”

Continue routine surveillance

Aggressive intervention

R

Many Questions Remain

• What are “circulating tumor cells”?• Are CTCs the same as in situ cancer?• How does cell separation process affect

gene expression?• How can CTCs be used in the drug

development process?• How can CTCs be integrated into routine

patient care?

The End