EBV reactivation and post transplant lymphoproliferative disorders following allogeneic SCT.

Fatal problems encountered in allogeneic stem cell transplantation include EBV reactivation and post transplant lymphoproliferative disorders (PTLDs) with high mortality rates. We performed a retrospective analysis in all consecutive adult and pediatric EBV reactivations and PTLD during a period of 8.5 years. There were 26 patients with EBV reactivation/PTLD out of a total of 854 transplantations giving an overall incidence of 3.0%. Specifically, the incidence of EBV-PTLD was 1.3%, whereas that of EBV reactivation was 1.8%. Median age was 46.0 and 11.0 years in the adult and pediatric patients, respectively. There were high rates (54%) of concomitant bacterial, viral, fungal and parasitic infections at the time of EBV manifestation. Variable treatment regimens were applied including in most cases an anti-CD20 regimen often in combination with virustatic compounds, polychemotherapy or donor lymphocytes. The mortality rates were 9 of 11 (82%) in patients with EBV-PTLD and 10 of 15 (67%) in patients with reactivation. Only 7 of 26 patients (27%) are alive after a median follow-up of 758 days (range 24-2751). The high mortality rates of EBV reactivation and of EBV-PTLD irrespective of multimodal treatment approaches emphasize standardization and optimization of post transplant surveillance and treatment strategies to improve control of these often fatal complications.

Absence of APOBEC-1 mediated mRNA editing in human carcinomas.

The transgene expression of the catalytic subunit APOBEC-1 of the apo B mRNA editing enzyme-complex can cause hepatocellular carcinoma in mice and rabbits. It has been proposed that aberrant editing of mRNA may represent a novel oncogenic principle. This investigation aimed to define whether such aberrant hyperediting mediated by APOBEC-1 occurs in human carcinomas. Editing and hyperediting of apo B, NAT1 or NF1 mRNA was not identified in any of 28 resected tumor specimens, including hepatocellular, bile duct, gastric, colorectal, pancreatic adeno- and neuroendocrine, lung adeno-, medullary thyroid and breast carcinoma, soft tissue sarcoma and neuroblastoma. In most types of carcinoma, significant levels for full-length APOBEC-1 mRNA could not be detected. Low level expression of APOBEC-1 was found in colorectal and gastric carcinoma where most of the APOBEC-1 mRNA is inactivated by alternate splicing. The 'auxiliary' components of the apo B mRNA editing enzyme-complex are missing in many tumors including colorectal and gastric carcinoma, but are highly expressed in hepatocellular, lung adeno- and breast carcinoma all of which lack APOBEC-1. Taken together, either APOBEC-1 or the 'auxiliary' components of the apo B mRNA editing enzyme-complex or both are missing in human carcinomas resulting in the absence of mRNA editing. Currently, there is no evidence that aberrant editing mediated by APOBEC-1 contributes to the tumorigenesis of natural human carcinomas.

Effective prevention of GVHD using in vivo T-cell depletion with anti-lymphocyte globulin in HLA-identical or -mismatched sibling peripheral blood stem cell transplantation.

To investigate the impact of anti-lymphocyte globulin (ATG-Fresenius) as part of the HLA-sibling transplantation, we evaluated 238 patients (median age 48 years) with different diagnoses (AML, ALL, CML and lymphoproliferative disorders). A total of 79 patients received ATG and 159 patients did not. In the ATG group, there were more HLA-mismatched donors (6% vs 1%, p=0.02), bad risk patients (70% vs 55%, P=0.04), reduced intensity conditioning (RIC) regimens (65% vs 34%, P=<0.001) and older patients (median age 51 vs 48 years, P=0.002). The median time to leukocyte engraftment was significantly faster in the non-ATG group (13 vs 15 days, P < 0.001). EBV reactivation was more often seen in the ATG group (9% vs 2%, P=0.05). Cumulative incidence of acute and chronic GVHD was less observed in the ATG group (27% vs 40%, P=0.004, and 33% vs 54%, P=0.002). The cumulative incidence rates of non-relapse mortality and of relapse at 5 years were 20 and 34%, respectively, for ATG and 34 and 29%, respectively, for non-ATG (P=0.06 and P=0.3). ATG can prevent GVHD without an obvious risk of relapse but should be confirmed in a randomized study.

Donor choice according to age for allo-SCT for AML in complete remission.

In a retrospective study of 168 patients with AML in CR who underwent allo-SCT, we compare the impact of young unrelated donors (UD) vs older matched related donors (MRD) on 5-year OS (5-yr OS). Median follow-up was 59 months and median donor age was 39 years, which was used as cutoff for young vs older donors. Kaplan-Meier-estimated 5-yr OS was better with UD ≤39 years vs MRD >39 years (66% vs 34%, P=0.001). In multivariate analysis, only donor age and cytogenetic risk impacted 5-yr OS. Compared with UD ≤39 years, both MRD >39 years (relative risk (RR): 4.31, P=0.001) and UD >39 years (RR: 2.14, P=0.03) were associated with poorer 5-yr OS. Standard-risk cytogenetics was associated with better 5-yr OS compared with bad-risk cytogenetics, (RR: 0.53, P=0.02). Subgroup analyses of patients ≥50 years (n=76) revealed similar results, with 5-yr OS of 62% for UD ≤39 yrs and 26% for MRD >39 yrs (P=0.022). In patients undergoing allo-HSCT for AML, young UD may improve outcome as compared with older MRD.

Inhibition of the apolipoprotein B mRNA editing enzyme-complex by hnRNP C1 protein and 40S hnRNP complexes.

The apolipoprotein (apo) B mRNA can be modified by a posttranscriptional base change from cytidine to uridine at nucleotide position 6666. This editing of apo B mRNA is mediated by a specific enzyme-complex of which only the catalytic subunit APOBEC-1 (apo B mRNA editing enzyme component 1) has been cloned and extensively characterized. In this study, two-hybrid selection in yeast identified hnRNP C1 protein to interact with APOBEC-1. Recombinant hnRNP C1 protein inhibited partially purified apo B mRNA editing activity from rat small intestine and bound specifically to apo B sense RNA around the editing site. The inhibition of apo B mRNA editing by hnRNP C1 protein was not due to masking of the RNA substrate as the mutant protein M104 spanning the RNA-binding domain of hnRNP C1 protein bound strongly to the apo B RNA, but did not inhibit the editing reaction. The apo B mRNA editing enzyme-complex of rat liver nuclear extracts sedimented in sucrose density gradients around 22-27S, but did not contain hnRNP C1 protein that was found exclusively within 40S hnRNP complexes. The removal of 40S hnRNP complexes increased the activity of the 22-27S editing enzyme-complex. Adding back 40S hnRNP complexes with hnRNP C1 protein resulted in an inhibition of the 22-27S apo B mRNA editing enzyme-complex, while addition of 18S fractions had no effect. In conclusion, hnRNP C1 protein identified by two-hybrid selection in yeast is a potent inhibitor of the apo B mRNA editing enzyme-complex. The abundant hnRNP C1 protein, which is contiguously deposited on nascent pre-mRNA during transcription and is involved in spliceosome assembly and mRNA splicing, is a likely regulator of the editing of apo B mRNA which restricts the activity of APOBEC-1 to limited and specific editing events.

Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex.

Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP). Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzyme-complex. In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzyme-complex with its substrate apoB RNA serving as an additional auxiliary component.