Quantitative real-time PCR (qPCR) has been proposed as a highly sensitive method for monitoring hematopoietic chimerism and may serve as a surrogate marker for the detection of minimal residual disease minimal residual disease in myelodysplastic syndrome (MDS), until specific methods of detection become available. Because a systematic comparison of the clinical utility of qPCR with the gold standard short tandem repeat (STR)-PCR has not been reported, we retrospectively measured chimerism by qPCR in 54 children transplanted for MDS in a previous study. Results obtained by STR-PCR in the initial study served as comparison. Because the detection limit of qPCR was sufficiently low to detect an autologous background, we defined the sample as mixed chimera if the proportion of recipient-derived cells exceeded .5%. The true positive rates were 100% versus 80% (qPCR versus STR-PCR, not significant), and mixed chimerism in most cases was detected earlier by qPCR than by STR-PCR (median, 31 days) when chimerism was quantified concurrently in peripheral blood and bone marrow. Both methods revealed a substantial rate of false positives (22.7% versus 13.6%, not significant), indicating the importance of serial testing of chimerism to monitor its progression. Finally, we propose criteria for monitoring chimerism in pediatric MDS with regard to the subtypes, specimens, PCR method, and timing of sampling.

Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc−, NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity against AML and RMS cells without causing GvHD.

CD33/34 lineage specific chimerism analysis (CD33/34 LCA) can improve the predictive value of chimerism monitoring after allo-SCT in AML. Recently, real time PCR (qPCR) has been proposed for highly sensitive and accurate quantitation of chimerism. However, data to compare clinical impact of qPCR versus gold standard conventional STR-PCR was not available. In 2011, we reported on the impact of STR chimerism monitoring in a pediatric AML multicenter study (Rettinger, Willasch et al., Blood 2011;118(20):5681-88). Hereby, we present a prospective analysis on CD33/34 LCA in bone marrow (BM) and mononuclear cell (MNC) chimerism in peripheral blood (PB) by qPCR in the pediatric AML cohort mentioned above. Pre-existing STR-PCR data were used for comparison. This analysis for the first time refers to the clinical impact of a combination of CD33/34 LCA and qPCR technology. Monitoring of 75 transplantations, performed in 72 patients at 11 German pediatric transplant centers between 05/2005 and 04/2009, included 26 (0-84) PB and 5 (0-21) BM samples per patient and covered 1.5 (0.1-4.3) years (median). PB was analyzed weekly and BM at days 30, 60, 100 and 6, 9, 12, 15 and 18 months post-transplant. Lowering the quantifiable limit by qPCR (here </=10E-3 (0.1%)) can result in detection of recipient derived (autologous) signals (AS) in virtually every sample. The challenge was to define a clinically relevant cut off level for diagnosis of complete (CC) or mixed chimera (MC). For qPCR, cut off levels of 0.1 and 0.5% for AS were evaluated and chimerism was defined according to tab. 1. Based on this definition, risk groups and profiles were specified (tab. 2). Allocation into risk groups 2 to 4 was done at day of first MC in either compartment. Risk profile I (RP I) defined each sample with MC in either compartment as HR, whereas risk profile II (RP II) defined each sample with MC in MNC as HR. 56/75 transplant follow-ups allowed for analysis of predictive value of chimerism (tab. 3). Sensitivity of RP I ranged from 100% (qPCR cut off 0.1) to 75% (STR-PCR); notably specificity was low and false pos. rate high, if this highly sensitive approach was applied. In RP II sensitivity was significantly lower compared to RP I, but specificity was considerably higher and false pos. rate lower. Relapse was predicted earlier by qPCR with a median of 42 days compared to STR-PCR in 71% if 0.1% cut off for definition of MC was applied (tab. 4). If 0.5% cut off was used, this rate dropped to 44.5%. Taken together, qPCR as well as STR-PCR allowed for monitoring of chimerism. Each method revealed its specific pros and cons according to its high (qPCR, </=10E-3) or moderate (STR-PCR, 10E-2) quantifiable limit. For the first time we describe clinically applicable cut off levels for qPCR to define complete and mixed chimeras. LCA in CD33/34 subpopulations increased the sensitivity of chimerism monitoring. Early and sensitive detection of impending relapse was possible by qPCR. However, the substantial proportion of false positives had to be kept in mind. Disclosures: No relevant conflicts of interest to declare.

No abstract