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TGCTs are highly apoptosis-prone cancers which can be cured by Cisplatin-based chemotherapy. We have previously shown that p53 plays a major role in this phenotype. Furthermore, we demonstrated that sensitivity of these tumors is dictated by p53 accumulation in general and is not restricted to Cisplatin. Hypersensitivity seems to be an intrinsic feature of pluripotent TGCTs, since both differentiation and loss of the pluripotency factor Oct-4 induce resistance. We sought to investigate the link between the Oct-4-mediated pluripotent context and the proapoptotic p53 response in these tumors. For this, we tested if loss of Oct-4 also reduces p53-mediated apoptosis upon non-genotoxic p53 activation. Indeed, RNAi-mediated silencing of Oct-4 significantly reduced sensitivity to the p53 activator Nutlin-3. Hence, we investigated a possible differential activation of p53 in the context of Oct-4-positive TGCT cells. For this, a qPCR system that allows simultaneous acquisition of 46 bona fide p53 target genes was used to detect p53’s transactivation capacity in NTERA-2D1 and 2102EP cells upon Cisplatin treatment in the presence and absence of Oct-4. In spite of p53’s central role in Cisplatin-induced apoptosis, no significant alterations in transactivation of p53 target genes were observed upon Cisplatin treatment in both cell lines. We also analyzed the cellular distribution of p53 upon Cisplatin treatment and found that p53 was not only accumulated in the nucleus but was increased to a similar extent in the cytoplasm. Importantly, RNAi-mediated silencing of Oct-4 did neither influence accumulation of p53 in the nucleus nor in the cytoplasm. These data indicate that Oct-4 does not directly modulate p53 activity but provides a cellular context that augments the proapoptotic activity of p53. We have previously proposed that Oct-4-dependent high constitutive Noxa protein levels account for the low apoptotic threshold in TGCTs. In contrast to Cisplatin or Nutlin-3, treatment with MG132 resulted in apoptosis in Oct-4-depleted NTERA-2D1 and 2102EP cells. This was due to simultaneous accumulation of p53 and Noxa, as RNAi-mediated silencing of either protein rescued the cells from death. It is of importance that p53 knockdown did not compromise Noxa accumulation upon MG132 treatment, indicating that both p53 activation and the Noxa-mediated apoptosis-prone context are required for high sensitivity. In conclusion, our data demonstrate that in spite of its impact on hypersensitivity, p53 activity is not altered in Oct-4-positive TGCT cells when compared to relatively resistant Oct-4-depleted cells. Although a robust p53 response is essential for hypersensitivity of TGCTs, its predominantly proapoptotic characteristic requires the apoptosis-prone cellular context of pluripotent Oct-4-positive TGCT cells, which includes high Noxa protein levels. Citation Format: Matthias Gutekunst, Thomas Mueller, Andrea Weilbacher, Michael A. Dengler, Moshe Oren, Walter E. Aulitzky, Heiko van der Kuip. Testicular germ cell tumors are hypersensitive to p53 activation based on their Oct-4/Noxa-mediated cellular context rather than on differential p53 activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1721. doi:10.1158/1538-7445.AM2013-1721

MCL is an aggressive type of non-Hodgkin lymphoma with poor prognosis and short survival. The genetic hallmark of MCL is the t(11;14) translocation leading to an aberrant expression of the oncogene cyclin D1. Several other deregulated pathways also contribute to the pathogenesis of MCL, including the DNA damage response-, B-cell receptor- and PI3K/mTOR pathway. Deregulated oncogenic pathways not only induce proliferation but also frequently lead to a constitutive stress signal. A common response to cellular stress is the transcriptional up-regulation of pro-apoptotic genes. By analyzing mRNA expression of these genes in MCL cells we found high levels of the pro-apoptotic Bcl-2 family member NOXA (PMAIP1). NOXA transcript was significantly higher in MCL cell lines as well as in samples from MCL patients when compared to other cancer cell lines and PBMCs. To analyze if these high transcript levels depend on activation of oncogenic pathways, we treated the MCL cells with a panel of inhibitors of different signaling pathways and found that the inhibition of the PI3K/mTOR pathway led to a significant reduction of NOXA mRNA levels. These results indicate that this signaling axis is not only acting pro-survival but also mediates up-regulation of NOXA mRNA. In contrast to the high transcript levels, NOXA protein is low in MCL cells. We found that NOXA protein is unstable with half-life times of 15-30 min. Inhibitors of the ubiquitin proteasome system (UPS) such as bortezomib, MG-132 and the cullin-ubiquitin ligase inhibitor MLN4924 stabilized NOXA and led to a strong accumulation of the protein. This was accompanied by a rapid induction of cell death. Interestingly, similar effects could be observed using the fatty acid synthase inhibitor orlistat indicating that fatty acid metabolism is also involved in the UPS. Reducing the high NOXA mRNA levels by treating the cells with the PI3K/mTOR dual-inhibitor Bez235 or using RNA interference prior to treatment with UPS inhibitors significantly reduced cell death and NOXA protein accumulation. These results indicate that the high NOXA mRNA levels are essential for the response of MCL cells to proteasomal inhibitors. In summary, our results show that MCL have a constitutive signal mediated by the PI3K/mTOR pathway resulting in a high expression of NOXA mRNA. The cells survive by rapidly degrading the NOXA protein. Therefore, an effective strategy to kill MCL cells is to target the high NOXA protein turnover in these cells by inhibiting the proteasome ubiquitin system. This mechanism apparently underlies the in vitro activity of bortezomib which is already used in the clinic for treatment of patients with mantle cell lymphoma. Moreover, also other inhibitors such as MLN4924 or increasing the metabolic stress by orlistat might be promising to selectively kill MCL cells. Citation Format: Michael A. Dengler, Andrea Weilbacher, Matthias Gutekunst, Annette M. Staiger, Heike Horn, German Ott, Heiko van der Kuip, Walter E. Aulitzky. High NOXA (PMAIP1) transcript levels combined with a short-lived NOXA protein primes mantle cell lymphoma (MCL) cells for death by inhibition of the ubiquitin proteasome system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2013-1717

Induction of apoptosis by growth factor deprivation or gamma-irradiation-induced DNA damage was directly studied in proliferating primary haemopoietic cells derived from CD34-positive cells of 13 CML patients and 12 normal controls. CD34-positive cells were cultured in the presence of appropriate concentrations of SCF and G-CSF for 5–7 d. After gamma irradiation with 500 rad or growth factor deprivation, the fraction of apoptotic cells was assessed by two independent methods applying either measurement of cells incorporating FITC-labelled dUTP by terminal transferase or assessment of the fraction of cells with a less than 2N DNA content in flow cytometry. Proliferating CML cells were not resistant to the induction of apoptosis either after gamma irradiation or subsequent to growth factor deprivation. A similar fraction of normal and CML cells underwent apoptosis 48 h after withdrawal of growth factors. CML cells displayed an increased susceptibility to induction of apoptosis after DNA damage. A significantly higher proportion of apoptotic cells were detected in samples derived from CML patients after irradiation with 0.5 Gy. These results, in conjunction with conflicting observations by other investigators, on the induction of apoptosis by gamma irradiation in various bcr-abl positive cells, suggest that bcr-abl-dependent effects on apoptosis strongly depend on the cells used. Our observations in CML cells derived exclusively from newly diagnosed CML patients demonstrate that bcr-abl expression per se is not sufficient to induce resistance to apoptosis.

Myeloproliferative disorders (MPD) are characterized by several common clinical and biological features, although at the molecular level, each disease entity exhibits distinct abnormalities. IFN-alpha exerts beneficial therapeutic effects in chronic myelogenous leukemia, polycythemia vera and essential thrombocythemia, resulting in control of hematopoietic hyperplasia and, in a minority of patients, in induction of cytogenetic remission. The mechanism of action of IFN-alpha in MPD is poorly defined. Recently published in vitro findings suggest that IFN-alpha interacts with the regulation of hematopoiesis by multiple ways. Its antiproliferative activity is well known for more than a decade, however, substantial growth inhibition is achieved only at relatively high concentrations. Defective adhesion of hematopoietic progenitor cells in CML to bone marrow stromal cells is corrected by IFN-alpha, which might expose CML progenitors to inhibitory cytokines produced by the bone marrow microenvironment. Recent work from our group demonstrated, that IFN-alpha potently interacts with the production of hematopoietic cytokines in bone marrow stromal cells. Expression of stimulatory cytokines, such as GM-CSF, G-CSF, IL-1 and IL-11 is inhibited by IFN-ct, whereas the production of negative regulators, such as IL-1RA and MIP-1 alpha, is stimulated. The combined action of IFN-alpha on paracrine expression of cytokines suggests an indirect antihematopoietic effect, which might contribute to its clinical activity in MPD.

Abstract 1481 Introduction Acute erythroleukemia (AEL) represents a rare type of acute myeloid leukemia accounting for less than 5% of all cases. So far, according to WHO classification this AML entity is thought to have a poor prognosis in itself. Design 3267 patients with newly diagnosed AML were treated according to the protocols of the AML96, AML2003 or AML60+ studies of the Study Alliance Leukemia (SAL). 116 of these patients had acute erythroleukemia (AEL). The median age both for patients with AEL and non-AEL was 57 years. We assessed the influence of relevant clinical and demographic parameters, FLT3-ITD, NPM1 status and cytogenetics on complete remission rates (CR), overall survival (OS) and event free survival (EFS) separately in AEL and non-AEL patients. Results Compared to non-AEL AML, significantly more AEL were due to secondary causes than non-AEL AML (31% versus 20.4%; p=0.024). NPM1 mutations were found in 11.1% (out of 99) of the patients with AEL and in 32.8% (out of 2693) of the patients with non-AEL AML (p 0.8 were found less frequently in AEL (FLT3-ITD mutation in 4.9% vs 23.3%; p=0.001; FLT3-ITD ratio >0.8 in 0% vs. 33.4%; p The cytogenetic aberrations +8, −7 and complex aberrant karyotype (>/= 3 independent aberrations) were found more often in the AEL cohort (14.8%, 10.3% and 26.7%) than in the non-AEL AML cohort (8.9%, 5% and 13.2%) (p=0.036, p= 0.01 and p Despite these differences, no significant differences in CR rates, OS and EFS were found between both groups. This finding was confirmed in a multivariate analysis including cytogenetics, molecular markers and clinical parameters (LDH, WBC, blast count, platelet count and ECOG). According to the analysis, ALE morphology was not an independent prognostic factor for OS and EFS. With the AEL group, patients with monosomy 7 (n=12) had a higher median blast count (NEC) of 62% and shorter median OS with 5.7 months compared to patients with AEL and no monosomy 7 (n=104) with a median blast count (NEC) of 43% (p=0.013) and an median OS with 15.7 months (p=0.016). A complex aberrant karyotype was found more often in patients with secondary AEL than in patients with de novo AEL (p=0.037). Significant differences were also seen in patients with AEL and complex aberrant karyotype compared to AEL without complex aberrant karyotype with respect to CR rates (54.8% versus 77.6%; p=0.016), OS (6.2 versus 17.4 months; p In patients with AEL and abn17p/-17 (n=15), lower median platelet counts (31 versus 48 ×106/μl; p=0.017), a worse OS (6.4 vs 15.7 months; p=0.011) and EFS were observed (1.7 vs 5.7 months; p=0.046). Conclusions According to our data, the characteristic morphological features of acute erythroleukemia do not confer an unfavorable prognosis in itself. Furthermore, we can confirm the relevant influence of established prognostic factors such as cytogenetics and disease status within the AEL subgroup. Disclosures: Off Label Use: sorafenib for the treatment of acute myeloid leukemia.

Abstract 144 Background: Sorafenib is a multi-kinase inhibitor with activity against several oncogenic kinases, which may play a role in the pathogenesis of acute myeloid leukemia (AML). In-vitro data and results from non-randomized clinical trials suggest that sorafenib might be an effective drug for the treatment of AML. So far, no randomized-controlled data are available for treatment of newly diagnosed AML patients up to the age of 60 years. We present the first results from the randomized placebo-controlled SORAML trial of the Study Alliance Leukemia (SAL). Patients and Methods: Between March 2009 and October 2011, 276 patients from 25 centers were enrolled in the SORAML trial (NCT00893373). The main eligibility criteria were: newly diagnosed AML, age from 18 to 60 years and suitability for intensive therapy. The treatment plan for all patients included two cycles of induction with DA (daunorubicin 60 mg/m2 days 3–5 plus cytarabine 100 mg/m2 cont. inf. days 1–7), followed by three cycles of high-dose cytarabine consolidation (3 g/m2 b.i.d. days 1, 3, 5). Patients without response after DA I received second induction with HAM (cytarabine 3 g/m2 b.i.d. days 1–3 plus mitoxantrone 10 mg/m2 days 3–5). Allogeneic stem cell transplantation was scheduled for all intermediate-risk patients in first complete remission with a family donor and for all high-risk patients with a matched donor. At study inclusion, patients were randomized to receive either sorafenib (800 mg/day) or placebo as add-on to standard treatment. Block randomization at a ratio of 1:1 was performed within cytogenetic and molecular risk strata, allocation was concealed and treatment was double blinded. Study medication was given on days 10–19 of DA I+II or HAM, from day 8 of each consolidation until 3 days before the start of the next consolidation and as maintenance for 12 months after the end of consolidation. The primary endpoint of the trial is event-free survival (EFS) with an event being defined as either failure to achieve a complete remission (CR) after induction, relapse or death. Secondary endpoints were overall survival (OS), CR rate and incidence of adverse events (AE). We present the results of the planned interim analysis (intent to treat) after the occurrence of 50% of EFS events. The O'Brien/Fleming adjusted significance level was set at p=0.0052. Results: Out of 276 randomized patients, 264 were evaluable for EFS, 132 in each arm. Demographic and disease characteristics were equally distributed between the two arms; the FLT3-ITD incidence was 16%. The median cumulative dose of administered study medication was equal in both arms. The CR rates were 56% versus 60% in the placebo versus sorafenib arm (p=0.622). By the time of analysis, a total number of 100 events had occurred. After a median observation time of 18 months, the median EFS was 12.2 months in the placebo arm and was not reached in the sorafenib arm, corresponding to a 1-year EFS of 50% versus 64% (p=0.023). The median OS had not been reached in both arms, the 2-year OS was 66% versus 72% in placebo and sorafenib arms, respectively (p=0.367). The most common reported AEs CTC Grade ≥3 were infectious complications including fever and pneumonia, followed by bleeding events, cardiac and hepatic toxicity, hypertension, skin toxicity and headache. The risk for hepatic toxicity (relative risk 6.2, p=0.025) and bleeding events (relative risk 3.6, p=0.016) was significantly higher in the sorafenib arm while the incidence of all other AEs showed no significant differences. Conclusions: In younger AML patients, the addition of sorafenib to standard chemotherapy is feasible but associated with a higher risk of liver toxicity and bleeding events. Sorafenib treatment resulted in a marked EFS prolongation; this difference is not significant according to the adjusted significance level of this interim analysis. Results from the final analysis including post-hoc exploration of molecularly defined subgroups are necessary for drawing final conclusions on the efficacy of sorafenib. Disclosures: Off Label Use: sorafenib for the treatment of acute myeloid leukemia. Serve:Bayer: Research Funding. Ehninger:Bayer: Research Funding.

Abstract 229 Background: The optimal timing of hematopoietic cell transplantation (HCT) in AML and its use in risk groups defined by genetic markers or early blast clearance is still under debate. We addressed this question in the AML 2003 study, a large multicenter, randomized, open-label study within the German SAL group. All patients received one cycle of induction therapy (IT) with a 3+7 regimen combining daunorubicin and continuous infusion cytarabine. Upfront cytogenetic and molecular characterization, HLA typing, related donor search and a preliminary search for an unrelated donor were performed for all patients. The subsequent transplant strategy was tailored to the biological risk and donor availability. Patients and Methods: Patients aged 18–60 years were randomly assigned upfront 1:1 to either of two transplant strategies: In the control arm allogeneic HCT was scheduled in first complete remission for patients with intermediate risk cytogenetics and an HLA-identical sibling and for patients with a complex karyotype (CK) and a related or unrelated HLA-compatible unrelated donor. In the experimental arm the indication for allogeneic HCT was extended to patients with an FLT3-ITD allelic ratio >0.8 (mutant/wild type), >10% marrow blasts on day 15 after IT1 and patients with adverse karyotypes, including: −7, −5, del(5q), inv(3q), t(3;3), t(6;9), t(6;11), t(11;19)(q23;p13.1). Furthermore, allogeneic HCT was scheduled at an early time-point, i.e. in aplasia after the first or the second cycle of IT in the experimental arm. Patients without an HLA-compatible donor assigned to the transplant strategy were scheduled for high-dose busulfan and cyclophosphamide followed by autologous SCT. Here, we present the final analysis of this study according to the intent-to-treat principle. Results: Between December, 1, 2003 and November, 26, 2009 1179 patients were randomized between the experimental (N=598) and the control intervention (N=581). The median age was 48 years (range, 18 to 60 years). The distribution of age, ECOG performance status, type of AML, cytogenetic risk groups, NPM1- and FLT3-ITD-mutations, LDH, white blood cell and platelet count was not statistically different between the two treatment arms. However, more males and patients with higher marrow blast counts at diagnosis were randomized to the experimental treatment arm. The median observation time for all patients was 52 months. The hazard ratio of the treatment effect (experimental versus control) was 0.92 (95% CI, 0.75 to 1.14; p=0.45) for primary endpoint overall survival and 0.85 (95% CI, 0.71 to 1.02; p=0.08) for the secondary endpoint event-free survival. Patients in both arms had an excellent long-term overall survival of 50% (95% CI, 46% to 54%) at 5 years after enrollment in the experimental arm and 47% (95% CI, 42% to 51%) in the control arm (see Figure). Across all risk groups the rate of early allogeneic HCT performed per protocol was 22% in the experimental arm and 8% in the control arm. Among patients with high risk cytogenetics this rate was 48% and 17%, respectively. These numbers point at the difficulty to deliver allogeneic HCT within two months after diagnosis of AML in the context of a multicenter trial. However, since HLA-typing and preliminary donor search was done for all patients and allogeneic HCT was standard for relapsed or refractory AML in both arms, the rates of allogeneic HCT as first post remission therapy or after induction failure/relapse were substantially higher than in previous studies and almost equal in the experimental and control arm (63% versus 56%). Conclusions: Although a survival benefit by early allogeneic HCT could not be demonstrated in this large randomized study in newly diagnosed AML, the study treatment resulted in excellent overall survival rates in both arms. Relatively small differences between the per-protocol transplantation rates and crossover effects may partially explain why a benefit of early allogeneic HCT could not be demonstrated. Early awareness of donor availability and the option of allogeneic HCT in patients with primary induction failure or AML relapse resulted in a high overall rate of allogeneic transplantation. This may have contributed to the excellent overall survival in both arms. Disclosures: No relevant conflicts of interest to declare.

Embryonal carcinoma (EC) cells are the pluripotent precursors of differentiated structures of testicular germ cell tumors (TGCT) characterized by expression of the embryonal transcription factor OCT-3/4. Loss of OCT-3/4 during differentiation abrogates the extraordinary high sensitivity to cisplatin in these cells. We previously observed a constitutively high expression level of NOXA and a major role of this pro-apoptotic BH3-only protein for cisplatin hypersensitivity in the EC cell lines NTERA-2D1 and 2102EP. We now investigated if these high NOXA protein levels may be directly linked to the OCT-3/4 status in TGCT cells. Therefore, we analyzed NOXA protein expression in an extended TGCT cell line panel of 5 OCT-3/4 negative and 5 OCT-3/4 positive cell lines. Importantly, NOXA protein was highly correlated with OCT-3/4 levels and also with cisplatin sensitivity. These data indicate that constitutively high NOXA levels might be responsible for the low threshold for cisplatin-induced apoptosis in OCT-3/4 positive pluripotent cells. A direct link between OCT-3/4 and NOXA could also be demonstrated by RNAi mediated silencing experiments performed in NTERA-2D1 and 2102EP cells. Silencing of OCT-3/4 resulted in a significant downregulation of NOXA transcript and an almost complete loss of NOXA protein accompanied by a loss of cisplatin sensitivity. This was observed in both differentiation competent NTERA-2D1 cells as well as in differentiation incompetent 2102EP cells pointing to a direct OCT-3/4 dependent NOXA regulation rather than a bystander effect of differentiation upon loss of OCT-3/4. We further validated our in vitro data by comparing OCT-3/4 and NOXA expression levels in patient samples derived from ECs with other non-seminomatous GCTs, seminomas, and tumor entities from lung, breast, and ovary. In agreement with our in vitro observations, OCT-3/4 and NOXA were found to be highly expressed selectively in seminomas and ECs. This could also be confirmed on protein level in primary tissue samples derived from 5 ECs and 8 seminomas by western blot analysis. In conclusion, our data for the first time demonstrate a close correlation between OCT-3/4 and NOXA protein levels and strengthen the previously hypothesized role of constitutively high NOXA levels for the exquisite sensitivity of TGCTs to cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2002. doi:1538-7445.AM2012-2002

Cyclin D1 over-expression has been linked to the development and progression of several types of cancer including mantle cell lymphoma (MCL), an aggressive type of B-cell lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation. Recent studies have shown that cyclin D1 is a multifunctional protein not only regulating the cell cycle but also affecting other cellular processes including DNA repair, apoptosis, as well as glucose, fatty acid, and lipid metabolism. In this study, we investigated the effects of the fatty acid synthase and lipase inhibitor Orlistat on cyclin D1 over-expressing MCL cell lines. In contrast to non-malignant peripheral blood lymphocytes and normal fibroblasts all MCL cell lines examined were sensitive to Orlistat. This enhanced sensitivity was dependent on cyclin D1 overexpression since siRNA mediated cyclin D1 knockdown almost completely rescued MCL cells from Orlistat induced apoptosis. Cell death upon Orlistat treatment was accompanied by loss of mitochondrial membrane potential and dependent on caspase activation since pre-incubation of the cells with the pan-caspase inhibitor zVAD-fmk completely blocked induction of apoptosis. We therefore investigated potential Orlistat-mediated changes in the expression levels of the pro- and anti-apoptotic Bcl-2 family proteins and found NOXA to be strongly induced whereas the expression of other Bcl-2 proteins did not change significantly. RNAi mediated knockdown of NOXA inhibited induction of cell death demonstrating the predominant role of this protein for the proapoptotic effect of Orlistat in MCL cells. Interestingly, silencing of cyclin D1 reduced the expression of NOXA upon Orlistat treatment further indicating a connection between cyclin D1, fatty acid metabolism, and the induction of NOXA. Since inhibition of fatty acid metabolism by Orlistat was found to disturb the balance between pro- and anti-apoptotic proteins in MCL cells we analyzed possible combinatory effects with Bcl-2 family modulators. Co-treatment of the cells with Orlistat and the BH3 mimetic ABT737 led to a rapid induction of cell death and an almost complete loss of cell viability in cyclin D1 over-expressing cells. A similar synergistic effect could be observed by combining Orlistat and 2-deoxy-D-glucose, a glycolysis inhibitor known to reduce MCL1 protein. These combinatory effects were selective for MCL cells as the same treatments had only minor or no effects on cell viability of primary PBMCs and fibroblasts from healthy donors. In summary, our results for the first time indicate that fatty acid metabolism may be an attractive target for therapy of cyclin D1 over-expressing MCL cells. Furthermore, these observations may contribute to the development of rational strategies combining fatty acid metabolism inhibitors with Bcl-2 family modulators for treatment of MCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4675. doi:1538-7445.AM2012-4675

One of the major molecular players in cellular stress responses is the p53 tumor suppressor protein. The vast majority of the research of p53 activities upon DNA damage has focused on its cell autonomous functions, i.e. its ability to induce cell cycle arrest, DNA repair, apoptosis, autophagy, or senescence in a homogenous cell population. However, data on the impact of tumor cells on p53 activation in their neighboring cancer associated fibroblasts (CAFs) and also vice versa are still limited. We investigated p53 status and responses to the p53-activating drug cisplatin both in tumor cells and in their adjacent CAFs in intact tumor tissues derived from 28 individual lung cancer patients. For this, 200µm thick patient derived tissue slices were cultivated ex vivo in presence or absence of cisplatin for 72 hours. By comparing cultivated control slices with the corresponding tumor tissue material immediately fixed after surgery (pathological routine material) we could show that morphology and p53 staining pattern were highly preserved during cultivation. TP53 mutations were detected in 16 of the 28 cases (57.1%). Independent of their p53 mutation status, the tumor samples could be divided into 3 categories according to their p53 immunostaining characteristics in the tumor cell compartment: (I) tumor cells with constitutively low p53 protein which was accumulated upon cisplatin (12 cases, 7 with mutated p53); (II) tumor cells with constitutively high p53 and no further accumulation upon cisplatin (8 cases, 7 with mutated p53); (III) tumor cells with constitutively no detectable p53 and no accumulation upon cisplatin (8 cases, 2 with mutated p53). Importantly, regardless of the p53 mutation status of the tumor cell compartment we detected a CAF specific p53 accumulation and induction of the p53 target p21 selectively in category I tumors (i.e. tumor tissue samples characterized by p53 accumulation in the tumor cell compartment). In sharp contrast, tumor tissues with no detectable p53 accumulation in tumor cells (categories II and III) showed no p53 accumulation or p21 induction in their adjacent fibroblast compartment. Furthermore, a CAF specific induction of TUNEL positive cells in cisplatin treated samples (7 of 28 cases) was selectively observed in tumor tissues characterized by a parallel induction of tumor cell death. These data for the first time show a close correlation of p53 response in tumor cells and adjacent fibroblasts in intact lung tumor tissues from patient derived material indicating that tumor cells control molecular and functional responses of their neighborhood fibroblasts upon DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2869. doi:1538-7445.AM2012-2869

The efficacy and immunomodulatory effects of low-dose gamma-interferon (gamma IFN) were investigated in an unselected population of patients with metastasising renal cell carcinoma. 36 patients suffering from metastasising renal cell carcinoma with a performance status exceeding Karnofsky index of 50 were entered into the open phase I/II trial. The majority of the patients recruited displayed a large tumour burden, and 28 patients (78%) had metastases involving two to six organ sites. Treatment was started with a 2-week cycle of either daily or weekly subcutaneous administration of either 100, 200 or 400 micrograms gamma IFN. After a therapy-free interval of 2 weeks treatment was switched to the alternate mode of administration. Subsequently, treatment was continued with the same dose applied once a week for a minimum of 3 months. Serum levels of neopterin and beta-2-microglobulin, as well as flow cytometric analyses of peripheral blood mononuclear cells, were used for the assessment of biological response. Minimal antitumour activity was observed in this high-risk patient group and only 1 patient experienced a partial response (PR) lasting 36 + months. Comparison of the patients' characteristics to those of other low-dose gamma IFN trials revealed a highly significant difference in the tumour burden and clinical response. We conclude that patient selection is a decisive parameter for the outcome of treatment with low-dose gamma IFN, and that patients with poor prognostic features and a large tumour burden are not likely to respond to this almost atoxic treatment.

Abstract 1656 Mantle cell lymphoma (MCL) is an aggressive type of B-cell lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation resulting in a constitutive over-expression of cyclin D1. Cyclin D1 is a multifunctional protein not only regulating cell cycle progression but also affecting other cellular functions including glucose and lipid metabolism. In this study, we investigated the effects of the fatty acid synthase (FASN) inhibitors orlistat and C75 on viability of different MCL cell lines including JEKO-1, MINO, Granta-519, JVM-2, and Rec-1. In all cell lines inhibition of FASN alone induced apoptosis. In contrast, normal peripheral blood lymphocytes were resistant to these compounds. This proapoptotic effect was dependent on cyclin D1 as silencing of cyclin D1 partially rescued cells from induction of cell death following FASN inhibition. Inhibition of FASN led to a strong induction of the proapoptotic protein NOXA (PMAIP) in all MCL cell lines investigated independent of the p53 status. Pre-treatment of cells with NOXA siRNA significantly reduced induction of cell death demonstrating the predominant role of this proapoptotic protein for the observed effects. We then analyzed the combined effects of inhibition of FASN with a panel of compounds with potential clinical relevance. Combination of FASN inhibitors with the BH3 mimetic ABT737, the proteasome inhibitor Bortezomib, and the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) led to an almost complete loss of viability. These combinatory effects were selective for MCL cells as the same treatments had almost no effects on cell viability of primary PBMCs or fibroblasts from healthy donors. Bortezomib, 2-DG, and ABT737 enhanced the proapoptotic effect of FASN inhibitors by further disturbing the balance of pro- and antiapoptotic Bcl-2 family proteins: FASN inhibitor mediated induction of NOXA was enhanced by Bortezomib whereas 2-DG significantly reduced the NOXA antagonist Mcl-1. In summary, our results suggest that FASN inhibitors exert significant antitumoral activity especially in combination with Bcl-2 family modulators in different MCL cell lines and may therefore represent an attractive model for the design of new therapeutic approaches for this entity. Disclosures: No relevant conflicts of interest to declare.

Abstract 493 Background: According to retrospective analyses, the presence of a mutated Nucleophosmin-1 gene (NPM1+) in acute myeloid leukemia (AML) is associated with a favorable prognosis, particularly in the absence of an FLT3-ITD mutation (FLT3-ITD-). Therefore, AML with NPM1+/FLT3-ITD- and normal karyotype has been classified as favorable risk in current prognostic classifications. In order to assess the predictive value with regard to allogeneic stem cell transplantation (allo SCT), we compared the clinical course of 309 NPM1+ AML patients eligible for allo SCT in a donor versus no-donor analysis. Patients and Methods: Patients diagnosed with AML, aged 18–60 years, and treated in the AML 2003 trial of the Study Alliance Leukemia (SAL) were analyzed. According to the risk-adapted treatment strategy of the trial, cytogenetically intermediate-risk (IR) and adverse-risk (AR) patients should receive an allo SCT as consolidation treatment if an HLA-identical-sibling donor (IR) or HLA-matched related or unrelated donor (AR) was available. Patients with no available donor received high-dose cytarabine-based consolidation or autologous SCT. In order to avoid selection bias in an as-treated analysis of transplanted versus non-transplanted patients, we compared relapse-free survival (RFS) and overall survival (OS) depending on the availability of a suitable donor in a donor-no-donor analysis. Survival analyses were performed using the Kaplan-Meier method including log-rank tests for significance testing. Cox regression models and Wald tests were used for multivariate analyses on the influence of potential prognostic variables on the outcomes. Results: Of 1182 patients enrolled in the AML 2003 trial between December 2003 and November 2009, 375 were NPM1+ (32%), and 309 patients were eligible for evaluation for the donor vs. no-donor analysis. Their median age was 49 years, 304 patients had an intermediate-risk karyotype according to MRC criteria (98%), and amongst them there were 277 patients with a normal karyotype (90%). The FLT3-ITD mutation was present in 144 patients (37%). A donor was identified for 77 patients (25%), of whom 57 actually received allo SCT as first consolidation (74%). The no-donor group consisted of 232 patients. Age, disease status, cytogenetic profile, and FLT3-ITD incidence were equally distributed between the two groups. Median follow up was 41 months (3.4 years). The 3-year RFS in the donor and no-donor groups was 72% (95%–CI 61%–82%) and 47% (95%–CI 40%–55%), respectively. (p=0.007). The OS in the donor and no-donor groups were 70% (95%–CI 59%–81%) versus 60% (95%–CI 54%–67%) after 3 years, and 70% (95%–CI 59%–81%) versus 53% (95%–CI 45%–61%) after 5 years (p=0.138). In multivariate analyses, the presence of a donor as a prerequisite for allo SCT retained its statistically significant favorable influence on RFS (HR=0.56) even after adjustment for established risk factors such as FLT3-ITD, cytogenetic risk, WBC, LDH, age, and disease status. In patients with normal karyotype and NPM1+/FLT3-ITD- (n=152), the 3-year RFS in the donor and no-donor groups was 87% (95%–CI 77%–97%) and 53% (95%–CI 42%–63%), respectively (p=0.001). Conclusions: According to our results, allo SCT leads to a significantly prolonged RFS in NPM1+ AML patients with a pronounced effect even in NPM1+/FLT3-ITD- patients. The absence of a statistically significant difference in OS is most likely due to the fact that relapsed NPM1+ patients responded well to salvage treatment, particularly to allo SCT from an unrelated donor. Our data suggest that patients with NPM1+ AML who have a well-matched donor benefit from allo SCT in first remission. This hypothesis is currently being tested prospectively in a randomized controlled trial (“ETAL-1”, NCT01246752) evaluating allo SCT in all intermediate-risk AML patients with a well-matched sibling or unrelated donor identified until the achievement of first CR. Disclosures: No relevant conflicts of interest to declare.

Abstract 255 Introduction: Standard treatment of acute myeloid leukemia (AML) comprises one or two cycles of chemotherapy to induce complete remission (CR) followed by postremission treatment in order to prevent relapse of the disease (consolidation therapy). In 2003, we initiated a prospective multicenter randomized trial to investigate the impact of different consolidation strategies on long-term outcome in AML patients ≤ 60 years. Consolidation options comprised upfront allogeneic stem cell transplantation (allo SCT) in aplasia after induction therapy, autologous SCT, and three cycles of standard high-dose-cytarabine-based consolidation. For patients receiving high-dose cytarabine, the main study aim was to evaluate the benefit of adding additional mitoxantrone and amsacrine to cytarabine consolidation. Design: From 2003 to 2009, 1182 patients (median age, 48 years; range 16–60 years) with untreated AML were randomly assigned at diagnosis to different consolidation strategies after classical 7+3 induction. According to the risk-adapted treatment strategy of the trial, cytogenetically or molecular intermediate-risk (IR) and adverse-risk (AR) patients should receive an allo SCT as consolidation treatment if an HLA-identical-sibling donor (IR) or HLA-matched related or unrelated donor (AR) was available. IR and AR patients with no available donor should receive autologous SCT. All favorable risk patients and patients with no available donor were scheduled for high-dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine alone while the other half received high dose cytarabine with the addition of amsacrine and mitoxantrone. Standard chemotherapy consisted of three cycles with high dose cytarabine (2 × 3 g/m2, day 1,3,5) whereas combined consolidation contained two cycles of MAC (cytarabine 2 × 1g/m2, day 1–6, mitoxantrone 10 mg/m2, day 4–6) plus one cycle of MAMAC (cytarabine 2 × 1 g/m2, day 1–5, amsacrine 100 mg/m2, day 1–5). In order to evaluate the effect of the two cytarabine based consolidation strategies, we determined overall survival (OS) and event free survival (EFS) using the method of Kaplan Meyer. Survival distributions were compared using the log rank test. Results: 1182 patients were randomized for further intervention (Arm A+B: n=582, 49.3%; Arm C+D: n=600, 50.7 %). Median follow-up was 41.4 months (95%-CI 39.3–43.6). A total number of 375 patients received allogeneic (n=322) or autologous SCT (n=53) and 807 patients were consolidated with cytarabine. Of these patients, 407 were randomized for cytarabine alone and 400were randomized to receive cytarabine plus mitoxantrone and amsacrine (MAC/MAC/MAMAC). Complete remission rate (CR) after second induction therapy was 59.1% (n=698). Between the four arms, there were no significant differences of the CR rates. Five-year OS of patients receiving high dose cytarabine alone was 47.1% (95%-CI 42.0–52.2%), for patients receiving MAC/MAMAC as consolidation therapy it was 46.8% (95%-CI 42.3–51.3%; p = 0.610). Three-year event free survival (EFS) was also not significant with 30.5% (95%-CI 26.6–34.4%) for patients receiving high dose cytarabine alone and 35.6% (95%-CI 31.7–39.5%; p = 0.059) for patients receiving MAC/MAMAC. Conclusions: According to our data, the addition of mitoxantrone and amsacrine to high dose cytarabine consolidation confers no benefit for treatment outcome in younger AML patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 497 Background: The ratio of the FLT3-ITD mutation to the wt-FLT3 allele has significant prognostic importance in FLT3-ITD mutated acute myeloid leukemia (AML). Patients with a high mutant/wt ratio above 0.78 have significantly shorter overall (OS) and disease-free survival (DFS), whereas survival in patients with ratios below 0.78 did not differ from those without FLT3 aberrations (Thiede C et al, Blood 2002). There is still uncertainty about the role of allogeneic transplantation (allo-SCT) in the treatment of patients with FLT-ITD mutations (FLT3-ITD+). In order to prove the presence of an allogeneic effect, we compared the survival after allo-SCT in first remission in patients with a mutant rate < 0.8 with those ≥ 0.8. For comparison, the results in patient cohorts treated with chemotherapy alone were analyzed. Patients and Methods: Patients diagnosed with AML, aged 18–60 years, and treated in the AML-2003 trial of the SAL Study Alliance Leukemia were analyzed. According to the risk-adapted treatment strategy of the trial, cytogenetically intermediate-risk (IR) and adverse-risk (AR) patients should receive an allo SCT as consolidation treatment if an HLA-matched-sibling donor (IR) or HLA-matched related or unrelated donor (AR) was available. Patients with no available donor received high-dose cytarabine-based consolidation or autologous SCT. Survival analyses were performed using the Kaplan-Meier method including log-rank tests for significance testing. Results: Of 1182 patients enrolled in the AML-2003 trial between December 2003 and November 2009, 257 were FLT3-ITD+ (22%). The ratio of the FLT3-ITD mutation to the wt-FLT3 allele was < 0.8 in 182 patients (low-ratio) and ≥ 0.8 in 75 patients (high-ratio). 47 (26%) of the low-ratio group and 30 (40%) of the high-ratio group received an allogeneic transplantation. In the cohorts having received an allogeneic transplantation, the 3-year DFS in the low- and high-ratio groups was 58% and 50%, respectively (p=0.53). The 3-year OS was 61% and 59%, respectively (p=0.47). In the cohorts having chemotherapy as consolidation, the 3-year DFS in the low- and high-ratio groups was 36% and 10%, respectively (p Conclusions: According to our results, allo-SCT in patients with a high allelic ratio of FLT3-ITD mutation led to an overall and event-free survival comparable with those with a low allelic ratio. In patients who received chemotherapy as consolidation and no allogeneic transplantation significant differences in DFS and OS could be shown between patients with a high vs. low ratio confirming our results obeserved in the previous AML96 study. These data point towards a strong graft versus leukemia effect after transplantation thus abrogating the negative impact of a high allelic ratio seen after conventional therapeutic appoaches. Disclosures: No relevant conflicts of interest to declare.

Abstract 765 Background: The combination of ATRA and idarubicin (AIDA) for induction therapy of acute promyelocytic leukemia (APL) yields complete remission (CR) rates of > 90%. If this is followed by intensive consolidation treatment, about 85% of patients are disease free and alive after 6 years. In fact, three cycles of consolidation treatment are considered the therapeutic standard; however, it is unclear how much treatment intensity is necessary for long-term survival. In order to address this question, we analyzed the clinical course of patients enrolled into the APL study of the German Study Alliance Leukemia (SAL), which contained only two courses of consolidation. Patients and Methods: All patients ≥ 18 years diagnosed with cytogenetically confirmed APL were eligible for inclusion in the risk-adapted SAL-AIDA-2000 trial. Enrolled patients received standard induction treatment with ATRA (45 mg/m2/d until CR) and idarubicin (12 mg/m2 for 4 doses every other day). After CR, non-high-risk patients (WBC ≤ 10,000/μL) received daunorubicin (45/60 mg/m2 days 1–3, dose depending on age) as first consolidation and mitoxantrone (10 mg/m2 days 2–4) as second consolidation. High-risk patients received additional cytarabine in both consolidation cycles (100/200 mg/m2 continuous infusion over 7 days in 1st consolidation and 1000/3000 mg/m2 twice daily on 4 days in 2nd consolidation, dose depending on age). After 2 cycles of consolidation, all patients were scheduled for 24 months of maintenance with 6-mercaptopurin (90 mg/m2 daily), methotrexate (15 mg/m2 weekly), and ATRA (45 mg/m2 for 15 days every 3 months). The following outcomes were analyzed: CR rate, induction deaths, disease-free survival (DFS), and overall survival (OS). Results: Between January 1999 and October 2010, 141 patients were enrolled in the trial. The median age at diagnosis was 51 years (range, 19–82), and 41 (29%) patients had a WBC >10,000/μL (high risk). The CR rate was 92% in the entire cohort; 95% in patients ≤ 60 and 86% in patients > 60 years (p=0.082). No significant differences in CR rates were seen between high-risk and non-high-risk patients (88% vs 94%, p=0.213). Three patients died during induction treatment (2%). After a median follow up of 55 months, the median DFS and OS were not reached. The estimated 6-year DFS was 80% (95%–CI 72%–88%) in all patients; 84% in patients ≤ 60 and 72% in patients > 60 years (p=0.140). The estimated 6-year OS was 77% in all patients; 84% (95%–CI 76%–92%) in the younger and 62% (95%–CI 47%–78%) in the elderly group (p=0.004). No significant survival differences between the high-risk and the non-high-risk patients were observed, neither for DFS (6-year DFS 78% (95%–CI 64–93%) vs 81% (95%–CI 72%–91%), p=0.625) nor for OS (6-year OS 71% (95%–CI 57%–86%) vs 79% (95%–CI 70–89), p=0.207). Conclusions: Our results confirm the efficacy of a risk-adapted approach both in high-risk and non-high-risk APL patients. The similarity for DFS and OS times between these two groups demonstrate the efficacy of cytarabine added to anthracyclines during consolidation in high-risk patients. Both CR rates and survival outcomes are comparable to the results obtained in the AIDA0493 and AIDA2000 trials by the GIMEMA group, which used three cycles of higher-dosed consolidation. In light of the data, modification in number and intensity of consolidation cycles may result in a less toxic but equally effective option for the treatment of APL and should be considered for further evaluation in a randomized clinical trial. Disclosures: No relevant conflicts of interest to declare.

Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis plays a central role in this hypersensitive phenotype. The role of the tumor suppressor p53 in this response, however, remains controversial. Here we show that siRNA-mediated silencing of p53 is sufficient to completely abrogate hypersensitivity not only to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. The close relationship between p53 protein levels and induction of apoptosis is lost upon short-term differentiation, indicating that this predominant pro-apoptotic function of p53 is unique in pluripotent embryonal carcinoma (EC) cells. RNA interference experiments as well as microarray analysis demonstrated a central role of the pro-apoptotic p53 target gene NOXA in the p53-dependent apoptotic response of these cells. In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to p53 activation. Furthermore, in the very specific cellular context of germ cell-derived pluripotent EC cells, p53 function appears to be limited to induction of apoptosis. © 2011 Gutekunst et al.

Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer such as NTERA-2D1 show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis is responsible for this hypersensitive phenotype. However, the role of the p53 pathway remains controversial. Using RNA interference-mediated gene silencing, we systematically investigated the impact of DNA damage kinases upstream of p53, p53 itself, as well as apoptosis-related p53 targets on this hypersensitivity. Unexpectedly, neither knockdown of ATM, ATR, or DNA-PK alone nor ATM/ATR double knockdown or ATM/ATR/DNA-PK triple knockdown had any significant protective effect on survival of NTERA-2D1 cells upon Cisplatin. However, siRNA-mediated silencing of p53 was sufficient to completely abrogate hypersensitivity demonstrating that in these cells p53 acts preferentially as a pro-apoptotic factor. This is also supported by the finding that NTERA-2D1 cells were not only hypersensitive to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. Screening of different p53 targets revealed that the pro-apoptotic function of p53 was dependent on NOXA, since silencing of this p53 target almost completely mimicked the effect of the p53 knockdown. Importantly, both constitutive and Cisplatin-induced levels of NOXA were significantly reduced in resistant NTERA-2D1 cells obtained by either short-term differentiation or cultivation with increasing Cisplatin concentrations. Furthermore, comparison of a panel of p53 wildtype tumor cell lines revealed exceptionally high NOXA levels in the most sensitive cell line NTERA-2D1. In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to pro-apoptotic functions of p53 mediated by exceptionally high levels of NOXA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4693. doi:10.1158/1538-7445.AM2011-4693

Cytokines have been widely tested in clinical trials during recent years and beneficial responses have been observed in a variety of malignant, infectious and autoimmune diseases. Interferon-alpha induces remissions in patients with certain hematological malignancies such as hairy cell leukemia and chronic myelogenous leukemia. A proportion of patients with chronic viral hepatitis is cured upon application of interferon-alpha. Treatment with interferon-gamma reduces the number of infections in patients with chronic granulomatous disease. In addition, several chronic infections with intracellular pathogens also respond to treatment with this cytokine. With the exception of some patients with renal cell carcinoma and malignant melanoma, solid tumors are largely resistant to administration of these cytokines. Cytokine treatment has changed the outlook for a small group of patients with selected chronic diseases. However, clinical experience with cytokines is still limited and only interferons have been tested for treatment of a variety of diseases. Thus, it seems reasonable to expect that more cytokine-responsive diseases might be identified by continued research efforts.

Abstract 2142 Background: VCR is an important component of the treatment of ALL, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, and other adult and childhood cancers. In part, because of the cell cycle specific activity of VCR, its anti-cancer activity is believed to be very exposure time and concentration dependent. Standard dosing of conventional VCR (1.4 mg/m2 with a 2 mg cap) is limited because of early onset peripheral neuropathy and fails to achieve sustained VCR delivery. VSLI (Marqibo) is a nano-particle encapsulated formulation of VCR designed to facilitate dose intensification, prolonged drug delivery and enhanced cancer penetration and concentration. Methods: In a pivotal, Phase 2, multi-national study (RALLY Trial), 65 adults with Philadelphia chromosome negative ALL who were either in second or greater relapse or who had progressed after two or more prior lines of treatment received single-agent intravenous VSLI 2.25 mg/m2 (without any dose cap) weekly over 1 hour as salvage therapy. First-dose PK was investigated in a representative subset of 13 study subjects. Blood for analysis was collected at 8 time points ranging from 5 minutes to 48 hours following infusion. Total VCR plasma levels were determined by HPLC-MS/MS. PK parameters were calculated with Phoenix WinNonlin. Results: The PK subject subset had a median body surface area (BSA) of 1.92 m2 (range 1.47 to 2.45 m2) and received a median VSLI dose (VCR component) of 4.32 mg (range 3.3 to 5.51 mg). Based on BSA and the 2 mg dose cap, all subjects in this study group would have been dosed with 2.0 mg of conventional VCR. The median cumulative induction dose of VSLI (VCR component) that was administered in this study was 18.8 mg (range 3.5 to 70.1 mg). Total VCR plasma concentration decreased rapidly from Cmax after the VSLI infusion in 5 subjects (38%); 8 subjects (62%) exhibited a delay of 4 to 10 hours before the total VCR plasma concentration began to decrease. The calculated Tmax was 1.3 ± 0.4 hours (range 1.1 to 2.0 hours). The Cmax was 1214 ± 233 ng/mL (range 919 to 1720 ng/mL). The apparent mean half-life was 7.1 ± 3.2 hours. The mean AUCinf was 13,993 ± 6,588 ng hr/mL with a range from 7,167 to 27,233 ng hr/mL. The mean clearance (CL) was 6.4 ± 2.6 mL/min. The mean volume of distribution (Vd) was 0.051 ± 0.018 L/Kg. There were no significant differences in the PK parameters between the male and female subjects participating in this study. The table below presents VSLI PK parameters in addition to historical PK parameters for conventional VCR dosed at 2 mg. Conclusions: VSLI clearly provides dose intensification and prolonged VCR delivery compared to conventional, non-encapsulated VCR. VSLI, as dosed in this adult ALL clinical trial, delivered individual and cumulative amounts of VCR that exceed those achievable with standard and approved dosing of conventional VCR. This translated into a median dose intensification of 116% (range 65 to 176 percent) calculated as the percent change in VSLI dose from a standard VCR dose. This dose intensification is believed to have contributed to the 35% overall response rate including 20% complete responses (with or without full blood count recovery) reported in this heavily pre-treated, multiply-relapsed/refractory population without apparent enhanced toxicity [J Clin Oncol 28:15s, 2010 (suppl; abst 6507)]. VSLI has a distinctly different PK profile than conventional VCR. The larger VSLI Cmax and AUCinf reflect the dose intensification afforded by a larger mg/m2 dose and lack of dose capping. Even in the absence of dose capping, the 2.25 mg/m2 VSLI dose represents a 61% dose escalation above conventional VCR. While Cmax and AUCinf are dose-dependent PK parameters, the observed differences between VSLI and VCR control cannot be explained by dose alone. The larger AUCinf also reflects prolonged circulation afforded by the sphingomyelin:cholesterol liposome encapsulation. The modest VSLI mean CL and small Vd reflect the retention of encapsulated VCR within the plasma compartment for an extended period of time so that VCR can better penetrate and accumulate in sites of cancer through fenestrated vasculature. The enhanced delivery of encapsulated VCR contributes to maintenance of VCR concentrations above the effective concentration. Disclosures: Silverman: Hana Biosciences: Employment. Deitcher: Hana Biosciences: Employment.

Abstract 3150 Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One mechanism of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to induction of a non-apoptotic cell death starting approximately 40 hours after Imatinib withdrawal. This cell death was preceded by a rapid and robust induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling. Over-activation of Bcr-Abl downstream signalling was accompanied by a significant enhanced glucose and amino acid metabolism and an elevated intracellular protein and nucleic acid content. As a result of this enhanced metabolisms we observed a massive increase in cell size, multinucleation, cytoplasmic vacuolization, and reduced intracellular ATP level. The phase-lucent vacuoles did not incorporate the endosome/lysosome tracker, Lyso Tracker Red indicating that the vacuoles were formed by dilation of ER cisternae. To determine if Imatinib deprivation induces an ER stress response concurrent with vacuolization we investigated typical ER stress markers. Upon removal of Imatinib a massive induction of CHOP expression and eIF2 alpha phosphorylation, as well as alternative splicing of XPB could be detected. It has been demonstrated that severe ER stress promotes cell death either by induction of a BIM-dependent apoptitic process or by a TRAF2-RIP1 dependent pathway. Despite of a robust induction of BIM and posttranslational modification of RIP1, siRNA-mediated suppression of BIM and RIP1 had no effect on Imatinib deprivation-induced cell death whereas downmodulation of CHOP partially rescued those cells. Using different small molecule inhibitor libraries, we also identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK, as well as glucocorticoids as potent compounds which not only completely prevented metabolic stress and induction of cell death upon removal of Imatinib but also partially repressed induction of CHOP upon Imatinib withdrawal. Importantly, in the presence of glucocorticoids, we found a significant enhanced number of cell clones with Bcr-Abl overexpression in lympoid cell lines upon de novo transfection with Bcr-Abl. In conclusion, acute over-activation of the oncogene Bcr-Abl led to metabolic stress and ER stress followed by a delayed induction of a necrosis-like cell death. These cellular responses to Bcr-Abl-mediated oncogenic stress could be completely blocked by glucocorticoids. Therefore, treatment of glucocorticoid resistant ALL may generate selective pressure towards Bcr-Abl overexpession. Disclosures: No relevant conflicts of interest to declare.

Abstract 333 Background: Standard chemotherapy for elderly AML patients results in a median overall survival of only about one year. Case reports and early phase I/II data have shown that the kinase inhibitor Sorafenib might show clinical benefit for Flt3-ITD-positive AML patients (Metzelder S Blood 2009; 113:6567) and that its addition to standard chemotherapy is feasible (Ravandi F JCO 2010; 28:1856). Sorafenib is a potent Raf, c-Kit and FLT3 inhibitor that may also affect AML blasts and bone marrow (BM) stroma cells via VEGFR and PDGFR-β inhibition. Therefore, we performed a multicenter, randomized, placebo-controlled, double-blind phase II trial in elderly (>60 y) AML patients analyzing the effect of Sorafenib in addition to standard chemotherapy and as a maintenance therapy for up to one year. Methods: 197 AML patients in 16 centers received up to two cycles of standard 7+3 induction chemotherapy plus two cycles of consolidation therapy with intermediate dose (6 × 1g/sqm) AraC. Before start of treatment, they were randomly assigned to receive either placebo or Sorafenib (400 mg bid between the cycles and after chemotherapy for up to one year after start of induction). The primary aim was to compare the event-free survival (EFS) of the two treatment groups. Secondary end points were to compare EFS and overall survival (OS) of predefined subgroups according to NPM and FLT3 mutation status and toxicity of treatment. Results: Among the 197 evaluable patients, 102 pts received Sorafenib and 95 pts placebo. EFS and OS were not significantly different between the two treatment groups (placebo vs. Sorafenib: EFS: Median: 7 vs. 5 months, hazard Ratio (HR): 1.261(p=0.13); OS: Median: 15 vs. 13 months, HR 1.025 (p=0.89)). CR or blast clearance without complete blood count recovery was observed in 49 (48%) and 9 (8.8%) Sorafenib patients and 57 (60%) and 4 (4.2%) placebo pts, respectively. Exploratory subgroup analyses did not reveal any significant difference between the treatment groups but showed a tendency towards decreased EFS in the Sorafenib arm for NPM1-wild type AML cases. Flt3-ITD mutations were found in 28 out of 197 patients (14.2%), in line with the reported incidence in the target population. No differences in EFS or OS were to be noted in this small patient population. Also, CR rate was not improved by the study drug in this subgroup of patients. Sorafenib was relatively well tolerated. The most frequent adverse events (AE) ≥grade 3 were febrile neutropenia, pneumonia in neutropenia, sepsis, diarrhea, skin rash, mucositis, hypertension (77 vs 74, 54 vs 35, 15 vs 15, 17 vs 6, 14 vs 7, 9 vs 6, 8 vs 5 events in the Sorafenib vs the placebo group). A hand-foot-skin reaction (≥grade 3) was noted in 5 vs 0 events in Sorafenib vs control pts. There was a trend of slower regeneration of leukocytes and thrombocytes within the Sorafenib arm compared to the control arm after the first and second induction course but not after consolidation cycles. Conclusion: Although the combination regimen appeared to be feasible and tolerable in elderly AML pts, Sorafenib treatment did not improve EFS or OS in this unselected elderly AML patient population. Further studies should focus on selected AML target populations for Sorafenib, especially FLT3-ITD+ AML patients. Disclosures: Off Label Use: Sorafenib (multikinase inhibitor) is given in combination with standard chemotherapy in elderly AML patients. (See title of the abstract!).

6507 Background: Management of adult ALL patients who relapse is challenging due to highly drug resistant disease and frequent residual therapy-induced toxicity. The increasing success of Salvage 1...

Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One of the major mechanisms of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to a huge induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling leading to an elevated cell size and cellular protein content. Massive induction of cell death was then observed at later time points (48 hours after Imatinib deprivation) accompanied by loss of outer and inner mitochondrial membrane integrity. Using a protein kinase inhibitor library, we identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK as the most potent compounds which completely prevented induction of cell death upon removal of Imatinib whereas the same inhibitors had synergistic effects with Imatinib in Imatinib-sensitive ALL cells. In contrast to GSK3- and p38, inhibition of PI3K had no effect on oncogenic stress upon Imatinib withdrawal in the resistant clones. Importantly, oncogenic stress-induced cell death could not be blocked by the pan-caspase inhibitor z-VAD-fmk whereas the RIP1 inhibitor Necrostatin partially rescued the cells upon Imatinib deprivation. We also detected a posttranslational modification of RIP1 upon Imatinib withdrawal that was completely absent in cells pre-treated with GSK3- or p38 inhibitors indicating a central role of RIP1 for oncogenic stress-induced cell death. In conclusion, we found a massive induction of a necroptosis-like cell death in Bcr-Abl overexpressing ALL cells as a consequence of oncogenic stress upon Imatinib deprivation. GSK3 and p38 turned out to play the most prominent role for oncogenic stress induced cell death. These data implicate that a discontinuous treatment with Imatinib (periods of Imatinib treatment followed by short periods without Imatinib) may prevent the appearance of cell clones with Bcr-Abl overexpression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4522.

Cancer associated fibroblasts (CAFs) show a molecular and functional phenotype that is different from their normal counterparts (normal fibroblasts, NAFs). It is a well established phenomenon that these modified and activated fibroblasts possess permissive and promoting properties for epithelial tumor cells and therefore play a central role in tumor progression, metastasis, and probably also in sensitivity to antitumor agents. We asked whether CAFs could be reverted back pharmacologically to normal homeostasis by inhibiting PDGFR signalling. We therefore evaluated the efficacy of the FDA approved PDGFR inhibitor Dasatinib on proliferation and gene expression profile of CAF strains isolated from different lung carcinoma specimens. Dasatinib significantly reduced proliferation without inducing cell death in all 40 CAF strains investigated. Genome-wide microarray analysis of 9 CAF strains cultivated in presence or absence of Dasatinib identified 491 genes whose expression was significantly changed at least twofold. 104 of these encoded cell cycle related proteins with a great majority of 97 being down-regulated by Dasatinib. To determine the relevance of the differentially regulated genes in Dasatinib treated cells, we compared the Dasatinib expression signature to the previously described signatures of NAFs and CAFs derived from breast cancer patients (Sadlanova et al., 2008). Importantly, 102 of the 491 genes regulated by Dasatinib were also shown to be differentially expressed in CAFs versus NAFs. The expression of 72 of the genes we identified as down-regulated by Dasatinib was also reduced in NAFs compared to CAFs (p In summary, our data suggest that Dasatinib may reverse the cancer-activated phenotype of CAFs to a normal fibroblast like phenotype. Dasatinib treatment may therefore also reverse the tumor-promoting properties of CAFs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1425.

The aim of this study was to investigate excretion of urinary and pancreatic juice neopterin in patients after combined pancreas-kidney transplantation and to relate it to the clinical course. The study design was a prospective observation study. Thirty consecutive patients with end-stage diabetic disease received a simultaneous pancreas-kidney transplant with pancreaticocystostomy and temporary exteriorization of pancreatic juice. In 30 patients urinary neopterin (UN) was measured daily from day +1 until hospital discharge by high-performance liquid chromatography; in ten of these 30 patients pancreatic juice neopterin (PJN) was additionally analyzed daily from day +1 for as long as pancreatic juice was diverted to the exterior. Elevated urinary neopterin levels were observed in acute cellular rejection (19/24 rejection episodes) and in bacterial infection (9/16 cases)--however, increments were more pronounced in acute cellular rejection. In contrast, pancreatic juice neopterin increased in all seven observed pancreatic graft rejection episodes. Pancreatic juice infection did not result in a rise in pancreatic juice neopterin excretion. Patients without postoperative complications exhibited stable and low urinary/pancreatic juice neopterin levels. The highest urinary neopterin levels were observed in CMV disease. Levels measured prior to discharge from hospital did not correlate with graft and patient survival. Evaluation of urinary and pancreatic juice neopterin, although nonspecific, helps identify patients with an uncomplicated or complicated clinical course. Pancreatic juice neopterin appears to be superior to urinary neopterin in early detection of pancreatic graft rejection. This may be of particular importance in monitoring nonuremic pancreas allograft recipients.

Abstract 3088 Poster Board III-25 Adult ALL is a disease of primarily young and middle-aged adults that is associated with a high relapse rate following initial remission induction and a short overall survival following relapse. Patients in second relapse must deal with the lingering toxicities of prior therapies, organ dysfunction secondary to extramedullary disease, and the absence of fully approved and standard of care therapies for this advanced disease setting. Realistic goals of treatment in second relapse include eradication of leukemia, bridging to stem cell transplant, and prolongation of survival. Vincristine is active against leukemia and is typically used as part of multi-agent regimens to treat relapsed disease, but the pharmacokinetic profile and dosing cap are suboptimal and contribute to poor disease control and abysmal overall survival. VSLI is a nanoparticle formulation of vincristine sulfate USP encapsulated in sphingomyelin/cholesterol liposomes called Optisomes™. The Optisomal formulation permits dose intensification beyond that attainable with conventional vincristine sulfate injection, USP (VSI). VSLI provides a long circulation time and slow release of encapsulated vincristine sulfate resulting in enhanced tumor penetration and concentration. Preclinical studies of VSLI showed enhanced efficacy versus VSI in a variety of solid and hematologic malignancies. VSLI has a maximum tolerated dose of 2.25 mg/m2 weekly with no dose cap, while conventional vincristine sulfate is dosed at 1.4 mg/m2 with a 2 mg dose cap. A previous study involving VSLI in relapsed ALL showed a complete response rate of 19%, suggesting activity in the relapsed setting and prompting further study. This international, multicenter, single-arm study has completed enrollment of its target 56 subjects at 21 centers in 4 countries over 27 months. Major endpoints include response rate (CR/CRi) and overall survival (OS). 56 adult subjects received single agent intravenous VSLI at a dose of 2.25 mg/m2 weekly with no dose cap. The highest single dose of VSLI given to date in this study was 5.22 mg contrasted to the 2 mg dose this subject would have received with conventional vincristine. Subjects were monitored for disease response every 4 weeks and could be treated until disease progression. Of the demographic data available for 48 subjects, 24 were female and 24 male with a mean age of 38.7 years (range 19.3-79.6). All subjects received at least one prior vincristine-containing regimen and 21 subjects (44%) received vincristine sulfate in two prior regimens. 18 subjects (37%) had a prior stem cell transplant and 2 subjects (4%) had 2 prior stem cell transplants before receiving single agent VSLI. 71% of subjects had an ECOG Performance Status of 1 or greater, with 21% ECOG 2 or 3. Seven subjects (15%) had evidence of extramedullary disease at study entry. At interim analysis, treatment with single agent VSLI produced morphologic and cytogenetic CR in several of these heavily pretreated subjects, suggesting significant activity of VSLI in second relapsed leukemia. Of investigator-reported responses on 48 subjects with data available to date, 71% have evidence of either disease response or disease stabilization and 29% have reported disease progression. Of the subjects experiencing CR/CRi, at least 5 subjects subsequently underwent allogeneic stem cell transplant after receiving VSLI. At the time of this abstract the median OS is estimated to be 4.7 months (Q1-Q3: 3.4-10.5) using Kaplan-Meier methodology. The 5 most frequently reported adverse event are peripheral neuropathy, 14.6% Grade 3 or 4 (100% attributed to study drug); constipation, 2.4% Grade 3 (100% attributed to study drug); nausea, no Grade 3 or higher; pyrexia, 9.8% Grade 3 (2.4% attributed to study drug); and febrile neutropenia, 34.2% Grade 3 or 4 (7.3% attributed to study drug). VSLI is a promising new agent for the treatment of acute lymphoblastic leukemia in second relapse, a situation where there is high unmet need and no approved therapies exist. VSLI may be used as a single agent as a bridge to transplant in these heavily pretreated, multiply-relapsed subjects, or considered as a substitute for conventional vincristine sulfate. The ability to complete enrollment rapidly internationally with compelling evidence of objective responses in this study underscores the enthusiasm for and importance of developing VSLI for the treatment of this orphan leukemia indication. Efficacy data and preliminary PK results from the target 56 subjects will be presented. Disclosures O'Brien: Hana Biosciences, Inc.: Consultancy. Hagey:Hana Biosciences, Inc.: Employment. Deitcher:Hana Biosciences, Inc.: Employment. Kantarjian:Hana Biosciences, Inc.: Consultancy.

Abstract 2652 Poster Board II-628 Background: Secondary acute myeloid leukemia (sAML) following a myelodysplastic syndrome (mdsAML) or deriving as therapy-related AML (tAML) is regarded as an entity with a poor prognosis and patients are normally treated as high risk AML. However due to progress in elucidating the impact of molecular and cytogenetic markers and therefore combining biological and clinical data for prognosis and treatment outcome the aim of this analysis was to provide a prognostic scoring system for this entity by including clinical and laboratory data from patients being treated in the prospective AML96 trial of the DSIL study group. Patients and methods: A total of 318 patients with sAML (mdsAML = 239 and tAML = 79) were treated within the AML96 trial with a median follow-up for patients alive of 5.66 years (95% CI 4.426 – 6.895). All patients received double induction chemotherapy. Consolidation therapy contained high-dose cytosine arabinoside and for patients ' 60 years of age the option of autologous or allogeneic hematopoietic stem cell transplantation (HSCT) according to donor availability. Prognostic factors for survival were analyzed in the whole group of sAML patients in a multivariate Cox regression model for overall survival (OS) stratified by treatment groups (chemo-consolidation vs. allogeneic HSCT). Model selection was performed by backward selection applying the Likelihood-Ratio-Test. Results: Complete remission (CR) rate for all patients was 30.8% (n = 96). CR rate was lower in patients with mdsAML compared to patients with tAML (25.9% vs. 44.3%, p=.003). Patients with mdsAML were older and had a higher percentage of CD34+ blasts at diagnosis but to a lower extend aberrant karyotypes than patients with tAML. OS and disease free survival (DFS) at three years for all patients was 15.8% and 20.6%, respectively. While disease status (mdsAML vs. tAML) had no independent influence on survival, the dichotomized prognostic factors platelet count in the peripheral blood at diagnosis [HR = 0.535 (95% CI .415 – .689), p=50 Gpt/l at diagnosis and the high risk group being NPM1 negative and having platelets of '50 Gpt/l at diagnosis. Three year OS for patients who received chemo-consolidation in the low risk group was 19.9% [95% CI = .128 - .270] and for patients in the high risk group 5.1% [95% CI = .014 - .088], p Conclusions: For patients with sAML we provide a new prognostic model for risk stratification: 1) NPM1+ or Platelets >50 Gpt/l defining a low risk group and 2) NPM1- and Platelets ' 50 Gpt/l defining a high risk group. Disclosures: No relevant conflicts of interest to declare.

Abstract 51 Background Allogeneic stem cell transplantation (allo SCT), a treatment modality based on transfer of immunocompetent donor lymphocytes offers curative potential to subjects with a variety of hematological cancers. In multiple myeloma (MM), high-dose melphalan followed by autologous stem cell transplantation (auto SCT) is adopted as a standard of care. However, it remains palliative since virtually all patients (pts) relapse and renders allo SCT an option of interest. Deletion of chromosome 13q14 (13q-) in MM has been shown to negatively impact prognosis. Therefore, improvement of therapy for 13q- pts is highly desirable. Patients and methods A prospective two-arm multi-center trial (DSMM V) was set up by our group to compare tandem high-dose melphalan 200 mg/m2 (HD Mel) with a reduced intensity conditioning allo-SCT after one cycle of HD Mel for 13q- MM. Eligibility criteria were 13q- on bone marrow FISH analysis; age up to 60 years; newly diagnosed MM in Salmon and Durie stages II and III; and measurable disease. Allocation to either treatment arm was by availability of an HLA-matched (one mismatch allowed) volunteer related (VRD) or unrelated donor (VUD). Initially, all pts received four cycles of anthracycline/dexamethasone-based induction followed by chemomobilization of peripheral blood stem cells (PBSCT) and one cycle of HD Mel. Allogeneic SCT was performed after preparation with fludarabine (30 mg/m2 for 3 consecutive days) and melphalan 140 mg/m2. ATG was administered for VUD transplants. Results 199 pts with a median age of 53 (range, 30 – 60) years were enrolled between October 2002 and March 2007 and included in this interim analysis. Sixty-seven percent had stage III disease. Allo SCT was performed in 126 of 199 pts (63%), 76 of whom (60%) received VUD allografts. The remaining 73 subjects uniformely received tandem HD Mel. Pts following allo SCT were more likely to achieve CR (59%) when compared to tandem HD Mel (32%; p=.003) within one year after end of therapy. Similarly, overall response rate was significantly higher with allo SCT (91% versus 86%; p=.003). Of note, depth of response to allo SCT was not associated with presence of acute graft-versus-host disease (GVHD): 62% CR with grades II to IV GVHD vs 58% CR with grades 0 and I (p=.75). Treatment-related mortality (TRM) at 2 years from allo SCT was 16/126 (12.7%). At a median follow up of 25 months for tandem HD Mel and 34 months for allo SCT, projected 3-year overall survival is 72% (auto) and 60% (auto/allo SCT; p=0.22), respectively. Conclusions This is the largest trial on first-line allogeneic stem cell transplant in MM so far. Our interim results show a higher CR rate in FISH 13q- subjects undergoing allo SCT when compared to tandem HD Mel. Despite a majority of allografts in our study being delivered from unrelated donors, TRM was comparable to trials confined to sibling transplants. At a relatively short follow-up, there is not yet a difference between both arms regarding OS, albeit longer follow-up may be important as previously described. This as well as analysis of the impact of donor type and chronic GVHD on outcome will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

The pharmacokinetics, toxicity and biological effects of subcutaneous and intramuscular treatment of cancer patients with recombinant tumour necrosis factor alpha (rTNF-alpha) was investigated. 17 patients suffering from refractory malignant disease were treated with either 1.0 micrograms/m2, 10 micrograms/m2 or 100 micrograms/m2 rTNF-alpha. Vital signs, peripheral blood cell counts, TNF and interferon (IFN) gamma serum levels, neopterin, beta 2-microglobulin, C reactive protein (CRP) and cortisol levels were measured immediately before and 2, 12, 24, 48 and 168 h after the first administration of rTNF-alpha. Tumour response was evaluated after 4 and 12 weeks of treatment. The pharmacokinetics followed the same characteristics as those reported for other cytokines. Major toxicities were dose dependent and comprised fever, constitutional symptoms and hypotension. TNF dependent changes were observed in serum levels of IFN-alpha, CRP, neopterin, beta 2-microglobulin, cortisol and white blood cell counts. No objective tumour response was observed. This study indicated that rTNF-alpha administered subcutaneously or intramuscularly results in measurable TNF serum levels, significant toxicity and biological response in absence of clinical efficacy in patients with advanced cancer.

BackgroundCarcinoma-associated fibroblasts (CAFs) can promote carcinogenesis and tumor progression. Only limited data on the response of CAFs to chemotherapy and their potential impact on therapy outcome are available. This study was undertaken to analyze the influence of chemotherapy on carcinoma-associated fibroblasts (CAFs)in vitroandin vivo.MethodsThein vivoresponse of stromal cells to chemotherapy was investigated in 22 neoadjuvant treated breast tumors on tissue sections before and after chemotherapy. Response to chemotherapy was analyzedin vitroin primary cultures of isolated CAFs from 28 human lung and 9 breast cancer tissues. The response was correlated toMdm2,ERCC1andTP53polymorphisms andTP53mutation status. Additionally, the cytotoxic effects were evaluated in anex vivoexperiment using cultured tissue slices from 16 lung and 17 breast cancer specimens.ResultsNine of 22 tumors showed a therapy-dependent reduction of stromal activity. Pathological response of tumor or stroma cells did not correlate with clinical response. Isolated CAFs showed little sensitivity to paclitaxel. In contrast, sensitivity of CAFs to cisplatinum was highly variable with a GI50 ranging from 2.8 to 29.0 μM which is comparable to the range observed in tumor cell lines. No somaticTP53mutation was detected in any of the 28 CAFs from lung cancer tissue. In addition, response to cisplatinum was not significantly associated with the genotype ofTP53norMdm2andERCC1polymorphisms. However, we observed a non-significant trend towards decreased sensitivity in the presence ofTP53variant genotype. In contrast to the results obtained in isolated cell culture, in tissue slice culture breast cancer CAFs responded to paclitaxel within their microenvironment in the majority of cases (9/14). The opposite was observed in lung cancer tissues: only few CAFs were sensitive to cisplatinum within their microenvironment (2/15) whereas a higher proportion responded to cisplatinum in isolated culture.ConclusionSimilar to cancer cells, CAF response to chemotherapy is highly variable. Beside significant individual/intrinsic differences the sensitivity of CAFs seems to depend also on the cancer type as well as the microenvironment.

Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment option for most high-risk patients with acute myeloid leukemia (AML). However, many high-risk patients regenerate with blasts or relapse early after induction therapy. Thus, consolidation with allogeneic HSCT in first CR is often not possible. Performing upfront allogeneic HSCT for remission induction as part of induction therapy has the potential to circumvent these problems and might furthermore reduce cumulative toxicity in high-risk patients. Therefore, in 2003 we started a prospective multicenter randomized trial that investigates both the feasibility and efficacy of upfront allogeneic stem cell transplantation for remission induction in high-risk AML patients. Methods: The AML2003 study compares in a randomized fashion an intensified treatment approach using upfront allogeneic transplantation in high risk patients as part of the induction therapy (IT) during marrow aplasia achieved by DA (daunorubicin 60 mg/ m2 – day 3–5; cytarabine 100 mg/m2 – day1–7) to a “conventional” treatment strategy, which allows for allogeneic transplantation only in patients achieving remission after two induction courses (DA). To do this, rapid analysis of cytogenetics, FLT3 status and HLA-DNA-typing of the patient and possible family donors is of utmost importance. This “fast search diagnostics” together with routine analyses of morphology and immunophenotyping is accomplished centrally in all enclosed patients. The dose-reduced preparative regimen for upfront allogeneic stem cell transplantation within induction therapy consisted of melphalan 150mg/m2 and fludarabine 150mg/m2. Results: Until the last update we recruited 679 patients Conclusions: These preliminary results show that rapid risk profiling and fast donor-search is feasible in a large multi-center study. This leads to a significant proportion of upfront allogeneic stem cell transplants as part of the induction therapy within the group of high-risk AML patients, which may improve the disastrous prognosis of this group of patients in the future.

We started a prospective randomized multicenter trial to examine the importance of the infusion schedule of Ara-C as part of the Mito-FLAG regimen in patients (pts) with relapsed or primary refractory acute myeloid leukemia (AML). The treatment consisted of mitoxantrone (7 mg/m2, days 1/3/5), fludarabine (15 mg/m2, q-12h, days 1–5), Ara-C as bolus (B) (1000 mg/m2 over 1 h, q-12h, days 1–5) or continuous infusion (CI) (125 mg/m2 over 24 h, days 1–5) and G-CSF (5 μg/kg/day, day 0 until a neutrophile count of 0.5 × 109/μl). Since 11/1999, 253 pts from 36 centers in Germany entered the study (131 men, 122 women, aged 19–75 years, median 59 years). 131 pts (52%) showed a primary refractory AML or had relapsed within 6 months after 1st complete remission (CR), whereas 120 pts had suffered their 1st relapse after 6–18 months (79 pts/31%) or later (41 pts/16%). Only 2 pts with a 2nd relapse were enrolled. The diagnoses according to FAB criteria were as follows: M0 - 17 pts, M1 - 55 pts, M2 - 71 pts, M3 - 2 pts, M4 - 44 pts, M5 - 27 pts, M6 - 9 pts, M7 - 2 pts, 26 pts were not classified. 62 pts (25%) had a poor risk karyotype (defined as aberrations on the chromosomes 5, 7, 8 or multiple). In 50 pts (20%) an elevated leukocyte count (> 20 × 109/μl) was observed. 22 pts (9%) had relapsed after prior hematopoietic stem cell transplantation (HSCT). Currently 249 pts are evaluable for response and 231 pts for toxicity and survival. Following Mito-FLAG, 120 pts (48%) achieved CR and 43 pts (17%) partial remission (PR), for an overall response rate of 65%. 206 pts experienced at least one episode of febrile neutropenia with a median duration of 8 (1–53) days. 19 pts (8%) suffered an early death because of toxicity (n=11) or progressive AML (n=8). Out of 163 responders, 8 pts were consolidated by high-dose therapy with autologous HSCT and 48 pts underwent allogeneic HSCT after dose-reduced conditioning. With a median follow-up of 40.2 months (0–93), the probabilities of event-free survival (EFS) and overall survival (OS) after 3 years were 13% and 19%, respectively. In responding pts the median duration of EFS and OS were 8.4 and 10 months, the 3-year-rates of EFS and OS were 28% and 33%, respectively. In conclusion, Mito-FLAG is an effective and well tolerated regimen in the salvage therapy of pts with AML.

Study objective: 1) to investigate serum levels of tumor necrosis factor-a in patients treated with recombinant interferon-γ and 2) to relate changes in TNF serum levels to other biological responses observed during treatment with interferon gamma (IFN-γ). Patients: Five patients suffering from metastasizing renal cell carcinoma. Intervention: Each patient received three treatment cycles of 10 μg, 100 μg and 500 μg IFN-γ applied three times at weekly intervals. The treatment cycles were separated by a therapy free interval of two weeks. The order of dose levels was randomly assigned to each patient. Measurements and main results: Tumor necrosis factor alpha (TNF-α), IFN-γ and neopterin serum levels, monocyte counts in the peripheral blood and body temperature were measured immediately before and 4, 24, 48, 72, and 168 h after each application of IFN-γ. Results indicated that elevated serum levels of TNF-α are induced by 100 Itg and 500 Itg IFN-γ. Repeated application of the same dose led to downregulation of TNF release into the serum. Changes in TNF serum levels did not correlate with the magnitude of febrile reactions, neopterin production or IFN-γ serum levels.

In a up-front randomized study, 939 adult patients up to the age of 60 years received a double induction therapy. One course of MAV (mitoxantrone 10 mg/m2 days 4–8, cytarabine 100 mg/m2 continuous infusion days 1–8, etoposide 100 m g/m2 days 4–8) was followed by one cycle of MAMAC (cytarabine 1 g/m2, every 12h days 1–5; amsacrine 100 mg/m2 days 1–5). Patients with intermediate risk cytogenetic (IRCG) and a HLA matched sibling received an allogeneic transplantation, those with poor risk cytogenetic (PRCG) were intended to be transplanted from a sibling or unrelated donor. All AML patients without an available donor received the randomly assigned first postremission therapy (PRT) mitoxantrone combined with intermediate-dose cytarabine (I-MAC; total dose 12 g/m2) or high-dose cytarabine (H-MAC; total dose 36 g/m2). As second PRT, patients with t(8;21) received an additional cycle of chemotherapy. An autologous transplantation was scheduled for IRCG and PRCG without an allogeneic donor. The CR rate was 88% for patients with t(8;21), with IRCG 71%, and 50% with PRCG. The 5-year-survival was 21% (95% CI: 16–27%) in the PRCG, 40% (95% CI: 36–45%) in the IRCG and 74% (95% CI: 60–88%) in the t(8;21) group. No difference was observed between the I-MAC and the H-MAC group. In a multivariate analysis, a significant (p50*10^9/L, and IRCG compared to PRCG. The relapse incidence was higher in patients without an allogeneic donor, a Flt3 mutant/wildtype ratio > 0.8 or PRCG. A risk score build out of the sum of the individual hazard ratios (SHR) was able to discriminate two groups for the IRCG with a marked difference in the 5-year-survival (low SHR: 55% [95% CI: 48–62%]; high SHR: 33% [95% CI: 28–38%]) was well as for the PRCG group (low SHR: 44% [95% CI: 32–56%]; high SHR: 13% [95% CI: 7–18%]). The risk score identified in this large patient cohort may allow individual tailoring of therapeutic interventions in future AML trials.

The majority of patients with acute myeloid leukemia (AML) are older than 60 years at diagnosis. However, treatment results for these elderly patients are still unsatisfactory. This is thought to be due to a more aggressive disease, preexisting co-morbidities or a decreased tolerance for intensive treatment approaches. As for younger patients there is growing evidence that elderly AML patients may be divided into prognostic subgroups. So far data on prognostic factors in this group of patients are still sketchy. Between February 1996 and March 2005 a total of 827 elderly AML patients with a median age of 67 (61–87) years were treated within the prospective AML96 trial of the German Study Initiative Leukemia (DSIL). 643 patients had de novo and 184 patients secondary disease. All patients were scheduled to receive a double induction therapy with Daunorubicin and Ara-C (DA3+7). The consolidation therapy consisted of one course of m-Amsacrine and intermediate-dose (10g/m2) Ara-C. 265 (32%) patients reached CR criteria after double induction therapy. Forty-two patients (5%) had only a PR, 307(37%) displayed refractory disease, 126(15%) died during induction therapy and 77(10%) received only one course of induction therapy due to severe toxicity. Out of the 265 patients in CR 120 (45%) patients received the consolidation course. The strongest independent prognostic factors for achieving a CR were less than 10% blasts in the day 15 bone marrow, the presence of a NPM mutation or a low-risk karyotype (p

Imatinib is highly effective in inducing remissions in chronic phase CML. However, complete eradication of the malignant clone by Imatinib monotherapy is rare. This prompted us to explore the efficacy of combination of Imatinib with the DNA damaging agent Cisplatin and with Nutlin-3, a compound which induces p53 accumulation by interfering with its binding to Mdm2. We used a Bcr-Abl positive cell line characterized by the loss of Imatinib sensitivity in the presence of optimal growth factor concentrations. Whereas combined treatment of Imatinib with either Cisplatin or Nutlin-3 in low and nontoxic doses induced ∼15–20% cell death, simultaneous treatment of all three compounds was highly effective (∼50% cell death) even in the presence of optimal growth conditions. Importantly, comparable effects were also seen in CFU assays performed with primary CD34 positive cells from 5 CML patients. To examine the molecular mechanisms of these effects we analyzed p53 dependent cellular response to Cisplatin in our cell line model in the presence or absence of Imatinib and/or Nutlin-3. The active Bcr-Abl kinase caused superinduction of p53 protein after DNA damage. We found both an upregulated p53 accumulation and enhanced p21 and Mdm2 RNA and protein levels upon Cisplatin treatment. This phenomenon could be reversed both by siRNA-mediated inhibition of Bcr-Abl expression and by Imatinib-induced inhibition of Bcr-Abl kinase activity: despite of optimal growth conditions inhibition of Bcr-Abl caused a significant reduction of p53 expression and activation. In the presence of Nutlin-3, p53 expression was significantly upregulated upon Cisplatin treatment both with and without Imatinib. However, Nutlin-3 was not capable to restore the Imatinib-induced blockade of the transcriptional activity of p53 on mdm2. The reduced p53 activation observed in Bcr-Abl positive cells treated with Imatinib was paralleled by a shift from cell cycle arrest to cell death both in the presence and absence of Nutlin selectively in Bcr-Abl positive cells rendering them hypersensitive to Cisplatin. In summary, combined treatment of Imatinib with low concentrations of a DNA damaging agent and a p53 activator is highly effective in vitro. Such combined treatment may prove to be clinically relevant for complete eradication of the malignant clone in CML, provided that conditions are found where it does not affect adversely normal hematopoietic cells in vivo.

Between 1996 and 2003, 994 patients aged under 60 years with de novo AML received a double induction chemotherapy consisting of MAV (mitoxantrone 10 mg/m2 day 4–8, Ara-C 100 mg/m2 continuous infusion day 1–8, etoposide 100 mg/m2 day 4–8) and MAMAC after an interval of at least 14 days (Ara-C 1 g/m2 q. 12 hrs day 1–5, amsacrine 100 mg/m2 day 1–5). A cytogenetic high risk profile (complex aberrations, −5/del(5q), −7/del(7q), hypodiploid karyotypes other than −5, −7, −X, −Y, abnl3q, abnl1q, abnl12p, t(6;9), t(9;22), t(9;11), +11, +13, +21, +22) was present in 252 patients (de novo AML [N=160], prior MDS [N=41], and secondary AML [N=51]). 119 out of 252 (47%) were in complete remission after 2 induction cycles. 35 (14%) additional patients showed complete remission after a further cycle. Patients with a HLA matched sibling donor (N=37) or an unrelated donor (N=34) received an allogeneic transplantation as consolidation therapy. The conditioning protocol consisted of 12 Gy TBI and cyclophosphamide. Cyclosporine and methotrexate were used as GvH prophylaxis. The actuarial survival at 5 years is 42% (CI: 26%–58%) in the related donor group and 53% (CI: 36%–70%) in the unrelated donor group. The disease free survival at 5 years is 40% (CI: 24%–56%) in the related group and 43% (CI: 26%–60%) in the unrelated group (for both, p=ns). In conclusion, the results after unrelated donor transplants are comparable with those after related donor transplants in patients with high risk AML. These data of the AML’96 study will be compared with the experience of our current upfront concept using allogeneic transplantation in aplasia after the 1st or 2nd induction cycle in high risk AML.

Imatinib inhibits cell proliferation and induces cell death in BCR-ABL positive leukemic cells in the absence of exogenic growth factors in vitro. In this study, we investigated the effects of physiological growth factors on cell survival and DNA damage response in Imatinib treated Bcr-Abl positive and Bcr-Abl negative cells from murine and human origin. To this end, we used 32D and 32D/bcr-abl, BaF3 and BaF3/bcr-abl, and M07 and M07bcr-abl cell lines. All bcr-abl positive cell lines were highly sensitive to Imatinib in the absence of growth factors. mIL3 protected Bcr-Abl transformed murine cell lines from Imatinib-induced cell death but was not capable to restore the Imatinib-dependent blockade of the p53 response to cisplatinum or gamma irradiation in Bcr-Abl positive cells. Despite prolonged pre-incubation with mIL3, Imatinib led to a transient inhibition of PI3K-, MAPK-, and STAT5-pathways selectively in Bcr-Abl positive murine cells. Importantly, this transient blockade of survival pathways by Imatinib was completely abolished after simultaneous siRNA-mediated suppression of Bcr-Abl synthesis. In contrast to the murine cell lines, Bcr-Abl positive human megakaryocytic M07 cells can not be rescued from Imatinib-induced cell death. Imatinib treated cells died even when pre-incubated simultaneously with a growth factor mix consisting of hGM-CSF, hSCF, and hIL3. Suppression of Bcr-Abl synthesis by RNAi using a breakpoint-specific siRNA selectively inhibited Bcr-Abl-dependent growth of M07/bcr-abl cells to an extent comparable to that observed with Imatinib. Importantly, growth factor stimulation enabled survival of M07/bcr-abl cells after suppression of Bcr-Abl synthesis by siRNA but not after Imatinib-mediated inhibition of Bcr-Abl kinase activity. In summary, our results indicate a dominant negative effect of kinase-inactive Bcr-Abl both on survival and on DNA damage response pathways.

In international multicenter trials, the 5-year overall survival (OS) rate of AML patients older than age 55 ranges from 5–16%. A similar outcome is seen for relapsed/refractory AML patients. As only a minority of these patients appear to benefit from myelosuppressive chemotherapy, new approaches are clearly warranted. Over 70% of AML-patients show expression of the tyrosine kinase (TK) c-Kit on myeloid blasts. c-Kit is involved in activation of proliferation pathways and plays an anti-apoptotic role in normal and malignant hematopoiesis. Imatinib mesylate (IM) inhibits protein TKs including c-Kit, PDGFRs and Abl. Two former studies have investigated IM as a single agent in patients with c-Kit positive AML and HR-MDS suggesting biological activity of IM in a subset of AML patients (Cortes, Cancer 2003; Kindler, Blood 2002). LDAC has been used for MDS and AML to induce differentiation in AML blasts with partial response rates of approximately 30% but without improvement in OS. As synergy between IM and Ara-C has been reported in vitro we conducted a phase II study applying a combination of these two agents. 40 patients at 4 centers with a median age of 72 years (range: 42–82 years with 95% of patients >60 y) were enrolled into this study. They either were not eligible for myelosuppressive therapy or had relapsed/refractory disease. Previous therapy included supportive care only, biological agents or < 3 cycles chemotherapy. In median, c-kit positivity of AML blasts from patients included was 67%. 34 patients (85%) were included with AML and 6 patients (15%) with HR-MDS (including CMML). 38 patients were evaluable for this analysis. The overall rate of biological activity was 45% (17 of 38 patients): In 6/38 (16%) patients, a blast response (reduction >50% in PB) was observed while 8/38 patients showed stable disease over a minimum period of 2 months. The objective response rate was low with each 1 patient (3%) showing hematologic improvement, PR and CR, respectively. While more than one third of patients included had a short term course of LDAC/IM only ( Patients over age 60 show a historical survival benefit of 2.5 months using myelosuppressive chemotherapy versus best supportive care and an early mortality rate of 25% within 6 weeks. Moreover, it has been pointed out that most of the ’benefit-time’ (>80%) is spent in hospital. Study medication offered in this trial was applied in an ambulatory setting with minimal hospitalization, an early mortality rate of 21% and a low toxicity rate. In conclusion, combination therapy of LDAC/IM does not appear to be inferior in comparison to myelosuppressive therapy in older AML patients. However, this trial shows that LDAC/IM does not improve substantially the clinical outcome of older patients with AML. Identification of molecular mechanisms for good and long-term responders will be part of further laboratory investigations.

The FIP1L1-PDGFRA positive myeloproliferative syndrome (MPS) represents a distinct clinicobiological entity with constitutively enhanced tyrosine kinase activity of the fusion protein and excellent response to treatment with imatinib. Most patients are initially diagnosed as idiopathic hypereosinophilic syndrome or chronic eosinophilic leukemia (CEL). A prominent clinical feature besides eosinophilia is the male predominance. However, the natural clinical course remains to be elucidated. Here we report on five male FIP1L1-PDGFRA positive patients (median age 58, range 46–64 years) with various subtypes of acute myeloid leukemia (AML) according to the FAB classification (M0, n=2; M2, n=1; M4eo, n=1; acute eosinophilic leukemia, n=1). All patients had a history of pronounced eosinophilia for a median interval of 6 mo (range 2–186) indicating the presence of a CML-like chronic phase disease evolving to blast crisis or secondary AML. One patient relapsed six years after conventional chemotherapy of a secondary FIP1L1-PDGFRA positive eosinophilia-associated AML M0 with typical features of CEL. Three patients had additional cytogenetic aberrations at diagnosis of AML similar to those frequently observed during clonal evolution of CML (trisomy 8, n=2; trisomy 9, n=1). All five patients received imatinib (100–300 mg/day) for a median time of 12 mo (range 1–23); three patients as monotherapy, one patient after one course and another patient after two courses of intensive chemotherapy. Three AML patients have been treated for more than 3 mo (12+, 18+, and 23+ mo) and are currently free of relapse. All three patients achieved a rapid complete hematological remission, defined as a complete clearance of blasts, normalization of peripheral blood and bone marrow eosinophil count, and a complete molecular remission 4, 5, and 14 mo, respectively, after start of treatment as determined by nested RT-PCR for the FIP1L1-PDGFRA transcript. In all cases treatment with imatinib was well tolerated. We conclude that the occurrence of the FIP1L1-PDGFRA fusion gene seems to be associated with a chronic phase disease which may progress to secondary AML. Therefore, all patients presenting with an eosinophilia-associated AML and normal karyotype should be screened for the cytogenetically invisible FIP1L1-PDGFRA fusion gene by RT-PCR and/or fluorescence in situ hybridization, in addition to the commoner CBFB-MYH11 fusion. Patients with a FIP1L1-PDGFRA positive MPS are excellent candidates for treatment with imatinib even if they present with secondary AML.

The increasing impact of targeted cancer treatment demands strategies to identify and evaluate resistance mechanisms toward kinase inhibitors prior to their therapeutic application. Point mutations within the Bcr-Abl kinase domain constitute the major mechanism of resistance toward imatinib mesylate in Philadelphia-positive (Ph+) leukemia. Using Bcr-Abl-transformed Ba/F3 cells, we established a cell-based screening strategy for the prediction of specific kinase mutations that cause resistance toward kinase inhibitors. With imatinib at clinically relevant concentrations, we generated 368 resistant Ba/F3 sublines that were derived from resistant colonies. Thirty-two different single point mutations within the kinase domain of Bcr-Abl were identified in twenty-five per cent (liquid culture conditions) and seventy-two per cent (solid culture conditions) of these lines at known and novel positions. Using imatinib, the pattern and relative frequency of mutations reflected matters observed in patients with imatinib resistance. We then applied this screen to the pyrido-pyrimidine PD166326 (PD16), an investigational Abl kinase inihibitor. Compared to imatinib, we observed a five to seven times lower frequency of resistant colonies with equipotent concentrations of PD16. In addition, PD16 produced a distinct pattern of Bcr-Abl mutations. P-loop, A-loop and the known imatinib contact site T315 were affected with both inhibitors, whereas C-helix and SH2 contact sites were affected in imatinib resistant colonies exclusively. In contrast to imatinib, where kinase domain mutations were still widely distributed over the kinase domain even at at 4μM, mutations observed with PD16 at a concentration of 100nM narrowed to the exchange at position T315 to iseulicine. We did not detect mutations outside the kinase domain. Some resistant sublines displayed increased Bcr-Abl activity. Mutations that were derived from the screen were cloned and examined for the extent of cross-resistance to both inhibitors. The majority of mutations were effectively suppressed by PD16 at 50–500nM. In contrast, only few mutations were inhibited by imatinib at 5–10μM. However, exchanges at position F317 mediated resistance toward PD16, but were inhibited by standard concentrations of imatinib. Since this cell-based system produced results that are clinically significant, it may be used to predict resistance mutations in Bcr-Abl and other oncogenic kinases like cKit, EGFR, FIP1L1-PDGFRalpha or FLT3 towards clinically applicated and investigational drugs. Thus, this robust and simple screening strategy provides a rational basis for combinatorial and sequential treatment strategies.

Fusion transcripts such as bcr-abl encoding pathological oncogenic proteins represent ideal targets for a tumor-specific RNA interference (RNAi) approach. The aim of the present study was to optimize the efficacy of bcr-abl RNAi. We evaluated several synthetic siRNAs targeting the fusion sites of all common bcr-abl transcript variants (e14a2, e13a2, or e1a2). Significant knock-down of p210Bcr-abl and p190Bcr-abl fusion proteins was successfully achieved in bcr-abl expressing 32D cells and human leukemic K562 and MEG-01 cells. Repeated application of siRNA proved to be significantly more efficient compared to single treatment. The optimized protocol led to a total decrease of up to 75–90% in Bcr-abl protein levels, which was accompanied by a loss of viability of up to 90%. The target specificity of the siRNA was high, and even a single point mutation in the siRNA-sequence led to significant, albeit not complete, loss of siRNA efficacy. To expend the duration of the knock-down effect, we explored the use of plasmids driving the stable expression of short hairpin RNA (shRNA). Efficient downregulation of Bcr-abl protein was achieved in K562 cells transfected with a pSUPER-based plasmid encoding bcr-abl shRNA.

4 malades en etat d'insuffisance renale due a une glomerulonephrite, dont 2 ayant deja rejete un premier rein transplante, recoivent un foie et un rein du meme donneur; ces malades presentaient aussi une insuffisance hepatique par hepatite virale et cirrhose avec encephalopathie. Les resultats sont tres encourageants, aucune mortalite et tres peu de morbidite

α-Interferon (IFN) has recently been evaluated for the treatment of chronic myelogenous leukemia (CML) [1, 2, 7–9]. Using natural and recombinant DNA-derived IFN-α preparations, significant improvement of hematological parameters was obtained in the majority of patients with Ph1-positive CML in the benign phase. Even in patients with CML in blastic crisis, a transient reduction of the blast cell counts and suppression of a secondary Ph1-positive clone has occasionally been observed [9]. Most notably, however, treatment with IFN-α has been reported to increase the proportion of Ph1-negative bone marrow cells [8].

Background Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. To study drug-sensitivity within different compartments of an individual tumor ex vivo, culture models directly established from fresh tumor tissues are absolutely essential. Methods We prepared 0.2 mm thick tissue slices from freshly excised tumor samples and cultivated them individually in the presence or absence of taxol for 4 days. To visualize viability, cell death, and expression of surface molecules in different compartments of non-fixed primary breast cancer tissues we established a method based on confocal imaging using mitochondria- and DNA-selective dyes and fluorescent-conjugated antibodies. Proliferation and apoptosis was assessed by immunohistochemistry in sections from paraffin-embedded slices. Overall viability was also analyzed in homogenized tissue slices by a combined ATP/DNA quantification assay. Results We obtained a mean of 49 tissue slices from 22 breast cancer specimens allowing a wide range of experiments in each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly in the presence of taxol in a dose-dependent manner. A three-color fluorescence viability assay enabled a rapid and authentic estimation of cell viability in the different tumor compartments within non-fixed tissue slices. Conclusion We describe a tissue culture method combined with a novel read out system for both tissue cultivation and rapid assessment of drug efficacy together with the simultaneous identification of different cell types within non-fixed breast cancer tissues. This method has potential significance for studying tumor responses to anticancer drugs in the complex environment of a primary cancer tissue.