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We retrospectively compared the incidence of virus infections and outcome in the context of immune reconstitution in two different HLA-haploidentical transplantation (haplo-HSCT) settings. The first was a combined T-cell-replete and T-cell-deplete approach using antithymocyte globulin (ATG) prior to transplantation in patients with hematological diseases (cTCR/TCD group, 28 patients; median age 31 years). The second was a T-cell-replete (TCR) approach using high-dose posttransplantation cyclophosphamide (TCR/PTCY group, 27 patients; median age 43 years). The incidence of herpesvirus infection was markedly lower in the TCR/PTCY (22 %) than in the cTCR/TCD group (93 %). Recovery of CD4+ T cells on day +100 was faster in the TCR/PTCY group. CMV reactivation was 30 % in the TCR/PTCY compared to 57 % in the cTCR/TCD group, and control with antiviral treatment was superior after TCR/PTCY transplantation (100 vs 50 % cTCR/TCD). Twenty-five percent of the patients in the cTCR/TCD group but no patient in the TCR/PTCY group developed PTLD. While 1-year OS was not different (TCR/PTCY 59 % vs cTCR/TCD 39 %; p = 0.28), virus infection-related mortality (VIRM) was significantly lower after TCR/PTCY transplantation (1-year VIRM, 0 % TCR/PTCY vs 29 % cTCR/TCD; p = 0.009). On day +100, predictors of better OS were lymphocytes >300/μl, CD3+ T cells >200/μl, and CD4+ T cells >150/μl, whereas the application of steroids >1 mg/kg was correlated with worse outcome. Our results suggest that by presumably preserving antiviral immunity and allowing fast immune recovery of CD4+ T cells, the TCR approach using posttransplantation cyclophosphamide is well suited to handle the important issue of herpesvirus infection after haplo-HSCT.

Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC). Relative fluorescent quantification (RFQ) analysis of HLA intron length polymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP) based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples.

Myeloid leukemic cells can differentiate into leukemia-derived dendritic cells (DC(leu)), presenting known/unknown leukemic-antigens. Induced anti-leukemic T-cell-responses are variable. To further elicit DC/DC(leu)-induced T-cell-response-patterns we performed (functional)flow-cytometry/fluorolysis-assays before/after mixed lymphocyte cultures (MLC) of matched (allogeneic) donor-T-cells (n=6), T-cells prepared at relapse after stem cell transplantation (n=4) or (autologous) patients'-T-cells (n=7) with blast-containing-mononuclear-cells ('MNC') or DC(leu)-containing DC ('DC'). Compared to 'MNC' 'DC' were better mediators of anti-leukaemic T-cell-activity, although not in every case effective. We could define cut-off proportions of mature DC, DC(leu), proliferating, CD4(+), CD8(+) and non-naive T-cells after 'MNC'- or 'DC'-stimulation, that were predictive for an anti-leukemic-activity of stimulated T-cells as well as a response to immunotherapy. Interestingly especially ratios >1 of CD4:CD8 or CD45RO:CD45RA T-cells were predictive for anti-leukemic function after DC-stimulation. In summary the composition and quality of DC and T-cells after a MLC-stimulating-phase is predictive for a successful ex-vivo and in-vivo anti-leukemic response, especially with respect to proportions of proliferating, CD4(+) and CD45RO(+) T-cells. Successful cytotoxicity and the development of a T-cell-memory after 'DC'-stimulation could be predictive for the clinical course of the disease and may pave the way to develop adoptive immunotherapy, especially for patients at relapse after SCT.

The Seventh International Symposium on graft-versus-host and graft-versus-leukemia reactions was held in Garmisch Partenkirchen (Germany, near Lake Riessersee) between January 22nd and 25th, 2006. A total of more than 100 invited participants (scientists and clinicians working in the area of allogeneic stem cell transplantation) discussed research in the area of lymphoid malignancies. Major topics of the 2006 meeting were lymphocyte biology, experimental systems, lymphoma pathogenesis, cellular therapy in vivo and vitro, idiotype-specific responses and graft-versus-malignancy reactions for lymphomas and multiple myeloma. Further highlights were immune responses to blasts of ALL, haploidentical transplantation, role of natural killer cells, clinical guidelines for allogeneic transplantation and adoptive immunotherapy in chronic lymphocytic leukemia and multiple myeloma, new antibody-mediated strategies. As can be seen in the summaries of the individual presentations, progress was made in the understanding of lymphoma biology and in the clinical application of graft-versus-lymphoma or graft-versus-myeloma effects. Each day was followed by round-table discussions, which summarized new data and challenged established concepts. The discussions resulted in new insights and projects for basic research and clinical transplantation.

T lymphocytes with self-destructive capacity are often found in healthy individuals, indicating efficient control mechanisms that prevent chronic autoimmune diseases. Since naive T lymphocytes do not circulate through extralymphoid tissues the concept has emerged that peripheral T cells ignore tissue-specific antigens unless they are presented by professional antigen-presenting cells in the lymphoid compartments. However, this view pays attention only to experiments performed in adult animals. This report reviews the evidence that tissues of neonatal mice, in contrast to adults, exhibit high accessibility for naive T cells, thereby allowing the direct contact with tissue-specific self-antigens on parenchymal cells during neonatal life and tolerance induction to such self-antigens. In mouse bone marrow chimeras generated at different ages, recent thymic emigrants were tolerized to a major histocompatibility class I antigen expressed on keratinocytes only during a neonatal period and not during adulthood. Blockade of T-cell migration neonatally prevented tolerance induction. The neonatally induced tolerance is maintained during adulthood, apparently by a dominant regulatory mechanism. Thus, parenchymal cells and T-cell migration in the neonate contribute to the control of autoreactive T cells.

In comparison with CD4 + regulatory T cells, the generation and function of immunomodulatory CD8 + T cells is less well defined. Here we describe the existence of regulatory anti-K b -specific CD8 + T cells that are rendered tolerant during neonatal life via antigen contact exclusively on keratinocytes. These regulatory T cells maintain tolerance during adulthood as they prevent K b -specific graft rejection by naïve CD8 + T cells. Third-party immune responses remain unaffected. Up-regulation of TGF-β1 and granzyme B in the regulatory CD8 + T cell population suggests the involvement of these molecules in common suppressive pathways shared with CD4 + regulatory T cells. In summary, CD8 + regulatory T cells can be induced extrathymically through antigen contact on neonatally accessible parenchymal cells and maintain tolerance throughout adult life.

Introduction: Monitoring of minimal residual disease (MRD) is commonly used after allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML) patients. Flow cytometry of leukemia-associated aberrant immunophenotypes (LAIP) and chimerism analyses are frequently used tools to monitor MRD. These methods have lower sensitivities and lower relapse detection rates in contrast to molecular markers monitored by quantitative RT-PCR. However, their clinical implications are still limited and only little evidence of single molecular markers is available. We report on a retrospective study of 144 patients with molecular MRD markers transplanted at the Ludwig-Maximilans-University Hospital of Munich-Grosshadern, and analyzed the prognostic values of molecular MRD Monitoring after SCT, baseline risk factors, and follow-up (FU) markers (e.g. chimerism analyses and LAIP). Patients and Methods: Between January 2000 and August 2012, 495 AML patients underwent SCT at our institution. 164 patients had molecular MRD markers and 144 patients had at least one sample with quantitative results prior and/or after SCT, and were included into the analyses. At transplantation, patients had a median age of 48 years (range, 18 -73), and 49 patients were in complete remission, while 95 patients were in a more advanced status of their disease (>CR2). 106 patients received SCT from a HLA matched (10/10) and 38 patients from a HLA mismatched donor. Grafts were obtained either from related (n=60) or unrelated (n=84) donors. Most patients received reduced intensity conditioning (n=142). In 338 bone marrow samples MRD was monitored prior to SCT, on day +30 and on day +100. During the FU period after day +100, MRD was monitored at individual intervals in 429 peripheral blood samples. Quantitative RT-PCR was performed for NPM1 mutation (n=52), MLL-PTD (n=31), RUNX1-RUNX1T1 (n=12), CBFß-MYH11 (n=14), MLL rearrangements (n=20), MDS-EVI1/EVI1 (n=10), and DEK-CAN (n=5). Sensitivities of the different RT-PCRs assays ranged between 10-4 and 10-6. Results: After a median FU of 41 months (range, 4 – 115), 43 patients (30%) relapsed. The MRD levels monitored by RT-PCR prior to SCT and on day +30 after SCT showed no significant impact on relapse-free survival (RFS) and overall survival (OS). At day +100 after SCT, MRD positivity was strongly associated with worse RFS (HR 3.1, p=0.001) and OS (HR 3.2, p=0.004). This was also reflected in a cumulative two-year incidence of relapse of 61% for MRD positive patients versus 15% for MRD negative patients (p Conclusions: Molecular MRD monitoring after SCT might be a useful tool to identify AML patients at high risk for relapse, in contrast to other FU markers, e.g. chimerism analyses and LAIP. In particular, MRD positivity on day +100 after SCT and the switch to MRD positivity during the FU period were significantly associated with worse RFS. As perspective, the individual molecular MRD course might be used as prognostic factor for the guidance of treatment post SCT. Patients with MRD positivity on day +100 after SCT or with a switch to MRD positivity in the FU period may be considered for donor lymphocyte infusions (DLI) or chemotherapeutic interventions, such as hypomethylating agents. Disclosures Subklewe: AMGEN Inc.: Research Funding.

Allogeneic hematopoietic stem cell transplantation (HSCT) has become a valuable therapeutic option for malignant and non-malignant hematological diseases. Engraftment of donor cells is confirmed by repetitive testing for donor chimerism. Since the underlying malignant disease is host derived, the decrease of donor chimerism might precede or indicate the imminent relapse, enabling early intervention and presumably better outcome. STR-assay (Short-Tandem-Repeats-Assay) and XY-FISH (XY based Fluorescence in Situ Hybridization) respectively are routinely performed after HSCT. Furthermore CD34+ cell- chimerism and quantification of minimal residual disease in patients with informative markers might be used to detect early relapse. In this prospective, non-interventional study we evaluated the accuracy, reliability and feasibility of a qPCR based commercially available assay (Allele SEQR® Chimerism Assay, Abbott). In addition, the early detection of hematological relapse was analyzed as a clinical readout. Between May 2011 and January 2013 95 patients received allogeneic HSCT for MDS or acute leukemia (AML = 84) at our transplantation unit and were therefore included in our analysis. According to our local standard bone marrow samples were collected at standardized time points including days 30, 90, 180. The qPCR based Allele SEQR® Chimerism Assay consists of two parts: screening for discriminating, informative markers and subsequent actual quantification of the host DNA. For assay validation 68 patients were suitable. In all patient/donor pairs at least one informative marker could be found and quantitative results could be achieved. In 65/68 pairs (95.6%) even ≥ 2 markers could be identified, however, among 28 related donors three (10.7%) only revealed one discriminating marker. The sensitivity of the assay was proven by means of artificially spiked DNA samples mimicking an amount of 0.1%, 0.05% and 0.01% of “host“ DNA respectively. Testing was performed with 100ng/well patient DNA. The overall time needed for testing was less than four hours. We identified 61 patients with at least two samples in cytomorphological complete remission (CR) after allogeneic HSCT. The overall hematological relapse rate in our cohort was 23% until March 2014. To enable relapse prediction we quantified donor chimerism in CR samples. Scoring for suspicious results by the increase of host chimerism by at least one percent point, but not less than one third as compared to previous testing, was highly predictive for imminent relapse (overall relapse rate 86% vs. 15%, p= The evaluated qPCR approach proved as a fast and highly sensitive tool for chimerism monitoring after HSCT. It may allow an early detection or exclusion of imminent relapse, enabling chimerism triggered therapy. In perspective we will validate this assay for peripheral blood samples, allowing more convenient and frequent testing. Disclosures Reibke: Abbott molecular Germany: Research Funding.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a valuable treatment option for patients with relapsed or refractory lymphoma including patients with relapse after a first autologous HSCT. However, outcome in non-remission patients is still poor. Here we report the feasibility and efficacy of a protocol using the concept of sequential therapy with clofarabine induction chemotherapy followed by a HLA-haploidentical HSCT in the treatment of patients with advanced non-remission lymphoma. 16 adults (11 male, median age: 53 years, median comorbidity index: 3) with MCL (n=7), CLL (n=1), AITL (n=2), secondary DLBCL (n=2), DLBCL (n=2), follicular lymphoma (n=1), B- lymphoblastic lymphoma (n=1) were treated between September 2010 and February 2013. After induction-chemotherapy with clofarabine (5x30 mg/m² IV), followed by 3 days of rest, a reduced intensity conditioning (RIC) consisting of fludarabine/cyclophosphamide plus melphalan 110 mg/m2 IV was performed prior to a T-cell replete HLA-haploidentical HSCT. High dose cyclophosphamide (Cy) was administered for post-grafting immuno-suppression followed by mycophenolate mofetil and tacrolimus. The median number of immuno-chemotherapy attempts prior to haploidentical HSCT was 4 including 8 autologous HSCT. None of the patients were in complete remission (CR) prior to haploidentical HSCT. After salvage therapy and prior to haploidentical HSCT, progressive disease (PD) was documented in 9 patients with one patient being primarily refractory, while in 7 patients a partial remission (PR) was observed. Neutrophil engraftment was achieved in 16 patients (100%) within a median of 21 days (range 14-36) and platelet engraftment in 14 patients (88%) within a median of 30 days (range 18-115). No primary graft rejection occured. By day +30 CR was achieved in 4 patients (25%) and PR in 11 patients (69%), whereas 1 patient (6%) showed PD. Full donor chimerism on day +30 was detected in all 16 patients. By day +100, 8 of the 14 evaluated 14 patients (57%) achieved CR, 4 (29%) PR, and only 2 (14%) had relapsed. Full donor chimerism was detected in 13 (93%) patients. The rate of acute GvHD grade II-IV was 31%, while chronic GvHD occurred in 3 of 14 (21%) evaluable patients (21%). Non hematologic regimen-related grade III-IV toxicity was observed in 9 patients (56%), and included most commonly transient elevation of liver enzymes (38%), mucositis (19%), and nausea and vomiting (19%). Creatinine elevation (6%), hand-foot syndrome (6%) and haemorrhagic cystitis (6%) were rare. After a median follow up of 14.5 months (range 5.7-35) 4 patients had relapsed, and 4 patients had died, while death was lymphoma-associated only in one patient. Causes of non-relapse mortality (NRM) were haemorrhagic cerebral bleeding in one patient (day +101), and toxicity/infection in 2 patients (day +141; day +156). Cumulative incidence of NRM was 0% on day +100 and increased up to 20% (7-50) on day +360. Estimated one-year overall survival and progression-free survival were 75% (95 CI 46-90) and 56% (95 CI 30-76), respectively. Sequential therapy combining cloforabine induction-chemotherapy and HLA-haploidentical HSCT using RIC, a T-cell replete graft and high dose Cy post-transplant is feasible, shows a favourable toxicity profile and furthermore achieves significant anti-lymphoid activity in patients with advanced non-remission lymphoma. These early results are promising, but longer follow up is needed. Disclosures: Off Label Use: Clofarabine off label use in adults. Hausmann:Sanofi: Travel support Other. Tischer:Sanofi: Travel support Other.

Abstract 3071 Background: Prognosis of relapse after first allogeneic hematopoietic stem cell transplantation (allo-HSCT) is poor, and no standard treatment is available. Second HSCT (HSCT2) is a valuable treatment option for selected patients. Change of donor for HSCT2 is a frequently used strategy to enhance the graft-versus leukemia-activity, although its role for improving the outcome is still unclear. However, identification and activation of a new donor for HSCT2 is time consuming and might delay or even prevent a second transplant. In contrast, haploidentical family donors are rapidly available in the vast majority of patients and might therefore be an alternative for HSCT2, once change to a new donor is planned. Based upon these considerations, HSCT2 from haploidentical family donors was used as preferred treatment for acute leukemia relapse after allo-HSCT in our transplant center since August 2009. According to the concept of sequential therapy (as introduced by the FLAMSA-RIC regimen), patients initially received cytoreductive chemotherapy, followed, after three days of rest, by reduced intensity conditioning (RIC). Based on the Baltimore protocol for haploidentical HSCT using RIC and post-transplant cyclophosphamide for depletion of allo-reactive T-cells, individual and disease-specific modification of this regimen were used for preparation before HSCT2. Patients and Methods: Fourteen adult patients (7 male, median age: 37 years) suffering from AML (n=11) or ALL (n=3) with an ECOG score of Results: Neutrophil and platelet engraftment was achieved in 12 (85,7%) and 10 (71,4%) patients, respectively. Median time to neutrophil engraftment was 32 days (range 24–90). No primary graft rejection was observed. Complete remission could be induced by day +30 in 11 patients (78,6%), whereas 1 patient died early and 2 patients had refractory disease. Full donor chimerism by day +30 was achieved in 10 patients (71,4%). After a median follow-up of 6 months (range 4 days–33 months) cumulative incidence of non-relapse mortality at day + 30, + 100 and +360 was 7%, 24% and 24 %, respectively. Rate of grade II-IV acute GvHD was 24,7%. Mild or moderate chronic GvHD was seen in 2 patients (14,3%). 8 patients died from relapse (n=4), infection (n=3) or infection and toxicity (n=1). Estimated overall survival and progression-free survival at one year from HSCT2 was 53 % (+/− 14 %) and 36 % (+/− 17 %), respectively. Infection was frequent detected in 13 (92,9%) patients with at least one episode of neutropenic fever in 8 patients, probably or proven invasive aspergillosis in 5 patients and a moderate rate of virus-reactivation/-infection (CMV 21,4%, HSV 14,2%, EBV 28,6%, HHV-6 28,6%, Polyomavirus 28,6%, Adenovirus 21,4%,). No patient developed a post-transplant lympho-proliferative disorder. Regimen related grade III-IV toxicity was observed in 9 (64,3%) patients with transient elevation of liver enzymes in 7 (50%), mucositis in 5 (35,7%), headache in 5 (35,7%), hand-foot syndrome in 3 (21,4%), hyper-bilirubinemia in 3 (21,4%) and severe deterioration of kidney function in 2 (14,3%) patients, respectively, as the most common observed toxicities. Conclusion: Haploidentical HSCT2 following initial cytoreduction with clofarabine, RIC and high-dose cyclophosphamide post-transplant is feasible in patients with acute leukemia relapsing after first allo-HSCT. Early results are promising, but long-term results are still pending. Disclosures: Off Label Use: Clofarabine is a novel purine nucleoside with immunosuppressive and anti-leukemic activity in hematologic malignancies, off-label use in adults.