Upload
stevenkcheng
View
59
Download
4
Tags:
Embed Size (px)
Citation preview
Clinical Flow Cytometry
Maryalice Stetler-Stevenson, M.D., Ph.D.
Primary Research Area(s)Our expertise
•The Flow Cytometry Laboratory is an international leader in diagnostic flow cytometry, minimal residual detection, quantification of antigen expression by specific cell populations and use of flow cytometric biomarkers for prognosis, decision making and surrogate endpoints in clinical trials• Cancer Steering Committee (CSC), Minimal Residual Disease (MRD) Project • Chair, Subcommittee on Clinical Applications of Flow Cytometric Immunophenotyping of Hematolymphoid
Cells, CLSI• Minimal Residual Disease Detection in Myeloma Consensus Initiative, Co-organizer • Expert consultant FDA, Clinical Flow Cytometry • NCI Leukemia Steering Committee (DCTD/NCI), special expert reviewer• NCI Pediatric Leukemia and Lymphoma Steering Committee, Ad hoc reviewer • CDC, NCEH. Consultant and Ad hoc reviewer
• New areas of investigation: Monitoring patients receiving CD19 CAR T-cell therapy, Myeloma MRD, CLL MRD, ALL MRD
• What technologies / approaches / methodologies do you utilize in your research area? Flow Cytometry!
CD19 CAR Therapy in Refractory B-ALL
• Patient’s lymphocytes removed, CD19 CAR T-cells manufactured, expanded and infused into patient in 11 days .
• Diagnostic Assessment:• Pre TX: ALL cells express CD19?• Post Tx:Response to therapy (MRD)
• Monitoring the CD19CAR T-cells:• Number of CD19CAR T-cells and the sites they reach
(BM, CSF, etc)-We developed a novel assay to detect CD19CART-cells with high sensitivity and specificity
• What type of T cells? CD4 vs. CD8 and naive, effector memory, central memory, central memory, T-memory stem cells and terminal effector T-cells
CD19 Chimeric Antigen Receptor
Signaling
CD19
Chimeric Antigen Receptors:•Specific for a surface protein•Free of MHC restriction - can be used in any patient•Signals for full activation are self-contained•Variable co-stimulatory domains
Subject # Target T-cell
infusion reached
SourceMaximum
% CAR+ T Cell
1 Yes
PB 0.07%
Marrow 0.4%
CSF 0%
2No
(28,000 CAR+/kg)
PB 15.3%
Marrow 5%
CSF 6.2%
3 YesPB 0.4%
Pleural Fluid 13%
4 YesPB 38%
Marrow 0.8%CSF 0%
5No
(416,000 CAR+/kg)
PB 1%Marrow 0.27%
CSF Indeterminate
PB = Peripheral blood; CSF = Cerebral spinal fluid
Subject # Target T-cell
infusion reached
SourceMaximum
% CAR+ T Cell
6 YesPB 0%
Marrow 0%
7 Yes
PB 0.5%
Marrow 0%CSF 0.3%
8 Yes
PB 1.5%
Marrow 0%
CSF 17%
9 YesPB 3.3%
Marrow 2.5%CSF 7.3%
10 Yes
PB PMarrow P
CSF 80%
We demonstrated that CD19CAR T-Cells Migrate to Extravascular Tissues
Patient with 1.3% of lymphocytes CD19CAR +T-cells in PB: Characterization of CD19CAR+ T-cell subsetsCD4+ naïve T-cells: 0.08CD4+ effector memory T-cells: 35.37CD4+ central memory T-cells: 1.36CD4+ T memory stem cells: 0.21CD4+ terminal effector T cells: 2.25 CD8+ naïve T-cells: 0.00CD8+ effector memory T-cells: 50.01CD8+ central memory T-cells: 0.75CD8+ T memory stem cells: 0.04CD8+ terminal effector T cells: 0.57
CD19CAR+ T Cells and Correlation with Response to Therapy
CD3 AH7
CA
R a
647
102
103
104
105
102
103
104
1050.00% 37.97%
0.02% 62.01%
CD3 AH7
CA
R a
647
102
103
104
105
102
103
104
1050.00%
0.95%
0.00% 99.05%
9% B-ALL in PB 0% B-ALL and 0% CD19+ B-cells in PB
Peripheral Blood
CD45 V500
CD
10 A
PC
102
103
104
105
102
103
104
105
CD3 AH7
CA
R C
D1
9 A
PC
102
103
104
105
102
103
104
105
83.24%0.00%
16.76%0.00%
0% B-ALL CSF
CD45 V500
CD
10
PE
102
103
104
105
102
103
104
105
6% B-ALL CSF
7/16/2013 7/22/2013
CSF
17% CD19CAR T’s
What type of T-cells are the CD19CAR T-cells?
Research ImplicationsHow does our research fit in the broader context?
•Our clinical/translational research contributes to the excellent hematolymphoid neoplasia group in LP which includes Hematopathology, Molecular Diagnostics, Cytogenetics and Flow Cytometry•Our research is a vital part of numerous clinical research protocols at NCI.
– We provide data that is correlated with outcome to try to determine prognosis and groups of patients that will receive optimal benefit from a specific therapy.
– We provide real time monitoring of response to novel therapies for kinetics, direct assessment of therapeutic agent and its acitivity.
– We provide data that is used to make clinical decisions (which branch of protocol decision tree, further rounds of therapy, treatment of additional sites, enroll on different protocol).
Future Direction • In the next 5-10 years we will develop new diagnostic and
prognostic tools, new biomarkers and continue to ascertain needs in clinical protocols and developing unique and novel flow cytometric approaches to meet these needs.
• What are the current challenges & obstacles beyond time, money, people, travel, and space?
• Purchasing regulations• Poor physical facility• Excessive regulations• Inadequate IT support• Difficult interaction with Phlebotomy• CRIS coordination within LP• Finding qualified people (HR)
CollaboratorsNIH•Ola Landgren, MOB, NCI•Robert Kreitman, LMB, NCI•Crystal Mackall, POB, NCI•Adrian Wiestner, NHLBI•Hematology, DLM•Mark Raffeld, LP, NCI•Diane Arthur, LP, NCI•Armando Filie, LP, NCI•Mark Roth, LP, NCI•Steve Pavletic, ETIB, NCI•Claude Sportes, ETIB, NCI
Extramural•Alan Wayne, Children’s Hospital, Los Angeles•Andy Rawstron, Leeds Teaching Hospital, Leeds, UK•Gerald Marti, FDA•Bruno Paiva, University Salamanca, Salamanca, Spain