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Diagnostic Molecular Pathology > 34 Myeloproliferative Neoplasms > Flashcards

34 Myeloproliferative Neoplasms Flashcards

1
Q

MPNs

A

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.

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2
Q

Is MPD and MPN the same?

A

Yes

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3
Q

Characteristics of MPNs

A

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

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4
Q

WHO classified MPNs into two classes

A

1) Philadelphia chromosome (BCR-ABL1) positive: CML;

2) Philadelphia chromosome (BCR-ABL1) negative:
– polycythemia vera (PV);
– essential thrombocythemia (ET) ;
– primary myelofibrosis (PMF);

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5
Q

Primary characteristics of PV and ET

A

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;

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6
Q

Primary characteristics of PMF

A

1). progressive marrow fibrosis;
2). variable degree of megakaryocyte and granulocyte proliferation;
3). more sever than PV and ET

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7
Q

MPNs can progress into what disease?

A

1). Myelodysplastic syndrome (MDS);
2). Acute myeloid leukemia (AML);

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8
Q

% risk of transformation of MPNs to MDS or AML

A

1). 20% for PMF;
2). 4.5% for PV;
3). <1% for ET

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9
Q

JAK2

A

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

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10
Q

JAK2 V617F mutation (Valine to Phenylalanine)

A

1) somatic mutation;

2) located on exon 14 nt. 1849;

3) G to T substitution

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11
Q

JAK2 protein domains

A

1). FERM domain;
2). SH2 domain;
3). pseudokinase (JH2) domain - negatively; regulates JAK function
4). tyrosine kinase domain (JH1)

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12
Q

Function of JAK2 V617F in MPNs

A

1) constitutive activation:
JAK-STAT, PI3K, and AKT pathways;
MAPK and ERK pathway;

2) cytokine hypersensitive;
cytokine-independent growth;

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13
Q

% MPNs with JAK2 V617F

A

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

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14
Q

What is uniparental disomy?

A

1) both members of a chromosome pair are inherited from one parent;

2) the other parent’s chromosome for that pair is missing

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15
Q

JAK2 exon 12 mutations

A

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

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16
Q

what kind of JAK2 exon 12 mutations are?

A

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

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17
Q

with respect to protein, JAK2 exon 12 mutations can be divided into 3 main types:

A

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

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18
Q

JAK2 exon 12 mutations can be detected in what blood cell types?

A

granulocytes;
monocytes;
platelets;

rarely in lymphocytes

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19
Q

What is the predominate feature of patients with JAK2 exon 12 mutations?

A

erythrocytosis 紅血球增多症

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20
Q

What is MPL gene

A

the myeloproliferative leukemia virus oncogene;

located on chr. 1p34;

12 exons;

encodes the thrombopoietin receptor

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21
Q

What MPNs associated with MPL mutations?

A

ET (3%) and PMF (10%);

MPL mutations Not seen in PV

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22
Q

MPL mutations in ET and PMF

A

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

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23
Q

amino acids affected by MPL mutations in ET and PMF

A

W515, S505

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24
Q

Majority of MPL mutations in ET and PMF

A

W515L, W515K

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25
Q

Less commonly reported MPL mutations in ET and PMF

A

W515A, W515R

S505N - reported as both germline and somatic

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26
Q

MPL mutations are found in which blood cell lineages?

A

myeloid;
lymphoid;

MPL is mainly expressed in the megakaryocytic/platelet lineage, but also in the stem cell and early hematopoietic progenitor compartment.

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27
Q

MPL and JAK2 mutations mutually exclusive?

A

No

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28
Q

Characteristics of patient with MPL mutations

A

more anemic;
older;
higher platelet counts;
higher risk of developing arterial thrombosis than those with JAK2 V617F

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29
Q

CALR

A

located on chr. 19p13.2;

encodes the ER associated, calcium-binding protein calreticulin

30
Q

Three domains of CALR protein

A

1). N-domain (residues 1-180);
2). P-domain (residues 181-290);
3). C-domain (residues 291-400);

31
Q

CALR mutations in MPNs

A

more than 36 mutations;

frameshift insertions or deletions;

on exon 9

32
Q

2 types of CALR mutations in MPNs

A

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

33
Q

% CALR mutations in MPNs

A

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

34
Q

Characteristics of patient with CALR mutations

A

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

35
Q

How is the prognosis of a PMF Patient with CALR and ASXL1 mutations?

A

unfavorable prognosis

36
Q

Can molecular tests for JAK2V617F, CALR, MPL mutations be used solely to classify or diagnosis MPNs?

A

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

37
Q

Which MPNs can be diagnosed with a JAK2 exon 12 mutations alone?

A

PV

38
Q

What kind of JAK2 mutations PV patients can have?

A

JAK2V617F ;
JAK2 exon12 mutations

39
Q

Which mutations are usually not used for PV diagnostic workup?

A

CALR mutations;
MPL mutations

40
Q

Is bone marrow examination essential for the diagnosis of PV?

A

No

41
Q

When MPNs suspected, molecular testing of which gene mutation is recommended?

A

JAK2V617F

42
Q

What is the hemoglobin (hematocrit) levels in a PV patient?

A

in men >18.5% g/dL;
In women >16.5% g/dL;

PV patient also has subnormal erythropoietin level

43
Q

If a patient suspected with PV but has JAK2V617F negative, what is the next molecular test recommended?

A

JAK2 exon 12 mutations

44
Q

What molecular test used for ET and PMF initial diagnostic workup?

A

JAK2V617F

45
Q

If a suspected ET or PMF patient is negative for JAK2V617F, what is the next molecular test recommended?

A

CALR mutations

46
Q

If a suspected ET or PMF patient is negative for both JAK2V617F and CALR mutation analysis, what is the next molecular test recommended?

A

MPL mutations

47
Q

Diagnostic of an MPN should be based on what kinds of assessment?

A
  1. combined assessment of
    1). Bone marrow morphology;
    2). laboratory values;
    3). clinical history;
  2. molecular tests are only used as supportive evidence
48
Q

% of PMF patients are triple negative of JAK2V617F, CALR and PML mutations

A

10%

49
Q

% of ET patients are triple negative of JAK2V617F, CALR and PML mutations

A

13%

50
Q

What types of specimens can be used for JAK2, CALR, and MPL mutation analysis?

A

peripheral blood;
bone marrow aspirate;

Cell types carries mutations: granulocytes

51
Q

Preferred sample collection tubes for molecular tests of JAK2, CALR and MPL mutation analysis

A

EDTA - lavender top tube

52
Q

Why heparinized tubes should be avoid for molecular tests of JAK2, CALR and MPL mutation analysis?

A

Heparin inhibits the polymerase enzyme in PCR

53
Q

Should blood be frozen for PCR tests?

A

No;

Freezing causes hemolysis, which interferes with DNA amplification

54
Q

Can we use decalcified bone marrow for PCR tests?

A

No.

DNA degraded

55
Q

What is necessary analytical sensitivity of a clinical JAK2 assay?

A

at least 1% to detect more than 90% of JAK2V617F

56
Q

What are the methods that can be used for JAK2V617F detection?

A

Sanger;
pyrosequencing;
high-resolution melting;

restriction fragment length polymorphism analysis;
amplification refractory mutation system;
allele-competitive blocker PCR;
allele-specific qPCR

57
Q

What is the analytical sensitivity of
Sanger
pyrosequencing
high-resolution melting

A

5-15%

58
Q

What is the analytical sensitivity of of qPCR can achieve?

A

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

59
Q

What is the FDA approved JAK1 and JAk2 inhibitor to treat myelofibrosis?

A

Ruxolitinib

60
Q

JAK2 exon 12 mutations detection methods

A

Sanger;

High-resolution melting (HRM) curve analysis;

Multiplex fragment length analysis

61
Q

What is high-resolution melting (HRM) curve analysis?

A

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

62
Q

Example of high-resolution melting (HRM) curve analysis

A

JAK2 exon 12 mutations in PV

63
Q

Why Multiplex fragment length analysis is used to detect JAK2 exon 12 mutations in PV?

A

JAK2 Exon 12 mutations are:
1). deletions of 3-12 bp;
2). duplications of 27-36 bp;
3). substitution leading to K539L

64
Q

What is Multiplex fragment length analysis?

A

PCR to target region, then run capillary electrophoresis

65
Q

What are the methods used to detect MPL mutations in MPNs?

A

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

66
Q

What are the methods used to detect CALR mutations in MPNs?

A

PCR then Sanger;
fragment length analysis;
HRM;
NGS

67
Q

Why Sanger is not a good method to detect CALR mutations in MPNs?

A

CALR mutation rate is <15%, thus Sanger might not be sensitive

68
Q

What is the sensitivity of fragment length analysis for CALR mutation analysis in MPNs?

A

2-5%;
May not be able accurately differentiate length-affecting polymorphisms from mutations ;

Thus NGS is better than fragment length analysis

69
Q

For diagnostic suspected PV, ET and PMF, which molecular test is the first to order?

A

JAK2V617F

70
Q

For diagnostic suspected PV, ET and PMF, which molecular tests are the second ones to order?

A

JAK2 exon 12 mutations (suspected PV);
CALR and MPL mutation analysis (suspected PMF or ET)

71
Q

For diagnostic suspected PV, ET and PMF, which MPNs is ruled out if the patient negative for JAK2 mutations?

A

PV

72
Q

If a patient lacks of JAK2 V617F, CALR, and MPL mutations, can we rule out ET or PMF for this patient?

A

No;

13% ET and 10% PMF cases are triple negative

Diagnostic Molecular Pathology (12 decks)

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