ALBERT

Description: BACKGROUND: LGL leukemia is a neoplasm arising from either CD3+ T-cells or CD3− NK-cells. Autoimmune-mediated anemia, neutropenia, and rheumatoid arthritis occur frequently in these patients and immunosuppressive agents are used for these associated clinical syndromes. In our previous studies, we found that patients display a constitutively activated Ras and MAPK/ERK signaling cascade that may drive leukemia survival. A multicenter phase 2 clinical trial was initiated to treat LGL leukemia patients with the farnysltransferase-inhibitor R115777 (tipifarnib, Zarnestra®, Johnson & Johnson) that inhibits Ras and other farnesylated proteins. One of the goals of this study was to determine the shifts in cytokine production during therapy. We found that LGL cells treated with tipifarnib in vitro displayed a switch to Th2 (IL-4 and IL-10)-polarized differentiation. After tipifarnib treatment of LGL patients, antigen-activated T-cells produced greater amounts of Th2 (IL4/IL-10) cytokines but less Th1 (IFNγ/TNFα). In this study, we determined the mechanism governing tipifarnib-mediated Th2 polarization in T-cells. METHODS: PBMCs were isolated from 10 healthy donors and from seven patients with T-LGL leukemia before and after treatment with tipifarnib 300 mg twice daily for 21 days of a 28- day cycle. LGL leukemia patients had increased numbers of αβ T lymphocytes and evidence of clonality in association with either neutropenia or transfusion-dependent anemia. Th1 and Th2 cytokines were determined by intracellular staining and flow cytometry after activation with anti-CD3 plus anti-CD28. In some experiments, Th1 polarization was induced by IL-12; whereas, Th2 was induced by IL-4. Expression of T-bet and GATA-3 transcription factors that regulate Th1 and Th2 polarization, respectively, phosphorylated (active) MAPK (ERK1 and ERK2), and total MAPK were measured by Western blots. FTI2153, tipifarnib, and geranylgeranyl transferase inhibitor(GGTI)-2417 were used compared to DMSO control. RESULTS: PBMCs from patients with T-LGL leukemia displayed a dose and time-dependent increase in IL-4 and IL-10 production after drug treatment (average increase to 100% and 43%, respectively). A dose-dependent increase in these Th2 cytokines in T-cells from healthy donors showed that the farnesylated protein targeted by tipifarnib was not selectively expressed in LGL leukemia. Culture with IL-12 induced Th1 differentiation associated with ERK phosphorylation and increased T-bet expression. Pre-treatment with tipifarnib and FTI2153 but not GGTI2417 prior to IL-12 inhibited T-bet induction with no change in anti-CD3-induced MAPK leading to enhanced IL-4 signaling and greater Th2 polarization. CONCLUSIONS: Our data reveal unique, previously unreported effects of FTIs on cytokine signaling in T-cells by inhibiting the induction of T-bet and blocking Th1 differentiation. These results are critical to determine the mechanism of action of tipifarnib in LGL leukemia and suggest that FTIs may be useful for autoimmune or lymphocyte-mediated disorders associated with excessive Th1 cytokine production.

Description: Background: Thalidomide which represents an effective treatment strategy for relapsed/refractory multiple myeloma, actually represents a standard of care also for newly diagnosed multiple myeloma patients. Methods: In the present study, we adopted a gene expression profiling (GEP) strategy in an attempt to predict response (〉 50% reduction in serum M protein) to primary therapy with thalidomide-dexamethasone for newly diagnosed multiple myeloma. Plasma cells (CD138+) were purified from bone marrow aspirates from 17 patients at diagnosis, before initiation of treatment with thalidomide-dexamethasone. GEP was performed using the Affymetrix U133 Plus_2 microarray platform. The Affymetrix output (CEL files) was imported into Genespring 7.3 (Agilent technologies) microarray analysis software, where data files were normalized across chips using GCRMA and to the 50th percentile, followed by per gene normalization to median. Criteria of response were those established by Bladè et al. Results: After sufficient follow-up, responders (n=9) and nonresponders (n=8) were identified, and gene expression differences in baselines samples were examined. Of the 11000 genes surveyed, Wilcoxon rank sum test identified 149 genes that distinguished response from non response. A multivariate step-wise discriminant analysis (MSDA) revealed that 14 of the 149 genes could be used in a response predictor model (see table). Of interest, the gene list encompasses WXSC1, known to be involved in the chromosomal translocation t(4;14) (p16.3;q32.3) in multiple myeloma. Conclusion: These results could be the first step to adopt microfluidic cards, in an attempt to select at diagnosis patients who will respond favourably to a particular treatment strategy. List of 14 genes able to predict response to primary therapy with thalidomide-dexamethasone for newly diagnosed multiple myeloma. Gene ID Gene Name Chromosomal location 212771_at C10orf38 10p13 229874_x_at LOC400741 1p36.13 219690_at U2AF1L4 19q13.12 202207_at ARL7 2q37.1 243819_at GNG2 14q21 203753_at TCF4 18q21.1 235400_at FCRLM1 1q23.3 211474_s_at SERPINB6 6p25 226785_at ATP11C Xq27.1 215440_s_at BEXL1 Xq22.1–q22.3 209054_s_at WXSC1 4p16.3 227168_at FLJ25967 22p12.1 213355_at ST3GAL6 3q12.1 223218_s_at NFKBIZ 3p12–q12

Description: The mammalian target of rapamycin (mTOR) is an intracellular protein that acts as a central regulator of multiple signaling pathways (IGF, EGF, PDGF, VEGF, amino acids) that mediate abnormal growth, proliferation, survival and angiogenesis in cancer. mTOR is a critical component of the PI3K/Akt pathway, a key cell survival pathway that is dysregulated in many cancers including multiple myeloma (MM). mTOR is an important therapeutic target because it is a “rate-limiting” bottleneck in the key signaling pathway that regulates cell survival, proliferation, and angiogenesis. RAD001 (everolimus) is a novel oral mTOR pathway inhibitor. Recent data suggests that RAD001 has direct effects on tumor cell proliferation and may have antiangiogenic activity due to inhibition of tumoral VEGF production and effects on vascular endothelial and smooth muscle cell biology. We first evaluated the in vivo effects of single agent RAD001 in mice bearing the human MM tumor LAGλ-1. Tumor-bearing mice receiving RAD001 at 3, 10, or 30 mg/kg once daily five times per week (M-F) via oral gavage showed marked inhibition of tumor growth at all doses (P

Description: Vascular endothelial growth factor (VEGF) is an important signaling protein that plays a critical role in vasculogenesis and angiogenesis, and serves as one of the contributors to physiological or pathological conditions that can stimulate the formation of new blood vessels. The uncontrolled growth of new blood vessels is an important contributor to a number of pathological conditions, including multiple myeloma (MM). In support of this, bone marrow angiogenesis has been shown to correlate with disease status and poor prognosis in MM. VEGF also directly induces myeloma cell proliferation. We previously evaluated the effects of single agent mouse/human anti-VEGF antibody G6.31 (Campbell et al, Blood (ASH Annual Meeting Abstracts), Nov 2006) in several of our mouse models of human MM. In this study, we evaluated the effects of the same anti-VEGF antibody in combination with bortezomib or lenalidomide. Severe combined immunodeficient (SCID) mice were implanted into the left superficial gluteal muscle with either a 2.0 – 4.0 mm3 fragment from a patient when she was bortezomib-sensitive, LAGκ-1A, or resistant, LAGκ-1B. The tumors were allowed to grow for 21 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups (n=10 mice/group). Treatment groups consisted of a control IgG antibody or anti-VEGF antibody administered via i.p. injection twice weekly at a dose of 2 mg/kg, bortezomib administered via intravenous injection at a dose of 0.25 or 0.5 mg/kg twice weekly, lenalidomide administered via i.p. injection at a dose of 50 mg/kg daily × 5 per week, anti-VEGF antibody (2 mg/kg) + bortezomib (0.25 or 0.5 mg/kg), and anti-VEGF (2 mg/kg) + lenalidomide (50 mg/kg). Mice receiving the combination therapy of anti-VEGF + bortezomib (0.5 mg//kg) antibody showed marked inhibition of tumor growth and reduction of paraprotein levels compared to mice receiving control antibody. Notably, this combination also produced much more marked anti-MM effects compared to bortezomib treatment alone, anti-VEGF antibody alone, or vehicle alone. This combination was well tolerated. In contrast, mice receiving the combination of anti-VEGF antibody + lenalidomide showed no significant differences in tumor volume or hIgG levels compared to single agent treatment or vehicle alone. The markedly improved anti-MM effects of the combination of bortezomib and anti-VEGF antibody compared to single agent treatment in this in vivo study of human MM is promising, and these results have provided the preclinical rationale for an ongoing randomized multi-center Phase II trial.

Description: PURPOSE: Although the use of highly active antiretroviral therapy (HAART) has led to improvements in the management of AIDS-associated Kaposi’s sarcoma (KS), KS may persist or progress despite HAART. It has been hypothesized that activation of KS-associated herpesvirus (KSHV, HHV8) lytic expression might render tumor cells susceptible to immune surveillance by cytotoxic T cells. Alternatively, it has been suggested that lytic induction could lead to tumor progression. In vitro, valproic acid (VA) and other histone deacetylase inhibitors induce KSHV lytic gene expression in primary effusion lymphoma cell lines. We investigated VA in AIDS/KS patients to assess its safety and its impact on lytic viral gene expression in tumor and viral copy number in blood. PATIENTS AND METHODS: VA was given orally to patients with AIDS and cutaneous KS on stable antiviral regimens; the dose was titrated to maintain trough concentrations between 50 and 100 mcg/mL. VA was given daily for 28 days followed by a rapid taper and patients were then followed for 6 months. Quantitative real time PCR was used to assess viral DNA in plasma and PBMC, and viral RNA in tumor specimens. Immunohistochemistry was used to assess viral antigen expression in tumor specimens. RESULTS: 18 patients were treated. 15/18 patients completed therapy; 3 patients discontinued therapy early, one secondary to grade 2 toxicity and 2 to patient preference. One patient showed a partial response and 17 showed stable disease at the completion of therapy. No patients progressed during treatment. There were no differences between KSHV copy number in plasma or PBMC before, during, or after therapy. Similarly, although serial biopsies in some patients showed an increase in lytic gene expression, these changes did not achieve statistical significance. However, in multivariate analyses, viral lytic RNA increased in tumor biopsies on day 8 as a function of VA level. There was no change in HIV viral load with VA treatment. CONCLUSION : VA was well tolerated in AIDS patients, was not associated with accelerated disease progression, but rarely induced tumor regression after short-term treatment. In patients who achieved the highest serum VA levels there was increased lytic viral RNA expression. These findings support investigation of more potent HDAC inhibitors over longer treatment courses in patients with AIDS-associated KS.

Description: BACKGROUND: Patients with acute myeloid leukemia (AML) and hyperleukocytosis are at high risk of early mortality due to pulmonary, renal, and central nervous system complications. Leukapheresis and low-dose continuous infusion of cytarabine and hydroxyurea (HU) have been used but their advantages and limitations are not well characterized. The University hospital in Casablanca, Morocco has a limited number of beds, and hence admissions must be prioritized. Here we report the effects of HU on the white blood cell (WBC) count and early mortality rate of patients with hyperleukocytic AML. METHODS: Between April 2003 and December 2006, patients with AML were enrolled on the AML-MA2003 protocol (2 induction courses of cytarabine and daunorubicin and 2 postremission courses that include intermediate-dose cytarabine). Patients with AML and hyperleukocytosis (WBC count 〉50 x 109/L) were immediately started on HU (50 mg/kg/day orally x 4 days), regardless of hospital bed availability. Response was evaluated after 4 days of HU; patients were considered responders if 〉50% reduction of the initial WBC count was observed. RESULTS: Ninety of 260 (34.6%) patients enrolled had hyperleukocytosis. Three patients were excluded, induction therapy started on the day of admission. Therefore, 87 patients (48 females, 39 males) were evaluable. The mean age was 32 years (range, 2–60); 29% were children (ages 2–20 years). The mean initial WBC count was 104x109/L (range 50–260 x109/L); 37 (42.5%) patients had WBC counts 〉 100x109/L. The French-American-British subtypes were M1 (45%), M2 (26%), M4 (12%), M5 (7%), and M0, M3, M6 and M7 (2.5% each); 5 cases were unclassified. Karyotypes determined for 65/87 cases revealed 13 (20%) favorable karyotypes [9 had the t(8;21); 3 had inv16; 1 had the t(15;17)], 30 (46%) intermediate-risk karyotypes, including normal karyotypes, and 22 (34%) unfavorable-risk karyotypes. Sixty-two (71%) patients were classified as responders. In an additional 3, the WBC count was reduced 25%–50%. In 22 (25%) patients, including 4 whose WBC counts increased, HU showed no cytoreductive effect. The mean WBC count after 4 days of HU was 24 x 109/L (range, 1.5–125 x109/L); 15 (17%) patients’ WBC counts remained 〉100x109/L. Four patients developed acute tumor lysis syndrome (TLS) (hyperuricemia and renal dysfunction) in response to HU. There were 5 (8%) early deaths (mean, 7 days after the start of HU; range, 4–14 days). All 5 patients had WBC counts 〉 100x109/L at diagnosis, and only 1 was a responder. This mortality rate does not differ from that (9%) observed among the 170 protocol patients who did not have hyperleukocytosis. Causes of death included infection (n=1), pulmonary and CNS leukostasis (n=1 each), TLS (renal failure and hyperkalemia, n=1), and intracranial hemorrhage (n=1). Among several factors (age, sex, FAB type, karyotype, WBC counts), only WBC count of ≤ 100x109/L was significantly associated with response to HU (P=0.01). The complete remission rate after first course of induction therapy was 43.5% for responders and 16% for non-responders (P=0.02). CONCLUSION: HU given orally for 4 days rapidly reduces the WBC count in pediatric and adult AML with hyperleukocytosis. The early mortality rate in this high-risk group treated with HU compares favorably with rates reported for similar patients. It remains to be determined whether initial response to HU is associated with overall outcome in AML.

Description: INTRODUCTION: Lymphoproliferative diseases of large granular lymphocytes (LGL leukemia) are clinical syndromes associated with increased circulating CD3+CD8+CD57+ T-LGL s. Chronic neutropenia, anemia, and arthritis are clinical manifestations mediated by autoimmune reactivity of this lymphocyte population. Previously, we found evidence that extracellular-regulated kinase (ERK) and Ras were constitutively active in the patient’s LGLs. Ablation of Ras activity by a dominant-negative form of Ras and pharmacologic inhibition with farnesyltransferase inhibitors FTI2153 and R115777 (tipifarnib, Zarnestra®, Johnson & Johnson) resulted in ERK inhibition and enhanced apoptosis of the leukemic LGLs. A multicenter phase 2 clinical trial using tipifarnib was initiated with a two-stage design. Here we provide the first report of the efficacy and safety of tipifarnib in patients with LGL leukemia. METHODS: Patients were to be treated in the first stage with four cycles of therapy at a dose of 300 mg twice daily for 21 days out of a 28- day intermittent cycle. Key patient entry criteria were neutrophil counts 〈 500 cells/μl, transfusion-dependent anemia, and clonal CD3+/CD57+ T-cells greater than 350 cells/μl in the peripheral blood. WBC, ALC, hemoglobin, platelet counts, and ANC were determined. Bone marrow biopsies were performed on all patients prior to therapy and then repeated when worsening cytopenias occurred. Tipifarnib was stopped when bone marrow cellularity was reduced by 50% compared to baseline. Bone marrow colony formation assessed using14-day CFU-GM and BFU-E assays in methycellulose (Stem Cell Separation Systems, Vancouver BC). Growth factor support with G-CSF was allowed during the 7 days off when ANCs were 〈 500 cells/μl. RESULTS: Five males and two females (mean 57 years old) received tipifarnib. Four patients failed to complete the study due to toxicity. Three patients were removed from study due to grade 3–4 bone marrow toxicity and/or infections requiring hospitalization and a tipifarnib-unrelated death occurred in one patient. Three patients completed four cycles and were evaluated for response with a dose reduction for bone marrow toxicity required in one of these evaluable patients. Leukemic LGL cells from the three evaluable patients displayed a dose-dependent increase in apoptosis in vitro and treatment was associated with reduced ALCs in two cases. None of the patients met the pre-determined criteria for response. However, one patient, shown to be growth factor unresponsive prior to therapy, had an increase in ANC from 0 at baseline to 8,600 cells/μl while receiving G-CSF on week 16. The number of bone marrow colonies also increased in this patient after therapy. All bone marrow biopsies, which were required in five cases, showed significantly improved erythroid or myeloid differentiation. CONCLUSIONS: Improvements in bone marrow differentiation suggest that tipifarnib may prove beneficial for the treatment of LGL leukemia if a safe dose and schedule is established and the treatment prolonged to allow for bone marrow recovery.

Description: Deacetylase (DAC) inhibitors represent a new class of anti-cancer therapeutics that inhibit DAC enzymes and have been shown to have multiple effects in tumor cell lines including decreased oncoprotein expression (Bcr-Abl, HER-2), decreased angiogenesis, induction of apoptosis, induction of cell-cycle arrest, and decreased tumor cell motility and invasion. Panobinostat (LBH589), a novel cinnamic hydroxamic acid analogue with potent histone DAC inhibitor activity, has recently been shown to have the potential to treat a wide range of solid and hematological malignancies including multiple myeloma (MM). In this study, we first evaluated the in vitro anti-MM effects of panobinostat alone and in combination with doxorubicin or melphalan using the MM cell lines RPMI8226, U266 and MM1S. Cells treated with the combinations of panobinostat + doxorubicin and panobinostat + melphalan showed marked synergistic anti-MM effects as determined by measuring proliferation with the MTS assay compared to treatment with single agent and untreated cells. Next, we evaluated the anti-MM effects of panobinostat alone and in these combinations in vivo using one of our SCID-hu mouse models of human MM, LAGλ-1. Each SCID mouse was implanted with a 2.0 - 4.0 mm3 LAGλ-1 into the left superficial gluteal muscle. In our panobinostat single agent study, tumors were allowed to grow for 7 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into panobinostat treatment groups. Panobinostat was administered via intraperitoneal (i.p.) injection once daily five times per week at 5, 10 and 20 mg/kg. Control mice were given sterile normal saline as vehicle. Mice receiving panobinostat showed marked inhibition of tumor growth (10 mg/kg, P 〈 0.003; 20 mg/kg, P 〈 0.009) and reduction of paraprotein levels (10 mg/kg, P 〈 0.0025; 20 mg/kg, P 〈 0.015) compared to mice receiving vehicle. Next, we evaluated the combination of low-dose panobinostat (5 mg/kg) with low doses of either liposomal doxorubicin (1 mg/kg) or melphalan (3 mg/kg) i.p. in mice bearing LAGλ-1. Tumors were allowed to grow for 10 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups. Panobinostat was administered as above, and liposomal doxorubicin was injected once daily for three consecutive days weekly and melphalan once weekly. Mice treated with the combination of panobinostat + liposomal doxorubicin showed markedly smaller tumors and reduced hIgG levels compared to treatment with the DAC inhibitor alone, and treatment with liposomal doxorubicin as a single agent produced no anti-MM effects. Mice bearing LAGλ-1 treated with the combination of low-dose panobinostat + low-dose melphalan also showed markedly smaller tumors and decreased hIgG levels compared to treatment with panobinostat alone whereas mice receiving melphalan alone showed similar results to vehicle-treated animals. These promising results support the further clinical development of panobinostat and suggest that combining this DAC inhibitor with low-dose chemotherapy (liposomal doxorubicin or melphalan) may enhance the efficacy of this novel agent for the treatment of MM patients.

Description: Pleiotrophin (PTN) is an important developmental cytokine that is highly expressed during embryogenesis but shows very limited expression in adult tissues, where it is largely restricted to the brain. High PTN serum levels are associated with a variety of solid tumors. We recently showed that patients with multiple myeloma (MM) also have elevated serum levels of this protein and the amount of PTN correlated with the patients' disease status and response to treatment. In this study, we demonstrate that MM cell lines and the malignant cells from MM patients' bone marrow produced PTN and secreted PTN protein into the supernatants during short-term culture. Moreover, Ptn gene expression correlated with the patients' disease status. Inhibition of PTN with a polyclonal anti-PTN antibody reduced growth and enhanced apoptosis of MM cell lines and freshly isolated bone marrow tumor cells from MM patients in vitro. Importantly, this antibody also markedly suppressed the growth of MM in vivo using a severe combined immunodeficiency (SCID)-hu murine model. This represents the first study showing the importance of PTN in the growth of any hematological disorder. Because the expression of this protein is very limited in normal adult tissues, PTN may represent a new target for the treatment of MM.