Tefferi, Ayalew

Abstract Abstract 2806 Background: A high degree of prognostic certainty is required to recommend or discourage high risk treatment procedures in primary myelofibrosis (PMF). The Dynamic International Prognostic Scoring System (DIPSS-plus) uses eight risk factors to predict overall survival (OS) in PMF: unfavorable karyotype, peripheral blood (PB) blast count ≥1%, platelet count <100 × 109/L, white blood cell count (WBC) >25 × 109/L, hemoglobin level <10 g/dL, red blood cell transfusion need, constitutional symptoms, and age >65 years (Gangat et al. JCO 2011;29 :392). The presence of four or more of these risk factors defines high-risk disease. purpose: The purpose of the current study was to enhance the prognostic weight of some of the DIPSS-plus risk factors with the intent to identify one or two parameters that can reliably predict death in the first two years of disease. Methods: An updated Mayo Clinic database of 884 karyotypically-annotated patients with PMF was used. Calculations of 2-year mortality rates and variables considered for prognostic value were from time of referral to the Mayo Clinic. Cytogenetic risk categorization per DIPSS-plus was further refined to capture additional prognostic information from monosomal karyotype (MK) (Vaidya et al. Blood 2011;117 :5612) and inv(3)/i(17q) abnormalities (Caramazza et al. Leukemia 2011;25 :82). Receiver operating characteristic (ROC) analysis was employed to define best discriminant levels. Results: To date, 564 (64%) deaths have been documented. Each one of the aforementioned DIPSS-plus risk factors was associated with a 2-year mortality rate that ranged from 42% (PB blast count ≥1%) to 60% (unfavorable karyotype). High-risk disease per DIPSS-plus was associated with 57% two-year mortality. The only risk factors that were associated with >80% two-year mortality were MK (n =19) and inv(3)/i(17q) abnormalities (n =8) and both were associated with significantly worse survival, compared to other unfavorable karyotype (n =102): HR (95% CI) of 5.1 (3.1−8.4) and 3.9 (1.7−8.8), respectively. ROC analysis identified PB blast counts of 2% and 9% (AUC 0.62) and WBC of 43 × 109/L (AUC 0.66) as best discriminant levels for predicting 2-year mortality; OS was significantly worse in the presence of PB blast >9% (HR 4.1, 95% CI 2.8–6.1) vs. 2% to 9% (HR 1.8, 95% CI 1.5–2.2) vs. <2%; the corresponding 2-year mortality rates were 73%, 46% and 25%. OS was also significantly worse in the presence of WBC ≥40 × 109/L (HR 2.8, 95% CI 2.2–3.6) vs. 26–39 × 109/L (HR 1.6, 95% CI, 1.2–2.1) vs. <26 × 109/L; the corresponding 2-year mortality rates were 63%, 42% and 28%. Two-year mortality rates exceeded 80% in the presence of any two of the following: PB blast count >9%, WBC ≥40 × 109/L or unfavorable karyotype other than MK or inv(3)/i(17q). Conclusions: A greater than 80% 2-year mortality in PMF is predicted by the presence of MK, inv(3)/i(17q) abnormalities, or any two of the following: PB blast >9%, WBC ≥40 × 109/L, other unfavorable karyotype. Such patients and those with high DIPSS-plus risk should be referred to allogenic stem cell transplantation earlier than later. Disclosures: No relevant conflicts of interest to declare.

Abstract BACKGROUND: Information regarding risk factors for leukemic transformation (LT) in primary myelofibrosis (PMF) is limited, although both leukocytosis and abnormal cytogenetics have previously been implicated. The current retrospective study examines clinical variables at diagnosis as well as specific treatment modalities for their effect on LT in PMF. METHODS: Diagnoses of PMF and LT were based on World Health Organization criteria. In addition, study inclusion required availability, for review, of bone marrow histology and clinical data obtained at or within 6 months of diagnosis but prior to treatment intervention. Statistical methods used were standard and included Cox regression analysis of leukemia-free survival (LFS). RESULTS: A master database of PMF patients seen at the Mayo Clinic from 1976 through 2006 was queried to identify 311 patients who fulfilled the aforementioned stipulated criteria for study inclusion; median age was 57 years and 41% were females. At presentation, 30% of the patients displayed a hemoglobin level (Hgb) of < 100 g/L, 20% a leukocyte count of < 4 or > 30 x 109/L, 18% a platelet count of < 100 x 109/L, 12% an absolute monocyte count (AMC) of ≥ 1 x 109/L, 28% hypercatabolic symptoms and 35% a peripheral blood (PB) blast percentage of ≥ 1; 27 patients (9%) had a PB blast percentage of ≥ 3. Dupriez prognostic scoring system distribution for high, intermediate, and low risk disease was 30 (10%), 94 (30%), and 187 (60%) patients. Among 182 and 139 evaluable patients, 74 (41%) and 80 (58%) displayed cytogenetic abnormalities and JAK2V617F, respectively. At a median follow-up of 27 months (range 0–282), 27 cases (9%) of LT were documented at a median time from diagnosis to LT of 26 months (range 0.8–266). Effect of clinical and laboratory variables at diagnosis on leukemic transformation By univariate analysis, LFS was negatively affected by anemia (p=0.04), leukocytosis (p=0.04), PB monocyte count ≥ 1 x 109/L (p=0.02), platelet count of < 100 x 109/L (p=0.004), hypercatabolic symptoms (p=0.03) and PB blast percentage of ≥ 3 (p<0.0001); LFS was not affected by PB blast percentage of > 1 but < 3 (p=0.49). By multivariable analysis, significance was sustained only for PB blast percentage of ≥ 3 (p = 0.0002) and platelet count of < 100 x 109/L (p = 0.02) with hazard ratios (HR) of 5.8 and 2.8 and 95% confidence intervals (CI) of 2.3–14.6 and 1.2–6.6, respectively. The presence of JAK2V617F did not affect LFS (p=0.98). Effect of specific treatment on leukemic transformation By univariate analysis, LT was significantly associated with history of splenectomy (p=0.01) and treatment with erythropoiesis stimulating agents (ESA; p=0.004), danazol (p=0.007), and androgens (p=0.03) but not with hydroxyurea (p=0.17), interferon alpha (p=0.95), thalidomide (p=0.26) or other drugs. On multivariable analysis, significance was sustained for both ESA (p = 0.005; HR 3.1, CI 1.4–6.8) and danazol (p = 0.01; HR 3.4, CI 1.3–8.5), even when the aforementioned prognostic indicators at diagnosis were added as covariates to the multivariable model. CONCLUSIONS: The current study identifies PB blast percentage of ≥ 3% and platelet count of < 100 x 109/L, at presentation, as independent risk factors for LT in PMF. Unexpectedly, the study also revealed an association between LT and treatment history with ESA or danazol. These observations are intriguing, considering recent reports on the possible harmful effects of ESA in certain solid tumors.

Abstract The JAK2V617F mutation is present in the majority of cases of myeloproliferative disease, including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is an attractive candidate for molecularly targeted therapy. However, the potential toxicities of JAK2 inhibition in vivo, and identification of appropriate surrogate endpoints for response, are challenges that may limit clinical usefulness in treatment of these relatively indolent diseases. We report efficacy and assessment of surrogate endpoints for response of a small molecule JAK2 inhibitor, TG101348 in a murine model of polycythemia vera. TG101348 is selective for JAK2 with an in vitro IC50 of ∼3 nM that is ∼334 fold more potent than for inhibition of JAK3. TG101348 showed therapeutic efficacy in the murine model of PV that included a statistically significant reduction in hematocrit, normalization of white blood cell count, a dose dependent reduction/elimination of extramedullary hematopoiesis in the spleen and liver, and marked attenuation of myelofibrosis. Consistent with its selective inhibition of JAK2 and not JAK3, there was no significant change in T-cell number in treated animals. These clinical responses correlated with surrogate endpoints for response, including reduction or elimination of JAK2V617F expressing clones based on quantitative genomic PCR, suppression of JAK2V617F positive endogenous erythroid colony growth of JAK2V617F MPD bone marrow, and inhibition of JAK-STAT signal transduction as assessed by phosphoflow cytometry for phosphorylated STAT5. Thus, TG101348 is efficacious in treatment of a murine model of PV, and surrogate endpoints have been identified that may be of value in clinical trials in humans.

Abstract Since the identification of the JAK family of intracellular tyrosine kinases and their downstream signaling partners, the STATs (Signal Transducers and Activators of Transcription), evidence has accumulated that activation of JAK signaling pathways can contribute to proliferation and survival of cancer cells. A direct link to oncogenesis was made when an activated form of JAK2 (JAK2 V617F) was identified in the majority of patients with myeloproliferative disorders (MPDs) including approximately half of patients with myelofibrosis (MF). Introduction of this JAK2 mutation into the bone marrow of mice induced erythrocytosis [Wernig et al. (2006) Blood107:4274] supporting JAK2 as a relevant target for treatment of myeloproliferative disorders. We have identified a novel small molecule inhibitor of JAK2, XL019, which may prove to have utility in treating MPDs. XL019 is a potent and reversible inhibitor of the JAK2 enzyme, with a Ki of 2 nM, and shows excellent selectivity (minimum of 50-fold selectivity against >120 protein kinases tested including other JAK family members). XL019 downregulates STAT signaling in cell lines expressing both wild type and activated forms of JAK2. IC50s for inhibition of STAT5 phosphorylation by XL019 ranged from 623 nM (HEL92.1.7) to 3398 nM (KG-1) in tumor cell lines. XL019 showed increased potency in primary human erythroid cells in vitro, where the IC50 for inhibition of EPO-stimulated phospho-STAT5 was 64 nM. XL019 inhibits proliferation in cell lines harboring activated or overexpressed JAK2, including certain lines derived from patients with Hodgkin’s Lymphoma (L-1236, 928 nM IC50), AML (MV4-11, 992 nM IC50), essential thrombocythemia (SET-2, 386 nM IC50), and erythroleukemia (HEL92.1.7, 6777 nM IC50). Preclinical single-dose pharmacodynamic studies demonstrate a potent effect of XL019 on JAK-STAT signaling in HEL92.1.7, CFPAC-1 and DU 145 xenograft tumors. Twice daily dosing (bid) led to substantial tumor growth inhibition in the DU145 and HEL models (maximum tumor growth inhibition of 86% and 60%, respectively), accompanied by increases in tumor cell apoptosis (4 – 4.4 fold increase) and decreases in tumor microvasculature (44% reduction in DU 145 xenograft tumors). Based on these encouraging data, XL019 is being evaluated in subjects with primary or post PV/ET MF in a Phase I dose escalation study, which has completed enrollment of the first cohort. The primary objectives of this study are to determine the safety and tolerability of XL019 when administered orally once daily for 21 days in 28 day cycles. Secondary objectives include determination of the pharmacokinetics and pharmacodynamics of XL019, and to evaluate response using the International Working Group for MF consensus response criteria. We have developed a flow-based mechanistic assay for JAK2 activity, which measures GM-CSF-stimulated phospho-STAT5 in peripheral granulocytes in whole blood samples. Additional pharmacodynamic assessments for this study include JAK2 V617F allele burden, erythropoietin-independent colony formation, cytokine levels, and changes in bone marrow histology. Our initial findings regarding the pharmacokinetics, pharmacodynamics, tolerability and efficacy of XL019 in MF patients will be presented.

Abstract Abstract 5148 INTRODUCTION. Patients presenting with a clinical picture of essential thrombocythemia (ET) can actually have an early/prefibrotic myelofibrosis (PMF), according to current WHO criteria, in about 18% of cases. Laboratory tests which are significantly different in early/prefibrotic PMF as compared with histologically confirmed ET (WHO-ET) include decreased gender-matched hemoglobin (Hb), increased white blood cell (WBC), platelet (PLT) counts and lactate dehydrogenase (LDH) values. AIM. To evaluate sensitivity (SE) and specificity (SP) of blood cell counts and LDH, at presentation, for the diagnosis of early/prefibrotic PMF vs. WHO-ET. METHODS. Five hundred thirty-six cases (50%) who had complete laboratory data measured at diagnosis constituted the exploratory set of our study and were derived from an international ET database. The discriminatory ability of Hb, WBC, PLT and LDH in correctly classifying patients in the early/prefibrotic PMF or WHO-ET groups was initially tested by plotting their Receiving Operating Characteristic (ROC) curves and comparing the relative Areas Under the Curve (AUC) with the value of 0.50 (which stands for the completely useless of the test). Three parameters with statistically significant discriminatory power were chosen (Hb, WBC and LDH) and thresholds searched in order to guarantee at least 90% of SE or SP. Finally, a diagnostic algorithm was designed. The validation set of this analysis was constituted by 321 patients with WHO-ET (n=62) or early/prefibrotic PMF (n=259) diagnosed by the same pathologist who confirmed the training set cohort and collected in the Institute for Pathology, University of Cologne, Germany. SE and SP for the same parameters and thresholds as well as the final diagnostic algorithm were applied to this set of patients to demonstrate the results' reproducibility. RESULTS. Sensitivity and specificity to recognize early/prefibrotic PFM have been evaluated by ROC curves. The best performance was found for LDH (AUC = 0.7059). WBC and Hb had super imposable curves, with AUC of 0.6279 and 0.6257, respectively. The worst performance was registered for PLT count: its AUC was only 0.5628, not significantly different from the reference value of 0.50 (p=0.154). Thresholds of Hb, WBC and LDH were searched to achieve at least 90% of SE or SP. HB < 12 g/dL for women or <13 g/dL for men, and WBC >= 13 x109/L had higher SP (92% and 91%, respectively). High SP is highly related to the presence of early/prefibrotic PMF (true positives). On the contrary, LDH < 200 mU/mL and WBC < 7 x109/L had good sensitivity (91% and 94%, respectively). High SE is highly related to the absence of early/prefibrotic PMF (true negatives). By applying these SE and SP values in a step-by-step algorithm, nearly half of patients (48%) could be classified as WHO-ET or early/prefibrotic PMF, assuming at each step a margin of error of less than 10%. For the remaining 50% of patients, laboratory results didn't allow to suspect or exclude the presence of early/prefibrotic PMF. In the validation set of 321 patients classified by WHO 2008 as true ET or early/prefibrotic PMF (Cologne cohort) SP of anemia was 84%, WBC < 7 x109/L or >= 13 x109/L had 91% and 81% of SE and SP, respectively. LDH values < 200 mU/mL had 85% of SE. By applying the same flow-chart, 46% of patients were classified as WHO-ET or early/prefibrotic PMF. CONCLUSIONS. The present study provides clinicians with laboratory parameters that should increase suspicion of early/prefibrotic PMF in a patient with a working clinical diagnosis of ET. In fact, while patients presenting clinically with ET can now be discriminated as true ET or early/prefibrotic PMF by adopting the WHO 2008 criteria that require bone marrow histology, an algorithm including baseline anemia, WBC count and LDH, allows this differentiation in about 50% of patients with a good approximation. However, for a definitive proof, bone marrow histology is still an integral part for final diagnosis. Disclosures: Vannucchi: Novartis: Honoraria.

Abstract Abstract 5154 Background Recent clinical trials with JAK or mTOR inhibitors in primary myelofibrosis (PMF) have identified pruritus as one of the most treatment-responsive disease traits. However, little is known about the prevalence of pruritus in PMF or its clinical and laboratory correlates. Methods The study population was selected among a consecutive series of patients with PMF seen at the Mayo Clinic and in whom bone marrow histologic and cytogenetic information was available for re-review. JAK2, MPL and IDH mutation status and plasma cytokine levels (IL-1b, IL-1RA, IL-2R, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, TNF-α, G-CSF, IFN-α, IFN-γ, MIP-1a, MIP-1b, HGF, IP-10, MIG, MCP-1 and VEGF) were recorded where available. Overall and leukemia-free survival analyses were performed from time of referral to the Mayo Clinic. Results I: Prevalence of pruritus and other baseline information Among 566 consecutive patients with PMF (median age 62 years; 368 males), the presence (n=90) or absence (n=146) of pruritus was documented in 236 patients. The prevalence of pruritus was 38% when considering only those patients in whom pruritus history was documented (n=236), and 16% when all study patients (n=566) were considered regardless of whether or not the absence of pruritus was documented in their history. Among the 90 patients with pruritus, it was severe enough to warrant specific therapy in 51 (57%) patients, was aquagenic in 29 (32%), and onset antedated PMF diagnosis in 15 (17%). All 236 patients had information on karyotype: 136 normal, 76 favorable and 24 unfavorable karyotype. Information on JAK2, MPL, and IDH mutations was available in 197 (125 positive), 147 (9 positive), and 131 (2 positive) patients, respectively. In addition, plasma levels of the aforementioned cytokines were available in 63 patients. Results II: Clinical correlates Parameters at diagnosis that were significantly associated with presence of pruritus included leukocytosis (p=0.03), absence of leukopenia (p=0.002), and absence of MPLW515 mutation (p=0.02). Borderline significance was noted for JAK2V617F positive status (p=0.08). The incidence of pruritus was 0% (0/9) vs. 41% (56/138) in MPL-mutated vs MPL-unmutated cases and 42% (52/125) vs. 29% (21/72) JAK2 mutated vs unmutated cases, respectively. No significant correlations were noted for age, DIPSS-plus scores, transfusion need, karyotype, or plasma cytokine levels. The association with leukocytosis/leukopenia was subsequently shown to be significant only in JAK2V617F-negative cases. To date, 124 deaths and 19 leukemic transformations have been documented. In JAK2-mutated cases (n=125), pruritus did not affect overall or leukemia-free survival. In JAK2-unmutated cases (n=72), a significant correlation between presence of pruritus and inferior leukemia-free survival was noted (p=0.04), and was independent of leukocytosis, thrombocytopenia or abnormal karyotype. Conclusions Pruritus accompanies PMF in a substantial proportion of patients and is more or less likely to occur in the presence of JAK2 or MPL mutations, respectively. In JAK2V617F-negative cases, PMF-associated pruritus clustered with leukocytosis and was independently associated with inferior leukemia-free survival. Disclosures: No relevant conflicts of interest to declare.

Abstract Myeloproliferative disorders (MPDs), diagnosed in 15,000 individuals annually, are characterized by overproduction of lineage-committed blood cells, thrombotic events, occasionally marrow fibrosis or progression to acute myelogenous leukemia and frequently a point mutation (V617F) in the JAK2 kinase. Mutant JAK2 expression results in erythrocyte overproduction in almost all cases of polycythemia Vera (PV). We investigated the molecular mechanisms driving erythroid skewed differentiation and the capacity of a selective JAK2 inhibitor (TG101348) to normalize PV progenitor differentiation. Both JAK2 V617F+ transduced cord blood and PV progenitor erythroid colony formation was potently inhibited by TG101348 (300 nM). Bioluminescent immunocompromised mouse transplantation studies revealed that TG101348 (150 mg/kg) treatment significantly reduced human erythroid engraftment by both PV and JAK2 V617F+ cord blood progenitors. Moreover, the imbalance between GATA-1 and PU.1 transcripts typical of JAK2 V617F+ progenitors normalized after treatment with TG101348. In addition, TG101348 inhibited both JAK2-mediated STAT5 phosphorylation and AKT-regulated GATA-1 phosphorylation in an erythropoietin responsive cell line suggesting a dual mechanism for the erythroid inhibitory effects of TG101348. Because of selective inhibition of JAK2 V617F+ progenitors both in vitro and in vivo, TG101348 may be an effective molecularly targeted inhibitor of JAK2 V617F+ driven MPDs in clinical trials.

Abstract We identified 68 patients with clonal T-large granular lymphocyte (T- LGL) proliferations who were seen at the Mayo Clinic between 1984 and 1992. Nineteen (28%) were asymptomatic at diagnosis, while the rest experienced fatigue (60%), B-symptoms (12%), and recurrent infections (15%). Associated comorbid conditions included rheumatoid arthritis (RA) in 26%. Severe anemia (hemoglobin [Hb] &lt; 8g/dL) and neutopenia (absolute neutrophil count [ANC] &lt; 500/microL) were seen in 19% and 40% of patients, respectively. Immunophenotypic studies showed CD3+, CD8+ phenotype in the majority (72%). Twenty-one patients (31%) have required no therapy, and remain relatively stable with a median follow- up period of 50 months. Treatment was required at either diagnosis (36 patients) or at subsequent follow-up (11 patients). Initial response rates were similar in patients treated with cyclophosphamide (CTX) with or without prednisone (69%), or prednisone alone (73%). Overall, 61 patients (90%) are alive with a median follow-up of 44 months. Actuarial median survival of this entire cohort is 161 months. The presence of anemia or symptoms does not appear to correlate with the tumor burden. In patients requiring therapy, a lower ANC and the presence of B-symptoms/infection were independently associated with a significantly lower probability of achieving a molecular or hematologic complete remission (H-CR). Intermittent immunosuppressive therapy is effective in achieving durable responses in a number of patients. T-LGL proliferations are associated with a favorable prognosis and response to therapy. However, significant heterogeneity exists in clinical presentation and associated comorbid conditions. These disorders should be included in the differential diagnosis of patients with unexplained cytopenias, particularly in the setting of RA and other autoimmune disorders. Analogous to the situation with monoclonal gammopathies, a term such as T-cell clonopathy of undetermined significance (TCUS) may be more appropriate to describe these patients.

AbstractLymphoproliferative disease of granular T lymphocyte (T-LDGL), also known as T-cell large granular lymphocyte leukemia, is a clonal disorder of cytotoxic T lymphocytes that is clinically manifested as chronic neutropenia and anemia. Association with autoimmune disorders is common. In 9 patients, T-LDGL is reported as presenting as aplastic anemia. The clinical characteristics were similar to acquired aplastic anemia. Morphologic evidence of increased granular lymphocytes in the peripheral blood and an excess of CD3+/CD8+/CD57+ cells in the bone marrow were found in most cases. Cyclophosphamide was ineffective, but noncytotoxic immunosuppressive agents generally produced a good response. After a median follow-up of 49 months, 5 patients had died from the disease or related complications. Median survival was 40 months. Aplastic anemia can be a presenting manifestation of T-LDGL, and T-LDGL should be considered in the differential diagnosis of acquired aplastic anemia.

Abstract Abstract 2807 CCDC26 is a retinoic acid-dependent modulator of myeloid cell differentiation and death (Yin W et al. Oncogene 2006 :25 :3735). CCDC26 maps to chromosome 8q24.21 and germline polymorphisms in the gene have been associated with low-grade gliomas (Jenkins RB et al. Cancer Genetics 2011;204 :13), which in turn have recently been shown to frequently express IDH1 mutations (Parsons D et al, Science 2008;321 :1807). More recently, we have discovered that CCDC26 SNP associations in gliomas are primarily restricted to patients with IDH1 mutations (Jenkins et al. unpublished observations). We therefore hypothesized that germline CCDC26 SNPS might be associated with patients with myeloid malignancies that harbor IDH1 mutations (Mardis et al. NEJM 2009;361 :1058). Seventy-two IDH -mutated patients with chronic or blast phase myeloproliferative neoplasms (MPN), myelodysplastic syndromes or acute myeloid leukemia (most constituting post-MPN events) were identified: 18 IDH1 R132, one IDH2 R172 and 53 IDH2 R140. These 72 patients and a control group of 153 IDH unmutated cases with myeloproliferative or myelodysplastic neoplasms (total n =225) were genotyped for CCDC26 risk alleles associated with IDH -mutated gliomas: rs10464870, rs891835 and rs4295627. The respective minor allele frequencies (MAF) were significantly different between IDH1 (11%, 8% and 6%) and IDH2 (30%, 27% and 24%) mutated cases (p=0.037, 0.040 and 0.021; OR 3.4, 3.7 and 6.1 with 95% CI of 1.1–10.5, 1.1–13.0 and 1.3–28.9) as well as between IDH- wild type (24%, 24% and 21%) and IDH1 -mutated (11%, 8% and 6%) cases (p=0.10, 0.043 and 0.035; OR 0.42, 0.29 and 0.21 with 95% CI of 0.15–1.19, 0.09–0.96 and 0.05–0.90). The MAF values in our IDH -unmutated cases were similar to that of population-based controls. However, the direction of SNP associations in IDH1 -mutated myeloid malignancies were opposite of those seen in IDH1 -mutated gliomas. Our observations suggest a differential effect of CCDC26 genetic polymorphisms in IDH1 -mutated myeloid malignancies compared to either their IDH2 R140-mutated or IDH1 -mutated gliomas. Such differences in genomic context might contribute to hitherto seen differences in prognostic impact between IDH2 R140 vs. IDH1 R132 vs. IDH2 R172, in AML (Green CL et al. Blood Published online before print May 19, 2011, doi: 10.1182/blood-2010-12-322479). Disclosures: No relevant conflicts of interest to declare.