Mesa, Ruben A.

Abstract BACKGROUND: THAL has been used as immunomodulatory therapy alone (Tefferi et. al. Blood 200;96:4007), or with steroids (Mesa et al. Blood2003;101:2534), in patients with primary myelofibrosis (PMF). Clinical benefit has been limited to improvements in cytopenias and/or splenomegaly. However, major histologic remissions of intramedullary manifestations of disease have not typically been observed. The mechanism of action of THAL in MF remains uncertain, and may involve cytokine inhibition or immunomodulation. We have previously observed increased numbers of CD8+ T lymphocytes on marrow trephines of patients who responded to THAL, and hypothesized that alterations in the T-cell distribution and population may provide insight into an immunomodulatory response and correspond to subsequent response in PMF patients. Methods: We analyzed a cohort of PMF patients who had received THAL therapy (alone or in combination with corticosteroids) from our institution and analyzed their clinical course and response to THAL therapy. Marrow trephines were evaluated in a systematic fashion (CYL in a blinded fashion to clinical course) for cellularity and degree of reticulin fibrosis (0–4+). Subsequently, megakaryocytes were assessed for quantitative (increased, decreased, or normal) and qualitative (size (large or small) and presence or absence of morphologic dysplasia). T cells were assessed by immunohistochemical staining for CD8 with an emphasis on number of T cell clusters per trephine, and the pattern of those clusters (large vs. small). Finally, an estimation of numbers of T cells per high power histology field was conducted in 3 distinct areas, and an average per marrow trephine obtained. All histologic features were then compared with PMF prognostic scores, and labs at presentation. Amongst those who received THAL, baseline histology was compared to response and in those patients with serial marrows longitudinal changes were assessed. Results: A cohort of 140 patients with PMF was analyzed, 65 having received THAL along the course of their illness (53 (82%) along with prednisone) and 75 from a comparison group who never received THAL. Patients from both groups had similar PMF prognostic scores and presentations. Comparison of baseline histologic features demonstrated that individuals with a baseline increases in T cell numbers (>100 per high power field) were significantly more likely to subsequently respond to THAL therapy (p<0.01). Additionally, analysis of megakaryocyte morphology demonstrated that those patients in which megakaryocytes were atypical for PMF (i.e. small) were significantly more likely to have thrombocytopenia and were less likely to respond to THAL (p=0.05). Independent assessment of the prognostic value of these individual histologic features upon survival failed to reveal any direct impact upon survival by Kaplan-Meier Analysis. Table 1. Comparison of Histologic Features in 140 patients with Myelofibrosis Group Cell Fibrosis ≥3 Meg Numbers Meg Size T Cell clusters T-Cells (per HPF) >100 Tcells (per HPF) Control (N=75) 72% (10–100) 33% 91% Increased 21% Small 4 (0–16) median 80 (13–258) 37% THAL ALL (N=65) 60% (5–95) 62% 79% Increased 38% Small 3 (0–14) median 93 (20–500) 43% -THAL Responders (N=28) 60% (5–95) 56% 82% Increased 22% Small (p=0.05) 5 (0–10) median 113 (45–198) 61% (p<0.01) -THAL Non Responders (N=37) 60 (5–95) 67% 76% Increased 48% Small 3 (0–14) median 70 (20–500) 26% A subgroup of patients who received THAL (N=12) had a second post therapy marrow available for analysis for comparison of THAL effects upon the marrow, although the numbers were small, there was no statistically significant differences upon histologic parameters analyzed including cellularity, degree of fibrosis, megakaryocyte morphology, or T cell clustering or numbers. Inclusion of prednisone in the regimen had no impact on histologic prognostic observations. Conclusions: THAL therapy for MF patients can be associated with significant clinical benefit for cytopenias and splenomegaly, and interestingly patients with increased marrow T-cell populations at baseline have a clear increased likelihood of response. Although this analysis is unable to gauge mechanism, it would suggests a greater potential for immunomodulation in patients with a robust T-cell population. Additionally, analysis of megakaryocytes suggests the presence of small megakaryocytes may in contrast represent a more resistant phenotype of disease with more thrombocytopenia and THAL resistance.

Abstract BACKGROUND: MF is a progressive illness associated with cachexia and weight loss (Mesa et al, Cancer2007;109: 68). These clinical signs, resulting from both hypercatabolism (secondary to increased pro-inflammatory cytokines) and MF associated splenomegaly, are associated with decreased survival (Dupriez et al., Blood1996;88:1013). Currently, there is no therapy that decreases the progressive cachexia of MF. INCB018424 is a selective JAK1/2 inhibitor which has the potential to improve both the aberrant myeloproliferation in MF through decreasing constitutively active JAK-STAT signaling, and nutritional status by decreasing both splenomegaly and the pathologically increased cytokines. METHODS: Symptomatic patients with MF enrolled in a phase I/II trial (www.ClinicalTrials.gov, NCT00509899) with INCB018424, were analyzed for the impact of therapy on nutritional status and cachexia. Specifically, in addition to traditional endpoints of IWG-MRT response (reported elsewhere) patients were assessed for changes in body mass index (BMI), serum cholesterol values, spleen size, and patient reports of early satiety and anorexia. Additionally, leptin, an adipose-derived protein hormone that plays a key role in regulating energy balance and circulates at levels proportional to body fat in health and disease (Mantovani et al, J Mol Med2001; 79:406), was assessed serially. RESULTS: Patients: Thirty four MF patients, treated for at least 2 months with 25 mg twice daily of INCB018424, were included in this analysis. Among this group 85% demonstrated splenomegaly (median 20 cm below left costal margin, range 4 cm to 32 cm; 2 patients had prior splenectomy) and had a median BMI of 24.8 (range 17.9 to 49.7). Although the median BMI at baseline would be considered in the “normal range” (18.5–24.9), loss of lean body mass at enrollment would be underestimated by the contribution of splenomegaly or edema. Appetite: At enrollment, a clearly positive correlation between the presence of anorexia and early satiety (by patient’s report) and significant splenomegaly was observed. Treatment with INCB018424 led to resolution of the symptoms of poor appetite and early satiety, along with the reduction in splenomegaly. Weight: MF patients on therapy initially lost weight, which reflects resolution of excess extravascular water (based on investigators reported decreases in peripheral edema, ascites, or splenomegaly). As the trial progressed MF patients on INCB018424 treatment progressively gained weight (mean increase of 0.40 kg @ 1 month, 2.93 kg @ 2 mo, 3.70 @ 3 mo), and exhibited improved appetite. Importantly, weight gain was more consistent, of greater magnitude and more durable in patients who entered the study in the lowest quartile for BMI. Cholesterol: We previously reported that hypocholesterolemia (total cholesterol <150 or 100 mg/dL; HDL < 60mg/dL) is associated with decreased survival in MF patients (Mesa et al, Blood2007; 2007;110:a2548), potentially from hypercatabolism and splenic consumption of lipids. At enrollment, median total cholesterol was 95 mg/dl (range), with 94% and 55% below 150 mg/dl and 100 mg/dl, respectively. Following treatment with INCB018424, median total cholesterol increased to 145 mg/dl (range 72–289 mg/dL) with 73% increasing to an improved range above their baseline (either to the >100mg/dl or >150 mg/dl range). Leptin: At enrollment, MF patients had very low leptin levels (mean = 2.55 ng/mL with 50% below 1 ng/mL vs. a range of 6–12 ng/mL for normal volunteers). Low plasma levels are associated with shortened survival in cancer patients. The plasma leptin levels increased 176% on average after one month of treatment with INCB018424, and continued to increase to levels matching healthy volunteers with time on study (mean = 7.04 ng/mL (range 0.25 – 35 ng/mL) after 2 months on INCB018424) and correlated to weight increases. CONCLUSIONS: Therapy with INCB018424 improves the nutritional status of MF patients, including improving pathologic weight loss, hypercatabolism associated hypocholesterolemia, and pathologically decreased serum leptin. The improved nutritional status of MF patients treated with INCB018424 may reflect the ability of JAK inhibition to target the underlying pathophysiology of MF cachexia by reducing the organomegaly, levels of pro-inflammatory cytokines, and pro-inflammatory cytokine signaling.

Abstract Background: Myelofibrosis (MF) shortens survival but also compromises quality of life from disease associated splenomegaly and constitutional symptoms (fatigue, weight loss, night sweats, and bone pain). No current validated instrument of patient reported outcomes (PRO) captures the spectrum of MF associated symptoms. In an era of rapid development and testing of therapeutic agents which may impact the natural history and symptoms associated with MF (such as JAK2 inhibitors) a uniform and validated instrument for capturing the presence, severity (and potentially improvement) of MF symptoms is needed. We sought to develop and validate such an instrument, the myelofibrosis symptom assessment form (MFSAF). Methods: Based upon the results in our MF QOL survey (Cancer2007;109(1):68–76), we developed the MFSAF to assess fatigue (via the brief fatigue inventory (BFI-Cancer1999;85:1186)), additional symptoms were independently assessed for presence and severity on a scale from 0 (absent) to 10 (worst possible) for splenomegaly associated symptoms (early satiety, pain, inactivity, cough) night sweats, itching, bone pain, fever, unintentional weight loss as well as an estimate of overall quality of life. Additionally, patients provided feedback on the quality and nature of the MFSAF, and also completed for comparison validated instruments including the Memorial Symptom Assessment Scale (MSAS), the Insomnia Severity Index (ISI), and the Brief Pain Index. Physician input was included including their assessment of patient’s fatigue, spleen symptoms, and quality of life (blinded to patient’s responses) as well as clinical history, lab and exam findings. Results: MFSAF Results 34 MPD patients were enrolled (24 MF, 10 in the comparison group (4 polycythemia vera (PV), 6 essential thrombocythemia (ET)). the MFSAF was rated by patients as easy to understand (median score 1, range 0–6), and “addressed most of my symptoms” (median score 1, range 0–6) both on a scale of 0 (as good as possible to 10 as bad as possible). When asked if a symptom was not addressed (open ended response) no single symptom was named more than once. As we have previously reported fatigue was common with BFI scores (median 3.6 (0–10) for MF and 2.5 (range 0.2–3.8) for the ET/PV group (increasing BFI score associated with worsening fatigue) worse than median score of published “healthy” controls 2.2. Additional MF associated symptoms were captured well by the MFSAF with splenic, constitutional symptoms, and QOL (median 3 (range 0–7) MF, median 2 (0–3) ET/PV) documented easily and worse than ET/PV controls. Physicians estimation of QOL was excellent (median 3 (0–8) for MF, median 2 (0–4) for ET/PV). MFSAF Comparison to other Instruments: The MSAS demonstrated more adverse values for MF (than ET/PV) in the PSYCH (psychologic stress; mean-1.27 (STD DEV 0.80), PHYS (physical symptoms; mean 0.90 (STD DEV 0.53)), GDI (Global Distress Index mean-1.29 (STD DEV 0.77)) and a total MSAS symptom score (mean 0.85 (std dev (0.49)) comparable with advanced renal disease (PSYCH 0.99, PHYS 0.99, GDI 1.27, and MSAS of 0.81; J of Pall Med2007;10(6):1266), as well as advanced lung disease, AIDS, and various advanced malignancies. Individual, previously validated MSAS questions were all highly correlated (all p<0.01) with MFSAF (in italic) counterparts including lack of energy (fatigue), cough (same), pain (both abdominal pain and bone pain), sweats (night sweats), itching (same), and weight loss (same). Further validation of pain measurements in the MFSAF came from comparison to the BPI where both individually the presence, and intensity of pain (both abdominal and bone) were highly correlated (all p<0.01). Insomnia, not yet included in the MFSAF, was present in 68% and 63% of patients (MSAS and ISI, respectively). Additionally, problems with sexual interest, or activity, was noted in 35% of patients to the MSAS. Conclusions: Currently available instruments of PRO and symptoms are lengthy, and individually incomplete for assessing the presence and severity of the diverse nature of symptoms associated with MF. The proposed MFSAF, was comprehensive and well understood by patients and the results for the individual symptoms assessed were highly correlated with previously validated instruments for other conditions as well as with physicians perceptions of fatigue and QOL. The MFSAF, with the addition of questions regarding insomnia and sexuality, should be utilized in all trials of therapeutic agents in MF patients in which symptomatic improvements are an endpoint.

Abstract Background: Current evidence links some of the disease manifestations in myelofibrosis (MF) to abnormal cytokines, likely produced by clonally involved megakaryocytes and monocytes. Furthermore, the recent discovery of JAK2/MPL mutations in MF suggests the contribution of abnormal JAK-STAT signaling to both clonal myeloproliferation and cytokine-driven debility. In order to gain additional pathogenetic insight regarding cytokine-phenotype correlations in MF, we looked into the plasma cytokine profile of MF before and after treatment with INCB018424, a selective JAK1/2 inhibitor. Methods: The current study includes subjects with MF enrolled in an ongoing phase 1–2 study of oral INCB018424 (doses ranging from 25 mg/day to 50 mg BID). Plasma samples were obtained prior to treatment and at intervals of 2 weeks, 1 month and 2 months following INCB018424 dosing. Samples were submitted to Rules Based Medicine Human MAP multiplexed immunoassay system to obtain plasma levels on a range of protein analytes. Results: Plasma cytokine levels in MF patients (n=53) vs. normal healthy volunteers (n=15): Compared to normal controls, plasma levels of pro-inflammatory cytokines and markers of inflammation were significantly increased in MF patients (Table; mean +/− SD values). Furthermore, the observed inflammatory cytokine levels in MF were often higher than those seen in patients with rheumatoid arthritis or cancer-associated cachexia (data to be presented at the meeting). Correlation of plasma cytokine levels in MF with JAK2 V617F mutational status, MF subtype and/or constitutional symptoms/cachexia: Comparison of JAK2V617F positive (n=40) and negative (n=13) MF cases suggested significantly (p<0.01) higher IL-1RA (mean +/− SD = 5575 +/− 917 vs. 1291 +/− 359 pg/ml) and CRP (17.4 +/− 1.6 vs. 6.7 +/− 1.9 μg/ml) levels for the former whereas the other cytokines were elevated to a similar extent. Plasma cytokine levels in PMF (n=30) vs. post-PV MF (n=15) vs. post-ET MF (n=8) were not significantly different. The presence of prior splenectomy did not appear to alter the specific MF cytokine profile (Table 1); the abnormal cytokine profile in MF is, therefore, not necessarily a consequence of marked splenomegaly. There was a direct correlation between the levels of pro-inflammatory cytokines and the presence or absence of constitutional symptoms/cachexia (Figure). Similarly, increased inflammatory cytokine levels in MF were accompanied with significantly decreased leptin levels, a surrogate for nutritional status (Table). Post-INCB018424 treatment cytokine levels: Treatment with INCB018424 induced a rapid decrease in MF-associated inflammatory cytokine levels, in parallel with the observed clinical benefit of both reduced splenomegaly and improvement in constitutional symptoms/cachexia (data to be presented at the meeting). Conclusion: The plasma cytokine profile of MF is reminiscent of a chronic inflammatory state with levels of pro-inflammatory cytokines that are possibly higher than those seen in other inflammatory/malignant conditions. Furthermore, the current study suggests a fundamental link between these cytokines and MF-associated constitutional symptoms/cachexia. Cytokine modulation through JAK-STAT inhibition appears to be a mechanism of action for INCB018424 in MF. Cytokine Normal volunteer (N = 15) MF with splenectomy (N = 6) MF without splenectomy (N = 47) IL-1b (pg/ml) 0.6 +/− 0.04 44 +/− 36 41 +/− 25 IL-1RA (pg/ml) 103 +/− 10 7759 +/− 3939 4111 +/− 763 IL-6 (pg/ml) 0 9.7 +/− 3.3 53.6 +/− 22.5 IL-8 (pg/ml) 7.6 +/− 1.17 1618 +/− 1165 2376 +/− 451 TNFa (pg/ml) 2.6 +/− 0.21 38 +/− 6.8 45 +/− 8.8 TNFRII (ng/ml) 3.1 +/− 0.12 27.3 +/− 7.4 24.7 +/− 2.5 CRP (mg/ml) 1.5 +/− 0.49 21.7 +/− 4.5 13.9 +/− 1.6 Leptin (ng/ml) 10.8 +/− 3.5 3.8 +/− 0.9 2.74 +/− 0.6

Abstract BACKGROUND: The potential risks of tumor growth promotion and thromboembolism associated with erythropoietin (Epo) therapy warrant cautious use of erythropoiesis stimulating agents (ESA). Recent data suggest a surprising and significant association between ESA use and increased incidence of leukemic transformation in primary myelofibrosis (PMF) (data from an analysis separately submitted for American Society of Hematology meeting, 2007). It is prudent, therefore, to avoid unnecessary ESA exposure in PMF, especially in patients with low likelihood of treatment response. METHODS: Diagnosis of PMF was based on World Health Organization criteria. Study inclusion required availability of treatment history with ESA and adequate follow-up. We excluded patients who were receiving hemoglobin-improving agents concomitantly with an ESA, such as prednisone, androgens, danazol, or thalidomide. Transfusion dependency was defined by a history of minimum 2 units of red blood cell transfusions in a 1-month period for a hemoglobin < 8.5 g/dL, not associated with bleeding. An adequate treatment response was defined as a minimum 2.0 g/dL increase in hemoglobin level or becoming transfusion-independent over a minimum 1-month period. RESULTS: A master database of PMF patients seen at the Mayo Clinic from 1976 through 2006 was queried to identify 43 patients (median age 67 years; 35% females) treated with ESAs and in whom adequate follow-up was available to determine response. Prior to ESA treatment, median hemoglobin was 9.2 g/dL (range 7.0–11.9), and 16 (37%) were red blood cell transfusion-dependent. In 25 patients, pre-treatment serum Epo level was available (median 34 mU/mL; range 2–323). Among the 43 study patients, 16 (37%) were previously treated with other PMF-specific therapies, and 18 (42%) were receiving cytoreductive treatment concurrent with an ESA. Overall, 23% of patients (10 of 43) responded to treatment with ESA. The response rate was 0% (0 of 16) in the transfusion-dependent group compared to 37% (10 of 27) in the transfusion-independent group (p=0.007). Among the transfusion-independent patients, the proportion of ESA responders was significantly higher in the presence of a baseline hemoglobin level < 10 g/dL prior to treatment (p=0.009). ESA response was not correlated with baseline serum Epo level in the context of either all 43 study patients (p=0.68) or the 27 transfusion-independent patients (p=0.39). ESA response was not affected by PMF-specific treatment history (p=0.46), use of concurrent cytoreductive therapy (p=0.28), cytogenetic findings (p=0.62), JAK2V617F presence (p=0.69) or its allele burden at time of initial PMF diagnosis (p=0.16). CONCLUSIONS: Clear benefit of ESAs in PMF is limited to red blood cell transfusion-independent patients and those with baseline hemoglobin levels less than 10 g/dL.

Abstract Background: Immunomodulatory therapy has been shown to be of benefit for the improvement of anemia, thrombocytopenia and splenomegaly in patients with myelofibrosis (MF) initially with thalidomide (Blood2000;96:4007), and further enhanced by combination therapy with corticosteroids (Blood2003;101:2534). We sought to further enhance our previously reported efficacy of single agent lenalidomide for MF patients (Blood2006;108:1158) by combination therapy with prednisone. Methods: Symptomatic patients with MF (all with baseline anemia, hemoglobin <10g/dL or transfusion dependent) were enrolled in the single arm Phase II trial. Adequate neutrophils (>1 x 109/L) and platelets (>100 x 109/L) were required for trial entry. Lenalidomide was administered at 10 mg daily with a 3 month prednisone taper (30mg/daily, 15 mg daily, 15 mg every other day months 1,2, and 3, respectively). Patients with at least stable disease remained on single agent lenalidomide for an additional 3 months. Responses were assessed by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) Response Criteria (Blood2006;108:1498). Results: Patients 28 patients were enrolled in the first step of the trial, and a total of 48 patients were enrolled (3 were ineligible because of proximity of prior therapy with 43 deemed evaluable). The enrolled eligible patients (56 % males) had a median age of 64 (range 18–83). At baseline, 52% were red cell transfusion dependent, and the median hemoglobin amongst those non transfusion dependent patients was 8.8 g/dL (range 5.9–12.4). By entry criteria neutrophils (median 5.1 x 109/L (range 1.0–79.4)) and platelets (median 234 x 109/L (range 40–1253)) were adequate. Therapy and Toxicity: A median of 6 cycles of therapy was administered, 10 patients received 3 months, and 25 patients the full 6 months of therapy. Premature discontinuation of therapy occurred in 19 patients; progressive disease (3), toxicity (7), patient withdrawal (5), death from disease (1), comorbidities (2), or seeking alternative therapy other (1). Myelosuppression was the main toxicity with 45% experiencing ≥ grade 3 hematologic toxicity (16%/2% and 23%/26%, grade 3/4 thrombocytopenia and/or neutropenia, respectively) and with 34% of patients having ≥ grade 3 non-hematologic toxicity. Less severe toxicities (< grade 3) were again mainly secondary to myelosuppression (34%) or gastrointestinal (i.e. diarrhea). No significant steroid induced toxicities were observed. Response: Among 43 patients eligible for response assessment the best confirmed response observed was a partial response in 11 (26%), stable disease (54%), and progression during trial in 8 (18%), 1 was not evaluable because no medication was taken. Among the latter responses improvement in anemia was seen in 9 (21%) (IWG-MRT clinical improvement (CI)-sustained normalization of hemoglobin, >2 g/dL increase, or transfusion independence for >2 months), and major reductions in spleen size in 4 (9%) (IWG-MRT CI - sustained >50% decrease). Serial marrow analysis was available in a subset which did not show any significant resolution of disease related fibrosis, hypercellularity or increased angiogenesis typical of MF. Conclusions: 1) Lenalidomide plus a corticosteroid taper was active in patients with MF primarily in those who suffer from anemia, but myelosuppression (particularly neutropenia is common) 2) Response rates observed with combination therapy are almost identical to our previously reported response rate seen with single agent lenalidomide (22% single agent, 21% in this trial), 3) Current results would suggest that given the myelosuppression observed, and rates of efficacy, thalidomide and prednisone would be first line for treating MF associated cytopenias with lenalidomide being considered second line (first line if a del(5q) present).

Abstract Background : Myelofibrosis (MF) is characterized by progressive bone marrow dysfunction, extramedullary hematopoiesis, massive splenomegaly, elevated levels of inflammatory cytokines, severe constitutional symptoms, cachexia and premature death. INCB018424 is a potent, selective inhibitor of JAK 1 and 2 which has demonstrated impressive clinical activity in the early portion of a phase 1/2 study in patients with PMF and Post-PV/ET MF. Methods : Following the initial dose escalation phase with a BID schedule (N=11), both twice daily (10 mg BID to 25 mg BID, N=56) and once daily (25 mg QD to 200 mg QD, N= 37) dose regimens were explored. Endpoints included spleen size assessed by manual palpation, constitutional symptoms assessed using the Modified Myelofibrosis Symptom Assessment Form (MFSAF) and JAK2 allele burden. Exercise capacity is being assessed using the Six Minute Walk Test (6MWT), and body mass and composition are being assessed using standard methods. Safety was assessed by collection of adverse events and monitoring of laboratory data. Results : Over 100 patients are currently enrolled in the study, with a mean exposure to drug of > 5 months. Twenty percent of patients have received INCB018424 for > 9 months. INCB018424 is associated with a rapid reduction of splenomegaly in over 93% of patients from a mean baseline spleen size >20 cm, with 50% or greater reduction being observed in 35% of patients dosed with 10 mg BID or 50 mg QD, and 59% of patients dosed with 25mg BID regimens. These declines nearly always occur within the first month of therapy, with further, slower decreases often observed thereafter. In responding patients, spleen size reduction persists for the duration of therapy (currently up to one year follow-up). Constitutional symptoms are reported with high frequency in the study population as assessed by the MFSAF: night sweats, pruritis and fatigue were reported by >60%, >45% and >90% of patients respectively. Improvements in self-assessed symptom scores (mean % improvement of 40% to 60%) occurred by the first assessment at week 2. Activity limitations are also reported by a high proportion of MF patients; 70% of respondents reported impaired ability to exercise or get around, with a mean score >5 on the 11-point MFSAF scale; INCB018424 therapy was associated with rapid (within 2 weeks) and marked (40% or more) improvement in self-assessed score. There was a dose-dependent weight gain, most pronounced in patients with the lowest body mass index (BMI) values at baseline. Profound reductions in inflammatory cytokines were observed by the first evaluation at week 2 in virtually all patients, and were maintained during therapy. INCB018424 is generally well tolerated with the primary toxicity being dose-dependent grade 3 or 4 reversible thrombocytopenia which occurred in 0%, 18% and 32% of patients dosed with 10 mg BID, 50 mg QD or 25 mg BID. Conclusions : INCB18424 shows unprecedented and durable clinical efficacy in reducing spleen size and improving constitutional symptoms in patients with MF. Results from proactive dose modification regimens, assessments of exercise capacity, MRI-based spleen volume assessment and body composition determination will be presented.

Abstract BACKGROUND: We sought to determine the prognostic relevance of aberrant serum lipid values in the myeloproliferative disorders (MPDs) of primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocythemia (ET). METHODS: The Mayo MPD patient institutional database was queried to identify clinical history and lipid profiles (within a year of diagnosis). Serum cholesterol (CHOL), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C), and triglycerides (TRI) were assessed compared to clinical outcomes. RESULTS: 558 patients with MPDs (PMF 28%, PV 27%, and ET 45%) (median age at diagnosis of 56 (range18–81)) were identified. A lipid profile obtained a median of 0.0 months from diagnosis (range (-8 to 12 months) from MPD patients displayed low CHOL, LDL-C, and higher HDL-C than typical for the USA. Amongst MPD disease types PMF patients had lower CHOL (p<0.001) and HDL (p<0.001) compared to ET and PV (see table). Amongst PMF patients, although hypocholesterolemia was not a surrogate for malnutrition (p=0.13) median serum albumin 4.1 g/dL (range 2.7–4.8)), there was correlation with the presence of weight loss, hypercatabolic symptoms, and worse Mayo-CBC myelofibrosis prognostic scores (Cancer2006;106:623; p<0.01). Amongst the study group PMF patients had the poorest survival (followed by PV then ET; p<0.001). Decreased cholesterol (CHOL <150mg/dL, or HDL <60 mg/dL) was associated with decreased survival (P<0.001), but only in PMF patients (see figure). Decreased PMF survival was also associated with traditional PMF risk factors of hemoglobin <10g/dL, leukocytes <4 or >30 × 10(9)/L, platelets <100 × 10(9)/L, and circulating blasts. In multivariate analysis (Cox-Proportional Hazard) only the Mayo-CBC score, hemoglobin <10g/dL, circulating blasts and decreased CHOL remained significant for decreased survival (p<0.001). CONCLUSIONS: The presence of hypocholesterolemia is very common in patients with PMF and is independently associated with inferior survival. Survival by HDL-C and Serum Cholesterol (CHOL) in 154 patient’s with Primary Myelofibrosis Survival by HDL-C and Serum Cholesterol (CHOL) in 154 patient’s with Primary Myelofibrosis Lipid Distributions in 558 MPD patients VALUE/ NCEP CATEGORY PMF (N=154) PV (N=149) ET (N=255) NHANES (USA MEANS;95th CI) NCEP: National Cholesterol Education Program: NHANES (National Health and Nutrition Examination Survey (values for all races ages 65–74) Serum Cholesterol (Median) 149 mg/dL 179 mg/dL 199 mg/dL 221 mg/dL (153–301) −>200mg/dl (Desirable) 10% 31% 48% −150–200mg/dL (Desirable) 39% 25% 41% −100–149mg/dL (Desirable) 41% 41% 9% −<100mg/dL (Low) 10% 3% 2% LDL Cholesterol (Median) 33 mg/dL 41 mg/dL 49 mg/dL 149 mg/dL (87–217) −>100mg/dL (Low to high risk) 1% 1% 1% −<100mg/dL (Optimal) 99% 99% 99% HDL Cholesterol (Median) 79 mg/dL 118 mg/dL 108 mg/dL 45.7 mg/dL (29–71) −>60mg/dL (Optimal) 74% 90% 91% −40–60mg/dL (Normal) 16% 10% 8% −<40mg/dL (Low) 10% 0% 1% Triglycerides (Median) 151 mg/dL 89 mg/dL 116 mg/dL 145 mg/dL (62–285) −>200mg/dL (High) 28% 0% 3% −150–199mg/dL (Borderline) 23% 6% 14% −<150mg/dL (low) 49% 94%% 83%

Abstract Background: Peripheral blood (PB) is sometimes used in place of bone marrow (BM) for cytogenetic studies in either primary (PMF) or post-polycythemia vera/essential thrombocythemia (post-PV/ET MF) myelofibrosis. In a retrospective study, we examined overall yield from PB cytogenetic studies in MF and where possible compared the results with those obtained by BM cytogenetic analysis. Methods: Standard laboratory techniques were used to perform cytogenetic studies in both the PB and BM. A successful study constituted the acquisition of at least two analyzable metaphases and an abnormal clone required the presence of a specific cytogenetic abnormality in at least two metaphases. Clinical and laboratory correlations were made using variables collected at the time of cytogenetic studies. Results I: A total of 242 PB cytogenetic studies were reviewed to identify 59 MF cases; 39 were PMF and 20 post-PV/ET MF. Median age was 60 years (range 36–77) and 37 were male patients. The median duration of the disease at the time of PB cytogenetic studies was 3.5 years (range 0.2–35). At least two analyzable metaphases (median 20, range 2–31) were obtained in 49 (81%) of the 59 study patients; in univariate analysis, this was predicted by presence and degree of circulating myeloid progenitor cells (p < 0.0001), higher PB CD34 count (p < 0.0001), higher leukocyte count (p = 0.0008), and absence of myeloid growth factor therapy (p = 0.005). In multivariable analysis, only the presence of circulating myeloid progenitor cells sustained its significance. Results II: Twenty two cases had concomitant PB and BM cytogenetic studies performed. Of these, 9 patients had abnormal BM cytogenetic findings; the concomitant PB cytogenetic studies were also abnormal in 5 (56%) patients but normal in the other 4. Conversely, PB cytogenetic studies were abnormal in 12 patients; the concomitant BM cytogenetic studies were abnormal in 5 (41%) and normal in the remaining 7. PB cytogenetic studies were not successful in 4 of the 22 total cases; BM studies were successful in 3 of these 4 cases and the results were normal in all. Conclusion: In the presence of circulating myeloid progenitor cells, PB can be considered as an alternative to BM for cytogenetic studies in MF patients, especially in view of the difficulty getting BM aspirates in this disease. However, considering the prognostic value of specific cytogenetic abnormalities in MF, the non-trivial discordance between the BM and PB cytogenetic findings, observed in the current study, warrants a prospective evaluation for further clarification.

Abstract BACKGROUND : Leukemic transformation (LT) eventually occurs in 10–30% of patients with myelofibrosis with myeloid metaplasia (MMM). Hoever, there is limited data regarding bone marrow histological and cytogenetic changes that accompany the process. METHODS : The study population consisted of 67 consecutive patients with MMM in whom the diagnosis of LT was documented at our institution by bone marrow examination. Serial comparisons of both histological and cytogenetic changes were performed to monitor longitudinal changes accompanying disease progression. RESULTS : I. Bone marrow histologic changes during LT : All 67 patients displayed acute myeloid leukemia (AML) and all FAB subtypes except M3 were represented (most common were M7 at 25.4% and M0 at 22.4%). Median bone marrow myeloblast percentage was 43% (range 20–95) and cellularity 80% (range 5–95). However, only 37.5% and 30.3% had marked increases (> grade 2) in either reticulin fibrosis or osteosclerosis, respectively. Serial bone marrow comparisons (available in 33 patients at a median interval of 24.5 months (range; 2.1–110.4) after the diagnosis of MMM) demonstrated that LT was occasionally a focal process, but was always accompanied by a marked increase in myeloblasts. However, overall bone marrow cellularity changed by >15% in only 25% of the cases, none of the LT was associated with a > grade 1 change in reticulin fibrosis, and only 12% had an increase in osteosclerosis. On the other hand, LT was frequently associated with morphologic changes in megakaryocytes from large, round and clumped at the time of diagnosis of MMM to small, condensed and non-clumped at the time of LT. II. Karyotypic changes of MMM to LT: Karyotypic analysis was available in 32 patients (48%) at the time of MMM (abnormal in 63%) and 56 patients (84%) at the time of LT (abnormal in 91%). At LT the majority of patients (n=34; 60.7%) displayed poor risk karyotypes with either complex karyotypes or single abnormalities of adverse prognostic significance. In contrast, only two patients (3.6%) manifested prognostically favorable karyotypes with a translocation or inversion involving one of the core binding factors (t(8;21)(q22;q22);Inv(16)(q22)). Twenty-eight patients had chromosome analysis performed both at diagnosis of MMM and at LT. At diagnosis of MMM, 20 patients had either normal chromosomes or a single chromosome anomaly while only 7 displayed a complex karyotype with 3 or more chromosome anomalies. However, at LT all twenty-eight patients had abnormal karyotypes, including 17 patients with markedly complex karyotypes and only 9 with simple clonal anomalies. The recurrent numeric and structural anomalies observed at transformation to AML most frequently involved chromosomes 5, 6, 7, 8, 17, 19 and 21. Additionally, amongst the 26/28 (93%) of patients whose karyotype changed from MMM to LT 11 patients (43%) displayed evolution of an existing clone, and 15 (57%) developed one or more additional abnormal clones. CONCLUSIONS: The progression of MMM to LT is characterized by the emergence of new cytogenetic abnormalities as well as morphological changes in megakaryocytes. Unfortunately, the karyotypic profile that accompanies clonal evolution in MMM is often of the poor-risk category that undermines the value of aggressive chemotherapy in such patients. However, such therapy might be of value to the occasional patient with good-risk cytogenetic lesions.