111 Circulating Tumor Cells (CTCs) in Cytology Malini Harigopal...
Transcript of 111 Circulating Tumor Cells (CTCs) in Cytology Malini Harigopal...
111 Circulating Tumor Cells (CTCs) in Cytology
Malini Harigopal MD
2011 Annual Meeting – Las Vegas, NV
AMERICAN SOCIETY FOR CLINICAL PATHOLOGY 33 W. Monroe, Ste. 1600
Chicago, IL 60603
111 Circulating Tumor Cells (CTCs) in Cytology This session will provide an introduction to CTCs. A brief history and background of CTCs will be presented, followed by a review of current technology, supporting literature and a demonstration of how the technology has been implemented in the presenter's laboratory. Key points of the session will include: instrumentation (CellTrack Autoprep system), enumeration of CTCs for predicting progression-free and overall survival in metastatic breast cancer patients, specimen collection, quality control, interpretation of results, limitations, clinical trial conclusions, and novel methods for CTC identification.
• To integrate the methods of CTC detection into the practice of cytopathology. • To be able to identify and evaluate CTCs in peripheral blood using CellSearch System. • To recognize the potential role of the cytopathologist in the process of measuring CTCs.
FACULTY: Malini Harigopal MD Entire Pathology Team New Techniques and Technologies New Techniques & Technologies 1.0 CME/CMLE Credit Accreditation Statement: The American Society for Clinical Pathology (ASCP) is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education (CME) for physicians. This activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education (ACCME). Credit Designation: The ASCP designates this enduring material for a maximum of 1 AMA PRA Category 1 Credits™. Physicians should only claim credit commensurate with the extent of their participation in the activity. ASCP continuing education activities are accepted by California, Florida, and many other states for relicensure of clinical laboratory personnel. ASCP designates these activities for the indicated number of Continuing Medical Laboratory Education (CMLE) credit hours. ASCP CMLE credit hours are acceptable to meet the continuing education requirements for the ASCP Board of Registry Certification Maintenance Program. All ASCP CMLE programs are conducted at intermediate to advanced levels of learning. Continuing medical education (CME) activities offered by ASCP are acceptable for the American Board of Pathology’s Maintenance of Certification Program.
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Malini Harigopal, MDAssistant Professor
Department of Pathology &
Kevin Schofield, CT(ASCP)Managerg
Department of Cytology
Yale School of MedicineNew Haven, CT
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Malini Harigopal, MDAssistant Professor
Department of Pathology &
Kevin Schofield CT(ASCP)
Financial Disclosure
Kevin Schofield, CT(ASCP)Manager
Department of Cytopathology
We have no financial disclosures.
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Basic Introduction to Circulating Tumor cells (CTCs)
& The CellSearchTM System
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- Evaluate the use of CTCs and CellSearch (Veridex, Raritan, NJ) in the management of patients with cancer
- Discuss the cytopathologist’s role in measuring
Objectives
Discuss the cytopathologist s role in measuring CTCs
- Describe the integration of CTC assessment into the practice of cytopathology
- Identify and evaluate CTCs in peripheral blood
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• Circulating tumor cells (CTCs): cancer cells shed from either the primary tumor or its metastases
Definition
metastases- Epithelial cells
derived from solid tumors
- Metastases are responsible for most cancer deaths.
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- Tumor cells were first identified in the blood stream of patients in (1869) by Thomas AshworthEngel 1955: cancer cells in the peripheral
History of CTCs
- Engel, 1955: cancer cells in the peripheral blood of pts with various types of cancer.
- Hematologists,Cytologists & Surgeons: background in Papanicolou & Romanowsky stains: morphologic criteria for cancer cells
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Slide Seminar The Circulating Cancer Cell Cooperative(CCCC): NCI(CCCC): NCIIdentification of CTC: Morphologic criteria
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• The Circulating Cancer Cell Cooperative 1962: Morphology, techniques and patient selection - Conclusion : “More extensive well-controlled studies, improved techniques, sharper criteria
National Cancer InstituteDiagnostic Research Branch
, p q , pfor recognition of tumor cells are required.”
• Immunofluorescence technique by Coons: labeling of antibodies with fluorochromes improved the specificity of detection of CTC.
• Value to cytologic diagnosis of CTC
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• CTC: Rare in healthy women and in patients with benign breast disease (<1 per 7.5ml blood).
• Monitoring CTC (counts) can predict prognosis in many solid tumors, breast, prostate and
l t l
Utility CTC Measurement
colorectal cancers.• Measuring changes in CTC counts help monitor
patient outcome.• Molecular characterization of CTCs enable
treatment to be tailored and limit metastases.
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• The role of CTC in blood is still under active investigation, biological significance/ therapeutic relevance (debated).
• Identification, enumeration and molecular
Utility CTC Measurement
characterization of CTCs could expand the understanding of the biology of metastases.
• Several strategies have been used for CTC enumeration.
• Nucleic-acid-based (PCR) and antibody-based cytometric assays (intact cells).
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• CellSearch system(Veridex, Warren, NJ): capture of epithelial cells (EpCAM) by ferrofluid
• CTC-chip:microfluid platform
Techniques for CTC Enumeration
• FAST(fiber-optic array scanning technology• Oncoquick: cellular density• MACS: capture of epithelial cells by
immunobeads• ISET: cellular size
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• CellSearch System (Veridex, Warren NJ): Technology
• Automated, standardized technology for CTC detection
CellSearch™ System
CTC detection• Based immunofluorescence • CellSearch system validated in clinical
trails• FDA approved for CTC detection
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• Instruments (CellTrack Auto autoprep system) • Specimen collection, processing and Quality con• Enumeration of CTCs for predicting progression-
free and overall survival in patients with
System Overview
free and overall survival in patients with metastatic breast, colorectal and prostate cancer
• Clinical trial background and conclusions• Interpretation of Results• Limitations
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CellSearch™ System
• Sample preparation system- Cell Search Epithelial kit (Veridex Corporation, Warren NJ)
Anti Epcam antibodies: Anticytokeratin antibodies conjugated to phycoerythrin (PE) 8,18 &19)Antibody to CD45 conjugated to allophycocyanin (APC): WBC,
Nuclear dye (DAPI, 4’6-diamidino-2-phenylindole)Controls: Breast cancer cell line (SKBr3)
- CellTracks AutoPrep system: Automated • Sample evaluation - CellSpotter Analyser (Veridex, immunocon): CTC Identification
and enumeration. • Interpretation of images: operators (cytotec &pathologist)
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Transfer to MagNest™
Aspirate fluid and un-labeled cells
Permeabilize andadd Staining
Reagents
Aspirate plasma Add buffer
Add ferrofluid.
Processing by the CellTracks™ AutoPrep SystemOff-Line
7.5 ml blood from CellSave™
Tube + Buffer
Immunomagnetic CTC Selection
Magnetic incubation
Remove magnets. Re-suspend target
cells in bufferCTC leukocyte
Centrifuge
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Anti-EpCAMFerrofluid
EpCAMNucleusDAPI
Immunomagnetic Labeling and Immunomagnetic Labeling and Immunofluorescent Identification of CellsImmunofluorescent Identification of Cells
FOR INTERNAL AND EXTERNAL USE MKG-1866, Rev. 1
Circulating Tumor Cell
Anti-CK-PE
CK
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Reproducibility of CTC Counts in Duplicate MCRC Samples (n=1627) with Average of <3 or ≥3 CTC per 7.5 mL of blood.
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Note: There may be more than one point superimposed over another. For example, on this plot,there are 975 instances (60%) where both tubes had 0 CTC, 116 instances (7%) where Tube 1 had 0 CTC and Tube 2 had 1 CTC, and another 109 instances (7%) where Tube 1 had 1 CTC and Tube 2 had 0 CTC.
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1MBC reference population information on page 7 of the clinical IFU.2MCRC reference population information on page 27 of the clinical IFU.3MPC reference population information on page 46 of the clinical IFU. 21
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Clinical Trial
3 Prospective multi-institutional clinical trials assessed the performance of the CellSearchTM
Assay• Metastatic Breast Cancer (MBC) > 5 cutoff( )• Metastatic Colorectal Cancer (MCRC) >3 cutoff• Metastatic Prostate Cancer (MPC) >5 cutoff• Selection of CTC cutoff : Prospectively identified
in patients in a training set and confirmed in a validation set
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Metastatic Breast Cancer (MBC) cutoff ≥≥ 55 CTC
Circulating tumor cells, Disease Progression,and Survival in Metastatic Breast Cancer, Cristofanilli et al, Sem Oncol. 2006
Cristofanilli M, Budd GT, Ellis MJ, et al: Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351:781-791, 2004
-Measurable disease, any type or line of therapy (first line, chemo Rx)
- 177 MBC (metastatic breast cancer)(20 centers) (67%ER/PR+, HER2 52%)
CTC analysis (performed in 7 centers)
MBC Clinical Trial Design
y (p )-145 healthy and 200 pts with benign disease- Imaging and CTC analysis (prior to initiation of therapy)CTC performed, 1 follow-up(~ 4 weeks)Duration of CTC: 6 months or until progressionClinical follow up: 50 monthsImaging and clinical progression of disease at 12 weeks**Circulating tumor cells, disease Progression,and Survival in Metastatic Breast
Cancer, Cristofanilli et al, NEJM 2004 24
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Predictive Value: OS of MBC Patients with <5 or >5 CTC at Baseline (N=177)
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Logrankp < 0.0001
Cox Hazards Ratio = 2.4chi-square = 19.54(p-value < 0.0001)
21.9 Months
CTC / 7.5mL Median OS in at Baseline N (%) Months (95% C.I.) <5 CTC 89 (50%) 21.9 (20.1 to 28.6) >5 CTC 88 (50%) 10.9 ( 7.0 to 15.2)
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%Pr
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Predictive Value: PFS of MBC Patients with <5 or ≥5 CTC at Baseline (N=177)
Cox Hazards Ratio = 1.9chi-square = 14.44(p-value = 0.0001)
7 0 Months
CTC / 7.5mL Median PFS in at Baseline N (%) Months (95% C.I.) <5 CTC 89 (50%) 7.0 (5.6 to 8.9) >5 CTC 88 (50%) 2.7 (2.1 to 4.4)
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Logrank p = 0.0001
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Cristofanilli M, Budd T,Ellis M, et al, N Eng J Med. 200426
A Reduction in CTC Below 5 After the Initiation of TherapyPredicts Longer OS whereas an Increase in CTC Count to 5 or
above Predicts a Shorter OS
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Curve LogrankComparison p-Value* 1 vs. 2 0.2023 1 vs. 3 0.0017 1 vs. 4 <0.0001
2 vs 3 0 1025
Median OS in Group Description N (%) Months (95% C.I.) 1 <5 CTCs at All Time Points 83 (47%) 22.6 (20.4 to >45) 2 >5 at Baseline & <5 CTC at Last Draw 38 (21%) 19.8 (14.6 to 31.6) 3 <5 at Early Draw & >5 CTC at Last Draw 17 (10%) 10.6 ( 6.1 to 16.2) 4 >5 CTCs at All Time Points 39 (22%) 4.1 ( 2.8 to 6.4)
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*p-values not adjusted for multiple hypothesis testsCristofanilli M, Budd T,Ellis M, et al, N Eng J Med. 2004 27
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Predictive Value: OS of MBC Patients with <5 or ≥5 CTC at different times of Follow-Up
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<5 CTCs at: 3- 5 Weeks (n= 92) 6- 8 Weeks (n= 77) 9-14 Weeks (n= 105)15-20 Weeks (n= 70)
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Time from Blood Draw (Months)0 5 10 15 20 30 35 40 45 5025
>5 CTCs at: 3- 5 Weeks (n= 40) 6- 8 Weeks (n= 22) 9-14 Weeks (n= 24)15-20 Weeks (n= 15)
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Metastatic Colorectal Cancers (MCRC) Cut off ≥≥ 3 CTCs
Cohen SJ, Punt CJ, Iannotti N et al: Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with meta-static colorectal cancer. J Clin Oncol 2008 Jul 1;26(19):
- Measurable disease, 1st or 2nd line of therapy (3rd line, only if anti-EGFR (chemo Rx)
- 430 MCRC (metastatic colorectal cancer) CTC analysis (performed in 4 centers)
MCRC Clinical TrialDesign
- 158 healthy and 55 pts with benign disease- Imaging and CTC analysis (prior to initiation of
therapy)- Frequency of CTC: 4 weeks- Duration of CTC: 12 months or until clinical
progression (imaging, clin eval)- Clinical follow up: 36 months
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Cox Hazard Ratio = 2.5chi-square = 31.48(p-value < 0.0001)
CTC / 7.5mL Median OS inat Baseline N (%) Months (95% C.I.)
<3 CTC 305 (74%) 18.5 (15.5 to 21.2)>3 CTC 108 (26%) 9.4 ( 7.5 to 11.6)
Predictive Value: OS of MCRC Patients with <3 or >3 CTC at Baseline (N=413)
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Cohen SJ, Punt CJ, Iannotti N et al: J Clin Oncol 2008.
Cohen SJ, Punt CJ, Iannotti N et al: J Clin Oncol 2008. 31
essi
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Cox Hazard Ratio = 1.6chi-square = 12.19(p-value = 0.0002)
7 9 Months
CTC / 7.5mL Median PFS in at Baseline N (%) Months (95% C.I.) <3 CTC 305 (74%) 7.9 (7.0 to 8.6) >3 CTC 108 (26%) 4.5 (3.7 to 6.3)
Predictive Value: PFS of MCRC Patients with <3 or ≥3 CTC at Baseline (N=413)
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%Pr
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7.9 Months4.5
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Cohen SJ, Punt CJ, Iannotti N et al: J Clin Oncol 2008.32
A Reduction in CTC Below 3 After the Initiation of Therapy Predicts Longer OS whereas an Increase in CTC Count to
3 or above Predicts a Shorter OS
Curve LogrankComparison p-Value* 1 vs. 2 0.0007 1 vs. 3 <0.0001 1 vs. 4 <0.0001 2 vs. 3 0.0078of
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Median OS inGroup Description N (%) Months (95% C.I.) 1 <3 CTC at All Draws 303 (70%) 18.6 (15.9 to 22.5) 2 >3 CTC at BL & <3 CTC at Last Draw 74 (17%) 11.7 ( 9.4 to 18.7) 3 <3 CTC at Early Draw & >3 CTC at Last Draw 29 ( 7%) 7.1 ( 6.3 to 10.8) 4 >3 CTC at All Draws 24 ( 6%) 3.9 ( 2.5 to 5.4)
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*p-values not adjusted for multiple hypothesis tests
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2 vs. 4 <0.0001 3 vs. 4 0.0001
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*p-values not adjusted formultiple hypothesis tests
Cohen SJ, Punt CJ, Iannotti N et al: J Clin Oncol 2008.33
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100%<3 CTC at: 1- 2 Weeks (n=316) 3- 5 Weeks (n=292) 6-12 Weeks (n=285)13-20 Weeks (n=172)
>3 CTC at: 1- 2 Weeks (n= 41) 3- 5 Weeks (n= 41) 6-12 Weeks (n= 25)13-20 Weeks (n= 21)
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%Pr
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Cohen SJ, Punt CJ, Iannotti N et al: J Clin Oncol 2008. 34
de Bono JS, Sher HI, Montogomery RB et al: Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer Clin Can Res 2008 oct
Metastatic Prostate Cancers (MPC)-K073338 Cut off ut off ≥≥ 5 CTCs5 CTCs
cancer, Clin Can Res 2008 oct.
- 231 MPC (metastatic prostate cancers), two consecutive increases in serum PSA, androgen independent, hormone resistant prostate cancer
- Bone metastases pos (90%), non-measurable disease 62%
MPC Clinical TrialDesign
- Baseline CTC count prior to initiation of new line of chemotherapy)
- Frequency of CTC:2- 4 weeks- Duration of CTC : 18 months or until progression- Clinical follow-up: 36 months.- Disease progression (PSA, imaging and clinical signs and
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Predictive Value: OS of MPC Patients with <5 or >5 CTC at Baseline (N=219)
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CTC / 7.5mL Median OS in at Baseline N (%) Months (95% C.I.) <5 CTC 94 (43%) 21.7 (21.3 to ------) >5 CTC 125 (57%) 11.5 ( 9.3 to 13.7)
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Cox Hazard Ratio = 3.3chi-square = 34.48(p-value < 0.0001)
de Bono JS, Sher HI, Montogomery RB et al, Clin Cancer Res. 2008. 37
Predictive Value: PFS of MPC Patients with <5 or >5 CTC at Baseline (N=219)
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CTC / 7.5mL Median PFS in at Baseline N (%) Months (95% C.I.) <5 CTC 94 (43%) 5.8 (5.0 to 7.9) >5 CTC 125 (57%) 4.2 (3.1 to 4.9)
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de Bono JS, Sher HI, Montogomery RB et al, Clin Cancer Res. 2008. 38
A Reduction in CTC Below 5 After the Initiation of Therapy Predicts Longer OS whereas an Increase in CTC Count to 5
or above Predicts a Shorter OS in MPC PatientsMedian OS in
Group Description N (%) Months (95% C.I.)1 <5 CTC at All Draws 88 (38%) >26 (21.4 to ------)2 >5 CTC at BL & <5 CTC at Last Draw 45 (20%) 21.3 (18.4 to ------)3 <5 CTC at Early Draw & >5 CTC at Last Draw 26 (11%) 9.3 ( 8.2 to 11.3)4 >5 CTC at All Draws 71 (31%) 6.8 ( 5.8 to 10.3)
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Curve LogrankComparison p-Value*
1 vs. 2 0.15281 vs. 3 <0.00011 vs. 4 <0.00012 vs. 3 <0.00012 vs. 4 <0.00013 vs. 4 0.5013
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*p-values not adjusted formultiple hypothesis tests
de Bono JS, Sher HI, Montogomery RB et al, Clin Cancer Res. 2008. 39
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Predictive Value: OS of MPC Patients with<5 or >5 CTC at different times of Follow-Up
<5 CTC at: 2- 5 Weeks (n=123) 6- 8 Weeks (n=110) 9-12 Weeks (n=100)13-20 Weeks (n= 99)
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>5 CTC at: 2- 5 Weeks (n= 80) 6- 8 Weeks (n= 53) 9-12 Weeks (n= 49)13-20 Weeks (n= 44)
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MCRC• 430 patients• Cut-off = ≥3 CTC• Patients with ≥3 CTC at
baseline = 26% (108/413 evaluable
MBC• 177 patients• Cut-off = ≥5 CTC• Patients with ≥5 CTC
at baseline = 50% (88/177 evaluable patients)
MPC• 231 patients• Cut-off = ≥5 CTC• Patients with ≥5 CTC
at baseline = 57% (125/219 evaluable patients)
MBC, MCRC, and MPC MBC, MCRC, and MPC Summary & ConclusionSummary & Conclusion
FOR INTERNAL AND EXTERNAL USEMKG-1866, Rev. 1
patients)• Should be used for
serial monitoring• Predicts PFS and OS• Combination of CTC
and imaging may provide the most accurate assessment of patient prognosis
patients)• Should be used for
serial monitoring• Predicts PFS and OS• Combination of CTC
and imaging may provide the most accurate assessment of patient prognosis
p )• Should be used for
serial monitoring• Predicts PFS and OS• Combination of CTC
and PSA may provide the most accurate assessment of patient prognosis
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MBC, MCRC, & MPCMBC, MCRC, & MPCMedian Overall Survival Comparison Median Overall Survival Comparison
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Summary
• Results should be used in conjunction with diagnostic tests (lab, imaging), physical exam and medical history.
CellSearch™ SystemLimitationsLimitations
• Not proven to affect overall health outcomes in patients with metastatic carcinoma
• Potential for monitoring patients• Insufficient evidence as a marker of
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Yale CTC Experience
CellSearch (Veridex device) 2006•>1000 CTC tests•Clinicians (Oncologists): breast, colorectal
d land lung cancers•Guide treatment, research use•CTCs investigated for HER2/neu protein expression in breast cancer patient’s
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• Pathologist and cytotechnologist (certified by Veridex)
• CTC are defined as:-Nucleated cells lacking CD 45 and expressing
t k ti (8 18 & 19)
InterpretationInterpretation
cytokeratin (8,18 & 19). -Morphology (round or oval with a nucleus within the cytoplasm).
-Size (4um)-Heterogeneity (morphology and size).
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Cell Analysis
CTC’s
CK-PE+/DAPI+/CD45-APC-
Leucocytes
CK-PE-/DAPI+/CD45-APC+
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Gallery of images HER2 + CTCs
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CTC REPORT
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Summary
CTC detection in peripheral blood in clinical practice
• Low frequency (rare)• Standardized methods with high degree of
SummarySummary
Standardized methods with high degree of reproducibility
• Currently, most data on the prognostic value, available for breast, prostate and colon cancers.
• Multicenter analysis and validation is needed to confirm clinical significance.
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Summary- Valuable tool for monitoring cancer patient status
and outcome. FDA approved.- Employs immunomagnetic-enrichment based
protocols focused on CTC number as the
Summary Summary CellSearchCellSearchTMTM SystemSystem
indicator of patient status or outcome.- Multi center trial: The number of CTCs was a
significant independent predictor of OS and PFS in patients with MBC, MCRC and MPC
- American Society of Clinical Oncology (ASCO):recommendation 2007: CTC test should not be used to make diagnostic or treatment decisions in patients with MBC
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• Guide prognosis: Metastatic and early stage cancer patients
• Measure response to anticancer Rx: predictive biomarker
Future Potential and Applications: CTCs
• Select patients for adjuvant chemotheray• Detect recurrent disease• ‘Real time biopsy’: Surrogate for Tumor biology• Molecular characterization: Discover and identify
new targets for therapeutic manipulation
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Summary and Conclusions
• CTC level (< 5): Favorable, this may imply a good response to treatment.
• Caution is warranted because of the lower
ConclusionConclusion
sensitivity of the CTC test. • Radiologic disease progression should not be
ignored on the basis of a favorable CTC level. • Favorable CTC level with overt radiologic
progression may still suggest a better outcome
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The CellSearch System (Veridex) • Morphology skills highly similar to those of the
Cytopathologist- Interpretation and Enumeration of CTCs.
ConclusionsConclusions
- Protein expression patterns of CTC (ER,PR,HER2, EGFR), additional prognostic information.
• Cytopathology lab with trained cytotechnologists and cytopathologists - Natural location for this technology in the healthcare delivery system.
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History
• Breast Group CEC/CTC Enumeration Study
What does it cost?What does it cost?
Lab cost around $175 per testCPT Codes: 88346 x 3 (immunofluorescent study) = $380 x 3 = $1140 88361 x 2 (morphometric analysis, IHC ) = $505 x 2 = $101088313 x 1 (special stain) = $210Charge $23602360 per test
Total CostsTotal Costs
Medicare Reimbursement Avg: $777.53Medicare Reimbursement Avg: $777.53Labor/Overhead: $386.00Labor/Overhead: $386.00
Labor/Overhead + Cost per test = $386.00 + $175.00 = $561.00 Labor/Overhead + Cost per test = $386.00 + $175.00 = $561.00
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Tests Requirements
• High Complexity Tests• Pathologist and cytotechnologist (certified
by Veridex)C ll I t t ti P fi i A t• Cell Interpretation Proficiency Assessment
• PT Test Requirement
Acknowledgements:Yale University School of Medicine, Dept of Pathology• David L. Rimm• David Chhieng• Diane Kowalski• Lab Manager: Kevin Schofield• Cytotechnologists: Brett Minger, Philip Galullo, Kristina Gordy, Rupay g g , p , y, p
VyasVeridexBrian ZuchelkowskiVera Gibson
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