Circulating Tumor Cells Minetta C. Liu, MD Associate Professor of Medicine and Oncology Director, Translational Breast Cancer Research Lombardi Comprehensive Cancer Center Georgetown University Medical Center

Era of Personalized Medicine need an individualized approach to treatment to maximize benefit and minimize costneed an individualized approach to treatment to maximize benefit and minimize cost  assessment of prognosis  measurement of treatment benefit  understanding of tumor biology

Biomarker Strategies serial biomarker assessments single biomarker assessment prognosis direct therapy prediction diagnosisprognosis

Improving Outcomes the enumeration and characterization of circulating tumor cells (CTCs) are useful in the clinical settingthe enumeration and characterization of circulating tumor cells (CTCs) are useful in the clinical setting alterations in CTC levels CTC phenotype and genotype identification of CTC prognosisprediction diagnosisprognosis direct therapy diagnosisprognosis

(Paterlini-Brechot et al. Cancer Letters :180.) Origin of CTCs

etiologyetiology  disseminated cancer cells  cancer stem cells  bystander cells rare cells in a dormant, nonproliferative staterare cells in a dormant, nonproliferative state  unaffected by chemotherapy  unrecognized by the host immune system  difficult to isolate Origin of CTCs

Isolation of CTCs enrichment detection characterization density gradient centrifugation filtration by size immunomagnetic labeling quantitation of growth factor expression gene expression profiling detection of epigenetic alterations Alix-Panabieres et al. Clin Cancer Res :5013.

Detection of CTCs antibody based markersantibody based markers  cytokeratins (CK8, CK18, CK19)  epithelial membrane antigen (EMA)  epithelial cell adhesion molecule (EpCAM) nucleic acid based markersnucleic acid based markers  CK19 by RT-PCR  mammoglobin by RT-PCR  erbB2 by RT-PCR or FISH  EGFR by RT-PCR

Available Technologies for Isolation of CTCs

Circulating Tumor Cell CK Y EpCAM Nucleus DAPI Anti- CK-PE Y Anti-EpCAMFerrofluid Immunomagnetic Capture Leukocyte CD45 Nucleus DAPI Y Anti - CD45-APC Immunomagnetic Labelingand Immunofluorescent Identification of Cells Immunomagnetic Labeling and Immunofluorescent Identification of Cells

Immunomagnetic Capture DAPIcytokeratincontrolCD45composite intact tumor cells

(Zheng et al. J Chromatogr A :154.) Parylene Filter Microdevice

(Nagrath et al. Nature :1235.) CTC Microchip

Immunomagnetic Labeling: Enumeration in MBC

Clinical Parameters CTCs in individuals with MBC   detected in ~70%   >5 per 7.5 mL blood in ~50% CTCs in individuals without a malignancy   detected in <10%   >5 per 7.5 mL blood in 0%

Prevalence of CTCs CTC threshold (per 7.5 mL blood) % patients at or above the CTC threshold cancer, baseline (n = 177) 3% 16% 21% 23% 27% 34% 36% 38% 42% 45% 46% 47% 49% 53% 58% 61% 71% cancer, first follow-Up (n = 163) 1% 8% 13% 15% 20% 21% 22% 23% 24% 26% 28% 29% 30% 32% 35% 40% 55% 6% healthy volunteers (n = 145) benign diseases (n = 200) 1% 8%

IMC-001 time points imaging blood patients (223 total)177 patients (223 total) metastatic breast cancermetastatic breast cancer measurable diseasemeasurable disease

(Hayes et al. Clin Cancer Res :4218.) IMC-001: CTCs at Baseline Probability of Progression Free Survival n = 177

(Cristofanilli et al. N Engl J Med :781.) p value < Cox Hazards Ratio = p value < Cox Hazards Ratio = IMC-001: CTCs at 1 st Follow-Up n = 177

(Cristofanilli et al. N Engl J Med :781.) % probability of PFS time from baseline (weeks) % 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 50 ~7.0 months ~2.1 months ~7.6 months p value < Cox Hazards Ratio = # patients (median PFS) < 5 CTC at baseline & at 1 st follow-up 81 (30.3 weeks) decrease in CTC to < 5 at 1 st follow-up 33 (32.9 weeks) > 5 CTC at 1 st follow-up 49 (8.9 weeks) IMC-001: Changes in CTC n = 177

(Cristofanilli et al. J Clin Oncol :1420.) n = 83 IMC-001: CTCs and Imaging

(Budd et al. Clin Cancer Res :6403.) n = 138 IMC-001: CTCs and Imaging

(Budd et al. Clin Cancer Res :6403.) n = 138 IMC-001: CTCs and Imaging

LCCC Validation Study imaging blood cycles evaluable patients74 evaluable patients metastatic breast cancermetastatic breast cancer measurable diseasemeasurable disease

(Liu et al. J Clin Oncol :5153.) Treatment at Time of CTC Result OR95% CIp-value Overall6.3(3.2, 13)<0.001 Chemotherapy6.3(2.9, 14)<0.001 Endocrine8.9(2.2, 35)0.002 CTCs Drawn at the Time of Radiographic Imaging Treatment at Time of CTC Result OR95% CIp-value Overall4.9(2.2, 118)<0.001 Chemotherapy6.9(3.0, 16)<0.001 Endocrine2.9(0.6, 14)0.2 CTCs Drawn 7-9 Weeks Prior to Radiographic Imaging Treatment at Time of CTC Result OR95% CIp-value Overall3.1(1.6, 5.8)0.001 Chemotherapy2.7(1.2, 6.3)0.02 Endocrine5.2(1.2, 23)0.03 CTCs Drawn 3-5 Weeks Prior to Radiographic Imaging LCCC: CTCs and Imaging

Recommendations for Use to assess for progression in patients with measurable metastatic disease to provide a guide by which to determine the timing of radiographic restaging studies to assess the feasibility and timing of drug holidays in patients with stable disease and intolerable drug related toxicities

Conclusions Conclusions

meaningful rate of detectionmeaningful rate of detection reliable thresholdreliable threshold correlation with clinical outcomes (validity)correlation with clinical outcomes (validity) ability to improve clinical outcomes (utility)ability to improve clinical outcomes (utility) Response/Futility Marker

CTCs – Present clinical validity   IMC-001   LCCC study clinical utility   assess disease status (with imaging)   guide the timing of radiographic studies   guide the timing of drug holidays   guide systemic therapy (?????)

blood drawn at baseline prior to first-line chemotherapy Arm B maintain first-line chemotherapy until progression Arm C1 maintain first-line chemotherapy until progression Arm C2 switch to alternate chemotherapy Arm A monitor for PFS & OS eligible for other first- line chemotherapy trials R CTC <5CTC ≥5 blood drawn three weeks after the first chemotherapy dose CTC ≥5CTC <5 target n = 120 Prospective Validation: Means to Improve Survival SWOG S0500

Prospective Validation: The Liquid Biopsy nucleuscytokeratincontrolleukocytecomposite HER2 and SE17 FISH Probes

Prospective Validation: The Liquid Biopsy CALGB blood weeks 212 paclitaxel and trastuzumab and lapatinib N = 400 paclitaxel and trastuzumab paclitaxel and lapatinib tissue

(Harris et al.J Clin Oncol :5287.) ASCO Tumor Marker Guidelines Circulating tumor cell assays as markers for breast cancerCirculating tumor cell assays as markers for breast cancer

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