AGA/ASCO/ASTRO/SSO Gastrointestinal Cancers Symposium Orlando, FL January 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 Genotyping 20%-70% of patients with colorectal cancer have detectable CTCs

Immunomagnetic Separation Magnetic Incubations ml Blood + 6.5mL Buffer Plasma Aspiration & Addition of EPCAM Ferrofluid Station 1Stations 2 & 3 Primary Magnetic Separation & Resuspension Station 4 - Addition of Cytokeratin-PE CD45-APC, & DAPI Station 5 Centrifuge Place on Instrument Described in: WJ Allard et al, Clin Cancer Res 10: , 2004

Immunomagnetic Separation Stations 6,7, & 8Station 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 CTC, green and red EGFR+, red WBC, blue Beads, 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 Eligibility Adults 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 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% >= 1>= 2>= 3>= 4>= 5>= 6>= 7>= 8>= 9>= 10 CTC / 7.5mL of Blood (Baseline Draw) Median OS from Baseline (Months) 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 % Progression Free Time from Baseline Blood Draw (Months) % 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 P = CTC/7.5 mLMedian in Months (95% CI) <3 CTCs7.9 ( ) ≥3 CTCs4.5 ( ) <3 CTCs ≥3 CTCs ASCO 2007

Baseline CTC: Overall Survival % Survival 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Time from Baseline Blood Draw (Months) <3 CTC ≥3 CTC CTC/7.5 mLMedian in Months (95% CI) <3 CTC18.5 ( ) ≥3 CTC9.4 ( ) P < ASCO 2007

CTC During Treatment: PFS %Probability of Progression Free Survival Time from Blood Draw (Months) % 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 NMedian PFS in Months (95% CI) <3 CTC ≥3 CTC 1-2 wks ( )3.8 ( ) 3-5 wks ( )1.9 ( ) 6-12 wks ( )2.0 ( ) P < at each timepoint ASCO 2007

CTC During Treatment: Overall Survival %Probability of Survival Time from Blood Draw (Months) % 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 Median OS in Months (95% CI) N<3 CTC ≥3 CTC 1-2 wks ( )6.1 ( ) 3-5 wks ( ) 4.4 ( ) 6-12 wks ( )3.3 ( ) P < at each timepoint ASCO 2007

Predictors of PFS and OS: Multivariable Model – Baseline (N=373) VariablePFSOS HRP P CTC number Line of therapy 1 st vs. 2 nd vs. 3 rd Age PS0 vs. 1 vs ASCO 2007

Predictors of PFS and OS: Multivariable Model – 3-5 Weeks (N=302) VariablePFSOS HRP P CTC number Line of therapy 1 st vs. 2 nd vs. 3 rd Age PS0 vs. 1 vs ASCO 2007

Circulating Tumor Cells at Time of 1 st Followup Image Add Prognostic Information %Probability of Survival Time from Baseline Blood Draw (Months) % 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 CTCResponseNOS in Months (95% CI) <3 CTCS/PR/CR ( ) <3 CTCPD/Death648.3 ( ) >3 CTCS/PR/CR137.1 ( ) >3 CTCPD/Death163.1 ( ) vs. P < ASCO 2007

Circulating Tumor Cells at Time of 1 st Followup Image Add Prognostic Information %Probability of Survival Time from Baseline Blood Draw (Months) % 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 CTCResponseNOS in Months (95% CI) <3 CTCS/PR/CR ( ) <3 CTCPD/Death648.3 ( ) >3 CTCS/PR/CR137.1 ( ) >3 CTCPD/Death163.1 ( ) vs. P = ASCO 2007

Circulating Tumor Cells at Time of 1 st Followup Image Add Prognostic Information %Probability of Survival Time from Baseline Blood Draw (Months) % 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 CTCResponseNOS in Months (95% CI) <3 CTCS/PR/CR ( ) <3 CTCPD/Death648.3 ( ) >3 CTCS/PR/CR137.1 ( ) >3 CTCPD/Death163.1 ( ) ASCO 2007

Decrease in CTC at 3-5 Weeks is Associated with Improved PFS in Patients with > 3 CTC at Baseline % Probability of Progression Free Survival Time from 3-5 Week Blood Draw (Months) % 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 20 Baseline3-5 wksNMed PFS (95% CI) ≥3 CTC<3 CTC526.2 ( ) ≥3 CTC≥3 CTC281.6 ( ) P = 0.02 ASCO 2007

CTC Association with PFS and OS by Lines of Therapy Cohen et al. AGA/ASCO/ASTRO/SSO GI Cancers Symposium, st line 2nd line PFS OS HR=1.44 ( ) HR=2.22 ( ) HR=1.90 ( ) HR=2.98 ( )

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