Lamin B1 regulates somatic mutations and progression of B-cell malignancies.

Somatic hypermutation (SHM) is a pivotal process in adaptive immunity that occurs in the germinal centre and allows B cells to change their primary DNA sequence and diversify their antigen receptors. Here, we report that genome binding of Lamin B1, a component of the nuclear envelope involved in epigenetic chromatin regulation, is reduced during B-cell activation and formation of lymphoid germinal centres. Chromatin immunoprecipitation-Seq analysis showed that kappa and heavy variable immunoglobulin domains were released from the Lamin B1 suppressive environment when SHM was induced in B cells. RNA interference-mediated reduction of Lamin B1 resulted in spontaneous SHM as well as kappa-light chain aberrant surface expression. Finally, Lamin B1 expression level correlated with progression-free and overall survival in chronic lymphocytic leukaemia, and was strongly involved in the transformation of follicular lymphoma. In summary, here we report that Lamin B1 is a negative epigenetic regulator of SHM in normal B-cells and a 'mutational gatekeeper', suppressing the aberrant mutations that drive lymphoid malignancy.

Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.

Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and are disrupted at the genomic level in a spectrum of human tumours including haematological malignancies. Using high-resolution single nucleotide polymorphism (SNP) arrays, we identified recurrent deletions of the SETD2 locus in 3% (8/261) of chronic lymphocytic leukaemia (CLL) patients. Further validation in two independent cohorts showed that SETD2 deletions were associated with loss of TP53, genomic complexity and chromothripsis. With next-generation sequencing we detected mutations of SETD2 in an additional 3.8% of patients (23/602). In most cases, SETD2 deletions or mutations were often observed as a clonal event and always as a mono-allelic lesion, leading to reduced mRNA expression in SETD2-disrupted cases. Patients with SETD2 abnormalities and wild-type TP53 and ATM from five clinical trials employing chemotherapy or chemo-immunotherapy had reduced progression-free and overall survival compared with cases wild type for all three genes. Consistent with its postulated role as a tumour suppressor, our data highlight SETD2 aberration as a recurrent, early loss-of-function event in CLL pathobiology linked to aggressive disease.

Monitoring of WT1 expression in PB and CD34(+) donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning.

Relapse of malignant disease remains the major complication in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) with reduced-intensity conditioning (RIC). In this study, we investigated the predictive value of disease-specific markers (DSMs), donor chimerism (DC) analysis of unsorted (UDC) or CD34(+) sorted cells and Wilms' tumor gene 1 (WT1) expression. Eighty-eight patients with AML or MDS were monitored after allogenic HCT following 2 Gy total-body irradiation with (n=84) or without (n=4) fludarabine 3 × 30 mg/m(2), followed by cyclosporin A and mycophenolate mofetil. DSMs were determined by fluorescence in situ hybridization (FISH) and WT1 expression by real-time polymerase chain reaction. Chimerism analysis was performed on unsorted or CD34(+) sorted cells, by FISH or short tandem repeat polymerase chain reaction. Twenty-one (24%) patients relapsed within 4 months after HCT. UDC, CD34(+) DC and WT1 expression were each significant predictors of relapse with sensitivities ranging from 53 to 79% and specificities of 82-91%. Relapse within 28 days was excluded almost entirely on the basis of WT1 expression combined with CD34(+) DC kinetics. Monitoring of WT1 expression and CD34(+) DC predict relapse of AML and MDS after RIC-HCT.

Bone marrow fibroblasts induce expression of PI3K/NF-kappaB pathway genes and a pro-angiogenic phenotype in CLL cells.

Microarray-based gene expression profiling (GEP) was used to study how stroma modulates the survival of CLL cells in an in vitro coculture model employing the murine fibroblast cell line M2-10B4. CLL cells cultured in direct contact with the stromal layer (STR) showed a significantly better survival than cells cultured in transwell (TW) inserts above the M2-10B4 cells. STR as compared to TW conditions induced a significant up-regulation of PI3K/NF-kappaB pro-survival pathway genes and mediated a pro-angiogenetic switch in the CLL cells by up-regulation of vascular endothelial growth factor (VEGF) and osteopontin (OPN) and down-regulation of the anti-angiogenetic molecule thrombospondin-1 (TSP-1).

Gene expression signatures separate B-cell chronic lymphocytic leukaemia prognostic subgroups defined by ZAP-70 and CD38 expression status.

B-cell chronic lymphocytic leukaemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of zeta-associated protein 70 (ZAP-70) and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70-CD38-) and poor (ZAP-70+CD38+) prognosis. DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70-CD38- B-CLL cases. The expression of 358 genes differed significantly between the two subgroups, including genes involved in B-cell receptor signaling, angiogenesis and lymphomagenesis. Three of these genes, that is, immune receptor translocation-associated protein 4 (IRTA4)/Fc receptor homologue 2 (FcRH2), angiopoietin 2 (ANGPT2) and Pim2 were selected for further validating studies in a cohort of 94 B-CLL patients. IRTA4/FcRH2 expression as detected by flow cytometry was significantly lower in the poor prognosis subgroup as compared to ZAP-70-CD38- B-CLL cells. In healthy individuals, IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. ANGPT2 plasma concentrations were twofold higher in the poor prognosis subgroup (P<0.05). Pim2 was significantly overexpressed in poor prognosis cases and Binet stage C. Disease progression may be related to proangiogenic processes and strong Pim2 expression.

X chromosomal and autosomal loss of heterozygosity and microsatellite instability in human cervical carcinoma.

The study analyzes tumor material and normal tissue from 27 patients with pure squamous cell carcinoma of the uterine cervix for loss of heterozygosity (LOH) and microsatellite instability (MSI) on 14 autosomal and 11 X chromosomal loci. Overall, 4-40% of the informative cases showed LOH at autosomal regions with the highest frequency at 3p (21-40%) and a marked frequency at 2q35-q37.1 (12.5%) and 17p13.3 (10%), representing regions with putative tumor suppressor gene (TSG) function. The frequency of X chromosomal LOH ranged from 4% to 20%, with a maximum at Xq28 (20%) and Xq11.2-q12 (17%), again indicating alterations in TSG. A 12% LOH was seen at Xq21.33-q22.3, a region encoding a protein with a regulatory function in the cell cycle via cyclin-dependent kinases. MSI was detected in autosomal regions in up to 7% in regions linked to the X chromosome in up to 11%, probably indicating alterations of mismatch repair mechanisms. Our results and those obtained from the literature suggest that autosomal LOH and MSI in carcinomas of the cervix uteri are predominantly found at regions with putative TSG function. Beside TSG alterations, X chromosomal LOH is probably more strongly connected to disturbances in cell cycle regulation.

Graves' disease and Hashimoto's thyroiditis in monozygotic twins: case study as well as transcriptomic and immunohistological analysis of thyroid tissues.

OBJECTIVE: To report on the rare simultaneous occurrence of Graves' disease (GD) and Hashimoto's thyroiditis (HT) in monozygotic twins. DESIGN: We compared the pattern of thyroid tissue-derived cDNAs to gain insight into previous and ongoing immune destruction and reconstruction processes using microarrays. The results were confirmed by immunohistology and real-time PCR. RESULTS: Destruction of thyroid tissue in HT reduced levels of thyrocyte-related cDNAs and cDNAs encoding extracellular matrix components, but increased levels of proteases involved in extracellular matrix degradation compared with GD. Lymphocytic infiltrates forming ectopic follicles replaced the thyroid tissue almost completely in HT. Thus, lymphocyte-related cDNA levels were higher in HT than in GD. The same was true for many chemokines and their receptors, which not only enable migration towards the thyroid but also maintain the lymphocytic infiltrate. HT also showed increased levels of cDNAs encoding molecules related to apoptosis than did GD. Surprisingly, the Th1- and Th2-specific cytokine profiles suggested for HT and GD respectively could not be confirmed. cDNAs encoding factors and receptors involved in angiogenesis were increased in GD compared with HT. CONCLUSIONS: Comparison of gene expression reflects the cellular differences between the two types of autoimmune thyroid disease in twins with identical genetic and similar environmental background.

Identity testing in cervical carcinoma in case of suspected mix-up.

The histopathologic diagnosis is the cornerstone of modern oncology. But mix-ups of specimens can occur at any stage. The resection of a 1.2 cm polypoid cervical mass in a 25-year-old woman showed a poorly differentiated adenocarcinoma prospectively staged as T1b1 (International Federation of Gynecology and Obstetrics IB1). Even after complete embedding and serial sectioning of the whole cervix of the hysterectomy specimen after radical hysterectomy, only adenocarcinoma in situ, but no invasive tumor, was seen. To exclude a mix-up of the specimens, identity testing of the paraffin-embedded material was performed by microsatellite analysis. For both materials, we established identical results after testing the microsatellite loci HumTH01, HumVWA, HumFGA, HumACTBP2, HumF13B, and HumD8S1132. The resulting probability of identity came to 99.9999%, excluding a mix-up of the specimens. Archival paraffin-embedded specimens can be used to establish identity and can prevent the wrong patient from having major surgery.

Second unrelated bone marrow transplantation without additional conditioning therapy after engraftment failure.

A 37-year-old female highly alloimmunized by multiple transfusions received a sex matched HLA-identical unrelated bone marrow transplant for hypoplastic MDS-RA with moderate myelofibrosis. Conditioning consisted of total body irradiation, cyclophosphamide and ATG, GVHD prophylaxis consisted of CsA, MTX and prednisolone. The CD34+ stem cell content of the first graft was relatively low due to an inadequate harvest. The patient appeared not to have engrafted by day 23 post-BMT. She therefore received a second sex mismatched HLA-identical unrelated bone marrow graft on day 25 after two days of 3.5 mg/kg methylprednisolone from a different donor. Over the ensuing days, the first marrow showed slow engraftment followed by engraftment of the second graft. The first graft was then rejected, as monitored by peripheral blood studies of chimerism. No signs of acute GVHD were observed. Despite successful trilineage engraftment and complete second donor chimerism, the patient died from disseminated toxoplasmosis encephalitis and pneumonia on day +104.

[Importance of secretory IgA in human body fluids. I. Development of an enzyme immunoassay for secretory IgA]. / Zur Bedeutung von sekretorischem IgA in menschlichen Körperflüssigkeiten. I. Aufbau eines Enzymimmunassays für sekretorisches IgA.

This paper describes an enzyme-immunoassay for the quantitative estimation of secretory IgA(S-IgA). As solid phase a monoclonal antibody against human secretory component was used, a polyclonal peroxidase labeled anti-IgA serum as conjugate. Thus, the method was specific for S-IgA. The test was done in microtiter plates, the upper limit was 0.5 ng S-IgA/ml. The standards of our test system ran in normal ranges, the coefficients of variation were 6.9% in the within-day-test and 12.2% in the day-to-day-test. The described test system is effective and sensitive. It is suitable for clinical use.