34 Myeloproliferative Neoplasms Flashcards
MPNs
1) Myeloproliferative Neoplasms;
2) also called myeloproliferative disorders, or MPDs;
3) are a collection of blood disorders that are caused by mutations in bone marrow stem cells.
Is MPD and MPN the same?
Yes
Characteristics of MPNs
1) abnormal and excessive production of one or more of myeloid cell lines:
granulocytes,
erythrocytes,
platelets,
2) No marked alteration of cellular maturation;
3) vast majority of MPN cases are sporadic: 5 in 100,000 individuals;
4) might have a familial predisposition in younger patients
WHO classified MPNs into two classes
1) Philadelphia chromosome (BCR-ABL1) positive: CML;
2) Philadelphia chromosome (BCR-ABL1) negative:
– polycythemia vera (PV);
– essential thrombocythemia (ET) ;
– primary myelofibrosis (PMF);
Primary characteristics of PV and ET
1). increased production of red blood cells and platelets;
2). night sweats;
3). Fever;
4). pruritus;
5). splenomegaly;
6). predispose to thrombosis or hemorrhage;
7). could be a longer-term illness;
8). may progress into myelofibrosis phase;
Primary characteristics of PMF
1). progressive marrow fibrosis;
2). variable degree of megakaryocyte and granulocyte proliferation;
3). more sever than PV and ET
MPNs can progress into what disease?
1). Myelodysplastic syndrome (MDS);
2). Acute myeloid leukemia (AML);
% risk of transformation of MPNs to MDS or AML
1). 20% for PMF;
2). 4.5% for PV;
3). <1% for ET
JAK2
1) Janus Kinase 2;
2) mediates signal transducing downstream of various cytokine receptors implicated in erythropoietin receptor signaling and hematopoiesis;
3) a cytoplasmic tyrosine kinase
JAK2 V617F mutation (Valine to Phenylalanine)
1) somatic mutation;
2) located on exon 14 nt. 1849;
3) G to T substitution
JAK2 protein domains
1). FERM domain;
2). SH2 domain;
3). pseudokinase (JH2) domain - negatively; regulates JAK function
4). tyrosine kinase domain (JH1)
Function of JAK2 V617F in MPNs
1) constitutive activation:
JAK-STAT, PI3K, and AKT pathways;
MAPK and ERK pathway;
2) cytokine hypersensitive;
cytokine-independent growth;
% MPNs with JAK2 V617F
1) 95% of PV patients have JAK2 V617F;
2) 55% of ET;
3) 65% of PMF;
4) PV and PMF patients can have biallelic JAK2 V617F due to uniparental disomy (UPD), not in ET cases
What is uniparental disomy?
1) both members of a chromosome pair are inherited from one parent;
2) the other parent’s chromosome for that pair is missing
JAK2 exon 12 mutations
1) the remaining 5% of PV patients that are JAK2 V617F negative have JAK2 exon12 mutations;
2) constitutive activation of erythropoietin signaling led to myeloproliferative phenotype;
3) only found in PV;
4) at least 37 different mutations located in codons 536-547;
5) mutually exclusive to JAK2 V617F
what kind of JAK2 exon 12 mutations are?
1) majority mutations are in-frame deletions of 3 to 9 bp;
2) 6-bp deletions are the most common;
3) substitution K539L;
4) in-frame duplications usually 33 bp in length
with respect to protein, JAK2 exon 12 mutations can be divided into 3 main types:
1). deletions that include E543;
2). amino acid substitution or deletion mutations that involve K539;
3). duplications of 10-12 amino acids that occur in the region of V536 to F547
JAK2 exon 12 mutations can be detected in what blood cell types?
granulocytes;
monocytes;
platelets;
rarely in lymphocytes
What is the predominate feature of patients with JAK2 exon 12 mutations?
erythrocytosis 紅血球增多症
What is MPL gene
the myeloproliferative leukemia virus oncogene;
located on chr. 1p34;
12 exons;
encodes the thrombopoietin receptor
What MPNs associated with MPL mutations?
ET (3%) and PMF (10%);
MPL mutations Not seen in PV
MPL mutations in ET and PMF
gain-of-function;
constitutive activation of thrombopoietin receptor without the binding of thrombopoietin ;
through JAK-STAT pathway;
on exon 10 (juxtamembrane domain);
two amino acids are affected: W515 and S505
amino acids affected by MPL mutations in ET and PMF
W515, S505
Majority of MPL mutations in ET and PMF
W515L, W515K
Less commonly reported MPL mutations in ET and PMF
W515A, W515R
S505N - reported as both germline and somatic
MPL mutations are found in which blood cell lineages?
myeloid;
lymphoid;
MPL is mainly expressed in the megakaryocytic/platelet lineage, but also in the stem cell and early hematopoietic progenitor compartment.
MPL and JAK2 mutations mutually exclusive?
No
Characteristics of patient with MPL mutations
more anemic;
older;
higher platelet counts;
higher risk of developing arterial thrombosis than those with JAK2 V617F
CALR
located on chr. 19p13.2;
encodes the ER associated, calcium-binding protein calreticulin
Three domains of CALR protein
1). N-domain (residues 1-180);
2). P-domain (residues 181-290);
3). C-domain (residues 291-400);
CALR mutations in MPNs
more than 36 mutations;
frameshift insertions or deletions;
on exon 9
2 types of CALR mutations in MPNs
Type 1: 52bp deletion, L367fs46;
Type 2: 5bp TTGTC insertion, K385fs47;
Type 1 and Type 2 CALR mutations accounts for 80% of all CALR mutations
% CALR mutations in MPNs
1) 20-30% in ET and PMF;
2) In JAK2 V617F negative patients:
CALR mutations incidence is 49-71% in ET;
CALR mutations incidence is 56-88% in PMF
Characteristics of patient with CALR mutations
lower hemoglobin levels;
lower leukocyte count;
higher platelet count;
lower risk of thrombosis than JAK-mutated patients;
more in male;
younger age;
better survival in PMF patients with CARL mutations
How is the prognosis of a PMF Patient with CALR and ASXL1 mutations?
unfavorable prognosis
Can molecular tests for JAK2V617F, CALR, MPL mutations be used solely to classify or diagnosis MPNs?
1) No;
2) MDS, CML, AML, and ALL can harbor a low frequencies of JAK2V617F, CALR and MPL mutations;
3) Lack of these mutations can’t rule out for ET and PMF;
4) Triple negative genotype accounts for 10-15% of MPNs patients
Which MPNs can be diagnosed with a JAK2 exon 12 mutations alone?
PV
What kind of JAK2 mutations PV patients can have?
JAK2V617F ;
JAK2 exon12 mutations
Which mutations are usually not used for PV diagnostic workup?
CALR mutations;
MPL mutations
Is bone marrow examination essential for the diagnosis of PV?
No
When MPNs suspected, molecular testing of which gene mutation is recommended?
JAK2V617F
What is the hemoglobin (hematocrit) levels in a PV patient?
in men >18.5% g/dL;
In women >16.5% g/dL;
PV patient also has subnormal erythropoietin level
If a patient suspected with PV but has JAK2V617F negative, what is the next molecular test recommended?
JAK2 exon 12 mutations
What molecular test used for ET and PMF initial diagnostic workup?
JAK2V617F
If a suspected ET or PMF patient is negative for JAK2V617F, what is the next molecular test recommended?
CALR mutations
If a suspected ET or PMF patient is negative for both JAK2V617F and CALR mutation analysis, what is the next molecular test recommended?
MPL mutations
Diagnostic of an MPN should be based on what kinds of assessment?
- combined assessment of
1). Bone marrow morphology;
2). laboratory values;
3). clinical history; - molecular tests are only used as supportive evidence
% of PMF patients are triple negative of JAK2V617F, CALR and PML mutations
10%
% of ET patients are triple negative of JAK2V617F, CALR and PML mutations
13%
What types of specimens can be used for JAK2, CALR, and MPL mutation analysis?
peripheral blood;
bone marrow aspirate;
Cell types carries mutations: granulocytes
Preferred sample collection tubes for molecular tests of JAK2, CALR and MPL mutation analysis
EDTA - lavender top tube
Why heparinized tubes should be avoid for molecular tests of JAK2, CALR and MPL mutation analysis?
Heparin inhibits the polymerase enzyme in PCR
Should blood be frozen for PCR tests?
No;
Freezing causes hemolysis, which interferes with DNA amplification
Can we use decalcified bone marrow for PCR tests?
No.
DNA degraded
What is necessary analytical sensitivity of a clinical JAK2 assay?
at least 1% to detect more than 90% of JAK2V617F
What are the methods that can be used for JAK2V617F detection?
Sanger;
pyrosequencing;
high-resolution melting;
restriction fragment length polymorphism analysis;
amplification refractory mutation system;
allele-competitive blocker PCR;
allele-specific qPCR
What is the analytical sensitivity of
Sanger
pyrosequencing
high-resolution melting
5-15%
What is the analytical sensitivity of of qPCR can achieve?
0.1% mutant allele;
currently used for JAK2 V617F;
False positive can occur since 0.1% mutant allele for JAK2 V617F can be observed in healthy patients
What is the FDA approved JAK1 and JAk2 inhibitor to treat myelofibrosis?
Ruxolitinib
JAK2 exon 12 mutations detection methods
Sanger;
High-resolution melting (HRM) curve analysis;
Multiplex fragment length analysis
What is high-resolution melting (HRM) curve analysis?
An efficient method to detect a variety of different mutations that cluster in a specific region:
1). PCR amplification with fluorescent dye;
2). Increase temperature to melt the double-stranded PCR products
3) Example JAK2 exon 12 mutations in PV
Example of high-resolution melting (HRM) curve analysis
JAK2 exon 12 mutations in PV
Why Multiplex fragment length analysis is used to detect JAK2 exon 12 mutations in PV?
JAK2 Exon 12 mutations are:
1). deletions of 3-12 bp;
2). duplications of 27-36 bp;
3). substitution leading to K539L
What is Multiplex fragment length analysis?
PCR to target region, then run capillary electrophoresis
What are the methods used to detect MPL mutations in MPNs?
1). Sanger, 10-15% mutant allele, examples:
c.1544 G>T (W515L);
C.1543_1544delinsAA (W515K)
2). Allele-specific PCR and capillary electrophoresis
S505N, W515L, W515K, W515A
What are the methods used to detect CALR mutations in MPNs?
PCR then Sanger;
fragment length analysis;
HRM;
NGS
Why Sanger is not a good method to detect CALR mutations in MPNs?
CALR mutation rate is <15%, thus Sanger might not be sensitive
What is the sensitivity of fragment length analysis for CALR mutation analysis in MPNs?
2-5%;
May not be able accurately differentiate length-affecting polymorphisms from mutations ;
Thus NGS is better than fragment length analysis
For diagnostic suspected PV, ET and PMF, which molecular test is the first to order?
JAK2V617F
For diagnostic suspected PV, ET and PMF, which molecular tests are the second ones to order?
JAK2 exon 12 mutations (suspected PV);
CALR and MPL mutation analysis (suspected PMF or ET)
For diagnostic suspected PV, ET and PMF, which MPNs is ruled out if the patient negative for JAK2 mutations?
PV
If a patient lacks of JAK2 V617F, CALR, and MPL mutations, can we rule out ET or PMF for this patient?
No;
13% ET and 10% PMF cases are triple negative
