Allogeneic stem cell transplantation for mantle cell lymphoma-update of the prospective trials of the East German Study Group Hematology/Oncology (OSHO#60 and #74).

Mantle cell lymphoma (MCL) is a non-Hodgkin's lymphoma with an often aggressive course, incurable by chemotherapy. Consolidation with high-dose therapy and autologous stem cell transplantation (autoSCT) has a low transplant-related mortality but does not lead to a survival plateau. Allogeneic stem cell transplantation (alloSCT) is associated with a higher early mortality, but can cure MCL. To investigate alloSCT for therapy of MCL, we conducted two prospective trials for de novo MCL (OSHO#74) and for relapsed or refractory MCL (OSHO#60). Fifteen and 24 patients were recruited, respectively. Induction was mainly R-DHAP alternating with R-CHOP. Conditioning was either Busulfan/Cyclophosphamide or Treosulfan/Fludarabin. Either HLA-identical siblings or matched-unrelated donors with not more than one mismatch were allowed. ATG was mandatory in mismatched or unrelated transplantation. Progression-free survival (PFS) was 62% and overall survival (OS) was 68% after 16.5-year follow-up. Significant differences in PFS and OS between both trials were not observed. Patients below 56 years and patients after myeloablative conditioning had a better outcome compared to patients of the corresponding groups. Nine patients have died between day +8 and 5.9 years after SCT. Data from 7 long-term surviving patients showed an excellent Quality-of-life (QoL) after alloSCT. AlloSCT for MCL delivers excellent long-term survival data. The early mortality is higher than after autoSCT; however, the survival curves after alloSCT indicate the curative potential of this therapy. AlloSCT is a standard of care for all feasible patients with refractory or relapsed MCL and should offer to selected patients with de novo MCL and a poor risk profile. For defining the position of alloSCT in the therapeutic algorithm of MCL therapy, a randomized comparison of autoSCT and alloSCT is mandatory.

Influence of reproductive history and exogenous hormone use on prevalence and frequency of circulating t(14;18)-positive cells in a population-based cross-sectional study.

PURPOSE: The t(14;18) translocation might represent an intermediate step in the pathogenesis of follicular lymphoma (FL), one of the most common subtypes of non-Hodgkin lymphoma. Circulating t(14;18)-positive cells can also be detected in 30-60 % of healthy individuals at low frequencies. Some studies found a negative association between reproductive factors or use of menopausal hormone therapy (MHT) with FL. The objective of this study was to evaluate whether there is an association between number of frequencies, oral contraceptive (OC) use, menopausal status and MHT, and t(14;18) prevalence and frequency in a representative population analysis based on an epidemiologic study in the northeastern part of Germany. METHODS: The analysis is based on results of buffy coat samples from 1,981 women of the Study of Health in Pomerania (SHIP-0) and data obtained in standardized face-to-face interviews. For prevalence, odds ratios (OR) and 95 % confidence intervals (CI) were calculated using unconditional logistic regression. Frequency data were analyzed using negative binomial regression. The multivariable models included age, number of pregnancies, menopausal status (premenopausal, natural, medical/surgical menopause), OC use and MHT as a measure for exogenous hormone exposure use. RESULTS: We found no association between reproductive history and combined exogenous hormone use on the prevalence of circulating t(14;18)-positive cells. Modeling MHT and OC use separately in a sensitivity analysis, the MHT parameter yielded statistical significance [OR 1.37 (95 % CI 1.04;1.81)]. t(14;18) frequency was associated with use of OC [incidence rate ratio (IRR) for ever use 3.18 (95 % CI 1.54;6.54)], current use [IRR 3.86 (1.56;9.54)], >10 years use [IRR 3.93 (1.67;9.23)] and MHT [restricted to postmenopausal women; IRR 2.63 (95 % CI 1.01;6.85)] in bivariate age-adjusted analyses. In the multivariable model, medical/surgical menopause [IRR 2.46 (1.11;5.44)] and the category ever use of OC and MHT were statistically significant [IRR 2.41 (1.09;5.33)]. CONCLUSIONS: Exogenous hormone use might be a risk factor for t(14;18) frequency rather than for t(14;18) prevalence. Further research on healthy individuals carrying a t(14;18) translocation and possible risk factors for malignant lymphoma is necessary to determine the additional molecular or immunological events that have to occur to develop FL.

Allogeneic stem cell transplantation for mantle cell lymphoma--final report from the prospective trials of the East German Study Group Haematology/Oncology (OSHO).

This study was conducted in order to evaluate allogeneic stem cell transplantation (alloSCT) as consolidation for patients with mantle cell lymphoma (MCL). Patients with MCL were included into two prospective trials OSHO #060 (refractory/relapsed) and #074 (de novo). Induction was rituximab and chemotherapy. Responding patients proceeded to alloSCT. Minimal residual disease was monitored by quantitative RT-PCR detecting either t(11;14) or clonospecific CDR-III regions. In case of circulating lymphoma cells, immunomodulation (cyclosporine A withdrawal, rituximab, donor lymphocyte infusion) was initiated. Thirty-three of 39 patients underwent alloSCT after myeloablative (n = 7) or toxicity-reduced (n = 26) conditioning. Leukocytes engrafted at day +16 (median, range 0-101) and platelets at day +14 (0-142). Acute graft-versus-host disease stages I-II occurred in 42 % and stages III-IV in 15 %. Five patients have relapsed after SCT. The overall mortality after SCT was 24 % (n = 8). Median follow-up after SCT was 2.8 years (range 0.0-10.9). Five-year progression-free survival was 67 %, and overall survival 73 % after SCT. The results were comparable for primary MCL and refractory/relapsed disease as well as for related vs. unrelated SCT. Younger patients had a significantly better outcome than the elderly. AlloSCT is a feasible and promising consolidation therapy for relapsed and refractory disease and an attractive option for young patients with de novo MCL of high risk.

Therapeutic platelet transfusion versus routine prophylactic transfusion in patients with haematological malignancies: an open-label, multicentre, randomised study.

BACKGROUND: Routine prophylactic platelet transfusion is the standard of care for patients with severe thrombocytopenia. We assessed the effect of a new strategy of therapeutic platelet transfusion on the number of transfusions and safety in patients with hypoproliferative thrombocytopenia. METHODS: We did a multicentre, open-label, randomised parallel-group trial at eight haematology centres in Germany. Patients aged 16-80 years, who were undergoing intensive chemotherapy for acute myeloid leukaemia or autologous haemopoietic stem-cell transplantation for haematological cancers, were randomly assigned via a computer-generated randomisation sequence to receive either platelet transfusion when bleeding occurred (therapeutic strategy) or when morning platelet counts were 10×10(9) per L or lower (prophylactic strategy). Investigators undertaking interventions were not masked to group assignment. The primary endpoint was the number of platelet transfusions. Analysis was by intention to treat. This trial is registered, NCT00521664. FINDINGS: 197 patients were assigned the prophylactic strategy and 199 the therapeutic strategy. Of 391 patients analysed, the therapeutic strategy reduced the mean number of platelet transfusions by 33·5% (95% CI 22·2-43·1; p<0·0001) in all patients (2·44 [2·22-2·67] in prophylactic group vs 1·63 [1·42-1·83] in therapeutic group), 31·6% (18·6-42·6; p<0·0001) in those with acute myeloid leukaemia (2·68 [2·35-3·01] vs 1·83 [1·58-2·10]), and 34·2% (6·6-53·7; p=0·0193) in those who had had autologous transplantation (1·80 [1·45-2·15] vs 1·18 [0·82-1·55]. We noted no increased risk of major haemorrhage in patients who had undergone autologous transplantation. In those with acute myeloid leukaemia, risk of non-fatal grade 4 (mostly CNS) bleeding was increased. We recorded 15 cases of non-fatal haemorrhage: four retinal in each transfusion group, and one vaginal and six cerebral in the therapeutic group. 12 patients died in the study: two from fatal cerebral haemorrhages in the therapeutic group, and ten (five in each treatment group) unrelated to major bleeding. INTERPRETATION: The therapeutic strategy could become a new standard of care after autologous stem-cell transplantation; however, prophylactic platelet transfusion should remain the standard for patients with acute myeloid leukaemia. The new strategy should be used by some haematology centres only if the staff are well educated and experienced in the new approach and can react in a timely way to first signs of CNS bleeding. FUNDING: Deutsche Krebshilfe eV (German Cancer Aid).

Upfront immunization with autologous recombinant idiotype Fab fragment without prior cytoreduction in indolent B-cell lymphoma.

Idiotype vaccination for follicular lymphoma is primarily being developed as remission consolidation after chemotherapy. We investigated idiotype vaccination as primary intervention for treatment-naive indolent B-cell lymphoma and in a separate cohort as remission consolidation after chemotherapy to assess immunization-induced immune responses in relation to progression-free survival (German Clinical Trials Register, DRKS00000227). Twenty-one patients in each cohort received 6 intradermal injections of adjuvanted recombinant idiotype Fab fragment (Fab(Id)); 76% of patients in both groups developed anti-idiotype antibodies and/or cellular immunity as measured by enzyme-linked immunosorbent assay and interferon-γ ELISpot. In treatment-naive patients, only cellular responses correlated with superior progression-free survival (P < .002) and durable objective remissions (P = .04). Immunization-induced T cells recognized hypermutated or complementarity-determining region 3 epitopes. After remission consolidation immunization, induction of anti-idiotype antibodies correlated with progression-free survival. Low B-cell counts after rituximab therapy predicted for failure to develop anti-idiotype antibodies. These results are similar to published trials showing an association of humoral immunity with control of residual lymphoma. In contrast, effective immunity against untreated lymphoma appears to be dependent on idiotype-specific T cells. Sustained remissions in patients with vaccination-induced cellular immunity suggest clinical benefit and warrant a randomized comparison of this vaccine with expectant management for asymptomatic follicular lymphoma.

Low-dose alemtuzumab vs. standard policy for prevention of graft-versus-host disease in unrelated and related allogeneic stem cell transplantation-a matched pair analysis.

Antibody-mediated in vivo T cell depletion is common prior to unrelated (URD) or mismatched allogeneic stem cell transplantation (alloSCT) and optional in HLA-identical sibling (FAM) alloSCT. While anti-thymocyte globulin (ATG) is the current standard, alemtuzumab is an alternative. The optimal dose of alemtuzumab has not been defined. This retrospective analysis compares low-dose alemtuzumab with ATG in URD alloSCT and with no antibody in FAM alloSCT. Twenty-eight patients treated with alemtuzumab (10 mg; HLA mismatch, 20 mg) were matched to 28 patients who have either received ATG (URD) or no antibody (noAB) according to disease, disease stage, age, transplant type and risk state. Both groups were compared for engraftment, outcome, disease-free (DFS) and overall survival (OS), graft-versus-host disease (GvHD), freedom from GvHD (ffGvHD) and transplant-related mortality (TRM). No significant differences were found between the groups for leukocyte engraftment, GvHD, ffGvHD, TRM, DFS and OS. There was a trend for reduction of cGvHD by alemtuzumab (p = 0.05). A transplant-type stratified subanalysis consolidated equivalency of alemtuzumab and ATG in URD-SCT and indicates possible superiority of low-dose alemtuzumab compared to noAB in FAM-SCT. Low-dose alemtuzumab, as part of conditioning regimen prior to alloSCT, is safe and comparable to standard ATG. Prospective trials, particularly comparing alemtuzumab vs. noAB in FAM alloSCT, should be conducted.

Severe disseminated coagulopathy caused by adenocarcinoma with bone marrow metastasis.

BACKGROUND: Patients with mucin-producing adenocarcinoma have an increased risk for venous and arterial thrombosis. When these patients present with thrombocytopenia, disseminated intravascular coagulopathy (DIC) is often the underlying cause. CASE REPORT: We report 2 patients who were admitted due to bleeding symptoms of unknown cause, in whom further workup revealed adenocarcinoma-induced DIC. CONCLUSION: In elderly patients presenting with signs of DIC, such as reduced fibrinogen levels, elevated prothrombin time, elevated D-dimer, and thrombocytopenia, without any obvious reason (e.g., sepsis), adenocarcinoma-associated coagulopathy should be considered as the underlying cause. Paradoxically, in these patients bleeding symptoms improve when the patient is sufficiently anti-coagulated with low molecular weight heparin. Treatment of the underlying disease is of central importance in controlling acute or chronic DIC associated with malignant diseases and chemotherapy should be started as soon as possible.

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC. METHODS: siRNA-mediated silencing of SRD5A1 was performed in A549 and NCI-H460 lung cancer cell lines in order to determine the impact on proliferation, on distribution during the different phases of the cell cycle, and on apoptosis/necrosis. In addition, lung cancer cell lines were treated with 4-azasteroids, which specifically inhibit SRD5A1 activity, and the effects on proliferation were measured. Statistical analyses using ANOVA and post-hoc Tamhane-T2-test were performed. In the case of non-parametric data, the Kruskal-Wallis test and the post-hoc Mann-Whitney-U-test were used. RESULTS: The knock-down of SRDA51 expression was very efficient with the SRD5A1 transcripts being reduced to 10% of control levels. Knock-down efficiency was furthermore confirmed at the protein level. However, no effect of SRD5A1 silencing was observed in the proliferation assay, the cell cycle analysis, and the apoptosis/necrosis assay. Treatment of lung cancer cell lines with 4-azasteroids did not significantly inhibit proliferation. CONCLUSIONS: In summary, the results suggest that SRD5A1 is not a crucial enzyme for the sustained proliferation of NSCLC cell lines.

Toxoplasmosis after allogeneic stem cell transplantation--a single centre experience.

Toxoplasmosis is a rare but possibly underestimated complication following allogeneic stem cell transplantation with a high mortality rate. One reason might be the limitation of the diagnostic instruments relying mainly on imaging and molecular-based techniques. In this report, we present three cases of toxoplasmosis identified among 155 allograft recipients treated at Greifswald University Hospital. Widely disseminated toxoplasmosis was detected post-mortem in two patients allografted for high-risk multiple myeloma. Clinical signs suspicious for toxoplasmosis occurred after days +32 and +75, respectively. In one case, serology and conventional Toxoplasma gondii PCR, targeting the B1 gene, revealed negative results, while in the other patient, toxoplasmosis was not investigated. Both patients received pentamidine for Pneumocystis jirovecii pneumonia (PcP) prophylaxis. The third patient, a 68-year-old woman allografted for AML, developed cerebral toxoplasmosis from day +395 after allogeneic SCT with typical signs in magnetic resonance tomography. Toxoplasma DNA was amplified from one of two samples of cerebrospinal fluid. The patient died of disseminated toxoplasmosis despite immediate initiation of therapy. Retrospective comparative testing of clinical specimens by the conventional T. gondii PCR and by a real-time PCR targeting a 529-bp genomic fragment suggests a higher sensitivity of the latter method in our patients. In conclusion, we suggest a rigorous real-time PCR monitoring for high-risk patients or patients with signs of infections suspicious for toxoplasmosis, even though low-copy results are presently difficult to interpret. Our reported cases might also encourage the use of trimethoprim-sufmethoxazole instead of pentamidine for PcP prophylaxis in those patients.

Fast approach for clarification of chromosomal aberrations by using LM-PCR and FT-CGH in leukaemic sample.

Chromosomal abnormalities, like deletions, amplifications, inversions or translocations, are recurrent features in haematological malignancies. However, the precise molecular breakpoints are frequently not determined. Here we describe a rapid analysis of genetic imbalances combining fine tiling comparative genomic hybridization (FT-CGH) and ligation-mediated PCR (LM-PCR). We clarified an inv(14)(q11q32) in a case of T cell acute lymphoblastic leukaemia with a breakpoint in the TRA/D in 68% of cells detected by fluorescence in situ hybridization. FT-CGH showed several mono- and biallelic losses within TRA/D. LM-PCR disclosed a TRA/D rearrangement on one allele. The other allele revealed an inv(14)(q11q32), joining TRDD2 at 21,977,000 of 14q11 together with the IGH locus at 105,948,000 and 3'-sequence of TRAC at 22,092,000 joined together with IGHV4-61 at 106,166,000. This sensitive approach can unravel complex chromosomal abnormalities in patient samples with a limited amount of aberrant cells and may lead to better diagnostic and therapeutic options.

Statistical analysis of results obtained by real-time PCR for improvement of absolute quantification of target sequences.

BACKGROUND: Real-time quantitative PCR is increasingly used in clinical laboratories. Genomic DNA or plasmids containing cloned target sequences are necessary to generate data for standard curves. These data must be analysed to obtain the relative or absolute quantity of the target concentration in a sample. The method chosen for data analysis can strongly influence results of the quantification. Absolute quantification is important especially in clinical settings. For different reasons estimating the copy number of the gene of interest based on DNA concentration measurements is vague and tends toward overestimation, especially if cell lines are used. METHODS: Data gained by limiting dilution and multiple-tube approach were analyzed using our new Poisson distribution based software and were compared with results from DNA concentration measurement. Data from different cell sources (peripheral blood mononuclear cells and two cell lines) were compared: RESULTS: Limiting dilution and multiple-tube approach analyzed by a Poisson distribution simplifies and improves the generation of standard curves for real time PCR if cell lines are used. The absolute target copy number in a sample, the standard deviation, and a 95% confidence interval are calculated by the software. CONCLUSIONS: With this easy to use program a target copy number can be reliably quantified. The program is available free of charge from: http://www.medizin.uni-greifswald.de/InnereC/index.php?id=18 (link will be activated after acceptance of the paper).

Transmission of cytomegalovirus (CMV) infection by leukoreduced blood products not tested for CMV antibodies: a single-center prospective study in high-risk patients undergoing allogeneic hematopoietic stem cell transplantation (CME).

BACKGROUND: Measures to prevent transfusion-transmitted cytomegalovirus (TT-CMV) infection after hematopoietic stem cell transplantation (HSCT) include transfusion of CMV antibody-negative blood units and/or transfusion of leukoreduced cellular blood products. We assessed the incidence of TT-CMV in CMV-seronegative patients receiving CMV-seronegative HSC transplants, who were transfused with leukoreduced cellular blood products not tested for anti-CMV. STUDY DESIGN AND METHODS: In a prospective observational study between 1999 and 2009, all HSCT patients received leukoreduced cellular blood products not tested for anti-CMV. Patients were screened for CMV serostatus and CMV-negative recipients of CMV-negative transplants were systematically monitored for TT-CMV clinically and by CMV nucleic acid testing. Anti-CMV antibodies (immunoglobulin [Ig]G and IgM) were assessed after three time intervals (Interval 1, study inclusion to Day +30 after HSCT; Interval 2, Day +30-Day +100; Interval 3, after Day +100). RESULTS: Among 142 patients treated with allogeneic HSCT, 23 CMV-negative donor-patient pairs were identified. These 23 patients received 1847 blood products from 3180 donors. All patients remained negative for CMV DNA and none developed CMV-associated clinical complications. This results in a risk for TT-CMV per donor exposure of 0% (95% confidence interval, 0.0%-0.12%). However, 17 of 23 patients seroconverted for anti-CMV IgG, but none for anti-CMV IgM. CMV IgG seroconverters received significantly more transfusions per week than nonconverters. CONCLUSION: The risk of TT-CMV is low in high-risk CMV(neg/neg) HSCT patients transfused with leukoreduced blood products not tested for anti-CMV. The cause of anti-CMV IgG seroconversion is most likely passive antibody transmission by blood products.

Mucositis after reduced intensity conditioning and allogeneic stem cell transplantation.

BACKGROUND: Therapyrelated mucositis is associated with considerable morbidity. This complication following allogeneic stem cell therapy (alloSCT) is less severe after reduced intense conditioning (RIC); however, even here it may be serious. METHODS: 52 patients (male: n = 35 (67%), female: n = 17 (33%)) at a median age of 62 years (35-73 years) underwent alloSCT after RIC. Conditioning was either total body irradiation (TBI)(2Gy)/±fludarabine (n = 33, 63.5%) or chemotherapy based. Graftversushost disease (GvHD) prophylaxis was carried out with cyclosporine A ± mycophenolate mofetil (MMF). 45 patients (87%) received shortcourse methotrexate (MTX). Mucositis was graded according to the Bearman and the World Health Organisation (WHO) scale. A variety of parameters were correlated with mucositis. RESULTS: The Bearman and WHO scales showed excellent correlation. Mucositis was significantly more severe after chemotherapybased conditioning compared to conditioning with TBI(2Gy)/±fludarabine (p < 0.002) as well as in cases with an increase in creatinine levels above the upper normal value (UNV) on day +1 after SCT (p < 0.05). Furthermore, the severity correlated with time to engraftment of leucocytes (correlation coefficient (cc) = 0.26, p < 0.02) and thrombocytes (cc = 0.38, p < 0.001). CONCLUSIONS: The conditioning regimen and increased creatinine levels at day +1 were identified as factors predicting the severity of mucositis after RICSCT. Creatinine levels on day +1 after SCT may help identify patients at risk for severe mucositis in the further course of transplantation.

Molecular characterization of a novel chromosomal translocation t(12;14)(q23;q11.2) in T-lymphoblastic lymphoma between the T-cell receptor delta-deleting elements (TRDREC and TRAJ61) and the hypothetical gene C12orf42.

Chromosomal aberrations have diagnostic, prognostic, and therapeutic relevance in hematologic malignancies. By combining fine-tiling comparative genomic hybridization (FT-CGH) and ligation-mediated PCR (LM-PCR), we established a fast, robust approach to precisely characterize chromosomal breakpoints. Using this approach, we clarified at the molecular level novel chromosomal translocation t(12;14)(q23;q11.2) in T-lymphoblastic lymphoma. The translocation occurred during the deletional rearrangement of the T-cell receptor delta gene (TRD), which is a pivotal step in T cell differentiation toward the alpha/beta vs. the gamma/delta lineage. We found that this rearrangement disrupted the hypothetical gene C12orf42 and brought the Achaete-scute complex homolog 1 gene into proximity of the TRA enhancer, which encodes a member of the basic helix-loop-helix family of transcription factors and is overexpressed in thyroid and lung cancers.

Prevalence and frequency of circulating t(14;18)-MBR translocation carrying cells in healthy individuals.

The t(14;18) translocation is a common genetic aberration that can be seen as an early step in pathogenesis of follicular lymphoma (FL). The significance of low level circulating t(14;18)-positive cells in healthy individuals as clonal lymphoma precursors or indicators of risk is still unclear. We determined the age dependent prevalence and frequency of BCL2/IgH rearrangements in 715 healthy individuals ranging from newborns to octo- and nonagenarians. These results were compared with number of circulating t(14;18)-positive cells in 108 FL patients at initial presentation. The overall prevalence of BCL2/IgH junctions in this large sample was 46% (327/715). However, there was a striking dependence upon age. Specifically, among individuals up to 10 years old, none had detectable circulating t(14;18)-positive cells. In the age groups representing 10-50 years old, we found a steady elevation in the prevalence of BCL2/IgH junctions up to a prevalence of 66%. Further increases of the prevalence in individuals older than 50 years were not seen. The mean frequency of BCL2/IgH junctions in healthy individuals > or =40 years (18-26 x 10(-6)) was significantly higher than in younger subjects (7-9 x 10(-6)). Four percent (31/715) of individuals carried more than one t(14;18)-positive cell per 25,000 peripheral blood mononuclear cells (PBMNCs). In comparison, 108 stage III/IV FL patients had a median number of circulating t(14;18)-positive malignant FL cells of about 9200/1 million PBMNCs (range 7-1,000,000). These findings will further improve the understanding of the relevance of t(14;18)-positive cells in healthy individuals as a risk marker toward the development into lymphoma precursors.

Hematopoietic stem cell differentiation affects expression and function of MRP4 (ABCC4), a transport protein for signaling molecules and drugs.

Several types of peripheral blood cells express ABC transporters. ABCC4 (MRP4) and ABCC5 (MRP5) localize to different cellular sites and fulfill lineage-specific functions such as mediator storage in platelets' dense granules. All mature blood cells originate from the same precursor and specific functionalities arise during differentiation. To characterize this process, expression, localization and function of MRP4 and MRP5 were assessed in differentiating human CD34+ progenitors and leukemia cell lines using real time polymerase chain reaction (PCR), immunofluorescence microscopy and cell viability assays. Median MRP4 mRNA copy numbers were significantly enhanced by megakaryocytic differentiation from 7.9 x 10(3) to 5.8 x 10(4) copies per nanograms of total RNA (p < 0.05) in CD34+ progenitors and in M-07e cells (MRP4 mRNA/18S rRNA ratios: 5.4 +/- 3.8 x 10(-4) vs. 2.7 +/- 0.9 x 10(-3) for native and differentiated cells, respectively, p < 0.05), and MRP4 protein was localized to granular structures and to the plasma membrane both in differentiated progenitors and bone marrow megakaryocytes. In contrast, expression of MRP4 decreased during maturation to leukocytes (MRP4 mRNA/18S rRNA ratios: 5.2 x 10(-3) for native vs. 3.5 x 10(-3) for CD34+ cells in the presence of G-CSF, p < 0.05) and was significantly reduced in mature monocytes and granulocytes compared with progenitors (MRP4 mRNA/18S rRNA ratios: 8.1 +/- 5.4 x 10(-5) and 2.8 +/- 1.6 x 10(-4) vs. 1.2 +/- 0.7 x 10(-3), respectively, p < 0.05). Expression of MRP5 was not significantly altered under all differentiation conditions. These results indicate that MRP4 expression is differentially regulated during hematopoiesis. The increase of MRP4 together with its specific localization during differentiation toward megakaryocytes supports the concept of platelet specific functions whereas decreased transporter expression in leukocyte differentiation may have implications for chemotherapy.

Low-dose total body irradiation-based regimens as a preparative regimen for allogeneic haematopoietic cell transplantation in acute myelogenous leukaemia.

Although much progress has been made in understanding the molecular basis of acute myeloid leukaemia (AML), this has not yet led to major improvements in the overall survival of patients. In particular, the treatment of elderly patients with AML remains one of the major challenges in haematology. Despite increases in complete remission rates, relapse remains a major obstacle and the major determinant of overall survival. Allogeneic stem cell transplantation (SCT) is the most efficient antileukaemic treatment for patients with AML, but eligibility for the treatment was confined for a long time to younger patients. More than 10 years ago, SCT protocols were initiated with reduced-intensity conditioning (RIC) rather than ablative chemoradiotherapy, with the intention of inducing a graft-versus-leukaemia effect also in elderly patients and patients with concomitant diseases. Operationally, all protocols below the conventional preparative regimens are referred to as RIC. Low-dose total body irradiation-based protocols result in minimal myelosuppression and are among the most popular. After extensive phase I and phase II studies, associated problems of graft rejection have been largely resolved and transplant-related mortality (TRM) evaluated in more than 3000 patients. TRM does not currently exceed 10-12% in related and 20% in unrelated SCT even in patients up to the age of 75 years, so that relapse after transplantation remains the major problem. A number of strategies for decreasing relapse rates has been developed. The most promising approach consists of monitoring CD34+ donor cell chimerism after transplantation. This has led to decreases in the relapse rate over the past few years. Randomized studies are now being initiated to define the role of SCT in the treatment of elderly patients with AML.

Myocardial and aortal involvement in a case of disseminated infection with Fusarium solani after allogeneic stem cell transplantation: report of a case.

We describe a 57-year-old woman suffering from acute erythroblastic leukaemia. After the first course of high-dose Ara-C containing consolidation therapy, the patient developed multiple skin lesions on the left foot. A skin biopsy revealed a Fusarium infection. The lesions regressed under therapy with caspofungin and voriconazole. Leukaemia relapsed after 1 year and an allogeneic stem cell transplantation was performed for consolidation of leukaemia in second remission. Again, the patient developed macular skin lesions located on the trunk and the extremities with central pallor. Clinical examination showed fever, tachyarrhythmia and a systolic murmur. Fusarium spp. was cultured from blood samples. An antimycotic therapy with amphotericin B, voriconazole and posaconazole failed completely. The patient died in a septic shock with consecutive multiple organ failure. The autopsy (SN 1/06, Institute of Pathology, University of Greifswald) revealed a disseminated infiltration with Fusarium solani including myocardial, endocardial and aortal infection. The involvement of the cardiovascular system is uncommon in fusariosis and has not been described so far. This case confirms other reports describing the high mortality of fusariosis after allogeneic stem cell transplantation. A rapid diagnosis and antimycotics with higher activity against Fusarium spp. are necessary for successful therapy of this severe mould infection in the immunocompromised host.

Low number of intrafollicular T cells may predict favourable response to rituximab-based immuno-chemotherapy in advanced follicular lymphoma: a secondary analysis of a randomized clinical trial.

BACKGROUND: First-line rituximab therapy together with chemotherapy is the standard care for patients with advanced follicular B-cell lymphoma, as rituximab together with chemotherapy prolongs progression-free and overall survival (Herold et al. 2007; Marcus et al. 2005). However, as not all patient subgroups benefit from combined immuno-chemotherapy, we asked whether the microenvironment may predict benefit from rituximab-based therapy. DESIGN: To address this question, we performed a retrospective immunohistochemical analysis on pathological specimens of 18 patients recruited into a randomized clinical trial, where patients with advanced follicular lymphoma were randomized into either chemotherapy or immuno-chemotherapy with rituximab (Herold et al. 2007). RESULTS: We show here that rituximab exerts beneficial effects, especially in the subgroup of follicular lymphoma patients with low intrafollicular CD3, CD5, CD8, and ZAP70 and high CD56 and CD68 expression. CONCLUSION: Rituximab may overcome immune-dormancy in follicular lymphoma in cases with lower intrafollicular T-cell numbers and higher CD56 and CD68 cell counts. As this was a retrospective analysis on a small subgroup of patients, these data need to be corroborated in larger clinical trials.

t(14;18) Translocations and risk of follicular lymphoma.

The chromosomal translocation t(14;18)(q32;q21) is characteristic of follicular lymphoma and a frequent abnormality in other types of non-Hodgkin lymphoma (NHL). In healthy individuals, the same translocation may also be found in a small fraction of peripheral blood lymphocytes, the biological significance of which is beginning to be explored. Translocation prevalence and frequency are potential risk factors for developing NHL. Here, we review the published data and describe recent and ongoing work on this promising biomarker. We have a series of studies in four major areas: 1) t(14;18) prevalence and frequency in healthy individuals; 2) maturation of translocation-harboring cells; 3) effect of rituximab treatment on t(14;18) carriage; and 4) predictive and clonotypic relationship between t(14;18) and follicular lymphoma or other NHL. Further studies are warranted to increase understanding of this crucial molecular event in the development of hematopoietic malignancies. Potential applications include determination of elevated risk for lymphoma, early detection of disease, and identification of molecular targets for preventive interventions.