Myeloproliferative Neoplasms: Diagnosis and Molecular Monitoring in the Era of Target Therapy

Transcription

1 Myeloproliferative Neoplasms: Diagnosis and Molecular Monitoring in the Era of Target Therapy C. Cameron Yin, M.D., Ph.D. Department of Hematopathology UT MD Anderson Cancer Center

2 Disclosures I do not have anything to disclose.

3 Overview Chronic myeloid leukemia, BCR-ABL1-positive JAK2/MPL/CALR-mutated myeloproliferative neoplasms Polycythemia vera Essential thrombocythemia Primary myelofibrosis Chronic neutrophilic leukemia and CSF3R mutation Chronic eosinophilic leukemia, NOS Myeloproliferative neoplasm, unclassifiable Mastocytosis and KIT mutation Myeloid/lymphoid neoplasms with eosinophilia and rearrangements of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2

4 An indolent clinical course Shared Features of MPNs Roughly equal gender predilection with a wide age range at presentation 1 hematopoietic lineage proliferation with minimal defect in maturation A somatic genetic change that activates a growth-promoting signaling pathway (e.g. BCR-ABL1, JAK2, MPL, CALR, CSF3R) Stepwise progression through chronic, accelerated and blast phases due to accumulation of shared genetic/epigenetic changes Disruption of bone marrow microenvironment leading to ineffective hematopoiesis and progressive myelofibrosis, myelodysplasia, or secondary leukemia

5 Chronic Myeloid Leukemia, BCR-ABL1-positive Accounts for 15-20% of adult leukemia Philadelphia chromosome t(9;22)(q34;q11)/bcr-abl1 constitutive tyrosine kinase activity essential for CML pathogenesis Additional chromosomal abnormalities during evolution from CP to AP/BP European LeukemiaNet (Blood 2013) Major route (>10%): +8, extra Ph, i(17)(q10), +19 Wang et al. (Blood 2016) Good px: +8, -Y, extra Ph Poor px: i(17)(q10), -7/del7q, 3q26

6 CML Chronic Phase

7 CML - Accelerated Phase WBC > 10 (x10 9 /L) Splenomegaly Platelet > 1,000 or < 100 (x10 9 /L) Basophils 20% in PB or BM Blasts 10-19% in PB or BM Additional clonal chromosomal abnormality in Ph+ cells at dx Major route (+8, extra Ph, i17q10, +19) Complex karyotype 3q26.2 abnormalities Any new clonal chromosomal abnormality in Ph+ cells occur during tx Provisional: response-to-tki Hematologic resistance to 1 st TKI Any hematologic, cytogenetic, molecular indication of resistance to 2 TKIs Occurrence of 2 mutations in ABL1 during TKI tx

8 CML Blast Phase Blasts 20% in PB or BM Extramedullary accumulation of blasts

9 Conventional Cytogenetics Karyotyping Detect additional chromosomal abnormalities Need metaphases Tedious ~95% of cases - t(9;22)(q34;q11) ~5% of cases - normal karyotype Cryptic translocation detected by FISH or RT-PCR

10 FISH Confirm BCR-ABL1 Interphase or metaphase PB or BM Touch prep or paraffin-embedded tissues Detect cytogenetically silent BCR- ABL1 rearrangement Fail to detect clonal evolution Vardiman, WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008

11 Molecular Testing BCR ABL1 e m-bcr M-bcr m-bcr a1 a2 a3 a4 a5 a6 a11 a12 Breakpoint region e1 b2 a2 e1 b2 b3 a2 e1 a2 e1 e19 a2 b2a2-p210 b3a2-p210 e1a2-p190 e19a2-p230

12 Establish the presence of BCR- ABL1 fusion Identify the type of fusion transcript which can be used for subsequent monitoring b3a2 or coexp a/w earlier and deeper molecular response and longer EFS (Jain et al. Blood, 2016) BM and PB are comparable but not interchangeable Molecular Testing Quantitative real-time RT-PCR TaqMan Fluorescence Resonance Energy Transfer (FRET) TaqMan - Single tube RT real-time PCR covering b2a2, b3a2 and e1a2 transcripts

13 Alternate BCR/ABL1 Transcripts Qualitative Multiplex PCR/Genescan b2a2 e19a2 e1a2 e6a2 b3a2 b3a3 b2a3 e1a3 ABL1

14

15 Molecular Monitoring Algorithm for CML Karyotypic analysis (sensitivity 5%) Pretreatment, every 3-6 months till CCyR and once a year thereafter FISH (sensitivity 0.5%) Pretreatment, every 6-12 months till achievement of CCyR Quantitative real-time RT-PCR (sensitivity 1/100,000) Pretreatment, every 3 months till achievement of MMR and every 6 months thereafter Alternate RT-PCR (for rare types of fusion transcripts, sensitivity 1/100,000) When an alternate transcript is suspected Rising level of bcr-abl1 fusion transcripts mandates more frequent monitoring

16 Tyrosine Kinase Inhibitors 1-G TKI: imatinib (STI571, Gleevec) Inhibitor for BCR-ABL1, PDGFR and KIT Competitively inhibiting ATP binding Most early-stage diseases achieve durable hematological and cytogenetic remission Responses are less stable in patients With advanced CML (primary resistance) After prolonged therapy (secondary resistance) 2-G TKI: dasatinib, nilotinib ATP P P P BCR-ABL P BCR-ABL 3-G TKI: ponatinib, bosutinib Gleevec

17 Mechanisms of TKI Resistance BCR-ABL1-dependent ABL1 kinase domain mutations ABL1 duplication/amplification Complex rearrangements of BCR-ABL1 transcripts resulting in altered BCR-ABL1 proteins BCR-ABL1-independent Clonal cytogenetic evolution Overexpression of other kinases (Src, Lyn) NFkB activation Drug inactivation by binding to plasma proteins or efflux Stem cell self-renewal

18 ABL1 Kinase Domain Mutations Most common cause of TKI resistance 40-90% of cases with resistance Incidence increases with stage activation/ catalytic domain imatinib P-loop ~100 point mutations Span >700bp in coding region Affect amino acids common mutations comprise ~85% of all mutations

19 ABL1 Mutation Analysis When and Who? Treatment naïve patients Restricted to AP or BP Patients on TKI therapy Inadequate initial responses Loss of previously attained responses Disease progression (AP or BP) Follow-up at 6-weeks, and then at 6-months after finding an ABL1 mutation

20 ABL1 Mutation Analysis How? Methods Sanger sequencing (sensitivity 20%) Current method of choice Pyrosequencing (sensitivity 1-5%) Emerging quantitative tool ASO-PCR (sensitivity 0.1%) Mainly a research tool NGS multiplex assay (sensitivity 5-10%) Also identify other gene mutations

21 Sanger Sequencing 1st round PCR amplification of BCR/ABL1 2nd round PCR amplification of ABL1 kinase domain (exon 2-10) Sequencing of codons T315I (ACT>ATT)

22 Pyrosequencing Short-length DNA: 10-30bp Quantitative High throughput 96-well plate Rapid 30min For defined mutations

23 ABL1 Y253H (TAC to CAC) A. Y253H TAC to CAC T=212.40, C=113.25, mutant/wild type=1.0:1.9 B. K562 wild type control

24 Mutated sequence T T C A A G Allelic discrimination by quantitative real-time PCR Wild type sequence G T C C A G

25 JAK2-mutated MPNs PV, ET and PMF A spectrum of disorders with a unitary molecular origin Cultured marrow from all three of these neoplasms is hypersensitive to stimulation by exogenous growth factors that bind to JAK-STAT-linked cytokine receptors, including EPO, TPO, GM-CSF and G-CSF Many of the presenting signs, symptoms, and laboratory findings are overlapping ET has been reported to evolve into PV, and both PV and ET can progress to MF Respond to common treatments

26 JAK-STAT Signaling Role in normal hematopoiesis, inflammation and immune function JAK2 cytoplasmic PTK that binds to cytokine receptors GM-CSF, G- CSF, EPO and TPO MPL Thrombopoietin receptor CALR Calreticulin Savona et al. Leuk Res 2014

27 Phenotypic Driver Mutations in MPNs PV ET PMF JAK2 V617F JAK2 exon 12 Others JAK2 V617F MPL W515X CALR TN JAK2 V617F CALR Genes % JAK2 V617F 95 JAK2 exon 12 4 Others 1 Genes % JAK2 V617F CALR MPL W515X 3-5 TN Genes % JAK2 V617F CALR MPL W515X 5-8 TN 5-15

28 JAK2 V617F (GTC to TTC, exon 14) JAK2 V617F positive Can be detected by pyrosequencing or ASO-PCR Seen in ~95% PV and 50-60% of ET and PMF Also seen in CMML, RARS-t, AML/MDS, etc JAK2 V617F negative

29 Detection of JAK2 V617F by ASO-PCR JAK2 Mutant JAK2 Wild-Type Positive Negative

30 MPL Mutation Thrombopoietin receptor Codon 515 (exon 10) W515K/L ~5% ET and PMF Detection: Pyrosequencing MPL wild type (TGG) MPL W515L (TGG to TTG) MPL W515K (TGG to AAG)

31 CALR Mutation CALR wild type Cause STAT5 activation and cytokineindep growth Seen in JAK2/MPL-negative ET/PMF Also seen in RARS-t, MDS, CMML, acml, CNL, etc Type I CALR mutation (52bp del) Frameshift mutations in exon 9 Type I: L367fs*46 (52bp del) Type II: K385fs*47 (5bp ins) A/w younger age, lower Hb and WBC, higher platelet, lower risk of thrombosis (ET) and better OS Type II CALR mutation (5bp ins) Detection PCR/CE screening (exon 9) Sanger confirmation (new cases)

32 How Does a Single JAK2 Mutation Give Rise to Different Phenotypes Different stem cell stage at which the JAK2 mutation occurs Different host genetic background Complementing genetic events Dosage effect: heterozygous vs homozygous Homozygous mutations seen in up to 70% of PV Homozygous mutations are almost never seen in ET Scott et al. Blood 2006

33 Functional Classification of Genes Mutated in MPN Signal transduction Driver: JAK2, MPL, CALR, SCF3R Others: CBL, NRAS, KRAS, FLT3, NF1 DNA methylation TET2, DNMT3A, IDH1, IDH2 Histone modification ASXL1, EZH2 Transcription factors RUNX1, TP53, CEBPA, ETV6, SETBP1 RNA splicing U2AF1, SF3B1, SRSF2, ZRSF2

34 Confounding Effect of Age-Related Clonal Hematopoiesis of Indeterminate Potential (CHIP) Some genes are frequently mutated in apparently healthy persons Most common: DNMT3A, TET2, ASXL1 Increased frequency as people age 10% of persons >65 vs 1% of persons <50 Increased risk of developing hematopoietic neoplasms 10-15x increased risk Estimated risk of 0.5-1% per year May represent early events in development of hematopoietic neoplasms High frequency of CHIP in the elderly limits the diagnostic value of somatic mutations Certain mutations, in appropriate clinical settings, helpful diagnostic adjuncts

35 Case 1 41 y/o man Thrombocytosis and mild splenomegaly Outside dx of ET Came for a second opinion WBC 7.8, Hb 16.3, platelet 504 LDH 454 IU/L (ref range, ) EPO 1.4 miu/ml (ref range, ) JAK2 V617F detected

36 A B C D

37 2016 Revised WHO Diagnostic Criteria for PV Major criteria Hgb >16.5 g/dl (men) or 16 g/dl (women), or Hct >49% (men) or 48% (women), or other evidence of increased RBC mass (>25% mean normal predicted value) Hypercellular BM with panmyelosis and pleomorphic mega JAK2 V617F or exon 12 mutation Minor criteria Subnormal serum erythropoietin level Diagnosis requires all 3 major or first 2 major plus minor

38 Case 2 58 y/o woman Found to have thrombocytosis incidentally Complained headache, finger and toe tingling and fatigue Outside dx of possible ET Came for a second opinion WBC 7.9, Hb 13.2, platelet 710 LDH 408 IU/L (ref range, ) EPO 3.0 miu/ml (ref range, ) JAK2 V617F 5%

39 A B C D

40 2016 Revised WHO Diagnostic Criteria for ET Major Sustained PB platelets 450x10 9 /L BM bx Minor Proliferation of mainly mega lineage with increased large mature megakaryocytes with hyperlobated nuclei No significant increase or left-shift of granulopoiesis or erythropoiesis No or rare minor (grade 1) reticulin fibers Not meeting WHO criteria for CML, PV, PMF, MDS or other myeloid neoplasm JAK2/MPL/CALR mutation Presence of a clonal marker or absence of reactive causes Diagnosis requires all 4 major or first 3 major plus minor

41 Case 3 51 y/o man Thrombocytosis found on PE, persistent and increasing Mild splenomegaly Outside dx of CALR-mutated ET Came for a second opinion WBC 10.8, Hb 13.9, platelet 957 LDH 1101 IU/L (ref range, ) EPO 19.4 miu/ml (ref range, ) CALR type I mutation detected

42 A B C D

43 2016 Revised WHO Diagnostic Criteria for PrePMF Major criteria Atypical mega hyperplasia without reticulin fibrosis > grade 1, with BM hypercellularity, granulocytic hyperplasia, often erythroid hypoplasia Not meeting WHO criteria for CML, PV, ET, MDS, or other myeloid neoplasm JAK2/MPL/CALR mutation, or other clonal marker, or exclusion of reactive causes of BM fibrosis Minor criteria (confirmed in 2 consecutive determinations) Anemia not attributed to a comorbid condition Leukocytosis 11x10 9 /L Palpable splenomegaly Increased serum LDH Diagnosis requires all 3 major and 1 minor

44 2016 Revised WHO Diagnostic Criteria for overt PMF Major criteria Atypical mega hyperplasia with reticulin and/or collagen fibrosis (grade 2 or 3) Not meeting WHO criteria for CML, PV, ET, MDS, or other myeloid neoplasm JAK2/MPL/CALR mutation, or other clonal marker, or exclusion of reactive causes of BM fibrosis Minor criteria (confirmed in 2 consecutive determinations) Anemia not attributed to a comorbid condition Leukocytosis 11x10 9 /L Palpable splenomegaly Increased serum LDH Leukoerythroblastosis Diagnosis requires all 3 major and 1 minor

45 Comparison of Demographic and Clinical Features of PV, ET and PMF Type Median age (years) Gender Incidence per 100,000 Median survival (years) Progression to MF at 10 years Incidence of AML at 10 years Comments PV ~70 M F 2.2 > % ~5% ET ~55 M = F 2.3 > % 1-2% Variants presenting in younger women PMF ~60 M = F 1.0 ~4 na 5-30%

46 Comparison of Bone Marrow Findings in PV, ET and PMF Feature PV ET PrePMF Overt PMF Cellularity Increased Normal or mildly Increased Decreased increased Reticulin fibrosis Minimal Minimal Minimal Marked Osteosclerosis Minimal Minimal Minimal Marked Megakaryocyte number Megakaryocyte morphology Increased, loose clusters Pleomorphic, minimal atypia Increased, scattered Large, hyperlobated Increased, dense clusters, adjacent to sinuses or bone trabeculae Markedly atypical (hyperchromatic, staghorn nuclei, cloud-like chromatin, bare nuclei) Granulocytes Increased Normal Increased Decreased Erythrocytes Increased Normal Decreased Decreased Iron storage Depleted Present Present Present

47 A B A Polycythemia vera B Essential thrombocythemia C Primary myelofibrosis C C

48 EUMNET Bone Marrow Fibrosis Grading MF-0 (normal) MF-1 MF-2 MF-3 Scattered linear reticulin with no intersections Loose network of reticulin with many intersections, especially perivascular Diffuse and dense increase in reticulin with extensive intersections, +/- focal collagen/osteosclerosis Diffuse and dense increase in reticulin with extensive intersections and coarse collagen bundles and/or osteosclerosis Barosi et al. Blood 2005;106:

49 MF-1 MF-2 MF-3

50 Molecular Genetic Features of PV, ET and PMF Type % JAK2 V617F % JAK2 exon 12 mutation % MPL W515L/K % CALR Chromosomal changes PV del(20q), +8, +9, del(13q) (20%) ET del(20q), +8, +9 (10%) PMF del(20q), +8, +9, del(13q), +1 (40%)

51 2016 Revised WHO Diagnostic Criteria for CNL PB WBC 25 x 10 9 /L Segmented neutrophils plus bands 80% of WBC Neutrophil precursors (promyelo, myelo, metamyelo) < 10% of WBC Myeloblasts rarely observed Monocytes < 1 x 10 9 /L No dysgranulopoiesis Hypercellular BM Neutrophil granulocytes increased in percentage and number Neutrophil maturation appears normal Myeloblasts < 5% Not meeting CML, PV, ET or PMF No rearrangement of PDGFRA, PDGFRB, FGFR1, or PCM1-JAK2 Presence of CSF3R T618I or other activating CSF3R mutation Or persistent neutrophilia ( 3m), splenomegaly w/o identifiable reactive causes Or demonstration of clonality of myeloid cells by molecular cytogenetic studies

52 CSF3R Mutation in CNL and acml Controls the production, differentiation and function of granulocytes Mutations seen in 50-60% of CNL and acml Truncation (cytoplasmic tail) activate SRC-TNK2 pathway sensitive to SRC inhibitor (dasatinib) Point mutation (membrane proximal) activate JAK pathway sensitive to JAK inhibitor (ruxolitinib) Detection Sanger sequencing Pyrosequencing: T618I (exon 14) CSF3R wild type CSF3R T618I (ACC to ATC)

53 Chronic Eosinophilic Leukemia, NOS Eosinophils 1.5 x 10 9 /L Not meeting CML, PV, ET or PMF, MDS/MPN No PDGFRA/PDGFRB/FGFR1 rearrangement Blast count <20% Clonal cytogenetic or molecular genetic abnormality, or blast >2% (PB)/5% (BM)

54 Myeloproliferative Neoplasm, Unclassifiable Fail to meet specific subtype criteria Features overlap two or more subtypes Three settings Early disease w/o well-developed characteristic features Late disease with severe fibrosis, osteoslerosis and/or increased blasts Co-existing neoplastic or inflammatory disorder obscuring the underlying disease

55 new MPN + BCR/ABL CML Ph-negative MPN JAK2 V617F PV ET PMF suspicious for PV JAK2 exon 12 mutation suspicious for ET or PMF MPL W515L/K and CALR PV other MPN ET or PMF CSF3R, FIP1L1-PDGFRA FISH for PDGFRA/PDGFRB/FGFR1 Other non-driver mutations

56 Molecular Testing in MPN at MDACC Quantitative real-time PCR for BCR-ABL1 NGS (81-gene) Additional eosinophilia work-up FIP1L1-PDGFRA (RT-PCR) FISH for PDGFRA, PDGFRB and FGFR1

57 Thank you! C. Cameron Yin, MD, PhD