Pathogenesis, diagnosis and monitoring of Acute Promyelocytic - - PowerPoint PPT Presentation

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Pathogenesis, diagnosis and monitoring of Acute Promyelocytic - - PowerPoint PPT Presentation

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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 &

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SLIDE 1

Pathogenesis, diagnosis and monitoring

  • f Acute Promyelocytic Leukemia

Laura Cicconi, MD

University Tor Vergata, Rome ,

7th INTERNATIONAL SYMPOSIUM ON ACUTE PROMYELOCYTIC LEUKEMIA Rome, 24-27 September 2017

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SLIDE 2

Outline

  • Pathogenesis & biology of APL
  • Monitoring of minimal residual disease
  • Diagnosis
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SLIDE 3

APL main presenting features

  • Usually abrupt onset, with rapidly progressing

coagulopathy (medical emergency)

  • Life-threatening hemorrhagic events
  • Infiltration by leukemic promyelocytes
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SLIDE 4
  • 1977. t(15;17) as a disease hallmark
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SLIDE 5

Molecular pathogenesis

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SLIDE 6

RARE Corepressors

PML-RARA oncoprotein functions

P M L PML

Tumor repression, Apoptosis Senescence, DNA repair Transcrip4onal repression of RARA target genes MYELOID DIFFERENTIATION BLOCK

R X R R X R

NB disrup4on

R A R A R A R A

R A R A PML R A R A PML R A R A PML RARA P M L RARA P M L RARA P M L R A R A PML

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SLIDE 7

Targeted therapies in APL

t(15;17)

J Rowley

ATRA

Complete remission through APL blast differentiation

First example of therapy with a genetic target

ATO

Most active single agent in APL (>70% cured); synergistic with ATRA

ATO+ATRA

>95% of APL patients cured

1970 1980 1990 2000 2010

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SLIDE 8

PML/RARα and ATRA

A model for differentiation therapy

RARE Coactivators

Transcrip4onal ac4va4on in RARE

R A R A R A R A

ATRA ATRA ATRA

Derivative of Vitamin A, at pharmacological dosis able to induce CR in >90% of APL patients (although short-lasted in the majority of cases)

R X R R X R

ATRA

Myeloid differen4a4on

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SLIDE 9

PML/RARα, ATRA and ATO

Synergistic effect of the two agents

PML RARA

RING B1 B2 Coiled-Coil DNA LBD

SUMO SUMO SUMO

NB reformation Transcriptional reactivation of RARA target genes

Proteasomal degrada4on

APL cure

U b U b U b U b

ATRA ATRA ATRA ATO ATO ATO K160

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SLIDE 10

PML RARA

Landscape of somatic mutations at APL relapse

PML and RARA mutations

Zhu, NEJM 2014; Lehmann-Che, NEJM 2014; Lou, Annals of Hem 2015; Chendamarai, Plos One 2015; Madan et al. Leukemia 2016;Iaccarino BJH 2016

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SLIDE 11

The PML/RARα 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
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SLIDE 12

Outline

  • Clinical & biological background
  • Monitoring of minimal residual disease
  • Diagnosis
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SLIDE 13
  • Start treatment with ATRA
  • Start intensive transfusion support
  • Send a marrow sample to reference lab for genetic diagnosis

Recommended actions in case of APL suspicion Consider the disease as a medical emergency

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SLIDE 14

Morphology (can be easy, can be tough)

  • Atypical promyelocytes
  • Bilobed nuclei
  • Cytoplasmic granules, Auer rods
  • Hypogranular
  • Bilobed nuclei
  • Monocytoid elements

25% of APLs

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SLIDE 15

Suspected Acute Promyelocytic Leukemia: the role of immunophenotype

Suspect of APL may arise from flow cytometry analysis

CD13 pos HLA-DR neg CD34 neg HLA-DR neg CD15 dim CD117 pos CD34 neg CD33 pos

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SLIDE 16

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)
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SLIDE 17

Methods for Genetic Diagnosis

Pros and Cons

Technique Target Advantages Pitfalls Karyotype for t(15;17) chromosomes Specific Time consuming; false negatives FISH for PML/RARα DNA Specific; rapid Poor sensitivity; No information on the PML/RARA isoform anti-PML MoAb Protein Rapid; low cost No information on the PML-RARα isoform RT-PCR for PML/RARα RNA Specific; rapid Artifacts; Contaminations

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SLIDE 18

Patterns of PML nuclear staining

Microgranular pattern PML/RARα +ve APL Nuclear body pattern PML/RARα -ve AML ATRA / ATO-responsive ATRA / ATO resistant

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SLIDE 19

RT-PCR amplification of PML/RARα transcripts

JM Van Dongen et al, Leukemia 1999 bcr1 (long) transcript

55 %

bcr2 (variable) transcript 5 % bcr3 (short) transcript

40 %

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SLIDE 20
  • Diagnostic confirmation at genetic level is mandatory:

PML-RARα 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/RARα fusion

  • Always send sample to a reference molecular biology lab

for RT-PCR of PML/RARα (required as baseline for MRD)

Recommended diagnostic work-up for APL

Sanz et al. (LeukemiaNet) Blood 2009; BSH guidelines, BJH 2006

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SLIDE 21

Gene fusions in APL variants: Implications for targeted therapy

A B C D E F RARα A B C D E F A B C D E F RARα

Sensitivity to targeted therapy ATRA ATO

  • ?
  • ?
  • ?

? ?

  • ?

Grimwade et al. Cancer Treat Rep. 2009; Yian et al. Natl Comp Canc Netw 2015; Yamamoto Y, Blood 2010

OBFC2A-RARα

DBD

  • ?

A B C D E F A B C D E F A B C D E F

NPM1-RARα

Oligomerisation

B C D E F

NPM1-RARα

Oligomerisation

B C D E F

Oligomerisation

B C D E F B C D E F

NuMA-RARα

Coiled-coil

B C D E F

NuMA-RARα

Coiled-coil

B C D E F

Coiled-coil

B C D E F B C D E F

PLZF-RARα

B C D E F

POZ Pro

Zn++ Zn++

PLZF-RARα

B C D E F B C D E F

POZ POZ Pro Pro

Zn++ Zn++ Zn++ Zn++ Zn++ Zn++ Zn++

B C D E F

Coiled-coil DBD SH3 SH2

STAT5b-RARα

B C D E F

Coiled-coil DBD SH3 SH2B C D

E F B C D E F

Coiled-coil DBD SH3 SH2 Coiled-coil DBD SH3 SH2

STAT5b-RARα

B C D E F

Coiled-coil

PRKAR1A-RARα

B C D E F

Coiled-coil

B C D E F B C D E F

Coiled-coil

PRKAR1A-RARα

B C D E F

FIP

FIP1L1-RARα

B C D E F

FIP

B C D E F B C D E F

FIP

FIP1L1-RARα

~0.5% of APL IRF2BP2 Exon 2

IRF2B2-RARα BCOR-RARα

?

  • ?
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SLIDE 22

Outline

  • Clinical & biological background
  • Monitoring of minimal residual disease
  • Diagnosis
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SLIDE 23

The PML/RARα 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
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SLIDE 24

Cheson et al. J Clin Oncol 2003

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SLIDE 25

PCR monitoring in APL: early studies

  • Patients treated with ATRA+CHT in long-term clinical remission

had consecutively negative RT-PCR tests

  • ATRA therapy alone did not induce molecular remission

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.

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SLIDE 26

Risk of relapse according to RT-PCR status

Post-consolidation time point

Diverio, et al. Blood 1998; Burnett et. Al, Blood 1999

RARα-PML o PML/RARα pos MRD neg MRD neg PML/RARα pos

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SLIDE 27

Salvage Therapy for Relapsed APL

Treatment in molecular vs hematologic relapse

Lo-Coco et al. Blood, 1999 Esteve et al. Leukemia, 2007

1 2 3 4 5 6 7 8

Years

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Probability

Hematological relapse (n=33; 24 events) Molecular failure (n=16; 5 events) p=0.008

1 2 3 4 5 6 7 8

Years

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Probability

Hematological relapse (n=33; 24 events) Molecular failure (n=16; 5 events) p=0.008

Molecular relapse Hematologic relapse

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SLIDE 28

Gabert et al., Leukemia 2003

Real-time Q-PCR for PML-RARA (EAC)

Gabert et al, Leukemia 2003

  • Sensitivity
  • Less prone to

contaminations

  • Reliable results in

low-quality samples

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SLIDE 29

Grimwade et al. JCO 2009

Median increment 1.1log/month

Kinetics of molecular/frank relapse in APL

Implications for optimal sampling time-points

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SLIDE 30

Sampling source: blood or marrow for MRD monitoring?

MR

∆ Post #1 #2 #3 #4 4 6 8 10 12 14 16 18 20 22 24 2 mo

0085 BM PB 0116 BM PB 0285 BM PB

26 28 30 32 34 36

0441 BM PB 0618 BM PB

MR MR HR MR PCR +ve PCR -ve MR Mol. relapse HR Hem. relapse

Adapted from Grimwade et al. JCO 2009

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SLIDE 31

Q-PCR in ATO-ATRA treated patients

Effective clearance of PML-RARA

Diagnosis Post Induction Post 3° cons

ATRA-CHT ATRA-ATO

Log-reduction PML/RARα Diagn-post ind P-value Post ind- post cons P-value ATRA-ATO 2.94 0.018 6.34 0.0024 ATRA-CHT 3.43 5.33

Similar clearance in ATO-ATRA and ATRA-CHT Low relapse % in ATO-ATRA pts questions cost- effectiveness of MRD monitoring

Cicconi et al. Leukemia 2016

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SLIDE 32
  • Use of standardized reference methods (Gabert et al

Leukemia 2003; Grimwade et al. J Clin Oncol 2009)

  • Bone marrow sampling more informative than PB
  • Send samples to reference labs experienced with molecular

testing of this rare disease

  • RQ-PCR advantageous over RT-PCR to better assess

sample quality and to investigate kinetics of MRD

Summary of Recommendations for Molecular Monitoring of APL

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SLIDE 33
  • M. Divona
  • C. Ciardi
  • A. Ferrantini
  • S. Lavorgna
  • T. Ottone
  • V. Alfonso
  • L. Iaccarino
  • G. Falconi
  • E. Fabiani

Acknowledgements

  • Prof. F. Lo Coco
  • Prof. MT Voso
  • Prof. W. Arcese
  • Prof. A Venditti
  • Prof. S. Amadori
  • M. Consalvo
  • P. Panetta
  • P. Curzi
  • D. Fraboni