Pathogenesis, diagnosis and monitoring of Acute Promyelocytic Leukemia Laura Cicconi, MD University Tor Vergata, Rome , 7 th INTERNATIONAL SYMPOSIUM ON ACUTE PROMYELOCYTIC LEUKEMIA Rome, 24-27 September 2017
Outline • Pathogenesis & biology of APL • Diagnosis • Monitoring of minimal residual disease
APL main presenting features • Usually abrupt onset, with rapidly progressing coagulopathy (medical emergency) • Life-threatening hemorrhagic events • Infiltration by leukemic promyelocytes
1977. t(15;17) as a disease hallmark
Molecular pathogenesis
PML-RARA oncoprotein functions L M P P RARA PML M Corepressors A R A R L PML A R R R R A R R R A PML R A PML A NB disrup4on X X A P P R R R R M M L L A A RARA RARA PML A R A R RARE Tumor repression, Apoptosis Transcrip4onal Senescence, DNA repair repression of RARA target genes MYELOID DIFFERENTIATION BLOCK
Targeted therapies in APL Most active single agent in APL (>70% cured); synergistic with ATRA ATO 1970 1980 1990 2000 2010 ATRA ATO+ATRA t(15;17) Complete remission J Rowley >95% of APL through APL blast patients cured differentiation First example of therapy with a genetic target
PML/RAR α and ATRA A model for differentiation therapy Derivative of Vitamin A, at pharmacological dosis able to induce CR in >90% of APL patients (although short-lasted in the majority of cases) ATRA Coactivators ATRA R R R R X A X A ATRA R R R R A A RARE ATRA Transcrip4onal Myeloid differen4a4on ac4va4on in RARE
PML/RAR α , ATRA and ATO Synergistic effect of the two agents PML RARA B1 B2 Coiled-Coil RING DNA LBD ATRA K160 b U ATRA SUMO ATO U b ATO ATO ATRA SUMO b U U b SUMO Proteasomal degrada4on NB reformation Transcriptional reactivation of RARA target genes APL cure
Landscape of somatic mutations at APL relapse PML and RARA mutations PML RARA Zhu, NEJM 2014; Lehmann-Che, NEJM 2014; Lou, Annals of Hem 2015; Chendamarai, Plos One 2015; Madan et al . Leukemia 2016;Iaccarino BJH 2016
The PML/RAR α fusion protein Why so important in diagnosis and treatment • Unique to APL • Strongly correlated with pathogenesis • Targeted by specific therapies • Detection predicts response to ATRA & ATO
Outline • Clinical & biological background • Diagnosis • Monitoring of minimal residual disease
Recommended actions in case of APL suspicion Consider the disease as a medical emergency • Start treatment with ATRA • Start intensive transfusion support • Send a marrow sample to reference lab for genetic diagnosis
Morphology (can be easy, can be tough) 25% of APLs • Atypical promyelocytes • Hypogranular • Bilobed nuclei • Bilobed nuclei • Monocytoid elements • Cytoplasmic granules, Auer rods
Suspected Acute Promyelocytic Leukemia: the role of immunophenotype Suspect of APL may arise from flow cytometry analysis CD13 pos CD34 neg CD34 neg CD15 dim HLA-DR neg CD33 pos HLA-DR neg CD117 pos
Immunophenotypic features of APL • CD34 and HLA-DR frequently absent • Strong and highly homogeneous CD33 expression • Heterogeneous expression of CD13 • Absence or very low expression of CD15, CD11a-b-c • Expression of CD56 and CD2 (M3v)
Methods for Genetic Diagnosis Pros and Cons Technique Target Advantages Pitfalls Karyotype Time consuming; chromosomes Specific for t(15;17) false negatives Poor sensitivity; FISH for No information on the DNA Specific; rapid PML/RAR α PML/RARA isoform Rapid; No information on the anti-PML Protein low cost PML-RAR α isoform MoAb RT-PCR for Specific; Artifacts; RNA PML/RAR α rapid Contaminations
Patterns of PML nuclear staining Microgranular pattern Nuclear body pattern PML/RAR α +ve APL PML/RAR α -ve AML ATRA / ATO-responsive ATRA / ATO resistant
RT-PCR amplification of PML/RAR α transcripts bcr1 (long) transcript 55 % bcr2 (variable) transcript 5 % bcr3 (short) transcript 40 % JM Van Dongen et al, Leukemia 1999
Recommended diagnostic work-up for APL Sanz et al. (LeukemiaNet) Blood 2009; BSH guidelines, BJH 2006 • Diagnostic confirmation at genetic level is mandatory: PML-RAR α predicts response to ATRA and/or ATO • FISH or PML staining allow rapid diagnosis (valid for pt eligibility for ATRA- or ATO-based protocols).They do not define the type of PML/RAR α fusion • Always send sample to a reference molecular biology lab for RT-PCR of PML/RAR α (required as baseline for MRD)
Gene fusions in APL variants: Implications for targeted therapy Sensitivity to targeted therapy ATRA ATO F RAR α F RAR α A B C D A B C D A B C D E E E F PLZF-RAR α PLZF-RAR α � Pro Pro Pro B C D B C D B C D E E E F F F ~0.5% of APL � POZ POZ POZ Zn ++ Zn ++ Zn ++ Zn ++ Zn ++ Zn ++ Zn ++ Zn ++ Zn ++ NPM1-RAR α NPM1-RAR α � B C D B C D B C D B C D E E E E F F F F ? Oligomerisation Oligomerisation Oligomerisation � NuMA-RAR α NuMA-RAR α ? B C D B C D B C D B C D E E E E F F F F Coiled-coil Coiled-coil Coiled-coil � STAT5b-RAR α STAT5b-RAR α ? SH2 B C D B C D B C D B C D E E E E F F F F Coiled-coil Coiled-coil Coiled-coil Coiled-coil DBD DBD DBD DBD SH3 SH3 SH3 SH3 SH2 SH2 SH2 PRKAR1A-RAR α PRKAR1A-RAR α B C D B C D B C D B C D E E E E F F F F ? ? Coiled-coil Coiled-coil Coiled-coil FIP1L1-RAR α FIP1L1-RAR α � B C D B C D B C D B C D E E E E F F F F ? FIP FIP FIP OBFC2A-RAR α � A B C D A B C D A B C D E E E F F F ? DBD IRF2B2-RAR α IRF2BP2 Exon 2 ? � BCOR-RAR α ? � Grimwade et al . Cancer Treat Rep. 2009; Yian et al. Natl Comp Canc Netw 2015; Yamamoto Y, Blood 2010
Outline • Clinical & biological background • Diagnosis • Monitoring of minimal residual disease
The PML/RAR α fusion protein Why so important in diagnosis and treatment • Unique to APL • Strongly correlated with pathogenesis • Targeted by specific therapies • Detection predicts response to ATRA & ATO • Ideal marker for residual disease monitoring
Cheson et al. J Clin Oncol 2003
PCR monitoring in APL: early studies • ATRA therapy alone did not induce molecular remission • Patients treated with ATRA+CHT in long-term clinical remission had consecutively negative RT-PCR tests Miller et al.Proc Natl Acad Sci USA 1992 ; Lo-Coco et al. Lancet 1992; Martinelli G BJH 1995; Laczika K et al. Leukemia 1994; Grimwade et al , Leukemia 1996.
Risk of relapse according to RT-PCR status Post-consolidation time point PML/RAR α pos RAR α - PML o PML/RAR α pos MRD neg MRD neg Diverio, et al. Blood 1998; Burnett et. Al, Blood 1999
Salvage Therapy for Relapsed APL Treatment in molecular vs hematologic relapse 1.0 1.0 Molecular relapse 0.9 0.9 Molecular failure (n=16; 5 events) Molecular failure (n=16; 5 events) 0.8 0.8 0.7 0.7 Probability Probability 0.6 0.6 Hematologic relapse 0.5 0.5 0.4 0.4 0.3 0.3 Hematological relapse (n=33; 24 Hematological relapse (n=33; 24 events) events) 0.2 0.2 0.1 0.1 p=0.008 p=0.008 0 0 0 0 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 Years Years Lo-Coco et al . Blood, 1999 Esteve et al . Leukemia, 2007
Real-time Q-PCR for PML-RARA (EAC) • Sensitivity • Less prone to contaminations • Reliable results in low-quality samples Gabert et al ., Leukemia 2003 Gabert et al , Leukemia 2003
Kinetics of molecular/frank relapse in APL Implications for optimal sampling time-points Median increment 1.1log/month Grimwade et al . JCO 2009
Sampling source: blood or marrow for MRD monitoring? MR 0085 BM PB MR 0116 BM PB MR PCR +ve 0285 BM PCR -ve PB MR Mol. relapse HR Hem. relapse HR 0441 BM PB MR 0618 BM Adapted from Grimwade et al . JCO 2009 PB ∆ Post #1 #2 #3 #4 2 mo 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
Q-PCR in ATO-ATRA treated patients Effective clearance of PML-RARA ATRA-CHT � Similar clearance in ATRA-ATO ATO-ATRA and ATRA-CHT � Low relapse % in ATO-ATRA pts questions cost- effectiveness of MRD monitoring Post 3° cons Post Induction Diagnosis Log-reduction PML/RAR α Diagn-post ind P-value Post ind- post cons P-value ATRA-ATO 2.94 6.34 0.018 0.0024 ATRA-CHT 3.43 5.33 Cicconi et al. Leukemia 2016
Summary of Recommendations for Molecular Monitoring of APL • Send samples to reference labs experienced with molecular testing of this rare disease • Use of standardized reference methods (Gabert et al Leukemia 2003; Grimwade et al. J Clin Oncol 2009) • Bone marrow sampling more informative than PB • RQ-PCR advantageous over RT-PCR to better assess sample quality and to investigate kinetics of MRD
Acknowledgements Prof. F. Lo Coco M. Divona Prof. MT Voso C. Ciardi Prof. W. Arcese A. Ferrantini Prof. A Venditti S. Lavorgna Prof. S. Amadori T. Ottone M. Consalvo V. Alfonso P. Panetta L. Iaccarino P. Curzi G. Falconi D. Fraboni E. Fabiani
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