Search

One of the pre-requisites for forensic DNA analysis is the fact that all nucleated cells of a person carry the same genetic information. However, this is not the case for individuals who have received an allogeneic hematopoietic stem cell or bone marrow transplantation, as all new cells formed by the bone marrow no longer show the genetic information of the recipient but that of the donor, while all other cells still carry the original information before transplantation. Thus, STR typing of a blood sample after successful transplantation yields a DNA profile that differs from the recipient’s original profile and corresponds to the donor genotype instead. Evidence from a routine case suggests that transplanted individuals may show donor alleles in skin swabs, as well. In order to examine this issue more closely, various skin swabs from 28 patients who have received an allogeneic hematopoietic stem cell transplantation were examined in this study. Swabs from the right and left palm, the back of the hand, one of the two upper arms, and the neck were collected from each person. Ninety-one of the 140 resulting swabs delivered useful results. All of those samples showed mixtures of recipient and donor DNA with different mixture ratios and the proportions of donor and recipient alleles revealed inter- and intra-individual differences. Those results were discussed with respect to graft versus host disease.

Prognosis is poor for patients with biologically aggressive Non-Hodgkin lymphoma (NHL), refractory to chemotherapy or relapsed after autologous transplantation, especially when no disease control before allogeneic transplantation is achieved. In 16 patients (median age 53, median prior regimes 5) with relapsed or refractory non-remission NHL, we analysed retrospectively the efficacy of a sequential therapy comprising clofarabine re-induction followed by a reduced-intensity conditioning with fludarabine, CY and melphalan, and T-cell-replete HLA-haploidentical transplantation. High-dose CY was utilized post-transplantation. All patients engrafted. Early response (day +30) was achieved in 94%. Treatment-related grade III-IV toxicity occurred in 56%, most commonly transient elevation of transaminases (36%), while there was a low incidence of infections (19% CMV reactivation, 19% invasive fungal infection) and GVHD (GVHD: acute III-IV: 6%; mild chronic: 25%). One-year non-relapse mortality was 19%. After a median follow-up of 21 months, estimated 1- and 2-year PFS was 56 and 50%, respectively, with 11 patients (69%) still alive after 2 years. In summary, sequential therapy is feasible and effective and provides an acceptable toxicity profile in high-risk non-remission NHL. Presumably, cytotoxic reinduction with clofarabine provides enough remission time for the graft-versus lymphoma effect of HLA-haploidentical transplantation to kick in, even in lymphomas that are otherwise chemo-refractory.

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.

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.

Haploidentical haematopoietic SCT (HSCT) using T-cell-replete grafts and post-transplant high-dose CY has found increasing acceptance. Our purpose was to evaluate the feasibility and outcome of this strategy as second HSCT incorporating donor change for acute leukaemia relapse after a first allogeneic transplantation. The courses of 20 consecutive adults (median age 37 years, 12 male) with AML (n=14), ALL (n=5) and acute bi-phenotypic leukaemia (n=1) were analysed retrospectively. Conditioning consisted of fludarabine, CY and either melphalan or TBI or tresosulfan+/-etoposide. Engraftment was achieved in 17 (85%), and a second remission was induced in 15 patients (75%) on day +30. The rate of grade II-IV acute GvHD was 35%, while chronic GvHD occurred in five patients. Most commonly observed grade III-IV toxicities were mucositis (30%), hyperbilirubinemia (20%), elevation of transaminases (20%) and creatinine (20%), while invasive fungal infection affected 30%. One-year non-relapse mortality (NRM) was 36%. At a median follow-up of 17 months, estimated 1-year OS was 45%, and 1-year relapse-free survival was 33%. This strategy was feasible and allowed for successful engraftment with a moderate rate of toxicity. Early outcome and NRM are at least comparable with results after a second HSCT from HLA-matched donors without donor change at HSCT2.

Recently it was shown that myeloid leukemic cells can be induced to differentiate into leukemia-derived dendritic cells (DCleu), regaining the stimulatory capacity of professional DCs while presenting the leukemic antigen repertoire. But so far, the induced antileukemic T-cell responses have varied in specificity and efficacy, or have even mediated opposite effects. In an attempt to further characterize the DC/DCleu induced T-cell response pattern, immunoscope spectratyping, a novel and powerful tool to detect T-cell receptor (TCR) rearrangements was used in combination with functional flow cytometry and non-radioactive fluorolysis assays. Human leucocyte antigen (HLA) matched donor T-cells were repeatedly stimulated, either with leukemic blasts (French-American-British, FAB M4eo) or the corresponding blast-derived DCs. Functional comparison revealed no significant difference in their T-cell stimulatory capacity, while the DC/DCleu fraction favored T-cells with a higher lytic activity, comprising a higher proportion of T-memory CD45R0+ cells. Stimulation with blasts and DC/DCleu induced a similar TCR restriction pattern, while stimulation with DC/DCleu favored the CD4 T-cell subset and seemed to cause a higher grade of restriction. In conclusion, a combined strategy using spectratyping with functional tests might not only provide useful information about the specificity and efficacy of the induced T-cell response, but also pave the way to gain effective T-cell clones for therapeutic use.

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.

Context/Objective: Approximately 15% of thyroid cancer patients develop subsequent metastases. The clinical course of patients with metastatic thyroid carcinoma is highly variable. We hypothesized that the metabolic activity of metastatic lesions, as defined by retention of 2-[18F]fluoro-2-deoxyglucose (FDG), would correlate with prognosis. Design/Patients: The initial FDG-positron emission tomography (PET) scans from 400 thyroid cancer patients were retrospectively reviewed and compared with overall survival (median follow-up, 7.9 yr). We examined the prognostic value of clinical information such as gender, age, serum thyroglobulin, American Joint Committee on Cancer (AJCC) stage, histology, radioiodine avidity, FDG-PET positivity, number of FDG-avid lesions, and the glycolytic rate of the most active lesion. Results: Age, initial stage, histology, thyroglobulin, radioiodine uptake, and PET outcomes all correlated with survival by univariate analysis. However, only age and PET results continued to be strong predictors of survival under multivariate analysis. The initial American Joint Committee on Cancer stage was not a significant predictor of survival by multivariate analysis. There were significant inverse relationships between survival and both the glycolytic rate of the most active lesion and the number of FDG-avid lesions. Conclusions: FDG-PET scanning is a simple, expensive, but powerful means to restage thyroid cancer patients who develop subsequent metastases, assigning them to groups that are either at low (FDG negative) or high (FDG positive) risk of cancer-associated mortality. We propose that the aggressiveness of therapy for metastases should match the FDG-PET status.

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.