Molecular Pathology of the Myeloproliferative Neoplasms Sudan MPN lecture to use.pdf · Molecular...
Transcript of Molecular Pathology of the Myeloproliferative Neoplasms Sudan MPN lecture to use.pdf · Molecular...
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Molecular Pathology of the
Myeloproliferative Neoplasms
XXIV International Academy of Pathology– Arab Division (IAPAD) Update on Molecular Pathology
Khartoum, Sudan – December 7, 2012
Adam Bagg, MD Director, Hematology Medical Director, Clinical Cancer Cytogenetics University of Pennsylvania
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Myeloproliferative neoplasms
Acute myeloid leukemias
Myelodysplastic syndromes
Myeloid neoplasms
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myeloid chronic myelogenous leukemia
erythroid polycythemia vera platelet essential thrombocythemia
2o fibroblast + primary myelofibrosis
transformed stem cell
• chronic eosinophilic leukemia/hypereosinophilic syndrome • mast cell neoplasms • chronic neutrophilic leukemia • unclassifiable
Myeloproliferative neoplasms
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Chronic myelogenous leukemia
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Chronic myelogenous leukemia
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The Philadelphia story
Goldman J, N Engl J Med 2001, 344:1084-1086. Copyright © 2001,
Massachusetts Medical Society. All rights reserved.
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Molecular testing in CML
Diagnosis
Monitoring
Resistance
Diagnosis
Monitoring
Resistance
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CML diagnosis: t(9;22) and BCR-ABL1
? CML cytogenetics
~95% +
CML
? t(9;22)
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? CML
~95% +
CML
cytogenetics
~5% -
? t(9;22)
CML diagnosis: t(9;22) and BCR-ABL1
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? CML
~95% +
CML
cytogenetics
CML
~5% -
molecular genetics
? t(9;22)
? BCR-ABL1 ~2.5% +
CML diagnosis: t(9;22) and BCR-ABL1
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? CML cytogenetics
CML ~2.5% - ~2.5% + CML
Avoid term “Ph-neg” CML
? CMML ? aCML
? BCR-ABL1
~95% +
CML
~5% -
molecular genetics
? t(9;22)
CML diagnosis: t(9;22) and BCR-ABL1
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? CML cytogenetics
CML ~2.5% + ? BCR-ABL1
~95% +
CML
~5% -
molecular genetics
? t(9;22)
? molecular genetics
YES! molecular target for: 1] Rx 2] MRD
CML diagnosis: t(9;22) and BCR-ABL1
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? CML cytogenetics
CML ~2.5% +
? molecular genetics
YES! molecular target for: 1] Rx 2] MRD
? cytogenetics
YES! - clonal “evolution” [ACAs]
[Ph+; Ph- with imatinib] ? BCR-ABL1
~95% +
CML
~5% -
molecular genetics
? t(9;22)
CML diagnosis: t(9;22) and BCR-ABL1
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What’s going on at the DNA level …
not really!
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Molecular testing in CML
Diagnosis
Monitoring
Resistance
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CML monitoring
Two major forms of therapy …
TKI
SCT
• Initial therapy of choice • Does not eradicate/cure CML • ? Long-term outcome • Minimal toxicity1
1 cytopenias (~40%), cardiotoxicity, ? mutagenicity [inhibit eph tumor suppressor, Ph (-) clones] 2 indicated when [i] very young, [ii] TKI failure, [iii] AP and BC 3 10-yr survival ~65% 4 10-20% mortality even when low risk
• No longer 1st line Rx2
• Only Rx that cures CML3
• Major toxicity and mortality4
BCR-ABL1 reduction
BCR-ABL1 negativity
Rx goal
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CML monitoring: definitions of response
complete hematologic
• platelet: < 450 • WBC: < 10 • diff: no immature granulocytes
• basos: < 5% • clinical: non-palpable spleen
cytogenetic # Ph+
• none: >95% • minimal: 66-95% • minor: 36-65% • partial: 1-35% • complete: 0%
molecular
• next slide please …
{ major
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diagnosis
complete hematologic remission
complete cytogenetic remission
major molecular response
“complete molecular remission” undetectable transcript
1012
<1010
<109
<106
100
<0.1
<0.0001
responses BCR-ABL1
ratio
… yet overall survival ~95%
imatinib responses
~98%
~85%
~75%
~10%
@ 5 years
log reduction
<1011
>3
40% @ 12mo 55% @ 24mo 75% @ 44mo
~25% with 800mg
# cells
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Molecular testing in CML
Diagnosis
Monitoring
Resistance
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Resistance to imatinib (and other TKIs …)
primary • failure to achieve an initial response
• seen in ~5% of pts
• bioavailability; not due to mutations
secondary • loss of response, occurs at ~1-4%/year
• ~10-15% of pts [in chronic phase, no prior Rx]
• more common if:
- prior interferon Rx
- accelerated phase [~75%]
- blast crisis [~95%]
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Mechanisms of resistance to imatinib
BCR-ABL1 independent
BCR-ABL1 dependent
1. Kinase domain mutations:
- most common cause of resistance [~40-90%]
- spans ~240 aa’s
2. BCR-ABL1 amplification:
- genomic > transcriptional [~10%]
3. Clonal evolution:
- other genetic/cellular pathways [LYN]
4. ↓Bioavailability:
- absorption
- metabolism [hepatic]
- plasma binding [a1acid glycoprotein sequestration]
- ↓influx ↑efflux [MDR1, PGP, BCRP2/ABCG2, hOCT1, MRP1]
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Krause DS, N Engl J Med 2005, 353:172-187. Copyright ©
2005, Massachusetts Medical Society. All rights reserved.
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Kinase domain mutations
P = P loop
- ATP-binding site
- ? worst mutations
B = Binding domain
- where imatinib binds
C = Catalytic domain
A = Activation loop
- conformation altered
- affects imatinib binding
- closed: inactive
- open: active
2-10% of patients
>10% of patients
green
red
> 100 different mutations
these 6 account for > ~65% of all mutations
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Kinase domain mutations
• most common cause of resistance
- 10% [early CP] 30% [late CP] 60% [AP] 90% [BC]
• not induced by Rx
- selected for by Rx [cf antibiotic resistance in bacteria]
• ? BCR-ABL1 induced
- via ROS [escape inhibition by self-mutation; ? add antioxidants to Rx]
• not all are created equally
- initially: all bad … currently: P-loop worst [not all agree]
- can overcome most with dose-escalation or other TKI’s [T315I exception]
- does not necessarily have a proliferative advantage
- presence does not always account for resistance
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Indication for mutation testing
treatment failure/ suboptimal response
loss of response
anyone in AP or BC
• no CHR by 3 months • no MCR by 6 months • no CCR by 12 months
• hematologic relapse • cytogenetic relapse • ↑ing BCR-ABL1 levels [5x]
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Genetic testing in CML: summary
Diagnosis
Monitoring
Resistance
CC BM ? PB FISH instead
RT-PCR PB qualitative vs quantitative with characterization
CC BM 3-6 monthly until CCR
6-12 monthly thereafter
FISH PB before achieve CCR
RQ-PCR PB 3 monthly
direct
sequencing
PB v
BM
Rx failure, loss of response, accelerated & blast phase
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Molecular and other testing in non-CML MPNs
karyotype genes PRV1 EEC mpl
megas GATA1
megas
circ
CD34+
PV 9p+, +8+9 ? + + + ? -
ET ? ? + [50%]
+ [50%]
+/- ? -
PMF del(13q14) ? - - +/- + +
PPMF 1q+ ? ? ? ? ? ?
pre-2005 …
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Molecular and other testing in non-CML MPNs
karyotype genes PRV1 EEC mpl
megas GATA1
megas
circ
CD34+
PV 9p+, +8+9 ? + + + ? -
ET ? ? + [50%]
+ [50%]
+/- ? -
PMF del(13q14) ? - - +/- + +
PPMF 1q+ ? ? ? ? ? ?
JAK2 V617F mutation (etc)
post-2005 …
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JAK2 is out of the box …
• Just Another Kinase - one of many cloned at the time (1989)
• Janus Kinase - two-headed Roman god of gates and
passages
• non-receptor tyrosine kinase (TK)
• has 2 TK domains (hence the name) - most TKs have only 1
• 4 members of JAK family - JAK1, JAK2, JAK3 and TYK2
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JAK-STAT pathway
Reproduced from Steensma , DP J Mol Diagn 2006, 8:397-411
with permission from the American Society for Investigative Pathology
and the Association for Molecular Pathology.
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JAK2 structure, function, mutation
mediates binding to receptor
autoinhibitory activity
*V617F*
*Mutation*
~50% ET ~50% PMF ~95% PV
phosphorylated after ligand
binding
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JAK2: wild-type and mutant
Campbell P, N Engl J Med 2006, 355:2452-2466. Copyright © 2006, Massachusetts Medical Society. All rights reserved.
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Eosinophilic neoplasms and mastocytosis
• myeloid & lymphoid neoplasms with eosinophilia & abnormalities of:
** PDGFRA (4q12): - most often cryptic FIP1LI-PDGFRA fusion on 4q12 [CHIC2 deletion]
- responds to imatinib
- rare variant translocations
** PDGFRB (5q31~33) - most often t(5;12), fused to ETV6
- typically CMML responds to imatinib
- numerous variant translocations
** FGFR1 (8p11) - most often t(8;13), fused to ZNF198 - typically associated AML and/or T-LBL - numerous variant translocations
- ~95% KIT mutation - D816V - imatinib resistant
Systemic mastocytosis
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Any questions …
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