The graft-versus-leukemia effect of prophylactic donor lymphocyte infusions after allogeneic stem cell transplantation is equally effective in relapse prevention but safer compared to spontaneous graft-versus-host disease.

Therapeutic donor lymphocyte infusions (tDLI) are used to reinforce the graft-versus-leukemia (GvL) effect in relapse after allogeneic stem cell transplantation (alloSCT). In contrast, the role of prophylactic DLI (proDLI) in preventing leukemia relapse has been less clearly established, although supported by retrospective, case-control, and registry analyses. We report a prospective, monocentric, ten year cohort of patients with high risk acute leukemias (AL) or myelodysplasia (MDS) in whom proDLI were applied beyond day +120 post alloSCT to compensate for lack of GvL.272 consecutive allotransplanted AL or MDS patients in complete remission and off immunosuppression at day +120 were stratified according to the prior appearance of relevant GvHD (acute GvHD °II-IV or extensive chronic GvHD) as a clinical indicator for GvL. Escalating doses of unmodified proDLI were applied to 72/272 patients without prior relevant GvHD. Conversely, 157/272 patients with prior spontaneous GvHD did not receive proDLI, nor did 43/272 patients with contraindications (uncontrolled infections, patient refusal, DLI unavailability).By day 160-landmark analysis (median day of first DLI application), proDLI recipients had significantly higher five-year overall (OS) and disease free survival (DFS) (77% and 67%) than patients with spontaneous GvHD (54% and 53%) or with contraindications (46% and 45%) (p=0.003). Relapse incidence for patients with proDLI (30%) or spontaneous GvHD (29%) was significantly lower than in patients with contraindications (39%; p=0.021). With similar GvHD incidence beyond day +160, non-relapse mortality (NRM) was less with proDLI (5%) than without proDLI (18%; p=0.036).In conclusion, proDLI may be able to compensate for lack of GvL in alloSCT recipients with high risk AL or MDS.

Proteomics for hematopoietic stem cell transplantation.

Introduction: After the genomic era, the analysis of the proteome has gained increasing importance. Peptides and/or proteins present in tissue or body fluids can depict health and are prone to change during disease, not only in configuration but also in abundance. Early on, high throughput proteome analysis was implemented in the diagnostic of therapy-linked or induced complications arising after allogeneic hematopoietic stem cell transplantation (HSCT). Several proteomic approaches are currently used in the prediction or diagnosis of acute and/or chronic graft-versus-host disease (GvHD).Areas covered: This review will report on two high throughput proteomics technologies used in the clinical setting to date, namely enzyme-linked-immunosorbent assays (ELISA) for key proteins involved in the pathogenesis of acute GvHD and on capillary electrophoresis coupled on-line to mass spectrometry (CE-MS). Here, we summarize the current data and discuss the strength as well as the limitations of each method and compare the usefulness and practicability in the post-HSCT setting for prediction and diagnosis of acute GvHD.Expert commentary: Both technologies are applied in the clinic and have been tested on several hundred patients after HSCT. The data from both technologies may complement each other in diagnosis of GvHD.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

Characterization of High-Avidity Cytomegalovirus-Specific T Cells with Differential Tetramer Binding Coappearing after Allogeneic Stem Cell Transplantation.

CMV reactivation is a major complication after allogeneic stem cell transplantation (SCT). Immune reconstitution of CMV-specific CTLs (CMV-CTLs) is essential for virus control. During CMV-CTL monitoring using mutated HLA/CMV tetramers selectively detecting high-avidity T cells, we observed coappearance of CMV-CTLs with low (CMV tetlow CTLs) and high tetramer binding (CMV tethigh CTLs) in 53/115 CMV IgG+ patients stem cell transplanted from CMV IgG+ donors. However, the relevance of these coappearing differentially tetramer binding ("dual") CMV-CTLs was unclear. In this study, we investigated the kinetics, properties, and clinical impact of coappearing CMV tetlow and tethigh CTLs after allogeneic SCT. Patients with dual CMV-CTLs had more CMV tethigh than tetlow CTLs. Chimerism analysis of isolated CMV tetlow and tethigh CTLs revealed their exclusive donor origin. CMV tetlow and tethigh CTLs had an identical effector memory CD45RA-CCR7- and CD45RA+CCR7- T cell distribution, equal differentiation, senescence, and exhaustion marker expression and were negative for regulatory CD8+ T cell markers. Isolated CMV tetlow and tethigh CTLs were equally sensitive to CMV peptides in IFN-γ release and cytotoxicity assays. However, CMV tethigh CTLs proliferated more in response to low CMV peptide concentrations than tetlow CTLs. TCR repertoire analysis revealed that CMV tetlow and tethigh CTLs use different TCRs. Finally, dual CMV-CTLs were not associated with CMV antigenemia. In conclusion, these data show for the first time, to our knowledge, that both CMV tetlow and tethigh CTLs are functional effector T cells differing by proliferation, numbers in peripheral blood, and probably by their precursors without increasing the CMV reactivation risk after allogeneic SCT.

Possible Impact of Cytomegalovirus-Specific CD8+ T Cells on Immune Reconstitution and Conversion to Complete Donor Chimerism after Allogeneic Stem Cell Transplantation.

Complete donor chimerism is strongly associated with complete remission after allogeneic stem cell transplantation (allo-SCT) in patients with hematologic malignancies. Donor-derived allo-immune responses eliminate the residual host hematopoiesis and thereby mediate the conversion to complete donor chimerism. Recently, cytomegalovirus (CMV) reactivation was described to enhance overall T cell reconstitution, to increase graft-versus-host disease incidence, and to reduce the leukemia relapse risk. However, the link between CMV and allo-immune responses is still unclear. Here, we studied the relationship between CMV-specific immunity, overall T cell reconstitution, and residual host chimerism in 106 CMV-seropositive patients transplanted after reduced-intensity conditioning including antithymocyte globulin. In accordance with previous reports, the recovery of CMV-specific cytotoxic T cells (CMV-CTLs) was more frequent in CMV-seropositive recipients (R) transplanted from CMV-seropositive than from seronegative donors (D). However, once CMV-CTLs were detectable, the reconstitution of CMV-specific CTLs was comparable in CMV R+/D- and R+/D+ patients. CD3+ and CD8+ T cell reconstitution was significantly faster in patients with CMV-CTLs than in patients without CMV-CTLs both in the CMV R+/D- and R+/D+ setting. Moreover, CMV-CTL numbers correlated with CD3+ and CD8+ T cell numbers in both settings. Finally, presence of CMV-CTLs was associated with low host chimerism levels 3 months after allo-SCT. In conclusion, our data provide a first indication that CMV-CTLs in CMV-seropositive patients might trigger the reconstitution of T cells and allo-immune responses reflected by the conversion to complete donor chimerism.

Bile proteomic profiles differentiate cholangiocarcinoma from primary sclerosing cholangitis and choledocholithiasis.

UNLABELLED: Early detection of malignant biliary tract diseases, especially cholangiocarcinoma (CC) in patients with primary sclerosing cholangitis (PSC), is very difficult and often comes too late to give the patient a therapeutic benefit. We hypothesize that bile proteomic analysis distinguishes CC from nonmalignant lesions. We used capillary electrophoresis mass spectrometry (CE-MS) to identify disease-specific peptide patterns in patients with choledocholithiasis (n = 16), PSC (n = 18), and CC (n = 16) in a training set. A model for differentiation of choledocholithiasis from PSC and CC (PSC/CC model) and another model distinguishing CC from PSC (CC model) were subsequently validated in independent cohorts (choledocholithiasis [n = 14], PSC [n = 18] and CC [n = 25]). Peptides were characterized by sequencing. Application of the PSC/CC model in the independent test cohort resulted in correct exclusion of 12/14 bile samples from patients with choledocholithiasis and identification of 40/43 patients with PSC or CC (86% specificity, 93% sensitivity). The corresponding receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.93 (95% confidence interval [CI]: 0.82-0.98, P = 0.0001). The CC model succeeded in an accurate detection of 14/18 bile samples from patients with PSC and 21/25 samples with CC (78% specificity, 84% sensitivity) in the independent cohort, resulting in an AUC value of 0.87 (95% CI: 0.73-0.95, P = 0.0001) in ROC analysis. Eight out of 10 samples of patients with CC complicating PSC were identified. CONCLUSION: Bile proteomic analysis discriminates benign conditions from CC accurately. This method may become a diagnostic tool in future as it offers a new possibility to diagnose malignant bile duct disease and thus enables efficient therapy particularly in patients with PSC.

Adoptive transfer of pp65-specific T cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation.

Cytomegalovirus (CMV) disease and infection refractory to antiviral treatment after allogeneic stem cell transplantation (allo-SCT) is associated with a high mortality. Adoptive transfer of CMV-specific T cells could reconstitute viral immunity after SCT and could protect from CMV-related complications. However, logistics of producing virus-specific T-cell grafts limited the clinical application. We treated 18 patients after allo-SCT from human leukocyte antigen-mismatched/haploidentical or human leukocyte antigen-matched unrelated donors with polyclonal CMV-specific T cells generated by ex vivo stimulation with pp65, followed by isolation of interferon-γ-producing cells. Patients with CMV disease or viremia refractory to antiviral chemotherapy or both were eligible for adoptive T-cell transfer and received a mean of 21 × 10³/kg pp65-specific T cells. In 83% of cases CMV infection was cleared or viral burden was significantly reduced, even in cases of CMV encephalitis (n = 2). Viral control was associated with in vivo expansion of CMV-specific T lymphocytes in 12 of 16 evaluable cases, resulting in reconstitution of antiviral T-cell responses, without graft-versus-host disease induction or acute side effects. Our findings indicate that the infusion of low numbers of CMV-specific T cells is safe, feasible, and effective as a treatment on demand for refractory CMV infection and CMV disease after allo-SCT.

Prognostic significance of combined MN1, ERG, BAALC, and EVI1 (MEBE) expression in patients with myelodysplastic syndromes.

Overexpression of MN1, ERG, BAALC, and EVI1 (MEBE) genes in cytogenetically normal acute myeloid leukemia (AML) patients is associated with poor prognosis, but their prognostic effect in patients with myelodysplastic syndromes (MDS) has not been studied systematically. Expression data of the four genes from 140 MDS patients were combined in an additive score, which was validated in an independent patient cohort of 110 MDS patients. A high MEBE score, defined as high expression of at least two of the four genes, predicted a significantly shorter overall survival (OS) (HR 2.29, 95 % CI 1.3-4.09, P= .005) and time to AML progression (HR 4.83, 95 % CI 2.01-11.57, P< .001) compared to a low MEBE score in multivariate analysis independent of karyotype, percentage of bone marrow blasts, transfusion dependence, ASXL1, and IDH1 mutation status. In a validation cohort of 110 MDS patients, a high MEBE score predicted shorter OS (HR 1.77; 95 % CI 1.04-3.0, P= .034) and time to AML progression (HR 3.0, 95 % CI 1.17-7.65, P= .022). A high MEBE expression score is an unfavorable prognostic marker in MDS and is associated with an increased risk for progression to AML. Expression of the MEBE genes is regulated by FLI1 and c-MYC, which are potential upstream targets of the MEBE signature.

Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease.

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

Adaptive NK cells undergo a dynamic modulation in response to human cytomegalovirus and recruit T cells in in vitro migration assays.

Human cytomegalovirus (HCMV) reactivation remains a relevant complication after hematopoietic stem cell transplantation (HSCT) despite the great progress made in prophylaxis and treatment. Adaptive Natural Killer (NK) cells undergo a persistent reconfiguration in response to HCMV reactivation however, the exact role of adaptive NK cells in HCMV surveillance is currently unknown. We studied the relationship between HCMV reactivation and adaptive NK cells in 70 patients monitored weekly until day +100 after HSCT. Absolute cell counts of adaptive NK cells increased significantly after resolution of HCMV-reactivation compared to patients without reactivation. Patients with HCMV-reactivation had an early reconstitution of adaptive NK cells ("Responders") and had mainly a single reactivation (75% Responders vs 48% Non-Responders). Adaptive NK cells eliminated HCMV-infected human foreskin fibroblasts (HFF) in vitro and recruited T cells in an in vitro transwell migration assay. An extensive cytokine/chemokine panel demonstrated strongly increased secretion of CXCL10/IP-10, IFN-α, IL-1α, IL-1ß, IL-5, IL-7 and CCL4. Thus, adaptive NK cells may control viral spread and T cell expansion and survival during HCMV-reactivation. Taken together, we have demonstrated the potential of adaptive NK cells in the control of HCMV reactivation both by direct cytotoxicity and by recruitment of other immune cells.

Human Cytomegalovirus pUL11, a CD45 Ligand, Disrupts CD4 T Cell Control of Viral Spread in Epithelial Cells.

Human cytomegalovirus (HCMV) encodes numerous immunomodulatory genes that facilitate its persistence. Previously described mechanisms by which HCMV avoids T cell control typically involve evasion of detection by infected cells. Here, we show that the virus also inhibits T cells directly via an interaction between the pUL11 glycoprotein on infected cells and the CD45 phosphatase on T cells. The antiviral functions of CD4 T cells are impaired as a result of this interaction, largely via induced interleukin 10 (IL-10) secretion in the CD4 T cell central memory compartment, resulting in enhanced viral spread. This establishes CD45 as an inhibitory receptor that regulates antiviral T cell functions and has parallels with the manipulation of natural killer (NK) cells by HCMV. By coculturing donor T cells with HCMV-infected epithelial cells, we observed that CD4 T cells can respond to epithelial cell antigen presentation and can control HCMV spread via cytolytic and cytokine-dependent mechanisms. pUL11 impairs both mechanisms. We showed that pUL11-induced IL-10 secretion requires IL-2, mTOR, and T cell receptor signaling. This characterization of the effects of the pUL11-CD45 interaction may allow for the development of new antiviral therapies and treatments for inflammatory disorders. IMPORTANCE Human cytomegalovirus (HCMV) is adept at avoiding its host's immune defenses, both by evading detection and by directly inhibiting immune cells. This can lead to a loss of control of the infection, and dangerous disease can result, particularly in cases in which an individual's immune system is immature, weak, or suppressed. T cells form a crucial part of the response to HCMV and are used in cellular HCMV therapies. We show that an interaction between a viral glycoprotein (pUL11) and a T cell surface receptor (CD45) impairs T cell memory functions and allows for increased viral spread. This defines a new immunomodulatory strategy for the virus as well as a new T cell regulatory mechanism. These results are important, as they increase our understanding of how T cells function and how HCMV disrupts them. This will allow for the development of new antiviral therapies that restore T cell functions and indicates a new target for controlling pathological T cell disorders.

Navigating preemptive and therapeutic donor lymphocyte infusions in advanced myeloid malignancies by high-sensitivity chimerism analysis.

Preemptive and therapeutic donor lymphocyte infusions (preDLI and tDLI) are widely used in relapsing and relapsed hematopoietic malignancies after allogeneic stem cell transplantation (alloSCT) to enhance the graft-versus-malignancy effect. However, in advanced myeloid malignancies, long-term survival after preDLI and tDLI remains low, reflecting our inability to master the double-edged sword of alloreactivity, balancing anti-neoplastic activity versus graft-versus-host disease (GvHD). We previously evaluated a quantitative PCR-based high-sensitivity chimerism (hs-chimerism) based on insertion/deletion polymorphisms instead of short tandem repeats, where increasing host chimerism in peripheral blood predicts relapse more than a month before clinical diagnosis, and declining host chimerism signals anti-host alloreactivity. Here we report 32 consecutive patients with advanced myeloid malignancies receiving preDLI or tDLI "navigated" by hs-chimerism ("navigated DLI"). We compared them to a historical cohort of 110 consecutive preDLI or tDLI recipients, prior to implementation of hs-chimerism at our institution ("controls"). Both groups were comparable regarding age, gender, conditioning, donor type, and time to DLI. With longer median follow-up of the navigated DLI group (8.5 versus 5 months), their landmark overall (64%) and disease-free survival (62%) at 2 years from first DLI compared favorably with controls (23% and 21%, respectively). Improved survival of navigated DLI was due to both reduced relapse incidence (38% versus 60%) and non-relapse mortality (17% versus 44%) at 2 years. Early relapse prediction by hs-chimerism allowed a preemptive approach in 28% of navigated DLI versus 7% in controls. Our results confirm hs-chimerism as a highly valuable tool for monitoring and steering immune interventions after alloSCT.

Intramedullary spinal cord implantation of human CD34+ umbilical cord-derived cells in ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with marginal therapeutic options. Degeneration of motor neurons in the primary motor cortex, brainstem and spinal cord lead to rapidly progressive paralysis and finally to death due to respiratory failure. As pharmacological therapies have failed to provide sufficient neuroprotective effects in ALS, transplantation of stem or progenitor cells is considered a promising treatment strategy. Cell transplantation approaches in ALS mainly aim to generate a neuroprotective environment for degenerating motor neurons by transplantation of non-neuronal cells, rather than to replace lost motor neurons. We present a 63-year-old male patient suffering from ALS who underwent intramedullary thoracic spinal cord implantation of human CD34(+) umbilical cord-derived haematopoietic progenitor cells with a three-year follow up after transplantation.

A multicenter prospective, randomized, placebo-controlled phase II/III trial for preemptive acute graft-versus-host disease therapy.

Acute graft-versus-host disease (aGvHD) contributes to about 50% of transplant-related mortality (non-relapse mortality) after allogeneic hematopoietic stem cell transplantation (HSCT). Here the predictive value of a urinary proteomic profile (aGvHD_MS17) was tested together with preemptive prednisolone therapy. Two-hundred and fifty-nine of 267 patients were eligible for analysis. Ninety-two patients were randomized upon aGvHD_MS17 classification factor above 0.1 to receive either prednisolone (2-2.5 mg/kg, N = 44) or placebo (N = 47; N = 1 randomization failure) for 5 days followed by tapering. The remaining 167 patients formed the observation group. The primary endpoint of the randomized trial was incidence of aGvHD grade II between randomization and day +100 post HSCT. Analysis of the short-term preemptive prednisolone therapy in the randomized patients showed no significant difference in incidence or severity of acute GvHD (HR: 1.69, 95% CI: 0.66-4.32, P = 0.27). Prednisolone as preemptive treatment did not lead to an increase in relapse (20.2% in the placebo and 14.0% in the prednisolone group (P = 0.46)). The frequency of adverse events was slightly higher in the placebo group (64.4% versus 50%, respectively). Taken together, the results of the Pre-GvHD trial demonstrated the feasibility and safety of preemptive prednisolone treatment in the randomized patients.

Urinary excretion of twenty peptides forms an early and accurate diagnostic pattern of acute kidney injury.

Early and accurate detection of acute kidney injury (AKI) is needed to prevent the progression to chronic kidney disease and to improve outcome. Here we used capillary electrophoresis-mass spectrometry to identify urinary peptides predictive of AKI in a training set of 87 urine samples longitudinally collected from patients in an intensive care unit. Within this patient cohort, 16 developed AKI while 14 maintained normal renal function. The sequence of twenty peptides significantly associated with AKI was identified. They were found to be degradation products of six proteins. These formed a diagnostic pattern. Peptides of albumin, α-1-antitrypsin, and ß-2-microglobulin were upregulated but fragments of fibrinogen α and collagens 1 α(I) and 1 α(III) were downregulated in AKI. After cross-validation of the training set, a good diagnostic performance of the marker pattern was found with an area under the ROC curve of 0.91. This was confirmed in a blinded validation set of 20 patients in the intensive care unit and 31 allogeneic hematopoietic stem cell transplantation patients, of which 13 had and 18 had not experienced an episode of AKI. In comparison to more established markers of AKI such as serum cystatin C and urinary kidney injury molecule-1, interleukin-18, and neutrophil gelatinase associated-lipocalin, the proteomic marker pattern was found to be of superior prognostic value, detecting AKI up to 5 days in advance of the rise in serum creatinine.

Capillary electrophoresis-mass spectrometry as a powerful tool in biomarker discovery and clinical diagnosis: an update of recent developments.

Proteome analysis has emerged as a powerful technology to decipher biological processes. One of the main goals is to discover biomarkers for diseases from tissues and body fluids. However, the complexity and wide dynamic range of protein expression present an enormous challenge to separation technologies and mass spectrometry (MS). In this review, we examine the limitations of proteomics, and aim towards the definition of the current key prerequisites. We focus on capillary electrophoresis coupled to mass spectrometry (CE-MS), because this technique continues to show great promise. We discuss CE-MS from an application point of view, and evaluate its merits and vices for biomarker discovery and clinical applications. Finally, we present several examples on the use of CE-MS to determine urinary biomarkers and implications for disease diagnosis, prognosis, and therapy evaluation.

IDH1 mutations in patients with myelodysplastic syndromes are associated with an unfavorable prognosis.

BACKGROUND: Myelodysplastic syndromes are a heterogeneous group of hematopoietic stem cell disorders with a high propensity to transform into acute myeloid leukemia. Heterozygous missense mutations in IDH1 at position R132 and in IDH2 at positions R140 and R172 have recently been reported in acute myeloid leukemia. However, little is known about the incidence and prognostic impact of IDH1 and IDH2 mutations in myelodysplastic syndromes. DESIGN AND METHODS: We examined 193 patients with myelodysplastic syndromes and 53 patients with acute myeloid leukemia arising from myelodysplastic syndromes for mutations in IDH1 (R132), IDH2 (R172 and R140), and NPM1 by direct sequencing. RESULTS: We found that mutations in IDH1 occurred with a frequency of 3.6% in myelodysplastic syndromes (7 mutations in 193 patients) and 7.5% in acute myeloid leukemia following myelodysplastic syndromes (4 mutations in 53 patients). Three mutations in codon R140 of IDH2 and one mutation in codon R172 were found in patients with acute myeloid leukemia following myelodysplastic syndromes (7.5%). No IDH2 R140 or R172 mutations were identified in patients with myelodysplastic syndromes. The presence of IDH1 mutations was associated with a shorter overall survival (HR 3.20; 95% CI 1.47-6.99) and a higher rate of transformation into acute myeloid leukemia (67% versus 28%, P=0.04). In multivariate analysis when considering karyotype, transfusion dependence and International Prognostic Scoring System score, IDH1 mutations remained an independent prognostic marker in myelodysplastic syndromes (HR 3.57; 95% CI 1.59-8.02; P=0.002). CONCLUSIONS: These results suggest that IDH1 mutations are recurrent molecular aberrations in patients with myelodysplastic syndromes, and may become useful as a poor risk marker in these patients. These findings await validation in prospective trials.

Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I-II study.

BACKGROUND: Procedures to prevent severe graft-versus-host disease (GVHD) delay immune reconstitution secondary to transplants of haploidentical haemopoietic stem cells for the treatment of leukaemia, leading to high rates of late infectious mortality. We aimed to systematically add back genetically engineered donor lymphocytes to facilitate immune reconstitution and prevent late mortality. METHODS: In a phase I-II, multicentre, non-randomised trial of haploidentical stem-cell transplantation, we infused donor lymphocytes expressing herpes-simplex thymidine kinase suicide gene (TK-cells) after transplantation. The primary study endpoint was immune reconstitution defined as circulating CD3+ count of 100 cells per muL or more for two consecutive observations. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00423124. FINDINGS: From Aug 13, 2002, to March 26, 2008, 50 patients (median age 51 years, range 17-66) received haploidentical stem-cell transplants for high-risk leukaemia. Immune reconstitution was not recorded before infusion of TK-cells. 28 patients received TK-cells starting 28 days after transplantation; 22 patients obtained immune reconstitution at median 75 days (range 34-127) from transplantation and 23 days (13-42) from infusion. Ten patients developed acute GVHD (grade I-IV) and one developed chronic GVHD, which were controlled by induction of the suicide gene. Overall survival at 3 years was 49% (95% CI 25-73) for 19 patients who were in remission from primary leukaemia at the time of stem-cell transplantation. After TK-cell infusion, the last death due to infection was at 166 days, this was the only infectious death at more than 100 days. No acute or chronic adverse events were related to the gene-transfer procedure. INTERPRETATION: Infusion of TK-cells might be effective in accelerating immune reconstitution, while controlling GVHD and protecting patients from late mortality in those who are candidates for haploidentical stem-cell transplantation. FUNDING: MolMed SpA, Italian Association for Cancer Research.

Proteomics studies after hematopoietic stem cell transplantation.

Complex biological samples hold significant information on the health status and on development of disease. Approximately 35,000 human genes give rise to more than 1,000,000 functional entities at the protein level. Thus, the proteome provides a much richer source of information than the genome for describing the state of health or disease of humans. The composition body fluids comprise a rich source of information on changes of protein and peptide expression. Here we describe the application of capillary electrophoresis (CE) coupled online to an electrospray-ionization time-of-flight mass spectrometer (ESI-TOF-MS) to analyze human urine for the identification of biomarkers specific for complications after allogeneic hematopoietic stem cell transplantation (HSCT). Sequencing of native proteins/peptides is necessary for the identification of possible new therapeutic targets.

Fast and Efficient Titration of Human Cytomegalovirus Stocks with a Self-Excisable Bacterial Artificial Chromosomes Cassette by Flow Cytometry.

The study of human cytomegalovirus (HCMV) has for long been challenging due to the inability of clinical strains to efficiently proliferate in vitro until adaptive mutations occur. These mutations lead to strains that differ considerably from clinical isolates, many of them showing altered cell tropism, a decrease in cell association and higher susceptibility to an innate immune response. These problems were recently solved by the use of bacterial artificial chromosome (BAC) vectors that allow for the conservation of an intact HCMV genome. Other characteristics that render HCMV difficult for in vitro study are related to its slow replication rate that leads to some constraints in its titration. During the cloning of HCMV into BAC vectors, many groups additionally inserted a fluorescent tag to facilitate the virus characterization. However, the methods used for titration of HCMV-BAC stocks are still relaying on the standard methods that are expensive and/or time consuming. In this study, we assessed the possibility of viral titration by fluorescence-activated cell sorting (FACS), making use of the fluorescent tags that many of the HCMV-BACs hold. We compared viral titers obtained by immunohistochemistry with FACS, a faster and inexpensive technique. We showed that viral titers are comparable using the techniques already mentioned, and that titration by FACS is an efficient, fast, and cost-effective method. The establishment of viral titration of BAC vectors by FACS can further simplify the study of HCMV.