Tefferi, Ayalew

Abstract Abstract 277 Background: Current information on the natural history of polycythemia vera (PV) is mostly derived from early studies based on the PV Study Group diagnostic criteria. The World Health Organization (WHO) diagnostic approach to PV is currently the gold standard. The main objective of the current study was to describe survival, disease complication rates and risk factors in WHO-defined PV. Methods: Under the auspices of an International Working Group, seven international centers of excellence for myeloproliferative neoplasms submitted a total of 1,430 patients, locally diagnosed with “WHO-defined PV”. The two principle investigators (AT and TB) reviewed all the cases and selected 1,263 patients who were age 18 years or older, diagnosed after 1970, and whose submitted data included diagnostically essential information. Results I: Presenting Features: Median age was 60 years (range, 18–95; 51% females). The number of patients diagnosed between 1970–1990, 1991–2000, and 2001–2011 were 136 (11%), 337 (27%) and 790 (62%), respectively. Arterial/venous thrombosis history before or at diagnosis was documented in 142/73 and 80/42 patients, respectively. Other features at diagnosis included pruritus (37%), microvascular disturbances (29%), palpable splenomegaly (37%), abnormal karyotype (13%), and leukoerythroblastosis (6%). History of hypertension (45%), hyperlipidemia (17%), diabetes (9%), and tobacco use (15%) was also obtained. Results II: Clinical Course: To date, 228 (18%) deaths, 42 (3%) leukemic progressions, and 101 (8%) fibrotic transformations have been recorded. Post-diagnosis arterial or venous thrombosis occurred in 139 (11%) and 85 (7%) patients, respectively. Cytoreductive therapy had not been used in 342 (27%) patients whereas 154 (12%) patients were exposed to two or more cytoreductive drugs. Results III: Risk Factors: Multivariable analysis identified advanced age, leukocytosis, venous thrombosis history and abnormal karyotype as significant (p<0.01) risk factors for overall survival (OS). Receiver operating characteristic (ROC) plots delineated ages 69 (AUC 0.83) and 59 (AUC 0.61) years and leukocyte count 13 × 109/L (AUC=0.61) as the best discriminant levels in this regard; HR (95% CI) were 11.4 (7.7–16.9) for age ≥70 years, 3.8 (2.6–5.7) for age 60–69 years, and 2.0 (1.5–2.6) for leukocyte count ≥15 × 109/L. Results IV: Risk Stratification: HR-based risk scores were assigned to age ≥70 years (3 points), age 60–69 years (2 points) and leukocyte count ≥15 × 109/L (one point), in order to create four risk groups: low (zero points; n=492; median survival 322 months), intermediate-1 (one or 2 points; n=353; median survival 208 months; HR 3.8, 95% CI 2.5–5.8), intermediate-2 (3 points; n=286; median survival 132 months; HR 10.7, 95% CI 6.9–16.5), and high (4 points; n=56; median survival 90 months; HR 22.7, 95% CI 13.9–37.1) (Figure 1). Similar results were obtained when analysis was restricted to the 1,011 JAK2 mutation-positive patients (Figure 2). The particular risk model was equally robust in predicting leukemia-free survival (p=0.001). Conclusions: Age and leukocyte count matter the most in predicting survival in PV. The current study suggests additional prognostic value from karyotype and venous thrombosis history. Disclosures: Vannucchi: Novartis: Honoraria. Gisslinger:Novartis: Speakers Bureau; Celgene Austria: Speakers Bureau; AOP-Orphan: Speakers Bureau.

Abstract Abstract 5158 Background: Vitamin D insufficiency is commonly observed in clinical practice. Observational studies have suggested an association of vitamin D insufficiency with increased risk of cancer development. Further, vitamin D insufficiency has been associated with poorer prognosis in solid tumors as well as hematologic malignancies, including non-Hodgkin lymphoma and chronic lymphocytic leukemia. In this study, we examined the clinical and prognostic correlates of low plasma levels of 25-hydroxyvitamin D (25[OH]D), which reflects whole-body vitamin D stores, in a cohort of myeloproliferative neoplasm (MPN) and myelodysplastic syndrome (MDS) patients. Methods: We studied a total of 409 patients: 247 (60%) had primary myelofibrosis (PMF), 74 (18%) de novo MDS, 63 (15%) polycythemia vera (PV) and 25 (6%) essential thrombocythemia (ET). Inclusion criteria required availability of archived plasma, bone marrow aspirate and biopsy and cytogenetic information at the time of first referral to our institution. Vitamin D measurements were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the purposes of this study, plasma 25(OH)D levels <10 ng/mL indicated severe deficiency, <25 ng/mL insufficiency, and 25–80 ng/mL optimal levels. Results: Patient characteristics were as follows: (i) PMF: median age=63 years, >65 years=41%, males=68% and serum creatinine above the upper normal limit (UNL)=15%; (ii) MDS: median age=72 years, >65 years=77%, males=69% and creatinine >UNL=12%; (iii) PV median age=61 years, >65 years=37% and males=50%; and (iv) ET: median age=48 years, >65 years=10% and males=28%. Median (range) plasma 25(OH)D levels (ng/mL) were as follows: PMF 25 (0–60), PV 27 (6.8–51), ET 31 (6–50) and MDS 30 (7.7–72) (p=0.005). The proportion of patients with 25(OH)D insufficiency were: PMF 48%, PV 43%, ET 28% and MDS 28% (p=0.01). Similarly, the proportion of patients with severe 25(OH)D deficiency were: PMF 9%, PV 3%, ET 12% and MDS 1% (p=0.05). In PMF and MDS, 25(OH)D insufficiency was not significantly correlated with age (≤65 vs. >65 years), gender, red cell transfusion need, DIPSS-plus/IPSS category, hemoglobin (<10 vs. ≥10 g/dL), thrombocytopenia (<100 vs. ≥100 × 109/L), circulating blasts, unfavorable karyotype, serum creatinine >UNL, or JAK2V617F (PMF only) (p>0.05). 25(OH)D insufficiency was not predictive of inferior overall survival (OS) in PMF, MDS, PV, or ET (p>0.05). Similarly, in PMF and MDS, 25(OH)D insufficiency was not predictive of inferior leukemia-free survival (LFS) (p>0.05). Severe 25(OH)D deficiency was infrequently observed in MDS or PV patients; in PMF or ET patients, there was no prognostic impact of severe 25(OH)D deficiency on OS or LFS (p>0.05). Conclusions: Vitamin D insufficiency is relatively common in PMF, PV, ET and de novo MDS however this finding had no prognostic value for OS in the aforementioned patients. In PMF and MDS patients, low 25(OH)D levels had no clearly identifiable clinical correlates and further did not have prognostic value for LFS. Disclosures: No relevant conflicts of interest to declare.

Abstract Two prospectively studied patients with polycythemia vera (PV) whose platelet counts showed marked periodic fluctuation during treatment with hydroxyurea (HU) are reported. Cycle lengths in both were approximately 28 to 30 days. In one patient, the cyclic process was no longer evident when treatment with HU was withheld, and it reappeared on treatment rechallenge. Circulating thrombopoietin (TPO) levels fluctuated out of phase with the platelet count despite markedly reduced TPO-receptor (c-Mpl) expression in bone marrow megakaryocytes. These observations suggest that the cyclic phenomenon may be related to both a transient state of HU-induced depletion of megakaryocytes and a concentration-dependent mitigation by TPO of the HU effect on megakaryocytes and their precursors. It is conceivable that the affected patients harbor a megakaryocyte progenitor pool whose apoptotic activity is differently modulated by either HU or high concentrations of TPO.

Background: Therapy-related MDS/AML (t-MDS/AML) is a common and devastating complication of treatment with alkylating agents and topoisomerase II inhibitors, often employed with curative intent, in standard or high-doses, in the treatment of hematological malignancies and solid tumors. Allogeneic HSC transplant is considered the standard treatment for t-MDS/AML but its applicability is limited by the unavailability of donors, comorbidities in the recipient and risk of acute and chronic graft-versus-host disease. Patients carrying diseases commonly treated with high-dose chemotherapy and autologous HSC transplant (such as multiple myeloma and non-Hodgkin lymphoma) who latter develop t-MDS/AML occasionally have stored HSC harvested prior to the diagnosis of the secondary malignancy (hereafter called early-stored HSC). The benefit of using early-stored HSC to support myeloablative therapy for the treatment t-MDS/AML is unknown. Methods: Retrospective, single center analysis of patients with t-MDS/AML receiving myeloablative chemotherapy with or without irradiation supported with early-stored HSC. Results: Six patients received early-stored HSC transplant for treatment of t-MDS/AML between 2000 and 2007. Median age at the time of the transplant was 57 years (range 15–71). Three patients had been primarily diagnosed with follicular lymphoma, 2 with multiple myeloma and 1 with Ewing’s sarcoma. One patient had received prior autologous HSC transplantation. Two patients developed AML, 1 patient CMML, 1 patient RCMD, 1 patient RAEB1 and 1 patient RAEB2. Median time from diagnosis of primary malignancy to HSC harvesting was 26 (2–122) months and from harvesting to development of t-MDS/AML 44 (7–79) months. Conditioning regimen was Cy/TBI in three patients, BuCy in 2 patients and Samarium + Melphalan in one patient. Five patients engrafted neutrophils and 4 patients engrafted platelets 10–34 and 13–126 days after transplant respectively. In 3/6 patients there was disappearance of the pre-transplant chromosomic abnormality (2 patients -7 and 1 patient -7 and t(1;7)). Two patients obtained hematologic CR and two patients obtained CR without full recovery of platelets. One patient died of sepsis 230 days after transplant, one died with refractory disease on day 103 after transplant and one patient is alive with recurrence of t-MDS. Three patients are alive and free of disease 7, 73 and 87 months after transplant. Conclusion: Myeloablative conditioning followed by autologous, early-stored HSC transplant can provide prolonged survival to patients with tMDS/AML not suitable to allogeneic SCT.

Abstract Background: The prognostic significance of lymphocyte counts and immune status has been carefully examined in lymphoid malignancies, but the importance of the lymphocyte count in chronic myeloid neoplasms is less clear. In a recent analysis of MDS cases associated with deletion of chromosome 5q (Holtan SG et al AmJHem 2008 Epub 12Jun), we observed that an ALC >1.2 × 109 cells/L at diagnosis is independently associated with improved survival. The prognostic value of ALC in MDS not associated with del(5q) is unknown. Methods: We reviewed the medical records of patients without del(5q) diagnosed with MDS at our institution over a 10-year period and gathered data on complete blood counts and leukocyte differentials, bone marrow findings, and clinical course. Inclusion criteria included diagnosis within 6 months prior to initial consultation at our institution and <20% marrow blasts. Patients were classified according to the International Prognostic Scoring System (IPSS) and the WHO-classification based Prognostic Scoring System (WPSS). Results: The median age of the 503 included patients (360 M, 143 F) was 69 years (range 20–91). Median follow-up was 14 months, and 65% of patients were known to have died with an overall median survival of 36 months. Cytogenetic results as classified by the IPSS were favorable in 326 (65%) patients, intermediate in 112 (22%), and poor in 65 (13%). Both IPSS and WPSS accurately stratified patients, but the WPSS was better at identifying the lowest and highest risk patients. Survival by IPSS risk group was as follows: low-risk disease (n=95, median survival 41.8 months), intermediate-1 (n=285, 23.6 months), 106 with intermediate-2 (n=106, 14.1 months), and high-risk (n=17, 8.05 months). WPSS risk groups included very low risk (n=87; median survival 71.9 months), low risk (n=149, 28.8 months), intermediate risk (n=85, 19.3 months), high risk (n=143, 14.4 months), and very high risk (n=39, 8.8 months). In univariate analysis, factors associated with poorer survival included advanced age (p = 0.0012), higher-risk WHO subtype (p<0.0001), increased marrow blast percentage (p<0.0001), poor-risk karyotype (p<0.0001), higher IPSS score or WPSS score (p<0.0001), lower hemoglobin (p<0.0001), lower MCV (p=0.03), lower ALC (p=0.001), and lower platelets (p=0.001). Factors not associated with survival included sex and ANC. In a multivariate model including only baseline CBC parameters, hemoglobin >10 g/dL (p < 0.001), MCV >96 fl (p = 0.0493), ALC >1.2 × 109/L (p = 0.0449), and platelet count >100 × 109/L (p = 0.0046) were associated with improved survival. A high baseline ALC also maintained independent prognostic significance in a multivariate model against the IPSS (RR 0.746, 95% CI: 0.598, 0.933, p = 0.0099), and the raw WPSS score (RR 0.791, 95% CI 0.633, 0.987, p=0.037) but not the WPSS group (p=0.0794). There was no significant difference in the distribution of IPSS scores between the ALC >1.2 × 109 cells/L and the ALC ≤ 1.2 × 109 cells/L groups, although there were significantly more WPSS high risk patients in the low baseline ALC group (p = 0.0163). The creation of an ALC-modified IPSS score by adding 0.5 point for an ALC ≤ 1.2 × 109 cells/L resulted in a significant increase in median overall survival of the low risk group (41.8 months versus 66.1 months, p < 0.001, Figure 1). Conclusion: ALC is a novel and easily obtained prognostic marker in MDS that provides information complementary to existing prognostic scoring systems. Figure 1. Kaplan-Meier curves based upon IPSS (a) and the ALC-modified IPSS (b), where 0.5 point was added for an ALC ≤ 1.2 × 109 cells/L. Figure 1. Kaplan-Meier curves based upon IPSS (a) and the ALC-modified IPSS (b), where 0.5 point was added for an ALC ≤ 1.2 × 109 cells/L.

Abstract In a 20-year period, 223 patients (median age, 64.8 years) with myelofibrosis with myeloid metaplasia (MMM) had therapeutic splenectomy at our institution. Primary indications for surgery were transfusion-dependent anemia (45.3%), symptomatic splenomegaly (39.0%), portal hypertension (10.8%), and severe thrombocytopenia (4.9%). Operative mortality and morbidity rates were 9% and 31%, respectively. The 203 survivors of surgery had a median postsplenectomy survival time (PSS) of 27 months (range, 0-155). Among preoperative variables, thrombocytopenia (platelet count less than 100 × 109/L) and nonhypercellular bone marrow were identified as independent risk factors for decreased PSS. Durable remissions in constitutional symptoms, transfusion-dependent anemia, portal hypertension, and severe thrombocytopenia were achieved in 67%, 23%, 50%, and 0% of the patients, respectively. Histologic or cytogenetic features of bone marrow obtained before splenectomy did not predict a response in cytopenias. After splenectomy, substantial enlargement of the liver and marked thrombocytosis occurred in 16.1% and 22.0% of the patients, respectively. The thrombocytosis was associated with an increased risk of perioperative thrombosis and decreased PSS. The rate of blast transformation (BT) was 16.3%, and the risk of BT was higher in the presence of increased spleen mass and preoperative thrombocytopenia. However, the PSS of patients with BT was not significantly different from that of patients without BT. We conclude that presplenectomy thrombocytopenia in MMM may be a surrogate for advanced disease and is associated with an increased risk of BT and inferior PSS. However, the development of BT after splenectomy may not affect overall survival and does not undermine the palliative role of the procedure for the other indications.

Abstract Abstract 971 Unlike the case in acute myeloid leukemia, there is limited information on the prognostic impact of IDH mutations in myelodysplastic syndromes (MDS). In the current study of 277 patients with MDS, IDH mutations were detected in 34 (12%) cases: 26 IDH2 (all R140Q) and 8 IDH1 (six R132S and two R132C). Mutational frequency was 4% (2 of 56) in refractory anemia with ring sideroblasts (RARS), 12% (16 of 130) in refractory cytopenia with multilineage dysplasia, 14% (2 of 14) in MDS-unclassifiable, 14% (6 of 42) in refractory anemia with excess blasts (RAEB)-1, and 23% (8 of 35) in RAEB-2. Normal karyotype was noted in all but one IDH1-mutated cases and 13 IDH2-mutated cases. Multivariable analysis identified presence of mutant IDH1 (p=0.0004; HR 4.0, 95% CI 1.9–8.8), revised International Prognostic Scoring System (IPSS-R) risk category (p<0.0001), and red cell transfusion need (p=0.002) as independent predictors of inferior survival (Figure 1). In a similar multivariable analysis, mutant IDH1 was the only variable associated with shortened leukemia-free survival (p=0.001; HR 7.0, 95% CI 2.3–20.8). The presence of IDH2 R140Q did not affect overall (p=0.54) or leukemia-free (p=0.81) survival (Figure 2). The current study suggests a powerful adverse prognostic effect for mutant IDH1 in MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 64 Megakaryocytes are one of the few cell types that undergo a modified form of the cell cycle termed endomitosis, in which cells skip the late stages of mitosis to become polyploid. Murine and human megakaryocytes commonly reach modal ploidy states of 32N and 16N, respectively, and can sometimes achieve DNA contents as high as 64N. Polyploidization is associated with upregulation of megakaryocyte lineage specific genes, proplatelet formation and expression of genes related to apoptosis. RNA expression array studies have shown that high ploidy states are strongly correlated with megakaryocyte differentiation and maturation. Importantly, the choice of a megakaryocyte to undergo polyploidization and differentiation is inextricably linked to exit from the proliferative cell cycle. Given that megakaryocytes in patients with essential thrombocythemia are hyperproliferative and that those in primary myelofibrosis fail to undergo normal differentiation or polyploidization, we hypothesized that small molecule inducers of polyploidization would drive these cells to exit the proliferative cell cycle and undergo terminal differentiation or death. In collaboration with the Broad Institute, we performed a high throughput screen and identified small molecules that induce polyploidization and proliferative arrest of malignant megakaryocytes, including those that express MPLW515L and JAK2 V617F. We have shown that these compounds, including the Rho kinase inhibitor dimethylfasudil (diMF), selectively increase polyploidization, expression of megakaryocyte cell surface markers, and apoptosis of murine and human megakaryocytic cell lines and primary cells. Furthermore, diMF blocked the growth of primary human AMKL blasts both in vitro and in vivo. With respect to MPNs, diMF showed potent activity against megakaryocytic cell lines and primary cells expressing either JAK2 V617F or MPL W515L alleles commonly associated with these disorders. diMF inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis of megakaryocytes transduced with JAK2 V617F or MPLW515L. diMF also significantly reduced megakaryocyte colony forming units (CFU-MK), and induced polyploidization and differentiation of bone marrow and fetal liver megakaryocytes from Gata1 mutant mice, which develop a PMF-like disease. Given that diMF induces growth arrest, polyploidization and apoptosis of cells that express activated mutants of JAK2 and MPL, we predicted that it, as well as other small molecule inducers of polyploidy, would be efficient at restraining aberrant megakaryocyte proliferation in both PMF and ET. To assay the effectiveness of diMF in these disorders, we treated peripheral blood mononuclear cells from patients with either PMF or ET with diMF and monitored growth and maturation of megakaryocytes. We discovered that diMF induced polyploidization and subsequent apoptosis of both types of MPN primary samples. diMF also reduced CFU-MK of these MPN patient samples. Next, we assessed the activity of diMF in a model of MPN in which congenic recipients of MPLW515L transduced Balb/C bone marrow cells develop a rapid MPN characterized by leukocytosis, thrombocytosis, bone marrow fibrosis, and death. diMF led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen of a mouse model of PMF. diMF also led to a significant reduction in the platelet count and a trend towards decreased white cell count, with no effect on hematocrit. Overall, diMF results were comparable to intermediate doses of INCB16562. These encouraging results strongly suggest that diMF induces a decline in megakaryocyte lineage, which leads to reduction in platelet count, and support pre-clinical development of diMF for megakaryocytic subtypes of MPNs. Of note, diMF did not inhibit the phosphorylation of Stat5 or Stat3, suggesting that it acts through a mechanism distinct from JAK2 inhibitors. Interestingly, combination of diMF with a selective JAK2 inhibitor greatly enhanced the efficacy of diMF to inhibit proliferation and induce apoptosis in MPLW515L transduced megakaryocytic cell line. These data support combining JAK inhibition and induction of megakaryocyte polyploidy as a new therapeutic strategy for MPNs. Disclosures: No relevant conflicts of interest to declare.