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

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

Background

• There are approximately 1,000,000 new cases of colorectal cancer and 500,000 deaths worldwide each year

• Multiple therapeutic options render the management of patients with metastatic disease complicated and nuanced

• A noninvasive prognostic and predictive marker could guide therapeutic decisions

• Circulating tumor cells can be isolated from the blood of patients with metastatic colorectal cancer (SJ Cohen et al, Clin Colorectal Cancer 2006)

Hypothesis: Circulating tumor cells can be used to guide therapy

• Baseline– prognostic

• During treatment– early indication of treatment effect– complementary to radiographic imaging– prognostic– Non-invasive source of tumor for in vivo

pharmacodynamic monitoring

Objectives

Primary Objective

To measure agreement between circulating tumor cell number and objective antitumor response defined by imaging

Secondary Objective

To determine the association between circulating tumor cell number and progression-free and overall survival

Eligibility

• Adults with measurable metastatic colorectal cancer

• Initiating first-, second-, or third-line therapy (if EGFR inhibitor)

• ECOG performance status 0-2

• Hemoglobin >8 g/dL

• Written informed consent

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

CTC Sample Preparation (1)

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

CTC Sample Preparation (2)

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

Training and Validation Sets

• Preplanned analysis after 100 patients to select cutoff for “favorable” vs. “unfavorable” CTC at 3-5 weeks

• Criteria for selection of cutoff:– CTC threshold must be above normal

background– Percent accuracy for agreement of CTC with

imaging and median PFS and OS must reach a plateau

– Frequency of CTC unfavorable patients must be at least 10%

• Based upon these criteria, > 3 CTC per 7.5 ml of blood was defined as “unfavorable”

Statistical Considerations for Primary Endpoint

• >99% power

• one-sided type I error () = 0.025

• Ho of <60% overall agreement between CTC (“favorable” vs. “unfavorable”) and radiographic response

• Ha of >75% agreement

• N = 400

Patient CharacteristicsTraining

Set (N=109)Validation

Set (N=321)p All Patients

(N=430)

Median Age (Range) 65 (25-86) 63 (22-92) 0.29 64 (22-92)

Female 42% 45% 0.58 45%

Performance Status

0

1

2

Unknown

46%

39%

9%

6%

45%

45%

7%

3%

0.49 46%

43%

7%

4%

First-line

Second-line

Third-line

55%

35%

10%

77%

18%

5%

<0.0001 72%

22%

6%

Liver Metastasis 71% 74% 0.62 73%

Median Followup, months (mean+/- SD)

14.5

(14.9 + 8.6)

9.7

(9.9 + 5.2)

0.001 10.2

(11.2 + 6.6)

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)

CTCs at 3-5 Weeks and Response

Radiographic Response

CTC at 3-5 Weeks

N (%)

Total

< 3 cells > 3 cells

CR/PR/SD 228 (93%) 18 (7%) 246 (77%)

PD or death 54 (73%) 20 (27%) 74 (23%)

Total 282 (88%) 38 (12%) 320 (100%)

Sensitivity = 27%Specificity = 93%

Overall Agreement = 78%

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


%P

rob

abil

ity

of

Su

rviv

al


0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20



vs. P = 0.0001


%P

rob

abil

ity

of

Su

rviv

al


0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

20



Baseline CTC: Progression Free Survival%

Pro

gre

ssio

n F

ree


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

Baseline CTC: Overall Survival%

Su

rviv

al

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%


<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

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

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

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

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

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

Change in CTC at 3-5 Weeks is Predictive of Overall Survival

%P

rob

ab

ilit

y o

f S

urv

ival

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 227 17.7 (14.7 - 19.9)≥3 CTC <3 CTC 53 11.0 (8.7 - 18.1)<3 CTC ≥3 CTC 9 10.9 ( 0.6 - ----)≥3 CTC ≥3 CTC 30 3.7 (2.4 – 8.4)

vs. P = 0.002 vs. P = 0.0002

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

• Elevated CTC number at 3-5 weeks has high specificity but low sensitivity for early disease progression

• 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

Participating Clinical Investigators

N. Iannotti, Hematology Oncology Associates of the Treasure Coast, Port Saint Lucie, FL; B.H. Saidman, Medical Oncology Associates, Kingston, PA; T.M. Fynan, Medical Oncology and Hematology, P.C., New Haven, CT; J. Picus, Washington University School of Medicine, St. Louis, MO; N. Gabrail, Canton, OH; M. Morse, Duke University Medical Center, Durham, NC; S.J. Cohen and N.J. Meropol, Fox Chase Cancer Center, Philadelphia, PA; M. Schwartz, Richmond VA; E. Mitchell, Thomas Jefferson University, Philadelphia, PA; F. Swan, Cancer Outreach Associates, Abingdon, VA; H.S. Jhangiani, Compassionate Cancer Care Medical Group, Inc, Corona, CA; K. Patel, Little Rock Hematology Oncology Associates, Little Rock, AR; R. Pelley, Cleveland Clinic Foundation, Cleveland, OH; C. Farber, Hematology-Oncology Associates of Northern New Jersey, Morristown, NJ; R.L. Robles, Bay Area Cancer Research Group, Concord, CA; V. Jones, Yakima Valley Memorial Hospital/North Star Lodge Cancer Center, Yakima, WA; H. Ghazal, Kentucky Cancer Center, Hazard, KY; P. Ross, St. Thomas Hospital, London, UK; G.A. Fisher, Stanford University Medical Center, Stanford, CA; R. Rangineni, St. Joseph Oncology, Inc, St. Joseph, MO; L. Bertoli, Clinical Research Consultations; Birmingham, AL; R.O. Giudice, New Mexico Oncology Hematology Consultants, Ltd., Alruquerque, NM; A. Khojasteh, Columbia Comprehensive Cancer Care Clinic, Jefferson City, MO; M.V. Pillai, Virginia Oncology Care, P.C., Richlands, VA; A. Pippas, John B. Amos Cancer Center, Columbus, GA; S. Berk, Hematology Oncology Associates of South Jersey, P.A., Mount Holly, NJ; A. Rakkar, Palo Verde Hematology Oncology, Inc, Glendale, AZ; M.B. Wax, Summit Medical Group/Overlook Oncology Center, Summit, NJ; K. Nahum, Oncology Physicians Network, Howell, NJ; A. Gonzalez, Cancer Specialists of South Texas, P.A., Corpus Christi, TX; R.K. Gamble, Cancer Care Specialists, Houma, LA; J.W. Jakub, Lakeland Regional Cancer Center, Lakeland, FL; R.J. Jacobson, Palm Beach Cancer Institute, Palm Beach, FL; J. Fleagle, Rocky Mountain Cancer Center – Thornton, Thornton, CO; G. E. Newman, Knoxville, TN; M. Woodson, Hematology Oncology Consultants, Inc., St. Louis, MO; P. Cobb, Hematology/Oncology Centers of the Northern Rockies, Billings, MT; D. Mukhopadhyay and R. Prabhu, Redrock Medical Group, Las Vegas, NV; A.M. Wierman, Nevada Cancer Center, Las Vegas, NV; W. Ho, St. Joseph Hospital, Orange, CA; A. Cohn, Rocky Mountain Cancer Center – Lone Tree, Lone Tree, CO.

Dutch Colorectal Cancer Group (DCCG):C.J.A. Punt, Radboud University Medical Center, Nijmegen; G.J. Creemers, Catharina Ziekenhuis, Eindhoven; P. Deprest, F.A. Valster and T.J. Roding, Ziekenhuis de Lievensberg, Bergen op Zoom; M.B. Polee, Medisch Centrum Leeuwarden, Leeuwarden; A.J. ten Tije, Ziekenhuis Gooi-Noord, Blaricum; D.J. Richel, University of Amsterdam, Amsterdam; M. Legdeur, Medisch Spectrum Twente, Enschede, J.J.M. van her Hoeven, Amstelveen, A. Honkoop, Isala Klinieken, Zwolle; Z. Erjavec, Delfzicht Ziekenhuis, Delfzijl; R.S. de Jong, Martini Ziekenhuis, Groningen, P. Slee, St. Antonius Ziekenhuis, Nieuwegein; J. Douma, Ziekenhuis Rijnstate, Arnhem; J. Sleeboom, Ziekenhuis Leyenburg, Den Haag.

Documents

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