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Intensive upfront therapy in newly-diagnosed multiple myeloma (MM) including induction therapy (IT), high-dose melphalan (MEL200), and autologous blood stem cell transplantation (ASCT) followed by consolidation and/or maintenance is mostly restricted to patients up to 65 years of age. Prospective phase III trial data in the era of novel agents for patients up to 70 years of age are not available. The GMMG-MM5 trial included 601 patients between 18 and 70 years of age, divided in three groups for the present analysis: ≤60 years (S1, n = 353), 61–65 years (S2, n = 107) and 66–70 years (S3, n = 141). Treatment consisted of a bortezomib-containing IT, MEL200/ASCT, consolidation, and maintenance with lenalidomide. Adherence to treatment was similar among patients of the three age groups. Overall toxicity during all treatment phases was increased in S2 and S3 compared to S1 (any adverse event/any serious adverse event: S1:81.7/41.8% vs. S2:90.7/56.5% vs. S3:87.2/68.1%, p = 0.05/p = 0.73), overall survival (log-rank p = 0.54) as well as time-to-progression (Gray’s p = 0.83) and non-relapse mortality (Gray’s p = 0.25), no differences were found between the three age groups. Our results imply that an intensive upfront therapy with a bortezomib-containing IT, MEL200/ASCT, lenalidomide consolidation, and maintenance should be applied to transplant-eligible MM patients up to 70 years of age.

The MM5 trial aimed at demonstrating a progression-free survival (PFS) difference in continued vs. response-adapted (in case of complete response, CR) lenalidomide (LEN) maintenance therapy (MT) in newly diagnosed, transplant-eligible multiple myeloma (MM). Patients were equally randomized to receive induction therapy with PAd (bortezomib/doxorubicin/dexamethasone) or VCD (bortezomib/cyclophosphamide/dexamethasone), high-dose melphalan and autologous blood stem cell transplantation, and LEN consolidation, followed by either LEN MT for a fixed duration of 2 years (LEN-2Y) or until achievement of CR (LEN-CR, intention-to-treat population n = 502): arms A1:PAd + LEN-2Y (n = 125), B1:PAd + LEN-CR (n = 126), A2:VCD + LEN-2Y (n = 126), B2:VCD + LEN-CR (n = 125). In the LEN-CR group (B1 + B2), n = 88/17.5% patients did not start or discontinued LEN MT due to CR. There was no PFS (p = 0.60, primary endpoint) nor overall survival (OS) (p = 0.15) difference between the four study arms. On pooled LEN MT strategies, OS (hazard ratio, hazard ratio [HR] = 1.42, p = 0.03) but not PFS (HR = 1.15, p = 0.20) was shorter in LEN-CR (B1 + B2) vs. LEN-2Y (A1 + A2) groups. PFS was shortened on landmark analyses from the start of LEN MT in patients being in CR in the LEN-CR group (LEN-CR vs. LEN-2Y, HR = 1.84, p = 0.02). OS from first progression was shortened in the LEN-CR vs. LEN-2Y group (HR = 1.60, p = 0.01). LEN MT should be applied beyond CR for at least 2 years.

The article Bortezomib-based induction, high-dose melphalan and lenalidomide maintenance in myeloma up to 70 years of age, written by Elias K. Mai, Kaya Miah, Uta Bertsch, Jan Dürig, Christof Scheid, Katja C. Weisel, Christina Kunz, Markus Munder, Hans- Walter Lindemann, Maximilian Merz, Dirk Hose, Anna Jauch, Anja Seckinger, Steffen Luntz, Sandra Sauer, Stephan Fuhrmann, Peter Brossart, Ahmet Elmaagacli, Martin Goerner, Helga Bernhard, Martin Hoffmann, Marc S. Raab, Igor W. Blau, Mathias Hänel, Axel Benner, Hans J. Salwender, and Hartmut Goldschmidt for the German-speaking Myeloma Multicenter Group (GMMG), was originally published Online First without Open Access. After publication in volume 35, pages 809–822, the author decided to opt for Open Choice and to make the article an Open Access publication. Therefore, the copyright of the article has been changed to © The Author(s) 2020 and the article is forthwith distributed under the terms of the Creative Commons Attribution. FUNDING Open Access funding enabled and organized by Projekt DEAL.

Natural killer (NK)/T-cell lymphomas and large granular lymphocyte (LGL) leukemia are rare lymphoproliferative malignancies with fundamental different prognosis. While NK/T-cell lymphoma and NK-cel...

Primary refractory or relapsed acute myeloid leukemia is associated with a dismal prognosis. Allogeneic stem cell transplantation is the only therapeutic option that offers prolonged survival and cure in this setting. In the absence of a matched sibling donor, transplantation from unrelated 10/10 HLA allele-matched or 9/10 HLA allele-mismatched donors and haploidentical donors are potential alternatives. The current study aimed to compare the outcomes of acute myeloid leukemia patients with active disease who received allogeneic stem cell transplantation from a haploidentical donor with post-transplant cyclophosphamide (n=199) versus an unrelated 10/10-matched donor (n=1111) and versus an unrelated 9/10-mismatched donor (n=383) between 2007 and 2014 and who were reported to the European Society for Blood and Marrow Transplantation registry. Propensity score weighted analysis was conducted in order to control for disease risk imbalances between the groups. The leukemia-free survival rates at 2 years of recipients of grafts from a haploidentical donor, an unrelated 10/10-matched donor and an unrelated 9/10-mismatched donor were 22.8%, 28% and 22.2%, respectively (P=NS). In multivariate analysis, there were no significant differences in leukemia-free survival, overall survival, relapse incidence, non-relapse mortality, or graft-versus-host-disease-free relapse-free survival between the three groups. Two predictive factors were associated with a higher relapse incidence: transplantation during first or second relapse compared to primary refractory acute myeloid leukemia and poor cytogenetics. Allogeneic stem cell transplantation may rescue about 25% of acute myeloid leukemia patients with active disease. Importantly, the outcomes of transplants from haploidentical donors were comparable to those from 10/10-matched and 9/10-mismatched unrelated donors. Therefore, a haploidentical donor is a valid option for acute myeloid leukemia patients with active disease.

Systemic mastocytosis (SM), a rare myeloid neoplasm, is caused by the accumulation of abnormal mast cells (MCs) in bone marrow, spleen, gastrointestinal tract, liver, and skin, and is characterized...

Introduction. Malignant diseases treated with allogeneic hematopoietic stem cell transplantation (alloHSCT) predominantly occur beyond the 7 th decade of life. Numerical age per se is not regarded an adverse risk factor in alloHSCT. In an aging society, interventions historically deemed high risk are increasingly used in elder patients. Methods. Epidemiology, outcomes and risk factors of patients aged ≥70 years undergoing alloHSCT in Germany 1999-2019 and registered with the DRST/EBMT database were analyzed retrospectively. Baseline patient, disease, and transplant data were collected from MED-A forms. Centers were contacted to provide additional treatment and follow-up information. Results. Between 1999 and 2019, 1648 patients aged ≥70 years (median 72, range 70-79.7; 585 female) were transplanted in 50 German centers. More than 90% of all patients were transplanted 2010-2019. Centers transplanted between 2 and 192 patients, with 14 centers contributing 100 patients each. Most patients suffered acute leukemia (1084, 65.8%) or MDS/MPN (410, 24.9%). Karnofsky index before start of conditioning was 100% (n=230, 14%), 90% (n=651, 39.5%), 80% (n=480, 29.1%), 70% (n=94, 5.7%), Conclusion. AlloHSCT is increasingly used to treat elder patients in Germany with a sharp increase during the last decade. Age per se is a modest adverse risk factor for adult patients after alloHSCT with slightly increased mortality in patients 70-80 versus those at 60-69. Further research might concentrate on patient selection and further reduction of procedural toxicity. Figure 1 Figure 1. Disclosures Schetelig: Roche: Honoraria, Other: lecture fees; Novartis: Honoraria, Other: lecture fees; BMS: Honoraria, Other: lecture fees; Abbvie: Honoraria, Other: lecture fees; AstraZeneca: Honoraria, Other: lecture fees; Gilead: Honoraria, Other: lecture fees; Janssen: Honoraria, Other: lecture fees . Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding. Stelljes: Pfizer: Consultancy, Research Funding, Speakers Bureau; Medac: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau. Dreger: AbbVie: Consultancy, Speakers Bureau; Bluebird Bio: Consultancy; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy; AstraZeneca: Consultancy, Speakers Bureau; Gilead Sciences: Consultancy, Speakers Bureau; BMS: Consultancy; Riemser: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Speakers Bureau. Wulf: Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Clinigen: Consultancy, Honoraria. Scheid: Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria; Roche: Consultancy; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bethge: Novartis: Consultancy, Honoraria, Speakers Bureau; Kite-Gilead: Consultancy, Honoraria, Speakers Bureau; Miltenyi Biotec: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

Introduction: Cytogenetic changes occur in 50% of patients (pts) with Myelodysplastic Syndromes (MDS). Complex aberrations (cA, = 3 or more) are associated with a very poor outcome. In about 50% of the cases with cA aberrations of the TP53 locus are detectable. Those pts show an even worse outcome with a significantly shortened median overall survival (OS) compared to pts with wildtype TP53 (wtTP53). One of the most common cytogenetic aberrations in MDS is an interstitial deletion of the long arm of chromosome 5 (5q). As an isolated aberration, it is associated with a rather favorable prognosis. As part of a cA, 5q deletions however are assumed to even worsen the prognosis further. We wanted to find out in which prevalence 5q deletions and TP53 changes appear together and how those two factors in combination or not influence the OS of pts with MDS and cA. Methods: 218 pts with MDS or sAML and cA were identified and extensively characterized. 126 of them were diagnosed with MDS, 89 with sAML and 3 with CMML. Cytogenetic analysis by chromosome banding (CBA) and fluorescence in situ hybridization (FISH) of the TP53 locus on 17p as well as sequencing of TP53 either by Sanger or by Next Generation Sequencing was available for all pts. Multicolour FISH (mFISH) was available for 146 pts, SNP array analysis for 42 pts. The median number of cytogenetic aberrations was 8 (range 3-50). At the time of first diagnosis with cA the median age was 72 (range: 29-95). Median OS of the entire cohort was 10.7 months (95% CI: 8.0-16.4). Results: In 146 of 218 pts we found alterations of TP53: a single hit mutation in 32 pts, a single deletion in 22 pts, a combined mutation and deletion in 67 pts and more than 1 mutation in 25 pts. The OS of those 146 pts was 6.6 months compared to 22 months of the pts with wtTP53 (p-value Conclusion: Mutations and/or deletions of TP53 show a strong association with del(5q). Both were frequent in our cohort of 218 pts with MDS and cA. There also was a large intersection of 130 pts with both del(5q) and TP53 alteration. The combination of both changes seems to further worsen the already poor prognosis of pts with MDS and cA. Our observation that those two factors appear together frequently supports the hypothesis that the presence of del(5q) may promote the acquisition of cA. This is in accordance with Hsu´s hypothesis that in small clones with a mono-allelic TP53 mutation a del(5q) may favor the loss of heterozygosity of TP53 which could in a next step lead to a higher complexity of cytogenetic aberrations (Hsu et al, 2019). It is remarkable that the presence of del(5q) in combination with a single hit status of TP53 confers the same bad prognosis compared to multi hit TP53 status (figure 1).We will continue analyzing pts with MDS and cA to examine the influence of different TP53 and 5q alterations on the prognosis, the disease progression and median OS of those pts with cA. Figure 1 Disclosures Platzbecker: Novartis: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Geron: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; BMS: Consultancy, Honoraria.

International audience; Background: Experience using post-transplant cyclophosphamide (PT-Cy) as graft-versus-host disease (GVHD) prophylaxis in allogeneic stem cell transplantation (HSCT) from matched sibling donors (MSD) or unrelated donors (UD) is limited and with controversial results. The study aim was to evaluate PT-Cy as GVHD prophylaxis post-HSCT from MSD and UD transplants. We analyzed 423 patients with acute leukemia who received PT-Cy alone or in combination with other immunosuppressive (IS) drugs as GVHD prophylaxis. Seventy-eight patients received PT-Cy alone (group 1); 204 received PT-Cy in combination with one IS drug—cyclosporine-A (CSA) or methotrexate (MTX) or mycophenolate-mofetil (MMF) (group 2), while 141 patients received PT-Cy in combination with two IS drugs—CSA + MTX or CSA + MMF (group 3). Transplants were performed from 2007 to 2015 and median follow-up was 20 months. Results: Probability of overall survival (OS) at 2 years was 50, 52.2, and 62.4%, for the three groups, respectively, p = 0.06. In multivariate analysis, in comparison to PT-Cy alone, the addition of two IS drugs was associated with reduced risk of extensive cGVHD (HR 0.25, p = 0.02). Use of bone marrow (BM) and anti-thymocyte globulin were independently associated with reduced risk of extensive cGVHD. Prognostic factors for non-relapse mortality (NRM) were the addition of two IS drugs to PT-Cy (HR 0.35, p = 0.04), diagnosis of AML, disease status at transplant, and patient CMV serology. Factors associated with increased OS were the use of PT-Cy with two IS drugs (HR 0.49, p = 0.02), AML, and disease status at transplant. Conclusion: For GVHD prophylaxis in MSD and UD HSCT, the addition of IS drugs to PT-Cy enhances its effect and reduces the risk of severe cGVHD, reducing mortality and improving survival.

Introduction: Most patients with FLT3-ITD-positive AML, who relapse after allogenic stem cell transplantation (allo-SCT) die from their disease. Whether prophylactic FLT3-ITD inhibition with sorafenib can prevent AML relapse and improve outcome of patients in complete hematological remission (CHR) after allo-SCT is unknown and was tested in the SORMAIN trial. Methods: This randomized, double blind, placebo-controlled study was done at 14 centers in Germany and Austria. Patients with FLT3-ITD+ AML, aged 18 years or older, who had undergone allogenic stem cell transplantation from a HLA-matched sibling donor, 10/10 or 9/10 HLA-matched unrelated donor, and who were in confirmed CHR at the time of screening between day +30 and day +100 post allo-SCT, were included. Patients were randomly assigned (1:1) to receive either sorafenib (starting dose: 2 x 1 tbl. [2 x 200mg] qd, increasing every 14d to up to 2 x 2 tbl. [2 x 400mg] qd according to tolerability) or placebo (2 x 1 or 2 tbl. qd) for up to 24 months. Randomization was done centrally. In case of drug related adverse events, study medication could be interrupted, stepwise reduced to a minimum of 2 x 1 tbl. qd, temporarily withheld and recommenced at a lower dose level. FLT3-ITD diagnostics was done centrally at baseline and at time of relapse. In relapsing patients, off-label compassionate use of sorafenib was possible. The primary endpoint was relapse-free survival (RFS) as defined by either hematological relapse or death from any cause. The secondary endpoint was overall survival (OS). We here report the final RFS analysis. The OS results will be unblinded only prior to the ASH meeting and will be reported there. The SORMAIN study was terminated prior to full recruitment because of slow accrual. SORMAIN was registered with the European Clinical Trials Database (EudraCT 2010-018539-16) and the German Clinical Trials Register (DRKS00000591). Results: Between October 29, 2010, and May 17, 2016, 83 patients (41 males, 42 females) were randomized and included in the primary analysis (placebo, n=40; sorafenib, n=43). Median age was 54 years (IQR 47.75 - 61.33) for the entire study population and not significantly different between sorafenib and placebo groups. With a median follow up of 41.8 months after randomization (IQR 24.1 - 42.5), median RFS was 30.9 months (lower bound of 95% CI 5.2 months) in the placebo group versus not reached in the sorafenib group, corresponding to a 2-year RFS of 53,3 % (95% CI 36.5-67.5) in the placebo versus 85.0 % (69.5-93.0) in the sorafenib group (hazard ratio [HR] 0.39, 95% CI; 0.18 -0.85; P=0.0135) (Fig. 1). Overall, sorafenib was well tolerated. The most common grade 3-4 adverse event in both groups was acute GvHD (seven [ 17.5%] in the placebo group vs. nine [20.9%] in the sorafenib group. Conclusion: Sorafenib maintenance therapy after allo-SCT is feasible and significantly reduces the risk of relapse or death in patients with FLT3-ITD positive AML. OS results will be presented at the meeting. Figure 1. Figure 1. Disclosures Burchert: Bristol Myers Squibb: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; AOP Orphan: Honoraria, Research Funding; Novartis: Research Funding. Bug:Amgen: Honoraria; Neovii: Other: Travel Grant; Novartis Pharma: Honoraria, Research Funding; Astellas Pharma: Other: Travel Grant; Jazz Pharmaceuticals: Other: Travel Grant; Celgene: Honoraria; Janssen: Other: Travel Grant. Finke:Riemser: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding; Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding. Stelljes:Pfizer: Consultancy, Honoraria, Research Funding; MSD: Consultancy; JAZZ: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Rollig:Bayer: Research Funding; Janssen: Research Funding. Wäsch:Pfizer: Honoraria. Lang:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Serve:Bayer: Research Funding. Kroeger:Neovii: Honoraria, Research Funding; JAZZ: Honoraria; Sanofi: Honoraria; Celgene: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Götze:JAZZ Pharmaceuticals: Honoraria; Novartis: Honoraria; Takeda: Honoraria, Other: Travel aid ASH 2017; Celgene: Honoraria, Research Funding. Schmid:Jazz Pharma: Honoraria, Other: Travel grant, Speakers Bureau. Wolf:BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bethge:Miltenyi Biotec GmbH: Consultancy, Honoraria, Research Funding; Neovii GmbH: Honoraria, Research Funding.

Angiogenesis has an important role in the pathogenesis and progression of multiple myeloma (MM). MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Therefore, we evaluated the anti-myeloma effect of VEGF small interfering RNA (siRNA) silencing in MM cells and whether it can be augmented by the additional inhibition of the mammalian target of rapamycin (mTOR) by everolimus. We shown that everolimus inhibits cell growth of MM cells and other leukemic cells at low concentrations in a dose-dependent manner. After transfection with VEGF siRNA we observed a reduction of cell growth and VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, JURKAT and RAJI. VEGF siRNA both significantly induced apoptosis and inhibited proliferation in OPM-2 cells (P

Introduction: Complex aberrant karyotypes (CK, ≥3 cytogenetic aberrations, CA) are associated with an unfavorable prognosis and an increased AML transformation rate in MDS. However, even MDS with CK (CK-MDS) are heterogeneous in terms of genetic profile and prognosis. Recently, we demonstrated that a high number of CA as well as mutations in TP53 (TP53mut) are associated with increased risk in CK-MDS (Haase et al, 2019). However, as there is a strong association between CK-MDS and TP53mut, it is still a matter of debate whether the karyotype and TP53mut are prognostically independent genetic markers. Furthermore, loss of heterozygosity (LOH) of 17p13 (TP53LOH), due to loss of genetic material or to copy number neutral LOH (CN-LOH), is also associated with a poor prognosis. We here aimed to characterize TP53mut andTP53LOH in CK-MDS and to elucidate the impact of cytogenetics, TP53mut and TP53LOH on the outcome of CK-MDS. Methods: We included 178 pts with MDS (N=138), CMML (N=5) and secondary AML after MDS (AML with myelodysplasia-related changes, N=35), all with CK. The median precentage of bone marrow (bm) blasts was 11% (range: 0-90%). The median age was 72 yrs (range: 30-95 yrs). The male:female ratio was 1.23:1. The number of CA was determined by banding analysis in all cases. The karyotype was confirmed by multicolor FISH in 134 cases. TP53LOH was verified by FISH analysis of the TP53 locus in 17p13 (146 analyses) and/or molecular karyotyping (MK, 41 analyses). In 144 cases further FISH probes in addition to TP53 were used. TP53mut was identified by NGS (54 cases) or Sanger sequencing (124 cases). Follow-up data for survival analyses were available for 127 pts with MDS and oligoblastic AML with less than 30% bm blasts. Results: The median number of CA was 7 (range: 3-46), 98/178 pts (55%) showed a TP53mut (median VAF: 34%, range: 8-93%) and 64/178 (36%) a TP53LOH (median FISH clone size: 65%, range: 6-99%), including 9 pts with a CN-LOH in 17p13. The CN-LOH was either identified by MK (5/41 pts (12%) where MK was available showed a CN-LOH, 4/5 with TP53mut) or by NGS (4/54 pts (7%) where NGS was available showed a VAF >70% and normal TP53-FISH). In total, a TP53mut and/or a TP53LOH was identified in 116/178 pts (65%). Overall survival (OS) did not significantly differ between CK-MDS with TP53mut only, TP53LOH only, and TP53mut+TP53LOH (Fig.1). Therefore, we merged TP53mut and TP53LOH to TP53altered in all further analyses. Regarding the cytogenetic characterization of pts with TP53altered, the number of CA was significantly higher in pts with TP53altered than in pts with normal TP53 (median 9 CA (range: 3-46) vs 5 CA (range: 3-24), P The number of CA as well as the TP53 status contributed significantly to OS (Fig.2). The presence of anemia (Hb Conclusions: The presence of ≥5 CA is associated with reduced OS in CK-MDS. A TP53mut as well as a TP53LOH both further segregate outcome. The impact of the clone size of TP53mut and TP53LOH on survival is currently being evaluated. Our data imply that the TP53 status (TP53mut and/or TP53LOH) and the complexity of the karyotype are independent prognostic markers. Based on the presence of anemia, the TP53 status (TP53mut and/or TP53LOH), and the number of CA, the individual risk of CK-MDS can be estimated more accurately. This will allow to better tailor treatment decisions for individual pts with CA. Funding (FS): 2017 SGR 288-GRC Disclosures Germing: Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding. Kaivers:Jazz Pharmaceuticals: Other: Travel Support. Kröger:Celgene: Honoraria, Research Funding; DKMS: Research Funding; JAZZ: Honoraria; Medac: Honoraria; Neovii: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Riemser: Research Funding; Sanofi-Aventis: Research Funding. Hertenstein:RS Media: Research Funding. Döhner:Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bug:Hexal: Membership on an entity's Board of Directors or advisory committees; Celgene Neovii: Other: travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Sanofi: Other: travel grants; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees. Nickelsen:Roche Pharma AG: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant; Janssen: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

We examined the molecular characteristics of monocytes of pregnant and non-pregnant women to investigate the molecular effects that are associated with immunoregulation at the maternal-fetal interface. We analyzed molecular features and target genes in monocytes of pregnant women using flow cytometry, real-time PCR and oligonucleotide microarray technology. CD14(high) monocytes and several immune gene members including CD200, CD200R, IDO, IFI27, IL-10 and G0S2 were found to be differentially expressed in monocytes throughout pregnancy. In addition, transcripts within components of the signaling cascade of immune cells (HLA-DRB4, HBEGF, IL-8, CD3D, CCL5), and of several transcription factors (SOCS1, CXCL10, ID1, ID2) were altered in the monocytes of pregnant women. Further studies will be needed to elucidate the biological significance of our observation.

Bronchiolitis obliterans (BO) is a late non-infectious pulmonary complication after allogeneic hematopoietic SCT. Among 982 patients after myeloablative hematopoietic SCT between January 2000 and October 2010, 68 were diagnosed with BO according to NIH criteria. The median onset of BO was 18 months post transplant, 5-year cumulative incidence was 5.8% and 5-year mortality 41%. BO prevalence rate was 10% among all long-term surviving hematopoietic SCT recipients and 12% among chronic GVHD-patients. Chronic GVHD, peripheral SCT and ABO blood group incompatibility were identified as risk factors associated with BO. IgG levels were significantly decreased at the onset of BO (6.7 g/L±0.7, P=0.001), the mean exhaled NO concentrations were lower in BO-patients than in stem cell recipients without BO (14 p.p.b.±0.9 vs 20 p.p.b.±2.1) or healthy controls (25 p.p.b.±2.4, P

A preventive effect of early human cytomegalovirus (HCMV) replication was evaluated in 136 non-Hodgkin lymphoma (NHL) patients with mature B-cell NHLs (n = 94), and mature T- and NK-cell NHLs (n = 42) after allogeneic stem cell transplantation (alloSCT). Most study-patients (85%) had received at least 2 cycles of chemotherapy and 60% had also received an autograft prior to alloSCT. First detection of CMV-replication by HCMV antigenemia/viremia was found at a median of day +33 after alloSCT. The cumulative incidence of relapse at 5 years after alloSCT was 38% (95% confidence interval [95%CI]: 26–49) in 82 patients without compared to 22% (95%CI: 8–37) in 54 patients with HCMV antigenemia/viremia (p = .013). A decreased relapse risk of HCMV replication was confirmed by multivariate analysis for HCMV antigenemia/viremia (Hazard ratio [HR]: 0.29, 95%CI: 0.11–0.76, p

Alpha-fetoprotein (AFP) is a tumor marker routinely used for the diagnosis and follow-up of malignant neoplasms. We report a case of extremely high serum levels of AFP associated with benign lesions of the liver and of the lungs with a spontaneous and gradual drop to normal values in the 9-month follow-up without any form of treatment. As a result a high serum level of AFP does not necessarily indicate a malignant tumor and every patient presenting with such finding must be thoroughly examined before making a definitive diagnosis.

In a prior multicenter randomized controlled trial, we found that a 12-week course of extracorporeal photopheresis (ECP) plus standard immunosuppressive therapy resulted in several beneficial outcomes in patients with corticosteroid-refractory/intolerant/dependent chronic graft-versus-host disease (GVHD). Here, we report the results of an open-label crossover ECP study in 29 eligible participants randomized initially to the standard of care non-ECP (control) arm. Eligible for the crossover ECP study were control arm patients who either (1) had progression of cutaneous chronic GVHD (cGVHD), defined as >25% worsening from baseline as measured by the percent change in the total skin score (TSS) at any time, or (2) had less than 15% improvement in the TSS, or had a ≤25% reduction in corticosteroid dose at week 12 of the initial study. ECP was administered 3 times during week 1, then twice weekly until week 12, followed by 2 treatments monthly until week 24. The median age of the study cohort was 43 (20-67) years and 90% had extensive cGVHD. The median months from onset of cGVHD to start of ECP were 26 (range: 4-79). Twenty-five of 29 patients (86%) completed the 24-week course of ECP. Complete or partial skin response at week 24 was noted in 9 patients (31%). The median percent of decrease in TSS from baseline to weeks 12 and 24 was −7.9 and −25.8, respectively. In 4 (17%) and 8 (33%) patients, a ≥50% reduction in corticosteroid dose at weeks 12 and 24 was observed. Extracutaneous cGVHD response was highest in oral mucosa with 70% complete and partial resolution after week 24. In conclusion, progressive improvement in cutaneous and extracutaneous cGVHD was observed after a 24-week course of ECP in patients who previously had no clinical improvement or exhibited worsening of cGVHD while receiving standard immunosuppressive therapy alone in a randomized study. These results confirm previous findings and support the notion that prolonged ECP appears to be necessary for optimal therapeutic effects in corticosteroid-refractory cGVHD patients.

The impact of early human cytomegalovirus (HCMV) replication on leukemic recurrence was evaluated in 266 consecutive adult (median age, 47 years; range, 18-73 years) acute myeloid leukemia patients, who underwent allogeneic stem cell transplantation (alloSCT) from 10 of 10 high-resolution human leukocyte Ag-identical unrelated (n = 148) or sibling (n = 118) donors. A total of 63% of patients (n = 167) were at risk for HCMV reactivation by patient and donor pretransplantation HCMV serostatus. In 77 patients, first HCMV replication as detected by pp65-antigenemia assay developed at a median of 46 days (range, 25-108 days) after alloSCT. Taking all relevant competing risk factors into account, the cumulative incidence of hematologic relapse at 10 years after alloSCT was 42% (95% confidence interval [CI], 35%-51%) in patients without opposed to 9% (95% CI, 4%-19%) in patients with early pp65-antigenemia (P < .0001). A substantial and independent reduction of the relapse risk associated with early HCMV replication was confirmed by multivariate analysis using time-dependent covariate functions for grades II to IV acute and chronic graft-versus-host disease, and pp65-antigenemia (hazard ratio = 0.2; 95% CI, 0.1-0.4, P < .0001). This is the first report that demonstrates an independent and substantial reduction of the leukemic relapse risk after early replicative HCMV infection in a homogeneous population of adult acute myeloid leukemia patients.

Introduction: Loss of the Y-chromosome (LOY) is frequent in myelodysplastic syndromes (MDS) and observed as a single aberration in 3-4% of male MDS patients (pts). It is often clonal and not only age associated and confers a very good prognosis and a very low risk for leukemic transformation (Greenberg et al, Blood 2012; Schanz et al, JCO 2012). But LOY does not prove a hematologic disease per se (Arber et al, Blood 2016). To facilitate a better discrimination between age-related and clonal LOY, the aim of this study was to identify molecular mutations and cytomorphological features which might be characteristic for MDS with isolated LOY. Methods: We included 291 pts in our analysis. The cohort comprised 199 pts with normal karyotype (NK) and morphologically proven MDS (excess blasts (EB) in 77/199 (38%) pts) and 92 pts with LOY. NK was defined by 20 normal metaphases or at least 10 normal metaphases and normal fluorescence in situ hybridization (FISH, Tab.1). Results from mutational analysis were available for all pts with NK and for 61 pts with LOY as single cytogenetic aberration in ≥3 metaphases. Seventeen core genes (Tab.2) were sequenced in all 260 pts by Sanger and/or next generation sequencing (NGS). In 134 pts further 28 genes (Tab.2) were analyzed using one of two NGS panels. In addition to these myeloid genes, the second NGS panel covered single nucleotide polymorphisms on the Y-chromosome which enabled determination of the LOY clone size. Detailed cytomorphology for the evaluation of dysplasia was performed by two experts (UG, UB) as previously described (Germing et al, Leuk Res 2012) in 41 pts, including pts with small LOY clones and with cytogenetic sub-clones. Results: Sequencing of 40 pts with LOY and morphologically proven dysplasia showed higher frequencies of mutations in TET2 (epigenetic regulator), ZRSR2 (splicing factor, located at Xp22.2), and CBL (kinase signaling) compared to MDS with NK (Fig.1). Amongst others, mutations in IDH1/2 (epigenetic regulators) and RUNX1 (transcription factor) were rare in MDS with LOY (Fig.1). The total number of mutated core genes did not significantly differ between MDS with LOY and MDS with NK and no EB (p=0.54), but it was significantly higher in MDS with NK and EB (p=0.014, Fig.2). To distinguish between LOY as ancestral or secondary mutation we sequenced 12 pts with MDS and cases we included as clonal cytopenia of undetermined significance (CCUS, pts with cytopenia(s) and molecular mutation and/or LOY≥75% of metaphases) (Wiktor et al, GCC 2000) using the second NGS panel that allowed determination of LOY clone size and detection of molecular mutations. Thereby, we identified four pts where LOY was most likely the founder aberration, two pts with LOY as secondary aberration in addition to ancestral molecular mutations, and six pts with co-dominance of LOY and a molecular mutation (Fig.3). Finally, we aimed to evaluate if the cut off of LOY≥75% (Wiktor et al, GCC 2000) can distinguish between age-related and clonal LOY in our cohort. In 41 pts analyzed in more detail, peripheral blood counts (hemoglobin: mean 10.4 vs. 9.7 g/dL; white blood count: 4.9 vs. 6.0x10(9)/L, platelets: 163 vs. 198x10(9)/L) and dysplasia of the individual cell lines (erythro-, granulo-, megakaryopoesis) did not differ significantly between LOY≥75% and Conclusions: Cytopenia with isolated LOY seems to comprise a heterogeneous mixture of cytomorphologic and molecular subtypes. We identified pts where LOY definitely is part of the MDS clone that also harbors molecular mutations, indicating that LOY is not only age-associated in these cases. The low total number of mutated genes corresponds to the previously described favorable prognosis of isolated LOY (Schanz et al, JCO 2012; Greenberg et al, Blood 2012). Our data support the current guidelines that LOY does not prove a hematologic disease in the absence of diagnostic morphologic features. In these cases LOY might fulfill the requirements of a clonal mutation as described for clonal hematopoiesis of indeterminate potential (CHIP) or CCUS. In summary, our data suggest that MDS with LOY shows a characteristic molecular mutation profile. Disclosures Germing: Janssen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Outcome of adult ALL has improved considerably during the past decades by intensive chemotherapy, which still remains a challenge in older pts. This may be partly due to comorbidities. So far there are no standards to differentiate pts who will be able to tolerate even age-adapted chemotherapy (fit vs unfit). In addition, little is known about the prevalence of comorbidities. Clinical trials with new compounds often represent a selection of pts w/o comorbidities. There is also no generally accepted tool for comorbidity scoring. The goal of this analysis is to provide reference data for pre-existing comorbidities in a large set of adult ALL pts, to compare two different tools and to evaluate the impact on early death (ED) in older pts. The German Multicenter Study Group for Adult ALL (GMALL) has collected data from trials for younger (18-55 y) and older (>55 y) pts and from a prospective registry. Trials had very limited exclusion criteria and in the registry there are no exclusion criteria. The Charlson Comorbidity Index (CCI) was assessed in the GMALL Elderly trial, whereas the Sorror Score (HCT-CI) was used in trials for younger pts and in the registry. 879 pts had a documented HCT-CI score from GMALL 08/2013 trial (N=282;group 1) and 3 groups from the registry: >55 y but eligible for intensive therapy (N=56, group 2), > 55 y in GMALL Elderly protocol (N=505, group 3) and >55 y in GMALL Frail protocol (N=36; group 4) (Table 1). In addition the CCI was documented in 333 pts treated in the GMALL Elderly Trial. HCT-CI-Score: The most frequent comorbidities were infections (17%), prior malignancies (16%), diabetes (16%), cardiac (14%) and moderate pulmonary disease (12%), obesity (11%) and mild liver disease (10%). Arrhythmias (55 y (group 2) considered eligible for intensive therapy (57%) compared to those considered for the Elderly protocol (76%) (group 3). The proportion of low risk (LR) scores decreased with age (54%, 43%, 25% and 8% resp.;p=.01), whereas high risk (HR) increased (18%, 25%, 50% and 59% resp; p=.01). CCI: The most frequent comorbidities were prior malignancy (14%), diabetes (25%) with (3%) or w/o (22%) end organ damage, cardiac (11%) and vascular disease (8%). The incidence of prior malignancy within the last 5 y was 7%. Risk classification was: LR (0) 51%, intermediate risk (IMR) (1-2) 42% and HR (≥3) 7%. HCT-CI vs CCI in pts >55 y: With HCT-CI the incidence of heart diseases (21% arrhythmias, cardiac disease or valve damage) was higher compared to CCI (9%), which differentiated better into cardiac failure (7%) and myocardial infarction (4%). Peripheral vascular disease (8% with CCI) is not assessed by HCT-CI. Liver disease was less frequent with CCI (1.5%) vs HCT-CI (14%) due to different definitions, whereas moderate pulmonary disease (12%) or infections (18%) are not assessed by CCI. The incidences of prior malignancies and diabetes were comparable. Of note, the overall incidence of distinct comorbidities e.g. cardiac was lower than the sum of subentities because some pts had several comorbidities. ED in pts >55 y: ED rates in pts >55 y in group 3 and in GMALL Elderly trial were comparable (13% vs 12% resp). In group 3 ED rates in risk groups (HCT-CI) were 7% vs 13% vs 15% (p>.05). In the GMALL Elderly trial ED in risk groups (CCI) were 9%, 12% and 35% (p=.05; p=.003 LR/IMR vs HR). Overall the analysis reveals a high incidence of comorbidities in older (57-92%) and even in younger pts (46%), which partly would represent contraindications in clinical trials with novel compounds; thus real world data in pts with comorbidities are required after marketing authorisation. HCT-CI and CCI have a different focus and shortcomings. For ALL pts a more specific score with different organ modules would be helpful. Comorbidity is significantly correlated to ED risk. CCI allows to identify a small HR group (7%) with a mortality of 35%. HCT-CI (24% of pts) and even more CCI (51% of pts) allow to identify LR groups with Disclosures Viardot: Gilead Kite: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Amgen: Consultancy; BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Fiedler:Teva: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSO: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; Daiichi Sankyo: Other: support for meeting attendance. Stelljes:JAZZ: Honoraria; MSD: Consultancy; Amgen: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Honoraria. Serve:Bayer: Research Funding. Goekbuget:Kite / Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Other: Travel support, Research Funding; Pfizer: Consultancy, Other: Travel support, Research Funding; Amgen: Consultancy, Other: Travel support, Research Funding.

Introduction Allogeneic hematopoietic stem cell transplantation (alloSCT) is curative therapy for chronic myeloid leukemia (CML), but its long-term outcomes regarding graft sources are not well described. Results Here the outcomes of CML after alloSCT with bone marrow (BM, n=134) were compared to those of peripheral blood stem cell (PBSC, n=172) in the HLA-compatible unrelated donor setting. Patients were transplanted in 1st CP (Bone marrow transplantation (BMT), n=100, and Peripheral blood stem cell transplantation (PBSCT), n=103), in >1st CP (BMT, n=24, and PBSCT, n=52) and in blast crises (BMT, n=10, and PBSCT, n=17). Median follow-up were 54 months after BMT and 106 months after PBSCT. No significant differences were found in the incidence of acute and chronic graft-versus-host disease (GvHD) between both study-groups. The 5-year estimated probability of hematological relapse was 11% for patients in 1st CP CML and 49% for patients in advanced disease after BMT (p 40 years), disease stage, acute GvHD, chronic GvHD and immunprophylaxis with use of ATG influenced OS significantly. For leukemia-free survival, the following risk factors were significantly in the multivariate analysis, graft source, patient age, gender constellation, disease stage, acute GvHD and chronic GvHD. Conclusions This large trial show significant difference between transplant recipients who received PBSC and those who received BM from unrelated donor. These finding may influence the selection of a graft source for alloSCT from unrelated donor. Disclosures Dührsen: Amgen: Research Funding; Gilead: Consultancy, Honoraria; Janssen: Honoraria; AbbVie: Consultancy, Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Beelen:Medac: Consultancy, Other: Travel Support.

Myelodysplastic syndromes (MDSs) often occur in older adults with significant comorbidities. Therefore, a reduced-toxicity conditioning regimen may be more suitable than standard conditioning regimens before allogeneic blood stem cell transplantation. Here, we retrospectively compare the outcome of a treosulfan-based conditioning regimen with standard myeloablative TBI-based conditioning regimens in patients (pts) with MDS. A total of 48 pts with MDS were included in the study, of which 29 (60%) pts received TBI-based and 19 (40%) pts received a treosulfan-based conditioning regimen. A significantly lower relapse incidence (5% vs 34% at 3 years, P=0.019) resulting in a better, but not statistically significant relapse-free survival (RFS) (57% vs 31%, P=0.086) was observed after treosulfan-based conditioning. In pts with increased risk for significant side effects due to comorbidities (haematopoietic stem cell transplantation specific comorbidity index), the estimated 3-year RFS was significantly better in the treosulfan group: 54% (95% confidence interval (CI), 17-90%) compared with pts in the TBI group: 11% (95% CI, 0-44%; log-rank test P=0.0455). Treosulfan-based conditioning therapy is a feasible and effective regimen for pts with MDS, especially in pts with preexisting comorbidities.

Chronic graft-versus-host disease (cGVHD) is a major limitation of successful hematopoietic cell transplantation. The safety and efficacy of extracorporeal photopheresis (ECP) for 12 to 24 weeks together with standard therapy was compared with standard therapy alone in patients with cutaneous manifestations of cGVHD that could not be adequately controlled by corticosteroid treatment. The primary efficacy end point was a blinded quantitative comparison of percent change from baseline in Total Skin Score (TSS) of 10 body regions at week 12. Ninety-five patients were randomized to either ECP and standard therapy (n = 48) or standard therapy alone (n = 47). The median percentage improvement in TSS at week 12 was 14.5% for the ECP arm and 8.5% for the control arm (P = .48). The proportion of patients who had at least a 50% reduction in steroid dose and at least a 25% decrease from baseline in TSS was 8.3% in the ECP arm at week 12 and 0% in the control arm (P = .04). The nonblinded investigator assessment of skin complete or partial responses revealed a significant improvement in favor of ECP (P < .001). ECP was generally well tolerated. These results suggest that ECP may have a steroid-sparing effect in the treatment of cGVHD. Clinical trials registered at www.ClinicalTrials.gov as NCT00054613.

Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM), and MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Therefore, we evaluated the anti-myeloma effect of VEGF small interfering RNA (siRNA) silencing in MM cells and whether it can be augmented by the additional application of bortezomib directed against the 26S proteasome. After transfection with VEGF siRNA, we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji, and Karpas-299, as well as in cells of MM and lymphoma patients. VEGF siRNA significantly induced apoptosis and inhibited proliferation in OPM-2 cells (P

Polymorphisms in cytokine genes can influence immune responses and inflammation and thereby affecting the outcome of hematopoietic stem-cell transplantation. We analyzed a single-nucleotide polymorphism in the gene for the interleukin-23 receptor (IL-23R) (1142G>A) in a cohort of 221 transplant recipients and their human leukocyte antigen (HLA)-identical sibling donors and in a second cohort of 186 transplant recipients and their HLA-identical unrelated donors. Genotypes were tested for an association with graft-versus-host disease (GVHD) by multivariate analysis. The donor's IL-23R genotype was significantly associated with a reduced risk of acute GVHD in both cohorts for patients after transplant. Analysis of all 407 transplant recipients showed that IL-23R (1142G>A, Arg381Gln) genotype of the donor was associated with a decreased risk of grades 2-4 acute GVHD (31.6 compared to 51.0%, P=0.02) and grades 3-4 severe acute GVHD (3.9 compared to 23.4%, P=0.003). Death in remission was significantly lower in patients transplanted from donors with variant IL23-R (11.7 versus 27.7%, P=0.028), whereas overall survival or relapse rates were not influenced significantly by the IL-23R genotype. Among recipients of hematopoietic cells from HLA-identical donors, the IL-23R (Arg381Gln) gene variant on the donor side has a protective effect on the occurrence of acute GVHD in recipients after transplantation.

The polymorphic gene expression of CYP2C19 causes individual variability in drug metabolism and thereby in pharmacologic and toxicologic responses. We genotyped 286 patients and their donors for the CYP2C19 gene who underwent allogeneic transplantation for various diseases and analyzed their outcome. Patients were classified as: poor metabolizers (PMs; 3.1%), intermediate metabolizers (IMs; 24.5%) and extensive metabolizers (EMs; 72.5%). Patients genotyped as PMs had significant higher hepato- and nephrotoxicities compared to IMs or EMs. Maximum bilirubin and serum creatinine levels measured after transplant were approximately twofold higher than those of EMs or IMs. The increased toxicity resulted in an increased 4-year estimate for transplant-related mortality (TRM) with 50+/-18.6% for PMs compared to 25.1+/-3.7% for EMs (P

RNA interference is referred to as the recently discovered process of sequence-specific, post-transcriptional gene silencing that is initiated by double-stranded RNA molecules known as small interfering RNAs (siRNA). We herein present a first report on the in vivo application of targeted non-virally delivered synthetic bcr-abl siRNA in a female patient with recurrent Philadelphia chromosome-positive chronic myeloid leukaemia (CML) resistant to imatinib (Y253F mutation) and chemotherapy after allogeneic haematopoietic stem cell transplantation. We found a remarkable inhibition of the overexpressed bcr-abl oncogene resulting in increased apoptosis of CML cells. In vivo siRNA application was well tolerated without any clinically adverse events. Our findings imply that the clinical application of synthetic siRNA is feasible, safe and has real potential for genetic-based therapies using synthetic non-viral carriers.

Bronchiolitis obliterans (BO) and bronchiolitis obliterans organizing pneumonia (BOOP) are late-onset non-infectious pulmonary complications (LONIPCs) following allogeneic hematopoietic stem cell transplantation (HSCT). In the present study 10 of 197 conventionally prepared stem cell recipients developed BOOP after 365 days and 6 patients developed BO 333 days post-transplant. No BOOP or BO was diagnosed following T-cell depletion (p

Cytomegalovirus (HCMV) reactivation occurs frequently after hematopoietic stem cell transplantation and is associated with an increased treatment-related mortality. Induction of apoptosis by HCMV is unusual because HCMV utilizes various strategies to prevent apoptosis in infected cells in order to delay cell death and maintain viral replication. Here we show that HCMV can infect the acute leukemia cell lines Kasumi-1 (AML) and SD-1 (BCR-ABL-positive ALL), which inhibited their proliferation and induced apoptosis in almost all cells after 14 days. Although HCMV induced a significant up-regulation of the anti-apoptotic gene cFLIP and the anti-stress gene Gadd45a, and simultaneously down-regulated the pro-apoptotic genes p53, Gadd45gamma in Kasumi-1 and SD-1 cells, we found that these anti-apoptotic mechanisms failed in HCMV-infected acute leukemia cells and apoptosis occurred via a caspase-dependent pathway. We conclude that HCMV can provide anti-leukemic effects in vitro. To determine if this phenomenon may be clinically relevant further investigations will be required.

Objective In the hematopoietic stem cell transplantation setting, granulocyte colony-stimulating factor (G-CSF) administration can reduce donor cell reactivity in vitro, but the clinical significance of this phenomenon was only sparsely defined. Methods We performed lymphocyte transformation tests in 28 related stem cell donors pre and 5 days post G-CSF treatment, respectively, and correlated proliferative responses of donor peripheral blood mononuclear cells with clinical parameters in the corresponding recipients. Results In vitro reactions towards 4 mitogens and 12 recall antigens at day 5 post G-CSF administration were predictive for the occurrence of chronic graft-vs-host disease (cGVHD). Here, proliferative responses towards the mitogen anti-CD3 monoclonal antibody (OKT3) above median were most informative; this threshold could be determined by discrimination and receiver operating curve (ROC) analyses. In the whole cohort (18 human leukocyte antigen [HLA]-identical and 10 partially mismatched donor-recipient pairs), OKT3 responses predicted cGVHD with an odds ratio of 33.0, a sensitivity of 79%, and a specificity of 90%. A subgroup analysis of HLA-identical pairs even yielded an odds ratio of 85.0. Furthermore, bivariate analysis defined HLA compatibility and responses towards OKT3 as independent risk factors for cGVHD ( p = 0.02 and p = 0.0007, respectively). Conclusion The proliferative capacity of G-CSF-mobilized donor cells appears as a graft factor that determines the future incidence of cGVHD in the corresponding recipient.

We compared the results of chimerism analyses with real-time SNP-PCR to those obtained by the classical STR-PCR method in 135 hematopoietic stem cell transplantation recipients. Using 10 different SNP gene loci, the SNP-PCR method was able to discriminate patient from donor cells in 125 of 135 cases (93%), whereas the use of 11 different STR gene loci with the STR-PCR analysis using agarose or polyacrylamide gel resolution resulted in accurate donor-host discrimination in all patients. Of the 470 analyzed samples we found in 74% concordant results for both chimerism methods. In all 26% discordant cases the SNP-chimerism method showed mixed chimerism (MC), whereas the STR-method found complete chimerism (CC). As a consequence, the SNP-PCR chimerism analysis method detected a MC prior to the occurrence of relapse significantly earlier than the STR-PCR chimerism method (120 vs. 30 days, P < 0.007). The probability of relapses was significantly higher in patients with increasing MC (70%) compared to 30% in patients with CC (P < 0.00001) associated with a significantly shorter overall survival in patients with increasing MC. The multivariate Cox model showed that chimerism analsis with SNP-PCR was the only significant risk factor predicting relapse (RR 6.08, P < 0.0001).Furthermore, we analyzed the chimerism status in male recipients with a female donor in 580 samples of 134 patients using quantitative real-time PCR of Y-chromosome-specific sequences and compared the results with interphase XY-fluorescent in situ hybridization (FISH). MC without signs of relapse was detected in 35% of samples using quantitative real-time PCR of Y-chromosome-specific sequences. The detected Y-DNA amounts were low compared to the amounts detected in 104 samples of 42 patients with leukemic relapse at the time of analysis (P < 0.0001). Quantitative real-time PCR of Y-chromosome-specific sequences detected therefore an increasing MC with high residual host DNA amounts approximately 143 days (mean) prior to the occurrence of relapse. By comparing the results of Y-chromosome PCR with the XY-FISH analysis we found concordant results in 73% in patients with myeloablative regimens. The XY-FISH could detect 12 relapses, whereas the Y-chromosome PCR detect 36 relapses by MC (P < 0.005). Residual host cells gradually decreased during the posttransplant period from a mean of 5.4 ng (first months) to 0.5 ng (above 5 years) without evidence of relapses. The probability of relapses was significantly higher in patients with increasing MC (100%) compared to 8% in patients with CC (P < 0.00001) associated with a significantly shorter overall survival in patients with increasing MC. The multivariate Cox model showed that chimerism analysis of Y-chromosome-specific sequences is an important risk factor for relapse (RR 17.0, P < 0.0001). We conclude that the use of real-time SNP or Y-PCR may be superior to the STR-PCR or interphase XY-FISH methods in detecting patients who are at high risk for relapse after transplant.

This study aimed to evaluate the outcome following myeloablative allogeneic hematopoietic stem cell transplantation (SCT) among patients older than 50 yr of age. A total of 215 patients with a median age of 57 yr underwent allogeneic hematopoietic SCT for early (41%) or advanced (59%) hematologic malignancies. After a median follow-up of 36 months a 10-yr survival estimate of 56 +/- 6% could be assessed for patients in early disease stages while patients with advanced diseases showed a significantly decreased survival probability of 31 +/- 5% (p < 0.0002). Transplant related mortality (TRM) at day 100 and 365 post-transplant was 13% and 30% for early but increased to 21% and 49% for advanced disease stages. As major determinants of TRM advanced disease stage (p < 0.0001) and occurrence of grades II-IV graft-vs.-host disease (GVHD) (p < 0.0001) were identified. These results show that hematopoietic SCT following myeloablative conditioning is also applicable to elderly patients whereas disease stage and high-grade GVHD represent the essential prognostic factors for outcome.

Background. To examine how killer-cell immunoglobulin-like receptor (KIR) ligand incompatibilities effect molecular relapse (MR), we compared the occurrence of bcr-abl-positive reverse-transcriptase polymerase chain reaction (RT-PCR) results in 236 CML patients (pts) after human leukocyte antigen (HLA)-identical (n= 158) (group 1), HLA class I antigen mismatched and KIR-ligand compatible (n=49) (group 2), and HLA class I antigen mismatched and KIR-ligand incompatible (n=29) (group 3) hematopoietic stem-cell transplantation. Methods. We performed a retrospective single-center study. MR was evaluated using the real-time RT-PCR method for the detection of bcr-abl transcripts. Results. In the first group, 133 of 158 (84%) pts were in the first chronic phase of CML, and the corresponding figures were 33 of 49 (67%) pts in group 2 and 19 of 29 (64%) in group 3 (P

It remains controversial whether alloreactive donor-derived natural killer (NK) cells display graft-versus-leukemia reactions after unmodified allogeneic hematopoietic stem cell transplantation (HSCT). The present study evaluated the role of inhibitory killer immunoglobulin–like receptor (KIR) ligand incompatibility using a well-defined and uniform setting of unmodified allogeneic HSCT in 374 patients with myeloid leukemias. The most striking finding was a significant heterogeneity in the 5-year estimates of hematologic leukemic relapse after human leukocyte antigen (HLA)–identical (n = 237; 22%), HLA class I–disparate (n = 89; 18%), and KIR ligand–incompatible transplantations (n = 48; 5%) (P < .04). Multivariate analysis confirmed that the relative relapse risk (RR) was influenced by HLA class I disparity alone (RR 0.49), but was lowest after HLA class I–disparate, KIR ligand–incompatible transplantations (RR 0.24) (P < .008). The primary graft failure rates, however, increased from 0.4% after HLA class I–identical to 2.3% after HLA class I–disparate, and to 6.3% after KIR ligand–incompatible transplantations, respectively (P < .02). Unlike some other reports, no beneficial effect of KIR ligand incompatibility on other major endpoints of allogeneic HSCT (transplantation-related mortality, and overall and event-free survival) was detectable in the present study. In conclusion, unmodified allogeneic HSCT from KIR ligand–incompatible donors provides a superior long-term antileukemic efficacy in patients with myeloid malignancies.

Caspofungin (CAS) is the first of a new class of antifungal agents, the echinocandins, that interfere with fungal cell wall synthesis by inhibition of glucan synthesis. Here, we report the results of 31 patients treated with CAS following allogeneic SCT. CAS was administered as a second-line agent to patients with invasive fungal infection (IFI) (n=15) or fever of unknown origin (n=16) who were recalcitrant to or intolerant of prior antifungal therapy. Unsuccessful first-line regimes included amphotericin B (n=17), liposomal amphotericin B (n=5), fluconazole (n=3), itraconazole (n=1), and voriconazole (n=2). All patients received concomitant immunosuppressive therapy for graft-versus-host disease. In 23 patients, cyclosporin A (CSA) and CAS were administered concurrently without any major side effects detected. Observed increases in GPT were not clinically significant. Normalization of serum creatinine and significant reductions in C-reactive protein were observed in response to CAS. Favorable outcome to CAS were documented in eight of 15 patients with IFI and in 15 of 16 patients with fever of unknown origin. CAS is a promising alternative in patients with IFI and fever of unknown origin in the setting of allogeneic SCT.

Atypical chronic myeloid leukemia (aCML) occurs rarely and is associated with a poor prognosis when treated with conventional chemotherapy. We evaluated the outcome of aCML after allogeneic hematopoietic stem cell transplantation (HSCT). Nine patients were transplanted from HLA-identical siblings (n = 4), HLA-compatible unrelated donors (n = 4) or twin brother (n = 1). Median follow-up was 55 months after transplant (range, 9.1-118.1 months). One patient who was transplanted in advanced disease with bone marrow from his twin brother relapsed 19 months post transplant. This patient was successfully retransplanted from the original donor. All patients remained in complete remission. Analysis of the leukocyte chimerism of peripheral white blood cells and bone marrow buffy coat cells by VNTR-polymerase chain reaction (PCR) and single-nucleotide polymorphism real-time PCR revealed complete chimerism in all patients who had received an allogeneic transplant. One patient suffering from cerebral toxoplasmosis died 9 months post transplant. All other patients were alive at the time of analysis. Our findings suggest that the outcome of allogeneic or syngeneic transplantation in patients with aCML may not be worse than the outcome of transplantation for BCR-ABL-positive CML.

Objective: To test whether the functional impairment of the host bone marrow (BM) microenvironment pre-existing at the time of transplantation could be overcome by the increased content of immature cells in allogeneic peripheral blood stem cell transplantation (PBSCT) when compared with bone marrow transplantation (BMT). Methods: Cobble stone area forming cells (CAFC) were assayed in normal BM and BM after allogeneic BMT and PBSCT after stable engraftment. Groups were compared by two-tailed t-test. Results: While BM from 11 normal controls contained an average of 778.8 CAFC-d35 per 106 low density bone marrow cells (LDBMC, range 453–1231 per 106 LDBMC), BM from patients after BMT contained an average of 123.7 CAFC-d35 per 106 LDBMC (range 38–257) per 106 LDBMC. BM from patients transplanted with PBSC after myeloablative conditioning contained 128.3 (range 46–305) CAFC-d35 per 106 LDBMC (P = 0.89 compared with BMT). Similar results were obtained when patients after PBSCT with non-myeloablative conditioning were included (P = 0.62 compared with BMT). CAFC numbers in patients transplanted in early stages of myeloid leukaemia (acute myeloid leukaemia first remission, chronic myeloid leukaemia first chronic phase) were significantly higher than CAFC numbers in patients transplanted in more advanced stages (P = 0.008) or myelodysplastic syndrome (P = 0.023). The lowest CAFC numbers were found in two cases of retransplantation. Conclusion: Our findings indicate that the functional state of the BM microenvironment rather than stem cell dose or source is limiting for the homing and engraftment of immature haemopoietic cells in clinical transplantation.

Purpose: The p53 family member p73 displays significant homology to p53, but data from primary tumors demonstrating increased expression levels of p73 in the absence of any gene mutations argue against a classical tumor suppressor function. A detailed analysis of the p73 protein in tumor tissues has revealed expression of two classes of p73 isoforms. Whereas the proapoptotic, full-length, transactivation-competent p73 protein (TA-p73) has a putative tumor suppressor activity similar to p53, the antiapoptotic, NH2-terminally truncated, transactivation-deficient p73 protein (ΔTA-p73) has been shown to possess oncogenic activity. The oncogenic proteins can be generated by the following two different mechanisms: (a) aberrant splicing (p73Δex2, p73Δex2/3, ΔN′-p73) and (b) alternative promoter usage of a second intronic promoter (ΔN-p73). The purpose of our study was to elucidate the origin of ΔTA-p73 isoforms in hepatocellular carcinomas. Experimental Design: We analyzed the underlying mechanisms of p73 overexpression in cancer cells by quantification of p73 transcripts from 10 hepatocellular carcinoma patients using isoform-specific real-time reverse transcription-PCR. Results: Our data demonstrate that only aberrantly spliced ΔTA-p73 transcripts from the TA promoter show significantly increased expression levels in the tumor whereas the ΔN-p73 transcript generated from the second promoter is not significantly up-regulated. Conclusions: Although we only analyzed 10 patient samples the results strongly suggest that the elevated activity of the first promoter (TA promoter) accounts for high-level expression of both full-length TA-p73 and aberrantly spliced ΔTA-p73 isoforms in hepatocellular carcinoma tissues.

Objective While retrovirally mediated gene transfer of dihydrofolate reductase mutants (mutDHFR) has convincingly been demonstrated to confer methotrexate (MTX) resistance to murine hematopoietic cells, clinical application of this technology will require high efficacy in human cells. Therefore, we investigated retroviral constructs expressing various point mutants of human DHFR for their ability to confer MTX resistance to human clonogenic progenitor cells (CFU-C) and to allow for in vitro selection of transduced CFU-C. Methods Primary human hematopoietic cells were retrovirally transduced using MMLV- and SFFV/MESV-based vectors expressing DHFR Ser31 , DHFR Phe22/Ser31 , or DHFR Tyr22/Gly31 . MTX resistance of unselected and in vitro–selected CFU-C was determined using MTX-supplemented methylcellulose cultures and gene transfer efficiency was assesed by single–colony PCR analysis. Results While less than 1% mock-transduced CFU-C survived the presence of ≥5×10 −8 M MTX, MMLV- and SFFV/MESV-based vectors expressing DHFR Ser31 significantly protected CFU-C from MTX at doses ranging from 2.5 to 30×10 −8 M. Vectors expressing DHFR Phe22/Ser31 or DHFR Tyr22/Gly31 were even more protective and MTX-resistant CFU-C were observed up to 1×10 −5 M MTX. Three-day suspension cultures in the presence of 10–20×10 −8 M MTX resulted in significant selection of mutDHFR-transduced CFU-C. The percentage of CFU-C resistant to 10×10 −8 M MTX increased fourfold to 20-fold and provirus-containing CFU-C increased from 27% to 79–100%. Conclusion Gene transfer of DHFR using suitable retroviral backbones and DHFR mutants significantly increases MTX resistance of human CFU-C and allows efficient in vitro selection of transduced cells using a short-term selection procedure.

Indoleamine 2,3-dioxygenase (IDO) is an interferon-γ (IFN-γ)-induced enzyme, which is suggested to play an important role in the prevention of allogeneic fetal rejection. IDO effects the suppression of T-cell activity by catabolizing the essential amino acid l-tryptophan. We studied IDO expression by reverse transcription polymerase chain reaction (RT-PCR) in dendritic cells and by real-time RT-PCR in monocytes of patients undergoing allogeneic transplantation for leukaemia, who developed acute graft-versus-host disease (aGvHD), and compared the IDO expression with that of pregnant women and healthy volunteers. A spontaneous IDO expression was detected in the monocytes of 20 pregnant women with an IDO/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio at a median of 1.0%, whereas none of 15 healthy volunteers or patients after allogeneic transplant had any detectable spontaneous IDO expression. The IDO expression increased by in vitro IFN-γ stimulation in pregnant women (median 116%), healthy volunteers (median 11.7%) and patients with a low-grade aGvHD (grades 0–II) 28 days after transplant (median 433%) but not in patients with a severe aGvHD (grades III–IV) (median 0%), which was highly significant (P

Outcomes of highly purified CD34+ peripheral blood stem cell transplantation (PBSCT) for chronic phase chronic myeloid leukemia (CML) (n = 32) were compared with those of PBSCT (n = 19) and of bone marrow transplantation (BMT) (n = 22) in the HLA-compatible sibling donor setting. Median follow-up was 18 months after CD34+-PBSCT and unmanipulated PBSCT and 20 months after BMT. CD34+-PBSCT was associated with delayed T-cell immune reconstitution at 3 months and 12 months after transplantation compared with PBSCT (P

Outcomes after peripheral blood stem cell transplantation (PBSCT) for chronic phase chronic myeloid leukemia (n = 37) were compared with outcomes after bone marrow transplantation (BMT) (n = 54) in the HLA-compatible unrelated donor setting. Median follow-up was 17 months after PBSCT and 29 months after BMT. Both neutrophil and platelet recovery were faster after PBSCT (P