Ken's Notes

alias JAK2 NM_004972.3:c.1849G>T (p.Val617Phe)
homozygous biallelic mutation in 1/3 of PV, 15% of CIMF, correlated with LOH at 9p
also seen in 8% of CMML / aCML, 4% of MDS, and 3% of AML

not seen so far in lymphoid or solid neoplasms

exon 12 mutation (~3% of PV) (in 2010, a minority of labs were testing for this)

at least 27 clinically verified mutations spanning codons 533 to 547
N542_E543del (~40% of V617F-negative PV)
F537_K539delinsL (~10% of V617F-negative PV)
K539L (~10% of V617F-negative PV)
R541_E543delinsK (~10% of V617F-negative PV)
E543_D544del (~10% of V617F-negative PV)
H538_K539delinsL (~5% of V617F-negative PV)
I540_E543delinsMK (~5% of V617F-negative PV)

exon 13 and 15 mutations, and other exon 14 mutations (exceptionally rare)

observed in a broader variety of disease types

JAK2 kinase is a non-receptor tyrosine kinase involved in cytokine receptor signaling
mutation is in the JH2 domain of JAK2 which is involved in inhibition of the kinase activity, and leads to constitutive activation of JAK2

cells no longer require cytokines to proliferate

useful as a diagnostic aid in these entities
The downstream effectors of JAK2 are the STAT transcription factor family (signal transducers and activators of transcription)
Not specific for MPN
Indications for testing:

Unexplained polycythemia
Neutrophilia (unexplained, BCR-ABL1 neg)
Thrombocytosis (unexplained, BCR-ABL1 neg)
Splanchnic vein thrombosis
RARS-T (RARS with thrombocytosis)
No clear role for follow-up testing or for predictive testing

Methods of testing:

Allele-specific qPCR

Most widely used for V617F
Analytical sensitivity <= 1% mutant alleles (only 2/22 labs in 2010, most reported 1-5% range)

Note however that very low levels (usually < 0.1%) have been described in peripheral blood of unaffected individuals, so an assay that detects < 0.1% is more likely to produce false-positive results

Quantification of the mutant as a percentage of all the JAK2 alleles (no clinical utility for this)
Watch out for false positive signal in negatives – laboratory needs to establish the minimal amount of signal needed to confidently call a sample positive

Other methods typically have an analytical sensitivity of 5 to 10% mutant alleles (including NGS)
Sanger sequencing has an analytic sensitivity of 20% and generates a significant number of false-negative results
NGS:

~1000-fold coverage needed to achieve 99% confidence of detecting more than 5% heterozygoud mutant alleles

Sample types for testing

Blood is preferred sample to test
Total WBCs is preferred type of cell population

Obtained by simply performing a red blood cell lysis procedure
Ficoll density centrifugation should be avoided because it depletes the granulocytic population (which carries the mutations)
Granulocyte isolation is not necessary

Allele burden varies greatly (1-100%) at first diagnosis

Low levels of JAK2 V617F are not uncommon

20-30% of PV patients and up to 75% of ET have < 25% mutant alleles

Homozygosity is most likely to occur in PV, uncommon in ET
High allele burden in PV and ET is associated with progression to myelofibrosis
Mutation may be present in a small fraction of the neoplastic clonal cell population

Reporting JAK2 mutation:

DETECTED or NOT DETECTED
Methodology summary including source information regarding any commercial primers and probes used

Statement of assay sensitivity
Clearly state the regions covered by the assay for sequencing assays

Mutations detected by sequencing should be reported using HGVS nucleotide and amino acid nomenclature
Interpretive comment

Whether the observed mutation has been previously reported in MPNs or other malignancies

COSMIC
dbSNP (NCBI)

estimate of mutant allele fraction (percentage)

particularly important for novel mutations

terms heterozygous and homozygous should generally be avoided

Positive controls:

AML cell lines:

HEL, MB-02, MUTZ-8, SET-2, UKE-1

Quantitative controls for JAK2 V617F are commercially available

Horizon Discovery, Cambridge)

Validation:

qPCR:

threshold for a positive result must be determined by running a series of normal DNA samples, due to cross-amplification of the wild-type allele
samples with a low-allelic frequency should be included
low-positive control should be included in each run

Proficiency testing:

CAP (JAK2 V617F)

o MPL mutation (1p34) (only tested by 2/22 labs in 2010)

§ Exon 10, codon 515 (majority) (~5% of PMF, ~3% of ET)

· W515L (most common)

· W515K (next most common)

§ Myeloproliferative leukemia virus oncogene

§ Thrombopoietin receptor

§ ET and PMF (not PV)

§ Less frequent than JAK2

§ May or may not coexist with JAK2 mutation

§ Mutually exclusive to BCR/ABL1 rearrangement

§ Not specific for MPN

§ Indications for testing:

· Suspicion of PMF or ET on biopsy/aspirate, negative for JAK2 V617F

· No clear role for follow-up testing

o TET2 mutation (rare)

o ASXL1 mutation (rare)

o IDH1/IDH2 mutation (rare)

o EZH2 mutation (rare)

o DNMT3A mutation (rare)

o +8 (?poor prognosis in CIMF)

o +9

§ May occur in conjunction with activating JAK2 mutations

§ Note: rare in MDS

o Chromosome 1 abnormalcies:

§ 1q gain

o del(13)(q12q22)

§ strongly suggestive of PMF (but not sensitive)

§ note: rare in MDS

o del(5)(q)

o der(6)t(1;6)(q21-23;p21.3)

§ strongly suggestive of PMF (but not sensitive)

o del(12)(p) (?poor prognosis in CIMF)

o +21 (3-4% of MPN)

o PDGFRA rearrangements (rare):

· Cryptic deletion at 4q12 with normal karyotype usually (most frequent abnormality in CEL – 40-60% of patients)

o 5’ FIP1L1 and 3’ PDGFRA creating a novel fusion tyrosine kinase FIP1L1-PDGFRA

o Breakpoints restricted to exon 12 in PDGFRA, always in-frame

o Easily detectable by RT-PCR

o Note also found in a subset of SM and eosinophilia

o Resistance to imatinib is extremely rare but emergence of a T674I kinase domain mutant has been described

§ M:F = 17:1

§ CEL with prominent involvement of mast cell lineage usually (neutrophils sometimes)

§ Also seen in AML or T-ALL with eosinophilia

§ Splenomegaly

§ B12 markedly elevated in serum

§ Tryptase elevated in serum

§ Responsive to imatinib

o PDGFRB rearrangements (5q31~q33) (rare):

§ t(5;12)(q32;p13)

· PDGFRB-ETV6

§ Multiple (>10) other fusion partners have been identified

§ Most often seen in CMML with eosinophilia, but also AML, maybe others (lymphoid?)

§ Proliferation of aberrant mast cells maybe

§ Responsive to imatinib

FGFR1 rearrangements (8p11) (extremely rare):

t(8;13)(p11;q12) – ZNF198-FGFR1 is most common
8p11 myeloproliferative syndrome (EMS)

Clinical phenotype with features of both eosinophilic MPD and lymphoma (T > B)
Rapid transformation to acute leukemia, mostly AML, within 1 or 2 years from diagnosis

8 fusion partners reported
Morphologies:

T-LBL with accompanying eosinophilia often
B-LBL
CEL
AML
CML – t(8;22) encoding BCR-FGFR1 fusion
PV – 2 patients with t(6;8)
Translocation is present in both myeloid and lymphoid cells (stem cell origin)

Poor prognosis:

Not responsive to imatinib

Other compounds have shown activity in vitro

Rapid transformation to acute leukemia, mostly AML, within 1 or 2 years from diagnosis

Complex karyotype (poor prognosis)

Other features:

increased peripheral blood counts (mature granulocytes, RBCs, and/or platelts)
Prognosis not influenced by presence of cytogenetically abnormal clone at diagnosis (several studies)

Some studies suggest shorter survival

Cytogenetic abnormalities may be associated with myeloxic treatments

References:

WHO blue book 2001
Heim S, Mitelman F. Cancer Cytogenetics. 3rd ed. Wiley-Blackwell; 2009.
Gong et al. Laboratory Practice Guidelines for Detecting and Reporting JAK2 and MPL Mutations in Myeloproliferative Neoplasms: a report of the Association for Molecular Pathology. J Molecular Diagnostics 2013;15(6):733-744