Multi-color polymer pen lithography for oligonucleotide arrays.

Multi-color patterning by polymer pen lithography (PPL) was used to fabricate covalently immobilized fluorophore and oligonucleotide arrays with up to five different components. The oligonucleotide arrays offer a virtually unlimited inventory of orthogonal binding tags for self-assembly of proteins as demonstrated by use of the arrays to monitor cell-protein interactions of MCF7 cells.

T-cell receptor Vß skewing frequently occurs in refractory cytopenia of childhood and is associated with an expansion of effector cytotoxic T cells: a prospective study by EWOG-MDS.

Immunosuppressive therapy (IST), consisting of antithymocyte globulin and cyclosporine A, is effective in refractory cytopenia of childhood (RCC), suggesting that, similar to low-grade myelodysplastic syndromes in adult patients, T lymphocytes are involved in suppressing hematopoiesis in a subset of RCC patients. However, the potential role of a T-cell-mediated pathophysiology in RCC remains poorly explored. In a cohort of 92 RCC patients, we prospectively assessed the frequency of T-cell receptor (TCR) ß-chain variable (Vß) domain skewing in bone marrow and peripheral blood by heteroduplex PCR, and analyzed T-cell subsets in peripheral blood by flow cytometry. TCRVß skewing was present in 40% of RCC patients. TCRVß skewing did not correlate with bone marrow cellularity, karyotype, transfusion history, HLA-DR15 or the presence of a PNH clone. In 28 patients treated with IST, TCRVß skewing was not clearly related with treatment response. However, TCRVß skewing did correlate with a disturbed CD4(+)/CD8(+) T-cell ratio, a reduction in naive CD8(+) T cells, an expansion of effector CD8(+) T cells and an increase in activated CD8(+) T cells (defined as HLA-DR(+), CD57(+) or CD56(+)). These data suggest that T lymphocytes contribute to RCC pathogenesis in a proportion of patients, and provide a rationale for treatment with IST in selected patients with RCC.

Hematopoietic stem cell transplantation for advanced myelodysplastic syndrome in children: results of the EWOG-MDS 98 study.

We report on the outcome of children with advanced primary myelodysplastic syndrome (MDS) transplanted from an HLA-matched sibling (MSD) or an unrelated donor (UD) following a preparative regimen with busulfan, cyclophosphamide and melphalan. Ninety-seven patients with refractory anemia with excess blasts (RAEB, n=53), RAEB in transformation (RAEB-T, n=29) and myelodysplasia-related acute myeloid leukemia (MDR-AML, n=15) enrolled in the European Working Group of MDS in Childhood (EWOG-MDS) 98 study and given hematopoietic stem cell transplantation (HSCT) were analyzed. Median age at HSCT was 11.1 years (range 1.4-19.0). Thirty-nine children were transplanted from an MSD, whereas 58 were given the allograft from a UD (n=57) or alternative family donor (n=1). Stem cell source was bone marrow (n=69) or peripheral blood (n=28). With a median follow-up of 3.9 years (range 0.1-10.9), the 5-year probability of overall survival is 63%, while the 5-year cumulative incidence of transplantation-related mortality (TRM) and relapse is 21% each. Age at HSCT greater than 12 years, interval between diagnosis and HSCT longer than 4 months, and occurrence of acute or extensive chronic graft-versus-host disease were associated with increased TRM. The risk of relapse increased with more advanced disease. This study indicates that HSCT following a myeloablative preparative regimen offers a high probability of survival for children with advanced MDS.

A novel HLA-C allele, HLA-Cw*06:20.

A novel HLA-C allele, HLA-Cw*06:20 differs from HLA-Cw*06:02 by one nucleotide exchange.

Rhabdomyosarcoma in patients with constitutional mismatch-repair-deficiency syndrome.

BACKGROUND: Biallelic germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 or PMS2 cause a recessive childhood cancer syndrome characterised by early-onset malignancies and signs reminiscent of neurofibromatosis type 1 (NF1). Alluding to the underlying genetic defect, we refer to this syndrome as constitutional mismatch repair-deficiency (CMMR-D) syndrome. The tumour spectrum of CMMR-D syndrome includes haematological neoplasias, brain tumours and Lynch syndrome-associated tumours. Other tumours, such as neuroblastoma, Wilm tumour, ovarian neuroectodermal tumour or infantile myofibromatosis, have so far been found only in individual cases. RESULTS: We analysed two consanguineous families that had members with suspected CMMR-D syndrome who developed rhabdomyosarcoma among other neoplasias. In the first family, we identified a pathogenic PMS2 mutation for which the affected patient was homozygous. In family 2, immunohistochemistry analysis showed isolated loss of PMS2 expression in all tumours in the affected patients, including rhabdomyosarcoma itself and the surrounding normal tissue. Together with the family history and microsatellite instability observed in one tumour this strongly suggests an underlying PMS2 alteration in family 2 also. CONCLUSION: Together, these two new cases show that rhabdomyosarcoma and possibly other embryonic tumours, such as neuroblastoma and Wilm tumour, belong to the tumour spectrum of CMMR-D syndrome. Given the clinical overlap of CMMR-D syndrome with NF1, we suggest careful examination of the family history in patients with embryonic tumours and signs of NF1 as well as analysis of the tumours for loss of one of the mismatch repair genes and microsatellite instability. Subsequent mutation analysis will lead to a definitive diagnosis of the underlying disorder.

The DNA methyltransferase inhibitors azacitidine, decitabine and zebularine exert differential effects on cancer gene expression in acute myeloid leukemia cells.

The three DNA methyltransferase (DNMT)-inhibiting cytosine nucleoside analogues, azacitidine, decitabine and zebularine, which are currently studied as nonintensive therapy for myelodysplastic syndromes and acute myeloid leukemia (AML), differ in structure and metabolism, suggesting that they may have differential molecular activity. We investigated cellular and molecular effects of the three substances relative to cytarabine in Kasumi-1 AML blasts. Under in vitro conditions mimicking those used in clinical trials, the DNMT inhibitors inhibited proliferation and triggered apoptosis but did not induce myeloid differentiation. The DNMT inhibitors showed no interference with cell-cycle progression whereas cytarabine treatment resulted in an S-phase arrest. Quantitative methylation analysis of hypermethylated gene promoters and of genome-wide LINE1 fragments using bisulfite sequencing and MassARRAY suggested that the hypomethylating potency of decitabine was stronger than that of azacitidine; zebularine showed no hypomethylating activity. In a comparative gene expression analysis, we found that the effects of each DNMT inhibitor on gene transcription were surprisingly different, involving several genes relevant to leukemogenesis. In addition, the gene methylation and expression analyses suggested that the effects of DNMT-inhibiting cytosine nucleoside analogues on the cellular transcriptome may, in part, be unrelated to direct promoter DNA hypomethylation, as previously shown by others.

T-cell receptor Vbeta CDR3 oligoclonality frequently occurs in childhood refractory cytopenia (MDS-RC) and severe aplastic anemia.

(Very) severe acquired aplastic anemia ((v)SAA) and myelodysplastic syndrome (MDS) are rare diseases in childhood. (V)SAA is a bone marrow (BM) failure syndrome characterized by immune-mediated destruction of hematopoietic progenitors. MDS is a malignant clonal stem cell disorder, of which the hypoplastic variant is, in case of absence of a cytogenetic clone, difficult to separate from (v)SAA. Recently, studies provided a molecular signature of autoimmunity in adult (v)SAA, by showing oligoclonality based on the length of the TCR Vbeta CDR3 region. We investigated retrospectively the frequency and the discriminative value of TCR Vbeta CDR3 oligoclonality in pediatric (v)SAA and MDS patients. Peripheral blood (PB) and/or BM mononuclear cell samples of pediatric patients with (v)SAA (n=38), refractory cytopenia (MDS-RC) (n=28) and 18 controls were analysed via TCR Vbeta heteroduplex PCR analysis of extracted RNA. A skewed TCR Vbeta CDR3 repertoire was found in 21/38 (v)SAA and in 17/28 RC patients in contrast to 2/18 in the control group. These data suggest an overlapping group of RC and SAA patients that may share a common immune-mediated pathogenesis. Prospective studies are required to establish the clinical value of TCR Vbeta CDR3 repertoire analysis to predict the clinical response in these patients.

Reduced intensity conditioning in unrelated donor transplantation for refractory cytopenia in childhood.

Myelodysplastic syndromes (MDS) are a heterogenous group of acquired hematopoietic stem cell disorders. Refractory cytopenia (RC) is the most common subtype of childhood MDS and hematopoietic stem cell transplantation (HSCT) is the only curative treatment. HSCT following a myeloablative preparative regimen is associated with a low probability of relapse and considerable transplant-related mortality. In the present European Working Groups of MDS pilot study, we investigated whether a reduced intensity conditioning regimen (RIC) is able to offer reduced toxicity without increased rates of graft failure or relapse. Nineteen children with RC were transplanted from an unrelated donor following RIC consisting of fludarabine, thiotepa and anti-thymocyte globulin. Three patients experienced graft failure. Neutrophil and platelet engraftment occurred at a median time of 23 and 30 days, respectively. Cumulative incidence of grade II-IV and grade III and IV acute graft-versus-host disease (GVHD) was 0.48 and 0.13, respectively; three patients developed extensive chronic GVHD. Although infections were the predominant complications, only one patient with extensive chronic GVHD died from infectious complications. Overall and event-free survival at 3 years were 0.84 and 0.74, respectively. In conclusion, our results were comparable to those of patients treated with myeloablative HSCT. Long-term follow-up is needed to demonstrate the expected reduction in long-term sequelae.