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Chapter 70 Primary Myelofibrosis 1129

IDH1/2 mutations are found at a frequency of approximately 4% in been documented in MPNs, most frequently in PMF patients, sug-
PMF and can coexist with mutations in JAK2, MPL, and TET2. The gesting a potential role of tumor suppressor gene silencing as a
specific IDH mutant variants seen in MPNs (IDH1 R132C, IDH1 mechanism in disease progression. In a study of 614 patients with
R132S, IDH2 R140Q) all affect arginine residues. IDH1/2 mutations myeloid malignancies, 42 cases were found to have a total of 49
are not predictive of survival in PMF but do have prognostic signifi- EZH2 mutations. Thirty PMF patients were analyzed in this cohort,
cance in multivariate analysis in blast-phase disease. It is not yet clear and 13% were found to have an EZH2 mutation. Microarray and
if different IDH mutation variants carry different biologic or prog- single-nucleotide polymorphism (SNP) analysis did not show an
nostic consequences in PMF. A significant association between association with copy number alterations or uniparental disomy.
IDH1/2 mutation and absence of the 46/1 JAK2 haplotype has been Because of small patient numbers and short follow-up, it has not been
observed, and supports a theory that PMF patients that are nullizy- possible to gauge the true prognostic significance of mutated EZH2
gous for the 46/1 haplotype are more susceptible to acquiring addi- in PMF. Retrospective studies using archived MPN BM samples to
tional molecular lesions that contribute to a poor prognosis. assess for EZH2 mutations have also not been shown to have prog-
The additional sex combs-like 1 (ASXL1) gene is located on nostic significance in PMF.
chromosome 20q11.1 and is responsible for encoding an enhancer The use of next-generation sequencing of 104 genes from a cohort
of the trithorax group (trxG) and polycomb repressive complex of 197 MPN patients by Skoda et al has provided insight into the
(PcG). The PcG proteins (repress) and trxG proteins (activate) regu- impact of clonal evolution on clinical phenotype. Somatic mutations
late gene expression of homeotic genes, such as Hox genes via histone were observed in 90% of patient samples in which approximately
methylation. ASXL1 mutations result in loss of PRC2-mediated H3 40% harbored mutations other than JAK2V617F and CALR.
lysine (H3K27me3) trimethylation. ASXL mutations have been JAK2V616F was the most common mutation (69%), followed by
identified in various myeloid malignancies and are in highest fre- CALR (15%), TET2 (12%), ASXL1 (5%) and DNMT3A (5%). The
quency in myelodysplastic syndrome (MDS) and CMML patients. presence of two or more acquired somatic mutations portended a
In a study of 64 MPN cases, ASXL1 mutations were found in five poor outcome and increased risk of leukemic transformation. By
MPN patients (three with PMF), all of whom were negative for the contrast, none of the patients with a sole mutation in JAK2, MPL,
JAK2 mutation. ASXL1 mutations can be found in the MPN HSC, or CALR evolved to AML. However, the rate of acquisition of new
suggesting acquisition early in the pathogenesis of MPNs. ASXL1 mutations was very low when assayed from serial samples (two muta-
mutations are associated with an adverse outcome and leukemic tions detected during a total follow-up of 133 patient-years). The
transformation in PMF patients. majority of mutations were present at low allelic levels from the time
The Casitas B-lineage lymphoma (CBL) proto-oncogene is located of diagnosis, and in the case of p53, rapid evolution to blast phase
on chromosome 11q23.3 and is believed to play a role in the main- occurred with loss of the WT allele. Mutations in p53 were frequently
tenance of the hematopoietic pluripotent stem cell pool. CBL has the present in a heterozygous state for a prolonged period of time, but
capacity to downregulate and upregulate tyrosine kinase activity by the loss of the WT allele either by chromosomal deletion or unipa-
either serving as an adaptor protein or recruiting downstream targets rental disomy was associated with leukemic transformation. These
of the pathway or by activation of E3 ubiquitin protein ligase activity data argue against the concepts of hypermutability and genomic
and the subsequent recycling of the receptor through internalization instability in MF. These data also support a complex clonal architec-
of the receptor/ligand complex or lysosomal-directed destruction. ture in MPNs, where some individuals may develop biclonal disease
CBL mutations were first appreciated in MPNs in the setting of and others develop a linear acquisition pattern. In general, mutations
mutational screening of acquired uniparental disomy of chromosome in TET2 and DNMT3A appeared earlier in the disease course,
11q, with a total of 27 CBL variants identified in MPNs. Within a whereas mutations in ASXL1, EZH2, and IDH1 were often acquired
cohort of 579 MPN patients screened, three patients with PMF were after JAK2V617F. The authors provide a model for clonal evolution
identified with CBL mutations at an overall frequency of 6%. The of MPNs and influence on disease course and risk of transformation
majority of the CBL mutations identified were located in the RING to leukemia (Fig. 70.1).
or linker domains, resulting in impaired ubiquitin ligase activity. The number of hematopoietic progenitor cells constitutively
Additionally, the oncogenic activity of selected CBL mutant variants mobilized into the blood of PMF patients is dramatically increased.
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was tested in vitro by monitoring its ability to confer growth factor The number of CD34 cells present in the peripheral blood in PMF
independence to the IL-3-dependent cell line 32D and by its ability is 360 times greater than in normal control participants and 18–30
to enhance the proliferation of transformed cells overexpressing WT times higher than in patients with PV or ET. These findings are so
FLT3. striking that some investigators have suggested that the quantitation
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Ikaros is a Kruppel-like zinc finger transcription factor that has of CD34 cells in the peripheral blood might serve as a means of
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pleiotropic functions important in the development of normal discriminating PMF from other MPNs. A level of 15 × 10 CD34
hematopoiesis and is encoded by the Ikaros family zinc finger 1 cells/L in peripheral blood allows differentiation of PMF from PV
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(IKZF1) gene located at 7p.12. Mutations involving IKZF1 have been and ET. The numbers of circulating CD34 cells tend to increase as
identified as a molecular event leading to transformation of chronic- the disease progresses, and there is a close correlation between patients
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phase MPNs to blast-phase disease, and appears to occur after the presenting with more than 300 × 10 CD34 cells/L of peripheral
acquisition of JAK2V617F. The mutational frequency in chronic- blood and imminent evolution to leukemia. These findings suggest
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phase MPN is very low at 0.2%, in contrast to 21% of blast-phase that PMF CD34 cell trafficking abnormality is caused by the inabil-
MPN patients, and is highly associated with del7p. The potential ity of these cells to be retained within the BM or their premature
effects of an IKZF1 mutation was studied in mouse primary progeni- release into the peripheral blood.
tor cells by creating IKZF1 deficiency with small hairpin RNA PMF is characterized not only by the constitutive mobilization of
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technology, which was associated with cytokine hypersensitivity and CD34 hematopoietic cells, but also endothelial progenitor cells into
increased p-STAT5 expression. the peripheral blood. Endothelial progenitor cell mobilization pre-
The enhancer of zeste homolog 2 (EZH2) gene located on chro- dominates during the prefibrotic phase of PMF, while hematopoietic
mosome 7q36.1 encodes the catalytic subunit of the histone meth- stem/progenitor cell mobilization occurs characteristically in more
yltransferase polycomb repressor complex 2 (PRC2), and mutations clinically advanced phases of the disease. This dysregulation of stem
in this gene have also been described in MPN patients. PRC2 is a cell trafficking likely ultimately leads to the seeding of extramedullary
multiprotein enzyme complex (EZH2, SUz12, EED, and YY1) sites with primitive hematopoietic and endothelial cells, which results
responsible for the trimethylation of lysine 27 on histone H3 in the production of EMH within the liver and spleen, as well as
(H3K27me3). Additionally, PRC2 can recruit other polycomb a variety of other organs. Several proteolytic pathways have been
complexes, DNA methyltransferases (DNMTs), and histone deacety- documented to play a role in cytokine-mediated stem cell mobili-
lases to the gene site, resulting in chromatin compaction and addi- zation. Proteases released by activated neutrophils cleave vascular
tional repressive activity. Upregulation of EZH2 gene expression has adhesion molecule-1 (VCAM-1) expressed by stromal cells, leading