Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate.

DNA polymerase mu (Pol mu) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol mu protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol mu in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol mu to mutation of G and C residues during SHM. In vitro analysis of Pol mu misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol mu to induce transient template/primer misalignments.

Identification of biological markers of sensitivity to high-clinical-risk-adapted therapy for patients with diffuse large B-cell lymphoma.

The aim of the project was to identify biological variables in high-clinical-risk patients with diffuse large B-cell lymphoma (DLBCL), treated with risk-adapted therapies. The study was performed in a series of high-clinical-risk patients with DLBCL treated with MegaCHOP or MegaCHOP + IFE followed by autologous stem-cell transplantation (ASCT). An initial reduced set of diagnostic tumoral samples was studied by gene expression profiling and gene-set-enrichment analysis. A set of potential biomarkers extracted from this study was then explored in tissue microarrays containing paraffin-diagnostic tissue from 50 patients. The statistical analysis identified 17 immunohistochemical markers associated with the clinical endpoints. A subsequent multivariate analysis identified FoxP3+ T-reg cells as an independent predictor of failure-free survival. Bcl6 expression, CG/ABC subclasses and IPI were found not to predict survival in this series. The increased presence of regulatory T-cells as a marker of adverse outcome highlights specific components of the tumoral microenvironment in the pathogenesis and treatment response prediction for high-clinical-risk patients with DLBCL.

Array comparative genomic hybridization identifies genetic regions associated with outcome in aggressive diffuse large B-cell lymphomas.

BACKGROUND: Diffuse large B-cell lymphomas (DLBCLs) are the most common type of non-Hodgkin lymphomas. With chemotherapy and progenitor stem cell transplantation, about 60% of patients with DLBCL are long-term survivors. The International Prognostic Index identifies patients with different outcomes. However, biologic characteristics also may help to discriminate different treatments groups. METHODS: DNA copy number changes identified by array comparative genomic hybridization (array-CGH) were studied in 40 patients who had DLBCL with a poor prognosis and who were treated uniformly with dose-escalated cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) and intensification before high-dose chemotherapy with autologous stem cell transplantation. RESULTS: In total, 722 copy number changes were observed (median, 5 copy number changes per patient; range, 0-75 copy number changes per patient), with a predominance of gains. Gains on 2p16 were present only in patients who failed to achieve a complete response after escalated CHOP therapy (P < .05). In univariate analysis, gains on 2p16 and losses on 17p13 (the tumor protein p53 gene TP53 gene) were associated with a poor response to the therapy. Furthermore, age >60 years and losses on 10q23.31 (the phosphatase and tensin homolog gene PTEN) or on 17p13 were associated with short survival. In multivariate analysis, only advanced age and losses on 10q23.31 retained an adverse prognostic impact. CONCLUSIONS: Array-CGH identified multiple regions with common copy number changes, some of which were associated with outcome in patients with DLBCL.

Multifocal intrafollicular granulosa cell tumor of the ovary associated with an unusual germline p53 mutation.

A 23-year-old woman presented with a 7 cm right multicystic mass in the ovary, which corresponded microscopically to an unusual lesion consisting of a multifocal granulosa cell tumor with intrafollicular ('in situ') growth involving two-thirds of mature follicles. Stromal invasion was found in only one area where neoplastic follicles coalesced. Granulosa cells had atypical, bizarre TP53 positive nuclei with hyperchromatism, abundant mitoses and numerous hyaline globules. The contralateral ovary was normal. From the age of 10 years, the patient had a complex medical history of multiple tumors, including telangiectatic osteosarcoma, typical and malignant phyllodes tumor, reticulohistiocytoma of skin, carcinomas of the breast and lipo- and leiomyosarcoma. The female genital tract also harbored myometrial leiomyomas and an early endometrial carcinoma. Retrospective histologic study of all mesenchymal neoplasms in this patient showed, the conspicuous presence of similar bizarre TP53 positive cells with hyaline globules in all the mesenchymal neoplasms. In the genetic study, a germline p53 gene mutation was detected in exon 10, codon 336, generating a stop codon in the oligomerization domain of the protein (E336X). A further p53 mutation was found in exon 7 in the granulosa cell tumor. Mutation occurred de novo since there was no history of tumors in any family members, all of whom had a wild-type p53. Although this patient shows a typical tumor phenotype of Li Fraumeni syndrome, the germline mutation corresponded to a highly unusual mutated domain, which is similar to the one found in childhood malignant adrenocortical tumor; also a rare neoplasm that originates in adrenocortical cells; which are closely related, both functionally and embryologically, to granulosa cells.

Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays.

Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.