BONE MARROW OCCURRENCE OF
CYTOTOXIC AUTOLOGOUS BCR-ABL SPECIFIC
T CELLS
DURING IMATINIB TREATMENT IN Ph+ ACUTE
LYMPHOBLASTIC LEUKEMIA PATIENTS WITH
PROLONGED DISEASE-FREE SURVIVAL
Dr. Leonardo Potenza, Department of Oncology, Haematology and Respiratory Diseases,
Section of Haematology
Background 1: Ph+ Acute Lymphoblastic Leukemia
Vignetti, M. et al. Blood 2007;109:3676
If these results have been obtained either
in virtue of the sole TKI activity of IM or
also with the contribution of a restored
immune system has not yet been
addressed in Ph+ALL setting.
Grammatico, S. et al. Leuk Res 2008; doi:10.1016/j.leukres.2008.11.009
Potenza, L. et al.
Haematologica/the hematology journal 2005; 90: 1275
Background 2: Immunity in Chronic Myeloid Leukemia
Development of Robust T-cell responses to CML under Imatinib treatment
Chen, I-U. et al. Blood 2008; 111: 5342
Background 3: Immunity in Chronic Myeloid Leukemia
Effect of a p210 multipeptide vaccine associated with imatinib in patients
with CML and persistent residual disease
Bocchia, M. et al. Lancet 2005; 367:657
Aim of the Study
We have evaluated the presence
of BCR-ABL specific T cell responses
in bone marrow (BM)
and peripheral blood (PB)
samples from 9 consecutive Ph+ ALL
patients under Imatinib alone
Patients
Characteristics
9
Patients
3 Relapses
6 CRs
Maintenance Treatment
Imatinib alone
Cytological
Monthly Bone marrow samples
Immunophenotypic
Molecular
Methods
• IFNγ-ELISPOT assay
• Multiparametric Flow
Cytometry/Cytokine Secretion
Assay
• 51 Chromium-releasing Cytotoxic
Assays
ANTIGENS for ELISPOT assay
Volpe, G. et al. Cancer Res 2007;67:5300
Peptides have been used
as a Mixture:
1. mix 1 int: including 4 decameric peptides
(d4d7)and one nonameric peptide (n4);
2. mix 1 ext: including 5 decameric peptides (d1d3, d8 e d9) and one nonameric peptide
(n9);
3. mix 2: including five eicosameric peptides
(v1-v5)
4. mix 3: peptides deriving from BCR-ABL alternate
splicing (OOF1-OOF6).
BM Anti BCR-ABL T cell responses
in 9 Ph+ ALL Patients
72.5% of the tested samples
Ph+ ALL Patients with MRD < 10-3
P < 0,001
Pt 4
BCR‐ABL/ABL
X
100
IFNγSFC/106
BMMCs
Pt 1
Pt 5
BCR‐ABL/ABL
X
100
IFNγSFC/106
BMMCs
Pt 2
Ph+ ALL Patients with MRD > 10-3
Pt 7
BCR-ABL/ABL X 100
IFNγSFC/106 BMMCs
Pt 8
BCR-ABL/ABL X 100
IFNγSFC/106 BMMCs
Pt 9
Ph+ ALL Patients with Relapse
BCR-ABL/ABL X 100
IFNγSFC/106 BMMCs
Pt 3
Molecular
BCR-ABL/ABL X 100
Hematologic
IFNγSFC/106 BMMCs
Pt 6
Flow Cytometric Analysis
Pt 2
Pt 3
Pt 4
Pt 5
Flow Cytometric Analysis
Pt 6
Pt 7
Pt 8
Pt 9
Flow Cytometric Analysis
Pt 1
Prevalent Expansion
of EMRA CD8+ and Naive CD4+
TEMRA cells
are generated upon
homeostatic proliferation in
the absence of the antigen
Geginat, J. et al. Blood 2003;101:4260
CSA Analysis
BCR-ABL specific T cells: cytokine production profiles in BM samples
of the 9 Ph+ LLA Patients.
BCR-ABL Specific EM CD8+ T Cells were mainly responsible for the IFNγ Production
IL2-producing subsets were equally distributed either in CD4+ or CD8+ Cells
BCR-ABL Specific EM CD4+ T Cells mainly produced TNFα
• 6 out of 9 patients showed a specific
cytotoxic response against target cells pulsed
with BCR-ABL derived peptides. No lysis was
observed with target cells pulsed with controlpeptides
7500
5000
• 5 Pts were also tested for direct cytotoxicity
against Ph+ autologous and heterologous
blasts and all showed high lysis of target cells
2500
0
BCR-ABL
peptides
Ph+ blasts
• Low level or no lysis was observed by using
heterologous Ph- blasts as target cells.
Ph- blasts
10000
7500
Cytotoxicity
Assay
LU10/106 cells
LU10/106 cells
10000
5000
2500
0
unmanipulated
CD8+
CD8-
Cytotoxicity Assay
in Patient n° 6
%
Specific
Lysis
40
30
20
10
0
20:1
10:1
1:1
0,1:1
0,01:1
PHA-Controls
PHA-Peptides Pulsed
Ph- Allogeneic Blasts
Ph+ Autologous Blasts
Conclusions
In Ph+ ALL patients:
• BM-resident, BCR-ABL specific autologous T
cells are allowed during IM therapy (72.5% of
the tested samples)
• BCR-ABL specific autologous T cells are rarely
identified in PB (20% of the tested samples)
• BCR-ABL specific autologous T cells are
statistically associated with lower values of MRD
(p 0.001) and may directly cause lysis of Ph+
Blasts
Perspectives
• BCR-ABL specific autologous T cells
possibly synergise with IM in maintaining a
prolonged complete remission and may be
predictive of the IM treatment response
• The possibility of developing
immunotherapeutic strategies against
Ph+ ALL should be further investigated
Acknowledgements
Experimental Hematology Lab. And Division of Hematology
University of Modena and Reggio Emilia, MODENA
(Prof. G. Torelli)
Lab: G. Riva, P. Barozzi, C. Quadrelli, D. Vallerini, E. Zanetti, R. Bosco,
S. Martinelli, P. Zucchini
Clinics: M. Luppi, M. Morselli, F. Forghieri, M. Maccaferri, F. Volzone, R.
Marasca, F. Narni
Statistics: C. Del Giovane, R. D’Amico
Division of Pediatric Hematology, IRCSS Policlinico S. Matteo
University of Pavia
(Prof. F. Locatelli)
Lab: S. Basso, P. Comoli