Ketterling, Rhett P.

Abstract Background: The FIP1L1-PDGFRA fusion, an imatinib-sensitive molecular target, is detected in a subset of cases with moderate-to-severe eosinophilia (eosinophil count ≥1500/μL). Using 2 FISH strategies to detect loss of the CHIC2 locus as a surrogate marker of the FIP1L1-PDGFRA fusion, we previously found 14% of cases to be CHIC2-deleted when studying bone marrow smears from 81 adult patients with moderate-to-severe, non-reactive eosinophilia at our institution (Blood 2004, 104:3038–3045). Since then, the FISH test has been made available for routine clinical use both within our institution, and to external physicians as a mail-in test, for the workup of patients with eosinophilic disorders, including systemic mast cell disease (SMCD). Study design: FISH data, subsequent to the test’s introduction into routine clinical practice (from Nov. 2003), were retrospectively analyzed for CHIC2-deletion. Data from the initial pilot study of 81 patients was not used in this analysis (Table). The imatinib treatment analysis, however, included all CHIC2-deleted patients treated at our institution, including those from the pilot study (Table). Results: A total of 741 patients were studied in the post-pilot phase. Of these, 20 cases were found to be CHIC2-deleted, with an overall prevalence of 2.7% (Table). Of the 219 patients seen at our institution, 13 (all male) were found to be CHIC2-deleted (Table) (prevalence=5.9%), and of these, 10 have been treated with imatinib (100 mg/day starting dose) to date. Nine patients are evaluable for response - all achieved a complete clinical and histological remission (7 patients were tested by FISH post-treatment - all achieved cytogenetic remission). One patient received concomitant corticosteroids for co-morbidities and could not be evaluated for response. After a median follow-up of 24 months (range 17 to 39 months), all 9 patients continue to be in remission, with a normal eosinophil count. At last follow-up, one patient had discontinued imatinib and the others were on maintenance therapy at the following dose: 100mg/wk (n=1), 100mg BIW (n=1), 100mg TIW (n=2), 50mg QOD (n=1), and 100 mg/day (n=3). Conclusions: FIP1L1-PDGFRA prevalence in non-selected patients with eosinophilia is lower than anticipated from prior studies. Patients with FIP1L1-PDGFRA exhibit durable responses to imatinib therapy. It may be feasible to maintain some patients in remission on an empirically derived, intermittent schedule of low-dose imatinib. Pilot Data Post-pilot Data (n=741) Total Mayo samples Mayo samples Mail-in samples Number of Patients 81 138 603 822 Number CHIC2-deleted 11 2 18 31 Prevalence 14% 1.4% 3.0% 3.8% Number Imatinib treated 8 2 n/a 10

Abstract Deletion 5q in Acute Myeloid Leukemia (AML): a Clinicopathological Description of 79 Cases From a Single Institution Rafael Santana-Davila, Shernan G. Holtan, Rhett P. Ketterling, Ryan A. Knudson, David P. Steensma, Dong Chen, James D. Hoyer, Curtis A. Hanson, Ayalew Tefferi. Background: Deletion 5q (del(5q)) is a lenalidomide-sensitive cytogenetic abnormality commonly seen in patients with myeloid disorders. In this study we analyze the clinicopathological characteristics of patients with AML and del(5q). Methods: Patients with del(5q) and AML were identified through a database query of our institutional cytogenetic database from May, 1992 through December, 2005. Both peripheral blood and bone marrow specimens from each patient were morphologically examined by a hematopathologist (DC, JDH and/or CAH). A morphologic diagnosis was given based on current WHO criteria. A critical review of the patient history was performed for each patient. Results: A total 79 patients were identified. Median age was 71 (range 39–87) years, 56% (n=43)were female. The most common subtype according to the FAB classification was M2 (n=23, 29%). In 35 (44%) patients AML developed a median of 9.4 (range 1–294) months since the diagnosis of a pre-existing myeloid malignancy, of which myelodysplastic syndrome accounted for 24 (9 cases of MDS NOS, 6 of RA, 6 of RAEB1, 2 of RAEB-2, and a single RARS), a myeloproliferative neoplasm in 10 (4 cases of a CMPD NOS, 4 cases of PV and a single case of ET and PMF each) and 1 case of a prior history of AML with a t(8;21). The del(5q) was documented in 7 of this 24 patients in the antecedent hematologic disorder. Neither a history of prior exposure to cytotoxic agents nor a prior myeloid disorder was found in 44% (n=35) of the patients. Del(5q) was commonly associated with a complex karyotype (n=67), however in 9 patients it was found to be the sole abnormality. The most frequent breakpoint was q13q33 (n=47) followed by q15q33 (n=12). Initial pathological description did not identify any morphological abnormalities in 6 cases, in contrast myelodysplasia involving at least two cell lines was appreciated in 58% (n=46) of the patients. The median survival was 2.8 months. The majority of the subjects (n=51, 65%) received palliative treatment, 7 patients were lost to follow up and no treatment information was available for review and the remaining 21 received induction chemotherapy with a curative intent, in this group median survival was 8.8 months with 52% (n=11) of patients achieving a complete response. Surprisingly the only factor that appeared to alter survival in the whole group was the presence of the breakpoint in chromosome 5 being distal to q15 (median 2.8 vs 4.1 months, p=0.025) Conclusions: This study constitutes the largest review of del(5q) in AML, its presence is currently associated with advanced age and a poor survival. Close to half of the patients in our series were classified as de novo AML. Although more commonly encountered as part of a complex karyotype it can also be present as a single abnormality. The different cytogenetic characteristics of the del(5q) may impact survival and this deserves further investigation. Figure. Survival of patients with AML and del(5q) in which the breakpoint was found to be distal to q15 (straight line) compared with patients in which the breakpoint was proximal to q13 (dotted line). Figure. Survival of patients with AML and del(5q) in which the breakpoint was found to be distal to q15 (straight line) compared with patients in which the breakpoint was proximal to q13 (dotted line).

Abstract Malignant plasma cell (PC) disorders are clonal diseases based on their immunoglobulin variable region sequence signature. However, intratumor phenotypic, genetic, and morphologic heterogeneity is often observed, fueling the notion that a subpopulation of the malignant PC clone may have extensive self-renewal properties whereas others may not. Little information exists regarding the relationship between these putative tumor subpopulations that is instrumental to our understanding of the growth, progression and therapeutic targeting of PC disorders. We recently described establishment of two novel cell lines from one patient initially diagnosed with primary amyloidosis (ALMC-1), who eventually progressed to a combination of AL and symptomatic multiple myeloma (MM; ALMC-2). Although both cell lines are clonally related, they exhibit a number of intriguing differences, including a marked variability in their makeup of mononuclear (MoN) and multinuclear cells (2 − >10 nuclei; MuN). The ALMC-1 cell line consistently displays a much larger fraction of MuN cells (~50%) than the ALMC-2 cell line (~20%). By contrast, the growth rate of the ALMC-2 cells is significantly higher than ALMC-1 cells suggesting an inverse relationship between growth rate and fraction of MuN cells. It has been demonstrated that MoN plasmablastic morphology is a predictor of poor survival in MM, however the clinical significance of MuN PCs, which are frequently observed in bone marrow biopsies, has yet to be determined. We therefore used the ALMC-1 and ALMC-2 cell lines to begin to test the hypothesis that the MoN fraction in both cell lines contains cells with extensive self-renewal properties whereas the MuN cells are no longer capable of cell division. The central questions become by what mechanism are MuN PCs generated and how are MoN PCs maintained? Two hypotheses that can explain the dual persistence of both types of cells are: random cell fusion; regulated or sporadic cell division of MoN cells into 2 daughter cells, one of which divides normally and the other one fails to undergo cytokinesis. To address the first possibility, each cell line was transfected with GFP or YFP. Single positive (GFP or YFP) cells were then mixed and cocultured for assessing the frequency of dual positive cells by flow cytometry at various time points. However, studies to date suggest this mechanism does not occur. To begin to address the second possibility and determine whether extrinsic factors, e.g., cytokines, may regulate the rate of generation of MuN PCs, we cultured both cell lines +/− IL-6 and IGF-I and assessed numbers of MoN and MuN PCs. Initial analyses suggest that IL-6, but not IGF-I, increases the proportion of MoN PCs in both cell lines. To address the hypothesis that MuN PCs are no longer capable of growth, cells were labeled with bromodeoxyuridine (BrDU) to assess the capability of MuN cells to synthesize DNA. We observed that MuN PCs incorporating BrDU did so uniformly in each nucleus present in the cell. We then assessed the ability of cell division of MuN cells by quantitating normal vs abnormal metaphases in both cell lines and observed that >90% of metaphases were present in MoN PCs, suggesting that MoN PCs largely give rise to MuN PCs and the latter are likely compromised in cytokinesis. As centrosomes play a critical role in regulating cell division and centrosome abnormalities have been noted in MM by others, we next used gamma-tubulin immunofluorescence to detect centrosomes. MoN PCs exhibited uniformly normal centrosome numbers and positioning while MuN PCs showed clear centrosomal abnormalities including accumulation of centrosomes, centrosome clustering, and multiple directional centrosome polarization, suggesting a mechanism by which the MuN cells may be generated. In summary, our data suggest that MuN PCs lack growth potential and that intrinsic and/or extrinsic factors may drive accumulation of these cells. These observations support the possibility that patients with a larger proportion of MuN PCs may have differences in clinical outcome from those patients with largely MoN PCs. To test this, we have begun hematopathological analysis of a large series of patients diagnosed with various plasma cell disorders. Knowledge of the signals that regulate the balance between MoN and MuN cells could lend itself toward identification of therapeutic modalities that may maximize differentiation of tumor cells into end-stage cells.

Abstract Background: For most patients with MDS, the disease-associated molecular abnormalities are unknown, which contributes to diagnostic uncertainty and has limited development of effective therapies. Chromosomal deletions such as del(5q) or monosomy 7 are more common than translocations in MDS, but balanced translocations are more likely to be pathobiologically informative. Here we report cloning of an MDS-associated translocation that revealed a novel recurrent molecular abnormality. Methods: We first studied a 71-year old man who presented with MDS and t(6;9)(p21.3;q34) as an isolated chromosomal abnormality in 18 of 20 metaphases. FISH assays for DEK/CAN and ABL rearrangements were unrevealing. We generated somatic hybrid murine/human cell lines from a buffy coat; microdissected and amplified derivative chromosomes; and hybridized to 6p/9q custom comparative genomic hybridization (CGH) arrays. The CGH results considerably narrowed the 6p and 9q breakpoints, which were then amplified by long-range PCR and sequenced to define the rearrangement. Results: The IER3 gene (Immediate Early Response 3; also known as IEX-1) at 6p21.3 was found to be separated from its upstream regulatory elements in this rearrangement and translocated to a transcript-poor region of 9q. RT-PCR confirmed marked downregulation of IER3 expression in the patient compared to healthy controls. IER3 is a plausible candidate for involvement in MDS because of its known role in regulating death receptor-induced apoptosis, interaction with NF-κb pathways, and its importance in response to genotoxic stresses such as ionizing irradiation. We then identified archival bone marrow cell pellets from 204 additional patients with various clonal hematological disorders and chromosomal rearrangements involving 6p21 or 6p22 (i.e., translocations, inversions, deletions, or additions), and designed a splitsignal FISH probe set to assay IER3. FISH results were abnormal in 8 additional patients (9/205 pts total abnormal, 4.4%): 3 split signals and 6 amplifications (one patient had both abnormalities); all 8 patients had MDS and 6p rearrangements as part of a more complex karyotype. FISH studies in MDS patients without 6p rearrangements are ongoing. RTPCR in MDS patients without 6p rearrangements demonstrated down-regulation of IER3 by >2-fold in 38% of patients (67% lower-risk) and up-regulation in 12% of patients (50% higher-risk) when compared to the mean for healthy controls. Direct sequencing of the IER3 coding region and the promoter region in 30 patients without 6p rearrangements revealed no point mutations, but the prevalence of non-synonymous single nucleotide polymorphism rs3094124 (p.A127P) was higher than expected in patients (variant in 28.3 % of MDS alleles vs. 8.6% of 116 HAP-CEU controls, p=0.0015). IER3−/− mice are known to have hypertension, cardiac hypertrophy, and epithelial proliferation, but are not anemic (Sommer SL et al J Appl Physiol 2006). We performed methylcellulose hematopoietic colony growth assays using bone marrow mononuclear cells from IER3−/− mice and wildtype controls, and noted no differences in BFU-E, CFU-E, and CFU-GM growth under unstressed conditions. Studies of IER3 expression induction under stressed conditions in primary MDS cells are ongoing. Conclusion: IER3 rearrangements represent a novel clonally-restricted recurrent genetic abnormality in MDS. Dysregulation of IER3 expression is common in MDS, even in patients without 6p rearrangements. This is the first time IER3 has been linked to human disease. Clarification of the role of IER3 in MDS is likely to yield new insights into MDS pathobiology.

Abstract The dic(9;12)(p12;p11.2) has been described as a rare cytogenetic abnormality in pediatric precursor B-cell ALL. Initial studies suggested that the rearrangement is associated with a favorable outcome, and recent studies demonstrated the presence of a PAX5-ETV6 fusion gene was associated with this cytogenetic abnormality. Twenty cases with a cytogenetic dic(9;12) were identified in the Children’s Oncology Group (COG) cytogenetics databases. FISH analysis with the ETV6-RUNX1 (TEL-AML1) probes was done on 12 of these samples. Five cases were positive for fusion, indicating a cryptic t(12;21)(p13;q22), and also had loss of the ETV6 probe from the chromosome 12 not involved in the t(12;21). Seven cases were negative for fusion and had loss of an ETV6 signal, although one of the latter had a diminished ETV6 signal identified. To determine whether both PAX5-ETV6 and ETV6-RUNX1 rearrangements occurred in some patients, a diagnostic sample from each patient was analyzed by RT-PCR for the PAX5-ETV6 and ETV6-RUNX1 fusion genes. Primers from exon 3 of PAX5 and exon 3 of ETV6 were used for the PAX5-ETV6 analysis and from exon 5 of ETV6 and exon 4 of RUNX1 for the ETV6-RUNX1 analysis. Of the 20 cases, only 8 were RT-PCR positive for the PAX5-ETV6 fusion with the above primers; however, an additional 2 were RT-PCR positive with alternate primers, and all 10 of these were negative for the ETV6-RUNX1 fusion by RT-PCR. Of the remaining 10 patients, 9 were RT-PCR positive for the ETV6-RUNX1 fusion, including all of the ETV6-RUNX1 cases positive by FISH. The gene rearrangement associated with the dic(9;12) in these cases is not known. One patient was negative for both fusions by RT-PCR, negative by FISH for ETV6-RUNX1 rearrangement, yet had loss of an ETV6 signal. No cytogenetic differences could be seen between the 2 groups, either in the appearance of the dic(9;12) or in the other abnormalities identified. These results demonstrate the presence of two mutually exclusive dic(9;12) rearrangements in pediatric ALL; one associated with ETV6-RUNX1 rearrangement and one resulting in PAX5-ETV6 fusion. Both PAX5-ETV6 and ETV6-RUNX1 rearrangements are associated with a favorable prognosis. However, molecular analysis of the dic(9;12) patients must be performed to determine whether the dicentric chromosome results in PAX5-ETV6 fusion or whether the case has ETV6-RUNX1 fusion.

Abstract BACKGROUND: DNA rearrangements that result in the inappropriate activation of the PDGFRA gene at 4q12 and the PDGFRB gene at 5q31-q33 occur rarely in patients with chronic myeloproliferative disorders. Approximately 10% of patients with systemic mast cell disease/hypereosinophilic syndrome have a unique mutational mechanism resulting in PDGFRA overexpression due to a novel microdeletion of the CHIC2 region resulting in the juxtaposition of FIP1L1 and PDGFRA. PDGFRB activation has been observed in patients with chronic myelomonocytic leukemia/atypical chronic myeloid leukemia and has been associated with 11 translocation partners. Since patients with demonstrable breakpoints in the PDGFRA and PDGFRB genes often have a dramatic disease response to the tyrosine kinase inhibitor imatinib, we searched the Mayo Clinic Cytogenetic database to identify additional translocation partners involving these regions. METHODS: Homebrew dual-color FISH probes were created which flank the PDGFRA gene at 4q12 and the PDGFRB gene at 5q31-q33. Archived bone marrow karyotypes analyzed in the Mayo Clinic Cytogenetics laboratory from a 15 year period (1989-2004) were reviewed to determine the frequency of specimens with breakpoints at 4q12 and 5q31-33. Of the 29,047 abnormal specimens, 64 possessed a 4q12 breakpoint and 164 possessed a 5q31-q33 breakpoint (excluding simple deletions). Of these 228 patients, residual bone marrow specimens were available from 170 patients for FISH analysis. RESULTS: Eleven of 50 patients with a 4q12 breakpoint yielded a break with the PDGFRA FISH probe. Eight patients had the previously described t(4;12)(q12;p13) which results in a reciprocal exchange between the TEL oncogene and has a break near, but not within, the PDGFRA gene. Three patients had breaks within the PDGFRA gene and had novel translocation partners including 1q44, 3q25 and 17q23. Twelve of 120 patients with a 5q31-q33 anomaly had a break detected with the PDGFRB FISH probe. Nine patients had the classic t(5;12)(q33;p13) involving PDGFRB and TEL. Three patients had novel PDGFRB translocation partners, including 1q21, 14q32 and 16p13.1. CONCLUSIONS: Breakpoints involving the PDGFRA and PDGFRB genes appear to be quite uncommon as only 23 patient samples were abnormal in our series of 29,407 abnormal bone marrow samples. With the description of three new translocations involving PDGFRB, at least 14 unique translocation partners have been identified with this gene. With the exception of the recurrent t(5;12) between PDGFRB and TEL, most translocations involving PDGFRB appear to be unique. The microdeletion of CHIC2 at 4q12 resulting in the juxtaposition of FIP1L1 and PDGFRA has been the sole mechanism thus far described resulting in the activation of this gene. The identification of the three translocations involving the PDGFRA gene represent the first classic cytogenetically visible rearrangements involving this novel gene region. While rare, we propose that all chromosome anomalies identified with breakpoints in the 4q12 and 5q31-q33 regions should receive appropriate FISH testing to determine the potential involvement of the PDGFRA and PDGFRB genes.

Abstract Background: Peripheral blood (PB) is sometimes used in place of bone marrow (BM) for cytogenetic studies in either primary (PMF) or post-polycythemia vera/essential thrombocythemia (post-PV/ET MF) myelofibrosis. In a retrospective study, we examined overall yield from PB cytogenetic studies in MF and where possible compared the results with those obtained by BM cytogenetic analysis. Methods: Standard laboratory techniques were used to perform cytogenetic studies in both the PB and BM. A successful study constituted the acquisition of at least two analyzable metaphases and an abnormal clone required the presence of a specific cytogenetic abnormality in at least two metaphases. Clinical and laboratory correlations were made using variables collected at the time of cytogenetic studies. Results I: A total of 242 PB cytogenetic studies were reviewed to identify 59 MF cases; 39 were PMF and 20 post-PV/ET MF. Median age was 60 years (range 36–77) and 37 were male patients. The median duration of the disease at the time of PB cytogenetic studies was 3.5 years (range 0.2–35). At least two analyzable metaphases (median 20, range 2–31) were obtained in 49 (81%) of the 59 study patients; in univariate analysis, this was predicted by presence and degree of circulating myeloid progenitor cells (p < 0.0001), higher PB CD34 count (p < 0.0001), higher leukocyte count (p = 0.0008), and absence of myeloid growth factor therapy (p = 0.005). In multivariable analysis, only the presence of circulating myeloid progenitor cells sustained its significance. Results II: Twenty two cases had concomitant PB and BM cytogenetic studies performed. Of these, 9 patients had abnormal BM cytogenetic findings; the concomitant PB cytogenetic studies were also abnormal in 5 (56%) patients but normal in the other 4. Conversely, PB cytogenetic studies were abnormal in 12 patients; the concomitant BM cytogenetic studies were abnormal in 5 (41%) and normal in the remaining 7. PB cytogenetic studies were not successful in 4 of the 22 total cases; BM studies were successful in 3 of these 4 cases and the results were normal in all. Conclusion: In the presence of circulating myeloid progenitor cells, PB can be considered as an alternative to BM for cytogenetic studies in MF patients, especially in view of the difficulty getting BM aspirates in this disease. However, considering the prognostic value of specific cytogenetic abnormalities in MF, the non-trivial discordance between the BM and PB cytogenetic findings, observed in the current study, warrants a prospective evaluation for further clarification.

Abstract Background: Deletion 5q is a lenalidomide-sensitive cytogenetic abnormality seen usually in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In the current study, we looked into the occurrence and clinicopathologic correlates of del(5q) in myeloproliferative neoplasms (MPNs) defined according to the World Health Organization (WHO) criteria. Methods: Patients with del(5q)-positive MPN were identified through database query involving over 2000 patients with myeloproliferative neoplasms (MPD) including no less than 500 patients with primary myelofibrosis (PMF), 600 patients with essential thrombocytosis (ET), 500 patients with polycythemia vera (PV), and the remaining comprised of patients with molecularly-undefined chronic eosinophilic leukemia, hypereosinophilic syndrome, systemic mastocytosis (SM), chronic neutrophilic leukemia, and “MPN, unclassifiable”. Results: A total of 23 patients with del(5q) were identified (12 at time of initial diagnosis), suggesting an overall prevalence rate of 1%. A central pathological review was conducted to confirm the diagnosis in each instance. Specific diagnoses included PMF (n=14; estimated prevalence of 3%), MPN, unclassifiable (n=4), PV (n=2), ET (n=1), post ET-myelofibrosis (n=1) and SM (n=1). Del(5q) was a sole abnormality in 5 patients, accompanied by one other abnormality in 6 patients, and part of a complex karyotype in 12 patients. The most common breakpoint was q13q33 (n=10) followed by q15q33 (n=6). Based on sample size adequacy, we focused on del(5q)-positive PMF patients and compared their bone marrow histological and clinical features with their del(5q)-negative counterparts seen at the Mayo Clinic during the same period of time (n=311). There was no difference in gender or age distributions. Histopathological review did not show the typical tight clusters of megakaryocytes seen in classic PMF. The megakaryocytes were not the predominate feature of the biopsy, in general were less lobulated and smaller in size, and had a higher grade of fibrosis (med = 3+) than classic PMF. Furthermore, del(5q)-positive PMF patients presented with significantly lower hemoglobin level (p=0.0009) and platelet count (p=0.0003). The percentage of patients presenting with a hemoglobin level of < 10 g/dL or a platelet count of < 100 × 109/L were 70% and 92% in del(5q)-positive patients as opposed to 43% and 18% in del(5q)-negative patients. However, the presence of del(5q) did not significantly affect either overall survival (Figure) or leukemic transformation in PMF (14% vs 9%). Conclusions: Del(5q) is generally rare in MPNs but might occur in as much as 3% of patients with PMF. The presence of del(5q) does not appear to affect overall survival in PMF despite its clustering with adverse prognostic features for the disease including anemia and thrombocytopenia. Finally, megakaryocyte morphology and distribution in del(5q)-associated PMF are characteristically different from classic PMF. Figure Figure

Abstract Background: Previous cytogenetic studies in polycythemia vera (PV) have included a relatively small number of patients (“n” ranging 10–64). In the current study (n=137), we describe cytogenetic findings at presentation and examine their relationship to clinical and laboratory features, including bone marrow JAK2V617F allele burden. Methods: The study consisted of a consecutive group of patients with PV who fulfilled the World Health Organization (WHO) diagnostic criteria and in whom bone marrow biopsy and cytogenetic studies were performed at diagnosis. Results I: cytogenetic details At diagnosis: A total of 137 patients (median age, 64 years; 49% females) were studied at diagnosis and had adequate metaphases for interpretation. Cytogenetics were normal in 117 patients (85%) and displayed either a sole -Y abnormality in 5 patients (7% of the male patients), and other chromosomal abnormalities in 15 (11%). The latter included trisomy 8 in five patients, trisomy 9 in three patients, two patients each with del(13q), del(20q), and abnormalities of chromosome 1, and one patient each with del(3)(p13p21), dup(13)(q12q14), and del(11)(q21). At follow-up: Repeat cytogenetic studies while still in the chronic phase of the disease were performed in 19 patients at a median of 60 months (range, 8–198) from diagnosis. Of these, 4 had aquired new cytogenetic clones including 3 with normal cytogenetics at time of initial PV diagnosis. The new abnormalities included del(20q), del(5q), del(1p), chromosome 1 abnormality, and inv(3)(q21q26.2). At time of disease transformation: Leukemic transformation was documented in 3 patients of whom cytogenetic information at the time was available in 2 patients; both patients had normal results at time of initial PV diagnosis and complex cytogenetic abnormalities at time of leukemic transformation. In contrast, among 6 patients with available cytogenetic information at time of fibrotic transformation, the results were unchanged from those obtained at time of diagnosis in 5 patients. ii) Correlation between cytogenetics at diagnosis and JAK2V617F allele burden: Allele-specific, quantitative PCR analysis for JAK2V617F was performed in 71 patients using genomic DNA from archived bone marrow obtained at the time of the initial cytogenetic studies. JAK2V617F mutation was detected in 64 of the 71 (90%) patients; median mutant allele burden was 16% (range 3–80%) without significant difference among the different cytogenetic groups: normal vs. –Y vs. other cytogenetic abnormalities (p=0.72). iii) Clinical correlates and prognostic relevance of cytogenetic findings at diagnosis: Among several parameters studied for significant correlations with cytogenetic findings at diagnosis, an association was evident only for age (p=0.02); all –Y abnormalities (n=5) as well as 13 of the 15 (87%) other cytogenetic abnormalities occurred in patients ≥ 60 years of age. Stated another way, the incidence of abnormal cytogenetics (other than -Y) was 4% for patients younger than age 60 years and 15% otherwise. The presence of abnormal cytogenetics at diagnosis had no significant impact on either overall or leukemia-free survival. Conclusions: Abnormal cytogenetic findings at diagnosis are infrequent in PV, especially in patients below age 60 years. Furthermore, their clinical relevance is limited and there is not significant correlation with bone marrow JAK2V617F allele burden.

Abstract BACKGROUND : Leukemic transformation (LT) eventually occurs in 10–30% of patients with myelofibrosis with myeloid metaplasia (MMM). Hoever, there is limited data regarding bone marrow histological and cytogenetic changes that accompany the process. METHODS : The study population consisted of 67 consecutive patients with MMM in whom the diagnosis of LT was documented at our institution by bone marrow examination. Serial comparisons of both histological and cytogenetic changes were performed to monitor longitudinal changes accompanying disease progression. RESULTS : I. Bone marrow histologic changes during LT : All 67 patients displayed acute myeloid leukemia (AML) and all FAB subtypes except M3 were represented (most common were M7 at 25.4% and M0 at 22.4%). Median bone marrow myeloblast percentage was 43% (range 20–95) and cellularity 80% (range 5–95). However, only 37.5% and 30.3% had marked increases (> grade 2) in either reticulin fibrosis or osteosclerosis, respectively. Serial bone marrow comparisons (available in 33 patients at a median interval of 24.5 months (range; 2.1–110.4) after the diagnosis of MMM) demonstrated that LT was occasionally a focal process, but was always accompanied by a marked increase in myeloblasts. However, overall bone marrow cellularity changed by >15% in only 25% of the cases, none of the LT was associated with a > grade 1 change in reticulin fibrosis, and only 12% had an increase in osteosclerosis. On the other hand, LT was frequently associated with morphologic changes in megakaryocytes from large, round and clumped at the time of diagnosis of MMM to small, condensed and non-clumped at the time of LT. II. Karyotypic changes of MMM to LT: Karyotypic analysis was available in 32 patients (48%) at the time of MMM (abnormal in 63%) and 56 patients (84%) at the time of LT (abnormal in 91%). At LT the majority of patients (n=34; 60.7%) displayed poor risk karyotypes with either complex karyotypes or single abnormalities of adverse prognostic significance. In contrast, only two patients (3.6%) manifested prognostically favorable karyotypes with a translocation or inversion involving one of the core binding factors (t(8;21)(q22;q22);Inv(16)(q22)). Twenty-eight patients had chromosome analysis performed both at diagnosis of MMM and at LT. At diagnosis of MMM, 20 patients had either normal chromosomes or a single chromosome anomaly while only 7 displayed a complex karyotype with 3 or more chromosome anomalies. However, at LT all twenty-eight patients had abnormal karyotypes, including 17 patients with markedly complex karyotypes and only 9 with simple clonal anomalies. The recurrent numeric and structural anomalies observed at transformation to AML most frequently involved chromosomes 5, 6, 7, 8, 17, 19 and 21. Additionally, amongst the 26/28 (93%) of patients whose karyotype changed from MMM to LT 11 patients (43%) displayed evolution of an existing clone, and 15 (57%) developed one or more additional abnormal clones. CONCLUSIONS: The progression of MMM to LT is characterized by the emergence of new cytogenetic abnormalities as well as morphological changes in megakaryocytes. Unfortunately, the karyotypic profile that accompanies clonal evolution in MMM is often of the poor-risk category that undermines the value of aggressive chemotherapy in such patients. However, such therapy might be of value to the occasional patient with good-risk cytogenetic lesions.