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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
Molecular Hematopathology
• Genetics
• Neoplasia
ENDOTHELIAL
INJURY
ABNORMAL
BLOOD FLOW HYPERCOAGULABILITY
THROMBOSIS
Genetic Hematopathology
Germline Mutations
Signs and Symptoms of Thrombosis
• DVT:
– Painful, swollen,
warm, and plethoric
extremity with
reduced pulse
volume
• PE:
– Cough, SOB,
Hemoptysis
– Tachycardia
Thrombosis
Hereditary thrombophilia
Acquired thrombophilia
Surgery trauma Immobility
Inflammation
Malignancy
Estrogens
Risk Factors for Thrombosis
Atherosclerosis
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
• 150-200% 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
Hereditary Thrombophilia
153 bp
116 bp
Exon 10
G->A
67 bp
37 bp
+/+ +/m m/m MW
MnlI sites
PCR primer
PCR primer
(+)
(Mut)
Mutation destroys an MnlI site
Agarose gel
Detection of Factor V Leiden (R506Q) Mutation by
PCR-RFLP
Thrombophilia Risk Assessment Based on
SNP Analysis
FII
FV
A
T
Mut probe Flap
A
A
Mutation present -> Cleavage
F Q
Complex formation
Fluorescence in plate well
indicates presence of mutation
F Cleavage
A
C
wt probe Flap
Normal sample (no cleavage)
Factor V Leiden (R506Q) Mutation Detection by
INVADERTM Assay
Invader Assay Set Up
Coagulation SNP Panel Detection by Allelic Discrimination
TAT < 2 hours
Hematopoiesis and Malignancy
Hematopoietic Malignancies
1. Chronic Lymphocytic Leukemia
2. Chronic Myeloid Leukemia
3. Myeloproliferative Diseases
4. Acute Promyelocytic Leukemia
5. Acute Myeloid Leukemia
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
• 2008 - WHO added several point mutations
• 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
Indications for Molecular Testing in Hemepath
Diagnosis of Hematopoietic Malignancies Historical
Prior to 1980 - Diagnosis based on
morphology
1980 - Introduction of immunopathology
1980’s - Rapid growth in immunopathology
1985 - Introduction of molecular genetics
1985 to present - Rapid growth in molecular
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
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
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.
Immunoglobulin and T Cell Receptor
Gene Rearrangements
IgH GR IgH GR + IgL GR IgH + IgL GR
Early B cell precursor Pre-B B cell Mature PC
TCR d and g GR TCR b and a GR
Early thymocytes Common
thymocytes
Cytotoxic T
Helper T
Lymphoid
stem cell
IMMUNOGLOBULIN AND T-CELL RECEPTOR GENE
REARRANGEMENTS
V D J C
V D J C
V D J C
V D J C
NH2 COOH VARIABLE CONSTANT
GERMLINE
DJ REARRANGEMENT
VDJ REARRANGEMENT
TRANSCRIPTION
AND SPLICING
TRANSLATION
5
5
5
5
3
3
3
3 D
D
D
Vn 5 Cn 3
IMMUNOGLOBULIN HEAVY CHAIN GENE
n=100-200 n=30
JH probe
Vn 5 D1 J J J J J J D2 J J J J J J J
CB2 3 CB1
TCR BETA CHAIN GENE
JB1 GROUP JB2 GROUP
JB1B2 PROBE
CHROMOSOME 7q34
CHROMOSOME 14q32
J J J J J J Dn
n=9
n=75-100
5 V D J C GERMLINE
(LANE A)
3
18 kb
V D J C DJ REARRANGEMENT
(LANE B)
5 3
12 kb
V D J C VDJ REARRANGEMENT
(LANE C)
5 3
21 kb
A B C
21 kb
18 kb
12 kb B-CELL SOUTHERN BLOT
JH PROBE
JH PROBE
JH PROBE
D
D
Southern Blot Detection of Gene Rearrangements
(1-5% sensitivity)
23.1
9.4
6.6
Two novel bands in 2 separate enzyme digests or
both present in the same enzyme digest.
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
GERMLINE
(LANE A) 3
VDJ REARRANGEMENT
(LANES B AND C)
5 V D J C
JH PRIMER
VH PRIMER
NO PCR PRODUCT
V D J C 5 3
JH PRIMER
VH PRIMER
PCR PRODUCT
A B C
GERMLINE POLYCLONAL MONOCLONAL
D
Polymerase Chain Reaction (PCR) Detection of Gene Rearrangements
(0.1-0.01% sensitivity)
1 2 3 4 5 6 7 8
Advantages of PCR
• FFPE and small specimens
• Less labor intensive
• More sensitive
• Fast TAT
Disadvantage = false negative rate proportional to primer sets used.
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-
CT98-3936
JJM van Dongen et al. Leukemia (2003) 17, 2257–2317.
TCRB
TCRG
IgH Polyclonal
IgH Clonal
Detection of BCR-ABL A Molecular Approach to Diagnosing and
Monitoring CML
Southern Blot Transfer Analysis Fluorescence In Situ Hybridization (FISH)
GeneXpert®
Compliance vs Resistance
Importance of CML Resistance
• Gleevec® (Imatinib Mesylate)
• Second generation TKIs – Nilotinib (Tasigna),
Dasatinib (Sprycel)
• T315I resistance mutation
• 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
BCR-ABL1 Negative Myeloproliferative Neoplasms
JAK2 Allelic Discrimination Plots and RFLP
RFLP
V617F
WT
WT
0
1
2
3
4
5
0 1 2 3
Fam (fluorescence)
Vic
(fl
uo
rescen
ce)
SNP Genotyping Assay
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
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
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
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
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
AML Prognostic Indicators
• Cytogenetic markers and molecularly determined mutation
status of FLT3 and NPM1
• Allows a risk-adapted treatment approach
Bad Prognostic Indicator
Complex karyotype
Monosomy 7
deletion of 7q
FLT3 ITD
Good Prognostic Indicator
t(15;17)
t(8;21)
inv(16)
NPM1 4bp insertion
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
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
Prognostic Stratification
Gale et al. (2008) Blood, 111, 2776_2784.
NPM1-ve/FLT3 ITD+ve
NPM1+ve/FLT3 ITD-ve
NPM1-ve/FLT3 ITD-ve
NPM1+ve/FLT3 ITD+ve
• In Normal Karyotype Leukemia (~40% of AML)
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)
FLT3 ITD and D835
ITD
D835
NPM1 Mutation Analysis
Wild type
Mut A
Mut B
Mut D
TCTG....GCAG
TCTGTCTGGCAG
TCTGCATGGCAG
TCTGCCTGGCAG
75-80%*
~10%*
~5%*
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
Fusion Genes/Transcripts Translocations Classification
Diagnosis: Signature LTx Leukemia Translocation Panel v2.0 Minh Hang T. Hoang
University of Connecticut
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
Whole Blood
Bone Marrow
Reverse-Transcription
ssRNA
cDNA
Total RNA
Emedicinehealth.com
Polymerase-Chain Reaction
BCR/ABL1
PML/RARα
AML1/ETO MLL/AF4
Hybridization Detection
Purple Bead =
TEL/AML
MFI ≈ Bound
Products
Prep 30 min.
RT 60 min.
Prep 30 min.
PCR 150 min.
Prep 10 min.
Hyb 30 min.
Prep 10 min.
Detection 30 min.
Signature LTx Assay Workflow
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
MicroRNAs and Prognostic Risk Groups In CLL
0.001
0.01
0.1
1
10
100
15a
0.0001
0.001
0.01
0.1
1
10
16-1
1E-10
1E-09
1E-08
0.0000001
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
10
100
29a
1E-11
1E-09
0.0000001
0.00001
0.001
0.1
10
181a
(Ward et al. Exp Mol Pathol Apr; 90(2):173-178, 2011)
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.
What if………..
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