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JAK2V617F as progression marker in CMPD and as cooperative mutation in AML with trisomy 8 and t(8;21): a comparative study on 1103 CMPD and 269 AML cases

Owing to the high incidence of the V617F point mutation in the JAK2 gene on 9p24 in the BCR-ABL-negative chronic myeloproliferative diseases (CMPD), molecular screening for the respective mutation achieved a key role for patients with polycythemia vera (PV), essential thrombocytosis (ET), chronic idiopathic myelofibrosis (CIMF) and in cases with the suspicion of a CMPD.1, 2, 3 The mutation was further detected in some cases of chronic myelomonocytic leukemia (CMML),1, 3, 4 and was reported in acute myeloid leukemia (AML) in lower frequencies, but the number of analyzed cases so far is limited.1, 3, 5, 6, 7 In addition, the association of JAK2V617F to certain cytogenetic aberrations remains to be clarified.

To further improve understanding of the cooperating role of JAK2V617F with other genetic markers, we screened 1372 patients with various CMPD and AML by a melting curve-based assay and correlated the results to cytogenetic data. The cohort was composed of 681 males and 691 females with a median age of 63.2 years (range=16.4–90.1). Within the total cohort, 1103 patients had a diagnosis of CMPD (PV: n=179 patients; CIMF: n=60; ET: n=205) and 632 cases had chronic myeloproliferative disorders, which could not be clearly classified (CMPD-U). In addition, 27 patients with the myeloproliferative form of CMML and 269 patients with AML were included (de novo AML: n=178; secondary acute myeloid leukemia (s-AML) after CMPD: n=27; s-AML after myelodysplastic syndrome (MDS): n=25; therapy-related AML (t-AML) after treatment of a previous malignancy: n=39) (median age=63.4 years, range=18.3–89.8). The group with de novo AML was not unselected, but selected to have a balanced cohort with all cytogenetic subgroups represented (normal: n=34; complex aberrant: n=32, t(8;21): n=20, inv(16): n=21, t(15;17): n=20, t(11q23): n=5, +8: n=46). Diagnoses were based on the WHO classification according to bone marrow and peripheral blood cytomorphology, histology and clinical criteria.

Chromosome banding analysis was performed – in part of cases combined with interphase fluorescence in situ hybridization (FISH) – in 820/1372 cases (PV: 83/179; ET: 93/205 cases; CIMF: 23/60, CMPD-U: 347/632, CMML: 23/27 patients; AML: 251/269). Chronic myeloid leukemia (CML) was excluded in all patients by interphase FISH and/or reverse transcriptase-polymerase chain reaction for BCR-ABL.

JAK2V617F mutational screening was performed either on bone marrow or on peripheral blood samples (in the CMPD, 30% on bone marrow and 70% on peripheral blood; in AML, 95% on bone marrow and 5% on peripheral blood). A melting curve-based LightCycler assay was performed with the forward primer as described previously.8 The sensitivity of this assay as estimated by limited dilution assays of JAK2V617F mutated in JAK2V617F unmutated cDNA was 1:100. Homozygosity was defined as JAK2mut/wild type ratios of >1. This remains an approximation and might be an underestimation; however, cases with apparent heterozygosity may be misinterpreted due to a mixture of homozygous and healthy cells in samples with lower percentages of JAK2V617F mutated cells.

In the patients with a CMPD, we found the JAK2V617F mutation in 662/1103 of all cases (60.0%). The mutation rate was highest in PV (153/179; 85.5%), being followed by ET (123/205; 60.0%), CIMF (36/60; 60.0%) and CMPD-U (345/632; 54.6%; Table 1). These frequencies were in the range of previous studies.1, 2, 3

Table 1 Frequency of JAK2 mutations in total and of homozygous mutations and of aberrant karyotypes in dependence on the JAK2 mutation status in cohorts with diverse CMPD and with AML

CMML showed a lower frequency of JAK2V617F with 5/27 cases (18.5%). In AML, the overall JAK2 mutation rate of all analyzed AML cases was not calculated because this group was selected showing an overrepresention of s-AML after CMPD. In de novo AML, 11/178 selected AML cases were JAK2V617F mutated, whereas in s-AML following a CMPD, the mutation rate was higher with 16/27 cases (59.3%) equally to the rate of JAK2 mutations in the CMPD. In the cohort with s-AML after MDS, there was no mutated case. In t-AML, the JAK2 mutation rate was 2/39 (5.1%). With two exceptions in the CMPD8 cohort, all mutations showed the previously described 1849g>t exchange leading to a valine to phenylalanine substitution at codon 617.

Homozygous mutations were most frequent in PV (106/153; 69.3%), closely followed by CIMF (20/36; 55.6%) and CMPD-U (166/632; 48.1%). In ET, the homozgyous mutation rate was lowest (19/123; 15.4%). In CMML, one of two mutated cases was homozygous. In de novo AML 7/11 cases were homozygously mutated, in s-AML following a CMPD 9/16 cases (56%), and in t-AML both JAK2 mutated cases were heterozygous.

In the cohorts with CMPD, aberrant karyotypes were most frequent in CIMF (12/23; 52.2%), being followed by PV (19/83; 22.9%). In CMPD-U, the karyotype aberration rate was 43/347 (12.4%). In ET, karyotype aberrations were found in 7/93 (7.5%) only. Overall JAK2 mutated cases were more frequently associated with aberrant karyotypes than nonmutated cases. This was significant or borderline significant in PV (P=0.028), CIMF (P=0.071), CMPD-U (P=0.063) and CMML (P=0.067; Table 1).

The specific chromosomal aberrations in the diverse cohorts are shown in Table 2. Subsuming the single entities, JAK2V617F mutations were most frequently associated with gain of the short arm of chromosome 9 either due to numerical gain of chromosome 9 or due to structural changes involving 9p: PV showed the respective aberrations in 6/18 cases (33%) – in four cases due to trisomy 9, in two cases due to an unbalanced rearrangement der(9;18)(p10;q10). CIMF demonstrated in the JAK2V617F mutated cases one case of a der(9;18)(p10;q10) leading to 9p trisomy. In the five chromosomally aberrant JAK2V617F mutated ET cases, there was one t(9;13)(p24;q22) involving the JAK2 locus and one case of a trisomy 9. JAK2V617F mutated CMPD-U demonstrated gain of chromosome 9 and/or 9p in 10/27 patients (37.0) – in 7/27 patients due to trisomy 9, and in three further cases due to structural changes of 9p (dup(9)(p13p21); t(1;9)(p11;p11); +der(1;9)(q10;p10). Chromosome 9 aberrations were found in 5/9 cases (67%) in de novo AML and in 2/9 (22%) in s-AML after CMPD. Trisomy 9 did not show up in unmutated JAK2 cases in the diverse CMPD.

Table 2 Specific chromosomal abnormalities in cases with JAK2V617F mutations and JAK2 wild type cases with chromosomal aberrations in the cohorts with diverse CMPD and AML

In 178 de novo AML cases, 11 cases were found to be positive for JAK2V617F. The total frequency could not be calculated because this cohort was selected to have all cytogenetic groups represented. Like in CMPD, aberrations of chromosome 9 (either as gain of the whole chromosome or as gain of 9p only) represented frequent chromosomal changes in JAK2V617F-mutated de novo AML in 5/9 cases (56%). However, in AML, trisomy 9 was never found in the context of a complex aberrant karyotype. The most frequent aberration associated with JAK2V617F in de novo AML was trisomy 8 (7/9; 77.8%) (in four cases showing an overlap with gain of 9p or +9). To verify an initially suspected association with +8, this group was extended to 46 cases, overall. The total frequency of JAK2V617F in AML with +8 was 7 in 48 cases (14.6%). In contrast, in the JAK2 wild type and chromosomally aberrant cases, complex aberrations (32/129; 24.8%) were more frequent than +8 (23/129; 17.8%) or +9. In contrast to the findings in de novo AML, chromosomally aberrant cases of JAK2V617F mutated s-AML after CMPD showed complex aberrant karyotypes in a high frequency (6/9; 67%), whereas trisomy 9 or 9p-rearrangements occurred in three cases only. In t-AML, both JAK2-mutated patients were positive for the t(8;21)/AML1-ETO. In contrast, all 18 de novo AML carrying t(8;21) were negative for the JAK2 mutation.7 Recently, an association of JAK2V617F with t(8;21) also has been described.5, 6 However, in both studies, there seemed to be no correlation to a previous chemotherapy.

These results contribute some interesting aspects in the discussion of the role of the JAK2 mutation in myeloid malignancies.

First, the increase of the JAK2V617F mutation rate from ET to CIMF and to PV suggests that these disorders overlap or represent a continuum.1, 3, 4 This is also in line with the difficult discrimination of the various CMPD with respect to the clinical course, cytomorphology and cytogenetics. The higher rate of homozygous JAK2 mutations in the PV and CIMF when compared with ET suggested a correlation of homozygous mutations with a more aggressive course of the CMPD or with disease progression. This is supported by analysis of the ratio JAK2mut/JAK2wt, which can be used for an approximate quantification of the JAK2-mutated clone, which was lower in ET.

Secondly, the high incidence of numerical gains of chromosome 9 or of 9p in patients with JAK2V617F mutations in the diverse CMPD as well as in s-AML after CMPD in contrast to JAK2 wild type CMPD suggests that acquisition of +9 may lead to progress or blast crisis in JAK2V617F-mutated CMPD. This theory is further supported by the high incidence of homozygous JAK2V617F mutations of at least 70% in the cases with trisomy 9 in the CMPD and in AML. A close association between trisomy 9 and V617F in the CMPD had been reported by Campbell et al.,9 who found the respective mutation in nearly all cases with trisomy 9. In addition, trisomy 9 represented not only the most frequent chromosomal aberration but also the only ‘specific’ chromosomal aberration in the CMPD in this study. All other chromosomal changes like trisomy 8 are found as additional changes in other myeloid malignancies in equal frequencies.

Almost all cases with del(20q) had JAK2V617F, whereas loss of chromosome 7 was more frequent in the JAK2 unmutated cohorts. Loss of chromosome 7 might, therefore, represent an aberration which does not cooperate with JAK2 mutations. These findings strongly support different patterns of cytogenetic aberrations in JAK2 mutated and unmutated cases.9

The high incidence of JAK2 mutations of 57% in s-AML after CMPD was in correlation to previous studies ranging from 36 to 100% in this subgroup.1, 3 As most of these s-AML cases were homozygously mutated, acquisition of the JAK2 V617F mutation probably plays an important role in the leukemic transformation of a CMPD.4 Further, s-AML after a CMPD in all cohorts showed the highest rate of complex aberrant karyotypes. Thus, the transformation from CMPD to AML seems to be a sequence of the JAK2V617F mutation, loss of JAK2wt, duplication of one chromosome 9 and acquisition of further cytogenetic aberrations leading to complex aberrant karyotypes.

Third, in de novo AML and in t-AML, JAK2V617F mutations were less frequent with <5% of all cases, respectively. This was in the range of other known activating mutations in AML like the FLT3-TKD or NRAS. The JAK2V617F mutation in de novo and in t-AML in contrast to s-AML after CMPD has no association with complex aberrations but with specific aberrations: There was a strong correlation of the JAK2V617F mutation with trisomy 8 (78% of all mutated cases) suggesting that V617F may cooperate with +8 (or a gene on chromosome 8) in leukemogenesis of AML. In addition, it may serve as a cooperating mutation in concert with t(8;21).

These results suggest that JAK2V617F may be involved in different leukemogenic mechanisms and plays two different roles depending on the history of AML. (1) It seems to be a typical cooperating and activating mutation in AML (specifically together with +8 and t(8;21)/AML1-ETO). (2) It seems to be part of a multistep mutagenesis in CMPD ending up with a complex aberrant karyotype in s-AML after CMPD.


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Schnittger, S., Bacher, U., Kern, W. et al. JAK2V617F as progression marker in CMPD and as cooperative mutation in AML with trisomy 8 and t(8;21): a comparative study on 1103 CMPD and 269 AML cases. Leukemia 21, 1843–1845 (2007).

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