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The optimal preoperative treatment for locally advanced pancreatic cancer is unknown. We aimed to compare the efficacy and safety of nab-paclitaxel plus gemcitabine with nab-paclitaxel plus gemcitabine followed by fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) as multidrug induction chemotherapy regimens in locally advanced pancreatic cancer.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMHmRNA were incorporated into adenoviral vectors to decrease AMHgene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMHshRNA-vector, (d) AMHshRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMHshRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMHexpression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.Anti-Müllerian hormone is a survival factor for preantral follicles in nonhuman primates, and promotes preantral follicle growth to the small antral stage with increased steroid hormone and paracrine factor production, as well as oocyte maturation.

Prevention of mother-to-child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested HIV-1–specific human neutralizing monoclonal antibodies (NmAbs) as a post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with the simian-human immunodeficiency virus SHIVSF162P3. On days 1, 4, 7 and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h after antibody administration. Replicating virus was found in multiple tissues by day 1 in animals that were not treated. All NmAb-treated macaques were free of virus in blood and tissues at 6 months after exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged after CD8+T cell depletion. These results suggest that early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs.

Real-time fluorescence microscopy is used to investigate the trafficking of metabolizable fluorescent fatty acid in unilocular adipocytes from adipose tissue of nonhuman primates. The study reveals novel cell biological features that may contribute to the mechanism of adipocyte hypertrophy.

Allogeneic hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning for hematologic malignancies depends on graft-versus-tumor effects for eradication of cancer. Here, we estimated relapse risks according to disease characteristics. Between 1997 and 2006, 834 consecutive patients (median age, 55 years; range, 5-74 years) received related (n = 498) or unrelated (n = 336) HCT after 2 Gy total body irradiation alone (n = 171) or combined with fludarabine (90 mg/m2; n = 663). Relapse rates per patient year (PY) at risk, corrected for follow-up and competing nonrelapse mortality, were calculated for 29 different diseases and stages. The overall relapse rate per PY was 0.36. Patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) in remission (CR), low-grade or mantle cell non-Hodgkin lymphoma (NHL) (CR + partial remission [PR]), and high-grade NHL-CR had the lowest rates (0.00-0.24; low risk). In contrast, patients with advanced myeloid and lymphoid malignancies had rates of more than 0.52 (high risk). Patients with lymphoproliferative diseases not in CR (except Hodgkin lymphoma and high-grade NHL) and myeloid malignancies in CR had rates of 0.26-0.37 (standard risk). In conclusion, patients with low-grade lymphoproliferative disorders experienced the lowest relapse rates, whereas patients with advanced myeloid and lymphoid malignancies had high relapse rates after nonmyeloablative HCT. The latter might benefit from cytoreductive treatment before HCT.

Allogeneic hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning for hematologic malignancies depends on graft-versus-tumor effects for eradication of cancer. Here, we estimated relapse risks according to disease characteristics. Between 1997 and 2006, 834 consecutive patients (median age, 55 years; range, 5-74 years) received related (n = 498) or unrelated (n = 336) HCT after 2 Gy total body irradiation alone (n = 171) or combined with fludarabine (90 mg/m2; n = 663). Relapse rates per patient year (PY) at risk, corrected for follow-up and competing nonrelapse mortality, were calculated for 29 different diseases and stages. The overall relapse rate per PY was 0.36. Patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) in remission (CR), low-grade or mantle cell non-Hodgkin lymphoma (NHL) (CR + partial remission [PR]), and high-grade NHL-CR had the lowest rates (0.00-0.24; low risk). In contrast, patients with advanced myeloid and lymphoid malignancies had rates of more than 0.52 (high risk). Patients with lymphoproliferative diseases not in CR (except Hodgkin lymphoma and high-grade NHL) and myeloid malignancies in CR had rates of 0.26-0.37 (standard risk). In conclusion, patients with low-grade lymphoproliferative disorders experienced the lowest relapse rates, whereas patients with advanced myeloid and lymphoid malignancies had high relapse rates after nonmyeloablative HCT. The latter might benefit from cytoreductive treatment before HCT.

The hematopoietic microenvironment, approximated in vitro by long-term marrow cultures (LTCs), consists of both nonhematopoietic-derived stromal elements and hematopoietic-derived monocyte/macrophages. To better understand the consequences of monocyte-stroma interactions, we compared gene expression profiles of CD14+ peripheral blood monocytes and HS-27a stromal cells cultured alone and together in cocultures. Results from 7 separate experiments revealed 22 genes were significantly up- or down-regulated in the cocultures, with osteopontin (OPN) up-regulated more than 15-fold. The microarray OPN data were confirmed by Northern blot, real-time polymerase chain reaction (PCR), and by detection of OPN protein. High levels of OPN gene expression were also detected in 2- to 3-week-old primary LTCs. Using Transwells we determined that stromal cells were secreting a factor that up-regulated OPN gene expression in CD14+ cells. When CD34+ cells were cultured in the presence of purified OPN, tyrosine phosphorylation of a 34-kDa molecule was increased 2- to 3-fold, an effect that was diminished in the presence of an OPN neutralizing monoclonal antibody. In addition, Notch1 gene expression was decreased 5-fold in OPN-treated CD34+ cells. We conclude that interactions between stroma and monocytes can result in activities that limit the role of Notch signaling in hematopoietic regulation. (Blood. 2004;103:4496-4502)

The hematopoietic microenvironment, approximated in vitro by long-term marrow cultures (LTCs), consists of both nonhematopoietic-derived stromal elements and hematopoietic-derived monocyte/macrophages. To better understand the consequences of monocyte-stroma interactions, we compared gene expression profiles of CD14+peripheral blood monocytes and HS-27a stromal cells cultured alone and together in cocultures. Results from 7 separate experiments revealed 22 genes were significantly up- or down-regulated in the cocultures, with osteopontin (OPN) up-regulated more than 15-fold. The microarray OPN data were confirmed by Northern blot, real-time polymerase chain reaction (PCR), and by detection of OPN protein. High levels of OPN gene expression were also detected in 2- to 3-week-old primary LTCs. Using Transwells we determined that stromal cells were secreting a factor that up-regulated OPN gene expression in CD14+cells. When CD34+cells were cultured in the presence of purified OPN, tyrosine phosphorylation of a 34-kDa molecule was increased 2- to 3-fold, an effect that was diminished in the presence of an OPN neutralizing monoclonal antibody. In addition, Notch1 gene expression was decreased 5-fold in OPN-treated CD34+cells. We conclude that interactions between stroma and monocytes can result in activities that limit the role of Notch signaling in hematopoietic regulation. (Blood. 2004;103:4496-4502)

Niederwieser: Daichii: Speakers Bureau; Cellectis: Consultancy. Scholl:Gilead: Other: Project funding; Daiichi Sankyo: Other: Advisory boards; AbbVie: Other: Advisory boards; Pfizer: Other: Advisory boards; Novartis: Other: Project funding. Zojer:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Sayer:Novartis: Other: none. Schwind:Daiichi Sankyo: Honoraria; Novartis: Honoraria, Research Funding. Maschmeyer:Gilead, Janssen Cilag, Astra Zeneca; BMS, Merk-Serono: Honoraria. Hochhaus:Pfizer: Research Funding; Novartis: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding. Al-Ali:Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; CTI: Honoraria.

IntroductionFor patients (pts) with aggressive B-cell lymphoma immunochemotherapy with R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, Prednisolone) represents the standard of care with curative intention. For elderly pts a dose-reduced R-mini-CHOP protocol is feasible. No standard of care exists for frail or very old (>80 yrs) pts not eligible for CHOP-like treatment. Here we present the final results of the prospective DSHNHL-phase-2 trial for old or frail pts receiving Rituximab/Bendamustine (RB) for 1st-line treatment of aggressive B-cell lymphoma. Our goal was to determine feasibility, toxicity of the BR regimen in this pt cohort, and to determine which pts groups might potentially benefit from this reduced-intensity approach.

Introduction:

Zettl: Roche: Other: travel grants, Research Funding; Mundipharma: Research Funding. Viardot:Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees. Korfel:Mundipharma: Consultancy, Research Funding. Dreyling:Janssen: Consultancy, Other: scientific advisory board, Research Funding, Speakers Bureau; Novartis: Other: scientific advisory board; Gilead: Consultancy, Other: scientific advisory board, Speakers Bureau; Sandoz: Other: scientific advisory board; Acerta: Other: scientific advisory board; Celgene: Consultancy, Other: scientific advisory board, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: scientific advisory board, Research Funding, Speakers Bureau; Bayer: Consultancy, Other: scientific advisory board, Speakers Bureau. Illmer:Roche: Other: travel support. Schmitz:Novartis: Honoraria; Gilead: Honoraria; Celgene: Equity Ownership; Riemser: Consultancy, Honoraria. Klapper:Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Poeschel:Hexal: Speakers Bureau; Roche: Other: Travel support; Amgen: Other: Travel support; Abbvie: Other: Travel support; Astra Zeneca: Other: Travel support. Truemper:Takeda: Consultancy, Research Funding; Roche: Research Funding; Seattle Genetics, Inc.: Research Funding; Mundipharma: Research Funding; Janssen Oncology: Consultancy; Nordic Nanovector: Consultancy.

Live-attenuated, spread-deficient rhesus CMV vectors retain T cell immunogenicity.

Hänel: Roche: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Sayer:RIEMSER Pharma GmbH: Honoraria. Niederwieser:Novartis: Research Funding; Miltenyi: Speakers Bureau. Al-Ali:Gilead: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Alexion: Honoraria; Novartis: Consultancy, Honoraria, Other: Travel support, Research Funding; Celgene: Honoraria, Other: Travel support, Research Funding.

The heterogeneity of AML requires a personalized treatment strategy to achieve a high initial response and translate this response into long-term survival. Based on a possible synergistic effect of azacitidine (AZA) and cytarabine (ARA-C) when AZA is given first through the induction of deoxycytidine kinase by AZA which phosphorylates ARA-C to its active compound, ara-CTP, priming with AZA was integrated with intensive chemotherapy (IC) in a response-based sequential approach in the multicenter RAS-AZIC (DRKS00004519) study for patients (pts) >60 years (y). The safety, the remission rate of 64%, and a low TRM of 10% up to day (d) 90 were previously reported (Jaekel et al, ASH 2017). The final results of outcome at two years are presented.

Niederwieser: Novartis Oncology Europe: Research Funding, Speakers Bureau; Amgen: Speakers Bureau. Hoffmann:Novartis Oncology Europe: Research Funding. Al-Ali:Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Hegenbart:Pfizer: Other: Travel grant; Janssen: Honoraria, Other: Travel grant. Sayer:Riemser Pharma: Consultancy. Hochhaus:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Fischer:Novartis: Consultancy, Honoraria. Dreger:Novartis: Consultancy; Janssen: Consultancy; Gilead: Consultancy; Gilead: Speakers Bureau; Roche: Consultancy; Novartis: Speakers Bureau. Hiddemann:Roche: Membership on an entity's Board of Directors or advisory committees; Genentech: Other: Grants; Roche: Other: Grants.

Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGRmRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4–6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGRmRNA and P levels. Addition of hCG increased (P< 0.05) PGRmRNA and medium P levels in controls. However, a time-dependent decline (P< 0.05) in PGRmRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P< 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization.

Al-Ali: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Wolf:Bayer: Honoraria; Geo Pharma: Honoraria. Hochhaus:ARIAD Pharmaceuticals, Inc.: Research Funding. Maschmeyer:Celgene: Consultancy.

Heidel: Novartis: Consultancy.

Off Label Use: Azacitidine is licensed in Germany for the treatment of adult patients with the following conditions, who are not eligible for haematopoietic stem cell transplantation: • intermediate-2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Scoring System (IPSS), • chronic myelomonocytic leukaemia (CMML) with 10-29% marrow blasts without myeloproliferative disorder, • acute myeloid leukaemia (AML) with 20-30% blasts and multi-lineage dysplasia,according to World Health Organisation (WHO) classification.. Wolf:Bayer: Honoraria; Geo Pharma: Honoraria. Sayer:Takeda: Other; Medac: Other. Al-Ali:Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Al-Ali: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Wolf:Bayer: Honoraria; Geo Pharma: Honoraria.

Purpose:To define prognostic factors for overall survival in adult patients (pts) with relapsed acute myeloid leukemia (AML).

The incidence of acute myeloid leukemia (AML) is age-dependent with the majority of patients (pts) being older than 60 years at diagnosis. Treatment of these pts needs to be well balanced between sufficient efficacy and tolerable toxicity. Here, we report long term follow-up of the OSHO AML97 study. Pts with AML older than 60 years were registered after informed consent and received age-adapted intensive chemotherapy treatment (curative arm; induction therapy with AraC 2 g/m2iv day 1, 3, 5, 7 and mitoxantrone 10 mg/m2iv day 1 to 3 to induce complete remission (CR), followed by 2 consolidation courses with AraC 240 mg/m2iv day 1 to 5 and mitoxantrone 10 mg/m2iv day 1 to 2), low dose chemotherapy (palliative arm; idarubicin 10 mg po day 1 and either thioguanine 40 mg po day 1-5, or AraC 80 mg sc day 1-5 or etoposide 100mg po day 1-5) or supportive therapy (best supportive care including transfusions). A total of 618 pts were enrolled (curative arm n=471, palliative treatment n=115 and supportive therapy n=32 pts). In the curative arm, CR was obtained in 66.8% of pts. Treatment related mortality (TRM) was 11.2% after induction and 4.5% after consolidation I, respectively. Median overall survival for all pts in the curative arm was 12 months, event free survival (EFS) at 12 years was 0.11±0.02%. In multivariate analysis, cytogenetics at diagnosis was the most important prognostic factor for CR (p=0.001). With a median follow up of 10 years (range 0.1 - 11.8) probability of overall survival (OS) at 5 years was 0.48±0.11; 0.13±0.03; 0.10±0.04 and 0.08±0.03 for pts with favorable, normal, other and unfavorable cytogenetics. Median survival for pts treated with palliative chemotherapy was 54 days.

Treatment of elderly patients with AML remains challenging. While increasing doses of induction and consolidation chemotherapy have failed to improve outcome, efforts to decrease relapse rates using the graft-versus-leukemia effect have shown promising results in phase II studies. In the present analysis of the prospective OSHO 2004 study we evaluated the effect of post-induction hematopoietic cell transplantation (HCT) in comparison to conventional consolidation chemotherapy (CT) on outcome in elderly patients with AML.

Myeloproliferative neoplasia (MPN) is a group of diseases which include polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) and is characterized by clonal proliferation of myeloid cells. In many cases there is also a striking inflammatory syndrome (CRP elevation, fever, elevation of pro-inflammatory cytokines in peripheral blood) in particular in advanced phases of the disease. Auto- and paracrine effects of pro-inflammatory cytokines may contribute to maintenance of the disease and aggravation of the inflammatory syndrome. Cold-shock proteins are multi-functional DNA/RNA-binding proteins and Ybx1 is the prototypic member. These proteins regulate the expression of a number of pro-inflammatory cytokines, as well as cytokine receptors, making them key players in the orchestration of inflammatory processes. Moreover, expression of Ybx1 is induced by cellular and oncogenic stress and mediates resistance to chemotherapy. As patients with MPNs may present with an inflammatory syndrome, we assessed for Ybx1 expression as a potential mediator of cytokine signaling.

In patients with relapsed acute myeloid leukemia (AML) > 60 years of age we analyzed age at relapse, interval from first complete remission (CR1) to relapse, cytogenetic risk at initial diagnosis, prior allogeneic stem cell transplantation (alloSCT) and FLT3/NPM1 mutational status as possible prognostic factors for overall survival (OS).After achieving CR1 more than 50% of elderly AML patients eventually relapse. Prognostic factors for OS are poorly defined in this patient population. For younger patients with relapsed AML a risk score has been described including age at relapse, interval from CR1 to relapse, cytogenetic risk at initial diagnosis and prior stem cell transplantation (SCT) as prognostic factors. We sought to investigate whether these are also prognostic factors in elderly patients with relapsed AML. In addition, we assessed the prognostic impact of FLT3- and NPM1 mutational status (wild-type (wt) or mutated (mut)) at diagnosis.In the ongoing multicenter OSHO trial #69 for AML patients > 60 years we evaluated data of all relapsed patients. Overall survival was calculated from the day of first relapse until the day of death using the Kaplan Meier method. Univariate analysis was performed to test for the influence of age at relapse, interval from CR1 to relapse, cytogenetic risk at initial diagnosis, prior alloSCT and FLT3/NPM1 mutational status. Subsequently, independent prognostic factors were defined in a multivariate analysis with age at relapse, time from CR1 to relapse, cytogenetic risk at initial diagnosis and prior alloSCT as covariates.From April 2005 until April 2013 904 patients were registered. 733 of these received intensive induction chemotherapy which resulted in CR1 in 447 (61%) pts. In this patient group 260 relapses were observed after a median interval, calculated from the day of CR1, for living patients of 2.7 years (range 0.1 to 7.5). Median age at relapse was 69 years (range 60 – 85) with 129 (49.6%) pts. being 60 to 68 years old, 102 (39.2%) pts. being 69 to 74 years old and 29 (11.1%) pts. being 75 to 85 years old. Median interval from CR1 to relapse was 0.58 years (0.07 – 6.28). 114 (43.8%) relapses occurred up to 6 months after CR1, 119 (45.8%) between 7 and 18 months after CR1 and 27 (10.4%) later than 18 months after CR1. Only five (1.9%) relapsed pts. showed good risk cytogenetics at diagnosis, whereas it was of intermediate risk in 159 (61.1%) pts., of poor risk in 68 (26.2%) pts. and unknown in 28 (10.8%) pts. Forty-one (15.8%) pts. had received prior alloSCT in CR1. Information on FLT3- and NPM1 mutational status at diagnosis was available in 194 (74.6%) pts. 110 (42.3%) pts. had FLT3/NPM1 wt/wt, 48 (18.5%) pts. had FLT3/NPM1 wt/mut, 23 (8.8%) pts. had FLT3/NPM1 mut/wt and 13 (5.0%) pts. had FLT3/NPM1 mut/mut.OS rate at 2 years of all relapsed pts. was 13 ± 2%. For patients younger than 69 years and for those 69 years of age or older OS rate at 2 years was 17 ± 4% and 9 ± 3%, respectively (p=0.03). The interval between CR1 and first relapse also affected 2 year-OS with 7 ± 3%, 15 ± 4% and 36 ± 12% for pts. with relapse up to 6 months, 7 to 18 months and later than 18 months after CR1, respectively ( 18 months: p=0.009). OS rate at 2 years was also influenced by cytogenetic risk at initial diagnosis with 17 ± 3% for pts. having good or intermediate risk cytogenetics and 3 ± 2% for those with poor risk cytogenetics (p< 0.0005). Prior alloSCT had a negative influence on OS. Two-year OS rate was 10 ± 5 and 13 ± 3% (p= .015) for patients with prior alloSCT vs. those without prior alloSCT, respectively. FLT3/NPM1 mutational status at diagnosis had no impact on OS.In univariate analysis age at relapse (p<0.04), interval from CR1 to relapse (p< 0.0005), cytogenetic risk at initial diagnosis (p<0.02) and prior alloSCT (p<0.02) were shown to be prognostic factors for OS, whereas FLT3/NPM1 mutational status was not significant (p=0.82). In multivariate analysis the same factors remained significant but only interval from CR1 to relapse (p<0.0005) and prior alloSCT (p=0.003) were independent.In AML patients >60 years in first relapse OS is poor. Longer interval from CR1 to relapse and no prior alloSCT are independent beneficial prognostic factors for OS. FLT3/NPM1 mutational status at diagnosis has no prognostic impact on OS.Wedding: Roche: Speakers Bureau; Amgen: Speakers Bureau; Chugai: Speakers Bureau; Janssen-Cilag: Speakers Bureau; Novartis: Speakers Bureau; Cephalon: Speakers Bureau; Prostarkan: Speakers Bureau; Pfizer: Speakers Bureau. Niederwieser:Novartis: Consultancy.

Hochhaus: Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria.

Treatment of refractory or relapsed non-Hodgkin lymphoma (NHL) remains challenging. Here, 88 patients with refractory or relapsed NHL received treosulfan and fludarabine as a reduced intensity conditioning for allogeneic hematopoietic stem cell transplantation (allo-HSCT).73/88 intensely pre-treated patients experienced a relapse (R) with 18/88 early relapses (ER < 6 months from the last chemotherapy). At the time of allo-HSCT, 26 patients were in complete (CR) and 43 in partial remission (PR), twelve patients had progressive disease (PD) and seven stable disease (SD). 47 patients received an autologous graft followed by allo-HSCT.After allo-HSCT, 69 of 88 patients were in CR, seven patients in PR, resulting in an overall response rate of 86.4% (76/88). Thirty-three patients achieved a CR from PR as well as six patients from PD and five from SD. 43/88 (49 %) patients were alive at the end of follow-up. Patients undergoing directly allo-HSCT without preceding auto-HSCT showed a better disease-free survival (DFS, p =.038) with a trend (p =.077) for better overall survival (OS). Patients with ER showed an OS of.35 ±.12 after three and seven years. Chronic graft-versus-host disease (cGvHD) had a positive impact on both OS and DFS (for limited cGVHD versus no cGvHD p =.002 and.004, respectively).Allogeneic stem cell transplantation following conditioning with treosulfan and fludarabine constitutes a good therapeutical option for patients with refractory or relapsed NHL and should be considered early in the course of salvage treatment.Schmitt: Medac: Participation in Conferences. Sayer:Medac: Travel grant to ASH 2009. Koenigsmann:Medac: Research Funding. Casper:Medac: Participation in Conferences. Beelen:Medac: Participation in Conferences. Freund:Medac: Honoraria, Research Funding.

Abstract 4106

Autologous stem cell transplantation (ASCT) after cytoreductive induction is considered standard of care for younger patients (pts) with multiple myeloma (MM). The previous standard of induction, the Vincristin-Adriamycin-Dexamethasone (VAD) combination, achieves inferior results compared with induction regimens which combine the proteasome inhibitor Velcade (V = Bortezomib) with Dexamethasone (D)(=VD) and a cytostatic drug such as Doxorubicin (PAD = VD plus Doxorubicin). Velcade-based induction therapy was shown to translate into better myeloma control after high dose melphalan and to lead to prolonged progression-free survival. In order to find a more efficacious and safer drug combination for induction therapy in MM, we tested the combination of Velcade with Cyclophosphamide and Dexamethasone (VCD).This trial was designed as an open, prospective, multi-center, uncontrolled, combined phase II/III study. As previously reported (Kropff M et al., Ann Hematol 2009), in the first 30 pts the optimal dose of iv Cyclophosphamide in combination with V and D was defined as 900 mg/m2 on d1. Between 03/2006 and 03/2009 we enrolled an additional 370 pts up to 60 years of age with untreated MM to receive three 3-week cycles of induction treatment with V 1.3 mg/m2 iv d1,4,8,11; D 40 mg/d orally d1,2,4,5,8,9,11,12; and C 900mg/m2 iv d1 before scheduled high dose melphalan and ASCT. The primary endpoint of the study is response rate on day 63 after 3 cycles of VCD according to EBMT and IMWG criteria.Final data from 400 pts from 39 German centers will be presented at the meeting. In the currently evaluable 300 pts (mean age 52.3 years; 1.7% stage I, 21.3% stage II, 77.0% stage III) molecular cytogenetic analysis showed a prevalence of 13q- in 38%, of t[4;14] in 13% and of 17p- in 12% of pts (no changes in 35%). All 300 pts (88.3% of whom completed three cycles) were included in the intent-to-treat analysis. Overall response rate (ORR = CR+PR) was 84%, with 10% CR and 74% PR, 5.7% MR, 7.3% NC and 2.3% PD. The negative prognostic impact of 13q- or t[4;14] was abrogated (ORR normal 87.3%, 13q- 83.7%, t[4;14] 90.0%), the unfavorable influence of p53 loss in the 17p- subgroup was still detectable (ORR 69.2%) but this did not reach statistical significance. VGPR rates will be reported at the meeting.Serious adverse events were documented in 78/300 (26.0%) patients. Death rate was remarkably low (1.3%, of which one was not related to the trial medication). 155/300 (52%) of pts experienced grade 3/4 non-serious AEs and of these leucopenia (93/300 pts= 31%), thrombocytopenia (7%), neutropenia (6%), anaemia (5%) were the most frequent events. 80 AEs grade 3 or 4 and 45 SAEs were of infectious origin and occurred in 47/300 pts. 80/130 SAEs (61.5%) were at least possibly related to Velcade. 101/300 pts (34%) developed episodes of peripheral neuropathy. PNP was grade 1 in 62/300 pt (20.7%), grade 2 in 31/300 pt (10.3%) and grade 3 in 7/300 pts (2.3%).This analysis demonstrates that proteasome inhibition by Velcade in combination with Dexamethasone and iv Cyclophosphamide (VCD) is an induction regimen for newly diagnosed MM which is highly effective in a short period of time, has a rather low toxicity profile and is feasible for administration in an outpatient setting. Based on these characteristics, VCD qualifies to become a new standard for MM induction therapy.Einsele: OrthioBiotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Bortezomib is licensed as monotherapy for use in relapsed/refractory MM and in combination with melphalan/Prednisone in the first-line treatment of MM pts ineligible for HD-MEL and ASCT. . Liebisch:OrthoBiotech: Consultancy, Honoraria. Langer:OrthoBiotech: Consultancy. Kropff:OrthoBiotech: Consultancy, Honoraria. Kröger:OrthoBiotech: Honoraria. Ostermann:OrthoBiotech: Honoraria. Mügge:OrthoBiotech: Honoraria. Wolf:OrthoBiotech: Honoraria. Gramatzki:OrthoBiotech: Consultancy, Honoraria. Maschmeyer:OrthoBiotech: Travel Grant. Sezer:OrthoBiotech: Consultancy, Honoraria. Heidemann:OrthoBiotech: Honoraria. Jäger:OrthoBiotech: Honoraria. Dechow:Celgene: Research Funding. Simon:OrthoBiotech: Honoraria. Straka:OrthoBiotech: Consultancy, Honoraria, Research Funding. Fingerle-Rowson:orthoBiotech: Employment. Knop:OrthoBiotech: Honoraria.

Abstract 131

Background: Viral infections contribute significantly to morbidity and mortality following allogeneic HSCT. Whereas incidences and risk factors for CMV infections are well studied data for other viral infections are more limited. Here, we present data on 202 alloHSCT recipients that received predominantly reduced intensity conditioning (RIC).

Background: Viral infections contribute significantly to morbidity and mortality following allogeneic HSCT. Whereas incidences and risk factors for CMV infections are well studied data for other viral infections are more limited. Here, we present data on 202 alloHSCT recipients that received predominantly reduced intensity conditioning (RIC). Patients and Methods: Data of 202 consecutive patients (pts) who received HSCT during 1/1999 to 12/2006 were retrospectively analysed with regards to viral infections caused by adenovirus (AdV), herpes simplex virus (HSV), varicella zoster virus (VZV), human herpesvirus 6 (HHV6), epstein-barr virus (EBV) and BK-virus (BKV). Other factors included were gender, age, underlying disease, disease status, year of diagnosis, year of HSCT, time from diagnosis to HSCT, donor type, gender-matching between donor and recipient, stem cell source, conditioning-regimen, T-cell-depletion, prior autologous HSCT, acute and chronic GvHD, prior fungal infection, and viral serostatus of the recipients. Results: Median follow-up was 9.2mths (mean 21.7mths, range 8days to 95.3mths). Median pt. age was 45yrs (range 15 to 69yrs). Underlying diseases were ALL (n=20), AML (n=63), CLL (n=7), CML (n=32), MDS (n=21), MM (n=17), NHL (n=28), OMF (n=5), others (n=9). 137 pts received RIC most commonly based on treosulfan/fludarabine (n=112). 65 pts received myeloablative conditioning mainly based on 12Gy TBI containing regimen (n=48). GvHD-prophylaxis consisted of CSA/MTX (n=106), other CSA based regimen (n=83) or other (n=13). The overall infection incidences of AdV were 4.0%, of HSV 7.4%, of VZV 5.9%, of HHV6 8.9%, of EBV 4.5% and of BKV 5.4%. Median time of onset of AdV infections were 64 days after HSCT, of HSV 34 days, of VZV 542 days, of HHV6 97 days, of EBV 83 days, of BKV 50 days. Pts with AdV or HHV6 infections had a inferior survival compared to pts w/o the according viral infection (p=0.024 and p=0.048, respectively). 3/8 pts (37%) with AdV and 5/18 pts (28%) with HHV6 infections died within 30 days after infection diagnosis. Pts with EBV or BKV infections tended to have an inferior survival compared to pts w/o the according infection (p=0.084, p=0.152, respectively). 4/9 pts (44%) with EBV and 2/11 pts (18%) with BKV infection died within 30 days after infection diagnosis. Whereas pts with HSV infections had a similar survival compared to pts w/o infections, mean survival for pts with VZV infections was significantly better compared to pts w/o VZV infections (p=0.003). In multivariate analyses VZV infections remained to be associated with better survival (p=0.046, HR 0.132, CI 0.018–0.962). The other viral infections did not remain significant risk factors when analysed adjusted for competing risk factors. In order to determine the influence of late viral infections we performed an additional multivariate analyses solely including pts surviving beyond day 90. EBV infections (p=0.024, HR 2.807, CI 1.147–6.873) and HHV6 infections (p=0.013, HR 2.594, CI 1.227- 5.484) were significant risk factors for inferior survival besides unrelated donor HSCT (p=0.013), acute GvHD III/IV (p=0.001) and fungal infection prior to HSCT (p=0.005). Conclusion: Non-CMV viral infections are commonly observed following myeloablative and RIC alloHSCT. Since they are associated with significant mortality early broad viral screening seems advisable if virus infections are clinically suspected.

Background and objective: Allogeneic transplantation can not be offered to many patients due to potential side-effects of conventional conditioning. Dose-reduced conditioning approaches improve tolerability, however, treatment intensity and efficacy might be reduced as well. We have, therefore, developed a dose intense, but toxicity reduced conditioning regimen based on treosulfan and fludarabine and report first results. Patients and methods: 65 patients with a median age of 50 years were transplanted from related (n=21) or unrelated donors (n=44) after conditioning with treosulfan (3x10, 3x12 or 3x14g/m2 i.v.) and fludarabine (5x30mg/m2 i.v.). 21 patients were in complete remission (CR) and 44 patients had not reached a CR at the time of transplantation. 59 of 65 patients were considered unfit for a conventional conditioning regimen. Results: After a median follow up of 3.2 years an actuarial overall survival (OS) of 59.2 % (at 3 years) and an event-free survival (EFS) of 40,1% has been reached. Patients with related donor or transplantation in CR had a better OS (85,4 resp. 74,2%) and EFS (52,2% resp. 61,9%). The cumulative incidence of relapse at 3 years was 26,2%. Transplantation in CR resulted in a lower relapse incidence than transplantation not in CR (17,9 vs. 30%). Treatment-related mortality (TRM) at day 100 is 17,4% (cumulative incidence). In stepwise Cox regression analyses for OS, EFS and TRM only the covariables transplantation in CR vs. not in CR and the donor status were shown to be influential. No effects were detected for age (under 50 years vs. 50 years and older), concomitant disease (present vs. not present) or intensity of pretreatment. The cumulative incidence for acute graft-versus-host disease (GvHD) °I-IV at day 100 was 43.3%. The cumulative incidence for chronic GvHD at 3 years is 36.1%. Conclusions: These results with a conditioning therapy of treosulfan and fludarabine indicate that patients despite higher age, concomitant disease or after intensive pretreatment can be successfully transplanted without increased treatment-related mortality.

For most hematologic malignancies treated by myeloablative conditioning, hematopoietic cell transplantation (HCT) from HLA-matched unrelated donors has been associated with greater non-relapse mortality (NRM) and worse overall survival (OS) than that from HLA-identical sibling donors. Poorer outcomes with unrelated grafts are largely related to more frequent or severe graft-versus-host disease (GVHD), and higher rates of graft failure. In this retrospective analysis we asked whether outcomes of patients with hematologic malignancies who had nonmyeloablative HCT also differed according to donor type. The analysis included all donor/recipient pairs for whom HLA-A, -B, -C, -DR, and DQ allele-level typing information was available. Single allele-level mismatches at HLA class I were allowed. Using these criteria, 308 patients given nonmyeloablative HCT (sibling donors, n=187; unrelated donors, n=121) and 849 patients with myeloablative HCT (sibling donors, n=576; unrelated donors, n=273) between 1997 and 2003 were included. The median ages (range) of nonmyeloablative and myeloablative HCT recipients were 54 (9–72) years and 41 (1–65) years, respectively. The nonmyeloablative regimen consisted of 2 Gy total body irradiation (TBI) with or without fludarabine followed by postgrafting immunosuppression with mycophenolate mofetil and cyclosporine (CSP). Recipients of myeloablative transplants were prepared with different high-dose (TBI-based, 45%; non-TBI-based, 55%) regimens, and the majority (78%) was given methotrexate and CSP for GVHD prophylaxis. G-CSF-mobilized blood was the source of stem cells for all nonmyeloablative transplant recipients, and for 43% of recipients of myeloablative transplants; 57% received bone marrow. Analyses of outcomes used proportional hazard models stratified on diagnosis/disease status, and adjusted for patient age and stem cell source (Table). Our results confirmed that myeloablative HCT from HLA-matched unrelated donors was associated with increased NRM compared to HCT from HLA-identical sibling donors, translating into significantly decreased overall survival for low-risk patients (acute leukemias in first remission, chronic myeloid leukemia in chronic phase, low-risk myelodysplasia). In contrast, a detrimental impact on outcome with unrelated grafts was not appreciable after nonmyeloablative conditioning. Within the limitations of a retrospective analysis, these data suggest that donor type does not influence outcome following nonmyeloablative HCT. Thus, depending on the likely duration and success of a donor search, and the availability of other treatment options, unrelated donor transplantation may be an option for patients who are candidates for nonmyeloablative HCT but who do not have a suitable sibling donor. OS and NRM with HLA-identical sibling (MSD) vs HLA-matched unrelated (MUD) donors according to type of conditioning Nonmyeloablative Myeloablative N (MSD/MUD) HR (95% CI) P N (MSD/MUD) HR (95% CI) P HR, hazard ratio; CI, confidence interval. HR <1 indicates more favorable, and >1 less favorable outcomes for MUD grafts. Low-Risk OS 21/17 0.23 (0.0–1.9) 0.17 288/152 1.89 (1.3–2.8) 0.001 NRM 0.33 (0.0–2.7) 0.30 2.12 (1.4–3.3) 0.001 High-Risk OS 166/104 1.08 (0.7–1.6) 0.71 288/121 1.09 (0.8–1.5) 0.59 NRM 0.93 (0.5–1.6) 0.80 1.54 (1.0–2.3) 0.05

Nonmyeloablative hematopoietic cell transplantation (NMHCT) has become an immunotherapeutic option for patients with a variety of malignant diseases. The susceptibility to malignancy-eradicating graft-versus-tumor effects, however, appears to depend on type and stage of the underlying diseases. Thus, the aim of this study was to estimate relapse risk after NMHCT according to pretransplant disease characteristics. Between 1997 and 2004, 595 consecutive patients (median age, 55; range, 5–74 years) received related (n=383) or unrelated (n=212) grafts after preparation with total body irradiation (TBI, 200 cGy, n=168) alone, or in combination with fludarabine (90 mg/m2, n=423). The relapse rate per person year (PY) at risk, which corrected for variation in follow-up time and the competing risk of non-relapse mortality, was calculated for 27 different disease and stage categories. No corrections were made for graft rejections, all of which resulted in relapse. The overall relapse rate per PY was 0.37. As shown in Table 1, patients with chronic lymphocytic leukemia, low-grade Non-Hodgkin lymphoma (NHL) and multiple myeloma had the lowest relapse rates per PY (0.00, 0.09, 0.09; “low-risk”). In contrast, patients with advanced myeloid diseases had a relapse risk per PY of >0.50 (“high-risk”). The increased incidence of graft rejection among patients with chronic myeloid leukemia and myelodysplastic syndrome, however, may have contributed to the higher relapse risk observed in this group. Patients with lymphoproliferative diseases not in remission (Hodgkin disease and high-grade NHL excluded), and myeloid malignancies in remission at transplant had relapse rates per PY of 0.20 – 0.50 (“standard-risk”). For patients deemed at “high risk” for relapse after NMHCT, intensification of the preparative regimens, and/or cytoreductive strategies before or after transplant need to be considered in the design of future transplant protocols. Relapse Risk Categories Low Risk (n) Relapse Rates per PY Standard Risk (n) Relapse Rates per PY High Risk (n) Relapse Rates per PY CLL, chronic lymphocytic leukemia; MM, multiple myeloma; MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; ALL acute lymphocytic leukemia; CMML, chronic myelomonocytic leukemia; CR, complete remission; CP/AP/BP, chronic-/accelerated-/blast-phase. CLL, CR (4) 0.00 Waldenstrom′s (7) 0.23 high-grade NHL, no CR (18) 0.61 MM, CR (23) 0.09 MM, no CR (109) 0.26 Hodgkin, CR (7) 0.67 low-grade NHL, CR (22) 0.09 CLL, no CR (52) 0.28 Hodgkin, no CR (26) 0.83 low-grade NHL, no CR (49) 0.19 Myelofibrosis (5) 0.28 MDS (RAEB/RAEB-t, 16) 0.51 MDS (RA/RARS, 18) 0.29 MDS, previous chemo (17) 0.65 AML > CR1 (31) 0.30 AML, no CR (10) 0.69 High-grade NHL, CR (16) 0.31 AML, antecedent MDS (31) 0.94 CML, CP1 (27) 0.34 CMML (7) 0.74 AML, CR1 (43) 0.42 CML, CP2 (7) 1.05 ALL, CR1 (9) 0.45 CML, AP/BP (11) 1.05 ALL, > CR1 (7) 0.88 ALL, no CR (4) 1.08

Forty-five patients (pts) with relapsed or chemotherapy refractory low-grade lymphomas were treated with HLA-matched related (n=22) and unrelated (n=23) HCT after nonmyeloablative conditioning using 2 Gy total body irradiation with or without fludarabine. Postgrafting immunosuppression consisted of cyclosporine and mycophenolate mofetil. Lymphoma histologies included follicular (n=38), small lymphocytic (n=5), and marginal zone (n=2). Seven pts had prior intermediate-grade lymphoma transformation. The median pt age was 54 (range 34–67) years and median time from diagnosis to allogeneic HCT was 4.3 (0.5–18.5) years. Twelve pts had failed and 3 received cytoreductive autologous HCT before nonmyeloablative HCT. Disease status at time of nonmyeloablative HCT included complete remission (CR, n=16), partial remission (PR, n=12), refractory (n=11) and untested relapse (n=6). Prognostic scoring at the time of HCT using the Follicular Lymphoma International Prognostic Index (FLIPI) identified 25 low-risk and 19 intermediate-risk pts and using the International Prognostic Index (IPI) identified 26 low-risk, 13 low-intermediate risk, and 5 intermediate-high risk pts. There were insufficient data to score 1 pt by the FLIPI or by the IPI. All pts received G-CSF mobilzed peripheral blood mononuclear cells. Six of the 23 unrelated donor graft recipients had human leukocyte antigen (HLA) mismatches: one at a single allele, 4 at a single antigen, and one at an antigen and an allele. The median follow up after nonmyeloblative HCT was 23.8 (range 2– 44.9) months. One pt experienced non-fatal graft rejection of a single antigen mismatched unrelated graft. The cumulative probabilities (CP) of acute grades II-IV, III-IV and chronic GVHD were 60%, 18%, and 51%, respectively. Responses [CR (n=14) and PR (n=6)] were seen in 20 of 28 (71%) pts with measurable disease at HCT while 3 had stable and 3 progressive disease, and 2 were not evaluable due to early nonrelapse death. None of the 20 pts who responded and 3 of the 17 transplanted in CR had disease relapse. At 2 years, the CPs of overall relapse/progression, and non-relapse mortality were 15% and 34%, respectively. The 2-year Kaplan-Meier estimates of overall and progression free survival were 58% and 51%, respectively. Higher IPI and FLIPI scores modeled as continuous variables were independently associated with worse PFS (HR 1.55, p=0.05 and HR 1.35, p=0.10 respectively). Use of a HLA mismatched unrelated donor was associated with a trend for worse OS and PFS (HR 3.0, p=0.08 for both). Allogeneic HCT after nonmyeloablative conditioning is a promising salvage strategy for pts with relapsed and refractory indolent lymphoma. The high response and low relapse rates with this approach suggest that relapsed indolent lymphomas are susceptible to graft-versus-tumor responses.

For most hematologic malignancies treated by myeloablative conditioning, hematopoietic cell transplantation (HCT) from HLA-matched unrelated donors has been associated with greater non-relapse mortality (NRM) and worse overall survival (OS) than that from HLA-identical sibling donors. Poorer outcomes with unrelated grafts are largely related to more frequent or severe graft-versus-host disease (GVHD), and higher rates of graft failure.

Nonmyeloablative hematopoietic cell transplantation (NMHCT) has become an immunotherapeutic option for patients with a variety of malignant diseases. The susceptibility to malignancy-eradicating graft-versus-tumor effects, however, appears to depend on type and stage of the underlying diseases. Thus, the aim of this study was to estimate relapse risk after NMHCT according to pretransplant disease characteristics.

Forty-five patients (pts) with relapsed or chemotherapy refractory low-grade lymphomas were treated with HLA-matched related (n=22) and unrelated (n=23) HCT after nonmyeloablative conditioning using 2 Gy total body irradiation with or without fludarabine. Postgrafting immunosuppression consisted of cyclosporine and mycophenolate mofetil. Lymphoma histologies included follicular (n=38), small lymphocytic (n=5), and marginal zone (n=2). Seven pts had prior intermediate-grade lymphoma transformation. The median pt age was 54 (range 34–67) years and median time from diagnosis to allogeneic HCT was 4.3 (0.5–18.5) years. Twelve pts had failed and 3 received cytoreductive autologous HCT before nonmyeloablative HCT. Disease status at time of nonmyeloablative HCT included complete remission (CR, n=16), partial remission (PR, n=12), refractory (n=11) and untested relapse (n=6). Prognostic scoring at the time of HCT using the Follicular Lymphoma International Prognostic Index (FLIPI) identified 25 low-risk and 19 intermediate-risk pts and using the International Prognostic Index (IPI) identified 26 low-risk, 13 low-intermediate risk, and 5 intermediate-high risk pts. There were insufficient data to score 1 pt by the FLIPI or by the IPI. All pts received G-CSF mobilzed peripheral blood mononuclear cells. Six of the 23 unrelated donor graft recipients had human leukocyte antigen (HLA) mismatches: one at a single allele, 4 at a single antigen, and one at an antigen and an allele. The median follow up after nonmyeloblative HCT was 23.8 (range 2– 44.9) months. One pt experienced non-fatal graft rejection of a single antigen mismatched unrelated graft. The cumulative probabilities (CP) of acute grades II-IV, III-IV and chronic GVHD were 60%, 18%, and 51%, respectively. Responses [CR (n=14) and PR (n=6)] were seen in 20 of 28 (71%) pts with measurable disease at HCT while 3 had stable and 3 progressive disease, and 2 were not evaluable due to early nonrelapse death. None of the 20 pts who responded and 3 of the 17 transplanted in CR had disease relapse. At 2 years, the CPs of overall relapse/progression, and non-relapse mortality were 15% and 34%, respectively. The 2-year Kaplan-Meier estimates of overall and progression free survival were 58% and 51%, respectively. Higher IPI and FLIPI scores modeled as continuous variables were independently associated with worse PFS (HR 1.55, p=0.05 and HR 1.35, p=0.10 respectively). Use of a HLA mismatched unrelated donor was associated with a trend for worse OS and PFS (HR 3.0, p=0.08 for both). Allogeneic HCT after nonmyeloablative conditioning is a promising salvage strategy for pts with relapsed and refractory indolent lymphoma. The high response and low relapse rates with this approach suggest that relapsed indolent lymphomas are susceptible to graft-versus-tumor responses.

Background and objective:

Sustained engraftment after marrow transplantation from major histocompatibility complex matched donors depends on the intensities of both pretransplant conditioning and postgrafting immunosuppression. Here we asked, both in a preclinical canine model and in the clinical setting, whether, additionally, the number of transplanted marrow cells had a role in the success of engraftment after minimally intensive conditioning. Thirty-four dogs received marrow from dog leukocyte antigen (DLA)-matched related donors after 1 Gy total body irradiation (TBI). Postgrafting immunosuppression included cyclosporine with or without added drugs, most often mycophenolate mofetil, for no more than 5 weeks. Median total nucleated cell (TNC) doses administered were 3.6 (range 1.7–7.98) x 108/kg. Donor chimerism reached median (range) peaks of 15 (2–75) % for granulocytes and 9 (2–55) % for mononuclear cells (MNC), respectively. Eventually, 31 dogs rejected their grafts, after a median of 10 (range 3–20) weeks. TNC doses had significant impacts on outcomes, after adjustment for immunosuppressive regimens received (Table 1); each increase by 1x108/kg decreased the hazard ratio (HR) of rejection by 0.7, prolonged time to rejection by 2 weeks, and increased maximal percentages of donor chimerism by 8% in granulocytes and by 5% in MNC. Eighteen patients with hematological malignancies received marrow grafts from 10/10 human leukocyte antigen (HLA)-matched unrelated donors, after conditioning with Fludarabine, 90 mg/m2, and 2 Gy TBI, followed by postgrafting mycophenolate mofetil and cyclosporine. Median (range) TNC doses were 3.3 (1.3–4.6) x 108/kg, CD34 cells were 2.8 (0.9–7.2) x 106/kg, and CD3 cells were 31 (16–62) 106/kg. Donor chimerism reached median peaks of 95 (0–100) % in granulocytes and 43 (0–100) % in T cells. Eight patients rejected their grafts after a median of 1 (range 1–4.5) month. The numbers of CD34 and CD3 cells infused were not significantly associated with the risk of rejection (Table 2). However, TNC doses significantly impacted on engraftment; each increase in TNC dose by 1x108/kg decreased the HR of rejection by a factor of 0.3 and increased percentages of donor T-cell chimerism by 18% (p=0.05); there was no correlation with granulocyte chimerism. This effect was not negated by adjustment for patient age, diagnosis group, and numbers of preceding cycles of chemotherapy. In conclusion, while allogeneic marrow grafts are possible following minimal intensity conditioning in dogs and human patients, eventual graft rejection is a major problem, the risk of which can be minimized by transplanting the largest number of nucleated marrow cells possible. Impact of an increase in TNC dose by 108 cells/kg on outcomes of allogeneic canine marrow transplantation Outcome p-value 95% CI Rejection HR = 0.7 0.04 0.5–1.0 Time to rejection Prolonged by 2 weeks 0.02 0.4–4.0 Granulocyte chimerism Increased by 8% 0.006 2.4–13.0 Mononuclear cell chimerism Increased by 5% 0.003 1.9–8.6 Impact of marrow cell doses on rejection after allogeneic human marrow transplantation Cell populations and doses Hazard Ratio of Rejection p-value 95% CI TNC 108/kg increase 0.3 0.02 0.1–0.8 CD34 106/kg increase 0.6 0.21 0.3–1.3 CD3 0.6 0.44 0.2–2.2

Efforts to replace total body irradiation (TBI) used for transplant conditioning with agents that have less acute and long-term toxicities require a better understanding of the biological effects of low dose TBI. We therefore retrospectively analyzed the role of radiation dose, stem cell source, and type of immunosuppression on both the stability and degree of donor chimerism in canine recipients of matched littermate hematopoietic cell transplants. Recipients were prepared with 200 cGy (n=26), 100 cGy (n=76) or 50 cGy (n=19) total body irradiation (TBI) at 7 cGy/min. Stem cell sources included bone marrow (BM) alone (n=58), BM plus G-CSF mobilized peripheral blood mononuclear cells (G-PBMC) (n=42), BM and CD14-depleted G-PBMC (n=13), or BM and T-cell-depleted G-PBMC (n=8). Posttransplant immunosuppression consisted of cyclosporin (CSP) only (n=53), CSP plus mycophenolate mofetil (MMF) (n=23), CSP and rapamycin (n=12), CSP, MMF and rapamycin (n=5); or CSP and MMF in combination with pretransplant immunosuppression (n=28). The percentage of donor granulocytes in the peripheral blood, as determined by PCR amplification of variable numbers of tandem repeats (VNTR), served as a marker for engraftment. TBI dose and stem cell source were both significantly associated with long-term (>26 weeks) stable engraftment in multivariate analysis (p=0.0001 and p=0.004, respectively). Among the 39 dogs with stable engraftment, however, TBI dose was the only factor examined that was associated with the degree of donor chimerism (mean % of donor granulocytes after 200 cGy, 100 cGy and 50 cGy of TBI: 65%, 52%, and 24%, respectively; p=0.008). To determine whether low-dose irradiation directly affected recipient stem/progenitor cell numbers and thereby conferred a competitive disadvantage to donor cells, CD34+ cells were isolated from two normal human donors. One preparation of CD34 cells was ex vivoirradiated (=200 cGy) and then injected into NOD/SCID beta2m-/- mice in combination with an equal number of unirradiated CD34 cells from the second donor. The contributions of each donor to human engraftment were assessed at 10 weeks by VNTR. After 200 cGy, the irradiated population contributed 74% less than expected, 24% less after 100 cGy, but only 6% less after 50 cGy. Flow analysis of Caspase-3 activation indicated that a significant percentage of cells irradiated with 200 cGy were apoptotic, and that this was associated with the loss of L-selectin and P-selectin glycoprotein ligand-1. In conclusion, our findings suggest that TBI, in addition to its well-characterized immunosuppressive effects, determines the degree of donor cell engraftment by directly compromising recipient stem cells, thereby providing a competitive advantage to donor stem cells.

Sustained engraftment after marrow transplantation from major histocompatibility complex matched donors depends on the intensities of both pretransplant conditioning and postgrafting immunosuppression. Here we asked, both in a preclinical canine model and in the clinical setting, whether, additionally, the number of transplanted marrow cells had a role in the success of engraftment after minimally intensive conditioning. Thirty-four dogs received marrow from dog leukocyte antigen (DLA)-matched related donors after 1 Gy total body irradiation (TBI). Postgrafting immunosuppression included cyclosporine with or without added drugs, most often mycophenolate mofetil, for no more than 5 weeks. Median total nucleated cell (TNC) doses administered were 3.6 (range 1.7–7.98) x 108/kg. Donor chimerism reached median (range) peaks of 15 (2–75) % for granulocytes and 9 (2–55) % for mononuclear cells (MNC), respectively. Eventually, 31 dogs rejected their grafts, after a median of 10 (range 3–20) weeks. TNC doses had significant impacts on outcomes, after adjustment for immunosuppressive regimens received (Table 1); each increase by 1x108/kg decreased the hazard ratio (HR) of rejection by 0.7, prolonged time to rejection by 2 weeks, and increased maximal percentages of donor chimerism by 8% in granulocytes and by 5% in MNC. Eighteen patients with hematological malignancies received marrow grafts from 10/10 human leukocyte antigen (HLA)-matched unrelated donors, after conditioning with Fludarabine, 90 mg/m2, and 2 Gy TBI, followed by postgrafting mycophenolate mofetil and cyclosporine. Median (range) TNC doses were 3.3 (1.3–4.6) x 108/kg, CD34 cells were 2.8 (0.9–7.2) x 106/kg, and CD3 cells were 31 (16–62) 106/kg. Donor chimerism reached median peaks of 95 (0–100) % in granulocytes and 43 (0–100) % in T cells. Eight patients rejected their grafts after a median of 1 (range 1–4.5) month. The numbers of CD34 and CD3 cells infused were not significantly associated with the risk of rejection (Table 2). However, TNC doses significantly impacted on engraftment; each increase in TNC dose by 1x108/kg decreased the HR of rejection by a factor of 0.3 and increased percentages of donor T-cell chimerism by 18% (p=0.05); there was no correlation with granulocyte chimerism. This effect was not negated by adjustment for patient age, diagnosis group, and numbers of preceding cycles of chemotherapy. In conclusion, while allogeneic marrow grafts are possible following minimal intensity conditioning in dogs and human patients, eventual graft rejection is a major problem, the risk of which can be minimized by transplanting the largest number of nucleated marrow cells possible.

Efforts to replace total body irradiation (TBI) used for transplant conditioning with agents that have less acute and long-term toxicities require a better understanding of the biological effects of low dose TBI. We therefore retrospectively analyzed the role of radiation dose, stem cell source, and type of immunosuppression on both the stability and degree of donor chimerism in canine recipients of matched littermate hematopoietic cell transplants. Recipients were prepared with 200 cGy (n=26), 100 cGy (n=76) or 50 cGy (n=19) total body irradiation (TBI) at 7 cGy/min. Stem cell sources included bone marrow (BM) alone (n=58), BM plus G-CSF mobilized peripheral blood mononuclear cells (G-PBMC) (n=42), BM and CD14-depleted G-PBMC (n=13), or BM and T-cell-depleted G-PBMC (n=8). Posttransplant immunosuppression consisted of cyclosporin (CSP) only (n=53), CSP plus mycophenolate mofetil (MMF) (n=23), CSP and rapamycin (n=12), CSP, MMF and rapamycin (n=5); or CSP and MMF in combination with pretransplant immunosuppression (n=28). The percentage of donor granulocytes in the peripheral blood, as determined by PCR amplification of variable numbers of tandem repeats (VNTR), served as a marker for engraftment. TBI dose and stem cell source were both significantly associated with long-term (>26 weeks) stable engraftment in multivariate analysis (p=0.0001 and p=0.004, respectively). Among the 39 dogs with stable engraftment, however, TBI dose was the only factor examined that was associated with the degree of donor chimerism (mean % of donor granulocytes after 200 cGy, 100 cGy and 50 cGy of TBI: 65%, 52%, and 24%, respectively; p=0.008). To determine whether low-dose irradiation directly affected recipient stem/progenitor cell numbers and thereby conferred a competitive disadvantage to donor cells, CD34+ cells were isolated from two normal human donors. One preparation of CD34 cells was ex vivo irradiated (=200 cGy) and then injected into NOD/SCID beta2m-/- mice in combination with an equal number of unirradiated CD34 cells from the second donor. The contributions of each donor to human engraftment were assessed at 10 weeks by VNTR. After 200 cGy, the irradiated population contributed 74% less than expected, 24% less after 100 cGy, but only 6% less after 50 cGy. Flow analysis of Caspase-3 activation indicated that a significant percentage of cells irradiated with 200 cGy were apoptotic, and that this was associated with the loss of L-selectin and P-selectin glycoprotein ligand-1. In conclusion, our findings suggest that TBI, in addition to its well-characterized immunosuppressive effects, determines the degree of donor cell engraftment by directly compromising recipient stem cells, thereby providing a competitive advantage to donor stem cells.