ALBERT

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: 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 mammalian target of rapamycin (mToR) plays a crucial role in cell growth due to its role as nutrient dependent regulator of important cytokine signalling pathways. In multiple myeloma (MM) mToR is involved in the AKT pathway which can be activated by the loss of the tumor suppressor PTEN or stimulation with growth and survival factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). mToR can be blocked by rapamycin or everolimus. When various human plasmacytoma lines were tested, rapamycin and everolimus induced a dose-dependent growth inhibition. The addition of bone marrow stromal cells, IL-6 or IGF-1 was unable to prevent the inhibitory activity. Growth inhibition was mediated not only by G1 cell cycle arrest but also by an induction of apoptosis as measured by propidium iodide staining. Further analysis was performed to elucidate the mechanism of apoptosis in the particular sensitive plasma cell line INA-6. mToR inhibition induced exclusively cleavage of pro-caspase 8 (extrinsic pathway) but not of pro-caspase 9 (intrinsic pathway). To confirm this observation, INA-6 cells were transfected with BCL-XL, an inhibitor of the intrinsic pathway, and treated with rapamycin. The transfectants showed similar sensitivity to rapamycin as the paternal cell line. Rapamycin was also tested in the INA-6 SCID mouse xenograft model. Rapamycin was given orally for two weeks starting 24 h after tumor inoculation. A significant survival benefit of the rapamycin treated group compared to control animals (p= 0.0004) was observed. Six out of 14 treated mice did not develop plasmacytomas during the observation period of 149 days. In addition, even short term treatment of plasmacytoma bearing mice led to a significant shrinkage of the tumor mass. Histological examination revealed an increase of apoptotic plasma cells according to morphology after 72 h of treatment compared to controls. Induction of apoptosis in tumor cells was confirmed by immunohistological staining using an antibody specific for the human cleaved form of poly (ADP-ribose) polymerase. Since drugs inhibiting mToR are inducing apoptosis in human malignant plasma cells not only in vitro but also in vivo, the inhibition of mToR may represent an attractive new concept for multiple myeloma therapy.

Description: Treatment of chronic myeloid leukemia (CML) with the tyrosine kinase inhibitor imatinib represents a successful application of a molecularly targeted therapy. A rapid hematologic and cytogenetic response can be induced for the majority of patients even in advanced disease. However, the time course of disappearance of leukemia cells, characterized by the expression of the BCR-ABL fusion protein, varies between patients, and a complete eradication of the malignant cells is a rare event. The reasons for the heterogeneous response and the persistence of the malignant clone in many patients are currently not known. We propose a mathematical model which consistently explains short and long-term dynamics of BCR-ABL transcript levels in populations of CML patients under imatinib monotherapy. The model is based on the concept that normal and malignant cell clones compete for growth environments in which they behave slightly differently with regard to homing and cell cycle activation/deactivation. This concept has been successfully applied for understanding time-dependent chimerism in mice [Roeder et al.: Blood 105(2):609]. Applying the model to data sets from two independent cohorts of imatinib treated CML patients, we demonstrate the potential of our model to quantitatively describe the typical biphasic decline in BCR-ABL transcript levels during the first year of treatment. Besides the median transcript dynamics in the patient population the model is able to represent the heterogeneity in individual transcript time courses. Qualitative differences in the imatinib response are explained by small quantitative differences in the drug effects regarding proliferation inhibition and/or induction of apoptosis for BCR-ABL positive cells. As demonstrated by comparison with five years follow-up data of 69 unselected newly diagnosed CML patients recruited into the IRIS trial in Germany [Mueller et al.: Leukemia 17(12):2392] the model also correctly describes long-term BCR-ABL dynamics. The observed median BCR-ABL transcript levels, including the vanishing decline after year four of treatment, can quantitatively be explained by a decreasing treatment efficiency in a subset of patients, potentially caused by imatinib-resistant clones. Sensitivity analyses show that moderate functional differences of the resistance mutations can lead to remarkable differences in long-term treatment efficiency. On the other hand, in patients not developing resistance mutations our model predicts the general chance of an eradication of the malignant clone in the long run. This is supported by data in a patient subgroup showing a continued decline of BCR-ABL transcript levels over five years of treatment. Beyond the consistent description of the clinically observed BCR-ABL dynamics we provide testable predictions for effects of different combination treatments. Based on the explanation of CML as a clonal competition of malignant and normal hematopoietic stem cells, our model particularly predicts that the therapeutic benefit of imatinib can be augmented by a combination with proliferation stimulating treatment strategies. In addition the model permits to describe the heterogeneity of the effect of resistance mutations with respect to specific treatment strategies. In summary, our model describes CML dynamics under imatinib therapy with potential implications for the design of future treatment strategies.