Abstract Abstract 3351 Poster Board III-239 Objectives: The majority of patients with chronic lymphocytic leukemia (CLL) who receive allogeneic hematopoietic cell transplantation (HCT) have fludarabine-refractory disease. The most active single agent in this disease stage is alemtuzumab. Alemtuzumab has a long half-life and induces profound T-cell depletion (TCD). Since TCD may mitigate graft-versus leukemia effects we evaluated „pre-conditioning“ with alemtuzumab followed by a washout period in order to minimize in vivo T-cell depletion of the graft in a phase II study (NCT 00337519). Methods: Patients received cytoreductive treatment with 3 × 30 mg alemtuzumab weekly prior to HCT. The scheduled interval between last dose of alemtuzumab and HCT was increased from two weeks to one month during the study. The antibody level at the day of HCT was measured with an ELISA with a lower limit of detection of 31.25 ng/mL (BioAnaLab lim., Oxford, UK). The conditioning regimen contained fludarabine (150 mg/m2) and busulfan (8 mg/kg). Cyclosporine (CSA) and methotrexate (MTX) were applied as GVHD-prophylaxis. Medically fit patients with relapsed CLL were elible. Results: 62 patients with a median age of 57 years were included between April, 2004 and October, 2008. A median of 3 prior regimens had been given. 55% of the patients had fludarabine-resistant disease. Two patients failed to reach HCT due to progressive disease during alemtuzumab therapy. Donors were matched siblings for 26 and matched unrelated donors for 34 patients. The median level of alemtuzumab in peripheral blood after a washout period of two weeks was 62 ng/mL (interquartile range, 45 to 196 ng/mL; minimum below the limit of detection; maximum 490 ng/mL) compared to a median level below the limit of detection after a delay of four weeks (interquartile range, between the limit of detection and 77 ng/mL, maximum 256 ng/mL) (p=0.005). Despite one month time between the last dose of alemtuzumab and HCT 4 out of 30 patients (13%) had alemtuzumab levels greater than 200 ng/mL. No primary or secondary graft failure occurred. A linear relationship between the alemtuzumab level at HCT and the time to complete CD4-T-cell chimerism (TCC) was observed (p=0.003). At day +100 a CD4 positive T-cell-chimerism (TCC) >95% had been achieved by 84% of patients with alemtuzumab levels <100 ng/mL, 83% of patients with antibody levels between 100 and 200 ng/mL and 25% of patients with antibody levels >200 ng/mL (p=0.006). All patients had a complete neutrophil-chimerism at day +100. After early taper of immunosuppression (N=2) or the application of donor lymphocyte infusions in incremental doses (N=5) mixed TCC has been converted to complete TCC in all patients. The median follow-up is 17 months (1 to 61 months). Day +100 non-relapse mortality was 2%. At two years non-relapse mortality and relapse incidence were 21% and 29%, respectively. Two-year overall survival and progression-free survival were 67% (95% CI, 51% to 83%) and 50% (95% CI, 31% to 69%). Conclusions: In patients who received alemtuzumab prior to HCT, residual drug levels may interfere with T-cell engraftment. Lineage specific T-cell chimerism should therefore be assessed prospectively in this group of patients. Persistent mixed T-cell chimerism can be converted by an early taper of immunosuppression and incremental doses of donor lymphocyte infusions. Disclosures: Schetelig: Bayer Schering: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Improper T-cell reconstitution with its consequences, graft-vs-host disease (GvHD) and outbreak of viral infections, is the major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). To determine the factors affecting reconstitution of naive T-cells after non-myeloablative HSCT (NM-HSCT), the T-cell receptor excision circle (TREC) content was measured on a weekly basis in 24 transplanted patients with various malignant diseases. We analysed correlations of the results with the development of GvHD. In addition, in 11 chronic myeloid leukaemia (CML) patients, we correlated TREC and BCR-ABL transcript numbers. After HSCT, in most patients (22/24) TRECs became undetectable. In 12 patients, TRECs reappeared 3–4 months after HSCT, in 1 patient TRECs reappeared 5 months after HSCT, and in 11 patients TRECs remained negative for more than a year. All 11 patients who remained TREC-negative, developed acute GvHD grade 2–3, while only 6 out of 13 patients who recovered TRECs developed GvHD. We show that after non-myeloablative HSCT, thymopoiesis takes place and is affected by GvHD. Our results indicate that no recovery of TRECs after NM-HSCT (which most likely reflect the expansion of host-reactive co-transplanted mature T-cells) correlates with the onset of GvHD.

Background Stem cell transplantation as therapy for hematological disorders is often hampered by severe graft-versus-host-disease. This may be reduced by umbilical cord blood transplantation, an effect that has been attributed to qualitative differences between neonatal and adult T cells. We compared levels of secreted proteins and cytokine mRNA induced in cord blood leukocytes (CBL) and adult blood leukocytes (ABL) by various stimuli. Results While interleukin-2 (IL-2) levels were similar in CBL and ABL, there was less induction of the Th1 cytokine interferon-γ in CBL. Production of the Th2 cytokines IL-4, IL-5, and IL-13 and the hematopoietic cytokine IL-3 was much lower in CBL versus ABL after T-cell receptor-mediated stimulation, whereas production of GM-CSF was comparable in the 2 cell types. The lower levels of Th1 and Th2 cytokines were maintained in CBL during a 4-day time-course study, while after 12 hours IL-3 and GM-CSF reached in CBL levels similar to those in ABL. For all cytokines except IFNγ, the IC 50 values for inhibition by cyclosporin A were similar in ABL and CBL. In contrast, there was less expression and activation of transcription factors in CBL. Activation of NF-κB by TPA/ionomycin was detected in ABL but not CBL. Furthermore, there was less expression of the Th subset-specific transcription factors T-bet and c-maf in CBL versus ABL, whereas GATA-3 expression was similar. Expression of T-bet and c-maf correlated with expression of the Th1 and Th2 cytokines, respectively. Time course experiments revealed that T-bet expression was stimulated in both cell types, whereas c-maf and GATA-3 were induced only in ABL. Conclusion The diminished capability of CBL to synthesize cytokines is probably due to decreased activation of NF-κB, whereas differences in Th subsets are due to differences in regulation of Th lineage-specific transcriptions factors. We propose that the reduced incidence and severity of GvHD after allogeneic transplantation of umbilical CB cells is due to lesser activation of specific transcription factors and a subsequent reduction in production of certain cytokines.

Human herpesvirus-6 (HHV-6) can infect blood cells and thereby may inhibit hematopoietic stem and progenitor cell expansion and differentiation. In this context, it has been discussed if early progenitor cells can be infected by HHV-6. CD46 was identified as one possible cellular surface receptor for HHV-6. The study presented here had been done to get insight into the susceptibility of various leukocyte subpopulations to HHV-6 (including early hematopoietic progenitors) by determining the amount of CD46 molecules expressed on their surfaces. Human cord blood cells, peripheral blood cells and G-CSF mobilised progenitor cells were analysed by flow cytometry. CD46 molecule number per cell was determined and compared to calibration beads conjugated with known ratio of PE per bead. Highest CD46 expression was detected on B- lymphocytes, whereas T-lymphocytes only showed about half of the amount found on B cells. Hematopoietic progenitors also carried CD46 at intermediate levels. Unexpectedly, CD46 expression on progenitors from G-CSF mobilised leukapheresis products was approximately 20% of that found on comparable cells from untreated cord blood. In conclusion, hematopoietic progenitor cells express CD46 on their surface, thereby fulfilling a basic requirement for the susceptibility of HHV-6 infection.

Purpose: To analyse hematopoietic engraftment after allogeneic stem cell transplantation (SCT) in patients with chronic lymphocytic leukemia (CLL) who had received Campath prior to SCT.

Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

BACKGROUND:We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients.METHODS:We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples.RESULTS:The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 10(6) genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <10(3) GE/mL and 100% for urine samples containing 10(9) GE/mL.CONCLUSIONS:UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.

We recently presented a real-time duplex PCR method to determine the amount of human cells in chimeric mice. Although this assay was suitable for the quantitative detection of 1% of human cells in the background of murine cells in tissue, blood and bone marrow of chimeric mice, there is legitimate interest in detecting even lower amounts of human cells in these animals reliably. Therefore, we developed a new assay that reliably detects as few as 5 human cells in 100,000 mouse cells (0.005%). The assay specifically amplifies a human and a murine endogeneous retroviral sequence in two separate reactions. Based on the same primers, the assay can be performed either with 5' nuclease probes (TaqMan assay) or with a pair of hybridization probes (LightCycler assay), enabling the use of various real-time PCR instruments. The use of immunodeficient mice in hematological studies has dramatically increased during the last years. One possible approach to quantify chimerism is based on specific detection of human DNA in chimeric tissue. Published PCR methods are either highly sensitive or allow reliable real-time quantification; however, sensitivity is usually reduced at the expense of quantification. For the investigation of putative bone marrow engrafting cell types, kinetic studies of early engraftment events or the colonization of individual mouse organs with human cells, the combination of both, high sensitivity and reliable quantification, is required. Therefore, we established a new real-time PCR based assay, which combines i) all advantages of real-time PCR, including quantification, reduced contamination risk and specificity, with ii) an extremely high sensitivity. The assay is based on separate amplification of the human specific endogenous retroviral sequence ERV-3 and the mouse specific endogenous retroviral sequence ERV-L pol. For the detection of human ERV-3 a recently published TaqMan assay was modified and suitable hybridization probes were designed, whereas the mouse ERV-L assay was a completely new development. Both assays were optimized to be run under identical reaction conditions, permitting a simultaneous analysis of human and murine cells. TaqMan PCR was performed in a Perkin Elmer 7700 Sequence Detection System in 96-well microtiter plates in a final volume of 25 μL including 2.5 μL 10x PCR reaction buffer, 4.5 mM MgCl2, 1.0 mM dNTP, 1 U Platinum Taq DNA polymerase (all Invitrogen, Karlsruhe, Germany), 5 pmol of each primer, 3 pmol 5’ nuclease probe and 1 μM ROX (6-carboxy-X-rhodamine). 2 μL template DNA were amplified starting with 3 min at 94°C, followed by 45 cycles of 94°C for 30 s and at 58°C for 30 s. LightCycler PCR (Roche Molecular Diagnostics, Mannheim) was performed in a final reaction volume of 20 μL including 2 μL LightCycler-Fast Start DNA Master Hybridisation Probes mix (Roche, Mannheim, Germany), 5 mM Mg, 6 pmol of each primer, 2 pmol of each hybridization probe and 2 μL template DNA. Cycling conditions were 10 min at 95°C followed by 45 cycles of 8 s at 95°C, 8 s at 51°C and 8 s at 72°C. Table 1. shows primers and detection probes used for TaqMan and LightCycler PCR. To prove the sensitivity of the assay, human MCF-7 cells were diluted stepwise in murine P388 cells from 100% human cells to 0.005% human cells. DNA preparation was performed as described previously. Both assays were specific for each species. As shown

BACKGROUND AND OBJECTIVES Patients with angioimmunoblastic T-cell lymphoma (AIL) have a poor prognosis with conventional treatment. DESIGN AND METHODS We initiated an EBMT-based survey studying the impact of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell transplantation in patients with AIL. Data on 29 patients, who were transplanted between 1992 and 1998 in 16 transplant centers, were collected on standardized documentation forms. RESULTS The median age at transplantation was 53 years. HDCT was given as part of 1st-line therapy (N=14; 48%) or 2nd/3rd-line therapy (N=15; 52%). Regimens for the mobilization of peripheral blood stem cells (PBSC) included VIPE (N=7; 26%), DexaBEAM (N=6; 22%), CHOP-like regimens (N=6; 22%), other regimens (N=5; 19%) or alternatively growth factor alone (N=3; 11%). The median yield of PBSC was 3.8x106 CD34+cells/kg. Two patients received autologous bone marrow. The HDCT consisted of BEAM-type regimens in 16 patients, ICE-type regimens in 7, and other regimens in 6 patients. There was one treatment-related death. The rate of complete remissions increased from 45% before HDCT to 76% after HDCT. As of January 2003, after a median observation time of living patients of 5 years (range 2.5 to 10 years), 14 patients have died (13 from progressive disease), and 15 patients are alive. The probability of 5-year overall and event-free survival was 44% (95% CI, 22% to 66%) and 37% (95% CI, 17% to 57%), respectively. Long-term disease-free survival was observed in patients transplanted during 1st-line treatment as well as in the context of 2nd/3rd-line therapy. INTERPRETATION AND CONCLUSIONS There is evidence that AIL is susceptible to high-dose chemotherapy. HDCT and autologous stem cell transplantation should be considered in selected patients with AIL.

在细胞免疫功能受损的患者中分支杆菌感染率是比较高的.造血干细胞移植后的患者细胞免疫功能严重受损,恢复缓慢,其并发分支杆菌感染的几率应该较大.然而国内迄今尚未见造血干细胞移植后并发分支杆菌感染的报道,国外也很少报道.细胞免疫功能低下的患者并发分支杆菌感染后诊断困难,病死率高,且这类患者的传染性强[1],因此及时地诊断和治疗非常重要.现报告1例异基因骨髓移植(allo-BMT)后并发分支杆菌感染,并对相关文献进行复习.

Cytomegalovirus (CMV) can compromise the life of patients who are immunosuppressed after organ transplantation or who have acquired immunodeficiency syndrome. CMV infections or reactivations can be successfully treated, provided treatment is instituted early in the course of developing disease. Current assays to detect the CMV pp65 antigen in peripheral blood leukocytes or CMV DNA by qualitative PCR of leukocytes or plasma can be slow and tedious (1)(2)(3). Antigen detection is limited by the availability of sufficient white blood cell numbers and is impossible to perform on leukopenic patients. In addition, blood for antigen detection cannot be stored for long periods because white blood cells need to be intact for staining procedures (4)(5). PCR techniques detect relatively stable double-stranded DNA present in the plasma or whole blood regardless of storage conditions or cell integrity, but qualitative PCR assays are not useful for follow-up studies of patients, and they do not allow the determination of viral load, which is useful for prognosis. In this study we compared quantitative real-time PCR (6)(7) with conventional pp65 antigen staining among 77 patients (1122 blood samples) surviving more than 30 days after stem cell transplantation (see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue10/). In a retrospective study, results of the two assays were compared, and their sensitivity and specificity for predicting CMV disease were calculated. All patients were transplanted in the Virchow Klinikum (Berlin, Germany) between January 1998 and March 2001 (see Table 1 in the online Data supplement). Five patients developed CMV organ disease, which included pneumonitis and hepatitis (n = 1), hepatitis (n = 1), enteritis (n = 2), and retinitis (n = 1). Seven additional patients developed thrombo- and leukopenia not explained by other conditions. …

Background and Objectives. We studied the toxicity and efficacy of reduced intensity conditioning followed by allogeneic stem cell transplantation in 50 patients over 50 years old or with relative contraindications against myeloablative regimens. Diagnoses were chronic myeloid leukemia (n=15), acute myeloid leukemia (n=9), myelodysplastic syndromes (n=9), lymphoma (n=11) and refractory solid tumors (n=6). Design and Methods. Donors were identical siblings (n=25), non-identical family members (n=6) and unrelated volunteers (n=19). Peripheral blood stem cells (n=36) or bone marrow (n=14) were transplanted. The conditioning regimen consisted of fludarabine 180 mg/m(2), busulphan 8 mg/kg and rabbit antithymocyte globulin 40 mg/kg (Fresenius). Graft-versus-host disease (GVHD) prophylaxis was carried out with cyclosporin A (CSA) alone (n=17) or in combination with methotrexate (n=18) or mycophenolate mofetil (n=15). Results. Neutrophil counts >0.5/nL and platelet counts > 20/nL were reached after 17 (range 0-66) and 19 days (range 0-111), respectively. Three graft failures occurred. Fever lasted for a median of 2 days (range 0-15). Six patients developed veno-occlusive disease of the liver. Acute GVHD grade II-IV occurred in 47% of the patients and chronic GVHD in 46%. The 1-year overall survival probability was 44% (95% CI: 30-58%). GVHD-related complications were a major cause of the probability of 1-year non-relapse mortality of 31% (95% CI: 16-46%). Interpretation and Conclusions. In conclusion, the regimen itself can be carried out safely in patients with relative contraindications against myeloablative conditioning. However, GVHD causes significant non-relapse mortality in high risk patients.

Objective:To study the efficacy and toxicity of ganciclovir(GCV) in the prophylaxis of cytomegalovirus(CMV) infection after allo-HSCT. Method:46 patients undergoing allo-HSCT were observed, in which either the patients or the donors or both are CMV-IgG positive. The twenty-two patients received placebo, and the other twenty-four received GCV prophylaxis at engraftment. The dose of GCV was 5 mg/Kg body weight administered intravenously twice daily for 5 days, followed by once daily 5 days per week until day +100 after transplant ation.Result:Until day +100, the incidence of CMV infection in GCV group and in placebo were 8% Vs32%, respectively, P 0.05; the incidence of CMV disease were 0% Vs18%, respectively, P 0.05. Up to day +100 and day +180, the mortality in GCV group versus in placebo group were 4% Vs 5%, P 0.05; 12.5% Vs 9%,P 0.05, respectively. The deaths in GCV group were caused by bacterial and fungal infection, CMV-interstitial pneumonia (CMV-IP), or relapse. And the deaths in placebo group were caused by CMV-IP. Conclusion:Ganciclovir given prophylactically after engraftment is effective in suppressing CMV infection and disease. Neutropenia is the major toxicity of GCV and is associated with an increased risk for fatally bacterial and/or fungal infection. The optimum dose, protocols and the duration of ganciclovir prophylaxis is further studied.

目的:探讨非骨髓清除性预处理方案(nonmyeloablativeconditioningregimen,NACR)加异基因造血干细胞移植(allo-HSCT)治疗血液系恶性肿瘤的可行性及其效果.方法:本报告用Fludarabine180mg/m2,ATG40mg/kg,马利兰8mg/kg加异基因外周血干细胞移植治疗复发性难治性淋巴瘤4例.4例供者均系患者亲属,其中1例供受者HLA有一个位点不合,其余3例供受者HLA完全相合.结果:4例患者对预处理方案耐受良好,移植后无1例发生移植物排斥.迄今4例患者已完全缓解8～15个月.结论:用非骨髓清除性方案作造血干细胞移植预处理,安全有效,毒性低,可应用于年老体弱或有实质内脏损伤者.

mixed and not already predominantly of donor type, as in our cases. Gardiner et al state that their technique using lineage-specific STR-PCR provides reliable results on the chimeric profile post-BMT and may be more useful than the FICTION assay in situations with low cell numbers. Although the important aspect of morphology and individual cell by cell analysis is lost with the PCR approach, we agree that STR-PCR is a most valuable tool in this context and therefore complementary to our FICTION analysis. However, we doubt that subtle changes in the degree of mixed chimerism can be seen with a semiquantitative technique according to which mixed chimerism is defined as a percentage of recipient cells between less than 90% and greater than 10%. Indeed, in the work of Gardiner et al, significant changes in every case were detected only concomitantly to the onset of clinical graft-versus-host disease (GVHD), a result similar to that obtained in our cases when using the AmpliType Polymarker PCR kit to document non–lineage-specific DNA chimerism. In contrast, when using our quantitative techniques, ie, FICTION and competitive differential bcr-abl RT-PCR as an additional diseasespecific marker, the beginning of the critical switch period could be detected several weeks before the onset of clinical GVHD. Although we therefore very much support a larger scale study to better understand the kinetics of the GVL response after DLI in T-cell–depleted and non—T-cell– depleted BMT recipients, we strongly suggest the use of true quantitative measures of lineage-specific chimerism. The FICTION method is especially helpful in small centers, because it can be performed without larger equipment or in situations in which morphological control of the selected population is of additional value. Another attractive tool is the combination of FACS sorting and quantitative multiplex STR-PCR with fluorescent primers. 3 This technique has been successfully applied by members of our group to study subtle changes of subset chimerism in patients undergoing nonmyeloablative stem cell transplantation and was shown to be predictive of response, GVHD, and disease recurrence. 4

Mitoxantrone is a new effective antineoplastic agent with activity against a wide range of tumors. Compared with the anthracyclin drugs doxo- and daunorubicin, it exhibits a clearly lower toxicity and, most importantly, a reduced cardiotoxicity. The analysis of the side-effects recorded after accidental overdosage of the drug gives additional insight into its tolerability.

The expression of p21ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21ras could be detected in most of the cell lines, no significant p21ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.