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Abstract Background: Debate continues as to the clinical relevance of subclassifying patients with the myeloproliferative disorder (MPD) MMM into the subgroups of agnogenic myeloid metaplasia (AMM), post-polycythemic myeloid metaplasia (PPMM), and post-thrombocythemic myeloid metaplasia (PTMM). We looked to validate previously described phenotypic variability amongst subtypes, and explore relevant and differences in constitutional symptoms. Methods: An detailed analysis of patients with MMM whom responded to an international web based survey of MPD patients which registered responses regarding diagnosis, disease course, complete blood counts, co-morbidities (Charlson Co-morbidity index), symptoms, and fatigue (Fact-An and Brief Fatigue Inventory (BFI)). Results: A total of 456 patients self described as MMM responded of which 32.3% had AMM (n = 148), 38.0% % had PPMM (n = 174), and 29.7% had PTMM (n = 136). Detailed breakdown of differences in demographics, clinical course, current laboratory studies, treatment history, and current symptoms are outlined in the table below. Phenotypic Variability Amongst 458 MMM Patients Category AMM (N=148; 32.3%) PPMM (N=174; 38.0%) PTMM (N=136; 29.7%) MMM Total (N=458) Δ between groups (p = ) Hb = Hemoglobin; SCT = Stem Cell Transplant Age (Median) 61 57 57 58 0.09 Sex (% Males) 60% 49% 30% 47% <0.01 Yrs from MPD Dx 5 6 8 6 <0.01 Labs/ Prior History Hb (g/dL) 11.0 13.7 11.6 12.1 <0.01 % with RBC Tx 43% 6% 11% 20% <0.01 Leukocytes (×10(9)/L) 8.0 7.6 7.2 7.7 0.64 Platelets (×10(9))/L) 200 352 396 321 <0.01 % with Splenomegaly 76% 49% 41% 54% <0.01 % with Prior Thrombosis 10% 22% 31% 21% <0.01 % with Prior Bleed 16% 20% 31% 22% 0.01 Treatment History for the MMM % with Prior Tx for MPD 64% 88% 77% 77% <0.01 % Medical Therapy 57% 70% 75% 67% <0.01 # Meds (Median (Range)) 1 (0–8) 2 (0–6) 2 (0–6) 2 (0–8) <0.01 % with Splenectomy 8% 5% 6% 6% 0.48 % with SCT 6% 1% 1% 3% <0.01 MMM Associated Constitutional Symptoms % with Fatigue 85% 81% 85% 84% 0.56 % with Bone Pain 51% 46% 44% 47% 0.51 % with Fevers 19% 17% 18% 18% 0.87 % with Pruritus 39% 69% 39% 50% <0.01 % with Night Sweats 55% 58% 53% 56% 0.67 % with Weight Loss 30% 15% 16% 20% <0.01 Overall the data suggests patients with AMM are more likely male, with greater burdens of anemia, splenomegaly, and weight loss. However they are less likely to have experienced thrombohemorrhagic complications and have received fewer MPD medications. In contrast PPMM patients (not unsurprisingly) have little trouble from anemia, but have the most pruritus. Patients with PTMM are most likely female, with the highest platelet counts, and the greatest likelihood of thrombohemorrhagic complications. Additionally, all three subgroups of MMM had significant fatigue compared to published controls for both fatigue instruments (P<0.0001) yet there was no difference between MMM subtypes for the severity of fatigue. Conclusions: A large unselected series of MMM patients supports that although constitutional symptoms are significant and similar amongst MMM subgroups, these patients have variable burdens of other MPD manifestations.

Abstract Background: Chronic myeloid leukemia (CML) is invariably associated with the reciprocal translocation of BCR and ABL to form the Philadelphia chromosome (Ph), t(9;22)(q34;q11). At diagnosis, a small proportion of patients display additional cytogenetic abnormalities and/or a variable proportion of cytogenetically normal metaphases that coexist with Ph-positive metaphases. The objective of the current study was to examine the prognostic relevance of these two scenarios. Methods: The study population consisted of 65 (47.7% female) consecutive, newly diagnosed CML patients seen at the Mayo Clinic with a median age of 59 years. 44.5 percent were initially treated with interferon alpha (IFN) resulting in complete cytogenetic remission in 10.7 percent for a median length of 5.83 years, partial cytogenetic remission in 3.57 percent for a median length of 2 years. 55.5 percent were treated with imatinib (Gleevec) resulting in complete cytogenetic remission in 54.1 percent for a median length of 3.08 years, partial cytogenetic remission in 13.5% for a median length of 1.08 years. Results: Survival at five years through Kaplan-Meier analysis was approximately 92 percent in patients demonstrating only the Ph chromosome (n = 53), versus 60 percent in patients with additional chromosomal abnormalities (n = 12; p < 0.0001 by both Logrank and Breslow-Gehan-Wilcox analysis). Furthermore, patients with additional chromosomal abnormalities demonstrated lower rates of either complete or partial cytogenetic remission with imatinib therapy. Similarly, five year survival of patients with less than 90% Ph-positive metaphases at diagnosis (n = 4) was approximately 60% compared to 92% in patients with greater than 90% Ph-positive metaphases (n = 61; p = 0.01 by Logrank and p = 0.001 by Breslow-Gehan-Wilcox analysis). Additionally, the former group of patients was significantly less responsive to imatinib. Finally, there was significant correlation between Ph-positive metaphase mosaicism and the presence of additional chromosomal abnormalities (p = 0.05). Conclusion: Although it is tempting to speculate the possibility that Ph-chromosome mosaicism and/or additional cytogenetic abnormalities at presentation of CML is a surrogate for the presence of Ph-negative imatinib-resistant clones, the preliminary results from the current study require validation from a larger study. Figure Figure Figure Figure

Abstract All-trans retinoic acid (ATRA) syndrome is a complex constellation of symptoms that arise in about 15% of patients who receive retinoic acid as therapy for acute promyelocytic leukemia (PML). This syndrome typically consists of fever, dyspnea, weight gain, pulmonary infiltrates, pleural or pericardial effusions, episodic hypotension, renal dysfunction, and leukocytosis. Rarely does this entity present as typical chest pain suggestive of an acute myocardial infarction. A 39-year-old man diagnosed with acute PML treated with idarubicin and ATRA was admitted for febrile neutropenia, nausea, vomiting, cough, and epigastric pain despite being on ciprofloxacin and metronidazole at home. He continued to have low-grade fevers despite being on antibiotics and antifungals. On hospital day three, he was transferred to the CCU for worsening non-radiating left substernal chest pain associated with nausea and vomiting, new inferior lead 2 mm ST segment elevation, lateral lead T wave inversion, troponin and CKMB elevation, and hypotension not responding to frequent fluid boluses. A Swan-Ganz catheter was placed and pressors were started. The patient was found to have a dramatic neutrophil-predominant leukocytosis only hours after a CBC showed neutropenia. He also developed anuric acute renal failure. A cardiac catheterization was considered but could not be performed because of thrombocytopenia and the patient’s renal status. Subsequent EKG’s revealed more impressive changes. New 3 mm ST segment elevations and T wave inversions were noted in the lateral leads, the inferior lead ST segment elevations persisted and new inferior lead T wave inversions were present. An emergent echocardiogram was obtained which showed a significantly decreased LVEF at 36% and global LV hypokinesis without pericardial effusion. The constellation of symptoms was more suggestive of ATRA syndrome versus acute coronary syndrome and intravenous dexamethasone was started. The patient’s chest pain resolved spontaneously over the next three hours, the dramatic EKG findings and elevated cardiac enzymes resolved over the next few days, and the patient was transferred back to the admitting hematology service. A follow-up echocardiogram before discharge revealed a normal LVEF at 74% and no residual wall motion abnormalities. He eventually required hemodialysis for continued renal failure, which was continued as an outpatient after discharge. Of note, the patient’s fever resolved after beginning dexamethasone and multiple blood, urine, stool, and sputum cultures obtained remained negative throughout the hospital stay. This clinical situation presents a very distinct dilemma in how to proceed with clinical care. The presenting symptoms, signs, diagnostics, and imaging alone were all suggestive of a worsening ischemic cardiac process. But in considering the clinical presentation as a whole, especially the concomitant renal failure, fever in the setting of broad-spectrum coverage, and dramatic leukocytosis, we were more convinced of a systemic reaction to recent ATRA use. We maintained a low threshold for beginning an acute coronary syndrome protocol involving antiplatelet agents but were supported in our diagnosis by the patient’s remarkable recovery shortly after beginning intravenous steroids.

Abstract Idiopathic HES is a myeloproliferative disorder (MPD) characterized by persistent overproduction of eosinophils resulting in organ damage. In a small subset of patients (pts), the disease is caused by the FIP1L1-PDGFR-α fusion tyrosine kinase. Its expression has been associated with complete clinical responses to therapy with tyrosine kinase inhibitor (TKI) imatinib mesylate. In contrast, there is no specific therapy for pts with HES lacking FIP1L1-PDGFR-α expression and corticosteroids are used for palliation. Alemtuzumab (Campath-1H™) is a humanized rat monoclonal antibody directed against the CD52 antigen. Since CD52 is expressed on eosinophils, we evaluated its efficacy in pts with FIP1L1-PDGFR-α-negative symptomatic HES. A total of 9 pts (5 male, 4 female) received alemtuzumab in weekly cycles at the dose of 30 (8 pts) or 10 (1 pt) mg 3 times a week intravenously or subcutaneously. During the first week alemtuzumab was given in escalating doses (3→10→30mg) to assure tolerance. Median age was 53 years (range 24–70), time from HES diagnosis to alemtuzumab therapy 34 months (range 4–195), median WBC 17×109/L (range 6.9–82.4), absolute eosinophil count (AEC) 9×109/L (range 0.7–30), and bone marrow eosinophils 23% (range 10–68). Three pts had abnormal cytogenetics: del11(q23), +8, and t(5;6). Pts had received a median of 3 prior therapies (range 2–6), including corticosteroids (prednisone; n=9), imatinib (n=7), dasatinib (n=3), interferon-alpha (n=3), hydroxyurea (n=3), nilotinib (n=2), and cladribine (n=2). A total of 121 weekly cycles have been administered (median 13 cycles) thus far. A complete hematologic response (CHR; i.e. AEC normalization) was observed in 8 pts (89%) within 4 weeks of therapy; peripheral blood eosinophils were undetectable in 6 of 8 CHR pts. Follow-up bone marrow specimens were available in 3 pts while in CHR (after a median of 5.5 weekly alemtuzumab cycles) showing complete response in 2 and partial response in 1 pt. In addition, 3 pts that were on prednisone and 2 on hydroxyurea at the start of alemtuzumab discontinued them within 2 weeks into therapy. One pt had a remarkable partial response with reduction of AEC from 47.5 to 1×109/L after 8 weekly cycles of alemtuzumab; this patient was on 3g of hydroxyurea and 80mg of prednisone at the start of alemtuzumab and was able to discontinue hydroxyurea and decrease prednisone to 10mg while on alemtuzumab. Five pts who stopped alemtuzumab experienced relapse after a median time of 3.5 weeks (range, 1 to 10) and 1 relapsed while receiving alemtuzumab after 14 weekly cycles. Three pts are currently on alemtuzumab: 2 receiving 30 mg weekly as maintenance while in CHR after 8+ and 19+ weeks, respectively, and 1 with partial response is receiving 30mg 3 times a week. One relapsed pt was rechallenged with alemtuzumab and normalized AEC again. Alemtuzumab was generally well tolerated. Grade 3–4 drug-related toxicities occurred in 2 pts, 1 with neutropenia and 1 with infusion-related fever, which led to transient and complete alemtuzumab discontinuation, respectively. Two pts experienced CMV reactivation that required therapy. In summary, alemtuzumab was well tolerated and has remarkable activity in pts with FIP1L1-PDGFR-α-negative HES. Different dose schedules of alemtuzumab that include prolonged maintenance therapy are warranted to improve the response duration. Updated clinical results will be presented.

Abstract Background: Scant data exists regarding the burden of fatigue and constitutional symptoms in patients with myeloproliferative disorders (MPDs) (essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis with myeloid metaplasia (MMM)). Methods: An Internet based survey of MPD patients regarding clinical course, blood counts, constitutional symptoms, and fatigue (FACT-AN and Brief Fatigue Inventory (BFI)). Results: Patient Demographics: A total of 1179 MPD patients (median age 56; 41.4% male) internationally (76% USA: 50 states, 30 countries, 6 continents) responded to the survey. Self reported diagnoses were PV (35%), ET (26%), or MMM (39%). Clinical History: 1025 patients (87%) had undergone MPD therapy (drugs 70.5%, phlebotomy 44.1%, other 16%). Thrombosis (n=261, 22%), hemorrhage (N=272; 23%), and splenomegaly (N=478 (43%) were frequently reported. Blood tests (available in 917) showed 39% were anemic, 7% transfusion dependent. Symptoms: Fatigue, pruritus, night sweats and bone pain were present across the majority of MPD patients (see table). Additionally it was observed that these symptoms appeared with nearly equal frequency in PV as in MMM patients. However MMM patients displayed more advanced features (fevers, night sweats and weight loss). MPD Associated Constitutional Symptoms in 1179 Patients SYMPTOM PV (N=405) ET (N=304) MMM (N=456) MISSING (N=14) TOTAL (N=1179) P VALUE Fatigue 344 (84.9%) 220 (72.4%) 381 (83.6%) 7 945 (81.1%) <0.0001 Pruritus 265 (65.4%) 120 (39.5%) 228 (50.0%) 3 613 (52.6%) <0.0001 Night Sweats 199 (49.1%) 123 (40.5%) 254 (55.7%) 4 576 (49.4%) 0.0002 Bone Pain 174 (43.0%) 125 (41.1%) 214 (46.9%) 4 513 (44%) 0.2480 Fevers 53 (13.1%) 26 (8.6%) 81 (17.8%) 2 160 (13.7%) 0.0013 Weight Loss 39 (9.6%) 22 (7.2%) 93 (20.4%) 0 154 (13.2%) <0.0001 Spleen Pain 17 (4.2%) 27 (8.9%) 30 (6.6%) 4 74 (6.3%) <0.0001 Fatigue (82% MPD linked) was significantly increased across all MPD subtypes compared to published controls for both the BFI (P<0.0001) and Fact-An (P<0.0001). Additionally 69% curtail social activity because of fatigue, 35% need assistance with activities of daily living, and 11% reported MPD associated medical disability. Fatigue strongly correlated with multiple MPD disease features including: anemia, bone pain, constitutional symptoms, pruritus, splenic pain, smoking, thrombosis, and hemorrhagic events (all p<0.0001). Interestingly, even in “asymptomatic” MPD patients (those without anemia, splenomegaly, or thrombohemorrhagic complications) (n=279)) 24% (41% PV, 31% ET, 28% MMM) described significant fatigue worse than published controls. Conclusions: Fatigue and other constitutional symptoms are a significant burden to MPD patients, despite current therapies employed. Additionally fatigue is also a significant problem even in MPD patients lacking other advanced disease features. Clearly, direct attempts to improve fatigue (through pharmacologic and non-pharmacologic interventions) are necessary.

Abstract Background: Constitutive activation of the JAK-STAT signaling pathway through acquisition of the JAK2V617F mutation plays a key role in the pathogenesis of related myeloproliferative disorders (MPD) including polycythemia vera (PV), agnogenic myeloid metaplasia (AMM), and essential thrombocythemia (ET). Hypothesis: Genetic variation in JAK2 and/or the cytokine receptor genes for erythropoietin (EPOR), thrombopoietin (MPL), and granulocyte colony stimulating factor (GCSFR), influences the expression of a specific disease phenotype (PV, AMM, or ET). Methods: We studied 179 Caucasian MPD patients (PV=84, AMM=58, ET=37) for genetic variation in 4 candidate genes (i.e. JAK2, EPOR, MPL, GCSFR) through single nucleotide polymorphism (SNP) and haplotype analyses. A total of 32 LD (linkage disequilibrium) tag-SNPs with a minimum allele frequency of at least 5% were selected from the HapMap CEU database (JAK2=13, EPOR=4, MPL=5, GCSFR=10). Genotyping was performed using archived DNA from enriched neutrophils. Results: Seventy six (94%), 26 (45%), and 14 (38%) patients with PV, AMM, and ET, respectively, carried the JAK2V617F mutation. Significant differences in allele frequencies was observed at 6 SNP loci (rs10758669, rs3808850, rs7849191, rs7046736, rs10815148, and rs12342421, p-values < 0.0005), all within the JAK2 gene, in comparing our overall study population with the founder Caucasian population in the HapMap database. In the additive genotype-phenotype association analysis adjusted for gender and age at diagnosis, 3 SNP loci in JAK2 (rs7046736, rs10815148, and rs12342421) were found to be significantly, but reciprocally associated with PV (p-values < 0.00006, odds ratio=0.37, 2.82, and 2.39, respectively) and ET (p-values < 0.002, odds ratio=2.50, 0.36, and 0.41, respectively), but not AMM, in terms of the minor allele being ’protective’. These three SNPs were all in high LD, with the ’r2’ measures of LD between 0.59 and 0.71. Furthermore, 2 additional JAK2 SNP loci (rs10758669, p-value = 0.0024 and rs10974947, p-value = 0.0046) were significantly associated with PV (odds ratio=1.88 and 0.47, respectively), but not ET or AMM. Similarly, presence of the minor allele at a single SNP locus in EPOR (rs318699, p-value = 0.0012) was significantly associated with PV only (odds ratio=2.16). For the phenotype-genotype intragene haplotype analyses, the EPOR intragene haplotypes GAAA and GGAA were significantly associated with PV (simulated global p-value = 0.058, simulated individual p-values 0.0013 and 0.0068, haplotype frequency of 35% and 56%, respectively). In addition to EPOR, 6 intragene haplotypes within JAK2 where significantly associated with PV (simulated global p-value < 0.0001, individual simulated p-values < 0.03). Conclusion: The current study demonstrates a genotype-phenotype association that involves JAK2 and EPOR in the setting of PV, but not that of AMM. Some JAK2 SNPs were found to be associated with both PV and ET but in opposite direction. Therefore, genetic variation among MPDs might contribute to phenotypic diversity in the presence of specific mutations.

Abstract The FIP1L1-PDGFR-α fusion kinase induces hypereosinophilic syndrome (HES) in a subset of patients (pts). Imatinib is a potent inhibitor of PDGFR-α and has been associated with durable hematologic responses in pts with HES. However, development of kinase domain mutations may hamper TKI activity, underscoring the need for novel agents to prevent or overcome resistance. We evaluated the efficacy of the novel TKI EXEL-0862 in FIP1L1-PDGFR-α-expressing cells. In 72-hour MTS assay, EXEL-0862 inhibited EOL-1 cell proliferation with an IC50 of 1.0 nM and markedly decreased phosphorylation of STAT3 and Erk1/2, suggesting inhibition of downstream targets of PDGFR-α. More important, EXEL-0862 induced dose-dependent inhibition of PDGFR-α phosphorylation in peripheral blood mononuclear cells from a pt with HES. Exposure of EOL-1 cells to EXEL-0862 10 nM for 24 hours caused apoptosis as evidenced by flow cytometry (annexin-V/PI staining) and transmission electron microscopy. This was coupled with time-dependent cleavage of caspases-3, caspase-9, and PARP and with mitochondrial damage as reflected by cytochrome c translocation into the cytosol, and a diminished mitochondrial transmembrane potential. Of note, apoptotic features were not evident after 16 hours of EXEL-0862 exposure. Immunoblots showed marked downregulation of Mcl-1, but not of Bcl-2, Bcl-XL, or Bax. To elucidate the mechanism of Mcl-1 cleavage, EOL-1 cells were exposed to 10 nM EXEL-0862 for 6 hours followed by cycloheximide (5 μg/ml), a potent translation inhibitor, which resulted in marked Mcl-1 turnover. Treatment with the proteasome inhibitor MG-132 failed to abrogate EXEL-0862-induced Mcl-1 cleavage, suggesting proteasome-independent degradation. Rather, a decrease of pro-form Mcl-1 (p42) and increase of the cleaved Mcl-1 peptide form of Mcl-1 (p28) coincided with caspase-3 activation. Pretreatment with 20 μM pan-caspase inhibitor Z-VAD abolished EXEL-0862-induced Mcl-1 cleavage, pointing to a caspase--dependent mechanism. Collectively, our results establish EXEL-0862 as a solid candidate for the targeted treatment of pts with FIP1L1-PDGFR-α-positive HES.

Abstract Background: Lenalidomide (Revlimid; Celgene Co. New Jersey, USA) has ameliorated cytopenias and induced cytogenetic responses in patients with MDS, especially those with del(5)(q13q33)) (List et. al. NEJM2005;352:549–57)., and myelofibrosis with myeloid metaplasia with del(5)(q13q33) (Tefferi et. al. Blood 2006 epub). However, no data exist regarding lenalidomide efficacy in patients with del(5)(q13q33) who have progressed to AML. Case: An 82 year old woman presented to our institution with a several year history of MDS associated with mild anemia, thrombocytosis, and isolated del(5)(q13q33). She had required no prior MDS-specific therapy, and had suffered no thrombohemorrhagic complications. Subsequently, in response to worsening anemia (hemoglobin 9.9 g/dL), leukopenia (1.5 × 109/L), and neutropenia (0.45 × 109/L), with the appearance of peripheral circulating blasts (2%), a bone marrow was obtained. The marrow demonstrated minimally differentiated AML (AML-M0) with 34% blasts and trilineage dysplasia. Karyotypic analysis showed 11 of 20 metaphases containing the del(5)(q13q33), while allele specific PCR demonstrated the presence of JAK2V617F. Therapy: After discussion of the therapeutic alternatives with the patient (including the high risk and likelihood of failure with conventional induction chemotherapy with AML that arises in the context of MDS in an elderly patient (median survival 3–4 weeks - DeLima et. al. Br J Haem1996;93:89), and the responses reported with lenalidomide for patients with interstitial chromosome 5q deletions and MDS, she elected to begin lenalidomide at a dose of 10mg/day. She tolerated the agent well, without any adverse effects. After three months of treatment, she had normal peripheral blood counts, and no evidence of circulating blasts. A repeat marrow exam showed a slightly hypercellular marrow with 5% blasts, improved granulocyte maturation, and a completely normal female karyotype (>20 metaphases counted). This would be considered a partial response by the International Working Group for Acute Myeloid Leukemia (Cheson et. al. JCO2003;21:4642–49). After two additional months of lenalidomide the patient had evidence of disease progression, with recurrent leukopenia, thrombocytopenia, and 10% blasts in the marrow. Additionally, karyotype showed clonal progression, with 10/30 metaphases having the del (5) (q13q33), and 7 of them with multiple other structural abnormalities including loss of 17p. Alternative therapy was initiated. Discussion: To our knowledge, this is the first report of lenalidomide monotherapy resulting in achievement a solid 5 month partial remission in an elderly patient with high risk AML . The patient reported a high quality of life during this time, and was free of transfusions, hospitalization, and infection. The co-existence of the JAK2V617F mutation, a finding more commonly associated with myeloproliferative disorders, is an additional intriguing aspect to this case. JAK2V167F has been described in 6.2% MDS patients with del(5)(q13q33) (Ingram et. al. Leukemia 2006 April 16), and the clinical significance is unknown. Conclusions: Few agents offer legitimate therapeutic benefit to elderly patients with secondary AML. Lenalidomide may have activity as monotherapy against AML arising from del(5q)-associated chronic myeloid disorders. However, the apparent ability of the disorders to undergo clonal evolution suggests that combination or sequential therapy of lenalidomide with other novel or traditional agents should be explored.

Abstract Background: Overall prognosis in CMML is unfavorable. The only curative option is ASCT, yet the limited data available from the largest reported series, reports 2 year overall (OS) and disease free (DFS) survival of only 21% and 18%, respectively [Kroger et al, BJH 2002, 118: 67]. Data on DLI for CMML are generally restricted to case reports. We retrospectively reviewed our experience of ASCT and DLI for adults with CMML. Patients: Seventeen consecutive patients (11 males) with CMML underwent ASCT from 1992 to 2004. At transplant, median (range) age was 50 years (26–60), duration of disease 7 months (2.5–44), and bone marrow (BM) blast count 6% (1–58%). Prior to transplant, 11 had evolved into acute myeloid leukemia (AML). Of these, 10 received standard induction chemotherapy but only one achieved complete remission (CR); 5 had persistent CMML and 4 refractory AML. Karyotype at transplant was either normal (n=9) or revealed isolated monosomy 7 (n=4), complex (n=1), or single, nonrecurrent abnormalities. ASCT: Conditioning was Cytoxan/Total Body Irradiation in all but one who received a non-myeloablative (NMA) regimen (cladrabine/ATG/thiotepa). Stem cell source was HLA-identical sibling in 14 patients and unrelated in 3 peripheral blood (PB) in 7, BM in 8, or both in 2. Median BM mononuclear cell/kg dose was 2.3 (1.7–4.15) x 108 and PB CD34 cell/kg was 5.12 (0.94–7.29) x 106. Graft versus host disease (GVHD) prophylaxis was cyclosporin alone (n=3), with methotrexate (n=12), or prednisone (n=1) or was tacrolimus/methotrexate (n=1). Post-transplant course: All but one patient who died on day 11 of sepsis engrafted (sustained ANC > 500) at a median of 19.5 days (13–31). CR was documented in 13 (76%) post-ASCT. Acute GVHD was grade II-IV (75%) and III-IV (37.5%) and chronic GVHD was extensive (n=37.5%). Seven (41%) demonstrated relapse or persistent disease at a median of 6 months (3–55.5) and 13 (76%) have died due to disease (n=6) or transplant related mortality (n=7). DLI: Six patients underwent DLI for morphologic (4 recurrent/persistent CMML, 1 early AML with extramedullary disease) or cytogenetic relapse (n=1), at a median of 19.5 days (6–52) post-relapse. Median BM blast count at DLI was 3% (1–22%). The median CD3+ cell dose infused was 30.45 x 107 cells/kg (11.2–45.5), given in a median of 3 infusions (range: 1–3). Four did not respond, but two (one with cytogenetic and one with morphologic relapse) achieved a durable CR of 15 months each, both had extensive CGVHD, from which one died of complications. The other relapsed but failed to respond to a second DLI and died of disease. Survival: For the entire cohort, median DFS and OS were 6 months and 7 months, respectively. At last follow up, 4 remain alive, 3 in continuous CR (18%), at 12, 29 (treated with the NMA regimen) and 116 months, respectively. Of the 3 patients surviving in remission, all had transformed to AML prior to ASCT and only one had achieved CR following induction chemotherapy. Multivariate analysis failed to demonstrate any significant effect of age, duration of disease, BM blast %, karyotype or induction chemotherapy on RFS or OS, likely reflecting the small numbers available for analysis. Conclusion: The current study demonstrates anti-CMML activity of both ASCT and DLI although overall outcome is less than encouraging and unpredictable. Novel approaches are needed.

Abstract BACKGROUND. Several studies have recently reported on the occurrence of a JAK2 V617F mutation in myeloid cells from the majority of patients with polycythemia vera (PV). The clinical relevance of this novel observation is currently under study. Similarly, there is limited information regarding the correlation of JAK2 V617F mutational status and neutrophil PRV-1 transcript level in PV, secondary polycythemia (SP) and other myeloproliferative disorders (MPD). METHODS. In a single institutional study, mutation screening for JAK2 V617F was performed in DNA derived from archived blood granulocytes from 63 consecutive patients with PV in whom current diagnostic criteria were strictly applied and diagnosis confirmed by bone marrow histology. Concomitant quantitative analysis of neutrophil PRV-1 expression was performed in 28 of the 63 patients and the results of both JAK2 V617F and PRV-1 analysis in these 28 patients were compared with those from another 22 patients with essential thrombocythemia (ET), 10 with myelofibrosis with myeloid metaplasia (MMM), 19 with SP, and 11 healthy volunteers. RESULTS. The 63 patients with PV were followed for a median of 33 months (range 3–324) from the time of initial diagnosis. During this period, 3 patients have died including 2 from acute myeloid leukemia (AML). Disease transformation into either AML or MMM has occurred in 2 and 4 patients, respectively. The median time from diagnosis to the time of mutation analysis was 12 months (range 0–306). JAK2 V617F was detected in 58 of the 63 patients with PV (92%; homozygous in 21%). The clinical phenotype of the 5 patients with wild-type allele was otherwise typical for the disease. A statistical comparison between JAK2 V617F heterozygotes (n=45) and homozygotes (n=13) in PV did not reveal significant associations in regards to age, gender, leukocyte or platelet count at diagnosis, disease duration, or the incidences of thrombosis or bleeding. However, JAK2 V617F homozygotes, compared to their heterozygote counterparts, displayed significantly higher hemoglobin level at diagnosis (p=0.001), increased incidence of pruritus (69% vs. 38%; p=0.04), and a higher rate of fibrotic transformation (23% vs. 2%; p=0.009). The overall (homozygous) JAK2 V617F mutational frequencies in the other study patients with ET, MMM, SP, and healthy controls were 55% (0%), 30% (0%), 0%, and 0%, respectively. The corresponding figures for increased PRV-1 expression in these patients were 18%, 20%, 21%, and 9% as compared to 89% in PV. In patients with either ET or MMM, the likelihood of detecting JAK2 V617F was significantly higher in the presence of an increased PRV-1 expression (83% vs. 38%; p=0.05). Similarly, in patients with PV, homozygous as compared to heterozygous JAK2 V617F correlated with higher levels of PRV-1 expression (p=0.07). CONCLUSIONS. The current clinical study demonstrates a mutant allele dose effect on erythrocytosis, PV-characteristic clinical features, and granulocyte PRV-1 expression in PV. Furthermore, compared to the PRV-1 assay, mutation screening for JAK2 V617F displayed greater specificity in distinguishing PV from SP.