We conclude that MSC remain of host type irrespective of the conditioning regimen and graft source.

We have previously identified NME2 (Nm23-H2) as a tumor antigen in a patient with chronic myeloid leukemia (CML). Here we investigated the association between NME2 and Bcr-Abl. NME2 protein was highly overexpressed in the cytoplasm of peripheral blood mononuclear cells from 29/30 patients with CML at diagnosis and 10/10 patients resistant to imatinib. Protein was overexpressed in the absence of increased levels of mRNA and was limited to Bcr-Abl + populations, being absent from Bcr-Abl - patient cells, normal donors and 14/15 acute myeloid leukemia (AML) samples. Furthermore, the Bcr-Abl dependent overexpression of NME2 protein was reversed specifically by tyrosine kinase inhibitor (TKI) treatment of Ba/F3 expressing wild-type and TKI-sensitive, but not TKI-resistant, mutants of Bcr-Abl. The post-transcriptional up-regulation of the tumor antigen NME2 is therefore a common and specific property of CML closely associated with Bcr-Abl activity.

Therapeutic effects of haematopoietic stem cell transplantation are not limited to maximal chemoradiotherapy and subsequent bone marrow regeneration, but include specific as well as unspecific immune reactions known as graft-versus-leukaemia (GvL) effects. Specific immune reactions are likely to be particularly relevant to the long-term treatment of diseases, such as chronic myeloid leukaemia (CML), in which residual cells may remain quiescent and unresponsive to cytotoxic and molecular therapies for long periods of time. Specific GvL effects result from the expression on leukaemic cells of specific tumour-associated antigens (TAAs) in the context of HLA proteins. As human leukocyte antigen (HLA) types vary widely, the development of broadly applicable tumour vaccines will require the identification of multiple TAAs active in different HLA backgrounds. Here, we describe the identification of NM23-H2 as a novel HLA-A32-restricted TAA of CML cells and demonstrate the presence of specifically reactive T cells in a patient 5 years after transplantation. As the NM23 proteins are aberrantly expressed in a range of different tumours, our findings suggest potential applications beyond CML and provide a new avenue of investigation into the molecular mechanisms underlying CML.

Minor histocompatibility antigens can be responsible for both GvHD and GvT effects after HCT, depending on their tissue distribution. HA-1 is expressed on hematopoietic cells and several types of solid tumors, but not on GvHD target cells. Two alleles have been defined, of which HA-1H is immunogenic in the presence of HLA-A*0201. Here, we investigate HA-1-specific immune responses in HLA-A*0201 HA-1H individuals after related, HLA-matched, HA-1 mismatched HCT. Methods: A total of 9 patients with hematological malignancies received HCT from HLA-matched, HA-1-mismatched donors, in seven cases following reduced intensity conditioning (RIC). The appearance of HA-1-specific T-cells was monitored with HLA-A*0201/HA-1H Tetramers and with the IFN-γ Elispot Assay at different time points before and after HCT (days 0, 28, 56, 84 and monthly thereafter). T-cell lines were generated from 2 patients at different time points after engraftment. Results: HA-1-specific immune reactions were detected in 3 patients in the months following HCT, 2 after RIC and 1 after conventional HCT. Frequencies of HA-1-specific T-cells increased further with time. Both RIC-transplanted patients had secondary AML. The first patient had a complete remission (CR) of his malignancy coincident with a grade I skin GvHD. HA-1-specific T-cells persisted for at least 3 years after engraftment at an average of 0.05% total mononuclear cells. After discontinuation of immunosuppression at day 1000 post HCT, frequencies of up to 0.24% were detected and strong HA-1-specific reactions were observed by Elispot. The second RIC patient required immunosuppression to control acute GvHD. On day 298 after HCT, CD34+ donor chimerism (DC) declined to 15% as an early indication of relapse. After reducing cyclosporine, a reversion to 100% DC occurred simultaneously with an increase of HA-1-specific T-cells from 0.05 to 0.14%. The patient went into remission, but again experienced GvHD. Increased immunosuppression was associated with a decrease in HA-1-specific T-cells. A similar decrease in CD34+ DC occurred at day 591 and 726 after HCT and was treated with low-dose chemotherapy and reduction of immunosuppression. On these occasions, the level of HA-1-specific T-cells increased from 0.08% to 0.21% and from 0.05% to 0.18% respectively, and was accompanied by the restoration of full DC. Interestingly, the fluctuations of HA-1-specific T-cells observed with Tetramer staining were not detected in the Elispot assay. T-cell lines were generated from PBMC of the 2 patients during periods of different HA-1 specific T-cell frequency. They reacted similarly with HA-1 peptide pulsed target cells and HA-1H EBV-LCL in the Elispot and Cr-release assay. Tetramer staining revealed up to 77,2% HA-1-specific CD8+ T-cells after 6 weeks of culture. Of the 2 patients with conventional HCT, 1 patient with CML was in CR with up to 0.17% HA-1 specific T-cells 557 days after tranplant. Conclusions: HA-1-specific immune reactions are observed in vivo after HA-1 mismatched HCT. Reactivity to HA-1 increases during the first 3 months after HCT and after reduction/discontinuation of immunosuppression. In one patient, increases of HA-1 specific T-cell frequency were associated with reductions in tumor cell burden (leukemic host CD34+ cells) and decreases with reappearance of tumor cells. Tetramer staining was clearly more sensitive than the Elispot assay in monitoring the endogenous HA-1-specific immune response. T cell lines can be established from PBMC independent of the frequency of HA-1-specific T cells.

4904 Background: Prognostic molecular markers are expected to be of increasing value in stratifying treatment of patients with AML lacking cytogenetic abnormalities. While specific DNA-level mutations such as those in the FLT-3 or NPM1 genes clearly identify informative genotypes, phenotypic characterization of gene expression should be a more direct indicator of cell function. However, of the prognostic mRNA levels identified to date, such as BAALC, ERG or NME1/2, none appear to be superior to DNA mutation as indicators of prognosis. Because of the lability of mRNA expression, it is common practice to quantify levels only in freshly processed or direct-lysed samples in order to provide a snapshot of gene expression as close as possible to that of fresh tissue. However, characteristics of leukemic cells relevant to prognosis might become more apparent under challenging conditions, such as the stress environment developing in a bone marrow sample during transport or storage. The NME1 and -2 mRNAs have been reported to be prognostic indicators in AML and are multifunctional proteins coordinating metabolism, signalling and gene expression. To see how storage related stress affects the prognostic power of NME mRNA, we have assessed their prognostic relevance in CN-AML bone marrow samples which had originally been processed either directly, or following ≥ 2 days of transport from collaborating centers. Methods: A total of 78 archived CN-AML BMMNC samples were available for this analysis. All samples were taken at presentation from patients under 60ys treated within the AML 96 (OSHO #033) or AML 2002 (OSHO #061) protocols of the East German Study Group (OSHO). Of the 78 samples, 25 (32%) originated locally and had been freshly processed to cryopreserved MNC, while the remaining 53 (68%) had been submitted as bone marrow from other centers with an associated delay of 2–3 days. NME1 and NME2 mRNA levels were determined by triplicate qRT-PCR determinations normalized to RPLP0. The mean NME1 and NME2 expression values from each patient were expressed relative to the corresponding mean NME expression of 17 healthy donor BM samples (control group).To investigate directly the effects of delayed processing on NME mRNA expression, aliquots of 11 fresh CN-AML bone marrow samples were removed for analysis either immediately or following storage at room temperature for ≥ 2 days. Results: Both NME1 and -2 mRNA were increased relative to controls in 84% (21/25) of fresh samples with no prognostic relevance of expression above or below the median. Comparable results were obtained from a cohort of 59 freshly processed CN-AML samples from a separate study. In contrast, the transported samples yielded a heterogeneous pattern of NME mRNA expression above and below control levels, with a significant correlation between NME2 mRNA and event-free survival (p=0.009) independently of FLT-3ITD status. Direct analysis of aliquoted AML bone marrow samples confirmed that a delay in processing of ≥ 2 days resulted in a universal decrease in NME1 mRNA with variable changes in NME2 mRNA. Conclusion: NME1/2 mRNA levels are not indicative of prognosis in freshly processed bone marrow from CN-AML patients <60ys. However, delayed processing is associated with the development of an NME2 expression pattern with high prognostic relevance, maintenance of high NME2 in samples with reduced NME1 being a strong indicator of increased event-free survival. These results suggest that subjecting leukemic cells to defined challenges ex-vivo may reveal phenotypic properties of high relevance to treatment optimization. Disclosures: No relevant conflicts of interest to declare.