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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.

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.

The CDKN2A gene located on chromosome region 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16/INK4A, a negative cell cycle regulator. We analyzed p16/INK4A expression in different types of non-Hodgkin's lymphoma to determine whether the absence of this protein is involved in lymphomagenesis, while also trying to characterize the genetic events underlying this p16/INK4A loss. To this end, we investigated the levels of p16/INK4A protein using immunohistochemical techniques in 153 cases of non-Hodgkin's lymphoma, using as reference the levels found in reactive lymphoid tissue. The existence of gene mutation, CpG island methylation, and allelic loss were investigated in a subset of 26 cases, using single-strand conformational polymorphism and direct sequencing, Southern Blot, polymerase chain reaction, and microsatellite analysis, respectively. Loss of p16/INK4A expression was detected in 41 of the 112 non-Hodgkin's lymphomas studied (37%), all of which corresponded to high-grade tumors. This loss of p16/INK4A was found more frequently in cases showing tumor progression from mucosa-associated lymphoid tissue low-grade lymphomas (31 of 37) or follicular lymphomas (4 of 4) into diffuse large B-cell lymphomas. Analysis of the status of the p16/INK4A gene showed different genetic alterations (methylation of the 5'-CpG island of the p16/INK4A gene, 6 of 23 cases; allelic loss at 9p21, 3 of 16 cases; and nonsense mutation, 1 of 26 cases). In all cases, these events were associated with loss of the p16/INK4A protein. No case that preserved protein expression contained any genetic change. Our results demonstrate that p16/INK4A loss of expression contributes to tumor progression in lymphomas. The most frequent genetic alterations found were 5'-CpG island methylation and allelic loss.

Purpose: The goal of this work was to perform a comprehensive exploration of the relationship between the clinical outcome of diffuse large B-cell lymphoma (DLBCL) and the expression of a panel of tumor suppressor and oncogenic proteins, which includes some cell-cycle regulator proteins involved in the p53 pathway., Patients and Methods: To this end, we collected the clinical data of 141 patients with DLBCL and immunohistochemically analyzed diagnostic tumoral tissue from each patient for the presence of Ki67 (MIB1, Immuno-tech, Marseille, France), bcl2, p53, p21/WAF1, MDM2, and retinoblastoma (Rb) proteins., Results: The results show that several proteins are associated with some of the clinical traits analyzed. Multivariate analysis showed that an extended overall survival (OS) time was associated with low growth fraction, high Rb protein, and low MDM2 expression, as well as with known clinical parameters. The probability of inducing a complete remission (CR) was only associated with clinical parameters, although univariate study showed that a low growth fraction was associated with a higher probability of inducing a CR. Univariate study of disease-free survival (DFS) showed that tumors with high bcl2 expression and nodal origin have a shorter DFS time, although multivariate study only confirmed the adverse effect of bcl2 expression., Conclusion: Taking all these results into consideration, it seems that although the overall outcome for patients with DLBCL is decided by a combination of different clinical and biologic variables, the expression of some of these cell-cycle regulator proteins appears to be specifically associated with the different clinical features of tumors.

Aims: To determine the expression of p21WAF1/CIP1 in relation to the expression of Ki67 and p53 in various normal adult and fetal tissues, and to investigate its distribution throughout the cell cycle., Methods: The expression of p21WAF1/CIP1 in relation to Ki67 and p53 was analysed in adult and fetal tissues using immunohistochemical techniques. Heat induced epitope retrieval techniques were used to characterise the presence of p21WAF1/CIP1 in different tissues, as well as to detect its distribution throughout the cell cycle. In addition, flow cytometry and western blotting were used to test whether the level of p21WAF1/CIP1 expression varied at different phases of the cell cycle in phytohaemagglutinin (PHA) stimulated lymphocytes., Results: p21WAF1/CIP1 expression varied from one tissue to another, and it was restricted mainly to the squamous and glandular epithelium, where it appeared in association with p53. Human tissues in which p21WAF1/CIP1 was found showed a mutually exclusive topographical sequential expression between p21WAF1/CIP1 and Ki67. This was confirmed by double labelling studies, which showed that p21WAF1/CIP1 positive cells were in the G0 phase. Unlike these findings of a decline in p21WAF1/CIP1 expression after the G0 phase, PHA stimulated lymphocytes showed a level of p21WAF1/CIP1 expression that rose as the cell progressed through the cell cycle., Conclusions: The analysis of p21WAF1/CIP1 expression in relation to the status of p53 should take into account the existence of variable p21WAF1/CIP1 expression in different tissues. This could provide an explanation for the varying frequency of p53 mutations in tumours of different cellular origin. In tissues characterised by regular p21WAF1/CIP1 expression, it appears in a pattern that is consistent with the proposed role of this inhibitor of cyclin dependent kinases in cell cycle arrest-that of inducing cell differentiation. The conflicting results of in vivo and in vitro studies could support the hypothesis that microenvironmental conditions may influence the location of p21WAF1/CIP1 in different phases of the cell cycle.

Widespread cutaneous necrosis (WCN) associated with antiphospholipid antibodies is rare. Its mechanisms have yet to be elucidated, and there are no well-established guidelines for its management. We describe a woman who had two episodes of WCN related to surgical manipulation for urinary tract obstruction and urinary tract infection. Lupus anticoagulant was always positive. In the second episode anticardiolipin antibodies were elevated, and protein C levels were temporarily decreased. We found only ten previously reported cases of WCN associated with antiphospholipid antibodies, none of which were related to surgical manipulation.