Mesa, Ruben A.

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

Abstract Background: An acquired point mutations of JAK2 (V617F) was recently described in a substantial proportion of patients with myeloproliferative disorders (MPD), including myelofibrosis with myeloid metaplasia (MMM). The current study examines i) JAK2 V617F mutational status at time of leukemic transformation (LT) of MMM and its clinical correlates, ii) the possibility of changes over time in mutational status in MMM patients that have either undergone leukemic transformation (LT) or experienced disease progression. Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from either peripheral blood mononuclear cells (PBMC) (i.e. fresh specimens) using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Fluorescent dye chemistry sequencing was performed using the same primers used for amplification. Results: The study population consisted of 68 patients including 33 patients with disease transformation into AML. i. MMM patient cohort with LT (n=33). The JAK2 V617F mutant allele was detected in 21 of the 33 patients (64%; homozygous in 6%) at the time of LT. As expected, PPMM was over-represented in the group with the mutation but the presence of the mutation affected neither the clinical presentation at time of LT or overall outcome. Furthermore, in 9 patients (7 heterozygotes and 2 with wild-type allele), archived bone marrow that was collected at a median of 23 months (range, 3–105) before LT was available and disclosed no change in mutational status. ii. MMM patient cohort with disease progression without LT (n=35; 19 AMM, 9 PPMM, 7 PTMM): Each patient in this group had at least two bone marrow or peripheral blood samples that were collected at two different time points separated by a median of 18.2 months (range (3–82). Of the total 35 patients in this group, 32 (91%) showed no change in JAK2 V617F mutational status over time including 3 patients in whom the first sample was collected before they transformed into MMM from antecedent PV or ET. In the remaining 3 patients, a switch from wild-type to heterozygous state (1 patient), and heterozygous to homozygous (2 patients), was documented after 78, 43, and 83 months, respectively. Conclusions: In most instances, JAK2 V617F mutational status in MMM remains unchanged over time including during leukemic transformation. Furthermore, the presence of the mutation in MMM-related LT does not appear to affect clinical outcome. These observations undermine the role of JAK2 V617F in either disease progression or LT in MMM.

Abstract Background: Little objective data exists regarding the impact that myeloproliferative disorders (MPDs) (essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis with myeloid metaplasia (MMM)) have on quality of life. Methods: An Internet based survey of MPD patients regarding clinical course, co-morbidities (through the Charlson Co-Morbidity Index), fatigue (through two validated fatigue instruments (FACT-AN and Brief Fatigue Inventory (BFI)), and exercise tolerance (Godin Leisure-Time Questionnaire). Results: Patient Demographics: A total of 830 MPD patients (median age 56; 60.8% male) from a broad geographic distribution (77% USA: 50 states, 30 countries, 5 continents) responded to the survey. Self reported diagnoses were PV (N=272; 33%), ET (N=221; 27%), or MMM (N=329; 40%) for a median of 5 years (range (0–53). Clinical History: 708 patients (87%) had undergone treatment for their MPD (drugs 84.6%, phlebotomy 49.5%, other 16%). A history of thrombotic (n=195, 24%) or hemorrhagic events (N=214; 26%) were frequently reported. Blood tests (available in 597 a median 1.65 months prior to survey) showed 37% were anemic (7% transfusion dependent). Additionally, 347 (44%) had splenomegaly, painful in 21%. Symptoms: Frequent symptoms reported included fatigue (90%), pruritus (57%), night sweats (54%), bone pain (48%), undesired weight loss (15%), and/or fevers (15%). 41% felt restricted in their activities, and 89 (11%) were medically disabled secondary directly to their MPD. 82% of patients felt their fatigue was linked to their MPD. Additionally 69% curtail social activity because of fatigue, and 41% need some degree of assistance with activities of daily living. Charlson co-morbidity index demonstrated 722 (87%) had minimal co-morbidities strengthening the association between fatigue and their MPD. The burden of fatigue decreased from MMM to PV to ET respectively (p=0.0002). Results from both fatigue assessment tools (FACT-AN and BFI) were highly correlated. Fatigue (FACT-AN fatigue subscale) strongly correlated with multiple MPD disease features including: anemia, bone pain, constitutional symptoms, pruritus, splenic pain, hemorrhagic events (all p<0.0001) and thrombosis(p=0.01). Additionally, currently smoking (as opposed to being a prior smoker) was highly associated with increased fatigue (p<0.0001). Interestingly, even in “asymptomatic” patients (those without anemia, splenomegaly, or thrombohemorrhagic complications (n=187)) 73% (n=128; 40% PV, 32% ET, 27% MMM; p=N.S.) described moderate to severe MPD associated fatigue. Reported physical activity was diminished (Godin Leisure Score) between MPD patients and published norms, with 38% describing minimal to no exercise. Overall, 86% stated fatigue was the major obstacle to exercise. Conclusions: Fatigue is an extremely common problem in patients with MPDs despite current therapies, and a minimal burden of co-morbidities in the majority of patients. Fatigue is also common in patients lacking advanced MPD disease features, or thrombohemorrhagic complications. Clearly, direct attempts to improve fatigue (through pharmacologic and non-pharmacologic interventions) are necessary.

Abstract BACKGROUND: Disease manifestations in myelofibrosis with myeloid metaplasia (MMM) that require therapy include anemia, thrombocytopenia, hepatosplenomegaly, and constitutional symptoms. We have previously reported on the therapeutic value of low-dose thalidomide in combination with prednisone for the treatment of both anemia and thrombocytopenia in MMM (Blood2003;101:2534), and of etanercept (tumor necrosis factor-alpha antagonist) for alleviation of constitutional symptoms (Blood2002;99(6):2252). In the current study, we investigated the value of combining all three drugs (PET). METHODS: Study eligibility criteria included a histologically confirmed diagnosis of MMM and either severe anemia (hemoglobin < 10 g/dL) or symptomatic splenomegaly. Protocol treatment was initiated with 50 mg of oral thalidomide (THAL) daily, etanercept (ETAN) 25 mg subcutaneously twice-a-week, and a three month prednisone taper starting at 0.5 mg/kg/day for the first month followed by a 50% reduction for the second months and a further 50% reduction for the third and final month of treatment with prednisone. Patients with a demonstrable clinical response after three months were able to remain on thalidomide and etanercept. RESULTS: 15 patients were enrolled to the trial (median age 66 years, range, 54–77; 14 males). Twelve patients (80%) completed the planned three cycles (i.e. 3 months) of treatment (progression (n=2) and patient choice (n=1) accounted for the withdrawals), nine (60%) patients with evidence of response continued on an additional three months of THAL/ETAN alone (of which 3 progressed prior to study completion). At enrollment, 11 patients (73%) were anemic (7 (47%) were red cell transfusion dependent), 7 (47%) were at least moderately thrombocytopenic (platelet count < 100 x 109//L), and 6 had severe constitutional symptoms (night sweats or disease related fever). Anemia response were seen in 6 (of the 11 eligible; 54%) patients (2 in the non-transfusion dependent (sustained increases of 1.5 and 4.9 g/dL hemoglobin); 4 in transfusion dependent (1 transfusion independent, 3 >50% decrease in transfusion requirements)). All thrombocytopenic patients experienced an increase in platelet counts (median 55% increase (range(10–229) with 2 returning to the normal range (>150 x 109/L)). A greater than 50% decrease in organomegaly was documented in 3 patients with splenomegaly (25% of eligible), and 1 patient with hepatomegaly (25%). Constitutional symptoms resolved, or signifcantly improved in all afflicted. Therapy overall was well tolerated with the majority of toxicities being transient. Neuropathy (grade 1; n=3; grade 2; n=1) and hyperglycemia (grade 2; n=1) were minimal. The only greater or equal to grade 3 toxicities were (grade 3: elevated bilirubin, infection, and blurred vision; grade 4: anemia). Follow-up bone marrow examination, when available, did not show post-treatment histological changes. CONCLUSIONS: This phase II study of combination therapy with thalidomide-prednisone-etanercept in patients with MMM confirms the previously recognized value of etanercept in alleviating constitutional symptoms. However, the PET regimen does not appear to be superior to thalidomide-prednisone combination in terms of therapeutic value for anemia, thrombocytopenia, or splenomegaly.

Abstract Background Estrogen-based hormone treatment (EBHT), in the form of either oral contraceptives or hormone replacement therapy, is generally felt to be associated with an increased risk of thrombosis in women with essential thrombocythemia (ET). However, there is insufficient evidence to support or refute such a concern. Methods Data was abstracted from medical records of a consecutive cohort of women with ET seen at the Mayo Clinic. Thrombotic risk was determined by the occurrence of a major thrombotic event namely myocardial infarction, angina, cerebrovascular accident, transient ischemic attack, peripheral arterial thrombosis, pulmonary embolism, or deep venous thrombosis at or after diagnosis of ET. Results i. Information regarding EBHT at or after the diagnosis of ET The study cohort included 307 women with ET (median age, 55 years; range 16–88) with a median follow-up of 133 months from diagnosis (range, 0.2–397). EBHT at or after diagnosis was documented in 93 women (30%); 59 patients were on EBHT at time of diagnosis and treatment was initiated after diagnosis in 34 patients. Among the 59 patients who were receiving EBHT at the time of diagnosis, treatment was discontinued within one month of their ET diagnosis in 17 patients but continued in the other 42 patients for a median of 48 months (range, 2–168). In the 34 patients with EBHT that was initiated after the diagnosis of ET, median treatment period was 44 months (range, 1–144). ii. Correlation between EBHT and thrombosis at diagnosis At diagnosis, major thrombosis was documented in a total of 74 patients (24%) that included 11 of the 59 patients (19%) on EBHT and 63 of 248 patients (25%) that were not receiving such therapy at the time of diagnosis (p=0.28). iii. Correlation between EBHT and thrombosis after diagnosis A total of 95 patients (31%) experienced a subsequent (i.e. post-diagnosis) thrombotic episode during the study period. These occurred in 65 of 214 patients (30%) that were not exposed to EBHT at or after diagnosis, 5 of 17 patients (29%) in whom EBHT was discontinued with the diagnosis of ET, 13 of 42 patients (31%) in whom EBHT was continued despite the diagnosis of ET, and 12 of 34 patients (35%) in whom EBHT was started after the diagnosis of ET (p=0.95). Furthermore, duration of EBHT, using a cutoff value of 1 year, did not significantly affect the incidence of thrombotic events (p=0.67). The different comparative groups were similar in age distribution as well as frequency of other cardiovascular risk factors including smoking, diabetes mellitus, hypertension, and hyperlipidemia. Conclusion The current study addresses the important issue of safety pertaining to the use of estrogen-based hormones in women with ET and did not reveal a significant alteration of thrombosis risk as a result of hormone therapy.

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: 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: 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.