Updates in Molecular Hematology Gregory J. Tsongalis, Ph.D.

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1 Updates in Molecular Hematology Gregory J. Tsongalis, Ph.D. Professor of Pathology Director, Molecular Pathology Dartmouth Medical School Dartmouth Hitchcock Medical Center Norris Cotton Cancer Center Lebanon, NH

2 Molecular Hematopathology Genetics Neoplasia

3 Genetic Hematopathology ENDOTHELIAL INJURY THROMBOSIS Germline Mutations ABNORMAL BLOOD FLOW HYPERCOAGULABILITY

4 Signs and Symptoms of Thrombosis DVT: PE: Painful, swollen, warm, and plethoric extremity with reduced pulse volume Cough, SOB, Hemoptysis Tachycardia

5 Risk Factors for Thrombosis Hereditary thrombophilia Atherosclerosis Acquired thrombophilia Immobility Thrombosis Surgery trauma Inflammation Malignancy Estrogens

6 Hereditary Thrombophilia FV Leiden Most common (40-50% of inherited thrombophilias) 5% of the Caucasian population 10-20% of patients with first episode of idiopathic DVT; 50% of patients with recurrent DVT FII (Prothrombin) Mutation in 3 -untranslated region % in prothrombin levels 2-3% of Europeans MTHFR Hyperhomocysteinemia implicated in both arterial and venous thrombosis Why is homocysteine thrombogenic? Direct toxicity to endothelial cells Inhibits Protein C activation Promotes endothelial tissue factor expression Surpresses endothelial cell surface heparin sulfate

7 Detection of Factor V Leiden (R506Q) Mutation by PCR-RFLP PCR primer Exon 10 MnlI sites PCR primer 153 bp 116 bp 67 bp 37 bp +/+ +/m m/m MW Agarose gel G->A (+) (Mut) Mutation destroys an MnlI site

8 Thrombophilia Risk Assessment Based on SNP Analysis FII FV

9 Factor V Leiden (R506Q) Mutation Detection by INVADER TM Assay Flap Mut probe A T Mutation present -> Cleavage A Complex formation Flap wt probe A C Normal sample (no cleavage) F Q A Cleavage F Fluorescence in plate well indicates presence of mutation

10 Invader Assay Set Up

11 Coagulation SNP Panel Detection by Allelic Discrimination TAT < 2 hours

12 Hematopoiesis and Malignancy

13 Hematopoietic Malignancies 1. Chronic Lymphocytic Leukemia 2. Chronic Myeloid Leukemia 3. Myeloproliferative Diseases 4. Acute Promyelocytic Leukemia 5. Acute Myeloid Leukemia

14 Molecular Classification of Tumors 1976 French-American-British classification Based on morphology Discovery of genetic lesions that predict outcome and clinical behavior better than morphology 2001-WHO Classification added t(15;17) for PML WHO added several point mutations

15 Indications for Molecular Testing in Hemepath Diagnosis Determination of B and T cell clonality Gene rearrangements Identification of gene mutations (translocations, point mutations) BCR-ABL1, BCL-2, BCL-1, JAK2 Prognosis Prediction of outcome based on detection of gene specific mutations FLT3, NPM1 Therapeutic monitoring Detection of minimal residual disease (quant assays). BCR-ABL1

16 Diagnosis of Hematopoietic Malignancies Historical Prior to Diagnosis based on morphology Introduction of immunopathology 1980 s - Rapid growth in immunopathology Introduction of molecular genetics 1985 to present - Rapid growth in molecular

17 Clonality Normal lymphocyte populations are polyclonal with respect to Ig and TCR genes. A leukemia or lymphoma is monoclonal with regard to Ig or TCR rearranged genes. Polyclonal Monoclonal

18 Determination of Clonality A population of cells with similar characteristics that are all derived from a single precursor cell. 1. Morphology - monomorphous cell population 2. Immunopathology - monotypic SIg 3. Cytogenetics - recurrent chromosomal alteration (e.g. translocation) 4. Molecular genetics - clonal B or T cell gene rearrangements

19 Gene Rearrangements (GR) Gene rearrangements are normal events that occur in lymphocytes. Antibody genes [immunoglobulin heavy chain genes, immunoglobulin light chain genes (k, l)] and T-cell receptor genes (a, b, g, d) rearrange. Rearrangement occurs independently in each cell.

20 Immunoglobulin and T Cell Receptor Gene Rearrangements Early B cell precursor Pre-B B cell Mature PC Lymphoid stem cell IgH GR IgH GR + IgL GR IgH + IgL GR TCR d and g GR TCR b and a GR Cytotoxic T Early thymocytes Common thymocytes Helper T

21 IMMUNOGLOBULIN AND T-CELL RECEPTOR GENE REARRANGEMENTS 5 V D J C 3 GERMLINE 5 V D J C 3 DJ REARRANGEMENT 5 V D J C 3 VDJ REARRANGEMENT 5 V D J C 3 TRANSCRIPTION AND SPLICING NH 2 VARIABLE CONSTANT COOH TRANSLATION

22 IMMUNOGLOBULIN HEAVY CHAIN GENE 5 Vn Cn 3 Dn CHROMOSOME 14q32 J J J J J J n= n=30 n=9 JH probe TCR BETA CHAIN GENE CHROMOSOME 7q34 5 Vn D1 J J J J J J C B1 D2 J J J J J J J C B2 3 n= J B1 GROUP J B2 GROUP J B1B2 PROBE

23 18 kb 5 V D J C JH PROBE 3 GERMLINE (LANE A) 12 kb 5 V D J C 3 DJ REARRANGEMENT JH PROBE (LANE B) 21 kb 5 V D J C 3 VDJ REARRANGEMENT (LANE C) JH PROBE A B C 21 kb 18 kb 12 kb B-CELL SOUTHERN BLOT

24 Southern Blot Detection of Gene Rearrangements (1-5% sensitivity) Two novel bands in 2 separate enzyme digests or both present in the same enzyme digest.

25 Limitations of the Southern Blot Transfer Analysis Fresh or frozen tissue Labor intensive Turn around time Radioactivity Advantage = large probes span large regions so few false negatives

26 Polymerase Chain Reaction (PCR) Detection of Gene Rearrangements ( % sensitivity) VH PRIMER 5 V D J C 3 GERMLINE (LANE A) JH PRIMER NO PCR PRODUCT VH PRIMER 5 V D J C 3 VDJ REARRANGEMENT (LANES B AND C) JH PRIMER A B C PCR PRODUCT GERMLINE POLYCLONAL MONOCLONAL

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28 Advantages of PCR FFPE and small specimens Less labor intensive More sensitive Fast TAT Disadvantage = false negative rate proportional to primer sets used.

29 Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4- CT JJM van Dongen et al. Leukemia (2003) 17,

30 TCRB TCRG

31 IgH Polyclonal IgH Clonal

32 Detection A Molecular of BCR-ABL Approach to Diagnosing and Monitoring CML Southern Blot Transfer Analysis Fluorescence In Situ Hybridization (FISH)

33 GeneXpert Compliance vs Resistance

34 Importance of CML Resistance Gleevec (Imatinib Mesylate) Second generation TKIs Nilotinib (Tasigna), Dasatinib (Sprycel) T315I resistance mutation

35 BCR-ABL1 Negative Myeloproliferative Neoplasms Polycythemia vera (PV) is a myeloproliferative disease (MPD) characterized by overproduction of red blood cells Essential Thrombocythemia (ET) and Chronic Idiopathic Myelofibrosis (CIMF) are related MPDs Current diagnosis based on clinical, laboratory, and pathologic findings A mutation in the JAK2 gene has been identified that is present in the majority of PV patients and approximately half of patients with ET and CIMF

36 Vic (fluorescence) JAK2 Allelic Discrimination Plots and RFLP SNP Genotyping Assay 5 RFLP V617F WT WT Fam (fluorescence)

37 Acute Promyelocytic Leukemia A unique subtype of acute leukemia AML M3 -French-American-British (FAB) system APL with t(15;17)- WHO 5-8% of AML Predominantly adults in mid-life Frequently associated with DIC

38 Retinoic acid alpha receptor gene (RAR alpha) Promyelocytic gene (PML) Is encoded by the long arm of chromosome 17 encoded by the long arm of chromosome 15 Mainly expressed in hematopoietic cells Expressed ubiquitously Important role in regulating gene expression Thought to be involved in apoptosis and tumor suppression In the presence of retinoic acid, the genes are activated and terminal differentiation of promyelocytes occurs In the absence of retinoid acid, the retinoid acid alpha gene is bound by nuclear corepressor factor, and this causes transcriptional repression

39 Retinoic Acid Alpha Receptor Gene (RAR alpha) and Promyelocytic Gene (PML) Breakpoint in chromosome 17 (RAR alpha gene ) is consistently found in intron 2 3 breakpoints on the PML gene can occur at intron 6 (bcr1,l form), intron 3 (bcr3,s form), and exon 6 (bcr2, V form) The most frequent sites are bcr1 and bcr3. Detection by FISH or RT-PCR

40 Importance of APL Diagnosis Diagnosis of APL with t(15;17) (PML-RARA) is important because of the availability of highly effective therapy Morphologic diagnosis of APL can be problematic, as there can be substantial variation in the morphologic appearance of the promyelocytes

41 Acute Myeloid Leukaemia (AML) Uncontrolled proliferation of immature myeloid cells (blast) Median age of onset ~60 years Analysis to look for ACQUIRED abnormalities in leukaemic clone (i.e. not constitutional) Abnormalities can evolve during disease progression For diagnosis and prognosis

42 AML Prognostic Indicators Cytogenetic markers and molecularly determined mutation status of FLT3 and NPM1 Allows a risk-adapted treatment approach Good Prognostic Indicator t(15;17) t(8;21) inv(16) NPM1 4bp insertion Bad Prognostic Indicator Complex karyotype Monosomy 7 deletion of 7q FLT3 ITD

43 Fms-related tyrosine kinase (FLT3) Encodes a tyrosine kinase receptor (13q12) involved in regulation of stem cell proliferation Internal Tandem Duplications (ITDs) cause constitutive activation of receptor Associated with elevated risk relapse and reduced overall survival Bad prognostic indicator Exon 14

44 NPM1 (nucleophosmin) Encodes a ubiquitously expressed nuclear protein (5q35) Involved in nuclear-cytoplasmic shuttling facilitating transport of ribosomal proteins 4bp insertion in exon 12 of the NPM1 gene Loss of the nucleolar-localisation signal and gain of a nuclear export signal motif at the C-terminus. Abnormal cytoplasmic accumulation Good prognostic indicator in AML

45 Prognostic Stratification In Normal Karyotype Leukemia (~40% of AML) NPM1-ve/FLT3 ITD+ve NPM1+ve/FLT3 ITD+ve NPM1-ve/FLT3 ITD-ve NPM1+ve/FLT3 ITD-ve Gale et al. (2008) Blood, 111, 2776_2784.

46 Testing Strategy DNA and RNA extracted from blood or bone marrow using the automated Qiagen EZ1 TNAI protocol PCR for FLT3 internal tandem duplication and run on AB3500 CE instrument PCR/RFLP for FLT3 D835 point mutation and AB3500 Multiplex RT-PCR for common NPM1 mutations and run on Luminex 200 (Asuragen Signature NPM1 Assay)

47 FLT3 ITD and D835 ITD D835

48 NPM1 Mutation Analysis Wild type Mut A Mut B Mut D TCTG...GCAG TCTGTCTGGCAG TCTGCATGGCAG TCTGCCTGGCAG 75-80%* ~10%* ~5%*

49 Diagnosis: Signature LTx Leukemia Translocation Panel v2.0 Classification Minh Hang T. Hoang University of Connecticut Translocations Fusion Genes/Transcripts Chronic Myelogenous Leukemia t(9;22) BCR/ABL1 (b2a2) & (b3a2) Acute Lymphoblastic Leukemia t(9;22) t(1;19) t(12;21) t(4;11) BCR/ABL1 (e1a2) E2A/PBX1 TEL/AML MLL/AF4 (e9/e5) or (e10/e4) Acute Promyelocytic Leukemia t(15;17) PML/RARα (L form) & (S form) Acute Myelogenous Leukemia inv(16) t(8;21) CBFβ/MYH11 (A type) & (D type) AML1/ETO

50 xmap Technology Microspheres are dyed to create 100 distinct colors Each microsphere has spectral address based on red/infrared content Microspheres are suspendable Microspheres are coated with capture reagent (oligo or antibody) Sample is added to microspheres Analyte is captured to microspheres Fluorescent reporter tag added

51 Whole Blood Bone Marrow Total RNA Emedicinehealth.com Signature LTx Assay Workflow Reverse-Transcription Purple Bead = TEL/AML BCR/ABL1 cdna ssrna Prep 30 min. RT 60 min. Detection Prep 10 min. Detection 30 min. MFI Bound Products AML1/ETO MLL/AF4 Hybridization Prep 10 min. Hyb 30 min. PML/RARα Polymerase-Chain Reaction Prep 30 min. PCR 150 min.

52 CLL - Clinical and/or Disease Heterogeneity Extremely variable course Survival ranging from months to decades Only some patients die from their disease Indolent disease vs aggressive disease

53 MicroRNAs and Prognostic Risk Groups In CLL (Ward et al. Exp Mol Pathol Apr; 90(2): , 2011) 15a a a E-08 1E-09 1E E-09 1E-11

54 Conclusions Molecular markers have very good clinical utility in the workup of hematopoietic neoplasms Molecular markers can be used to monitor these malignancies Novel therapeutics require that laboratories be capable of this form of testing.

55 What if..

56 DHMC Molecular Pathology Laboratory and Translational Research Program Samantha Allen Betty Dokus Susan Gallagher Carol Hart Arnold Hawk Claudine Lefferts, Ph.D. Joel Lefferts, Ph.D. Rebecca O Meara Elizabeth Reader Mary Schwab Heather Steinmetz Laura Tafe, M.D. Brian Ward Brendan Wood Eric York