ALBERT

Description: Abstract 1843 Poster Board I-869 Introduction: The mammalian target of rapamycin (mToR) plays a crucial role in cell growth due to its role as nutrient dependent regulator of important cytokine signaling pathways. In multiple myeloma, mToR is involved in the phosphoinositide-3-kinase (PI3K)-AKT pathway which can be activated by the loss of the tumor suppressor phosphatase and tensin homolog (PTEN) or by stimulation with growth and survival factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). Inhibitors of the mToR pathway (sirolimus/rapamycin, everolimus and temsirolimus) are approved for immunosuppression and/or cancer treatment. However, the clinical activity of mToR inhibitors may be limited by the fact that, after inhibition of the rapamycin-sensitive mToR-Raptor complex, AKT is activated by the rapamycin-insensitive mToR-Rictor complex. In this regard, the inhibitory effect of mToR inhibitors was evaluated in combination with PI3K inhibitors in vitro and in vivo. Results: Rapamycin and everolimus induced a dose-dependent growth inhibition in six human malignant plasma cell lines. Growth inhibition was mediated by G1 cell cycle arrest and in a subset of cell lines by induction of apoptosis. Overexpression of Bcl-XL or Mcl-1 proteins did not prevent from apoptosis induction by mToR inhibitors, nor did sensitivity to rapamycin-induced apoptosis correlate with the p53 mutation status. In the INA-6 SCID mouse xenograft model, treatment with rapamycin resulted in a significant survival benefit compared to untreated mice. Six out of 14 rapamycin treated mice did not develop plasmacytomas during the observation period of 149 days. Remarkably, short term treatment of plasmacytoma bearing mice led to a significant shrinkage of the plasma cell tumor. Explanted tissue showed apoptotic plasma cells, a finding confirmed by immunohistological staining using an antibody specific for the human cleaved form of poly (ADP-ribose) polymerase (PARP). The combination of rapamycin and the PI3K inhibitor Ly294002 led to an increase of growth inhibition in all tested plasma cell lines. The additional growth inhibition by Ly294002 appeared to be due to AKT activation upon mToR inhibition by the rapamycin-insensitive Rictor complex, indicated by increased AKT phosphorylation at Ser473 as determined by Western blot analysis. Conclusion: Clinical trials currently evaluate mToR inhibitors for their potential to expand treatment options for myeloma patients. The data presented here suggest that a combination of mToR inhibitors with PI3K inhibitors may lead to additive therapeutic chances. Disclosures: Guenther: Novartis: Consultancy, Research Funding. Gramatzki:Novartis: Consultancy, Research Funding, Speakers Bureau.

Description: Hematopoietic stem cells (HSCs) show pronounced heterogeneity in self-renewal and differentiation behavior, which is reflected in their repopulation kinetics. Here, a single-cell–based mathematical model of HSC organization is used to examine the basis of HSC heterogeneity. Our modeling results, which are based on the analysis of limiting dilution competitive repopulation experiments in mice, demonstrate that small quantitative but clonally fixed differences of cellular properties are necessary and sufficient to account for the observed functional heterogeneity. The model predicts, and experimental data validate, that competitive pressures will amplify small clonal differences into large changes in the number of differentiated progeny. We further predict that the repertoire of HSC clones will evolve over time. Last, our results suggest that larger differences in cellular properties have to be assumed to account for genetically determined differences in HSC behavior as observed in different inbred mice strains. The model provides comprehensive systemic and quantitative insights into the clonal heterogeneity among HSCs with potential applications in predicting the behavior of malignant and/or genetically modified cells.

Description: Background: Activating mutations of the catalytic subunit of class IA phosphoinositide 3-kinase alpha (PIK3CA) are clustered in small hot-spot regions of the PIK3CA gene, including exon 9 within the helical domain and exon 11 within the kinase domain. They have been linked to several human neoplasias, including colorectal, breast and hepatocellular cancers. In acute leukemias, PIK3CA mutations have not been investigated in larger chorts and so far only been observed in a few patients. Since the PI3K/Akt/GSK3beta pathway is an important signaling cascade of receptor tyrosine kinases (e.g. Flt3R, Kit) which are frequently activated in acute leukemias we investigated the functional activity of PIK3CA mutants. Materials and methods: We transfected early hematopoietic cells (Ba/F3 cell line) with PI3KCA exon 9 and 11 mutations and investigated the cells in an in vitro factor-independent growth assay and pharmacologic inhibition experiments. Results: We demonstrate that mutations in the helical or kinase domain of PIK3CA lead to the constitutive activation of PI3Kalpha in Ba/F3 cells, inducing factor-independent growth of the IL3-dependent cells. The frequency of IL3-independent Ba/F3 cells after tranfection with exon 9 and 11 PIK3CA mutants was equivalent to the frequency confered by PIK3CA mutants containing the membrane localization signal of either src or ras. Proliferation and survival of the cells were inhibited by the PI3K inhibitors LY294002 and Quercitin or an inhibitor of the PI3K downstream target Akt. Inhibition occurred in a dose- and time-dependent manner and could be reverted by addition of IL-3. One of the major targets of PI3K/Akt signaling is GSK3beta which becomes inactivated after Akt-mediated phosphorylation. By using a GSK3beta-specific inhibitor or LiCl we could show that the inactivation of GSK3beta alone did not result in factor-independent growth of Ba/F3 cells. However, GSK3beta inhibition led to a delay in the induction of cell death after IL3-withdrawal. Conclusion: Activating mutations of PIK3CA, associated with several human neoplasias and acute leukemia are functionally active in hematopoietic cells, confer factor independency and respond to PI3K/Akt inhibition.

Description: We report a retrospective review of 18 children receiving haplocompatible related donor hematopoietic peripheral blood SCT and consecutively enrolled at four U.S. transplant centers. The median age was 8 yrs (range 1–20). Patients with malignancy (n=13) included: AML-CR1 (primary induction failure, failed cord blood transplant) [1], CR2 [3]; MDS-RA/RARS [2], RAEB [2]; AML and Fanconi anemia [1]; CML-CP2 [1]; ALL-CR3 [2]; NHL-CR2 [1]. Patients with non-malignant diseases included severe aplastic anemia [n=4] and Wiskott-Aldrich syndrome [n=1]. Thirteen donors were a 3/6 HLA match and 5 were a 4/6 match. CD34 positive selection was used to select stem cells and deplete T lymphocytes. Conditioning for 13 of the patients consisted of TBI 12–14 Gy in 6 fractions, thiotepa, fludarabine and ATG. Fractionated TBI was replaced by single fraction TBI (n=2) or melphalan (n=3). Cyclophosphamide replaced thiotepa for 1 patient with FA. No post-transplant graft-versus-host disease (GVHD) prophylaxis was given. Patients received a median of 18 × 106 CD34+ cells/kg (range 6–28) and 3 × 104 CD3+ cells/kg (range 0.3–11). Sustained primary engraftment occurred in 15/18 (83%) patients. Primary graft failure occurred in one patient. Two patients had immunological rejection following HHV-6 reactivation. Both engrafted after a second transplant; therefore the overall engraftment success was 94%. The median time to an ANC 〉0.5 × 109/L was 12 days (range 9–21). Platelet recovery occurred in 16/18 at a median of 17 days (range 9–22). Primary (occurring after SCT but prior to DLI) grade II acute GVHD was seen in 4/17 patients (24%). Grade III-IV GVHD was seen in 1 patient (6%) manifest as overlap syndrome in association with HHV-6 reactivation. One pt had primary extensive chronic GVHD. Of nine patients who received DLI and/or stem cell boosts, 4 had grade II GVHD (3/4 had prior acute GVHD), none had grade III-IV GVHD, 2 developed chronic GVHD and 1 developed overlap syndrome. Infections were common but manageable. All patients were at risk for CMV reactivation based on CMV serology: recipient/donor +/+ (9), +/− (3), −/+ (6). Seven patients (39%) reactivated CMV. All cases were responsive to anti-viral therapy and/or DLI. No CMV disease was seen. Seven patients had adenovirus reactivation and 6 had HHV-6 reactivation. EBV reactivation occurred in 5/18 (28%) patients. Rituximab (5) and DLI (2) yielded rapid resolution of EBV in all patients. Four of 13 (31%) at risk patients have relapsed: 1 pt with cytogenetic relapse remains in CR2 〉 6 mo later and another who recently relapsed is undergoing salvage therapy. The 100 day and 1 year transplant-related mortality was 11% and 19%, respectively. The overall survival is 72% with a median follow-up of 31 months (range 7–89). Among patients transplanted for malignant diseases (13) and non-malignant diseases (5), overall survival is 69% and 80%, respectively. The K-M 2 year survival was 70% +/− 22%. All survivors were complete donor chimeras by DNA methods. The use of megadose CD34-selected PBSC without post-transplant GVHD prophylaxis yielded rapid engraftment, low 100-day mortality and incidence of severe GVHD, and excellent survival. The overall survival compares favorably with MSD and MUD HSCT.

Description: A significant proportion of individuals undergoing treatment for lymphoma are of working age and are in employment prior to diagnosis and during subsequent chemotherapy treatment. This small-scale study seeks to explore the impact that chemotherapy treatments have on employment. Specifically, the reasons for the decision to continue to work or not, and any perceived benefits or disadvantages encountered as a result are explored. Methods: All individuals presenting with newly diagnosed lymphoma, over a 12 month period, were identified retrospectively. Individuals of official working age, and who were receiving out-patient chemotherapy (i.e. who had the ability to be working at the time) were invited to participate. Participants were asked to complete a questionnaire that was designed to explore their experiences of employment during this time. Results: A total of 33 patients were invited to participate. A return rate of 70% was achieved. Of those who responded, 5 (22%) had already retired prior to diagnosis. A total of 12 patients (52%) continued to work during their chemotherapy treatment. Of these, 6 (50%) continued to work the same hours in the same conditions, whereas 6 (50%) worked an altered or flexible pattern. 6 patients (26%) stopped working for the duration of treatment, one of whom has not returned since. There were no obvious differences in patients with Hodgkin’s Lymphoma as opposed to Non-Hodgkin’s Lymphoma. The response rate was much higher in those who had received intravenous chemotherapy than oral preparations, preventing analysis of any potential variation in experiences. The qualitative data obtained gives a clearer insight into the many issues faced by the patient when undergoing chemotherapy treatment. Familiar themes were identified in many cases, and these were grouped into 5 main theme categories: Diversion from the reality of diagnosis and treatment Psychological Issues Issues of retained normality Practical Issues Physical or medical issues. Difficulty in coping with the side-effects of treatment, particularly fatigue, were commonly cited. Emotional effects such as stress and anxiety were also alluded to. Despite this, those who continued to work reported benefit from retaining a normal lifestyle, gained support from co-workers and experienced a diversion of focus from treatment. Several of those who continued to work cited financial reasons for this decision. Feedback suggested that advice given by the health-care team was often lacking or inconsistent. This did not reflect our perception of current practice, so it may suggest that it is the way that information is given that is ineffective. Conclusion: The majority of patients continued to work during their treatment and there appear to be many benefits to this. Difficulties encountered related mainly to treatment side-effects, logistical and practical problems associated with undergoing chemotherapy. It is envisaged that a greater understanding of the impact that lymphoma and its treatment has on employment may improve the level of support that can be offered by the multi-disciplinary team. The curative nature of many Lymphomas demands that long-term complications of the disease and treatment are fully addressed at the time of diagnosis. Social, economic and employment welfare are integral aspects to be considered, especially in a group of individuals who are likely to be long-term survivors.

Description: Von Recklinghausen’s neurofibromatosis type 1 (NF1) is the most common genetic disease in man with a predisposition to cancer, a disorder caused by mutations of the NF1 tumor suppressor gene that functions as a GTPase for p21ras. Affected individuals are predisposed to plexiform neurofibromas, congenital tumors that arise from cranial and peripheral nerves for which no effective therapies are available. We have recently utilized a genetically engineered murine model to demonstrate that mast cells and the c-kit/kitL pathway have a key role in plexiform neurofibroma formation. In the present study, we utilized this model to determine whether imatinib mesylate, an FDA approved drug that targets the c-kit ligand in mast cells and other cell lineages in the tumor microenvironment in inducing tumor regression. Krox20; Nf1flox/− mice begin to develop plexiform neurofibromas by 6 months of age, especially in the dorsal root ganglia involving the sciatic nerve. Fluoridinated PET/CT images were utilized to verify that experimental mice had developed plexiform neurofibromas and to evaluate tumor progression. Following a 12 week treatment, Krox20; Nf1flox/− mice who received the vehicle had an increase in FDG uptake in the tumor growth area. In contrast, mice received imatinib mesylate had a dramatic reduction in the FDG uptake. At autopsy, the spinal cords were examined and sections were prepared for histologic examination. Grossly, tumors from the vehicle control treated mice were consistent with plexiform neurofibromas, as previously described (Zhu, Science 2002). In contrast, dissection of the spinal cords from Gleevec treated mice revealed that the mean volume of the tumor was significantly reduced. In addition, though there is still hyperplasia observed in the dorsal root ganglia from imatinib mesylate treated mice, the overall architecture of the nerve is improved as compared to the hematoxylin and eosin stained sections from the placebo treated control. Serial sections from 10 tumors isolated from placebo treated mice and serial sections from all dorsal root ganglia in the FDG positive affected regions of imatinib mesylate treated sections were scored. There is variability in the number of mast cells from the placebo treated tumors. However, there is a consistent, significant reduction in the number of mast cells scored in the dorsal root ganglia from the imatinib mesylate treated mice, suggesting that imatinib mesylate is inhibiting the c-kit receptor kinase and thus reducing mast cell numbers in the neurofibroma. Finally, since there are anatomic reductions in tumor size of imatinib mesylate treated mice as compared to placebo control treated animals, we hypothesized that the FDG affected regions would have an increase in apoptosis as compared to the amount of apoptosis observed in tumors from placebo treated mice. While only rare TUNEL positive cells were observed in tumors treated with the placebo, up to 10% of cells were TUNEL positive in tumors received imatinib mesylate. Collectively, these studies provide direct evidence that targeting the microenvironment induces regression of a plexiform neurofibromas and provides the basis for conducting clinical trials for a tumor that affects approximately 10–15 thousand patients in the US alone.

Description: Purpose: Cidofovir has efficacy in the treatment of adenovirus and cytomegalovirus (CMV) infection in immunocompromised individuals. It is licensed for the treatment of CMV retinitis in AIDS patients over the age of 12 years, and is nephrotoxic and contra-indicated with the use of other concomitant nephrotoxic agents. Its use has been further limited due to concerns regarding an individual’s ability to tolerate the hydration regimens associated with cidofovir administration. Despite this it is a valuable anti-viral agent particularly in the setting of haemapoietic stem cell transplantation. This study assessed the safety of cidofovir in a high-risk paediatric population. Methods: A retrospective review of all episodes of cidofovir administration at the Oncology Unit at the Children’s Hospital at Westmead from 2000–2005 was undertaken. The dose of cidofovir and renal function (measured by urea and creatinine) pre and post infusion were recorded, and the medical notes reviewed for adverse events and concomitant use of other nephrotoxic drugs. Results: A total of 233 cidofovir infusions in 23 patients were studied. Patients: Age range 5 months - 19 years (mean 6.6 years). Sex: male 17 (74%), female 6 (26%). Clinical setting: 16 (70%) were post allogeneic stem cell transplantation, 4 (17%) were post autologous stem cell transplation, 3 (13%) had other malignancies. Indications for cidofovir: In 20 (87%) for proven adenovirus identified in stool, urine, nasopharyngeal aspirate, CSF, or tissue biopsy; 2 (9%) for adenovirus prophylaxis as part of haplo-identical stem cell transplant conditioning, 1 (4%) for extended coverage in sepsis without an identified organism. Cidofovir doses: 111 (48%) were 1mg/kg, 75 (32%) were 3mg/kg, 47 (20%) were 5mg/kg. All episodes of cidofovir administration received the same standard recommended regimen of oral probenecid 40mg/kg at hour -3, 20ml/kg of intravenous 0.9% NaCl from hour −1 to 0, cidofovir infusion with 20ml/kg 0.9% NaCl from hour 0 to +1, maintenance intravenous fluids from hour +1 to +3, followed by oral probenecid 20mg/kg at hour +3 and +9. All patients received concomitant nephrotoxic agents: 4 (17%) received 1, 4 (17%) received 2, 5 (22%) received 3, and 10 (43%) received 4 or more agents. In 19 (83%) patients with normal renal function (defined by urea/creatinine within normal range for age), there was no evidence of nephrotoxicity (defined as 〉150% increase in urea/creatinine from baseline) following cidofovir therapy - mean change in urea/creatinine pre and post infusion −7.5%/+5.6%. There was no deterioration in renal function in 4 (17%) patients with pre-existing renal impairment (defined by urea/creatinine above the upper limit of normal) - mean change in urea/creatinine pre and post infusion −10.9%/+3.0%. Symptomatically cidofovir was tolerated by 20 (87%) patients without adverse events. Cidofovir therapy was associated with significant morbidity in 3 (17%) patients - 1 developed blindness and axonal degeneration of unknown aetiology, and in 2 patients, both with pre-existing pneumonitis post allogeneic stem cell transplantation, the fluid regimen precipitated a respiratory deterioration secondary to fluid overload. Conclusion: Cidofovir is a safe and well tolerated drug in a high-risk paediatric population, even with the use of concomitant nephrotoxic agents. An aggressive fluid regimen may not be necessary, with particular care required in patients with significant pulmonary disease in whom the fluid regimen may precipitate respiratory compromise.

Description: The phosphoinositide 3-kinase (PI3K)-AKT pathway is a relevant signal transduction axis which regulates survival, growth and proliferation of hematopoietic cells through a variety of downstream targets. Constitutive activation of the PI3K-AKT pathway is observed in up to 70% of acute myelogenous leukemia (AML), primarily due to activating mutations in receptor tyrosine kinases (e.g. c-KIT) or RAS and is also present in T-cell-non-Hodgkins-lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Hodgkins Lymphoma and myelodysplastic syndrome (MDS). We analyzed the effect of activating point mutations in the catalytic subunit p110alpha of class IA PI3K (PIK3CA) in hematopoietic cells. We transfected early hematopoietic, IL-3 dependent cells (Ba/F3 cell line) with point mutations in the helical (exon 9, E542K, E545A) and kinase domain (exon 20, H1047R) of the p110alpha catalytic subunit and determined the effect of PI3K-AKT pathway activation on cellular proliferation, survival and leukemogenic expansion in vitro and in vivo. We demonstrate that the p110alpha point mutations constitutively activated the PI3K-AKT pathway and resulted in factor-independent growth of Ba/F3 cells. Proliferation and survival of the cells were inhibited in a time- and dose-dependent manner using inhibitors of either PI3K or AKT. The mammalian target of rapamycin (mTOR) was demonstrated to be important for mitogenic, but not anti-apoptotic signaling of mutant PIK3CA. In a syngenic mouse model, hematopoietic cells expressing mutant p110alpha induced a leukemia-like disease. FACS analysis demonstrated a median chimerism of 68% in the bone marrow and 35% in peripheral blood of diseased mice, which were characterized by anemia, leukemic infiltration of hematopoietic organs, and 90% mortality within 5 weeks. No substantial differences were observed between E542K, E545A and H1047R. Mice, which were transplanted with an activating c-KIT point mutation (D814V), showed a significantly reduced survival and 100% died within 9 days. In conclusion our data show, that PIK3CA point mutations, by activating the PI3K-AKT pathway, confer factor-independence to hematopoietic cells in vitro and induce leukemogenic activity in vivo. As mutant c-KIT (D814V) showed enhanced leukemogenic potential, signaling through c-KIT may involve additional downstream pathways which cooperate with activated PI3K in leukemia progression. Our model is useful for the differential investigation of the leukemogenic relevance of the PI3K-AKT pathway and pharmacological inhibitor studies.

Description: Purpose: Signal transduction pathways, such as the PI3K/AKT cascade, are frequently activated in acute myeloid leukemia and stimulate the proliferation and survival of leukemic cells. Mutations in upstream genes such as Class III receptor tyrosine kinases are frequent but not exclusive causes of that activation. An important downstream target of PI3K/AKT is the key regulator GSK-3β. It controls anti-apoptotic genes such as NF-kappaB and Cyclin D1, is involved in wnt-pathway activation and drug resistance of leukemic cells. Deregulated signaling by GSK-3β occurs through inhibition by AKT-mediated phosphorylation. We have observed that constitutive phosphorylation of GSK-3β occurred in hematopoietic cells with pro-leukemogenic PI3K-mutations. We wanted to evaluate the relevance of GSK-3β inactivation in the transformation process of hematopoietic cells. Methods and Results: We used an in vitro factor-independent growth assay, GSK-3b inhibitors (Lithium, BIO) and established a second hit model using retroviral gene transfer of the weak oncogene Bcl XL. Signaling cascades were analysed by western blot. We demonstrate that inactivation of GSK-3b alone was not sufficient to induce factor-independent growth in IL-3 dependent early hematopoietic cells (Ba/F3). Induction of apoptosis upon growth factor withdrawal was reduced, but not prevented, in the presence of GSK-3b inhibitors, leading to a delayed Caspase 3 activation, PARP cleavage and DNA fragmentation. Overexpression of Bcl-XL also did not result in a prevention of apoptosis. GSK-3b inhibition in synergy with Bcl-XL overexpression resulted in the establishement of several growth factor-independent cell lines, which were characterized by the activation of multiple signaling cascades including AKT, MAPK, STAT5, but not STAT3. Also Cyclin D1 was overexpressed in contrast to other cyclines (D2, D3) which are no substrates of GSK-3b. Conclusions: Our data show that GSK-3β is part of the apoptotic response to growth factor withdrawal and suggest that GSK-3β is causaly involved in the transformation process of hematopoietic cells and seems to have an synergistic role in addition to other pro-leukemic mutations.

Description: The Raf/MEK/extracellular signal-regulated kinase (ERK) kinase cascade and the Ras-PI3-K-Akt pathways are intricately regulated and evolutionarily conserved pathways that have been implicated in specialized cellular functions including proliferation, differentiation, survival and degranulation. Recent data suggest that the strength and duration of these signals is maintained by extracellular growth factors and integrin stimuli as well as intracellular protein scaffolds. In the present study, we investigated the role of Kinase suppressor of Ras (KSR), a scaffold that appears to regulate both Ras-Erk and Ras-PI3-K activity in influencing mast cell function. In vivo, KSR−/− mice have a 2–3 fold reduction of resident mast cells in multiple organs including the peritoneum and the skin as evaluated by scoring Alcian blue positive cells. To evaluate the mechanistic underpinnings of these in vivo observations, bone marrow derived mast cells (BMMCs) were generated and proliferation, survival, degranulation, and migration was examined. A 3–4 fold reduction in kit-ligand mediated proliferation as measured by [3H]thymidine incorporation was observed in KSR−/− BMMCs as compared to WT BMMCs. In addition, a 50% increase in apoptosis was observed in KSR−/− mast cells as compared to that in WT cells as measured by flow cytometeric analysis using Annexin/PI staining. Given that Erk and Akt are established molecular targets control proliferation and survival, respectively; we next performed western blots to evaluate if the changes in biological activity was associated with these signaling pathways. Importantly, a reduction in phosphorylation of ERK and phosphorylation of AKT was observed in the KSR −/− BMMCs as compared to that in WT BMMCs. Given the role of PI3-K signals in mediating cytoskeletal organization in mast cells, we next tested whether the reduction in PI3-K signals was associated with a reduction in degranulation and migration. Following stimulation with kit-ligand and cross-linking of the IgE receptor, KSR−/− mast cells were found to have a 30–50% decrease in b-hexosaminidase release. Moreover, KSR−/− mast cells have up to a 5 fold reduction in migration to kit-ligand as measured over a range of kit-ligand concentrations. Collectively, the in vivo and in vitro studies suggest that KSR is an important regulatory kinase that may be a viable molecular target for modulating inflammatory mast cell functions.