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1296 Part X: Malignant Myeloid Diseases Chapter 84: Polycythemia Vera 1297

involving the expansion of erythrocytes, granulocytes, or leukocytes. level is not pathognomonic of PV; patients with primary familial and
The primary lesion associated with these disorders was discovered congenital polycythemia (PFCP) have levels of erythropoietin that are
in exon 14: A single nucleotide change JAK2 G1849T resulting in the as low or lower than in PV, and instances of normal erythropoietin
147
amino acid substitution V617F. Detection of the JAK2 V617F mutation levels occur in PV patients without apparent explanation. The latter is
provides a qualitative diagnostic marker for the identification of the more likely to occur in PV patients with exon 12 JAK2 mutations. 44
Philadelphia-chromosome-negative subgroup of MPNs, and differen-
tiates them from congenital and acquired reactive hematopoietic dis-
orders. In general, the JAK2 V617F allele burden is lower in ET patients ERYTHROID COLONY CULTURES
than in either PV or primary myelofibrosis (PMF). 131,132 In many, but In vitro assays of erythroid progenitor cells permit the study of their
not all, JAK2 V617F -positive PV patients, at least some progenitors exist responsiveness to erythropoietin. In PV patient samples, erythroid
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that became homozygous for the JAK2 V617F mutation by UPD acquired BFU-E progenitors grow in culture without added erythropoietin,
by mitotic recombination. 37,133 forming colonies that are termed EECs. Detection of EECs in cultures
Allele-specific polymerase chain reaction (PCR) is widely used for of blood or marrow had previously been the most specific test for
single nucleotide polymorphism (SNP) genotyping; the technique is PV. 12,14,148 In one study, all patients with PV, but none with secondary
based on amplification of DNA by an allele-specific primer matching or other causes of polycythemia, formed EECs in vitro. Rare EECs
149
the polymorphism at the 3′ position. This approach is directly appli- may, at times, be observed in PFCP and in congenital disorders of
cable to analysis of JAK2 V617F because the mutation (G1849T) is analo- hypoxia sensing, but unlike EECs in PV, these are abrogated by pre-
gous to a SNP. To improve the specificity, sensitivity, and reliability for treatment with erythropoietin and erythropoietin receptor-blocking
quantitating the JAK2 V617F allele burden, two modifications of technique antibodies. 150,151
were incorporated: Inclusion of a second mismatch at the –1 position, In experienced hands, the EEC assay is a specific and sensitive
and substitution of a modified locked nucleic acid at the –2 position. A means for detecting PV. It may be useful in diagnosing patients with
study comparing 11 different techniques was undertaken and carried unusual presentations of PV, such as Budd-Chiari syndrome, 85,89,152,153
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out in 16 laboratories using various testing platforms. Although five of isolated thrombocytosis, or the rare JAK2 V617F -negative PV patient. It
the 11 techniques were similarly reliable for quantification of JAK2 V617F has not been fully standardized, however, and is expensive and labori-
loads that were equal to or greater than1 percent of total JAK2 V617F , the ous, so it is now used primarily in a research setting where it remains
allele-specific quantitative PCR technique could detect 0.2 percent of informative.
JAK2 V617F .
The majority of laboratories analyze quantitative assays of JAK2 V617F
allele burden from (clonal) granulocytes; nonquantitative analyses use MARROW FINDINGS
total leukocytes, whole blood, or marrow for screening. A proportion In PV, the marrow is characteristically hypercellular, with an increase
of JAK2 V617F -negative assays are positive using sensitive quantitative in erythroid and granulocytic precursor cells and megakaryocytes.
analyses. Plasma has been used for detection of the JAK2 V617F DNA Whereas marrow morphology is part of the World Health Organization
134
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and mRNA mutation and zygosity state, 135,136 but plasma analysis is not (WHO) diagnostic criteria of PV, the morphologic features have not
reliable. 137 yet been validated and may be subject to inter- and intraobserver vari-
ation. Marrow morphology in patients with JAK2 exon 12 mutations
JAK2 Exon 12 Mutations may be subtly different, with subtle or no megakaryocytic clustering
45,155
Although the most common JAK2 mutation is a single SNP in exon 14, and a lack of panmyelosis. Absent or decreased iron stores are seen
many MPN cases negative for the exon 14 JAK2 V617F mutation may carry in the marrow of most PV patients. Various cytogenetic findings have
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one of a number of mutations in exon 12. Almost 40 different muta- been reported, but they are not sufficiently specific to be of diagnostic
tions in exon 12 have been identified within codons 536 to 547, includ- utility (Chap. 13).
ing substitutions, deletions, and duplications. 45,138–142 In addition to the
various mutations observed in this region, the proportion of mutations CLONALITY IN FEMALE SUBJECTS USING
within a given sample may be small and therefore difficult to detect in ASSAYS EMPLOYING X-CHROMOSOME–BASED
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the high background of a normal sequence. JAK2 exon 12 mutations POLYMORPHISM
have been observed primarily in younger patients and in patients with
isolated erythrocytosis, and thus may represent a somewhat different PV results from an acquired mutation in a pluripotent hematopoietic
phenotype from JAK2 V617F -positive PV. stem cell. Clonality studies based on the phenomenon of X-chromosome
inactivation show that red cells, granulocytes, platelets, monocytes,
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and B lymphocytes are all part of the neoplastic clone. 13,158 The majority
ERYTHROPOIETIN LEVELS of T lymphocytes and natural killer cells are polyclonal, but a small
PV is distinguished by the fact that erythroid cells proliferate even in proportion of these cells are also derived from the PV clone ; this is
9
the absence of normal levels of erythropoietin; thus, one would expect presumed to be the result of the presence of long-lived, normal T cells
that at high hematocrit levels, the production of erythropoietin would that preceded the development of the clone and younger, clonal cells.
be inhibited and serum levels consequently reduced. Indeed, several Unfortunately, the applicability of X-chromosome inactivation for the
studies have documented serum erythropoietin levels below the normal differential diagnosis of PV is hampered by the many methodologic and
159
reference range in patients with PV. 143–145 conceptual differences that have drawn conflicting conclusions. Some
160
Patients with secondary polycythemia usually have normal to ele- of these discrepancies result from using two different approaches to
vated erythropoietin levels, although considerable overlap exists in the distinguish the active from inactive X-chromosomes (Chap. 10). 161–164
range of erythropoietin levels between patients with PV and those with In a study of approximately 100 female PV patients, their reticulocytes,
secondary polycythemia rendering the test of marginal value in distin- platelets, and granulocytes were always clonal, with the exception of a
guishing between diagnostic possibilties. 144,146 An elevated erythropoie- few patients who converted to polyclonal hematopoiesis after therapy
tin level generally excludes the diagnosis of PV, but a low erythropoietin with IFN-α. 14

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