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