Li, Chin‐Yang

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: Current therapy in adult systemic mastocytosis (SM) includes observation alone, topical therapy for cutaneous disease, symptomatic non-cytoreductive therapy and cytoreductive therapy. The latter involves several drugs whose individual merit in the treatment of SM has not been well characterized. Patients and Methods: Diagnosis of SM was according to the 2001 WHO criteria (World Health Organization Classification of Tumours of Hematopoietic and Lymphoid Tissues, p 1–351. Lyon, France: IARC Press; 2001). Response to cytoreductive therapy was assessed by consensus criteria (Eur J Clin Invest.2007;37:435). The study population was selected from a series of 342 adult SM patients referred to our institution and seen between 1964 and 2008. KITD816V mutation analysis was performed by DNA sequencing. Results: A total of 134 study patients received treatment that included at least one cytoreductive drug as either first-line (n=105) or second-line (n=29) therapy. Interferon-alpha with or without prednisone (n=58), hydroxyurea (n=43), imatinib mesylate (n=42) and 2-chlorodeoxyadenosine (n=26) were the most frequently used cytoreductive agents accounting for 120 of the 134 study cases; the remaining 14 patients received other cytoreductive drugs, none of which were effective. i. Interferon-alfa (IFN-a) with or without prednisone IFN-a with or without prednisone (24 and 34 patients, respectively) was given to 12 patients with indolent SM (ISM), 14 aggressive SM (ASM), and 32 SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD). For all 58 patients receiving therapy, complete (CR), major (MR), and partial (PR) response/regression were achieved in 3, 7, and 14 patients, respectively, for an overall response rate (ORR) of 41%. ORR in ISM, ASM, and SM-AHNMD was 50%, 43%, and 38%, respectively. Median duration of response was 12 months (range, 1–117 months). Anemia and elevated ESR were significantly associated with inferior ORR; 29% vs. 56% (p=0.04) and 14% vs. 57% (p=0.01), respectively. BM detection of KITD816V did not appear to affect response rates (p=0.4). ii. Hydroxyurea (HU) HU was given to 2 ASM, 40 SM-AHNMD and 1 mast cell leukemia (MCL) patients. The drug was used as first-line therapy in 30 patients. MR and PR were achieved in 1 and 8 SM-AHNMD patients, respectively (ORR=21%). Median duration of response was 37 months (range, 5–84 months). iii. Imatinib mesylate (IM) in patients with SM and associated eosinophilia IM was given to 22 SM patients who had associated eosinophilia (SM-eos): 2 had ISM, 2 ASM, and 18 SM-AHNMD. The drug was used as first-line therapy in 9 patients. Ten of the 22 patients with SM-eos were known to harbor the FIP1L1-PDGFRA fusion mutation and all responded to IM (ORR=100%). None of 5 FIP1L1-PDGFRA-negative SM-eos patients had a response to IM. In the remaining 7 patients with unknown mutation status, 4 patients had a response to IM (1 CR, 1 MR, and 2 PR). iv. IM in patients with SM not associated with eosinophilia IM was administered to 20 SM patients without associated eosinophilia (7 ISM, 2 ASM, 10 SM-AHNMD, and 1 MCL). Response was documented in 4 (20%) patients (1 CR, 1 MR, and 2 PR) including 1 with ISM, 2 with ASM and 1 with SM-AHNMD. Median duration of response was 19.5 months (range, 9–69 months). Two patients developed interstitial pneumonitis. v. 2-Chlorodeoxyadenosine (2-CdA) 2-CdA was given to 10 ISM, 3 ASM, and 13 SM-AHNMD patients; the drug was used as first-line therapy in 8 patients. CR, MR and PR were achieved in 1, 7, and 4 patients, respectively (ORR=46%). ORR in ISM, ASM, and SM-AHNMD was 50%, 33%, and 46%, respectively. Median duration of response was 11 months (range, 3–74 months). Presence of leukocytosis (WBC > 10 x 109/L), monocytosis (absolute monocyte count > 0.9 x 109/L) or circulating immature myeloid cells was significantly associated with inferior response; 13% vs. 61% (p=0.02); 11% vs. 69% (p=0.006) and 0% vs. 71%, (p=0.001), respectively. Response was not affected by BM detection of KITD816V (p=1.0). Conclusions: Both IFN-a and 2-CdA are reasonable treatment options, with comparable efficacy, for symptomatic SM including the subcategories of ISM, ASM and SM-AHNMD. IFN-a response was significantly better in the absence of anemia or elevated ESR. 2-CdA response was similarly better in the absence of leukocytosis, monocytosis or circulating immature myeloid cells. The therapeutic value of IM was limited to FIP1L1-PDGFRA-positive disease.

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: Systemic mastocytosis (SM) has a varied presentation ranging from indolent forms with limited morbidity over many years to the rapidly fatal mast cell leukemia (MCL). This heterogeneity complicates clinical decision making regarding choice and timing of therapy. The aim of this study was to evaluate the prognostic value of the 2001 World Health Organization (WHO) classification of SM, and to refine risk stratification within SM sub-groups to facilitate clinical decision making. Information on KITD816V and JAK2V617F mutation analysis and clinical correlates is also provided. Methods: Patient records in the institutional electronic database from January 1976 to October 2007 were reviewed and SM patients 18 years of age or older identified; the date of diagnosis ranged from July 1964 to October 2007. Only those patients where diagnosis was confirmed by bone marrow (BM) histology were included in the analysis. KITD816V mutation analysis was performed by DNA sequencing. JAK2V617F was screened by allele-specific quantitative PCR analysis. Results: i. Clinical characteristics at presentation: A total of 342 SM patients were identified (154 female; median age at diagnosis=57 years; range 19 to 87 years). Per the 2001 WHO classification, 159 had indolent SM (ISM; median age=49 years), 41 aggressive SM (ASM; median age=65 years), 138 SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD; median age=65 years), and 4 MCL (median age=55 years) (p-value for age <0.0001). 140 (41%) patients had urticaria pigmentosa (UP) (63% with ISM; p<0.0001), 160 (47%) mast cell (MC) mediator release symptoms (69% with ISM; p<0.0001), 107 (31%) skeletal symptoms, and 142 (42%) constitutional symptoms (61% with ASM/SM-AHNMD versus 19% with ISM; p<0.0001). 123 patients (36%) had palpable splenomegaly (57% with SM-AHNMD; p<0.0001). 56 patients (16%) exhibited prominent eosinophilia (absolute eosinophil count >1500/mcl) – 31% and 22% with SM-AHNMD and ASM, respectively, versus 3% with ISM (p<0.0001); 12 of 23 patients (52%) with eosinophilia who were tested carried the FIP1L1-PDGFRA mutation. Serum tryptase (normal <11.5 ng/mL) was measured in 160 patients (47%) and almost all (96%) had an elevated level (median=64 ng/mL; range=4 to 2000 ng/mL; 33 patients had >200 ng/mL). ii. Disease course and prognostic factors After a median follow-up of 20.7 months (range=0–417), 153 deaths were recorded. 17 patients (5%) transformed to acute myeloid leukemia (AML; n=14) or MCL (n=3), most frequently with SM-AHNMD (11% versus 0% for ISM; p<0.0001). The median overall survival was 63 months (ISM=198 months, ASM=41 months; SM-AHNMD=24 months, and MCL=2 months; p<0.0001). Multivariate analysis of parameters at the time of diagnosis showed a significant and independent association between inferior survival and WHO sub-type (p<0.0001), age at diagnosis (p<0.0001), history of weight loss (p=0.01), anemia (p=0.007), thrombocytopenia (p=0.0008), hypoalbuminemia (p=0.0008), and excess BM blasts (>5%; p=0.004). A similar analysis in ISM identified anemia (p=0.04), hypoalbuminemia (P=0.002), and BM MC ≥ 30% (p=0.03) as independent predictors of inferior survival; the corresponding parameters in ASM were age at diagnosis (p=0.002) and history of weight loss (p=0.003). iii)KITD816V andJAK2V617F analysis Archived bone marrow was available in 165 patients for KITD816V and JAK2V617F analysis. By DNA sequencing, overall KITD816V detection rate was 28%: ISM 20%, ASM 29%, SM-AHNMD 33% (p=0.4). In patients with ASM (p=0.003) and SM-AHNMD (p=0.001), detection of KITD816V was associated with inferior survival. Greater than 1% (range 1–57) JAK2V617F allele burden was detected in 6 patients (4%); all belonged to SM-AHNMD including 4 with non-MC lineage myeloproliferative neoplasm. Conclusions: The current study confirms the prognostic value of the WHO subclassification of SM and identifies advanced age, weight loss, anemia, thrombocytopenia, hypoalbuminemia, and excess BM blasts as additional adverse prognostic factors. In ISM, presence of anemia, hypoalbuminemia, or BM MC ≥ 30% is associated with inferior survival. In ASM and SM-AHNMD, BM KITD816V allele burden might influence survival. JAK2V617F is rare in SM and when found, it is almost always in the context of SM-AHNMD.

Abstract Background: Systemic mastocytosis (SM) sometimes coexists with an associated clonal hematological non mast cell lineage disease and such an occurrence is recognized by the 2001 World Health organization (WHO) classification system as SM-AHNMD (Leuk Res. 2001 25:603). However, SM-AHNMD has not been further stratified into prognostically relevant subgroups. This was the major objective of the current study, which also provides information on prevalence and relevance of detecting KITD816V and JAK2V617F mutations. Methods: Patient records in the institutional electronic database from January 1976 to October 2007 were reviewed and SM patients 18 years of age or older identified; the date of diagnosis ranged from July 1964 to October 2007. Only those cases where diagnosis was confirmed by bone marrow (BM) histology were included in the analysis. SM- and AHNMD-components were classified according to WHO criteria. Results: AHNMD sub-classification: Of 342 consecutive cases of SM that were screened, 138 (40%) were identified as having SM-AHNMD (median age 65 years, range 20–87). The most frequent AHNMD was chronic myelomonocytic leukemia (SM-CMML) seen in 38 (28%) patients. 23 (17%) patients had SM associated with myelodysplastic syndrome (SM-MDS), 22 (16%) with hypereosinophilic syndrome/chronic eosinophilic leukemia (SM-HES/CEL) and 33 (24%) with another myeloproliferative neoplasm (SM-MPN). The number of cases with other SM-AHNMD was much lower and included lymphoma 8 (6%), acute leukemia 3 (2%), MDS/MPN 3 (2%), and 8 (6%) cases of multiple myeloma, primary amyloidosis or chronic lymphocytic leukemia. Of the 33 SM-MPN patients, 2 had polycythemia vera, 6 essential thrombocythemia, and 2 myelofibrosis; the remaining 23 patients had an unclassified MPN in association with SM. Clinical characteristics at presentation: The combined incidence of urticaria pigmentosa (UP) was 18%, mast cell mediator release symptoms 28% and constitutional symptoms 62%. 76 patients (57%) had palpable splenomegaly. 99 patients (72%) had anemia, 18 (13%) leukopenia (43% with SM-MDS; p<0.0001) and 76 (55%) thrombocytopenia. 70 (51%) patients had leukocytosis and 63 (55%) displayed circulating immature myeloid cells (78% with CMML; p=0.001). 59 (54%) patients had elevated serum alkaline phosphatase level: SM-CMML 70%, SM-MPN 60%, SM-HES/CEL 40%, and SM-MDS 30% (p=0.01). Where evaluated, serum tryptase was elevated in 92% of the patients (median=75 ng/mL; range=4–1360). BM blasts were ≥5% in 25 patients (22%), including 48% with SM-MDS (p=0.004). 12 of 35 patients tested carried the FIP1L1-PDGFRA mutation (all had SM-HES/CEL; p<0.0001). Disease course and prognostic factors At a median follow up of 15 months (range 0–182), 99 deaths were recorded and the combined median survival was 24 months: SM-CMML 15 months; SM-MDS 15 months, SM-MPN 30 months; and SM-HES/CEL 87 months (p=0.003). Fifteen patients (11%) transformed to acute leukemia or mast cell leukemia. Multivariate analysis of parameters at the time of diagnosis showed a significant and independent association between inferior survival and SM-AHNMD sub-type (p=0.007), presence of constitutional symptoms (p=0.04), thrombocytopenia (p=0.01), presence of circulating immature myeloid cells (p=0.006), and hypoalbuminemia (p<0.0001). In SM-CMML, hypoalbuminemia was associated with inferior survival (p=0.03). KIT D816V and JAK2 V617F analysis: Archived BM was available in 88 patients for KITD816V and JAK2V617F analysis. By DNA sequencing, overall KITD816V detection rate was 33%: SM-CMML 48%, SM-MDS 21%, SM-HES 8%, and SM-MPN 45% (p=0.03). In patients with SM-HES/CEL (p=0.0009) and SM-MPN (p=0.04), detection of KITD816V was associated with inferior survival. Greater than 1% (range 5–57) JAK2V617F allele burden was detected in 6 patients (9%): 4 in SM-MPN, 1 in SM-CMML and 1 in SM-MDS. Conclusions: The WHO-defined SM-AHNMD comprises prognostically diverse subcategories among which SM-CMML is the most frequent. Survival is favorable in SM-HES/CEL and significantly worse in SM-CMML and SM-MDS. Additional independent predictors of inferior survival include constitutional symptoms, thrombocytopenia, circulating immature myeloid cells, and hypoalbuminemia. In SM-HES/CEL and SM-MPN, BM KITD816V allele burden may influence survival. JAK2V617F occurs infrequently in SM-AHNMD and when it does, it clusters with SM-MPN.

Abstract Background: Cytotoxic T-cell infiltrates are a nearly universal finding in the bone marrow of patients with multiple myeloma. It has been postulated that presence of T-cells in the bone marrow of multiple myeloma (MM) patients represents an immune response against the tumor and therefore, might be associated with an improved prognosis. However, the impact of bone marrow T-cells on the prognosis of multiple myeloma patients has not been studied systematically. Methods: Bone marrow biopsies of patients with newly diagnosed multiple myeloma were stained by immnohistochemistry for the CD8 antigen and reviewed by a blinded hematopathologist. Three high power fields are reviewed for each biopsy and the total number of CD8 positive cells counted and reported. For patients with more than 300 cells per 3 fields, results were reported as >300. The number of bone marrow CD8 positive cells was then correlated with patients’ clinical data, including other prognostic factors and overall survival. Results: Bone marrow biopsy specimens from 100 patients, performed within the week of a diagnosis of multiple myeloma and collected between May 1998 and January 2001 were evaluated. The median number of CD8 positive cells was 270 (33 – >300). Patients’ characteristics are shown in Table 1. Median follow up was 30 months (0–80). The number of cytotoxic T-cells as a continuous variable was a risk factor for shortened overall survival, HR 1.86 (95% CI 1.11–3.35). Using minimal p value approach, the cutoff of 270 cells (the median) risk stratified patients into two groups: the median survival of patients with > 270 CD8 positive cells was 16 months vs. 48 months in patients with ≤270 cells, p=0.005 (Figure). In multivariate analysis including age, B2M, albumin, CRP, bone marrow plasma cell percentage and plasma cell labeling index, the number of cytotoxic T-cells was an independent predictor of overall survival was HR 3.1, p=0.0017. Conclusion: We show that the number of cytotoxic T-cells in the bone marrow is a strong and independent prognostic factor in patients with newly diagnosed multiple myeloma. Our observation does not contradict the hypothesis that cytotoxic T-cells participate in an immune response against the tumor since our findings may represent a higher level of immune response associated with baseline aggressive disease biology. However, our study suggests for the first time that increased marrow cytotoxic T-cells have an adverse effect on outcome in myeloma, and suggest that these cells may have a direct facilitating effect on tumor growth and on the marrow microenvironment. Further studies of the biology of behind this observation are warranted. Characteristic N Median (range) Gender male 61 CRP 81 0.4mg/L (0.01–11.2) Albumin 99 3.6 g/dL (2.6–5.4) B2microglobulin 94 4.0 (0.9–28) μg/mL Marrow PC% 90 45% (11–99) PC labeling index 90 high (>1%) 36 BM CD8 cells 100 270 (33 – >300) ISS 94 1 19 2 41 3 34 Figure Figure

Abstract Background: Previous cytogenetic studies in polycythemia vera (PV) have included a relatively small number of patients (“n” ranging 10–64). In the current study (n=137), we describe cytogenetic findings at presentation and examine their relationship to clinical and laboratory features, including bone marrow JAK2V617F allele burden. Methods: The study consisted of a consecutive group of patients with PV who fulfilled the World Health Organization (WHO) diagnostic criteria and in whom bone marrow biopsy and cytogenetic studies were performed at diagnosis. Results I: cytogenetic details At diagnosis: A total of 137 patients (median age, 64 years; 49% females) were studied at diagnosis and had adequate metaphases for interpretation. Cytogenetics were normal in 117 patients (85%) and displayed either a sole -Y abnormality in 5 patients (7% of the male patients), and other chromosomal abnormalities in 15 (11%). The latter included trisomy 8 in five patients, trisomy 9 in three patients, two patients each with del(13q), del(20q), and abnormalities of chromosome 1, and one patient each with del(3)(p13p21), dup(13)(q12q14), and del(11)(q21). At follow-up: Repeat cytogenetic studies while still in the chronic phase of the disease were performed in 19 patients at a median of 60 months (range, 8–198) from diagnosis. Of these, 4 had aquired new cytogenetic clones including 3 with normal cytogenetics at time of initial PV diagnosis. The new abnormalities included del(20q), del(5q), del(1p), chromosome 1 abnormality, and inv(3)(q21q26.2). At time of disease transformation: Leukemic transformation was documented in 3 patients of whom cytogenetic information at the time was available in 2 patients; both patients had normal results at time of initial PV diagnosis and complex cytogenetic abnormalities at time of leukemic transformation. In contrast, among 6 patients with available cytogenetic information at time of fibrotic transformation, the results were unchanged from those obtained at time of diagnosis in 5 patients. ii) Correlation between cytogenetics at diagnosis and JAK2V617F allele burden: Allele-specific, quantitative PCR analysis for JAK2V617F was performed in 71 patients using genomic DNA from archived bone marrow obtained at the time of the initial cytogenetic studies. JAK2V617F mutation was detected in 64 of the 71 (90%) patients; median mutant allele burden was 16% (range 3–80%) without significant difference among the different cytogenetic groups: normal vs. –Y vs. other cytogenetic abnormalities (p=0.72). iii) Clinical correlates and prognostic relevance of cytogenetic findings at diagnosis: Among several parameters studied for significant correlations with cytogenetic findings at diagnosis, an association was evident only for age (p=0.02); all –Y abnormalities (n=5) as well as 13 of the 15 (87%) other cytogenetic abnormalities occurred in patients ≥ 60 years of age. Stated another way, the incidence of abnormal cytogenetics (other than -Y) was 4% for patients younger than age 60 years and 15% otherwise. The presence of abnormal cytogenetics at diagnosis had no significant impact on either overall or leukemia-free survival. Conclusions: Abnormal cytogenetic findings at diagnosis are infrequent in PV, especially in patients below age 60 years. Furthermore, their clinical relevance is limited and there is not significant correlation with bone marrow JAK2V617F allele burden.

Abstract Background: Pruritus in polycythemia vera (PV) has been associated with a high JAK2V617F allele burden (Tefferi et al. Cancer. 2006;106:631, Vannucchi et al, Leukemia2007, in press). However, there is limited information regarding clinical correlates of pruritus per se in PV. Accordingly, we conducted a large (n=418) retrospective study in PV to accurately assess the prevalence and severity of pruritus as well as its relationship with presenting clinical features, bone marrow JAK2V617F allele burden, and prognosis. Methods: The study cohort consisted of a consecutive group of patients with PV who fulfilled the World Health Organization (WHO) diagnostic criteria and in whom information regarding the presence or absence of pruritus at diagnosis was fully documented. Pruritus was graded as being mild, moderate, or severe based on careful review of patient history. Results: Prevalence and severity of pruritus: A total of 418 patients fulfilled the above stipulated criteria for study inclusion (median age of 60 years; 56% males; median follow-up of 73 months). Of these, 131 (31%) experienced pruritus at time of initial diagnosis: mild in 97 patients (74%), moderate in 22 (17%), and severe in 12 (9%). Most patients with mild pruritus (n=92) received no specific therapy for pruritus whereas all 12 patients with severe pruritus required treatment; all 7 patients who received treatment with paroxetine responded. Pruritus and JAK2V617F allele burden: Quantitative measurement of JAK2V617F was performed in 64 patients using genomic DNA from archived bone marrow obtained at the time of PV diagnosis. Mutational frequency was 97%; median mutant allele burden was 25.4% (range 0.2 - 93.3%). Among the 62 V617F-positive patients, 24 (39%) had pruritus; the proportion of patients in the upper and lower quartile allele burden ranges were 50% and 0% in the presence of pruritus and 18% and 34% in its absence (p=0.002). Clinical correlates of pruritus: Pruritus was more prevalent in non-smokers (35% vs. 19%; p=0.004) and non-diabetics (33% vs. 16%; p=0.04) and was associated with a lower rate of arterial thrombosis at diagnosis (8% vs. 17%; p=0.01) as well as during follow-up (16% vs. 30%; p=0.003). These significant associations remained intact during multivariable analysis. Pruritus did not have an impact on the rate of leukemic or fibrotic transformation. Conclusions: Pruritus in PV is independently associated with a lower risk of arterial thrombosis despite high JAK2V617F allele burden and is also more prevalent in non-smokers. These observations suggest that pruritus might be a surrogate for an underlying platelet pathology with functional relevance.

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.