Decrease In Spleen Size Research Articles

Rapid Decrease in Overall Tumor Burden On Ibrutinib (PCI-32765) in CLL Despite Transient Increase in ALC Indicates a Significant Degree of Treatment Induced Cell Death.

AbstractAbstract 2899 Introduction:Bruton's tyrosine kinase (BTK) is essential for B cell receptor (BCR) signaling. Ibrutinib (PCI 32765) is a covalent inhibitor of BTK that has demonstrated durable antitumor activity in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (Byrd ASCO 2011, O'Brien ASH 2011). Ibrutinib induces a transient increase in the absolute lymphocyte count (ALC) occurring during a period of rapid reduction in lymphadenopathy. This is thought to be due to inhibition of CLL cell adhesion and migration leading to a release of CLL cells from tissue sites. Patients and Methods:This phase II trial (NCT01500733) using ibrutinib 420 mg daily on 28 day cycles enrolled two cohorts of CLL/SLL patients; 1.) >65 years old; and 2.) having deletion of chromosome 17p). Treatment naive and previously treated patients in need of treatment were eligible. Response was assessed after 2 cycles using CT scans. Bone marrow (BM) biopsies in a subset of patients were obtained at baseline and either on day 28 or 56. Whole blood viscosity was measured. Here we report on effects of ibrutinib in 26 patients (n=11, >65 yo; n=15, del 17p) who completed >2 cycles. Results:Fourteen patients (54%) had a partial response (PR) by IWCLL criteria, and 62% achieved a >50% reduction in lymphadenopathy measured as the sum of the products of the greatest diameter (SPD) of the 4 largest lymph nodes as assessed by CT, irrespective of ALC. The effects of ibrutinib on total tumor burden were measured by concomitantly assessing lymph node (LN), spleen, blood, and BM disease. The median reduction in lymphadenopathy was 52% (range 22–72%) on day 56. Despite at times massive reduction in tumor burden, there was no evidence of tumor lysis. The rates of nodal responses in subgroups of patients defined by deletion 17p, non deletion 17p, treatment naive and relapsed/refractory patients was comparable. There was an overall reduction in splenic disease with a median decrease in spleen size (measured as longest cranio-caudal extension) of 11% (0.5–40%; P<0.01). A trend towards more rapid resolution of splenomegaly in treatment naïve patients was seen. BM evaluations are complete in 10 patients: cellularity decreased in 3 of 7 and remained unchanged in 4 on day 28; on day 56 cellularity was decreased in 3 of 3 patients. Using immunohistochemistry for CD79 we confirmed that the reduction in BM infiltration was due to reduced tumor infiltration. As seen in other studies, the increase in ALC peaked within the first 2 months followed by a slow decline. The median ALC increased from 75 k/μl (3.5 – 308) to 102 k/μl on day 28 (P<.01). However, only 62% of patients had an increased ALC on treatment days 14 and 28 compared to baseline and the change in ALC at the end of the first cycle (day 28) was very heterogeneous (>95% reduction to ∼700% increase). Treatment naïve patients showed more pronounced relative increases in ALC and the most dramatic increase occurred in patients with a lower starting ALC. The rise in ALC correlated with an increase in whole blood viscosity (r=0.52; P<0.05, n=18). However, all viscosity measurements, both at baseline and on treatment, were within the normal range (3.6–6.0 cP) and there were no clinical signs or symptoms of a hyperviscosity syndrome. Conclusion:Reduction in lymphadenopathy with a transient increase in ALC has been reported in other studies and is thought to reflect a redistribution of tumor burden into the blood. While a treatment induced rise in ALC was common in our patients, up to a third had resolution of blood disease within a few weeks. This and the often dramatic reduction in nodal disease with or without an increase in ALC, and the concomitant reduction in tumor infiltration of BM and spleen indicate a significant contribution of cell death, the mechanism of which remains to be defined. Interestingly resolution of disease in the BM appears to be slower than in the LN. This may reflect more active BCR signaling in the LN and consequently more pronounced changes in tumor biology on ibrutinib in this site. In addition, treatment naïve patients appear to respond more rapidly raising the question whether untreated CLL is more dependent on BTK-dependent signaling pathways in the tissue microenvironment.This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for participating in these research studies. Disclosures:No relevant conflicts of interest to declare.

RNAi-Mediated Inhibition of Tmprss6 Ameliorates Anemia and Secondary Iron Overload in a Mouse Model of β-Thalassemia Intermedia and Decreases Iron Overload in Hfe−/− Mice

AbstractAbstract 1018β-Thalassemia intermedia (TI), an inherited hemoglobinopathy caused by partial loss of β-globin synthesis, is characterized by anemia, extramedullary hematopoiesis and ineffective erythropoiesis as well as secondary iron overload. Hereditary hemochromatosis (HH) is most frequently caused by mutations in HFE and is marked by excess uptake of dietary iron with concomitant tissue iron overload. In both diseases, increased iron absorption is due to inappropriately low levels of the liver hormone, hepcidin (encoded by Hamp1). The membrane serine protease Matriptase-2 (encoded by Tmprss6) attenuates BMP-mediated Hamp1 induction by cleaving the BMP co-receptor, hemojuvelin. Previously, it has been shown that elevating Hamp1 expression by genetic inactivation of Tmprss6 reduces disease severity in the Hbbth3/+ mouse model of TI and prevents iron overload in Hfe−/− mice. Therefore, a therapeutic approach comprising specific inhibition of Tmprss6 could prove efficacious in TI and HH.Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to >80% inhibition of Tmprss6 mRNA in the livers of Hbbth3/+ and Hfe−/− mice with concomitant >2-fold elevation in Hamp1 expression. In the TI model, Tmprss6 silencing leads to ∼30% reductions in serum iron and non-heme liver iron. In Hfe−/− mice, serum iron and non-heme liver iron are similarly reduced, and Perls staining of peri-portal iron is diminished. Remarkably, the partial iron restriction induced by Tmprss6 inhibition in Hbbth3/+ mice leads to dramatic improvements in the hematological aspects of the disease phenotype: the severity of the anemia is decreased as evidenced by an approximately 1 g/dL increase in total hemoglobin and a 50% decrease in circulating erythropoietin levels. As in the human disease, Hbbth3/+ mice exhibit the hallmarks of ineffective erythropoiesis including splenomegaly, decreased erythrocyte survival and marked reticulocytosis. Treatment with LNP formulated Tmprss6 siRNA leads to a dramatic 2–3 fold decrease in spleen size, a 3–4 fold decrease in reticulocyte counts and a >7-day increase in RBC half-life. Histological analysis of spleens from Tmprss6 siRNA treated animals demonstrates restoration of normal splenic architecture, as well as a reduction in the number of Tfr1-positive erythrocyte precursors in the spleen. Furthermore, as evidenced by the near normalization of blood smears, the overall quality of erythropoiesis in treated animals is vastly improved.Taken together, these data demonstrate that RNAi-mediated silencing of liver Tmprss6 elevates Hamp1 expression and reduces iron overload in both TI and HH model mice. More significantly, Tmprss6 siRNA treatment ameliorates all aspects of the disease phenotype in the TI mouse model. These results support the development of an RNAi therapeutic targeting TMPRSS6 for the treatment of TI, HH and potentially other disorders characterized by excess iron absorption due to physiologically inappropriately low levels of hepcidin. Disclosures:Racie:Alnylam Pharmaceuticals: Employment. Butler:Alnylam Pharmaceuticals, Inc.: Employment, Equity Ownership. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment, Equity Ownership.

The Activation of STAT3 and 5 in t(6;9)(DEK/CAN)-Positive Acute Myeloid Leukemia Models Is Inhibited by Exposure to Arsenic Trioxide

AbstractAbstract 1384▪▪This icon denotes a clinically relevant abstractIn acute myeloid leukemia (AML) chimeric genes derived from specific chromosomal translocations, such as t(15;17), t(8;21) or t(6;9) encode aberrant fusion proteins (FPs). These Fps, i.e. PML/RARα (PR), AML-1/ETO (AE) or DEK/CAN (DC), recapitulate the leukemic phenotype in the mouse. In about 50% af AMLs constitutive activation of STAT3 and/or 5 is observed and it is postulated that the aberrant stem cell capacity in leukemia is correlated to the activation of STATs. Furthermore it has been shown in models of the t(15;17)(PR)-positive AML that there is a relationship between STAT-activation and response to arsenic trioxide (ATO). t(6;9)(DC)-positive AML is classified as a separate entity, because of its young age of onset and poor prognosis. The mechanisms by which DC induces leukemia are nearly completely unknown.In order to investigate whether the STAT-activation plays a role for the pathogenesis of t(6;9)-positive AML we investigated the activation of STAT3 and 5 in models of DEK/CAN positive leukemia.As models of DC-induced AML we used either primary Sca1+/lin- murine hematopoietic stem and progenitor cells (mHSPC) retrovirally transduced with DC or as controls with PR. Samples of DC-positive pre-leukemia as well as primary leukemia and 2° leukemia from established DC-induced AML were used for the in vivo studies. Expression and activation of STAT3 and 5 was assessed by either intracellular FACS or Western blotting using antibodies against the phosphorylated (activated) and total STAT3 or 5. The effect of constitutively activated STAT3 (STAT3*) on the stem cell capacity was studied by the retroviral expression of STAT3* in mHSPC which then were subjected to serial replating and colony-forming unit spleen day 12 (CFU-S12) assays. ATO treatment was performed on sublethally irradiated recipients (8 mice/group), from day 5 after inoculation of DC-positive primary leukemic spleen cells for the induction of a 2° leukemia for 14 days. Leukemia development and response to ATO was assessed 30 days post-transplantation by spleen size and determination of the proportion of leukemic cells in the peripheral blood (PB), spleen and bone marrow (BM) in comparison to solvent treated controls.Here we show that i.) both DC and PR strongly activated both STAT3 and 5 in primary blasts from mouse leukemia as as well as in retrovirally transduced primary HSCPs; ii) the expression of constitutively activated STAT3 (STAT*) in HSC led to an increased replating efficiency in the presence of mIL-3, mIL-6 and SCF; iii.) STAT* increased CFU-S12 as a read out for short-term stem cell and early progenitor potential to a similar extent as PR. iv.) the pre-leukemic state of DC-positive AML was characterized by the only activation of STAT3 but not of STAT5; v.) STAT5 activation appeared only in the full blown leukemia; v.) exposure to ATO inhibited phosphorylation of STAT3 and STAT5 in vivo in the PB of the leukemic mice; vi.) the treatment with ATO significantly decreased spleen size and the proportion of leukemic cells in the BM and spleen as compared to solvent treated controls.In summary, our results strongly suggest a direct relationship between the expression of the leukemogenic oncogenes DC or PR and the activation of STATs, which seems to play an important role for the maintenance of the leukemic stem cell. Furthermore ATO seems to represent a novel therapeutic option for the t(6;9)-positive AML. Disclosures:No relevant conflicts of interest to declare.

A Transient Decrease in Spleen Size Following Stroke Corresponds to Splenocyte Release into Systemic Circulation

The splenic response to stroke is a proinflammatory reaction to ischemic injury resulting in expanded neurodegeneration. Splenectomy reduces neural injury in rodent models of hemorrhagic and ischemic stroke, however the exact nature of this response has yet to be fully understood. This study examines the migration of splenocytes after brain ischemia utilizing carboxyfluorescein diacetate succinimidyl ester (CFSE) to label them in vivo. The spleen was found to significantly decrease in size from 24 to 48 h following middle cerebral artery occlusion (MCAO) in rats compared to sham operated controls. By 96 h post-MCAO the spleen size returned to levels not different from sham operated rats. To track splenocyte migration following MCAO, spleens were injected with CFSE to label cells. CFSE positive cell numbers were significantly reduced in the 48 h MCAO group versus 48 h sham and CFSE labeled cells were equivalent in 96 h MCAO and sham groups. A significant increase of labeled lymphocyte, monocytes, and neutrophils was detected in the blood at 48 h post-MCAO when compared to the other groups. CFSE labeled cells migrated to the brain following MCAO but appear to remain within the vasculature. These cells were identified as natural killer cells (NK) and monocytes at 48 h and at 96 h post-MCAO NK cells, T cells and monocytes. After ischemic injury, splenocytes enter into systemic circulation and migrate to the brain exacerbating neurodegeneration.

PMO-129 Relaxin reduces portal hypertension through stimulation of hepatic nitric oxide production

IntroductionWe have previously reported that the multifunctional hormone relaxin (RLX) downregulated the activation state and contractility of hepatic myofibroblasts and reduced portal hypertension (PHT) in cirrhotic rats (Fallowfield J et...

Abstract 2494: A Transient Decrease in Spleen Size Following Stoke Corresponds to Splenocyte Release into Systemic Circulation

Objective: Splenectomy is protective in ischemic injuries to renal, hepatic, and gastrointestinal systems, showing that the splenic response promotes cellular degeneration. Removal of the spleen is also protective in ischemic and hemorrhagic stroke. Therefore, we investigated the cellular response of the spleen to brain ischemia. Hypothesis: Immune cells are released from the spleen following cerebral ischemia contributing to neural injury. Methods: A time course was conducted to investigate splenic size changes in rats over time following permanent middle cerebral artery occlusion (MCAO) and sham-MCAO (n≥3). In a second experiment, splenocytes were labeled in situ with carboxyfluorescein diacetate, succinimidyl ester (CFSE). Five evenly spaced splenic injections, 4 mg/ml in DMSO, were given to all groups five days prior to MCAO or sham-MCAO. The experimental groups were: 48 hr post-MCAO (n=4), 48 hr post-sham-MCAO (n=4), 96 hr post-MCAO (n=6), 96 hr post-sham-MCAO (n=4), and a CFSE only group that was euthanized five days following injection of CFSE without undergoing MCAO (n=4). Spleen, brain, thymus, and blood smears were collected for all groups. Results: The spleen was found to decrease in size at 48 hrs following MCAO in rats compared to controls (p&lt;0.01). However, by 96 hrs post-MCAO the spleen size returned to levels not different from sham operated rats. CFSE was found to be non-toxic at the 4 mg/ml concentration and five days following injection 15% of the splenocytes were labeled. In the spleen, there was a significant increase (p&lt;0.0001) in CFSE positive cells in the 48 hr sham group versus the 96 hr sham and both 48 and 96 hr MCAO groups. Blood smears showed a significant increase in total CFSE positive cells (p&lt;0.0007) at 48 hrs post-MCAO. CFSE positive cells in the blood were identified by Giemsa staining. A significant increase of lymphocytes (p&lt;0.005), monocytes (p&lt;0.02), and neutrophils (p&lt;0.0005) was found at 48 hrs post-MCAO when compared to the other groups. Conclusion: These results demonstrate that CFSE is a viable and safe way to track immune cells in situ in an animal model of stroke. The spleen transiently decreases in size post-MCAO in rats and releases splenocytes into systemic circulation at 48 hrs following MCAO. These cells are migrating to the brain or other lymphoid organs, affecting the overall immune response to ischemia. Brains and thymi from the CFSE injected animals will be analyzed for the presence of these cells.

Progression of Noncirrhotic Portal Hypertension in a Pediatric Population

The optimal management of children with noncirrhotic portal hypertension is controversial. Some groups suggest early and aggressive surgical intervention, while others report long-term success with conservative management. We conducted a retrospective study of 26 patients with noncirrhotic portal hypertension treated at our institution. We compared platelet counts, white blood cell (WBC) counts, spleen size, hospital admissions, gastrointestinal bleeds, and longitudinal trends of specific clinical parameters using standard univariate and time-trend analytic techniques. Mean age at the time of diagnosis was 5.2 years. Portal vein thrombosis was found in 84.6% of patients (n=22). There was one mortality related to malignancy. There was not a progression of hypersplenism in patients that did not receive a shunt and conversely, we did not notice a significant decrease in spleen size following shunt surgery (P=0.2). Platelet and WBC counts trended downward among patients managed medically, while platelets increased and WBC counts remained stable in surgical patients. There was a significant decrease in hospital admissions for gastrointestinal bleeding following surgical intervention in the shunt group compared with nonshunt (P=0.0009). While our analysis was limited given small sample sizes and selection bias, it suggests that the majority of pediatric patients with noncirrhotic portal hypertension will do well long-term without surgical intervention.

Therapy with the Histone Deacetylase Inhibitor Sb939 for Patients with Myelofibrosis,

Abstract 3863▪▪This icon denotes a clinically relevant abstract Background:The discovery of the JAK2V617F mutation has triggered the development of agents that target the activity of this mutant enzyme. It has been recently shown that both wild-type JAK2 and JAK2V617F kinases translocate to the nucleus of various human leukemic cell lines and primary CD34+ hematopoietic progenitors and phosphorylate histone H3, resulting in loss of affinity for the transcriptional repressor heterochromatin protein 1α (HP1α), which in turn, it causes aberrant gene expression. These results provide the rationale to use histone modifying agents for the treatment of MF. SB939 is a small molecule (dialkyl benzimidazole) competitive inhibitor of histone deacetylase (HDAC) that has >1000-fold selectivity for HDAC Class 1 and 2 versus Class 3. We are conducting a study of SB939 in pts with MF. Methods:This is a phase 2, prospective, open-label study to determine the efficacy and safety of SB939 in subjects with MF (primary or post-ET or –PV) with intermediate-1, intermediate -2 or high risk disease according to the IWG prognostic scoring system, and for those with low risk and symptomatic splenomegaly ≥5 cm below left costal margin. Initial SB939 dose was 60mg every other day three times weekly for three weeks. Response is assessed using the International Working Group (IWG) criteria. After 22 pts started SB939, the study was put on hold to assess toxicity with a provision of terminating the trial if ≤4 pts responded after being treated for 6 months.23 pts were accrued (77Results: 23 pts were accrued (77% male, 81% JAK2V617F-positive). Median age was 67 years (range, 51–74), WBC 13×109/dL (1.2–103.2), Hg 9g/dL (6–26.6), platelets 143×109/dL (21–572). Eighteen (82%) pts carried the JAK2V617F mutation, with a median allele burden of 59.6% (19.9–94.95%). Seven (33%) pts had abnormal cytogenetics. Ninety-one percent of pts were symptomatic at study entry with a performance status of 0 (n=5%), 1 (73%), or 2 (22%). Ninety-five percent had splenomegaly with a median spleen size of 13cm (range, 0–29cm). Twenty-two pts are assessable for response and toxicity. Six (27%) pts experience a decrease in spleen size (median 3 cm, range 1–4). Two pts had anemia clinical improvement by IWG criteria (Hg increased from 9.1g/dL at baseline to 11.1g/dL at last follow-up and from 7.9g/dL to 15.3g/dL). Five (50%) of 10 pts with hepatomegaly reduced their liver size (median 3cm, range, 1–6cm). Three (17%) of 18 JAK2V617F-positive pts had reductions of allele burden (median 11.4%). The most frequent side effect was fatigue, which occurred in 20 (91%) pts: grade 1 (n=17) and grade 2 (n=3). Other toxicities included pain (n=5), peripheral edema (n=4), and diarrhea (n=3), all grade 1. Rates of grade 3–4 neutropenia, anemia, and thrombocytopenia were 13%, 0%, and 21%, respectively. No pt has died during the conduction of the study and 3 remain on the study receiving SB939 at 60mg (n=1) or 50mg (n=2); 19 pts are off study due to lack of response (n=9), disease progression (n=4), pt’s request (n=2), unrelated medical problems (n=3, surgery for aortic aneurism, prostate cancer, infection), fatigue (n=1). Conclusion:SB939 has modest activity as single agent in pts with MF. Most responses consisted of reductions in spleen and/or liver size with minimal activity on anemia. Combination studies at lower doses are planned with JAK2 inhibitors. Disclosures:No relevant conflicts of interest to declare.

Oral administration of artemisinin analog SM934 ameliorates lupus syndromes in MRL/lpr mice by inhibiting Th1 and Th17 cell responses

SM934, an artemisinin derivative, possesses potent antiproliferative and antiinflammatory properties. The aim of this study was to examine the effects and explore the mechanisms of SM934 to treat autoimmune disease in lupus-prone female MRL/lpr mice. In vitro, the effects of SM934 on the activation of polyclonal CD4+ T cells and the differentiation of naive CD4+ T cells were examined. In vivo, the preventative or therapeutic effects of SM934 in MRL/lpr mice were investigated. Ex vivo, the mechanisms of treatment were explored according to the immunologic correlates of disease. In vitro, SM934 inhibited interferon-γ (IFNγ) and interleukin-17 (IL-17) production from polyclonal CD4+ T cells activated by T cell receptor engagement and the differentiation of naive CD4+ T cells into Th1 and Th17 cells, but not Treg cells. In vivo, 12-week-old MRL/lpr mice treated with SM934 for 4 weeks showed significantly ameliorated proteinuria and renal lesion severity; decreased levels of blood urea nitrogen, serum IFNγ, and serum anti-double-stranded DNA antibodies; decreased spleen size; and a lower percentage of CD3+B220+CD4-CD8- T cells; 16-week-old MRL/lpr mice treated with SM934 for 8 weeks avoided severe proteinuria and survived longer. Ex vivo, SM934 treatment elevated the percentage of Treg cells, inhibited the development of Th1 and Th17 cells, and impeded the comprehensive activation of STAT-1, STAT-3, and STAT-5 proteins in splenocytes. Taken together, the results of this study demonstrated that the artemisinin analog SM934 had therapeutic effects in lupus-prone female MRL/lpr mice by inhibiting both Th1 cell and Th17 cell responses. Moreover, this study indicated that both IFNγ and IL-17 are required for the elicitation and development of murine lupus.

A potential role of ruxolitinib in leukemia

Introduction: An increased understanding of cellular signaling pathways, like the JAK–STAT pathway, and the identification of the JAK2 V617F mutation in the classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), has generated great interest in the development of targeted JAK2 inhibitors. In a recently completed Phase I–II study, ruxolitinib, a selective orally available JAK1 and JAK2 inhibitor, has shown efficacy in patients with advanced myelofibrosis. Constitutive activation of the JAK–STAT pathway has also been implicated in other hematological malignancies suggesting a potential role of JAK kinase inhibitors in these malignancies.Areas covered: This article reviews the chemistry, pharmacodynamics, pharmacokinetics, clinical efficacy, safety and tolerability of ruxolitinib. The literature for this article was retrieved from PubMed database searches using the keywords ‘ruxolitinib’, ‘INCB 018424’, ‘JAK2 inhibitors’ and ‘leukemia’.Expert opinion: The JAK–STAT signaling pathway plays a vital role in leukemogenesis. Ruxolitinib, a potent JAK1 and JAK2 inhibitor, known to decrease spleen size and alleviate constitutional symptoms in myelofibrosis, represents a potentially promising agent for the treatment of leukemias by inhibiting the JAK–STAT signaling. Further studies of ruxolitinib, in patients with acute and chronic leukemias, are now needed to establish the clinical usefulness of this promising drug.

Analysis of the Ex Vivo and In Vivo Antiretroviral Activity of Gemcitabine

Replication of retroviral and host genomes requires ribonucleotide reductase to convert rNTPs to dNTPs, which are then used as substrates for DNA synthesis. Inhibition of ribonucleotide reductase by hydroxyurea (HU) has been previously used to treat cancers as well as HIV. However, the use of HU as an antiretroviral is limited by its associated toxicities such as myelosuppression and hepatotoxicity. In this study, we examined the ribonucleotide reductase inhibitor, gemcitabine, both in cell culture and in C57Bl/6 mice infected with LP-BM5 murine leukemia virus (LP-BM5 MuLV, a murine AIDS model). Gemcitabine decreased infectivity of MuLV in cell culture with an EC50 in the low nanomolar range with no detectable cytotoxicity. Similarly, gemcitabine significantly decreased disease progression in mice infected with LP-BM5. Specifically, gemcitabine treatment decreased spleen size, plasma IgM, and provirus levels compared to LP-BM5 MuLV infected, untreated mice. Gemcitabine efficacy was observed at doses as low as 1 mg/kg/day in the absence of toxicity. Higher doses of gemcitabine (3 mg/kg/day and higher) were associated with toxicity as determined by a loss in body mass. In summary, our findings demonstrate that gemcitabine has antiretroviral activity ex vivo and in vivo in the LP-BM5 MuLV model. These observations together with a recent ex vivo study with HIV-1[1], suggest that gemcitabine has broad antiretroviral activity and could be particularly useful in vivo when used in combination drug therapy.

Lenalidomide and prednisone for myelofibrosis: Eastern Cooperative Oncology Group (ECOG) phase 2 trial E4903

A multicenter Eastern Cooperative Group (ECOG) phase 2 trial assessed whether adding prednisone to lenalidomide would improve previously reported responses in persons with myelofibrosis (MF). Forty-eight subjects with anemia (42 evaluable) received lenalidomide, 10 mg/d, with a 3-month low-dose prednisone taper. Ten subjects received 3 months, and 25 received 6 months of therapy. Myelosuppression was the main toxicity with 88% with ≥ grade 3 hematologic toxicity and 45% ≥ grade 3 nonhematologic toxicity. There were responses in 10 subjects (23%) using the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT)-defined clinical improvement of anemia in 8 (19%) and/or decreased spleen size in 4 (10%). Serial bone marrow analysis showed no resolution of disease-related fibrosis or angiogenesis. With a median follow-up of 2.3 years, 23 subjects are alive. Lenali-domide and prednisone for myelofibro-sis evaluated through a multicentered-cooperative group mechanism is only modestly active and myelosuppre-sive. This study was registered at http://clinicaltrials.gov as NCT00227591.

Disruption of Laminin-γ1 Expression Induces Rapid Decrease In Spleen Size.

AbstractAbstract 2596Laminins are heterotrimeric proteins composed of an alpha, beta, and gamma subunit, and are expressed in a majority of organ systems, including hematopoietic and lymphoid tissues. Specific laminin subunits have been identified in prognostic stromal gene expression profiles from diffuse large B-cell lymphomas (Lenz et al. 2008). Laminin g1 is the gamma-subunit present in a majority of physiologically expressed laminin heterotrimers. Global laminin-g1 deficiency in mice is embryologically lethal at E5.5 and results in part from failure of the primordial basal lamina to form (Smyth et al. 1998). Conditional knockout mouse lines have therefore been developed to examine the role of laminin-g1 in specific tissues. Disruption of Schwann cell-specific expression of laminin-g1 prevents basal lamina formation within peripheral nerves, decreases glial cell proliferation, and increases glial cell apoptosis leading to a severe dysmyelinated phenotype (Chen and Strickland 2003; Yu et al. 2005).Here we present data that global disruption of laminin- g1 expression in adult animals induces involution of the spleen and suggests a fundamental role for laminins in maintenance of spleen integrity. Mice homozygous for the LoxP flanked laminin-g1 allele (floxed allele) were crossed with mice expressing a global Cre-ERT2 transgene. Mice homozygous or heterozygous for the floxed laminin-g1 allele that carry the inducible Cre transgene are hereafter referred to as homozygous and heterozygous mutant mice, respectively. Adult heterozygous and homozygous mutant animals were treated with either tamoxifen (TM) or corn oil (control), and three weeks afterwards, tissues were harvested for analysis. Treatment of homozygous mutant mice with TM, but not corn oil, induced laminin-g1 gene recombination. There was a significant decrease in spleen size in TM-treated but not control-treated homozygous mutant animals. Neither TM nor control treatment induced changes in the spleen size of heterozygous mutant animals. Similarly, TM treatment did not alter spleen size of animals that expressed Cre-transgene but were wild type with respect to the floxed laminin-g1 gene, or which were homozygous for the floxed laminin-g1 gene but lacked the Cre-transgene (Figure 1). [Display omitted] This is the first animal model to show that continued expression of laminins are critical for maintenance of the normal spleen and suggests a potential new target for disorders of lymphoid or splenic hyperproliferation.Chen ZL, Strickland S. 2003. Laminin gamma1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve. J Cell Biol 163(4):889-99.Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, Xu W, Tan B, Goldschmidt N, Iqbal J and others. 2008. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 359(22):2313–23.Smyth N, Vatansever HS, Meyer M, Frie C, Paulsson M, Edgar D. 1998. The targeted deletion of the LAMC1 gene. Ann N Y Acad Sci 857:283–6.Yu WM, Feltri ML, Wrabetz L, Strickland S, Chen ZL. 2005. Schwann cell-specific ablation of laminin gamma1 causes apoptosis and prevents proliferation. J Neurosci 25(18):4463–72. Disclosures:No relevant conflicts of interest to declare

Phase II Study of the JAK2 Inhibitor, INCB018424, In Patients with Refractory Leukemias Including Post-Myeloproliferative Disorder (MPD) Acute Myeloid Leukemia (sAML)

AbstractAbstract 509 Background:The JAK family of enzymes has a central role in normal hematopoiesis and immune response. Mutations of JAK2 gene that result in constitutive activation of JAK-STAT signaling and enhanced proliferation and survival signals have been described. This aberrant signaling pathway has been clearly demonstrated in myeloproliferative disorders (MPDs), but also reported in other hematological cancers including acute leukemias. Aim:To explore the activity of INCB018424 in patients (pts) with advanced hematological cancers Method:We are conducting a phase II study of INCB018424 in pts with relapsed/refractory leukemias for which no standard therapies are anticipated to result in a durable remission. Pts with performance status 0 – 2 with adequate organ function and no active, uncontrolled intercurrent illness or infection, receive INCB018424 orally at 25 mg twice daily for 4 weeks (one cycle). Response is assessed after every 2 cycles of treatment. Responding pts or pts with stable disease are allowed to continue until progression. Dose escalation to 50 mg twice daily is permitted in pts demonstrating a benefit. Result:Thirty eight pts [median age, 69 years; (range, 45–88)] with relapsed and refractory leukemias (15 sAML, 13 AML, 3 ALL, 1 MDS, 4 CMML & 2 CML) have been treated. The median number of prior therapies is 2 (range, 1 to 6). Eleven pts (7 with sAML, 2 with AML, and 2 with CMML) had the JAK2 V617F mutation. Cytogenetic analysis showed diploid karyotype in 11, chromosome 5 and 7 abnormalities in 7, t(2;9) in 1, Philadelphia chromosome in 2 and other various abnormalities in 17. Pts who completed 2 cycles of INCB018424 are defined as having stable disease (SD). Pts have received a median of 2 cycles of therapy (range, 1 –18 cycles) with 15 pts having SD or better (5 for 2 cycles, 4 for 3 cycles, 3 for 4 cycles, 2 for 5 cycles and 1 for 18 cycles). Among these, 3 pts (2 with sAML and 1 with CMML with JAK2 mutation) have had a significant reduction in their bone marrow blasts (to < 5%) associated with a significant decrease in spleen size and clinical improvement. Three pts are too early for assessment of response. Pts having benefit include sAML (9, or 60% of pts with sAML), AML (2, 15%), CMML (3, 75%) & MDS (1, 100%). Overall, 22 pts have had no response or progressed (12 after 1 cycle, 4 after 2 cycles, 2 after 3 cycles, 2 after 4 cycles, and 2 after 5 cycles). Nine pts died from causes unrelated to the treatment with INCB018424. Four pts continue with treatment (1 after 18 cycles, 1 after 3 cycles, & 2 after 2 cycles). One pt had a successful allogeneic SCT after 3 cycles with SD. Two pts came off protocol for unrelated complications. Overall INCB018424 was tolerated very well. One pt had elevated liver enzymes (G3) along with neutropenia (G3), another pt had G3 liver enzyme elevation and 2 pts developed G2 thrombocytopenia. 1 pt had G5 intracranial hemorrhage. Conclusion:INCB018424 is well-tolerated and has activity particularly in pts with sAML and CMML but also in AML and MDS, with or without JAK2 V617F mutation. Clinical studies combining INCB018424 with chemotherapy in sAML are warranted. Disclosures:Off Label Use: Use of arsenic trioxide in frontline therapy of APL. Verstovsek:INCYTE: Research Funding. Cortes:NOVARTIS: Research Funding. Newton:Incyte Corporation: Employment. Kantarjian:NOVARTIS: Research Funding. Ravandi:Incyte Corporation: Research Funding; Novartis: Honoraria, Speakers Bureau.

Effective management of accelerated phase myelofibrosis with low‐dose splenic radiotherapy

The main cause of hepatosplenomegaly in primary (PMF), post polycythemia vera (post-PV MF), and post essential thrombocythemia (post-ET MF) myelofibrosis (MF) is extramedullary hematopoiesis (EMH). Drug-refractory symptomatic splenomegaly in MF is usually managed by splenectomy or involved-field radiotherapy. The latter is most effective in the treatment of MF-associated bone pain and pulmonary hypertension.Our previous experience with hepatosplenic radiotherapy in MF showed efficacy in the majority of treated patients but its utility was limited by the transient nature of its benefit and the occurrence of treatment-related pancytopenia. In an effort to address these issues,we have adopted an induction-maintenance treatment strategy that utilizes lower radiation doses-induction with 100 cGy total in four daily doses of 25 cGy and maintenance with either the same or lower intensity regimen. Herein, we report our most recent experience using this treatment plan in two cases, who in addition to their expected response from the standpoint of splenomegaly, also unexpectedly showed a marked response of their underlying accelerated phase disease,including clearance of circulating blasts and basophilia.

Selective blockade of BAFF for the prevention and treatment of systemic lupus erythematosus nephritis in NZM2410 mice

To determine whether BAFF or combined BAFF/APRIL blockade is effective in a mouse model of systemic lupus erythematosus (SLE) nephritis characterized by rapidly progressive glomerulosclerosis. NZM2410 mice at early and late stages of SLE nephritis were treated with a short course of BAFF-R-Ig or TACI-Ig fusion protein. Proteinuria and serologic profile were evaluated every 2 weeks. Immunohistochemical, flow cytometric, and enzyme-linked immunospot analyses of the spleen, kidney, and bone marrow were performed after 8 weeks and after 33 weeks. A short course of selective blockade of BAFF alone was sufficient to prevent and treat SLE nephritis in NZM2410 mice, despite the formation of pathogenic autoantibodies. Decreases in spleen size and B cell depletion persisted for more than 33 weeks after treatment and resulted in secondary decreases in CD4 memory T cell formation and activation of splenic and peripheral monocytes. Immune complex deposition in the kidneys was dissociated from renal damage and from activation of renal endothelial and resident dendritic cells. Selective blockade of BAFF alone, which resulted in B cell depletion and splenic collapse, was sufficient to prevent and treat the disease in this model of noninflammatory SLE nephritis. This shows that the inflammatory microenvironment may be a determinant of the outcome of B cell modulation strategies.

Meisoindigo is a promising agent with in vitro and in vivo activity against human acute myeloid leukemia

Meisoindigo, a derivative of Indigo naturalis, has been used in China for chronic myeloid leukemia. In vitro cell line studies have shown that this agent might induce apoptosis and myeloid differentiation of acute myeloid leukemia (AML). In this study, we explored its mechanisms and potential in AML. NB4, HL-60, and U937 cells and primary AML cells were used to examine its effects and the NOD/SCID animal model was used to evaluate its in vivo activity. Meisoindigo inhibited the growth of leukemic cells by inducing marked apoptosis and moderate cell-cycle arrest at the G0/G1 phase. It down-regulated anti-apoptotic Bcl-2, and up-regulated pro-apoptotic Bak and Bax and cell-cycle related proteins, p21and p27. Furthermore, it induced myeloid differentiation, as demonstrated by morphologic changes, up-regulation of CD11b, and increased nitroblue tetrazolium reduction activity in all cell lines tested. In addition, meisoindigo down-regulated the expression of human telomerase reverse transcriptase and enhanced the cytotoxicity of conventional chemotherapeutic agents, cytarabine and idarubicin. As with the results from cell lines, meisoindigo also induced apoptosis, up-regulated p21 and p27, and down-regulated Bcl-2 in primary AML cells. The in vivo anti-leukemic activity of meisoindigo was also demonstrated by decreased spleen size in a dose-dependent manner. Taking these results together, meisoindigo is a potential agent for AML.

Relevance of the mTOR signaling pathway in the pathophysiology of splenomegaly in rats with chronic portal hypertension

Splenomegaly is a frequent hallmark of portal hypertension that, in some cases, can be very prominent and cause symptoms like abdominal pain, splenic infarction, and cytopenia. This study characterizes the pathogenetic mechanisms leading to spleen enlargement in portal hypertensive rats and focuses on mTOR pathway as a potential modulator of splenomegaly in portal hypertension. Characterization of splenomegaly was performed by histological, hematological, immunohistochemical and Western blot analyses in rats with portal hypertension induced by portal vein ligation, and compared with sham-operated animals. The contribution of the mTOR signaling pathway to splenomegaly was determined in rats with fully developed portal hypertension and control rats by treatment with rapamycin or vehicle. Our results illustrate that splenomegaly in portal hypertensive rats arises as a consequence of the interplay of several factors, including not only spleen congestion, as traditionally thought, but also enlargement and hyperactivation of the splenic lymphoid tissue, as well as increased angiogenesis and fibrogenesis. Since mTOR signaling plays a central role in immunological processes, angiogenesis and fibrogenesis, we next determined the involvement of mTOR in splenomegaly. Interestingly, mTOR signaling was overactivated in the spleen of portal hypertensive rats, and mTOR blockade by rapamycin profoundly ameliorated splenomegaly, causing a 44% decrease in spleen size. This effect was most likely accounted for the inhibitory action of rapamycin on lymphocyte proliferation, neovascularization and fibrosis. These findings shed light on the pathogenesis of splenomegaly in portal hypertension, and identify mTOR signaling as a potential target for therapeutic intervention in this disease.

Eliglustat tartrate

Eliglustat tartrate (Genz-112638) is a novel, orally administered agent currently in development for the treatment of lysosomal storage disorders, including type 1 Gaucher disease and Fabry disease. This glucosylceramide analogue acts as an inhibitor of glucosylceramide synthase, a Golgi complex enzyme that catalyzes the formation of glucosylceramide from ceramide and UDP-glucose and is the first step in the formation of glucocerebroside-based glycosphingolipids. Pre-clinical pharmacological studies demonstrate that the agent has a high therapeutic index, excellent oral bioavailability and limited toxicity. Phase I studies in healthy volunteers revealed limited toxicity with an excellent pharmacodynamic response, as measured by decreased plasma glucosylceramide concentrations. Phase II studies in patients with type 1 Gaucher disease have demonstrated promising clinical responses, as measured by decreases in spleen size, improvement in hemoglobin concentrations and increased platelet counts. Two randomized phase III trials testing the efficacy and safety of eliglustat tartrate are currently in progress.

Mesoportal bypass for extrahepatic portal vein obstruction in children: close to a cure for most!

Aim Extrahepatic portal vein obstruction (EPVO) is a common cause of portal hypertension in children and can lead to life-threatening bleeding, thrombocytopenia, and coagulation disorders. Mesoportal bypass (MPB) restores normal physiologic portal flow to the liver and corrects portal hypertension. There is, however, little long-term outcome data after MPB. The aim of our study was to analyze the long-term outcome after MPB in children. Methods Retrospective single-center review of all MPB with more than 5-year follow-up was performed in children between 1998 and 2003. Results Thirty children underwent MPB, which was successful in 29. Long-term follow-up is available for 24. Median age at the time of bypass was 8.5 years (range, 0.4-14.2 years). Material used for bypass was as follows: left internal jugular vein (n = 20), recanalized umbilical vein (n = 2), gastric vein, and a large colic vein (n = 2). Median time since MPB is 8 years (range, 5.3-8.8 years). One MPB using recanalized umbilical vein thrombosed at 4 months but was successfully refashioned using Gortex. One MPB with left internal jugular vein was thrombosed at 1 year after cardiac surgery. Overall, 23 of 24 children have a patent bypass and resolution of portal hypertension. All showed an decrease in spleen size. Recurrent blood flow in the cavernoma with secondary increase in spleen size occurred in 5 children (median time, 3.4 years after MPB). Further evaluation of these 5 revealed no recurrence of portal hypertension. Conclusion The MPB provides long-term correction of portal hypertension owing to EPVO. Where technically feasible, MPB is the preferred surgical procedure for treatment of EPVO.