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TO THE EDITOR: BCR-ABL + acute myeloid leukemia (AML) has recently been listed in the 2016 revised World Health Organization (WHO) classification of myeloid malignancies as a provisional entity.[1][1] BCR-ABL + AML comprises a group of de novo AML in patients without evidence of an underlying

Introduction Pseudomonas aeruginosa (PA) has historically been one of the major causes of severe sepsis and death among neutropenic cancer patients. There has been a recent increase of multidrug-resistant PA (MDRPA) isolates that may determine a worse prognosis, particularly in immunosuppressed patients. The aim of this study is to establish the impact of antibiotic resistance on the outcome of neutropenic onco-haematological patients with PA bacteraemia, and to identify the risk factors for MDRPA bacteraemia and mortality. Methods and analysis This is a retrospective, observational, multicentre, international study. All episodes of PA bacteraemia occurring in neutropenic onco-haematological patients followed up at the participating centres from 1 January 2006 to 31 May 2018 will be retrospectively reviewed. The primary end point will be overall case-fatality rate within 30 days of onset of PA bacteraemia. The secondary end points will be to describe the following: the incidence and risk factors for multidrug-resistant and extremely drug-resistant PA bacteraemia (by comparing the episodes due to susceptible PA with those produced by MDRPA), the efficacy of ceftolozane/tazobactam, the rates of persistent bacteraemia and bacteraemia relapse and the risk factors for very early (48 hours), early (7 days) and overall (30 days) case-fatality rates. Ethics and dissemination The Clinical Research Ethics Committee of Bellvitge University Hospital approved the protocol of the study at the primary site. To protect personal privacy, identifying information of each patient in the electronic database will be encrypted. The processing of the patients’ personal data collected in the study will comply with the Spanish Data Protection Act of 1998 and with the European Directive on the privacy of data. All data collected, stored and processed will be anonymised. Results will be reported at conferences and in peer-reviewed publications.

Contains fulltext : 190745.pdf (Publisher’s version ) (Closed access) Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD(+) leukemia cells. This synergized with the allogeneic CD8(+) T cell response, leading to long-term survival in six mouse models of FLT3-ITD(+) AML. Sorafenib-related IL-15 production caused an increase in CD8(+)CD107a(+)IFN-gamma(+) T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD(+) AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8(+) T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

The adult cerebral inflammatory form of X-linked adrenoleukodystrophy is a rapidly progressive neurodegenerative disease, as devastating as childhood cerebral adrenoleukodystrophy. Allogeneic haematopoietic stem cell transplantation has been demonstrated to provide long-term neurological benefits for boys with the childhood cerebral form, but results in adults are sparse and inconclusive. We analysed data from 14 adult males with adult cerebral adrenoleukodystrophy treated with allogeneic haematopoietic stem cell transplantation on a compassionate basis in four European centres. All presented with cerebral demyelinating lesions and gadolinium enhancement. Median age at diagnosis of adult cerebral adrenoleukodystrophy was 33 years (range 21-48 years). In addition to cerebral inflammation, five patients had established severe motor disability from adrenomyeloneuropathy affecting only the spinal cord and peripheral nerves (Expanded Disability Status Scale score ≥ 6). Eight patients survived (estimated survival 57 ± 13%) with a median follow-up of 65 months (minimum 38 months). Death was directly transplant-/infection-related (n = 3), due to primary disease progression in advanced adult cerebral adrenoleukodystrophy (n = 1), or secondary disease progression (n = 2) after transient multi-organ failure or non-engraftment. Specific complications during stem cell transplantation included deterioration of motor and bladder functions (n = 12) as well as behavioural changes (n = 8). Arrest of progressive cerebral demyelination and prevention of severe loss of neurocognition was achieved in all eight survivors, but deterioration of motor function occurred in the majority (n = 5). Limited motor dysfunction (Expanded Disability Status Scale score < 6) prior to transplantation was associated with significantly improved survival [78 ± 14% (n = 9) versus 20 ± 18%(n = 5); P < 0.05] and maintenance of ambulation (Expanded Disability Status Scale score < 7) post-transplant (78% versus 0%; P = 0.021). In contrast, bilateral involvement of the internal capsule on brain MRI was associated with poorer survival [20 ± 18% (n = 5) versus 78 ± 14% (n = 9); P < 0.05]. This study is the first to support the feasibility, complications and potential long-term neurological benefit of allogeneic haematopoietic stem cell transplantation in adult cerebral adrenoleukodystrophy. Further studies are warranted to attempt to improve outcomes through patient selection and optimization of transplantation protocols.

We hereby report a case of acute myeloid leukemia with translocation t(2;3) and involvement of the ectopic virus integration site-1 (EVI1) gene. Like most other 3q26-related disorders reported thus far, we describe a phenotype with elevated platelet counts and dysmegakaryopoesis. The clinical course of our patient was complicated by symptomatic thrombophilia and chemoresistance. In addition, our case exhibited FLT3 (Fms-related tyrosine kinase 3) internal tandem duplication. Although anagrelide was successful in controlling elevated platelet counts, allogeneic stem cell transplantation failed to overcome chemoresistance due to simultaneous graft-versus-host-disease and relapse of acute myeloid leukemia. Given the dismal outcome of our case and previously reported cases, we propagate the implementation of targeted therapies to newly diagnosed patients with acute myeloid leukemia t(2;3). Preclinical models indicate drugs that plausibly target the EVI1-related molecular vulnerability as candidates for basket trials. Anagrelide exhibited a hopeful signal of activity in 3q26-related thrombocytosis and should be evaluated for implementation as supportive care.

Chronic graft-versus-host disease (cGVHD) associated morbidity and mortality remain major barriers for successful allogeneic hematopoietic stem cell transplantation (alloHSCT). Currently, no reliable measures are established to monitor cGVHD activity changes for use in clinical trials. The Human Activity Profile (HAP) patient self-report was proposed by the National Institutes of Health (NIH) cGVHD consensus project as an independent measure of patients' functional status that could also indirectly reflect improvement of cGVHD, but that has not been validated in an alloHSCT patient population. One hundred seventy-six patients (median age 44 years [range: 18-72 years] after alloHSCT were evaluated with a German translation of the HAP, the NIH criteria-based cGVHD activity assessment, the Lee cGVHD Symptom-Scale, FACT-BMT, SF36, Berlin Social Support Scale, 24-Item Adjective Measure (24-AM), Hospital Anxiety and Depression Scale, and the NCCN-Distress-Thermometer. Enrollment occurred a median of 286 (range: 85-4003) days after alloHSCT. Follow-up surveys were conducted at 1, 2, 3, 5, 8, and 12 months after the baseline survey. Although 117 patient had cGVHD at time of enrollment (mild n = 33, moderate n = 50, or severe n = 34), 59 patients were included into the study in the absence of cGVHD between days 85 and 395 after transplantation. The maximum activity score (MAS) and adjusted activity score (AAS) of the HAP correlated inversely with grading of cGVHD severity (mild, moderate, or severe) (r = −0.25 for MAS and −0.24 for AAS). Lung manifestations of cGVHD correlated with AAS (r = 0.17), but not with MAS. HAP scores correlated with subscales from other instruments measuring physical domains, especially the physical functioning scale of the SF36. Performance was improved by use of an HSCT-modified HAP scoring system that excluded activities prohibited within the first year after alloHSCT. No significant correlation of the HAP was found with personality, age, sex, symptom burden, or social functioning or social well-being. Moreover, the HAP displayed a higher sensitivity to change of cGVHD activity compared to the SF36 and the FACT-BMT. In addition, steroid myopathy correlated with both HAP scores, but not the SF36. The HAP is a simple and valid questionnaire for the evaluation of the physical activity in patients after alloHSCT, with the advantage of detecting changes in cGVHD status independently of other quality-of-life measures and with a superior sensitivity compared to the SF36.

Disease stage is the most important prognostic parameter in allogeneic hematopoietic cell transplantation (HCT) for acute lymphoblastic leukemia, but other factors such as donor/host histocompatibility and gender combination, recipient age, performance status and comorbidities need to be considered. Several scoring systems are available to predict outcome in HCT recipients; however, their prognostic relevance in acute lymphoblastic leukemia is not well defined.In the present study we evaluated a modified EBMT risk score (mEBMT) and the HCT-specific comorbidity index (HCT-CI) in 151 adult acute lymphoblastic leukemia patients who received allogeneic HCT from 1995 until 2007 at our center.Disease status was first complete remission (CR1) (47%), CR1 (21%) or no CR (32%). Overall survival (OS) at one, two and five years was 62%, 51% and 40% and non-relapse mortality (NRM) was 21%, 24% and 32%. Median mEBMT was 3 (0-6). Higher mEBMT was associated with inferior OS (hazard ratio per score unit (HR): 1.50, P0.001), higher NRM (HR: 1.36, P=0.042) and higher relapse mortality (HR: 1.68, P0.001). Disease stage was the predominant prognostic factor in this score. Comorbidities were present in 71% of patients with mild hepatic disease (29%), moderate pulmonary disease (28%) and infections (23%) being the most common. Median HCT-CI was 1 (0-9). In univariate analysis a trend for inferior OS (HR: 1.08, P=0.20) and higher NRM (HR: 1.14, P=0.11) with increasing HCT-CI was observed but the level of significance was not reached. In additional analyses we found that reduced Karnofsky Performance Status (KPS) was associated with inferior OS (HR: 1.34, P=0.023) and higher relapse mortality (HR: 1.71, P=0.001) when analyzed univariately. However, KPS was associated with disease stage and significance was lost in multivariate analysis.The mEBMT was prognostic in our patient cohort with predominant influence of disease stage, whereas a trend but no significant prognostic value was observed for the HCT-CI.

P14(ARF) (p19(ARF) in the mouse) plays a central role in the regulation of cellular proliferation. Although the capacity of p14(ARF) to induce a cell cycle arrest in G1 phase depends on a functional p53/p21-signaling axis, the G2 arrest triggered by p14(ARF) is p53/p21-independent. Using isogeneic HCT116 cells either wild-type or homozygously deleted for p21, 14-3-3sigma or both, we further investigated the cooperative effect of p21 and 14-3-3sigma on cell cycle regulation and apoptosis induction by p14(ARF). In contrast to DNA damage, which induces mitotic catastrophe in 14-3-3sigma-deficient cells, we show here that the expression of p14(ARF) triggers apoptotic cell death, as evidenced by nuclear DNA fragmentation and induction of pan-caspase activities, irrespective of the presence or absence of 14-3-3sigma. The activation of the intrinsic mitochondrial apoptosis pathway by p14(ARF) was confirmed by cytochrome c release from mitochondria and induction of caspase-9- (LEHDase) and caspase-3/7-like (DEVDase) activities. Moreover, 14-3-3sigma/p21 double-deficient cells were exceedingly sensitive to apoptosis induction by p14(ARF) as compared to wild-type cells or cells lacking either gene alone. Notably, p14(ARF)-induced apoptosis was preceded by an arrest in the G2 phase of cell cycle, which coincided with downregulation of cdc2 (cdk1) protein expression and lack of its nuclear localization. This indicates that p14(ARF) impairs mitotic entry by targeting the distal DNA damage-signaling pathway and induces apoptotic cell death, rather than mitotic catastrophe, out of a transient G2 arrest. Furthermore, our data delineate that the disruption of G2/M cell cycle checkpoint control critically determines the sensitivity of the cell toward p14(ARF)-induced mitochondrial apoptosis.

Long-term survival after allogeneic hematopoietic stem cell transplantation requires intact immunosurveillance, which is hampered by lymphoid organ damage associated with conditioning therapy, graft-versus-host disease, and immunosuppression. Our study aimed to identify the mechanisms contributing to sustained low memory B cell numbers after transplantation. Peripheral B and T cell subset recovery and functional marker expression were investigated in 35 acute leukemic patients up to 1 year after transplantation. Apoptosis of B cells after CD40/TLR-9, CD40/BCR, and CD40/BCR/TLR-9-dependent stimulation and drug efflux capacity were analyzed. One half of the patients suffered from infections after day 180. All patients had strongly diminished CD27+ memory B cells despite already normalized total B cell numbers and fully recovered CD27−IgD− memory B cells, putatively of extra-follicular origin. Circulating memory follicular helper T cells were reduced in the majority of patients as well. Naive B cells exhibited a decreased expression of CXCR5, which mediates follicular B cell entry. Additionally, a lower HLA-DR expression was found on naive B cells, impairing antigen presentation. Upon CD40/TLR-9–dependent activation, B cells underwent significantly increased apoptosis paralleled by an aberrant up-regulation of Fas-L on activated T cells and Fas on resting B cells. Significantly increased B cell apoptosis was also observed after CD40/BCR and CD40/BCR/TLR-9–dependent activation. Drug efflux capacity of naive B cells was diminished in cyclosporin A–treated patients, additionally contributing to an apoptosis-prone phenotype. We conclude that B cell survival and migration and T cell communication defects are contributing candidates for an impaired germinal center formation of memory B cells after allogeneic hematopoietic stem cell transplantation. Follow-up studies should evaluate effectiveness of revaccinations on the cellular level and should address the long-term sequelae of B cell defects after transplantation.

s / Biol Blood Marrow Transplant 21 (2015) S147eS170 S167 overall survival (OS) was 54% (95% CI, 41e66%) for the entire cohort with an OS for NNGF of 73% (95% CI, 55 e 85%) and for NGF OS was 30% (95% CI, 14e47%) (p

In contrast to the initial notion that the biological activity of p14(ARF) strictly depends on a functional mdm-2/p53 signaling axis, we recently demonstrated that p14(ARF) mediates apoptosis in a p53/Bax-independent manner. Here, we show that p14(ARF) induces breakdown of the mitochondrial membrane potential and cytochrome c release before triggering caspase-9- and caspase-3/7-like activities in p53/Bax-deficient DU145 prostate cancer cells expressing wild-type Bak. Re-expression of Bax in these cells failed to further enhance p14(ARF)-induced apoptosis, suggesting that p14(ARF)-induced apoptosis primarily depends on Bak but not Bax in these cells. To further define the role of Bak and Bax in p14(ARF)-induced mitochondrial apoptosis, we employed short interference RNA for the knockdown of bak in isogeneic, p53 wild-type HCT116 colon cancer cells either proficient or deficient for Bax. There, combined loss of Bax and Bak attenuated p14(ARF)-induced apoptosis whereas single loss of Bax or Bak was only marginally effective, as in the case of DU145. Notably, HCT116 cells deficient for Bax and Bak failed to release cytochrome c and showed attenuated activation of caspase-9 (LEHDase) and caspase-3/caspase-7 (DEVDase) upon p14(ARF) expression. These data indicate that p14(ARF) triggers apoptosis via a Bax/Bak-dependent pathway in p53-proficient HCT116, whereas Bax is dispensable in p53-deficient DU145 cells. Nevertheless, a substantial proportion of p14(ARF)-induced cell death proceeds in a Bax/Bak-independent manner. This is also the case for inhibition of clonogenic growth that occurs, at least in part, through an entirely Bax/Bak-independent mechanism.

The death ligand TRAIL has been suggested as a suitable biological agent for the selective induction of cell death in cancer cells. Moreover, TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation (IR). Here, we show that synergy of TRAIL and IR, that is, crosssensitization between TRAIL and IR for induction of apoptosis, entirely depends on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to activate caspase-3 and -9 when exposed to TRAIL and IR. In contrast, TRAIL sensitized for IR-induced apoptosis and vice versa upon reconstitution of Bax expression. Notably, both DU145 and HCT116 still express significant levels of the multidomain proapoptotic Bcl-2 homolog Bak. This indicates that Bak is not sufficient to mediate crosssensitization and synergism between IR and TRAIL. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade upon DNA damage by IR.

The human INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16 INK4a and p14 ARF (p19 ARF in the mouse), which are frequently inactivated in human cancer. Both the proapoptotic and cell cycle-regulatory functions of p14 ARF were initially proposed to be strictly dependent on a functional p53/mdm-2 tumor suppressor pathway. However, a number of recent reports have implicated p53-independent mechanisms in the regulation of cell cycle arrest and apoptosis induction by p14 ARF. Here, we show that the G1 cell cycle arrest induced by p14 ARF entirely depends on both p53 and p21 in human HCT116 and DU145 carcinoma cells. In contrast, neither loss of p53 nor p21 impaired apoptosis induction by p14 ARF as evidenced by nuclear DNA fragmentation, phosphatidyl serine exposure, and caspase activation, which included caspase-3/7- and caspase-9-like activities. However, lack of functional p21 resulted in the accumulation of cells in G2/M phase of the cell cycle and markedly enhanced p14 ARF-induced apoptosis that was, nevertheless, efficiently inhibited by the cell permeable broad-spectrum caspase inhibitor zVAD-fmk (valyl-alanyl-aspartyl-(O)-methyl)-fluoromethylketone). Thus, loss of cell cycle restriction point control in the absence of p21 may interfere with p14 ARF-induced apoptosis. Finally, these data indicate that the signaling events required for G1 cell cycle arrest and apoptosis induction by p14 ARF dissociate upstream of p53.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.

Deregulation of cell-cycle G(1)-restriction point control by disruption of Rb-pathway components is a frequent event in cancer. In concert with the inactivation of cell death pathways, such events not only contribute to tumor development but also determine the intrinsic and acquired resistance to cancer therapy and, ultimately, disease prognosis. We previously observed that the cyclin-dependent kinase inhibitor p16(INK4a) and the proapoptotic Bcl-2 homolog Bax are positive prognostic factors and identify patients with good prognosis in esophageal squamous cell carcinoma (SCC). In the present study, we therefore extend our analysis to additional genes controlling the G(1) restriction point and apoptosis, respectively. This retrospective analysis was performed in a cohort of 53 patients undergoing surgery for esophageal SCC with curative intent, i.e., R0 resection. Protein expression profiles of cyclin D1, p16(INK4a), Rb, p21(CIP/WAF-1), p53, Bax and Bcl-2 were analyzed by immunohistochemistry and compared to p53 mutational status, as determined by SSCP-PCR of exons 5-8. Loss of p16(INK4a), Rb, p21(CIP/WAF-1) or Bax and overexpression of cyclin D1 were associated individually with shorter overall survival, while Bcl-2 expression and p53 mutation were not of prognostic relevance. The longest survival was observed in a subgroup of patients whose tumors bore a combination of favorite genotypes, i.e., low cyclin D1 and high Rb, p21(CIP/WAF-1), p16(INK4a) and Bax protein expression. These results show that multigene analyses based on limited sets of functionally linked genes reliably identify patients with good vs. poor prognosis.

For patients with acute myeloid leukemia (AML) early achievement of remission during induction treatment is an important predictor for long-term outcome irrespective of the type of consolidation therapy employed. Here, we retrospectively examined the prognostic impact of early remission (ER) versus delayed remission (DR) in a cohort of 132 AML patients with an intermediate risk karyotype undergoing allogeneic stem cell transplantation (alloSCT) in first complete remission (CR1). In contrast to patients showing DR, patients achieving ER had a significantly higher 3-year overall survival (OS) and disease-free survival (DFS) of 76% versus 54% (p=0.03) and 76% versus 53% (p=0.03). Likewise, three years after alloSCT the cumulative incidence of relapse (CI-R) was significantly lower in the ER subgroup as compared to patients achieving DR, i.e. 10% versus 35% (p=0.004), whereas non-relapse mortality (NRM) did not differ significantly. Multivariate analysis identified DR as an independent prognosticator for an inferior DFS (HR 3.37, p=0.002) and a higher CI-R (HR 3.55, p=0.002). Taken together, these data may indicate that the rapid achievement of remission predicts a favorable outcome in patients with intermediate risk AML undergoing alloSCT in CR1. In turn, the adverse effect of DR may not be fully overcome by alloSCT.

Background Allogeneic stem cell transplant recipients are prone to infections by various organisms. Tuberculosis (TB) represents a rare infectious complication, especially in countries non-endemic for TB. Case report Here, we report the case of a German patient with exposure to TB decades before he was diagnosed with disseminated TB as well as synchronous Epstein-Barr virus associated lymphoproliferative disorder and cytomegalovirus infection after allogeneic stem cell transplantation for refractory acute myeloid leukemia. Tuberculostatic and virostatic therapy was administered and the patient could be discharged with no apparent signs of infection two weeks after initiation of therapy. Conclusion This case illustrates the need for awareness of mycobacterial infections in patients from non-endemic regions undergoing stem cell transplantation even if other reasons for fever are present.

B-cell immune dysfunction contributes to the risk of severe infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Delayed B-cell regeneration is found in patients with systemic graft-versus-host disease (GVHD) and is often accompanied by bone marrow (BM) suppression. Little is known about human BM GVHD. We analyzed the reconstitution kinetics of B-cell subsets in adult leukemic patients within 6 months after allo-HSCT. B-cell deficiency already existed before transplant and was aggravated after transplant. Onset of B-cell reconstitution characterized by transitional B-cell recovery occurred either early (months 2-3) or late (from month 6 on) and correlated highly positively with reverse transcription-polymerase chain reaction quantified numbers of κ-deleting recombination excision circles (KRECs). Delayed recovery was associated with systemic acute GVHD and full-intensity conditioning therapy. Histological analysis of BM trephines revealed increased T-cell infiltration in late recovering patients, which was associated with reduced numbers of osteoblasts. Functionally, late recovering patients displayed less pneumococcal polysaccharide-specific immunoglobin M-producing B cells on ex vivo B-cell activation than early recovering patients. Our results provide evidence for acute BM GVHD in allo-HSCT patients with infiltrating donor T cells and osteoblast destruction. This is associated with delayed B-cell reconstitution and impaired antibody response. Herein, KREC appears suitable to monitor BM B-cell output after transplant.

Introduction: Allogeneic stem cell transplantation (alloSCT) has become an integral part in the therapy of patients with malignancies of the lympho-hematopoietic system. One of the main reasons for treatment failure after alloSCT is relapse of the underlying disease, which, in the majority of cases, is associated with a poor prognosis. Adoptive immunotherapy by the use of donor lymphocyte infusions (DLI) was shown to be effective in this setting. However, the conditions and the optimal timing of DLI administration for prophylaxis or treatment of (impending) relapse remains controversial. Patients and Methods: We retrospectively analyzed 160 consecutive patients (median age: 48 (range: 17-69) years) who received DLI after previous alloSCT performed at our center between 1998 and 2014. Indications for alloSCT were: acute myeloid leukemia (AML) (N=68), acute lymphoblastic leukemia (ALL) (N=49), myelodysplastic syndrome/myeloproliferative neoplasia (MDS/MPN) (N=26), or myeloma/lymphoma (N=17). The disease risk index (DRI) was low (N=1), intermediate (N=101), high (N=43), or very high (N=6) (unknown: N=9). Comorbidities, as specified by the hematopoietic cell transplantation-specific comorbidity index (HCT-CI), were low (N=38), intermediate (N=79), or high (N=38) (unknown: N=5). In N=71 patients a 10/10 human leukocyte antigen (HLA) matched-related donor was chosen, whereas N=89 patients were transplanted from an unrelated donor, either matched (N=73) or mismatched (N=16). Conditioning was either myeloablative (MAC) (N=71) or reduced-intensity (RIC) (N=89). The median interval from alloSCT to first DLI was 7.1 (range: 1.0-93.2) months. Indication for DLI was prophylactic (e.g. high-risk of relapse or active disease at the time of alloSCT) (N=28), pre-emptive (e.g. persistent or increasing mixed chimerism/molecular relapse) (N=86), or hematologic relapse (N=46). Pre-treatment before DLI was none/cessation of immunosuppression (N=129), lymphodepleting chemotherapy (N=16), or other (N=15). The median number of DLI units given was 2 (range: 1 - 6) and the median cumulative CD3+ cell dose/kg body weight given was 1.1 x 10E7 (range: 5.0 - 16.0 x 10E7). Results: The median follow-up of all patients from day of alloSCT was 37.3 (range: 3.0 - 202.6) months, whereas the median follow-up from day of first DLI administration was 21.2 (range: 0.3 - 200.5) months. Overall survival (OS) of the entire cohort at 1, 3, and 5 years after alloSCT was 80.5%, 63.8%, and 57.7%. Calculated from the day of first DLI OS at the same time points was 68.8%, 61.0%, and 55.8%. At five years after alloSCT OS in the group of patients with AML or ALL was significantly lower as compared to patients with MDS/MPN or myeloma/lymphoma, i.e. 52.0% versus 66.2% (p=0.043). Furthermore, OS in the group of patients receiving pre-emptive DLI was virtually identical to patients who received prophylactic DLI, i.e. 70.4% versus 69.8% at 5 years. In contrast, patients with hematologic relapse prior to DLI had an inferior outcome, i.e. an OS of 23.3% at 5 years. In addition to indication for DLI administration, i.e. prophylactic or pre-emptive versus therapeutic, the occurrence of chronic graft-versus-host disease (cGvHD) was the strongest predictor for outcome, i.e. long-term survival. Conclusions: Taken together, our data indicate that adoptive immunotherapy by the use of DLI is capable of inducing long-term remissions in patients after alloSCT. As pre-emptive and prophylactic treatment yielded virtually identical results, latter may be reserved for selected patients with (very) unfavorable disease characteristics, e.g. AML or ALL with active disease at the time of transplant. The optimal type of pre-treatment needs to be determined by investigating larger patient cohorts. Disclosures No relevant conflicts of interest to declare.

Using a dominant-negative mutant receptor (DNR) approach in transgenic mice, we have functionally inactivated transforming growth factor-beta (TGF-beta) signaling in select epithelial cells. The dominant-negative mutant type II TGF-beta receptor blocked signaling by all three TGF-beta isoforms in primary hepatocyte and pancreatic acinar cell cultures generated from transgenic mice, as demonstrated by the loss of growth inhibitory and gene induction responses. However, it had no effect on signaling by activin, the closest TGF-beta family member. DNR transgenic mice showed increased proliferation of pancreatic acinar cells and severely perturbed acinar differentiation. These results indicate that TGF-beta negatively controls growth of acinar cells and is essential for the maintenance of a differentiated acinar phenotype in the exocrine pancreas in vivo. In contrast, such abnormalities were not observed in the liver. Additional abnormalities in the pancreas included fibrosis, neoangiogenesis and mild macrophage infiltration, and these were associated with a marked up-regulation of TGF-beta expression in transgenic acinar cells. This transgenic model of targeted functional inactivation of TGF-beta signaling provides insights into mechanisms whereby loss of TGF-beta responsiveness might promote the carcinogenic process, both through direct effects on cell proliferation, and indirectly through up-regulation of TGF-betas with associated paracrine effects on stromal compartments.

The p14(ARF) tumor suppressor triggers cell death or cell cycle arrest upon oncogenic stress. In MCF-7 breast carcinoma cells, expression of the tumor suppressor gene p14(ARF) fails to trigger apoptosis but induces an arrest in the G1 and, to a lesser extent, in the G2 phase in the cell division cycle. Here, inhibition of cell cycle arrest resulted in apoptosis induction in caspase-3 proficient MCF-7 cells upon expression of p14(ARF) . This occurred in the absence of S-phase progression or mitotic entry. In contrast, syngeneic, caspase-3-deficient MCF-7 cells remained entirely resistant to p14(ARF) -induced apoptosis. Thus, cell cycle checkpoint abrogation overcomes resistance to p14(ARF) -induced cell death and promotes cell death via a caspase-3-dependent pathway. Cell death coincided with dissipation of the mitochondrial membrane potential, release of cytochrome c, and was inhibitable by pan-caspase inhibitors and the caspase-3/7 inhibitor zDEVD-fmk. Of note, mitochondrial events of apoptosis execution depended entirely on caspase-3 proficiency indicating that caspase-3 either acts "up-stream" of the mitochondria in a "non-canonical" pathway or mediates a mitochondrial feedback loop to amplify the apoptotic caspase signal in p14(ARF) -induced stress signaling.

The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.

Introduction: In acute myeloid leukemia (AML) genetic risk factors are among the strongest predictors for overall outcome. Recently, the European Leukemia Net (ELN) proposed a revised classification based on the presence or absence of specific cytogenetic and/or molecular aberrations. Here, we evaluated the prognostic significance of this system in patients with AML undergoing allogeneic stem cell transplantation (alloSCT). Patients and Methods: A total of 363 patients transplanted at our center between 2004 and 2014 was retrospectively evaluated. According to the ELN classification genetic risk was favorable (N=51), intermediate-1 (N=120), intermediate-2 (N=98), or adverse (N=94). Remission status at the time of alloSCT was first complete remission (CR1) (N=204), CR>1 (N=61), or refractory (N=98). In 107 patients standard myeloablative conditioning (MAC) was used, whereas reduced intensity conditioning (RIC) was applied in 256 patients. Grafts were from either related (N=103) or unrelated (matched: N=191, mismatched: N=69) donors. The median age was 52 (range: 18-75) years. Results: For the surviving patients the median follow-up was 30 (range: 3-129) months. Whereas in the subgroup of patients aged ≥60 years (N=98) no significant differences in disease-free survival (DFS) or cumulative incidence of relapse (CI-R) between the 4 ELN subgroups were found, the ELN classification was highly predictive for in the subgroups aged Conclusions: Taken together, our data suggest that the ELN classification of genetic risk is suitable for predicting relapse and overall survival of patients with AML aged Disclosures No relevant conflicts of interest to declare.

Induction of cell death by p14(ARF) is mediated through a Bax/Bak-dependent mitochondrial apoptosis pathway. To investigate the upstream signaling events required for the activation of Bax and/or Bak and to determine the functional impact of de-regulated cell cycle restriction point control in this context, we genetically dissected the impact of BH3-only proteins and the role of the cyclin-dependent kinase (cdk) inhibitor p21(CDKN1). Using isogenic HCT116 colorectal cancer cells, either wild-type or homozygously deleted for the BH3-only protein Puma/bbc3 and/or p21(CDKN1) or p53-reconstituted DU145 prostate cancer cells, we show that p14(ARF)-induced apoptosis is attenuated in the absence of Puma. Upon expression of p14(ARF) in HCT116 cells, Puma is rapidly induced at both the mRNA and protein level. Puma-proficient HCT116 cells undergo apoptotic (nuclear) DNA fragmentation, which is preceded by the N-terminal conformational change of Bax, the breakdown of the mitochondrial membrane potential, and induction of caspase-9 (LEHD)-like and caspase-3/7 (DEVD)-like activities. In contrast, p14(ARF)-induced apoptosis is markedly attenuated in isogenic HCT116 cells bi-allelically deleted for puma. The sensitivity of Puma-deficient cells to p14(ARF)-induced apoptosis is fully restored by functional reconstitution of Puma using a conditional adenoviral expression vector. Notably, the concomitant deletion of p21(CDKN1) strongly enhances p14(ARF)-induced apoptosis in Puma-proficient cells, but not in isogenic Puma-deficient cells. These results indicate that p14(ARF)-induced mitochondrial apoptosis critically depends on the BH3-only protein Puma. In the presence of a functional p53/Puma/Bax-signaling axis, p14(ARF)-triggered apoptosis is enhanced by loss of p21(CDKN1)-mediated cell cycle checkpoint control.

B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)–only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1–Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.

Purpose: Acute myeloid leukemia (AML) is a highly heterogeneous disease. In addition to patient-related factors (co-morbidities, age, physical performance) and disease-specific variables (genetic risk profile) response to treatment is an important prognostic factor. In particular, the rapid achievement of hematologic remission by induction therapy was shown to predict overall outcome irrespective of the type of post-remission therapy employed. Here, we specifically investigated the impact of achieving early remission (ER), i.e. absence of leukemic blasts in the bone marrow at the end of the first course of induction therapy, as compared to delayed remission (DR) on the outcome of patients with AML who underwent allogeneic stem cell transplantation (alloSCT) in first complete remission (CR1) at our center. Patients and Methods: We retrospectively analyzed 132 consecutive patients (ER: N=79, DR: N=53) with AML and an intermediate risk karyotype transplanted at our center between 1994 and 2013. Median age at alloSCT was 48 years. Before referral to transplantation, all patients were treated in a German multicenter AML trial. Patients aged Results: After a median follow-up of 56 (4-220) months for the surviving patients, 87 patients (66%) are alive and in CR. 26 patients (20%) relapsed after a median interval of 8 (1-133) months, whereas 19 patients (14%) died from NRM. Projected overall survival (OS) of the entire cohort after 1, 3, 5 and 10 years was 81%, 68%, 65%, and 61%. At the same time points the cumulative incidence of relapse (CI-R) or non-relapse mortality (CI-NRM) was 12%, 19%, 22%, and 22% or 13%, 13%, 13%, and 17%. In contrast to patients showing DR, patients achieving ER had a significantly higher 3-year OS and disease-free survival (DFS) of 76% versus 54% (p=0.03) and 76% versus 53% (p=0.03). Likewise, 3 years after alloSCT the CI-R was significantly lower in the ER subgroup as compared to patients achieving DR, i.e. 10% versus 35% (p=0.004). In contrast, NRM did not differ significantly between the ER and the DR subgroup. Multivariate analysis identified DR as an independent prognosticator for an inferior DFS (HR 3.37, p=0.002) and a higher CI-R (HR 3.55, p=0.002). Notably, there was no significant difference in OS, DFS, or CI-R between patients treated with MAC versus RIC in the ER or the DR subgroup. Conclusions: Taken together, these data indicate that rapid achievement of remission may predict a favorable outcome in patients with intermediate risk AML undergoing alloSCT in CR1. In turn, the adverse effect of DR may not be necessarily overcome by alloSCT. Disclosures No relevant conflicts of interest to declare.

There is an ongoing controversy regarding the relevance of apoptosis induction by ionizing irradiation as compared with other end points including transient or permanent cell cycle arrest of damaged cells. Here, we show that such permanent cell cycle arrest and apoptosis represent two sides of the same coin. MCF-7 cells fail to express procaspase-3, which results in resistance to apoptosis induced by anticancer drugs. Conversely, restoration of procaspase-3 sensitizes MCF-7 cells to chemotherapeutics including epirubicine, etoposide and taxol. In contrast, irradiation does not trigger apoptotic cell death but results in prolonged arrest in the G2 phase of the cell division cycle regardless of procaspase-3 expression. This suggested that the propensity of MCF-7 cells to arrest at the G2 checkpoint results in resistance to apoptosis upon gamma-irradiation. This G2 arrest was associated with upregulation of p21CIP/WAF-1. Inhibition of DNA-damage-induced stress kinases and p21CIP/WAF-1 expression by caffeine abrogated G2 arrest and induced apoptosis of the irradiated cells in a caspase-3-dependent manner. Inhibition of cell cycle progression by adenoviral expression of the cyclin dependent kinase inhibitor p21CIP/WAF-1 prevented apoptosis upon caffeine treatment indicating that cell cycle progression, that is, G2-release, is required for induction of apoptosis. Likewise, cells homozygously deleted for p21CIP/WAF-1 (HCT116 p21-/-) display enhanced irradiation-induced apoptosis via a caspase-3-dependent mechanism. These data indicate that the disruption of G2 checkpoint control overcomes cell cycle arrest and resistance to gamma-irradiation-induced cell death. Thus, DNA damage may trigger a permanent G2 arrest as an initial inactivation step of tumor cells where the phenomenon of apoptosis is hidden unless cell cycle arrest is overcome. The efficient induction of apoptosis upon G2 release thereby depends on the propensity to activate the key executioner caspase-3. This finding is of crucial importance for the understanding of molecular steps underlying the efficacy of ionizing radiation to delete tumor cells.

Comorbidity after allogeneic hematopoietic stem cell transplantation (alloHSCT) significantly impairs quality of life (QoL), physical functioning (PF), and survival. Therefore, we evaluated a newly developed measure of comorbidity after transplantation, the Post-transplant Multimorbidity Index (PTMI) within a multicenter trial validating the NIH consensus criteria for severity assessment of chronic graft-versus-host disease (cGVHD). Methods 189 patients (pts) (median age 44 years, range 18-72) after alloHSCT were prospectively evaluated using the NIH consensus criteria, including the NIH-cGVHD grading form, the FACT-BMT, the Human Activity Profile (HAP), and the SF-36 v.2. The PTMI and the Hematopoietic Cell Transplantation-Comorbidity Index (HCT-CI) (Sorror et al) was retrospectively documented by chart review. Enrolment occurred a median of 275 (range: 85–4003) days after alloHSCT. Based on NIH criteria, 121 pts had cGVHD (mild n=24, moderate n=59, severe n=38). Sixty eight pts without cGVHD were included as controls. Results The most prevalent comorbidities were infections treated as outpatient during the past 3 months (n=53; 28%) followed by mild renal function impairment (n=42; 22%), osteoporosis (DEXA t-score< -1.5) (n=27; 14%) and severe infections requiring hospitalization during the past 3 months (n=23; 12%). Applying the PTMI 47 (24%) pts had no comorbidity, while 107 (56%) pts. had 1-3 comorbidities, 24 (13%) pts 4-6 comorbidities, and 11 (6%) pts > 6 comorbidities. In the subgroup of pts with cGVHD (n=72) enroled after more than 1 year post alloHSCT 12.5% of pts had > 6 comorbidities detected by the PTMI. In contrast the HCT-CI classified only 4% of the 72 pts. as multimorbid with > 6 comorbidities. Chronic GVHD and time after transplant were significantly associated with PTMI, while age was not significant in a multivariate analysis. In contrast, the HCT-CI was not associated with the presence of cGVHD. None of the single comorbidities alone were significantly associated with having cGVHD, although osteoporosis (p=0.084) and moderate infections not requiring inpatient treatment during the last 3 months (p=0.07) approached significance. Examining the association between the PTMI sum score and QoL (FACT-BMT), physical and mental health (SF-36) and activity (HAP), a significant correlation was detected between PTMI sum score with all subscales of the FACT-BMT. Similarly, significant correlations were observed between the PTMI sum score and 6 of the 8 subscales of the SF-36 (physical functioning, role limitations due to emotional problems, mental health, vitality, general health perception and change in health), while comorbidity did not impair social functioning and role limitation due to physical problems. The PTMI sum score was also correlated with the HAP adjusted activity score (AAS) but not the maximal activity score (MAS). Osteoporosis was a significant independent predictor of impairments in QoL, physical and mental health, and activity. Infectious complications treated as outpatient during the past 3 months significantly impaired social/family and emotional wellbeing as captured by the FACT, a similar effect was detected by the social functioning and general health perception subscales of the SF36. The HCT-CI score was significantly associated with FACT-BMT and HAP-AAS, and with only 2 of the 8 SF-36 subscale scores (vitality and mental health). Conclusions Comorbidity burden in alloHSCT survivors is significant, and the summative score from a new measure of post-transplant comorbidity captured a wider range of comorbid conditions and was significantly associated with the presence of cGVHD, and with impairments in QoL, physical and mental health, and PF. The HCT-CI also demonstrated associations between comorbidity and QoL, although as a measure developed primarily to predict early post-transplant mortality it missed common long term comorbidities like metabolic bone diseases and does not cover a recent history of infectious complications. Our results provide preliminary support for a new measure of comorbidity developed specifically for the post-transplant population. A multicenter trial prospectively validating the PTMI is ongoing. Disclosures: No relevant conflicts of interest to declare.

Graft-versus-host disease (GvHD) and severe infections are main complications limiting the success of allogeneic hematopoietic stem cell transplantion (alloHSCT). Delayed B cell reconstitution followed by B cell immune dysfunction considerably contributes to an increased risk for life-threatening infections. Several studies have shown that B cell regeneration is impaired in patients with systemic GvHD. Bone marrow (BM) suppression is often observed in parallel as GvHD symptoms appear suggesting the BM as a target of GvHD. Thus far, little is known about mechanisms of BM dysfunction during GvHD in alloHSCT patients. In this study, we investigated the reconstitution kinetics of peripheral blood B cell subsets in adult acute leukemic patients (n=52) before and within six months after alloHSCT by flow cytometry and correlated the data with RT-PCR quantified numbers of kappa-deleting-recombination-excision-circles (KREC), which are stable episomal plasmids generated during BM B cell development. Furthermore, we determined specific B cell antibody responses after in vitrostimulation with CpG, CD40L and T cell cytokines by EliSPOT analysis. To investigate BM as a direct target of allo-reactive T cells we performed histopathological stainings of BM biopsy samples obtained 3-4 weeks after alloHSCT. T cells were detected by specific anti-CD3 antibody staining and osteoblasts were morphologically evaluated. We observed in all patients a profound B cell immune deficiency already pre-transplant that proceeded within the first months post alloHSCT (mean B cells/ml blood±SEM: 11±3 pretransplant, 3±1 day14, 3±1 day28 post alloHSCT; 83±13 healthy control). Onset of B cell reconstitution is characterized by transitional B cell recovery representing the first B cell subset which emigrates from the BM. B cell reconstitution occurred either early (37% of patients) with a strong increase of transitional B cells between days 60-90 (mean transitional B cells/ml blood±SEM: Day 60, 36±10) or late (33% of patients) with delayed recovering transitional B cells (Day 180, 5±2). KREC copy numbers correlated highly positive and significantly with transitional B cell numbers (Spearman 0.94, p=0.017). Less correlation was obtained with naïve and CD27+ memory B cell recovery. Delayed onset of B cell reconstitution was significantly associated with both presence of systemic acute GvHD and full-intensity conditioning therapy (GvHD 71% vs non-GvHD 32%, Fisher´s exact p=0.044; full-intensity 41% vs reduced-intensity 5%, p=0.016). Supporting the hypothesis of bone marrow GvHD we could show a stronger infiltration of CD3+ T cells in the BM in late than in early recovering patients (≥5% T cell infiltration: 64% vs 17%, p=0.010). This increased T-cell infiltration was associated with reduced numbers of osteoblasts, known in mice to support B cell lymphopoiesis (no/few osteoblasts: 65% vs 17%, p=0.011). Impaired B cell lymphopoiesis further resulted in a delayed naïve and IgM memory B cell recovery compared to early recovering patients. No recovery of switched-memory B cells was seen for both patient groups within the analyzed time-period. Functionally, ex vivoactivation of patient B cells revealed higher numbers of IgM producing B cells specific for pneumococcal polysaccharide (PnP) at day 180 post alloHSCT in early than in late recovering patients. Polyclonal IgG producing B cells were significantly diminished in all patients. We conclude from these data, that early onset of B cell reconstitution is characterized by strong increase in regenerating transitional B cells within three months after alloHSCT. Herein, KREC appears as a suitable biomarker to monitor BM B cell output post-transplant. B cell regeneration is significantly delayed in patients showing increased occurrence of systemic acute GvHD and stronger T cell infiltration with loss of osteoblasts in the BM. Thus, delayed onset of B cell reconstitution might result from acute BM GvHD in which alloreactive T cells lead to an osteoblast niche destruction. Increased PnP specific IgM antibody responses are most likely result of higher numbers of early reconstituted transitional and IgM memory B cells but not naïve B cells that were shown not to produce IgM upon CpG stimulation (Capolunghi F et al. 2008). Thus, early B cell reconstitution might provide a first natural antibody immunity after alloHSCT, emphasizing the importance of a functional bone marrow niche. Disclosures: No relevant conflicts of interest to declare.

Stem cell transplantation (SCT) has contributed to improved overall survival particularly in high risk adult ALL patients and the indication for SCT is a matter of ongoing discussion. We report the results obtained over a period of 18 years in a large center. Patients received first line treatment according to three consecutive trials of the German Multicenter Study group for Adult ALL (GMALL) and risk stratification was performed accordingly. In the most recent trial patients with high risk and very high risk factors are candidates for allogeneic stem cell transplantation (SCT) in CR1 from matched sibling or unrelated donors. The risk factors are: very high risk (VHR): Philadelphia chromosome positive ALL (Ph+ALL); high risk group (HR) of B-lineage ALL (HR-B): leukocytes > 30/nl at diagnosis and/or delayed CR > 3 weeks (other B) or pro B-ALL and of T-lineage ALL (HR-T): early-T-All and mature T-ALL. In addition standard risk patients were candidates for SCT in case of molecular failure or molecular relapse (SR-Mol). 106 high risk ALL patients in CR1 underwent allogeneic SCT between 1995 and 2012 at our center. Median age was 38 years (17 – 67). 40/106 patients had Ph+ALL (VHR), 57/106 patients were HR patients (HR-B n = 32: pro-B-ALL n = 15, other B n = 17; HR-T n = 25: early T n = 17, mature T n = 4, thymic n = 4). 9/106 patients belonged to the SR-Mol group. Stem cell donors were matched related donors (MRD) in 42 patients and compatible unrelated donors in 64 patients. The conditioning regimen was 12 Gy TBI ± Cyclophosphamide or VP16 in 86 patients, other n = 1, and in patients > 55 years 8 Gy TBI + Fludarabine (n = 5) or Fludarabine + Busulfan (n = 14). ATG as GvHD and rejection prophylaxis was given since 5/2005 in all patients with unrelated SCT and in reduced intensity conditioning regimens (RIC). Results 60/106 (57 %) patients with high risk ALL transplanted in CR1 are alive, 46/106 (43 %) patients are dead. The median follow-up of the surviving patients is 64 months (5 – 172). Causes of death were leukemia in 16/106 patients (15%) and transplant-related mortality (TRM) in 30/106 patients (28%), mainly infections ± GvHD (n = 24), secondary malignancies (n = 2) and other (n = 4). In Ph+ALL 19/40 patients (47 %) are alive in CR (CCR), 12/40 are dead due to TRM and 9/40 due to leukemia. In HR-B ALL 23/32 patients (71 %) are in CCR, 8/32 are dead due to TRM and 1/32 due to leukemia. In HR-T ALL 11/25 patients (44 %) are in CCR, 10/25 died due to TRM and 4/25 due to leukemia. In SR-Mol patients 7/9 patients (77 %) are in CCR, 2/9 died due to leukemia. Probability of survival (OS) for all patients at 96 months is 0.48, probability of disease free survival (DFS) at 96 months is 0.47, probability of TRM at 96 months is 0.34. OS was 0.50 for patients with myeloablative conditioning (MAC) (n = 86) and 0.38 for patients with RIC (n = 19), n.s. OS was different for the 4 risk groups: VHR 0.42, HR-B 0.67, HR-T 0.27, SR-Mol 0.74, but not significant (0.095). DFS: VHR 0.42, HR-B 0.68, HR-T 0.24, SR-Mol 0.76 (0.041) and TRM: VHR 0.34, HR-B 0.26, HR-T 0.58, SR-Mol 0. (0.026). In HR-B OS was better for pro-B-ALL than other B-lineage (0.73.vs. 0.66, resp.). OS was lower in early-T-ALL (0.24). In mature T-ALL 3/4 patients are dead, in thymic ALL 1/4 patients. In Conclusion Long term survival and probably cure can be reached in high risk ALL patients after allogeneic stem cell transplantation in CR1. OS in pro-B-ALL and B-lineage-HR-ALL patients is better than in Ph+ALL or in HR-T-ALL due to lower relapse rate and lower TRM. Whether the quality of remission before allogeneic SCT could be improved in Ph+ALL and HR-T-ALL, has to be studied. Allogeneic SCT in SR with molecular failure or relapse show promising data, though in a small patient cohort. For all patients the TRM-rate due to GvHD and infection has to be improved due to better supportive care. Disclosures: No relevant conflicts of interest to declare.

Purpose Cytogenetic abnormalities as detected by conventional karyotyping are among the strongest predictors for the long-term outcome of patients (pts) with acute myeloid leukemia (AML). However, up to 50% of pts are cytogenetically normal (CN) and the screening for mutations of the FLT3 and the NPM1 genes allowed to further dissect this heterogeneous group. Allogeneic stem cell transplantation (alloSCT) has become an integral part of the post-remission therapy for pts with intermediate or high-risk AML in case an HLA-compatible donor is available. Here, we analyzed the impact FLT3 and NPM1 mutations on the outcome of pts with CN-AML who underwent alloSCT at our center between 2006 and 2013. Pts and Methods Follow-up data of all pts were prospectively collected in a computer database and retrospectively analyzed as of June 30th, 2013. 101 pts (46 female, 55 male) with a median age of 54 (range: 18-75) years with CN-AML were included. 71 pts had de novo AML, whereas 30 pts had secondary or therapy-related AML. All pts were treated in a German multicenter AML trial and received at least two courses of induction therapy. The FLT3 and NPM1 mutational status was as follows: 14 pts were FLT3-mutated/NPM1 wild-type, 15 pts were NPM1 mutated/FLT3 wild-type, 22 pts were FLT3-mutated/NPM1 mutated, and 50 pts were wild-type for both FLT3 and NPM1. At alloSCT, 62 pts were in first complete remission (CR1), 16 pts were in CR2, and 23 pts had refractory disease. In 96 pts alloSCT was performed using peripheral blood stem cells (PBSCs), 5 pts received a bone marrow (BM) graft. Conditioning consisted of standard myeloablative conditioning (MAC) (6x2 Gy TBI and 2x60 mg/m2 cyclophosphamide) in 24 pts. 77 pts received reduced intensity conditioning (RIC) (2x4 mg/kg oral busulfan, 6x30 mg/m2 fludarabine and 4x10 mg/kg ATG). A matched related donor was available for 27 pts, whereas 55 pts or 19 pts were transplanted from a matched-unrelated or mismatched unrelated donor. Results After a median follow-up of 11 (range: 1-83) months for the surviving pts, 64 pts are alive and in CR. Causes of death were relapse or NRM in 26 pts or 12 pts, respectively. At 1, 3 and 5 years projected overall survival (OS) or disease-free survival (DFS) of the entire cohort was 67% (57-77%), 58% (46-69%), and 54% (42-67%) or 60% (51-71%), 57% (47-68%), or 50% (37-54%). At the same time points the cumulative incidence of relapse (CIR) or non-relapse mortality (CINRM) was 27% (19-38%), 30% (22-43%), and 37% (36-52%) or 12% (6-21%) remaining stable thereafter. Subgroup analysis showed that the presence of a NPM1 mutation in the absence of mutated FLT3 is associated with a significantly lower CIR, i.e. 8%, at 3 years. In the other subgroups the CIR ranges between 32% for pts lacking FLT3 and NPM1 mutations and 37% for pts carrying both mutations (p=0.002). In univariate analysis, pts with refractory disease had a significantly lower DFS (p=0.0002) and a higher relapse incidence (p=0.0001) as compared to pts transplanted in CR. All other factors examined, i.e. AML subtype, stem cell source, type of conditioning, donor/HLA-match were not associated with OS, DFS, or relapse incidence. Notably, there was no correlation between FLT3 or NPM1 mutational status and remission status at the time of alloSCT. OS did not differ significantly between the four subgroups due to an increased NRM in patients with NPM1-mutated CN-AML. Finally, in multivariate analysis only remission status was identified as an independent prognosticator for DFS and relapse incidence, whereas alloSCT from a mismatched unrelated donor predicted a higher NRM. Conclusions Taken together, our results indicate that pts with high-risk AML, as defined by the presence of a FLT3-mutation, may achieve durable long-term remissions following either MAC and RIC-alloSCT from related an unrelated donors. In turn, the presence of a mutation of the FLT3 gene may not per se predict a poor outcome in this setting. In contrast, remission status is among the strongest predictors for long-term outcome in patients with CN-AML undergoing alloSCT. Disclosures: No relevant conflicts of interest to declare.

TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In ‘type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting ‘type II' to ‘type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the ‘type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

Abstract 1998 Purpose: In patients with newly diagnosed acute myeloid leukemia (AML) rapid achievement of remission by induction chemotherapy is an important predictor for long-term disease control. In turn, patients who fail to attain early blast clearance after the first chemotherapy course have an inferior outcome. Here, we investigated the impact of early blast clearance on the overall outcome of patients with AML undergoing allogeneic stem cell transplantation (alloSCT) in first complete remission (CR1) as consolidation therapy. Patients and Methods: 169 (90 female, 79 male) patients with AML who underwent alloSCT in CR1 at our center between 1994 and 2011 were included. Data were prospectively recorded in our transplant data base and retrospectively analyzed as of December 31st, 2011. In detail, 107 patients (64%) had de novo AML, 31 patients (18%) had AML evolving from myelodysplastic syndrome (MDS), and 31 patients (18%) had therapy-related AML. According to the criteria of the SWOG/ECOG, cytogenetic risk was either favorable (6 patients, 4%), intermediate (104 patients, 62%), or poor (47 patients, 27%). Prior to alloSCT all patients were treated in a German multicenter AML trial and received at least two courses of induction chemotherapy, i.e. either standard “7+3” (daunorubicin 60 mg/m2, day 3–5 and Ara-C 100 mg/m2, day 1–7) or a “high-dose Ara-C” containing regimen (Ara-C 1–3 g/m2). In 98 patients (58%) induction chemotherapy resulted in blast clearance after the first course, whereas 71 patients (42%) failed to achieve early remission, but entered remission after 1 or 2 subsequent courses. Median age at transplantation was 47 years (range: 17–69 years). In 146 patients (86%) alloSCT was performed using peripheral blood stem cells (PBSCs), whereas 23 patients (14%) received a bone marrow (BM) graft. Conditioning consisted of standard myeloablative conditioning (MAC: 6 × 2 Gy TBI and 2 × 60 mg/m2 cyclophosphamide) in 81 patients (48%), whereas 86 patients (52%) received reduced intensity conditioning (RIC: busulfan 2 × 4 mg/kg, fludarabine 6 × 30 mg/m2 and ATG 4 × 10 mg/kg). A matched related donor was available in 82 patients (49%), whereas 68 patients (40%) or 19 patients (11%) were transplanted from a matched-unrelated or mismatched unrelated donor. Results: After a median follow-up of 45 months (range: 3–196 months) for the surviving patients, 91 patients (54%) are alive and in continuous remission. Causes of death were relapse in 38 patients (22%) or NRM in 33 patients (19%). At 1, 3 or 5 years projected overall survival (OS) was 72±6%, 58±6%, or 54±8% for all patients. Probability of relapse or non-relapse mortality (NRM) at 1, 3, and 5 years was 20±10% (20±11%), 31±12% (20±11%), and 34±12% (20±11%). Although there was no statistically significant difference in OS at 3 and 5 years between patients who achieved early blast clearance as compared to patients who failed to do so (p=0.09), disease-free survival (DFS) and probability of relapse differed significantly between the two groups at 3 years (77±8% vs 55±14%) or 5 years (75%±9% vs 52%±14%) following alloSCT (p=0.02). There was no significant difference in NRM between the two subgroups. Likewise, there was no statistically significant difference between patients conditioned with either MAC or RIC. In multivariate analysis cytogenetic risk group and remission status were identified as independent prognostic factors for DFS and probability of relapse. Conclusions: These results suggest that in patients with AML undergoing alloSCT in CR1 early blast clearance, i.e. following the first course of induction chemotherapy, predicts a very favorable outcome. Disclosures: No relevant conflicts of interest to declare.

Abstract 1449 Despite unprecedented success of tyrosine kinase inhibitors (TKI) in treating chronic myeloid leukaemia (CML) patients, optimum clinical management is still impeded by lack of reliable predictive and prognostic markers to identify those at risk of progression from chronic phase (CP) to advanced disease (AD), i.e. accelerated phase (AP) and blast crisis (BC). Significantly, SHP-1 (Src homology 2 domain-containing phosphates-1) is reported to bind to BCR-ABL1. Furthermore, SHP-1 knockout mice fail to thrive and develop a myeloproliferative-like disease, while point mutations have been detected in patients with acute myeloid leukaemia (AML) disorder. These observations are consistent with SHP-1 function as a negative regulator of PI3K-Akt pathway and a tumour suppressor. More recently data implied SHP-1 expression levels were prognostic and predictive of TKI response. Therefore we quantified SHP-1 mRNA levels in CML patients to assess these findings. In addition, we included acute myeloid leukaemia (AML) and adult blood donor samples controls. SHP-1, BCR-ABL1 and GUSβ (endogenous control gene) and MSI2 mRNA levels were retrospectively assessed by Taqman quantitative real time polymerase chain reaction in 78 highly heterogeneous CML patients [median age of 50 years (20–76); M: 44; F: 34]; 54 in CP; 24 in AD (6 in AP and 18 in BC). Among the 78 patients 59 were treated with two or more agents. But 19, all in CP, were prescribed single drug only; Imatinib (n=11); nilotinib (n=6); dasatinib (n=1) and interferon + cytarabine (n=1). Sanger sequencing of the BCR-ABL1 kinase domain in 40 (CP: 27; AD: 13) of the 78 patients identified 18 (CP: 9; AD: 10) with one or more mutations. In 8 patients the mutations mapped to the P-loop, 3 had T315I in isolation or in combination with another KD variant and 7 had non-P-loop variants. Seventyseven diagnostic samples from AML patients (M: 42; F: 35) with a median age of 63 years (8–85) were included and 18 normal control (NC) samples from blood donors with median age 44 years (35–61). Target gene expression levels were reported as ratio to GUSβ. Samples with SHP-1 expression was significantly higher (p= We report here differential expression of the putative tumour suppressor SHP-1 in CML patients in CP and AD, with lower levels in the latter. However, SHP-1 expression in these two groups was higher than that detected in AML and NC samples. In contrast to earlier reports we did not observe a significant difference between those achieving and failing MMR within 18 months nor between patients with different degrees of response to TKI therapy. This variance may have been affected by the different time points of sample acquisition during the course of the treatment. Our data imply SHP-1 regulates or is regulated by BCR-ABL1. Moreover, it is reported that SHP-1 may counteract oncogenic effect of BCR-ABL1, however, we observed no correlation in mRNA levels between them in our 78 CML patients. The data presented here warrant a prospective study to assess whether SHP-1 expression levels is able to identify patients at risk of progression prior to other markers, e.g. BCR-ABL1 copy numbers. Disclosures: le Coutre: Novartis: Consultancy.

Abstract 4547 Introduction: The 2005 NIH consensus criteria (NCC) for acute and chronic graft-versus-host disease (aGVHD, cGVHD) are the gold standard for classifying GVHD in trials but they are not routinely used in the clinic, in part due to uncertainty about their prognostic value. To address this limitation we analyzed NIH-defined GVHD in 147 consecutive acute lymphoblastic leukemia (ALL) patients who received a first myeloablative transplant at our center between 1995 and 2009. Methods: Median age was 31 years (range: 17–56). Disease status was CR1 (50%), CR>1 (22%) or no CR (28%). Myeloablative conditioning consisted of 12 Gy TBI ± etoposide ± cyclophosphamide. Donors were HLA-matched related (42%), -matched unrelated (46%) or -mismatched (12%) and peripheral blood stem cells (74%) or bone marrow (26%) were given. GVHD-prophylaxis consisted of CSA/MTX (72%), CSA/prednisolone (19%) or other CSA-based regimens (9%). ATG was given in HLA-mismatched transplants (12%) and since 2005 also in matched unrelated transplants (11%), according to GMALL study protocols. Preemptive donor lymphocyte infusions (DLI) were a treatment option in case of mixed chimerism or minimal residual disease. Results: Median follow-up was 60 months (8–185). Projected overall survival (OS) at 1, 2 and 5 years was 64%, 56% and 49%. 5-year cumulative incidence of relapse was 33% and of non-relapse mortality (NRM) 25% (7% infections, 9% aGVHD, 7% cGVHD, 2% other). Median time until onset of classic aGVHD was 20 days (5–95). Cumulative incidence of classic aGVHD was 41% for grade I/II and 29% for grade III/IV. Among patients with classic aGVHD, skin, liver or gut involvement was seen in 94%, 32% and 34%. Late aGVHD was observed in 12% at a median of 100 days (range 100–240). Among late aGVHD cases 82% were subclassified as persistent or recurrent classic aGVHD. Median time until onset of chronic GVHD was 115 days (14–294) measured from transplant or DLI with a cumulative incidence for mild, moderate and severe forms of 13%, 15% and 25%. Mouth, skin, eyes, liver, joints and fascia, gut, lung and other organs were involved in 91%, 70%, 61%, 48%, 27%, 19%, 10% and 6% of cGVHD cases. cGVHD was subclassified as classic cGVHD and overlap syndrome in 40% and 60% of cases. 62% had progressive or quiescent type of onset. In multivariate Cox regression analysis with GVHD as time-dependant covariate (table 1) classic aGVHD grade III/IV was associated with inferior OS due to higher NRM. Comparable effects were seen for late aGVHD. In contrast, moderate and severe cGVHD were associated with superior OS due to lower relapse incidence. Classic and overlap cGVHD had no differential prognostic impact. 34 patients without GVHD after cessation of immunosuppression received preemptive DLI. In this subgroup cumulative incidence of classic aGVHD, late aGVHD or cGVHD was 62%, 6% and 62%. Organ involvement was comparable to non-DLI associated GVHD. In a time-dependant multivariate analysis, patients who developed NIH-defined cGVHD after DLI had improved OS (HR 0.21, 95%CI: 0.054–0.81, P=0.023) due to lower relapse incidence (HR 0.26, 95%CI: 0.043–0.91, P=0.048) compared to patients without cGVHD after DLI. Conclusions: This is the first study on the natural history and prognostic impact of NIH-defined GVHD in ALL patients. We found that severe classic aGVHD leads to higher NRM and inferior OS with similar effects seen for late aGVHD. cGVHD had a positive impact on OS and relapse rate, both after transplant and after preemptive DLI, indicating a potent graft-versus-leukemia effect. Although our cohort contains some heterogeneity we believe that this data supports the use of the NCC as diagnostic and prognostic tool in ALL patients. Multivariate Cox regression analysis for OS, relapse and NRM with GVHD as time-dependant covariate. Only results for GVHD are shown. Disclosures: No relevant conflicts of interest to declare.

Abstract 2028 Purpose: A monosomal karyotype, as defined by the presence of two or more autosomal monosomies or a single autosomal monosomy in the presence of at least one structural chromosomal abnormalities (core binding factor abnormalities excluded), was shown to confer to a highly unfavorable prognosis in patients (patients) with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) treated with conventional chemotherapy. Here, we investigated the prognostic impact of a monosomal karyotype on the outcome of patients with AML or MDS following allogeneic stem cell transplantation (alloSCT). Patients and Methods: 254 patients who underwent alloSCT at our center between 1994 and 2010 were retrospectively analyzed. 204 patients (80%) had AML (de novo AML: 167 patients, therapy-related AML (tAML), AML evolving from MDS: 37 patients) and were in CR1 (157 patients (77%) or CR>1 (47 patients (23%). 50 patients had MDS (RA/RCMD: 36 patients, RAEB-I: 7 patients, RAEB-II: 9 patients). Median age was 47 years (range: 17–72 years). 223 patients (88%) received peripheral blood stem cells (PBSCs), 31 patients (12%) received bone marrow (BM). Conditioning consisted of standard myeloablative conditioning (MAC) in 134 patients (53%), whereas 120 patients (47%) received reduced intensity conditioning (RIC). 13 patients (5%) had a core-binding factor leukemia (CBF group), 117 patients (46%) were cytogenetically normal (CN group), 79 patients (31%) had an unfavorable risk MK-negative karyotype (MK– group), 26 patients (10%) had a highly unfavorable MK-positive (MK+ group). In 19 patients (8%) the karyotype was unknown/not evaluable. Results: After a median follow-up of 51 months (range: 3–191 months) for the surviving patients, 134 patients (53%) are alive and in remission. Causes of death were relapse in 53 patients (21%) or NRM in 58 patients (23%). At 1, 3 or 5 years projected OS (DFS) was 70±6% (66±6%), 57±6% (56±6%) or 54±7% (54±7%). At 3 years patients in the MK+ group had a statistically significantly lower OS (DFS) of 29% (29%) as opposed to 52% (52%) in the MK– group, 68% (66%) in the CN group, or 67% (55%) in the CBF-group (p Conclusions: These data indicate that karyotypic abnormalities remain the most important prognostic factors predicting the outcome of patients with AML or MDS. In particular, the presence of a monosomal karyotype provides a strong negative prognostic prediction for these patients undergoing alloSCT. Therefore, our data suggest that these patients should be referred to alloSCT in CR (AML) or early stage disease (MDS). Disclosures: No relevant conflicts of interest to declare.

Abstract 899 Introduction: The classic definition of acute (aGVHD) and chronic graft-versus-host disease (cGVHD) was based on a cut-off day 100 after transplantation, but this did not reflect that aGVHD can occur later and that symptoms of aGVHD and cGVHD can occur simultaneously. In 2005 a NIH consensus classification was proposed which included 1) classic aGVHD, occurring before day 100, 2) persistent, recurrent or late aGVHD occurring thereafter, 3) classic cGVHD and 4) an overlap syndrome with simultaneous features of aGVHD and cGVHD. Only few studies have evaluated this classification and no studies have determined the differential impact of reduced intensity (RIC) and myeloablative conditioning (MAC). Method: We retrospectively analyzed 202 AML patients who were transplanted between 1999 and 2008. 102 patients received RIC (generally 6×30 mg/m2 FLU, 4×4 mg/kg BU, 4×10 mg/kg ATG) and immunosuppression with CSA/MMF and 100 patients received MAC (generally 6×2 Gy TBI and 2×60 mg/kg CY) and CSA/MTX. Donors were HLA-matched related (n=82), -matched unrelated (n=88) or -mismatched (n=32). Result: Leukocyte recovery was faster after RIC than after MAC (14 vs. 19 days, P Conclusion: This study in AML patients shows that previously established GVHD risk factors remain valid for the new NIH classification. It also confirms the major impact of conditioning intensity on GVHD incidence, the negative prognostic impact of severe aGVHD and the benefit of moderate cGVHD. The new category late aGVHD may only include few patients but will allow more adequate allocation to therapies or clinical trials. Whether the subgroups classic and overlap cGVHD are clinically relevant remains to be determined. Disclosures: No relevant conflicts of interest to declare.

Abstract 3470 Introduction: Allogeneic stem cell transplantation (alloSCT) represents a curative treatment option for the post-remission therapy of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The presence or absence of distinct karyotypic abnormalities has a substantial impact on the risk of relapse and overall outcome. In particular, complex, i.e. ≥3, chromosomal aberrations are associated with a poor outcome. Patients and Methods: We retrospectively analyzed 91 patients (47 female, 44 male) with secondary AML or therapy-related AML (n=50; CR1: n=22, CR>1: n=2, no CR: n=24) or MDS (n=41; RA/RCMD: n=14, RAEB-1: n=11, RAEB-2: n=16) who underwent alloSCT at our center between 1995 and 2008. An intermediate-risk karyotype was present in 47 patients, whereas 38 patients had a poor-risk karyotype. Among those, 22 patients had aberrations of chromosome 5 and/or 7. 45 patients were treated with standard myeloablative conditioning (MAC) (12 Gy total body irradiation, 2×60 mg/kg cyclophosphamide) prior to alloSCT, whereas 46 patients received reduced intensity conditioning (RIC) (fludarabine 6×30 mg/m2, busulfan 2×4 mg/kg, anti-thymocyte globulin 4×10 mg/kg). 35 patients had a matched-related donor (MRD) and 37 or 13 patients had a matched-unrelated (MUD) or a mismatched-unrelated (MMUD) donor. Results: After a median follow-up of 58 (12-170) months, 42/91 patients (46%) are alive and in CR. 20/91 patients (22%) succumbed to relapse and 27/91 patients (30%) died from treatment-related mortality (TRM). Projected OS (DFS) at 1, 3, or 5 years was 60% (55%), 52% (53%), or 50% (48%) and TRM was 27%, 27%, or 31%. Patients with a poor-risk cytogenetic profile had a significantly lower DFS as compared to patients with an intermediate-risk cytogenetics, i.e. 57% versus 36% at 5 years (p=0.04). However, within the group of patients with a poor-risk karyotype, those with aberrations of chromosome 5 and/or 7 had significantly lower OS (30% versus 50% at 5 years; p=0.03), or DFS (26% versus 50%; p=0.01) as compared to patients with complex aberrations. In contrast, the OS of patients with complex chromosomal aberrations excluding those who have chromosome 5 and/or 7 aberrations had a similar OS, DFS, or probability of relapse as compared to patients with an intermediate risk karyotype. Of note, in this subgroup there was no statistically significant difference in OS or DFS between patients conditioned with either standard MAC or RIC. Likewise, there was no statistically significant difference between patients transplanted from a MRD or a MUD. Conclusions: Taken together, these results indicate that the presence of a poor-risk karyotype has a substantial prognostic impact in patients with secondary AML, therapy-related AML or MDS undergoing alloSCT. In particular, patients with aberrations of chromosome 5 and/or 7 are highest risk of relapse. Nonetheless, a substantial proportion of these patients may achieve durable remissions following either standard or RIC-alloSCT. Disclosures: No relevant conflicts of interest to declare.

Abstract 4639 Due to the lack of reproducibility and predictive value of former classifications, a new classification for cGVHD was proposed by the NIH in 2005. The former cut off day 100 required to diagnose cGVHD has been omitted and now the diagnosis relies on clinical characteristics. The NIH classification is now evaluated in different transplant centers. Purpose Diagnose and classify cGVHD in a retrospective fashion using the NIH criteria. Evaluate the incidence and clinical characteristics of cGVHD in AML transplanted with a myeloablative conditioning regimen (MAC) or with a reduced intensity conditioning (RIC). Patients and Methods Allogeneic stem cell transplantations for treatment of AML were performed at Charite Berlin, Germany between 12/1994 and 12/2008. 219 patients (pts) were included in the study. cGVHD was diagnosed, classified and graded according to the NIH criteria by Filipovich et al, Biol Blood Marrow Transplant. in 2005. Prognostic value of cGVHD was analyzed using the landmark method. Results The earliest diagnostic feature of cGVHD was found on day 84 post transplantation. 30 pts who died or had active leukemia before day 80 were excluded from the cGVHD analysis, 189 pts could be evaluated. Pts for whom the cGVHD status was unclear after first examination (20% of the sample) were reanalyzed by an independents second examiner. In the RIC group, 55 (62%) pts were transplanted in CR1 and 34 (38%) >CR1. In the MAC group, 56 (56%) pts were transplanted in CR1 and 44 (44%) >CR1. HLA compatibility between donor and recipient was as follows: 35 (39%) pts received a match related transplant (MRD), 36 (41%) pts had a match unrelated transplant (MUD) and 18 (20%) pts had a mismatch unrelated transplant in the RIC group. In the MAC group, 49 (49%) pts received MRD, 1 (1%) patient had a mismatch related transplant, 42 (42%) pts had MUD and 8 pts had a mismatch unrelated transplantation. Bone marrow stem cells were infused in 5 (6%) pts in the RIC group and in 20 (20%)pts in the MAC group. The other pts received peripheral stem cells. The median follow up was 19 months (range 3-96) in the RIC group and 47 months (range 3-146) in the MAC group. In the RIC group 29 (32%) pts developed cGVHD, including 9 pts who progress from late acute GVHD (aGVHD) and in the MAC group, 52 (52%) pts, including 3 pts who progressed from late aGVHD. Median time to develop cGVHD was 165 days (range 95-2385) in the RIC group and 236 days (range 84-1793) in the MAC group. In those pts who developed cGVHD, 39 had a history of aGVHD in the MAC group and 18 in the RIC group. Eleven (38%) pts developed classic and 18 (62%) pts overlap cGVHD in the RIC group, and 19 (37%) pts classic and 33 (63%) pts overlap in the MAC group. Mild, moderate and severe cGVHD was present in 12 (42%), 14 (48%), 3 (10%), and in 14 (27%), 27 (52%), 11 (21%) pts of the RIC and MAC group respectively. In Kaplan Meier Analysis the overall risk for cGVHD was higher in the MAC group in comparison to RIC (p=0.038). The first diagnostic feature of cGVHD was found in the mouth in 23 (79%) pts, skin in 4 (14%) pts and the lung in 2 (7%) of the RIC group and the mouth in 43 (83%) pts, skin in 7 (14%) pts, lung in 1 (1%) pts and muscle in 1(1%) patient in the MAC group. cGVHD was diagnosed on clinical criteria without biopsy in 28 (97%) pts in the RIC group and in 50 (96%) in the MAC group. 19 patients in both groups, who were considered affected by cGVHD in the past according to Shulman classification did not fulfill NIH criteria: 11 pts had late acute GVHD and 8 pts had non diagnostic features. The cumulative incidence of cGVHD at 1 and 2 years was 34% and 46% in the RIC group and 54% and 65% in the MAC group (p=0.038). In the entire sample, overall survival after two years was 61.1% (95%CI 53.5% - 68.7%). No prognostic benefit of cGVHD could be shown for overall survival and only moderate benefit of late cGVHD for disease free survival. Conclusion It is feasible to apply the NIH criteria retrospectively. Most patients can be diagnosed on clinical criteria without biopsy. Nearly 20% of pts diagnosed with cGVHD in the past did not fulfill the NIH criteria. Retrospective studies might show a delay in the onset of cGVHD compared to the expected timeframe of 4-6 months due to the requirement of diagnostic features not considered in the past and the category of late acute GVHD. Overlap and moderate cGVHD were the most common categories. A positive prognostic effect of cGVHD could not be detected for overall survival in our patient group. Disclosures: No relevant conflicts of interest to declare.