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1320 Part X: Malignant Myeloid Diseases Chapter 86: Primary Myelofibrosis 1321

polycythemia and primary myelofibrosis. At least four other modifying HMGA2, a gene on chromosome 12, normally is not expressed
factors have been proposed to account for the different phenotypes and in humans and is implicated in mesenchymal tumors. HMGA2 was
the apparent absence of the mutation in a high proportion of patients expressed in 12 of 12 patients with idiopathic myelofibrosis studied,
with primary myelofibrosis: (1) gene dosage, (2) germline modifiers, implying that, if confirmed, expression of this gene in myeloid cells may
(3) predisposition alleles, and (4) additional somatic mutations. 66,68,73 play a role in the disease. 87
An example of each influence follows. There is an increasing JAK2 V617F
allele burden from essential thrombocythemia, to polycythemia vera, CENTRALITY OF CD34+ CELL EGRESS AND
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to primary myelofibrosis. For example, the JAK2 V617F allele burden
may be a key determinant of the degree of myeloproliferation and mye- NEOPLASTIC MEGAKARYOCYTOPOIESIS
loid metaplasia reflected by significantly higher levels of white blood Neoplastic megakaryopoiesis is the most prominent alteration in this
cell counts, CD34+ cell counts, lower platelet counts, and a higher fre- clonal myeloid disease and is responsible for most of its major mani-
quency of splenomegaly in homozygous polycythemia vera patients festations. Constitutive mobilization and circulation of CD34+ cells are
compared to their heterozygous counterparts. These findings are con- prominent features of the clonal expansion. This phenomenon, prob-
sistent with JAK2 V617F -positive chronic myeloproliferative disorders as ably, is the result of epigenetic methylation of the CXCR4 promotor, a
a biologic continuum with phenotypic presentation in part influenced resultant decrease in CXCR4 mRNA, decreased expression of CXCR4
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by JAK2 V617F mutational load. Myeloproliferative neoplasm predispo- on CD34+ cells, and their resultant enhanced migration into the blood
sition alleles could provide a selective advantage for the development of in primary myelofibrosis patients. 85
mutations in the JAK2 signaling pathway. A number of pre-JAK2 alleles, Circulating CD34+ cells in patients with primary myelofibrosis
which arise in cells prior to JAK2 mutation, may contribute to the phe- generate about 24-fold the number of megakaryocytes in culture than do
notype displayed. 68 CD34+ cells from normal subjects, express increased levels of BCL-XL,
A mutation in the thrombopoietin receptor gene, MPL, (MPL 515L/K ) and have delayed apoptosis. 86,87 Media conditioned with CD61+ cells
has been found in some mutant JAK2-negative patients with pri- (presumptive megakaryocytes) elaborated greater quantities of growth
mary myelofibrosis. The finding of an activating JAK2 (~50 percent factors and proteases, including TGF-β and metalloprotease-9, than did
of patients) or MPL (~4 percent of patients) mutation, which employs CD61+ cells generated from normal CD34+ cells.
JAK2 for signaling, reinforces the critical role of unregulated activation Circulating CD34+ cells in patients with primary myelofibrosis
of the JAK-STAT (signal transducer and activator of transcription) sig- also had a higher expression of eight genes (CD9, GAS2, DLK1, CDH1,
naling pathway in the pathogenesis of primary myelofibrosis. 75–77 Using WT1, NFE2, HMGA2, and CXCR4) than did normal CD34+ cells. These
next-generation gene whole-exome sequencing, primary myelofibrosis genes or subsets of them are likely related to disease pathogenesis and
patients who did not have mutated JAK2 or MPL genes (approximately were shown to be related to specific manifestations in patients (e.g.,
half of all patients) were only found to have an occasional somatic increased expression of CD9 and DLK1 with platelet count and WT1
mutation. 78 with severity score). 88
It came as a surprise that nearly a decade after the description of
the JAK2 mutation in primary myelofibrosis and related myeloprolif-
erative diseases, two groups reported, simultaneously, the presence of ENHANCED ANGIOGENESIS AND SPLENIC
CALR mutations in approximately 70 percent of patients with primary ENDOTHELIAL CELLS
myelofibrosis who did not carry the JAK2 or MPL mutations (35 percent Microvessel density and marrow blood flow are increased in patients
of total population). 79, 80 The CALR mutations were either from deletions with myelofibrosis. These changes may be related to an increase in
or insertions, explaining the failure to identify them by Sanger sequenc- circulating endothelial cell progenitors. The endothelial cells exam-
89
ing technology. The 52-bp deletion (type 1 mutation) or 5-bp insertion ined by laser microdissection, cell sorting, or cell culture from spleen
(type 2 mutation) are the most frequent types, accounting for approx- capillaries and splenic vein contained the JAK2 V617F mutation in 12 of
imately 85 percent of the CALR mutations. The CALR mutations are 18 patients studied who had the mutation in their granulocytes. A
missense for the gene’s final domain, encoding the calcium binding site. definitive explanation for this finding has not been established. 90
Patients with primary myelofibrosis and JAK2, CALR, or MPL
mutations may have an additional two to four somatic mutations. Some
of the principal genes mutated are TET2, ASXL1, DNMT3A, EZH2, DYSFUNCTION OF HEMATOPOIESIS
and IDH1, which are implicated in epigenetic regulation, and TP53 Neoplastic myeloproliferation usually is the dominant marrow abnor-
and CBL. As many as five somatic mutations could be identified in mality in the granulocytic and megakaryocytic lineages resulting in
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3 percent of patients, whereas 12 percent of patients with primary mye- intensely cellular marrows and mild to moderate blood granulocyto-
lofibrosis did not have an identifiable somatically mutated gene, using sis and thrombocytosis. Hypocellular hematopoiesis, resulting from
7
targeted next-generation whole-exome gene sequencing. However, exaggerated apoptosis of very early precursors, can be present initially
the proportion of these additional somatic mutations was less that that or emerge later, leading to granulocytopenia and/or thrombocytope-
detected by similar studies in polycythemia vera, suggesting that these nia. Anemia is a frequent finding and results from a combination of
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sequencing technologies, capable of uncovering only approximately decreased erythropoiesis, shortened red cell survival, and the effects of
1 percent of the genome, may leave unidentified additional somatic and splenomegaly on the distribution of red cells in the circulation. Hemo-
germline mutations present in a much greater proportion of primary lysis can be a prominent factor in some cases. Neoplastic megakary-
myelofibrosis patients. ocyte expansion and intense dysmorphogenesis of megakaryocytes
Isolated genetic findings in individual patients have included are constant features of the disease. Even in intensely fibrotic marrows
(13q14) deletions, mutation or overexpression of the retinoblastoma with severe decreases in erythroid and granulocytic precursors, clus-
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gene, 82,83 NF1 (17q11) deletions, RAS mutations in approximately one ters of megakaryocytes are easily found interspersed between collagen
in 20 patients studied, and occasional patients with mutations in KIT. bundles. The term megakaryocytic myelosis, one of the many synony-
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Uniparental disomy has been found on chromosomes 9p (site of JAK2, mous terms for the disease, exemplifies the constancy of this finding.
creating a second allele of V617F) and 1p. 83 The dominance of megakaryopoiesis may relate to the average fivefold

Kaushansky_chapter 86_p1319-1340.indd 1321 9/18/15 10:22 AM