Citations

Abstract Background: The occurrence of an activating JAK2 mutation (JAK2 V617F) has been reported in a variable proportion of patients with bcr/abl-negative myeloproliferative disorders including myelofibrosis with myeloid metaplasia (MMM). In the current study, we report mutational frequencies in each of the 3 MMM variants as well as examine the prognostic relevance of the mutation in a large group of patients from a single institution. Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from peripheral blood mononuclear cells (PBMC) from all study patients (n=157). Concomitant analysis was also performed in DNA derived from purified granulocytes (n=57), CD34-positive cells (n=25), and T cells (n=19). Genomic DNA was amplified by PCR and fluorescent dye chemistry sequencing was performed using the same primers used for amplification. Results: The study cohort included 157 consecutive patients with MMM including 117 with agnogenic (AMM), 22 with post polycythemic (PPMM), and 18 with post thrombocythemic (PTMM) myeloid metaplasia. The overall detection rate for JAK2 V617F in PBMC was 51% (homozygous in 5.7%) and was significantly (p=0.0002) higher in PPMM (91%; homozygous in 18%) compared to either AMM (45.3%; homozygous in 2.6%) or PTMM (38.9%; homozygous in 11.1%). In AMM (n=117), the presence of JAK2 V617F was significantly associated with an older age at diagnosis (p=0.03), and a history of both thrombosis (p=0.01) and pruritus (p=0.005). No other statistical associations were evident and the presence of the mutation did not predict leukemic transformation. Furthermore, multivariate analysis for overall survival of patients with AMM identified only age and the Dupriez prognostic score as independent prognostic factors; JAK2 V617F had no prognostic significance. Finally, the mutation was not found in T cells and its detection rate in either granulocytes or CD34-positive cells was similar to that in PBMC. Conclusion: JAK2 V617F is a myeloid lineage-specific event, its incidence in MMM is significantly higher with an antecedent history of polycythemia vera, and its presence in AMM does not affect prognosis but is associated with PV-characteristic clinical features.

Abstract Background: Conventional cytogenetic studies in myeloproliferative neoplasms (MPNs) have provided pathogenetic, diagnostic and prognostic information and in certain instances guide therapeutic decisions. It is therefore reasonable to hypothesize that a higher resolution examination of chromosomes might reveal additional changes that are scientifically as well as clinically relevant. Methods: Granulocyte DNA from 70 patients (median age 56 years, range 18–84; 43 males) with classic BCR-ABL-negative MPNs were studied: 27 with primary myelofibrosis (PMF), 22 with polycythemia vera (PV), 12 with essential thrombocythemia (ET), 6 with post-ET MF and 3 with post-PV MF. Twelve healthy volunteers were used as controls. Cytogenetic information, collected at variable times before the current study, was available in 57 patients. Samples were analyzed by comparative genomic hybridization (CGH) with a gender matched normal control using oligoarrays with either 44,000 or 105,000 oligonucleotides. Data were analyzed with CGH Analytics (Agilent). Copy number changes were considered significant (CNCs) if they were defined by 4 or more adjacent oligonucleotides spanning at least 150,000 base pairs, contained at least one gene, and were not identified in the Database of Genomic Variants. Results: Array CGH results were equivocal in 6 cases, which were therefore excluded from further analysis. Among the 12 normal controls, granulocyte DNA displayed no CNCs. The percentage of patients with CNCs was 52% for PMF, 32% for PV and 17% for ET. In addition, 2 of 6 patients with post-ET MF and 1 of 2 patients with post-PV MF displayed CNCs. There was no correlation between the occurrence of CNCs and JAK2V617F mutational status in PMF and ET. Array CGH revealed CNCs in 16 of 49 patients (33%) with normal and 7 of 8 patients (88%) with abnormal cytogenetic findings; one patient with t(2;12)(p13;q14) in 19 of 20 metaphases detected at 9 months prior to sample collection for the array CGH study displayed no CNCs. In patients with abnormal cytogenetic findings, array CGH also detected imbalances at chromosomal translocations, including t(1;6), t(9;11), and t(1;18), as well as additional abnormalities that were not detected by cytogenetic analysis. A total of 23 single occurrence CNCs were documented and included all the ET [del(2)(q31.3q32.1), del(10)(q11.21q11.22)] and post-ET MF [del(2p12), del(Xq12)] associated CNCs. In contrast, several specific abnormalities were shared between PV and PMF including del(6p22) and dup(15q11.2). As expected, del(13q) and del(20q) abnormalities were recurrent in PMF. Conclusions: Conventional cytogenetic analysis underestimates the extent of genomic gains and losses in BCR-ABL-negative MPNs. The current study suggests that oligonucleotide array CGH is more sensitive to detect small genomic alterations and we are currently studying higher resolution arrays in a larger group of patients to detect additional genomic alterations as well as assess overall clinical utility. The current data also suggest that chromosomal translocations are commonly associated with imbalances.

Abstract Background: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal stem cell disorder characterized by intravascular hemolysis, thrombosis, bone marrow failure and reduced survival. In this current single institution study of patients with PNH, we investigate the risk factors predictive of survival and thrombosis. Methods: Data were abstracted from medical records of a consecutive cohort of patients with PNH seen at the Mayo Clinic. Diagnosis was confirmed by the presence of intravascular Hemolysis and either a positive Ham’s test, and/or by the presence of a GPI-deficient hematopoietic clone. Follow-up data were obtained by contacting the patients and by reviewing the social security death index. Results: i. Patients and treatment: The study cohort included 68 patients with PNH (median age 32 years, range 10–77; 33 females) with a median follow up of 84.5 months (range, 1–538 months). Presenting features included infection (n=9), thrombosis (n=9), neutropenia (absolute neutrophil count (ANC) < 1000) (n=32) severe intravascular hemolysis (n=16) and splenomegaly (n=11). Prior to the diagnosis of PNH, 6 patients (8.8%) carried a diagnosis of aplastic anemia (AA) and 2 (2.9%) myelodysplastic syndrome (MDS). Complications documented after diagnosis included major thrombosis (n=11), major hemorrhage (n=4), and development of AA (n=7), MDS (n=4), and non-hematologic cancer (n=4) but none experienced leukemic transformation. Treatment included prednisone (n=30), other immunosuppressive agents (n=6), androgens (n=10), and bone marrow transplantation (n=4). Review of treatment with systemic anticoagulation disclosed only 2 patients that were receiving prophylactic warfarin therapy and both patients remain free of thrombosis after 1 and 2 years of therapy. ii. Thrombotic events and risk factors: Overall, 20 patients (29.4 %) experienced one or more episodes of major thrombosis. Among several clinical and laboratory parameters evaluated at presentation, only the detection of urine hemoglobin and infection were found to be significantly associated with the occurrence of thrombosis (p=0.05) iii. Risk factors for survival To date, 18 patients (27%) have died and median survival for the study cohort is projected at 21.1 years. Multivariate analysis identified thrombosis (p=0.003) and an ANC of > 1000 (p= 0.05) at presentation as independent risk factors for inferior survival. In addition, the occurrence of major hemorrhage was significantly associated with poor survival (p= 0.04). Conclusion The current study provides natural history information in a relatively large cohort of patients with PNH and identifies certain disease features that could be further explored for their potential as risk factors for both thrombosis and survival.

Abstract Background: Copper deficiency is an established cause of hematological abnormalities, traditionally described in malnourished infants and persons with malabsorption or short bowel syndromes, particularly those requiring long-term parenteral nutrition. With the recent increase in bariatric (weight reduction) surgery, acquired copper deficiency may be increasing in incidence but the diagnosis is frequently delayed. Methods: To outline the natural history of copper deficiency we searched the Mayo Clinic database and our heavy metals laboratory for patients diagnosed with low serum copper during the years 1974–2005. Copper deficiency was defined as a serum level < 0.75 μg/ml. Results: We identified 123 patients with a low serum copper. 77 patients were excluded as the the hypocupremia was either transient or due to other factors such as Wilson’s disease. The remaining 46 patients were all symptomatic. 25 patients had hematological abnormalities, 20 of these patients also had neurological involvement. 5 had isolated hematological manifestations and 21 patients had isolated neurological findings, most commonly myeloneuropathy. 13 presented with anemia and leukopenia, 5 with pancytopenia, 6 with anemia and 1 with isolated neutropenia. In 10 of the 25 cases, a secondary contributing cause for the hematological findings was identified. These included possible anemia of chronic disease (4), renal insufficiency (2), iron deficiency (1), B12 deficiency (1), GI hemorrhage (1), and splenomegaly (1) while copper deficiency was the only identifiable cause in the remaining 15 patients. Hematological parameters at diagnosis are outlined in the table. 15 of the 25 patients with hematological manifestations had a history of GI surgery, including 6 patients who had bariatric surgery. The median time from surgery to diagnosis was 11.5 years (range 8 months - 45 years) and was shorter for bariatric surgery (10 years, range: 8 months - 25 years) than for non-bariatric surgery (18 years, range: 2–45 years). 1 patient had a history of excessive zinc ingestion, 1 had malabsorption secondary to chronic pancreatitis and 1 patient had celiac disease. In 7 cases, no explanation was found. 24 of 25 patients received treatment with oral or intravenous copper, 5 were lost to follow up. In 16 patients the hematologic parameters either improved significantly or normalized, while in the remaining 3 patients there was no improvement. Factors that may explain the lack of response in these patients included splenomegaly (1), renal insufficiency (1) and multiorgan illness (1). Conclusion: Copper deficiency is an increasingly frequent cause of hematological abnormalities, and can manifest with refractory cytopenias with or without associated neurological symptoms. Thrombocytopenia is relatively rare. Previous GI surgery, especially bariatric surgery, appears to be a major causative factor. Most patients will have an improvement in hematological parameters after copper supplementation. Recognition of copper deficiency is therefore assuming greater importance in view of the increasing frequency of bariatric surgery, ease of treatment and potential for irreversible neurological damage if diagnosis is delayed. Hematologic parameters at diagnosis HgB (g/dL) MCV (fL) WBC (x109/L) ANC(x109/L) Plts(x109/L) Median 10.6 99 3.0 1.25 215 Range 7.9-13.6 83.9-114.1 1-5.6 0.18-4.0 54-554

Abstract We previously have reported that autograft absolute lymphocyte count (A-ALC) is a possible prognostic factor for survival after autologous peripheral blood stem cell transplant (ASCT) for myeloma (MM). Factors affecting A-ALC in MM are unknown. We hypothesize that method of stem cell mobilization, hematopoietic growth factor (HGF) vs. HGF+Cytoxan chemotherapy (C+HGF), directly affects A-ALC collection. 191 consecutive MM patients between 1994 and 2004 were analyzed retrospectively. Patients generally were mobilized with C+HGF prior to 2003. Thereafter, C+HGF was reserved largely for those with ≥4% circulating peripheral blood plasma cells (PC), a negative prognostic indicator. No patients were transplanted in disease relapse or refractory disease. Patients also were matched for age, sex, β2-microglobulin, conventional cytogenetics, LDH, c-reactive protein, number of prior therapies, plasma cell labeling index (PCLI), pre-mobilization ALC, and % bone marrow (BM) PC. The groups HGF (n=80) and C+HGF (n=111) differed with respect to the conditioning regimen (p < 0.0001), and presence of (≥4%) circulating peripheral blood PC (p<0.005). The primary end-point of the study was to assess the correlation between HGF vs C+HGF, and A-ALC. The secondary endpoint was to determine if HGF vs C+HGF affected survival post-ASCT. Patients mobilized with HGF had a higher A-ALC compared to those mobilized with C+HGF [0.764 x 109 lymphocytes/kg (range: 0.146–1.803) vs. 0.212 (range: 0.016–1.26), p<0.0001]. No association was identified between A-ALC and conditioning regimens (p = 0.19) and PC (p = 0.31). Median overall survival (OS) and progression-free survival (PFS) were longer in those mobilized with HGF vs. C+HGF (not reached vs. 48 months, p<0.0150; not reached vs. 21 months, p<0.007, respectively). Multivariate analysis demonstrated that age ≥50 vs age ≤50 (p<0.05) and A-ALC ≥0.5 vs <0.5x109 lymphocytes/kg (p<0.0397) were independent predictors of OS. Factors influencing PFS in the multivariate analysis included circulating PC ≥4% vs <4% (p<0.0157), PCLI ≥ 1% vs PCLI ≤ 1% (p<0.0107), and A-ALC ≥0.5 vs <0.5x109 lymphocytes/kg (p<0.0042). On multivariate analysis, the method of stem cell mobilization and the conditioning regimen did not have a statistically significant effect on either OS or PFS. We hypothesize that the differences in PFS and OS seen between the HGF vs C+HGF mobilization groups are mediated through the A-ALC. These data suggest that mobilization regimens should not only collect CD34+ stem cells, but also be optimized to collect an A-ALC target which may impact on PFS and OS post-ASCT.

Abstract Background: Myelofibrosis with myeloid metaplasia (MMM) currently has limited therapeutic options. BAY 43-9006 (sorafenib; Bayer Pharmaceuticals) is a oral multikinase inhibitor with both inhibitory properties against the Raf/ MEK survival pathway and VEGF that are a potential therapeutic targets for the myeloproliferation and apoptotic resistance seen in this disorder. We evaluated the activity of this agent through inhibition of aberrant in vitro myeloid colony growth, as we have previously successfully utilized in this disorder (Leukemia2003;17(5):849). Methods: Peripheral blood mononuclear cells (PBMC) were isolated and plated on the Methocult™ (StemCell Technologies) colony assay media. Dilutions of BAY 43-9006 (0–10 μM) (Phase I trial sustained concentrations of 10 μM (JCO2005;23:965) was added for either a 24 hour incubation, or for 14 days. Myeloid colonies were then quantified according to standards established by the manufacturer. Subsequent assays were performed combining BAY 43-9006 with the pro-apoptotic agents arsenic trioxide (ATO) or adaphostin (ADA) (both at 1.25μM). Finally, immunoblotting was performed on protein extracted from treated primary MMM cells to quantify the downregulation of Mcl-1 (shown to be crucial for the pro-apoptotic effects of BAY 43-9006; Yu et. al. Oncogene 2005). Results: PBMC were isolated from 12 MMM patients and 10 controls (normal n=5, other myeloproliferative (MPD) n=5). Twenty-four hour exposure of cells to the agent was insufficient to suppress aberrant colony growth. Continuous exposure (for 14 days) of the agent led to a median inhibitory concentration-50% (IC50) of 12 μM for MMM (compared to 7.1μM for normals, and 5μM for MPD controls). Although no specificity could be deduced from these latter results, 4 MMM patients had an IC50 of <10μM (range 1.25 to 8.6μM). In contrast, the other 8 patients were quite resistant IC50 of > 10μM (higher than clinically achievable ranges). Subsequently we evaluated whether there might be benefit to use of BAY43-9006 in combination with a pro-apoptotic agent (i.e. ATO or ADA). No significant activity was seen combining ADA with BAY43-9006. When BAY43-9006 was combined with ATO, the median IC 50 for the BAY compound decreased to 2μM (range 1.25 to 16), however there was not a significant increase in colony inhibition over ATO alone. In order to evaluate whether the lack of activity of BAY43-9006 observed in colony formation assays was secondary to a lack of downregulation of the established target Mcl-1, we immunoblotted for Mcl-1 (after in vitro incubation) in a subset of MMM patients. Western blots demonstrated successful downregulation of Mcl-1 by Bay 43-9006 in MMM samples, even in samples with clear in vitro resistance to the drug (i.e. IC50 > 10 μM). Conclusions: The multi-kinase inhibitor BAY43-9006 appears to have minimal activity against primary cells isolated from MMM patients. Although, suppression of aberrant colony growth was noted in a subset of patients the sensitivities would not support specificity when compared to controls. Although BAY 43-9006 was observed to down regulate the anti-apoptotic target Mcl-1, in patient samples, no detectable inhibition of colony formation was observed in 8/12 MMM samples. These preliminary results do not appear to support a clinical trial of BAY 43-9006 in MMM, however combination of this multi-kinase inhibitor with other pro-apoptotic agents should be evaluated.

Abstract Background: Microcytosis is a characteristic laboratory feature for both iron deficiency anemia and thalassemia. Microcytosis is also infrequently seen with “anemia of chronic disease”. A partial list of the latter include rheumatoid arthritis, polymyalgia rheumatica, diabetes mellitus, connective tissue disease, chronic infection, Hodgkin’s lymphoma, Castleman’s disease, and renal cell carcinoma. In the current study, we show that microcytosis is a frequent laboratory feature in myelofibrosis with myeloid metaplasia (MMM) and investigate its clinical relevance in the particular setting. Methods: In order to minimize the confounding effects of nutritional deficiencies, red blood cell transfusion, and drug therapy on mean corpuscular volume (MCV), the current study was limited to young patients (age < 60 years) that had not received either red blood cell transfusion or drug therapy for MMM during the time of their initial laboratory investigation. Results: The study population consisted of 102 consecutive patients with MMM (median age 51 years, range 18–60) including 60 males. At diagnosis, the median (range) values for hemoglobin, leukocyte count, and platelet count were 11 gm/dL (7.4–14.9), 8.3 x 109/L (1–40) and 357 x 109/L (14–1512), respectively. Spleen measurements were documented in 78 patients (median 6 cm below the left costal margin, range 0–24) and the incidence of hypercatabolic symptoms was 18% (12 of 67 evaluable patients). The Dupriez prognostic score distribution included low-risk (n=73), intermediate-risk (n=19), and high-risk (n=10) disease categories. The overall incidence of microcytosis (MCV < 80 fL) was 28%; median MCV was 84.3 fL (range, 60.6–99.1). Among the 28 patients with microcytosis, MCV was less than 70 fL in 4 patients. Concomitant serum ferritin levels were available in 44 patients (median, 92 μg/L; range, 4–6440) and the levels were below 30 μg/L in 7 patients including 4 with microcytosis. Bone marrow iron stains were sporadically performed and showed normal iron stores in the majority of evaluable cases. The study cohort was followed for a median of 70 months (range 0–300). Multivariate analysis of variable that were measured in all study patients identified anemia, thrombocytopenia, and age but not MCV as independent prognostic indicators of inferior survival. The addition of variables that were measured in a variable proportion of the study population disclosed additional prognostic value for hypercatabolic symptoms and abnormal cytogenetics. Interestingly, microcytosis was significantly associated with the absence of cytogenetic abnormalities (p=0.04); among the 10 patients with abnormal cytogenetic findings, 9 had an MCV of ≥ 80 fL. Microcytosis did not correlate with several other clinical and laboratory features including gender, presence of hypercatabolic symptoms, spleen size, hemoglobin level, platelet count, leukocyte count, serum lactate dehydrogenase level, and the Dupriez prognostic score. Conclusion: Microcytosis with a normal or elevated serum ferritin level is a frequent laboratory feature in MMM but does not appear to influence survival. The observation regarding the low incidence of cytogenetic abnormalities in MMM patients with microcytosis is intriguing but requires validation by a prospective study. Whether or not MMM-associated microcytosis represents an acquired defect of globin synthesis, as might be the case in some patients with myelodysplastic syndrome, remains to be elucidated.

Abstract BACK GROUND Primary myelofibrosis (PMF) leads to weight loss, splenomegaly and constitutional symptoms (Cancer2007;109:68–76) and hypocholesterolemia associated with decreased survival (Blood2007;110:a2548). Initial trials of JAK2 inhibitors have demonstrated improvements in these (JCO2008;26:a7004), yet little objective data exists regarding the dynamic changes in weight and spleen size during the course of PMF. METHODS We analyzed the Mayo PMF database for patients with information on disease prognosis, presentation, therapies, height and weight at diagnosis, and outcomes. Additionally, when available, we analyzed additional weights during the clinical course, the body mass index (BMI- (weight/(height*height)), spleen size, and peripheral blood studies including lipids. Results: 370 patients with PMF were identified with 193 (52%) having a weight at the time of diagnosis, the remainder had a weight obtained a median of 10.0 months (range 1.8–159) after diagnosis. Additionally, 210 patients (57%) had a weight value available from 1–3 additional time points during the course of their disease. At PMF diagnosis patients had a median BMI of 25.3 (range 17.6 to 38.4), with 53% considered overweight (USA Norms (NHANES 2004): 66% and 32.2% are considered overweight or obese). Although weight lost prior to diagnosis unclear, only 2% of patients would be deemed “underweight” at diagnosis. Serial analysis demonstrated 67% experienced a decrease in weight over time, with 23% and 6% experiencing a >10% or 20% decrease, respectively. Overall during the course of illness 65% of patients remained in the same BMI category with 27% dropping to a lower category and 8% gaining in a category (Table 1). Table 1. Distribution of Body Mass Index and Spleen Size in 370 PMF patients followed serially DX of PMF Weight 2 Weight 3 Weight 4 Underweight BMI<18.5 2% 1% 2% 2% Normal BMI=18.5–24.9 45% 41% 41% 54% Overweight BMI=25–29.9 33% 42% 41% 35% Obese BMI>30 20% 16% 16% 10% Spleen Size (cm BLCM) (median: range) 5.0 (0–30) 6 (0–33) 7 (0–33) 7.5 (0–28) Time from PMF DX (median(range) months) 0 10.3 (1.8–159) 28.3 (6–191) 57 (15–258) Major weight increases were rare <1%, and might include buildup of fluid or splenic mass. Indeed spleen sizes generally increased during the course of the illness, with 51% having “massive” splenomegaly (either >10 (45%) or >20 (20%) cm below left costal margin or requiring splenectomy (19%)). Massive splenomegaly was not associated with weight loss or weight gain, suggesting increasing splenic mass came at the expense of alternate tissue mass (muscle and or fat). Median survival for the entire cohort was 77 months and PMF prognostic scores being valid (Lille PMF score p<0.01; IWG-MRT PMF Score - Submitted separately Cervantes et. al. p<0.01), however BMI number and category was not independently predictive of survival. Additionally, neither percentage of weight loss nor changing BMI category were associated with decreased survival. Although splenomegaly was not independently associated with decreased survival, the need for splenectomy clearly was (p=0.02). Consistent with our prior observation hypocholesterolemia (total <150 (median of 151 (range 12–366) or a HDL cholesterol of <60 (median 77 (range 25–248)) were associated with decreased survival (p<0.01 and =0.05, respectively). Decreasing weight in PMF patients was most highly correlated with decreases in HDL (p=0.03). Hypocholesterolemia was highly associated with splenomegaly (p<0.001 for spleen size >10 cm at any point during disease course). Conclusions: Weight loss is common in most PMF patients, despite the confounders of edema and increasing splenomegaly which could mask the degree of loss of lean body mass. Although few PMF patients are considered “underweight” by BMI standards, the distribution compared to USA norms suggest a lower weight distribution. Additionally, patients during the course of their illness are most likely to fall from a BMI overweight category to a “normal” category which may underestimate the true degree of catabolism of muscle mass occurring. Although not independently associated with decreased survival, weight loss remains common and a sign of advancing disease in this terminal illness. More precise methodology to estimate true lean muscle mass lost or gained may yield a better understanding of the hypercatabolic state seen in PMF. Efficacious therapy (such as potentially a JAK2 inhibitor) will likely reverse the trend of weight loss in these patients.

Abstract Background: Leukocytosis has recently been implicated as an adverse prognostic feature for thrombosis in both ET and PV. Such an association would be therapeutically most relevant in the context of “low-risk” disease. In the current study, we sought to clarify the relationship between leukocytosis at diagnosis and the subsequent occurrence of either arterial or venous thrombosis, in “low-risk” patients with ET or PV. Methods: Data was abstracted from the medical records of a consecutive cohort of patients with WHO-defined ET or PV seen at the Mayo Clinic. Low-risk disease was defined by the absence of both thrombosis history and age 3 60 years. Cox proportional hazards model was utilized to determine the impact of clinical and laboratory variables on thrombosis-free survival (TFS). Arterial- or venous-specific TFS curves were constructed by Kaplan-Meier method. Results: i) Patient characteristics and outcome A total of 407 “low-risk” patients were studied; 153 had PV (median age 48 years; females 43%) and 254 ET (median age 42 years; females 71%). A total of 46 thrombotic events (22 arterial and 24 venous) were recorded in 41 (27%) patients with PV during a median follow up of 130 months (range 2–562 months). The corresponding figures in ET were 54 total thrombotic events (41 arterial and 14 venous) in 47 (19%) patients at a median follow up of 104 months (range 0.25–424 months). Cytoreductive therapy was avoided in the presence of < 1000 x 109/L platelet count but, at the discretion of the treating physician, some patients with higher platelet counts received prophylactic cytoreductive therapy. ii) Correlation between leukocytosis and thrombosis A leukocyte count of 3 15 x 109/L at diagnosis was documented in 42 (27%) patients with PV and 21 (8%) patients with ET; 102 patients (40%) with ET had > 9.4 x 109/L leukocyte count. Leukocyte count considered as either a continuous or categorical variable (using cutoff levels of 15 x 109/L for PV and either 15 x 109/L or 9.4 x 109/L for ET) was not significantly associated with either arterial or venous thrombosis (Figure 1). iii) Correlation between other risk factors and thrombosis In univariate analysis, presence of the JAK2V617F was significantly associated with arterial thrombosis in ET (p=0.049) but significance was lost during multivariable analysis that included age as a covariate. Only advanced age was found to be significantly associated with arterial thrombosis in PV (p=0.04) and higher hemoglobin level with venous thrombosis in ET (p<0.0001). Conclusion: The current study does not identify leukocyte count at diagnosis as a marker of increased thrombosis risk in low-risk patients with either ET or PV. Instead, the study found an association between arterial thrombosis and advanced age in PV and venous thrombosis and higher hemoglobin level in ET. Figure Figure

Abstract BACKGROUND: In myelodysplastic syndromes (MDS) without excess blasts, red blood cell (RBC) transfusion requirement has been associated with poorer overall (OS) and leukemia-free (LFS) survival, suggesting that transfusion dependency is a marker of more severe disease (JCO2005;23:7594). We recently reported similar results in refractory anemia with ring sideroblasts (RARS): RBC transfusion requirement was an IPSS-independent adverse prognostic factor, but neither serum ferritin nor transfusion burden had prognostic value (AJH2008;83:611). Here, we examine the prognostic relevance of serum ferritin, need for RBC transfusions at time of diagnosis, and total transfusion burden in primary myelofibrosis (PMF). METHODS: We reviewed medical records to ascertain the clinical and laboratory features of 185 consecutive patients diagnosed with PMF according to the 2001 World Health Organization (WHO) criteria. Patients were excluded if ferritin measurements at time of diagnosis were unavailable. OS and LFS curves were constructed by Kaplan-Meier method, taking the interval from the date of diagnosis to death, leukemic transformation, or last contact. Log-rank test was used to test the homogeneity of survival curves over different groups. Cox proportional hazards model was utilized to determine the impact of various clinical and laboratory variables on OS and LFS. RESULTS: Clinical characteristics at diagnosis: Median age was 58 years (range 15–81; 110 males). Median serum ferritin level at diagnosis was 164 ng/mL (range 1–3903) and was ≥1000 ng/mL in 22 patients (12%). 101 (55%), 65 (35%) and 19 (10%) patients were assigned low, intermediate- and high-risk disease category (Blood1996;88:1013). In addition, 32 (17%) patients required RBC transfusions, 33% had constitutional symptoms and 39% displayed ≥ 1% circulating blasts. Where evaluated, 40% of the patients had cytogenetic abnormalities and 56% JAK2V617F. Events during the disease course: At a median follow-up of 28 months (range 0.5–231), 79 (43%) deaths and 15 (8%) leukemic transformations were documented. During this period, 126 (68%) patients required some form of therapy other than transfusion, including splenectomy in 33 (18%); only 4 underwent allogeneic stem cell transplantation. Causes of death were documented in 33 instances and none were attributed to iron overload. Median peak serum ferritin level during the disease course was 231ng/mL (range 9–13,080); 144 (78%) patients had peak levels <1000 ng/mL, 28 (15%) between 1000 and 3000 ng/mL, and 13 (7%) above 3000 ng/mL. Number of total RBC transfusions ranged from none to 121. Iron chelation therapy was reported in 62 patients (34%). Prognostic factors for OS and LFS: Kaplan-Meier projected median survival was 72 months. By univariate analysis, increased serum ferritin level at diagnosis considered as either a continuous (p<0.0001) or categorical (≥ 1000 ng/mL) variable (p<0.0001), RBC transfusion requirement at diagnosis (p<0.0001) and higher number of total RBC units transfused during the course of the disease (p=0.004) were all associated with inferior survival. However, only RBC transfusion requirement at diagnosis sustained its prognostic significance when age and conventional prognostic risk scores were added to the multivariable model as covariates. Similarly, a peak serum ferritin level of > 3000 ng/mL documented during the disease course was not detrimental to survival. History of iron chelation therapy was associated with shortened survival (p=0.003). Multivariable analysis that included previously described risk factors for LFS also identified RBC transfusion requirement at diagnosis as an additional and independent risk factor. CONCLUSIONS: Serum ferritin level at time of diagnosis or during the disease course of PMF lacks independent prognostic value for either OS or LFS. The same is true for total transfusion burden. However, although hemoglobin <10 g/dL is a component of all currently utilized prognostic scoring systems for PMF, the presence of a more severe erythropoietic defect as indicated by RBC transfusion need at time of diagnosis has an additional adverse prognostic value for both OS and LFS.