Molecular Diagnostics of Myeloid and Lymphoid Neoplasms Molecular Pathology: Principles in Clinical Practice - 2012 John Greg Howe Ph.D. Department of Laboratory Medicine Yale University School of Medicine May 8, 2012
Outline General Neoplastic Hematopathology Clonality - Gene Rearrangement Chronic Lymphocytic Leukemia- Somatic Hypermutation Myeloproliferative diseases- JAK2- Additional mutations Acute Myeloid Leukemia and Acute Lymphocytic Leukemia- AML and ALL translocation screen Acute Promyelocytic Leukemia- RT-PCR of t(15;17) translocation; therapy Normal Karyotype Acute Myeloid Leukemia FLT3 and NPM1 testing - testing algorithm for normal karyotype AML
Oncogene (2005) 24, 5676 5692 Hierarchy of Hematopoietic Cells
Harrison s Manual of Medicine 2008
Molecular Classification of Tumors WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues Swerdlow, S.H., Campo, E., Harris, N.L., Jaffe, E.S., Pileri, S.A., Stein, H., Thiele, J. 2008 Edition Although the French-American-British morphologic classification of myeloid neoplasms has been accepted for many years (1976), the discovery of a number of genetic lesions that predict clinical behavior and outcome better than morphology alone necessitates the incorporation of genetic information into the classification scheme. (2001 Edition) 1. 2001 edition changed classifications due to translocations [i.e. t(15;17)] translocation in promyleocytic leukemia] 2. 2008 edition- changed classifications due to point mutations (i.e. AML with mutation of CEBPA)
Neoplastic Hematopathology Molecular Diagnostic Testing 1. Molecular diagnostics laboratory provides information about the molecular basis of patient s disease. 2. This information is used in combination with the patient s history and symptoms, other clinical laboratory results, histopathological findings, and other diagnostic information. a. Diagnosis Identifying hematopathology disease based on the presence of specific gene mutations (i.e. translocations and JAK2). b. Prognosis Prediction of likely patient outcomes based on presence of a specific gene mutation. (i.e. translocations). c. Therapy Selection Prediction of response to specific therapies based on presence of gene mutation. (i.e. BCR-ABL). d. Therapy Outcome Monitoring by testing for minimal residual disease. (i.e. BCR-ABL).
Clinical Neoplastic Hematopathology Tests Chronic myeloproliferative diseases Chronic myeloid leukemia- BCR-ABL (9;22) (Quant RT-PCR); Kinase (sequencing) Polycythemia vera- Jak2 (V617F) (Quant); JAK2 exon 12, 13 mutations Essential thrombocytemia- MPL mutation analysis Eosinophilia- FiP1L1-PDGRFA; PDGRFB and FGRF1 translocations Systemic mastocytosis- c-kit (D816V) (Quant) Myelodysplastic/myeloproliferative diseases Myelodysplastic syndromes Acute myeloid leukemia Translocation screen (15;17) (Quant), (8;21) (Quant), inv16, (9;22) Normal karyotype- FLT3 and NPM1 mutations Precursor B-and T- neoplasms (Acute lymphoblastic leukemia) Translocation screen (12;21), (4;11), (1;19), (9;22) (FISH or RT-PCR) B and T cell gene rearrangement Mature B-cell neoplasms B cell gene rearrangement Chronic lymphocytic leukemia- IgV hypermutation (Sequencing) Mantle cell leukemia- (11;14) bcl-1 diagnostics Follicular lymphoma- (14;18) bcl-2 (Quant) Mature T-cell and NK- cell neoplasms T cell gene rearrangement; EBV (in situ hybridization) Hodgkin lymphoma- Epstein Barr Virus (EBV) Immunodeficiency associated lymphoproliferative disorders Epstein Barr Virus (Quant) Histiocytic and dendritic cell neoplasms
Monoclonal Cell Population Determination (Clonality) Definition: Population of cells with similar characteristics that are all derived from a single precursor cell. Morphology: Monomorphous cell population Immunopathology: Monotypic SIg (light chain kappa and lambda). ~1/3 of B-cell non- Hodgkin s neoplasms do not have surface antibody. T-cells have no equivalent of light chains. Cytogenetics: Recurrent chromosomal alteration (e.g., translocation). Molecular genetics: Clonal B- or T-cell gene rearrangements (Southern or PCR). http://www.bioweb.uncc.edu/1110lab/notes/notes1/labpics/lab6pics.htm /www.cytologyparis2005.com/pdf/ss/ss07_veronika_kloboves-prevodni_case_2
IgH and TCR Gene Rearrangements VH VH primer JH primer RNA Splicing V DJ C Leukemia (2003) 17:2257-317 Leukemia. (2007) 21:201-6
Immunoglobulin Gene Rearrangement Southern Blot and PCR 1. Southern blot is the gold standard, but is labor intensive, long turnaround time and requires high quality DNA. 2. PCR method is gradually becoming the standard however its sensitivity (60%) is significantly lower compared to the Southern (95%) method. The sensitivity of PCR can be increased with additional reactions. Enzyme Southern Control Patient E B H E B H 10% 1 2 3 4 5 6 7 FR3/JH Primers PCR FR2/JH Primers 10% 1% N W A B C M 10% N W A B C - 23kb - 9.4kb 1 2 3 4 5 6 7 8 9 10 11 12 13 14-2.3kb
Biomed-2 Gene Rearrangement and Capillary Electrophoresis Instead of using gel electrophoresis use capillary electrophoresis on a DNA sequencing instrument. Large number of primers are used. 123bp Leukemia (2003) 17, 2257 2317 bp Leukemia. 17:2257-317, 2003
Gene Rearrangement (Biomed-2) Leukemia. (2007) 21:201-6 Complementarity of Ig for clonality detection in B- cell malignancies (% clonality) Complementarity of TCR for clonality detection in T- cell malgnancies (% clonality)
Biomed-2- Problems 1. Although the number of labs using Biomed-2 has increased, there are several issues with their use. Too many reactions. Fourteen reactions (8 for IgH/K/L and 6 TCR) Too expensive Confusion as to what constitutes a positive Patent and RUO status 2. By using the in-house and BIOMED-2 TCRG reactions with a total of four tubes, clonal TCR-GR the reagent cost were one-third of that for the six BIOMED-2 reactions and the detection rate was also higher than the latter alone (91% vs 85%). J Clin Pathol (2011);64:536-542 Euroclonality consortium Dec 2009
Neoplastic Hematopathology Commercial Molecular Kits/Probes Used in Clinical Labs FDA approved Abbott, B-Cell Chronic Lymphocytic Leukemia (B-CLL), CEP12 DNA Probe pharmdx Kit, FISH on FFPE Abbott, Chromosome 8 Enumeration (CML, AML, MPD, MDS), CEP8 DNA Probe Kit, FISH ASR Abbott, Numerous translocations probes, FISH DakoCytomation, Numerous translocation probes, FISH Asuragen, Leukemia translocation Panel, RT-PCR RUO Gene rearrangement InVivoScribe (CE), Patent JAK2 InVivoScribe, Ipsogen, Invitrogen, Patent BCR/ABL Quantitiative Roche, Ipsogen (CE), Cepheid FLT3 InVivoScribe, Patent BCL2 Roche, InVivoScribe (CE) Various translocations Ipsogen (some CE), Hemovision Somatic hypermutation InVivoScribe NPM1 Ipsogen, Asuragen, Patent
B-Cell Differentiation Immunoglobulin Gene Rearrangement Nature Rev Immuno. (2003) 3: 801-812
Chronic Lymphocytic Leukemia (CLL) Somatic Hypermutation Heterogeneous prognosis differences based on chromosome abnormalities, IGH somatic mutations, ZAP-70 expression, and CD38 expression. IgVH Hypermutation Percentage vs. Survival <97% homology Patients with unmutated IgVH genes have a median survival of 8 years, those with mutated IgVH genes (>3%) a median survival of 25 years. The mutational status of the IgVH genes segregates CLL patients into more benign and more malignant subsets. 100%.homology Months between 99.9% and 97.9% Therapy: Aggressive-Early and frequent during the clinical course Indolent-None or late need during the clinical course British J Haematology, (2007) 140, 320 323
CLL Somatic Hypermutation Test 1. Direct Sequencing Direct sequencing works best for samples with no background amplification and one clonal product. 2. Gel Extraction Gel extraction is used with weak clonal bands in a polyclonal background, o more than one clonal band. 3. Cloning and Sequencing Clone the band extracted PCR product and sequenced. InVivoScribe
IGHV3-11*03 IGHV3-h*01 IGHV3-h*02 IGHV3-21*01 CLL Somatic Hypermutation Patient Using Mix 2 Analyzed by the IMGT, International ImMunoGeneTics information system IgH Rearranged Sequence IGHV3-11*03 Identity= 90.95% (221/243nt)
Myeloproliferative Disorders (MPD) The myeloproliferative disorders are a heterogeneous group of diseases characterized by excessive production of blood cells by myeloid precursors in the bone marrow. Thrombotic and bleeding complications, leukemic transformation can occur. Myeloproliferative disorders encompass the following entities Classic Atypical Chronic Myeloid Leukemia (CML) Polycythemia Vera (PV) Primary Myelofibrosis (PMF) Essential Thrombocythemia (ET) Chronic Eosinophilic Leukemia (CEL), not classified Hypereosinophilic Syndrome (HES) Systemic Mastocytosis (SM) Myeloproliferative Neoplasms, unclassifiable
Classification and Molecular Pathogenesis of the MPD Nature Reviews Cancer (2007) 7; 673
JAK2 Mutation and Myeloproliferative Diseases Polycythemia vera arises in a hematopoietic stem cell and increases red blood cell production that is independent of erythropoietin control. In 2005, four research groups discovered a mutation, Val617Phe, in the JAK2 gene is associated with certain chronic myeloproliferative diseases. JAK2 is a cytoplasmic tyrosine kinase and functions in signal transduction the erythropoietin receptor and others. Binding of erythropoietin leads to phosphorylation of JAK2 and leads to phosphorylation of signal transducers (Stat). Erythropoietin Receptor Thrombopoietin Receptor Nature Reviews Cancer (2007) 7; 673
Marker Positive control Negative control Water Frequency of JAK2 V617 Mutation in Myeloproliferative Disorders V617F J2 J1 Diagnosis Frequency of JAK2 Mutation (%) Frequency of Homozygos ity (%) Polycythemia vera (PV) >95 24-27 Essential Thrombocythemia (ET) ~50 2-4 Primary Myelofibrosis (PMF) >50 6-18 Chronic Myeloid Leukemia (CML) Rare Not Done Atypical Chronic Myeloid <20 Not Done Leukemia Human Pathology (2008) 39, 795 810 Assay can detect 1% V617F containing cells in the background of normal cells. Control Mutant
Additional Mutations in PV, ET and PMF Patients JAK2 exon12 mutations in Polycythemia Vera (PV) Additional mutations of JAK2 exon 12 have been found in JAK2 (V617F) negative patients with polycythemia vera. Exon 12 mutations can activate pathways associated with erythropoietin signaling. Other mutations in Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) 1-5% in JAK2(-) ET and PMF patients have W515L and K mutations in the thrombopoietin receptor (TPO-R, MPL). Results in the constitutive activation of JAK-STAT signaling and cytokine-independent proliferation of hematopoietic cells. Leukemia (2008) 22, 1299 1307
Quantitation of JAK2 V617F Allele DNA Assessment of V617F allele burden as a prognosis indicator V617F allele burden >50% represents a risk factor for progression to myelofibrosis in polycythemia vera. Higher V617F allele burden correlated with more advanced myelofibrosis, greater splenomegaly, and higher white blood cell count in patients with essential thrombocythemia. Higher V617F allele burden is associated with an increased risk of first thrombosis and recurrent risk in patients with essential thrombocythemia. Assessment of V617F allele burden as a means to measure response to therapy in polycythemia vera patients. Early studies indicate that the V617F allele burden decreases with successful therapy, disappears in some patients, and reappears during relapse. However more recent studies show that V617F allele burden did not change significantly in treated patients with serial V617F analyses Leukemia Research. (2011) 35:177-82 Annals of Hematology. (2010) 89:141-6 Leukemia. (2010) 24:1574-9 Leukemia (2008) 22, 1299 1307
Acute Myeloid/Lymphoid Leukemia (AML/ALL) WHO 2008 Classification Clinicopathologic Subcategories Molecular Signatures ACUTE MYELOID LEUKEMIA 1. Acute Myeloid Leukemia with Recurrent Genetic Abnormalities 2. AML with t(8;21)(q22;q22), RUNX1-RUNX1T1 (CBFA/ETO) 100% CBFA/ETO (+) 3. AML with inv(16)(p13q22) or t(16;16)(p13;q22), CBFB-MYH11 100% CBFB-MYH11 (+) 4. APL with t(15;17)(q22;q11-12), PML-RARA 100%PML-RARA(+) 5. AML with t(9;11)(p22;q23), MLLT3-MLL and others 100% 11q23 (MLL) 6. AML with t(1;22)(p13;q13), RBM15-MKL1 100% RBM15-MKL1 (+) 7. AML with t(9;22)(q34;q11), BCR-ABL1 100% BCR-ABL1 (+) 8. AML with normal cytogenetics 55% NPM-1 mutations; 40% FLT-3 mutations 9. AML with mutation of CEBPA 100% CEBPA ACUTE LYMPHOID LEUKEMIA B Lymhoblastic Leukemia/Lymphoma with recurrent Abnormailities 1. B Lymhoblastic Leukemia/Lymphoma with t(9;22) 100% BCR-ABL1 2. B Lymhoblastic Leukemia/Lymphoma with t(v;11q23) 100% MLL rearranged 3. B Lymhoblastic Leukemia/Lymphoma with t(12;21) 100% TEL-AML1 (ETV6-RUNX1) 4. B Lymhoblastic Leukemia/Lymphoma with hyperdiploidy 5. B Lymhoblastic Leukemia/Lymphoma with hypodiploidy 6. B Lymhoblastic Leukemia/Lymphoma with t(5;14) 100% IL3-IGH 7. B Lymhoblastic Leukemia/Lymphoma with t(1;19) 100% E2A-PBx1 (TCF3-PBX1) T Lymhoblastic Leukemia/Lymphoma Adapted from Tefferi and Gilliland Cell Cycle 6: 550-566 (2007); Best Practice & Research Clinical Haematology 19:365 385, (2006) J Mol Diagn (2010), 12:3 16
Translocation Screen 1. Screen for 8 different translocations using two different PCR mixtures. A Europe Against Cancer Program standardization study. 2. Faster than cytogenetics 3. Quantitative real time RT-PCR assays 4. Sensitivities are 10-3 to 10-5 5. Eventually use to follow outcomes similar to BCR-ABL. Myelocytic Screen Lymphocytic Screen t(9;22) BCR/ABL 210 t(9;22) BCR/ABL 190 t(15;17) PML/RARA t(12;21) TEL/AML1 t(8;21) AML1/ETO t(4;11) MLL/AF4 Inv16 CBFB-MYH11 t(1;19) E2A/PBX1 Leukemia (2003) 17: 2318-57
+ p210, PML, AML, INV16 1% + p210, PML, AML, INV16 0.1% Normal Control Reagent Control Patient A Patient A 1% 0.1% PCR SETUP SHEET ABI-7900HT for Acute Leukemia Screen -- MYELOCYTIC RUN DATE: Master Mix AML Translocation Screen PML-RARa Real-time qpcr Data Last Name First Name 10-2 10-3 Sample Date Accession/Flow # Unit# Sample type Diagnosis Physcian PCR Tube Number 2 3 4 5 6 ABI 7900HT Row # 2 3 4 5 6 BCR-ABL CT's p210 (Row-A) 26.3 29.8 PML-RARA t(15,17) CT's (Row B) 30.4 35.8 23.4 AML-ETO t(8,21) CT's (Row C) 25.60 28.60 INV16 Myelo-Mono CT's (Row D) 34.60 40.30 B-2 micro CT's (Row- E) 15.50 15.50 15.70 39.40 16.25 RESULTS Pos Neg Neg pos/neg Translocation Identified PML-RARA
Quantitative RT-PCR Testing in Patients with Acute Promyelocytic Leukemia Uses for minimal residual disease (MRD) monitoring by reverse transcription-polymerase chain reaction (RT-PCR) methods: to assess the risk of hematological relapse (HR) in patients in clinical remission after the completion of consolidation therapy to identify patients at high risk of HR as a basis for initiating salvage therapy on the premise that this will provide a more favorable outcome while persistent or recurrent disease is still sub-clinical. Leukemia Research (2009) 33: 1170 1172 National Comprehensive Cancer Network Guidelines Version 2.2011 Acute Myeloid Leukemia
Acute Myeloid Leukemia with Normal Karyotype Mutations present in AML cases with a normal karyotype (50% AML) FLT-3 ITD NPMMut 53% NPM mutations 31% FLT3-ITD mutations 11% FLT3 tyrosine kinase mutations NPMneg 14% CEBP-alpha mutations 8% MLL tandem duplication mutations FLT3-ITDMut 13% ras mutations Prognostic groups within intermediate-risk category: AML with internal-tandem duplication of FLT3 High-risk category, high relapse risk AML with mutated nucleophosmin (NPM1) gene If not associated with FLT-3 ITD mutation, a good-prognosis subgroup Current Opin Opinion Hematol in 14:90 97. Hematology 2007 (2007), 14:90 97 FLT-3 ITD
FLT3 FLT3 is a class III receptor tyrosine kinase contains five immunoglobulin-like domains in the extracellular region and an intracellular tyrosine kinase domain split in two. Mutations of the FLT3 receptor cause constitutive activation of receptor. Internal tandem duplication (ITD) of the juxtamembrane domain (40% AML). Point mutation of the aspartic acid residue D835 in the activation loop of the kinase domain (8% AML). FLT3 Mutation Assays Normal- 329bp Internal tandem duplication Schnittger et al., (2004) Blood 100: 59 Kiyoi et al (1997) Leukemia 11:1447 Yamamoto et al (2001) Blood 97:2434
J Mol Diagnostics, 10, 2008 Nucleophosmin (NPM1) Nucleophosmin (NPM) is a nucleus-cytoplasm shuttling protein. The phosphoprotein functions in ribosome biogenesis, response to stress stimuli, maintenance of genomic stability, regulation of activity and stability of p53, and transcriptional regulation. Mutations are insertions/deletions in NPM exon 12 that encodes a nucleic acid binding domain. More than 26 different NPM1 mutations have been identified. The most common mutation is an insertion of a TCTG at positions 956 through 959 NPM1mut genotype associated with a favorable prognosis. NPM1mut genotype does not affect the poor prognosis of a FLT3-ITD mutation.
AML with Normal Karyotype and Different NPM1 and FLT3 Combinations Blood. (2005);106: 3733-3739
Proposed Schema for Molecular Screening in AML Patients in Routine Practice Poor Poor 20% NPM1pos/FLT3 ITDpos Poor 25% NPM1pos/FLT3 ITDneg Good 20% NPM1neg/FLT3 ITDpos Poor 45% NPM1neg/FLT3 ITDneg 1. Additional mutations a. FLT-3-TKD b. IDL1 c. DNMT3A d. MLL-PTD e. RAS f. WT1 RT-PCR SCREEN Normal Karyotype- AML (40-50%) CEBPA= CCAAT/ enhancer binding protein, alpha 2. Minimal Residual Disease a. NPM1 b. FLT-3 c. WT1 Leukemia (2008) 22, 915 931 Good
Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia Identification of novel recurrent somatic mutations in TET2, ASXL1, IDH1 or IDH2, DNMT3A, MLL and PHF6 in patients with AML. Retrospective analyses suggest that a subset of these mutations may have prognostic significance in AML, although these findings have not been validated with detailed clinical and mutational annotation in large, homogeneously treated cohorts of patients with AML. Using 502 specimens from a phase 3 clinical trial from the Eastern Cooperative Oncology Group they hypothesized that integrated mutational analysis of all known molecular alterations occurring in more than 5% of patients with AML would allow for the identification of novel molecular markers of outcome in AML and to identify molecularly defined subgroups of patients who would benefit from dose-intensified induction chemotherapy. Used bidirectional Sanger sequencing to sequence the entire coding regions of TET2, ASXL1, DNMT3A, CEBPA, PHF6, WT1, TP53, EZH2, MLL, RUNX1, and PTEN and the regions of previously described mutations for FLT3, NPM1, HRAS, KRAS, NRAS, KIT, IDH1, and IDH2. N Engl J Med (2012);366:1079-89.
Integrated Mutational Analysis on Risk Stratification in the Test Cohort of Patients with AML N Engl J Med (2012);366:1079-89.
Revised Risk Stratification of Patients with AML on the Basis of Integrated Genetic Analysis Inv16, t(8;21) t(6,9), Monosomy 7, Del 5q, NEJM (2012); 366:1079-89.
Current Clinical Evaluation of Acute Myeloid Leukemia and Potential Future Testing NEJM (2012) 366 p1152
Future of Molecular Diagnostics of Neoplastic Hematopathology Molecular classification of leukemia Monitoring of leukemia markers by qrt-pcr Whole gene, exon and genome sequencing of leukemias and lymphomas Protein analysis by mass spectrometry