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The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera
Version of Record online: 20 DEC 2005
Copyright © 2005 American Cancer Society
Volume 106, Issue 3, pages 631–635, 1 February 2006
How to Cite
Tefferi, A., Lasho, T. L., Schwager, S. M., Strand, J. S., Elliott, M., Mesa, R., Li, C.-Y., Wadleigh, M., Lee, S. J. and Gilliland, D. G. (2006), The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera. Cancer, 106: 631–635. doi: 10.1002/cncr.21645
- Issue online: 20 JAN 2006
- Version of Record online: 20 DEC 2005
- Manuscript Accepted: 18 AUG 2005
- Manuscript Revised: 17 JUL 2005
- Manuscript Received: 17 JUN 2005
- polycythemia vera;
- mutant allele;
- myeloid cells;
Several studies have recently reported on the occurrence of a JAK2V617F mutation in myeloid cells from the majority of patients with polycythemia vera (PV). The clinical relevance of this novel observation currently is under study.
In a single institutional study, mutation screening for JAK2V617F was performed in DNA derived from archived blood granulocytes from 63 consecutive patients with PV in whom current diagnostic criteria were strictly applied and the diagnosis confirmed by bone marrow histology.
The JAK2V617F mutant allele was detected in 58 of the 63 patients (92%) with 21% homozygosity. The clinical phenotype of the five patients with the wild-type allele was otherwise typical for the disease. A statistical comparison between JAK2V617F heterozygotes (n = 45 patients) and homozygotes (n = 13 patients) did not reveal any significant associations with regard to age, gender, leukocyte or platelet count at the time of diagnosis, duration of disease, or the incidences of thrombosis or bleeding. However, compared with their heterozygote counterparts, JAK2V617F homozygote patients displayed a significantly higher hemoglobin level at the time of diagnosis (P = 0.001), an increased incidence of pruritus (69% vs. 38%; P = 0.04), a higher rate of fibrotic transformation (23% vs. 2%; P = 0.009), and higher PRV-1 transcript levels in their blood granulocytes (P = 0.07).
The results of the current clinical study support previous laboratory observations that link JAK2V617F with the PV phenotype by demonstrating a mutant allele dose effect on erythrocytosis and clinical and laboratory features characteristic of PV. Cancer 2006. © 2005 American Cancer Society.
An activating JAK2 mutation recently has been associated with a wide spectrum of myeloproliferative disorders (MPD), both typical1–5 and atypical.6, 7 The newly identified somatic point mutation is a G-C to T-A transversion, resulting in the substitution of valine by phenylalanine at codon 617 (JAK2V617F). To our knowledge, to date this particular mutation has not been detected in either control subjects or germline tissue, confirming that the allele is not a common polymorphism in the general population. The JAK2V617F mutation in MPD is usually heterozygous whereas molecular cytogenetic and polymerase chain reaction (PCR)-based studies have indicated that homozygosity for the mutant allele is a result of mitotic recombination rather than loss of heterozygosity. Functional studies have consistently demonstrated that the mutation results in constitutive JAK2 activity and enhanced JAK2-STAT signaling.2–5 Furthermore, JAK2V617F induces cytokine hypersensitivity in cell lines as well as erythrocytosis in mice, both of which are reminiscent of the polycythemia vera (PV) phenotype in humans.2–4
Consistent with the above mentioned in vitro and animal-based observations, the mutational frequency for JAK2V617F is reported to be the highest in PV. However, this particular association appears to be neither invariable nor specific because the mutation may not be detected in up to 35% of patients with PV,3 whereas a substantial minority of patients with both classic and atypical MPD might display the same mutation, even in the absence of a shared phenotype.6, 7 Conversely, JAK2V617F homozygosity is relatively frequent in patients with PV (approximately 25–33%), although it seldom occurs in other MPD patients.6, 7 Such observations warrant a closer examination of the clinical phenotype that is associated with different JAK2V617F mutational profiles in patients with PV.
MATERIALS AND METHODS
The current study was approved by the Institutional Review Board (IRB) and the study patients were selected based on the availability of archived peripheral blood granulocytes. The diagnosis of PV, acute myeloid leukemia (AML), and myelofibrosis with myeloid metaplasia (MMM) was made according to the World Health Organization (WHO) and Italian Consensus criteria.8, 9 Fibrotic transformation in particular was defined by a constellation of clinical and bone marrow histologic findings that included the appearance of leukoerythroblastosis and dacryocytosis in the peripheral blood accompanied by a substantial decline in the hemoglobin level and progressive splenomegaly. In addition, these clinical and peripheral blood indicators of disease transformation required confirmation by bone marrow examination (a substantial increase in overall bone marrow cellularity associated with marked megakaryocytic dysplasia and an increase in reticulin fibrosis). In all instances, bone marrow histology was reviewed by hematopathologists at the Mayo Clinic and whenever possible was rereviewed by one of the authors (C.Y.L.). In addition, the senior author (A.T.) reviewed all cases to confirm the diagnosis and accuracy of the patient data. Peripheral blood samples were collected under an IRB-approved protocol between February 2001 and March 2005 in a consecutive cohort of patients with PV with either a new or previously established diagnosis. Laboratory parameters for statistical analysis were established from the time of the initial diagnosis in all instances. All patients had complete follow-up information and episodes of disease complications were reviewed carefully for accuracy.
Granulocytes were prepared by double-density gradient centrifugation (Histopaque-1077™ layered over Histopaque-1119™; Sigma Diagnostics, St. Louis, MO).10, 11 Genomic DNA was extracted using the QIAamp Blood Mini Kit (Qiagen, Valencia, CA) and amplified with PCR. Successful amplification was confirmed by electrophoresis on an ethidium bromide-impregnated 1.5% agarose gel. Each 50-uL PCR reaction contained approximately 25 ng of DNA template, 5 uL of 10X Roche Buffer (final concentration of MgCl2 of 1.5 mM), 1.5 U of Taq polymerase (Roche, Indianapolis, IN), 0.8 mM dNTPs (Roche), and 20 pM each of sense and antisense primers (5′-TGCTGAAAGTAGGAGAAAGTGCAT-3′ and 5′-TCCTACAGTGTTTTCGTTTCAA-3′, respectively).6 PCR cycling parameters were 1 cycle of 94 °C for 2 minutes; and 35 cycles of 94 °C for 30 seconds, 52 °C for 40 seconds, and 72 °C for 40 seconds; followed by 1 cycle of 72 °C for 2 minutes. PCR products were cleaned using the QIAquick PCR purification Kit (Qiagen). Fluorescent dye chemistry sequencing was performed, using the same primers used for amplification, on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Foster City, CA). Sequencher 4.2 (Gene Codes Corporation, Ann Arbor, MI) and GenBank Accession NM_004972 (JAK2 mRNA) and the corresponding region from the NC_000009 chromosome 9 contig were used for sequence analysis (GenBank, National Institutes of Health, Bethesda, MD).6 Quantitative measurement of granulocyte PRV-1 expression was made according to a previous publication.12
Descriptive and statistically analyzed data noted at the time of diagnosis were obtained from the entire cohort of patients. Both three-way (wild-type vs. heterozygous vs. homozygous mutation) and two-way (heterozygous vs. homozygous) comparisons were performed. Categoric variables were compared using chi-square statistics. Comparison between categoric and continuous variables was performed using either the Mann–Whitney U test or the Kruskal–Wallis test. Because only three deaths occurred during the study period, survival analysis was not performed. A separate dataset of 171 patients with PV who were accrued to a previously published, Internet-based protocol study was used to validate some of the findings from the current study.4 Such analysis was restricted to patient demographics and thrombohemorrhagic events because all other data were not available from the Internet protocol. All data were analyzed by using SAS software (SAS Institute, Inc., Cary, NC).
The study cohort was comprised of 63 consecutive patients (with a median age of 55 yrs [range, 18–83 yrs]; 28 females) with PV who were evaluated at the Mayo Clinic either at or after the time the initial diagnosis was made. In all instances, laboratory parameters at the time of diagnosis were recovered and are outlined in Table 1. Because of issues with the reliability of outside information regarding spleen size, this particular information was not included in the current analysis. At the time of last follow-up, the median follow-up for the entire cohort was 33 months (range, 3–324 mos) from the time of the initial diagnosis. During this follow-up period, three patients had died (two from acute leukemia and one from metastatic nonsmall cell lung carcinoma). Disease transformation into either AML or MMM occurred in two patients and four patients, respectively (Table 1). The median time from diagnosis to the time of mutation analysis was 12 months (range, 0–306 mos).
|All patients (n = 63)||Wild-type JAK2V617F (n = 5)||Heterozygous JAK2V617F (n = 45)||Homozygous JAK2V617F (n = 13)||P value, three-way||P value, two-way|
|Median age in yrs (range)||55 (18–83)||35 (19–82)||57 (16–83)||56 (27–76)||0.28||0.93|
|Median duration of disease in mos (range)||33 (3–324)||42 (10–92)||27 (3–324)||60 (3–240)||0.32||0.14|
|Median time from diagnosis to JAK2V617F analysis in mos (range)||12 (0–306)||10 (0–92)||9 (0–306)||35 (0–185)||0.31||0.14|
|Median hemoglobin at the time of diagnosis in g/dL (range)||18.2 (15.8–24)||19.4 (17–24)||17.6 (15.8–21.5)||20.7 (15.8–22.5)||0.001||0.001|
|Median leukocyte count at the time of diagnosis, × 109/L (range)||10.9 (5.2–25)||9.1 (7.7–12)||11.7 (5.2–25)||10.8 (6.2–19.2)||0.19||0.37|
|Median platelet count at the time of diagnosis, × 109/L (range)||493 (221–1345)||700 (292–819)||515 (221–1345)||460 (339–702)||0.57||0.38|
|% with decreased erythropoietin level||78% (n = 58)||100% (n = 5)||79% (n = 42)||64% (n = 11)||0.26||0.31|
|Median PRV-1 transcript level (range)||1.01 (0.83–1.28) (n = 28)||1.14 (n = 1)||1.02 (0.87–1.28) (n = 20)||0.97 (0.83–1.12) (n = 7)||0.10||0.07|
|% with thrombosis history||18%||20%||20%||8%||0.58||0.30|
|% with bleeding history||8%||20%||9%||0%||0.33||0.27|
|% with pruritus||54%||60%||38%||69%||0.10||0.04|
|% with cytogenetic abnormalities||10% (n = 48)||0% (n = 4)||12% (n = 34)||10% (n = 10)||0.77||0.88|
|% with transformation into myelofibrosis||8%||0%||2%||23%||0.02||0.009|
|% with transformation into acute leukemia||3%||0%||4%||0%||0.66||0.44|
The JAK2V617F mutation was detected in 58 of the 63 patients (92%), 13 of whom (22%) were homozygous for the mutant allele. The number of patients with the wild-type allele was too small (five patients) to allow a valid comparative analysis involving this group of patients. Nevertheless, as outlined in Table 1, the patient demographics and clinical features of these patients were not significantly dissimilar from those in whom the mutant allele was detected.
A comparison between the homozygote patients and the heterozygote patients revealed significantly higher hemoglobin levels at the time of diagnosis in the former group (P = 0.001). Similarly, a significantly higher proportion of patients with homozygous JAK2V617F, compared with those with the heterozygous mutation, reported disease-associated pruritus at some time during their clinical disease course (69% vs. 38%; P = 0.04). Patients who were homozygous for JAK2V617F also were more likely to experience disease transformation into MMM (three of four patients with fibrotic transformation had the homozygote mutation; P = 0.009) but not AML (both patients with leukemic transformation were heterozygotes; P = 0.44). Information regarding bone marrow reticulin stain at the time of diagnosis was available in 28 patients, including 2 with the wild-type allele, 20 with a heterozygous mutation, and 6 with a homozygous mutation. The respective number of patients with > Grade 1 reticulin fibrosis were 0, 2, and 0 (P = 0.9) (grading system described in the study by Yoon et al.13). Granulocyte PRV-1 expression studies were performed in 28 patients and demonstrated a trend toward a significantly higher transcript level in the homozygote patients (Table 1). Conversely, the incidences of thrombosis and bleeding were found to be similar between the three mutational categories (Table 1). A complete blood count was available at the time of mutation analysis and demonstrated no significant difference in either the hemoglobin level or platelet count, but there was a trend toward a significant difference in the leukocyte count that registered higher in those patients who were homozygous for the mutant allele (P = 0.06).
With regard to correlations between JAK2V617F and gender, age, duration of disease, and thrombohemorrhagic events, the results of the current study were compared with those obtained from a separate database of 171 patients accrued to an Internet-based protocol study sponsored by the Harvard Institutes of Medicine (Table 2). Both the Mayo Clinic and the Harvard studies disclosed a longer duration of disease in patients with homozygote JAK2V617F compared with patients with the heterozygote mutation (Tables 1 and 2). This difference was found to be statistically significant in the Harvard study (P = 0.02), but not in the Mayo study (P = 0.14).
|All patients (n = 171)a||Wild-type (n = 43)||JAK2V617F heterozygotes (n = 80)||JAK2V617F homozygotes (n = 41)||P value|
|Median age in yrs (range)||59 (32–84)||58 (33–85)||59 (33–80)||62 (43–85)||0.16|
|M/F ratio, no. (%)||82/89 (48/52)||29/14 (67/33)||35/45 (44/56)||16/15 (39/61)||0.97|
|Median duration of disease in mos (range)||60 (4–433)||57 (9–249)||51 (4–264)||89 (11–433)||0.02b|
|Thrombosis history, no. (%)||31 (18)||9 (21)||17 (21)||4 (10)||0.23|
The current findings regarding the significantly higher hemoglobin levels as well as the increased risk of fibrotic transformation in patients with homozygous JAK2V617F are in keeping with in vitro observations regarding the link between JAK2V617F and the proliferative phenotype in MPD that favors erythrocytosis. It should be remembered that, in vitro, JAK2V617F induces erythropoietin hypersensitivity in cell lines2, 4, 5 and that such JAK2V617F–induced cell proliferation signals were inhibited by small molecule inhibitors of JAK2.4 Similarly, this particular mutation has been shown to induce erythrocytosis in mice transplanted with murine bone marrow that was transduced with a retrovirus containing JAK2V617F.2 Therefore, although the JAK2V617F mutational event is ubiquitous among patients with chronic myeloid disorders, the extent of its presence in patients with PV (single vs. double mutant allele dose) might influence the degree of both erythroid and megakaryocytic proliferation that translates clinically into increased levels of hemoglobin and myelofibrosis, respectively. Our finding of an association between the homozygous mutation and a higher leukocyte count during the course of the disease further supports this contention. By the same token, it is conceivable that JAK2V617F heterozygosity in PV patients attenuates the complete phenotype otherwise forced by homozygosity for the mutation. Such a contention is supported by the in vitro demonstration of a negative dominant effect for wild-type JAK2 during contransfection experiments involving both wild-type and mutant JAK2V617F.2 Conversely, the results of the current study suggest that a double dose of JAK2V617F might not contribute directly to leukemic transformation in PV patients, although the number of events was too small to be certain.
An alternative explanation for the observations made in the current study considers the overlap in clinical phenotype between PV and essential thrombocythemia (ET). Accordingly, the right tail of the hematocrit-based Gaussian distribution for the population of patients with ET is likely to include some patients who are biologically more akin to PV patients whereas the opposite is expected with regard to the left tail of the population curve in PV. At the same time, it has become evident that homozygote JAK2V617F is rare in ET despite the fact that approximately 50% of such patients are heterozygous for the mutant allele.1–5 Conversely, greater than 20% of patients with PV are homozygous for JAK2V617F. Therefore, the findings from the current study might reflect the inadequacy, from the biologic standpoint, of current diagnostic criteria in distinguishing PV from ET that is now being exposed at the molecular level. The demonstration of a near-significant correlation between JAK2V617F mutational status and neutrophil PRV-1 expression in the current study lends further support for such an explanation and is consistent with the findings from other studies.7, 14
The current study patients with the wild-type allele were phenotypically indistinguishable from those carrying the mutant allele (Table 1). Another study by Baxter et al. has similarly commented on the lack of atypical features in JAK2V617F-negative PV patients.1 The particular observation suggests the presence of either a biologically equivalent abnormality of other JAK/STAT pathway molecules or their regulatory elements in JAK2V617F-negative patients or a complex pathogenetic relevance for the particular mutation. In this regard, it is possible that JAK2V617F represents a modifier subclone rather than a disease-causing mutation. Regardless, this particular observation confirms the infrequent absence of JAK2V617F in bona fide cases of PV, although it is legitimate to question the role of assay sensitivity in this regard.1, 7
The clinical relevance of JAK2V617F was examined in a limited fashion by previous studies as an addition to the main objective, which was the original description of the mutation. To our knowledge to date, seven studies have reported on the association between JAK2V617F and both typical and atypical MPD.1–7 Six of these studies included patients with PV; however, clinical correlations were attempted in only three of these six studies.1, 3, 4 In one study,4 significant associations were demonstrated between the presence of a mutant allele and female gender in patients with PV (83% vs. 64%). The number of patients in the current study with the wild-type allele was too small to make a valid statement concerning this specific finding. Conversely, an association between gender and JAK2V617F was not apparent in either a group of patients with ET1, 4 or in a general MPD patient group.3 The study by Levine et al. also reported a significant association between homozygous JAK2V617F and a longer duration of disease in patients with either PV or ET.4 Restricting the analysis to patients with PV only did not appear to alter the particular observation (Table 2). The current study also demonstrated a trend toward an association between homozygous JAK2V617F and a longer disease duration (P = 0.14). It is noteworthy that a third study3 also found a similar pattern of difference with regard to the median duration of disease between homozygote and heterozygote MPD patients and suggested the existence of a two-step process to acquire homozygosity as an explanation.3 However, the study by Baxter et al.1 did not disclose a time of diagnosis-dependent difference in the prevalence of homozygous JAK2V617F and longitudinal studies with serial mutation screening are needed to clarify the issue further.
The study by Kralovics et al.3 suggested a higher incidence of myelofibrosis, hemorrhage, and thrombosis in MPD patients with the JAK2V617F mutation compared with those without it. Subset analysis was not available in that particular study, although two other studies did not report any apparent association between the presence of the mutant allele and thrombohemorrhagic events in patients with either ET1, 4 or PV.4 Similarly, both the current study and a reanalysis of the patient database from the study by Levine et al.4 (Table 2) failed to disclose a difference in the frequency of thrombotic events in PV patients carrying either the heterozygous or homozygous JAK2V617F mutation. It is possible that the results of the study by Kralovics et al.3 might have been confounded by the inclusion in the analysis of patients with all three diagnoses (PV, ET, and MMM). Nevertheless, a larger study is needed to determine whether the observations from the current study indeed represent a biologic effect of the mutant allele dose or an artefact of a diagnostic overlap between ET and PV.