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The constitutively active JAK2V617F somatic point mutation is a hallmark discovery in understanding the molecular pathogenesis of Ph- chronic myeloproliferative disorders (cMPDs). It is reported as a recurrent genomic lesion occurring in up to 97% of patients (pts) with PV. It is also found in 50–70% of pts with essential thrombocythemia (ET) and 30–50% of pts with idiopathic myelofibrosis (IMF). Thus, a significant proportion of ET and IMF pts are JAK2V617F negative. However, in addition to JAK2-negative PV pts they can have recurrent chromosomal abnormalities (Vizmanos et al Leukemia 20: 534, 2006). Moreover, JAK2 has been shown to form fusion hybrid proteins with three different genes: TEL/ETV6, BCR and PCM1 in cMPDs and lymphoid neoplasms. We therefore hypothesized that pts showing 9p24 chromosomal abnormalities have additional JAK2 structural rearrangements. Among 10,000 pts with hematological malignancies we detected 15 pts (MDS=5, AML=3, NHL=5, IMF=1, MM=1) with 9p24 chromosomal rearrangements. Additionally, 23 pts: 18 with PV [10 with a normal karyotype, 4 with +9, 2 with +i(9)(p10), 1 with der(9)t(1;9), and one with del(9)(p21)] and 5 pts with IMF showing del(13q) and del(20)(q12), were also included in the study. Two JAK2 BAC FISH probes: RP11-3H3 and RP11-28A9 (provided by N. Cross, Salisbury, England) did not show structural rearrangements in 2,000 nuclei from 4 controls and mapped to the 9p24 region in 40 metaphase cells, confirming 100% specificity for the probe. A NF-E2 BAC probe (provided by H. Pahl, Freiburg, Germany) did not show rearrangements in 1,000 nuclei and mapped to the 12q13.1–13.2 region in 20 metaphase cells. Among 38 evaluated pts, FISH studies showed gain of JAK2 in 13 pts (34%): 3 to 7 copies of JAK2 were observed in 10 pts (26%) and amplification of JAK2 was demonstrated in 3 pts (8%): 2 with PV and 1 with B-cell lymphoma. Two pts with PV had two JAK2 clonal populations: gain of 4 copies of JAK2 as well as JAK2 amplification (~20 copies). The original karyotype of B-cell lymphoma pt was t(2;9)(q14;p13) but metaphase FISH revealed the complete material attached to 9p was highlighted by the JAK2 probe. Five pts demonstrated JAK2 structural rearrangements. Three MDS pts showed JAK2 translocation from 9p24 to chromosome 12: 1 pt had JAK2-NF-E2 fusion, 1pt had a JAK2-TEL/ETV6 fusion and in the 3rd pt JAK2 moved from 9p24 to 12q13, where NF-E2 was located. A pediatric MDS pt and der(9)inv(9)(p23q34)t(9;14)(q22;q13) karyotype demonstrated JAK2 translocation to chromosome 14 within close proximity to IgH locus. In the 5th pt (AML) JAK2 moved to chromosome 4, band region q27. Our observations demonstrated that numerical gain of JAK2 is present in 6 of 18 pts with PV (33%). Moreover, rare amplification of JAK2 is a recurrent phenomenon in PV (11%) and also rarely occur in other hematological malignancies. The novel observation of JAK2-NF-E2 fusion as well as TEL/ETV6-JAK2 fusion and JAK2 translocation to chromosomes 4, 12 and 14 demonstrate that JAK2 not only has multiple fusion partners but also indicate its important role in the molecular pathogenesis of cMPDs, MDS and rare B-lymphoid malignancies. These findings also support the notion that JAK2V617F mutation may not be the only molecular JAK2 genomic defect in cMPDs.

The genetic hallmark of MDS is a gain or loss of chromosomal loci identified in bone marrow (BM) cells in ∼50% of pts at diagnosis. We previously demonstrated in longitudinal chromosome study a modulating effect of chronic AZA C therapy on the MDS clone which subdivided patients into five distinct cytogenetic groups with statistically significant differences in survival (P=0.0003) (Najfeld et al, ASH 2004). Our goal was, therefore, to investigate whether PB cells can be used in substitute of BM cells for detection of genomic defects to monitor the clone during AzaC-based therapy. Chromosomal and interphase (I-) FISH studies were performed using BM and PB cells at baseline and following AzaC-based therapy. I-FISH studies were evaluated with a panel of six probes [EGR1 (5q31), D7S522 (7q31), D8Z2 (8p11.1-q11.1), MLL (11q23), Rb1 (13q14), D20S108 (20q12)]. Of the 47 pts studied, 25 (53%) had a normal karyotype and disomy for the MDS panel of six probes. The other 22 pts (47%) were cytogenetically abnormal, showing concordant results in 16 of 22 pts (73%) for cytogenetic and I-FISH genomic defect in BM cells (showing ≤20% frequency difference). The remaining 6 pts had a mean of 77% (range 37.5–100%) of abnormal metaphase cells and a mean of 42% (range 1.8–73.3%) of abnormal BM interphase nuclei. The 35% difference in frequency seen between metaphase and interphase BM cells is attributed to the proliferative advantage of metaphase cells with a complex karyotype. The frequency of the abnormal MDS-marked clone in BM and PB cells was concordant in 13 of 20 pts (65%). Hematological response of these pts was PR=1, hematological improvement=3, stable disease=2, too early for evaluation=6, and 1 patient had no hematological response after 10 months of AZA C treatment. The remaining 7 pts had a mean of 59% (range 48–68%) of abnormal BM metaphase cells and a mean of 21% (range 7–41%) of abnormal PB interphase nuclei. The MDS abnormal clone was detectable in more than two-fold higher frequency in BM compared to PB prior to treatment in 35% of pts. Preliminary sequential studies in discordant pts revealed a transition between the BM and PB cells to concordant frequencies within 4–5 months after AzaC-based therapy. These early observations suggest that monitoring AzaC-based therapy can be achieved using peripheral blood cells in 80% of pts. Remarkably, one pt with monosomy 7, a notoriously poor IPSS indicator, demonstrated a hematological improvement, full cytogenetic and 97% FISH remission, both in the BM and PB after 4 months of AzaC-based therapy. To examine AzaC’s response on cell lineage involvement in MDS, purified BM and PB cells were subjected to FISH analysis before and during treatment from patients who were cytogenetically abnormal at baseline. Similar frequencies of genomic imbalances were seen in purified BM and PB derived CD34+ (97% vs. 96%) and CD15+ cells (94% vs. 98%), indicating that these cell populations had a similar response to AZAC therapy, as monitored in PB or BM, during the initial treatment period (4–5 months). Purified BM and PB-derived T-lymphocytes (CD3+/4+/8+) had normal disomic patterns before and during AzaC-based therapy, indicating that T-cells were not involved in the MDS clone in these pts. In contrast, BM and PB derived B-cells (CD19+) had slightly discordant results, showing a mean of 83% vs. 56% respectively of MDS-marked clone, indicating a trend towards greater response in PB- derived B-cells when compared to BM-derived CD19+ cells. In summary, our results demonstrates that although the MDS abnormal clone may be detected in both the BM and PB at the start of therapy, due to the proliferative advantage of the abnormal clone the optimal tissue should be bone marrow. This is the first study demonstrating that tracking the MDS-marked clone during the AZA-C therapy is feasible in peripheral blood cells.