Citations

Abstract Background: One of the utilities of molecular markers in hematological malignancies is their potential to predict response to drug therapy. Accordingly, we inquired about the effect of both cytogenetic profile and JAK2V617F mutational status on drug therapy response in myelofibrosis with myeloid metaplasia (MMM). Methods: Mutation analysis for JAK2V617 was performed in DNA derived from peripheral blood mononuclear cells, granulocytes, or both. Genomic DNA was amplified by PCR and fluorescent dye chemistry sequencing was performed using the same primers used for amplification (Levin et al. Cancer Cell2005;7:387). Abnormal cytogenetic findings were considered to be either favorable (sole 13q- or 20q- abnormalities) or unfavorable (other abnormalities) (Tefferi et al. BJH2001;113:763). Results: i. Patients and treatment: A total of 69 patients with MMM (median age 62 years, range 38–75; 47 males) received the following drugs as first-line therapy for either anemia or symptomatic splenomegaly; erythropoietin (Epo) alone or in combination with hydroxyurea (n=25), hydroxyurea alone (HU; n=17), interferon alpha (IFN; n=11), combination of thalidomide and prednisone (ThalPred; n=7), androgen preparations (Andro; n=5), and etanercept (n=4). ii. Results of cytogenetic studies and JAK2 mutation screening: Cytogenetic findings were abnormal in 35 patients (51%); favorable in 9 and unfavorable in 26. JAK2V617 mutation analysis revealed wild-type allele in 31 patients (45%) and either heterozygous (n=31) or homozygous (n=7) mutation in the remaining 38 patients (55%). iii. Correlation of response to cytogenetic/molecular markers: Overall, 17 patients (25%) achieved either a major (n=8) or minor (n=9) response in anemia from treatment with one of the aforementioned drugs. Regardless of either JAK2V617 mutational status or cytogenetic profile, treatment-induced responses in anemia were poor for IFN (0%) and HU (12%). In contrast, the best anemia responses were documented for ThalPred (57%), Andro (40%), and Epo-based therapy (32%). Among the 25 patients that received Epo-based therapy, all 3 patients with favorable cytogenetic abnormalities achieved major responses in anemia whereas such responses were seen in none of the 9 patients with unfavorable cytogenetic abnormalities and only 1 of 13 patients with normal cytogenetics (p=0.004). Furthermore, 3 of the 4 major responders were heterozygous for JAK2V617 and one carried the wild-type allele. In contrast, none of the 3 JAK2V617 homozygotes showed any type of response but all 3 also displayed unfavorable cytogenetics. None of the 7 ThalPred-treated patients carried favorable cytogenetics and yet 3 achieved major responses including one with unfavorable cytogenetics. JAK2V617 mutational status was heterozygous in 1 and wild-type in the other two. There were no major responses among the 5 Andro-treated patients despite the presence of favorable cytogenetic abnormalities in 2 patients. In order to investigate the relationship between cytogenetic profile and JAK2V617 mutational status further, we referred to an expanded database of 116 patients and found the incidence of unfavorable cytogenetics to be higher in JAK2V617 homozygotes compared to non-homozygotes (57% vs. 31%; p=0.16) Conclusion: The current study suggests clustering of favorable cytogenetic abnormalities in MMM with anemia response to Epo therapy and unfavorable cytogenetic abnormalities with homozygosity for JAK2V617 mutation.

Abstract Background: The FIP1L1-PDGFRA fusion, an imatinib-sensitive molecular target, is detected in a subset of cases with moderate-to-severe eosinophilia (eosinophil count ≥1500/μL). Using 2 FISH strategies to detect loss of the CHIC2 locus as a surrogate marker of the FIP1L1-PDGFRA fusion, we previously found 14% of cases to be CHIC2-deleted when studying bone marrow smears from 81 adult patients with moderate-to-severe, non-reactive eosinophilia at our institution (Blood 2004, 104:3038–3045). Since then, the FISH test has been made available for routine clinical use both within our institution, and to external physicians as a mail-in test, for the workup of patients with eosinophilic disorders, including systemic mast cell disease (SMCD). Study design: FISH data, subsequent to the test’s introduction into routine clinical practice (from Nov. 2003), were retrospectively analyzed for CHIC2-deletion. Data from the initial pilot study of 81 patients was not used in this analysis (Table). The imatinib treatment analysis, however, included all CHIC2-deleted patients treated at our institution, including those from the pilot study (Table). Results: A total of 741 patients were studied in the post-pilot phase. Of these, 20 cases were found to be CHIC2-deleted, with an overall prevalence of 2.7% (Table). Of the 219 patients seen at our institution, 13 (all male) were found to be CHIC2-deleted (Table) (prevalence=5.9%), and of these, 10 have been treated with imatinib (100 mg/day starting dose) to date. Nine patients are evaluable for response - all achieved a complete clinical and histological remission (7 patients were tested by FISH post-treatment - all achieved cytogenetic remission). One patient received concomitant corticosteroids for co-morbidities and could not be evaluated for response. After a median follow-up of 24 months (range 17 to 39 months), all 9 patients continue to be in remission, with a normal eosinophil count. At last follow-up, one patient had discontinued imatinib and the others were on maintenance therapy at the following dose: 100mg/wk (n=1), 100mg BIW (n=1), 100mg TIW (n=2), 50mg QOD (n=1), and 100 mg/day (n=3). Conclusions: FIP1L1-PDGFRA prevalence in non-selected patients with eosinophilia is lower than anticipated from prior studies. Patients with FIP1L1-PDGFRA exhibit durable responses to imatinib therapy. It may be feasible to maintain some patients in remission on an empirically derived, intermittent schedule of low-dose imatinib. Pilot Data Post-pilot Data (n=741) Total Mayo samples Mayo samples Mail-in samples Number of Patients 81 138 603 822 Number CHIC2-deleted 11 2 18 31 Prevalence 14% 1.4% 3.0% 3.8% Number Imatinib treated 8 2 n/a 10

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: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. Studies have shown elevated expression of anti-apoptotic proteins of the Bcl-2 family in MM cells, which appear to correlate with resistance to therapy with certain drugs. Hence, accelerating the apoptotic process by targeting the Bcl-2 family of proteins appears to be an attractive strategy for the treatment of MM. AT-101 is an orally bioavailable derivative of gossypol in cancer clinical trials, and is being developed by Ascenta Therapeutics. AT-101 behaves as a small molecule inhibitor of Bcl-2 and Bcl-XL, binding to the BH3-binding pocket of these proteins and inhibiting their ability to suppress the activity of pro-apoptotic proteins, resulting in apoptosis. Methods and Results: AT-101 was cytotoxic to several different myeloma cell lines with a median effect observed at around 5μM concentration using an MTT cell proliferation assay. Additionally, at similar doses AT-101 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. AT-101 was cytotoxic to myeloma cells under these conditions as well with a median effect at concentrations of 5–10μM. AT-101 was able to induce apoptosis in myeloma cells in a dose- and time dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. AT-101 also resulted in cytotoxicity of freshly isolated primary patient myeloma cells, inducing apoptosis in a dose dependent manner. We also studied the effect of AT-101 on levels of different pro- and anti-apoptotic proteins using flow cytometry on permeabilized cells. A time-dependent increase in the level of BAX was observed following treatment with AT-101 without any associated change in levels of Bcl-xL or Bcl-2. Further studies evaluating the combination of AT101 with other active myeloma agents as well as a detailed evaluation of its mechanisms in myeloma are ongoing. Conclusion: AT-101 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. More importantly, it has activity against cell lines resistant to conventional anti-myeloma agents. In addition, Phase I studies with this agent are currently ongoing in patients with solid tumors. The results from these studies form the rationale for early phase clinical trials in MM, either alone or in combination with other active therapies.

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

Abstract Background: Copper deficiency is increasingly being diagnosed in adults with malabsorption syndromes, especially following GI surgery such as gastric and bowel resections and bariatric surgery. The most common hematologic manifestations are refractory anemia and neutropenia and patients have been misdiagnosed as having myelodysplasia. Certain findings on bone marrow examination are felt to be characteristic for copper deficiency such as maturation arrest in the myeloid lineage, vacuolization of the erythroid and myeloid precursors and ringed sideroblasts. Methods: We reviewed all cases of copper deficiency diagnosed at the Mayo Clinic from 1974 – 2005. We also reviewed all cases of low serum copper (< 0.75 μg/ml) identified in our heavy metals laboratory. Results: 123 patients with hypocupremia were identified. 77 patients were excluded as the hypocupremia was either transient or due to other factors such as Wilson’s disease. Twenty-five patients had hematologic manifestations, including anemia and leukopenia (13), isolated anemia (6), pancytopenia (5), and isolated neutropenia (1). Eighteen patients had a bone marrow biopsy. We were able to retrieve and review bone marrow slides in 11 cases. The bone marrow examination findings are shown in the table. The marrow cellularity was decreased in four cases. All cases had granulocytic hypoplasia but preserved megakaryopoiesis. Nine cases had relative erythroid hyperplasia. All but one bone marrow had cytoplasmic vacuoles in pronormoblasts and myelocytes. Increased iron within macrophages was seen in 9 cases and increased iron in plasma cells was noted in 8 cases. This was especially prominent in the cases with low peripheral white blood count. Ringed sideroblasts were seen in 4 cases and were more commonly seen in anemic patients. The initial bone marrow examination commonly suggested toxic effect (drugs or alcohol), myelodysplasia or were non-diagnostic. Conclusion: We have described the bone marrow findings in 11 copper deficient patients with abnormal blood counts. None of these patients was suspected of copper deficiency based on the initial marrow examination. The initial impression was commonly toxic effect due to the presence of cytoplasmic vacuolization. Patients with ringed sideroblasts were suspected of having MDS. Copper deficiency should be considered in patients with cytopenias and the described bone marrow findings, especially if there is evidence of malabsorption or a history of GI surgery such as gastric resection or bariatric surgery. Bone marrow findings Patient Cellularity Ery/gran/meg Vacuoles Iron (mø/PC) RS Hgb/Plt WBC/ANC nl: normal, 0: absent, Ery/gran/meg: erythro-/granulo-/megakaryopoiesis, Iron (mø/PC): Iron in macrophages and plasma cells, RS: Ringed sideroblasts 1 ↓ ↑/↓/nl Yes ↑/↑ No 11.3/203 3.2/1.1 2 Nl ↑/↓/nl Yes 0/0 No 11.4/192 5.6/4.0 3 Nl ↑/↓/nl No ↑/0 No 10.6/95 3.0/2.2 4 ↓ ↑/↓/nl Yes ↑/↑ No 13.6/128 4.2/0.92 5 Nl ↑/↓/nl Yes ↑/↑ 10% 9.8/215 1.4/0.6 6 ↓ ↑/↓/nl Yes ↑/↑ No 7.9/292 1.8/0.18 7 Nl ↑/↓/nl Yes ↑/↑ No 10.6/280 2.7/1.4 8 Nl nl/↓/nl Yes ↑/↑ 20% 8.1/330 1.5/0.24 9 Nl ↑/↓/nl Yes ↑/↑ <1% 9.4/252 1.3/0.25 10 ↓ Nl/↓/nl Yes 0/0 No 10.5/167 2.4/1.67 11 Nl ↑/↓/nl Yes ↑/↑ yes 12.5/224 4.2/3.9

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 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: Green tea has long been touted as a health promoting substance. The active chemical compounds in green tea are called polyphenols or catechins. Epigallocatechin gallate(EGCG) is the major catechin in green tea. We previously reported the in vitro ability of EGCG to induce apoptotic cell death in chronic lymphocytic leukemia(CLL) B-cells in vitro (Blood 104:788). After publication of our findings, clinical activity in individuals using over the counter green tea extracts were reported (Leuk Res 30:707). Based on this information, we opened a phase I/II trial of green tea extracts for patients with asymptomatic, early stage CLL in fall of 2005. METHODS: The purpose of the phase I portion of this trial was to determine the optimal dose of EGCG in the Polyphenon E preparation for chronic daily administration and define tolerability in CLL patients. Previously untreated patients with asymptomatic, Rai stage 0–II CLL not currently meeting National Cancer Institute(NCI) Working Group(WG) Criteria for treatment were eligible for participation. Polyphenon E with a standardized dose of EGCG was obtained from NCI. The phase I portion of the trial was designed with 8 dose levels(range 400–2000 mg orally BID) using the standard 3 patient per dose level design. Patients remained on study up to 6 months. Grade 2 adverse events attributed to study treatment that did not respond to supportive care were considered dose limiting toxicity. The trial was designed to administer Polyphenon E in the fasting state. After accrual to dose levels 1 and 2, the U.S. FDA mandated all U.S. trials of Polyphenon E administer drug in the fed state. Accordingly, drug was administered in the fed state for dose levels 3–8. Trough plasma EGCG levels were measured 1 month after initiation of therapy. Response was classified using the NCI WG Criteria. RESULTS: As of August 2007, 33 patients have been accrued to dose levels 1–8. The maximum tolerated dose(MTD) has not been reached. Side effects have generally been mild. The most common toxicities were nausea(grade 1: 42%; grade 2: 3%), elevation in SGOT (42%; all grade 1), and abdominal pain (36%; all grade 1). To date, no patient has had a sustained 50% reduction in both absolute lymphocyte count (ALC) and lymphadenopathy that would meet the NCI WG criteria for partial response. A majority of patients have had a reduction in ALC(Table). Among the 10 patients who had palpable adenopathy at study enrollment, 7 patients experienced at least a 50% reduction in the sum of the products of all nodal areas at some point during treatment. Trough plasma EGCG levels after 1 month of treatment ranged from 2.93974 ng/mL(median 40.5 ng/mL). Plasma levels did not clearly relate to the degree of reduction in ALC suggesting sensitivity to Polyphenon E may relate more to characteristics of the leukemic clone than plasma EGCG levels. CONCLUSION: Daily oral EGCG in the Polyphenon E preparation was well tolerated by CLL patients in this phase I trial. The MTD has not been reached. As classified by the NCI WG criteria, no partial or complete remissions have been observed to date, however declines in ALC and lymphadenopathy have been observed in the majority of patients. The phase II portion of this trial will open at Mayo Clinic Fall 2007. Best reduction in ALC n % of patients At least 10% decline 25 76% At least 20% decline 14 42% At least 30% decline 8 24% At least 40% decline 4 12% At least 50% decline 2 6%