Flow cytometry has become a highly valuable method to monitor minimal residual disease (MRD) and evaluate the depth of complete response (CR) in bone marrow (BM) of multiple myeloma (MM) after therapy. However, current flow-MRD has lower sensitivity than molecular methods and lacks standardization. Here we report on a novel next generation flow (NGF) approach for highly sensitive and standardized MRD detection in MM. An optimized 2-tube 8-color antibody panel was constructed in five cycles of design-evaluation-redesign. In addition, a bulk-lysis procedure was established for acquisition of ⩾107 cells/sample, and novel software tools were constructed for automatic plasma cell gating. Multicenter evaluation of 110 follow-up BM from MM patients in very good partial response (VGPR) or CR showed a higher sensitivity for NGF-MRD vs conventional 8-color flow-MRD -MRD-positive rate of 47 vs 34% (P=0.003)-. Thus, 25% of patients classified as MRD-negative by conventional 8-color flow were MRD-positive by NGF, translating into a significantly longer progression-free survival for MRD-negative vs MRD-positive CR patients by NGF (75% progression-free survival not reached vs 7 months; P=0.02). This study establishes EuroFlow-based NGF as a highly sensitive, fully standardized approach for MRD detection in MM which overcomes the major limitations of conventional flow-MRD methods and is ready for implementation in routine diagnostics.

The presence of cytogenetic aberrations on mesenchymal stem cells (MSC) from myelodysplastic syndrome (MDS) patients is controversial. The aim of the study is to characterize bone marrow (BM) derived MSC from patients with MDS using: kinetic studies, immunophenotyping, fluorescent in situ hybridization (FISH) and genetic changes by array-based comparative genomic hybridization (array-CGH). In all 36 cases of untreated MDS were studied. MDS-MSC achieved confluence at a significantly slower rate than donor-MSC, and the antigenic expression of CD105 and CD104 was lower. Array-CGH studies showed DNA genomic changes that were proved not to be somatic. These results were confirmed by FISH. To confirm that genomic changes were also present in freshly obtained MSCs they were enriched by sorting BM cells with the following phenotype:They also showed genomic changes that were confirmed by FISH. To analyze the relationship of these aberrations with clinicalbiological data an unsupervized hierarchical cluster analysis was performed, two clusters were identified: the first one included the 5qÀ syndrome patients, whereas the other incorporated other MDS. Our results show, for the first time that MSC from MDS display genomic aberrations, assessed by array-CGH and FISH, some of them specially linked to a particular MDS subtype, the 5qÀ syndrome.

It is an open question whether in multiple myeloma (MM) bone marrow stromal cells contain genomic alterations, which may contribute to the pathogenesis of the disease. We conducted an array-based comparative genomic hybridization (array-CGH) analysis to compare the extent of unbalanced genomic alterations in mesenchymal stem cells from 21 myeloma patients (MM-MSCs) and 12 normal donors (ND-MSCs) after in vitro culture expansion. Whereas ND-MSCs were devoid of genomic imbalances, several non-recurrent chromosomal gains and losses (>1 Mb size) were detected in MM-MSCs. Using real-time reverse transcription PCR, we found correlative deregulated expression for five genes encoded in regions for which genomic imbalances were detected using array-CGH. In addition, only MM-MSCs showed a specific pattern of 'hot-spot' regions with discrete (<1 Mb) genomic alterations, some of which were confirmed using fluorescence in situ hybridization (FISH). Within MM-MSC samples, unsupervised cluster analysis did not correlate with particular clinicobiological features of MM patients. We also explored whether cytogenetic abnormalities present in myelomatous plasma cells (PCs) were shared by matching MSCs from the same patients using FISH. All MM-MSCs were cytogenetically normal for the tested genomic alterations. Therefore we cannot support a common progenitor for myeloma PCs and MSCs.

Summary:Hemorrhagic cystitis (HC) is a common complication following hemopoietic stem cell transplantation (HSCT), its incidence ranging from 7 to 52% of all patients. Late occurring HC frequently results from viral infections. We describe a patient who developed severe polyomavirus-associated HC, which responded dramatically to a single dose of intra-muscular vidarabine. Previous studies show an improvement in HC with vidarabine therapy, but to date only the intravenous route of administration has been described and responses described take from several days to weeks. This report confirms the safety and efficacy of vidarabine administered intramuscularly when used in patients with an adequate platelet count, thereby making its use feasible when intravenous vidarabine is not available. Bone Marrow Transplantation (2000) 26, 1229-1230. Keywords: hemorrhagic cystitis; vidarabine; intra-muscular; transplantation Hemorrhagic cystitis (HC) is a common complication following hemopoietic stem cell transplantation (HSCT), its incidence ranging from 7 to 52% of all patients. 1,2 Early onset HC is associated with high-dose cyclophosphamide conditioning regimens and is prevented by the widespread use of the acrolein chelator mesna, hyperhydration or continuous bladder irrigation, while late occurring HC frequently results from viral infections (eg BK virus, adenovirus type 11). [3][4][5] BKV is a ubiquitous virus which infects more than 60% of the population worldwide at an early age. 6 Antibodies to BKV are demonstrated in the serum of 80 to 100% of adult patients before transplant. Since post-transplantation viruria only occurs in patients who were seropositive pre- transplantation, reactivation, probably associated with immune deficiency, is postulated to be the likely cause. 7 The purine analogue vidarabine (adenine arabinoside) has been shown to have in vitro antiviral activity against human polyomavirus by selectively inhibiting viral DNA polymerase. Successful treatment of polyomavirus-associated HC occurring after HSCT with disappearance or reduction in viruria has already been reported using intravenous vidarabine. 8 We describe a patient who developed severe polyomavirus-associated HC and who responded dramatically to a single dose of intra-muscular vidarabine, thereby demonstrating the efficacy and safety of this drug when employed intramuscularly. Case reportA 13-year-old boy was diagnosed with severe aplastic anemia and underwent allogeneic peripheral blood stem cell transplantation from an HLA-identical brother. After conditioning with cyclophosphamide (50 mg/kg for 3 days with mesna 50 mg/kg 0, 4 and 8 h after cyclophosphamide) and anti-thymocyte globulin (30 mg/kg for 3 days), 8.3 × 10 6 CD34 + cells/kg were infused. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporin A at 1.5 mg/kg/12 h from day −4 onwards and methotrexate without folinic rescue on days +1, 3, 6 and 11.More than 500 granulocytes/mm 3 and 20 × 10 9 platelets/l were achieved on days +14 and +12, respectively. Complete chime...