Lenalidomide in combination with R-ESHAP in patients with relapsed or refractory diffuse large B-cell lymphoma: A phase 2 study from GELTAMO.

Diffuse large B-cell lymphoma (DLBCL) patients with relapsed or refractory (RR) disease have poor outcomes with current salvage regimens. We conducted a phase 2 trial to analyse the safety and efficacy of adding lenalidomide to R-ESHAP (LR-ESHAP) in patients with RR DLBCL. Subjects received 3 cycles of lenalidomide 10 mg/day on days 1-14 of every 21-day cycle, in combination with R-ESHAP at standard doses. Responding patients underwent autologous stem-cell transplantation (ASCT). The primary endpoint was the overall response rate (ORR) after 3 cycles. Centralized cell-of-origin (COO) classification was performed. Forty-six patients were included. The ORR after LR-ESHAP was 67% (35% of patients achieved complete remission). Patients with primary refractory disease (n = 26) had significantly worse ORR than patients with non-refractory disease (54% vs. 85%, p = 0.031). No differences in response rates according to the COO were observed. Twenty-eight patients (61%) underwent ASCT. At a median follow-up of 41 months, the estimated 3-year PFS and OS were 42% and 48%, respectively. The most common grade ≥3 adverse events were thrombocytopenia (70% of patients), neutropenia (67%) and anaemia (35%). There were no treatment-related deaths during LR-ESHAP cycles. In conclusion, LR-ESHAP is a feasible salvage regimen with promising efficacy results for patients with RR DLBCL.

Dissection of DLBCL microenvironment provides a gene expression-based predictor of survival applicable to formalin-fixed paraffin-embedded tissue.

Background: Gene expression profiling (GEP) studies recognized a prognostic role for tumor microenvironment (TME) in diffuse large B-cell lymphoma (DLBCL), but the routinely adoption of prognostic stromal signatures remains limited. Patients and methods: Here, we applied the computational method CIBERSORT to generate a 1028-gene matrix incorporating signatures of 17 immune and stromal cytotypes. Then, we carried out a deconvolution on publicly available GEP data of 482 untreated DLBCLs to reveal associations between clinical outcomes and proportions of putative tumor-infiltrating cell types. Forty-five genes related to peculiar prognostic cytotypes were selected and their expression digitally quantified by NanoString technology on a validation set of 175 formalin-fixed, paraffin-embedded DLBCLs from two randomized trials. Data from an unsupervised clustering analysis were used to build a model of clustering assignment, whose prognostic value was also assessed on an independent cohort of 40 cases. All tissue samples consisted of pretreatment biopsies of advanced-stage DLBCLs treated by comparable R-CHOP/R-CHOP-like regimens. Results: In silico analysis demonstrated that higher proportion of myofibroblasts (MFs), dendritic cells, and CD4+ T cells correlated with better outcomes and the expression of genes in our panel is associated with a risk of overall and progression-free survival. In a multivariate Cox model, the microenvironment genes retained high prognostic performance independently of the cell-of-origin (COO), and integration of the two prognosticators (COO + TME) improved survival prediction in both validation set and independent cohort. Moreover, the major contribution of MF-related genes to the panel and Gene Set Enrichment Analysis suggested a strong influence of extracellular matrix determinants in DLBCL biology. Conclusions: Our study identified new prognostic categories of DLBCL, providing an easy-to-apply gene panel that powerfully predicts patients' survival. Moreover, owing to its relationship with specific stromal and immune components, the panel may acquire a predictive relevance in clinical trials exploring new drugs with known impact on TME.

Lithocholic acid downregulates vitamin D effects in human osteoblasts.

BACKGROUND: Osteoporosis is a common complication in chronic cholestasis. It has been proposed that retained substances such as bile acids may produce a damaging effect on bone cells. This study analyses the effects of lithocholic acid (LCA) on cell survival and vitamin D metabolism in human osteoblasts (hOB). MATERIALS AND METHODS: Human osteoblasts cultures were performed with or without foetal bovine serum (FBS) or human albumin (HA) at different LCA concentrations and times with or without vitamin D. RESULTS: Lithocholic acid at concentrations higher than 10(-5 )M decreased cell survival. This effect was partially prevented by the presence of FBS or HA. Vitamin D stimulated CYP24A, BGLAP and TNFSF11 expression in hOB and these effects were modified by nontoxic LCA concentrations. LCA significantly decreased vitamin D stimulation of CYP24A, BGLAP and TNFSF11 gene expression at 72%, 79% and 56% (respectively). LCA alone has an agonistic effect, as has vitamin D, thus partially increasing CYP24A and BGLAP expression, but with no changes on TNFRSF11B expression. Equivalent effects of the LCA were observed by performing gene reporter assays using MG-63 cells transfected with constructs containing CYP24A1 promoter regions. CONCLUSIONS: Lithocholic acid decreases the stimulatory effect of vitamin D on CYP24A, BGLAP and TNFSF11 expression in hOB. This effect is produced through vitamin D response elements (VDREs), located in the promoter regions of these genes, suggesting that LCA acts as a mild analogous of vitamin D, interacting with the vitamin D receptor. These results may explain the potential deleterious effects of retained bile acids on hOB.

Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Integrating genomic alterations in diffuse large B-cell lymphoma identifies new relevant pathways and potential therapeutic targets.

Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

Effect of smoking and smoking cessation on bone mass, bone remodeling, vitamin D, PTH and sex hormones.

OBJECTIVE: To assess the effect of smoking and smoking cessation on bone density, bone remodeling markers, sex hormones, and vitamin D-PTH axis in healthy young subjects. MATERIALS AND METHODS: We studied 74 healthy people (31 men, 43 women; mean age 32.2 (7) years) divided into 52 never smokers and 22 smokers, 15 of which stopped smoking for one month. RESULTS: Male smokers compared with never smokers showed lower BMD (0.971 (0.11) g/cm(2) vs. 1.069 (0.09) g/cm(2), P=0.042); higher plasma estrone levels (32.37 (10.13) pg/mL vs. 20.91 (5.46) pg/mL, P=0.001); and lower serum iPTH levels (16.2 (3.5) pg/mL vs. 28.8 (2.0) pg/mL, P=0.008). In women, BMD values were similar in smokers than in never smokers, but 25-hydroxyvitamin D levels were lower in smokers (31.9 (15.1) ng/mL vs. 16.8 (9.9) ng/mL, P=0.002). After adjusting by age and coffee consumption, female smokers had higher urinary-NTX levels than never smokers. After smoking cessation, statistically significant decreases of 25-hydroxyvitamin D and SHBG plasma levels were observed in men and women, respectively. CONCLUSIONS: Tobacco increases bone resorption and affects bone mass by some alterations in sex hormone metabolism, but also importantly by alterations on the vitamin D-PTH axis.

COL1A1, ESR1, VDR and TGFB1 polymorphisms and haplotypes in relation to BMD in Spanish postmenopausal women.

INTRODUCTION AND HYPOTHESIS: Genetic studies of osteoporosis have focused on analysing single polymorphisms in individual genes - with inconclusive results. An alternative approach may involve haplotypes and gene-gene interactions. The aim of the study was to test the association between the COL1A1, ESR1, VDR and TGFB1 polymorphisms or haplotypes and bone mineral density (BMD) in Spanish postmenopausal women. METHODS: Sixteen polymorphisms were analysed in 719 postmenopausal women. ANOVA, ANCOVA and Xi2 tests were used to perform the statistical analysis. RESULTS: COL1A1 -1997G > T (p=0.04) and TGFB1 Leu10Pro (p=0.02) were found to be associated with adjusted lumbar spine (LS) BMD. Interactions were observed between: the COL1A1 -1997 G/T and Sp1 polymorphisms (p < 0.01 for LS BMD) and the COL1A1 -1663 indelT and VDR ApaI polymorphisms (p < 0.01 for femoral neck (FN) BMD). The COL1A1 GDs and ESR1 LPX haplotypes were associated with FN BMD (p=0.03 and p=0.03). CONCLUSIONS: Polymorphisms at COL1A1 and TGFB1 and haplotypes at COL1A1 and ESR1 were found to be associated with BMD in a cohort of postmenopausal Spanish women. Moreover, COL1A1 polymorphisms showed significant interactions among them and with the VDR 3' polymorphisms.

Inactive and temperature-sensitive folding mutants of aldehyde dehydrogenase of Escherichia coli.

Aldehyde dehydrogenase, encoded by the aldA gene in Escherichia coli, is inactive in nitrosoguanidine induced mutant strain ECL40 and temperature-sensitive in spontaneous mutant strain JA104. Both mutants were proven, by complementation experiments, to have a functional aldA regulator and promoter. In spite of no immunodetection of the aldA product, its specific transcript was present in the mutant extracts. It was subsequently proven that the immunodetection of aldehyde dehydrogenase in these mutants required denaturation, revealing that cells lacking the enzyme activity had the inactive protein in their extracts. Thus, the mutations seemed to affect the protein conformation. The temperature-sensitive aldehyde dehydrogenase did not show, neither in vivo nor in vitro, a different thermal stability compared to the wild type enzyme. In this temperature-sensitive strain, the recovery of active aldehyde dehydrogenase, in the presence of rifampicin but not of chloramphenicol, when cells grown at 37 degrees C were shifted to 30 degrees C indicated that this mutation affected the folding process of the protein at the restrictive temperature. Sequencing of the two mutant aldA corresponding genes determined a single amino acid change of Pro to Leu at position 182 for strain ECL40, and of Val to Met at position 145 for strain JA104. These mutations were thought to possibly promote changes in the local flexibility in the first case, and to perturb the packing of residues by steric hindrance in the second case.

[Prevalence of tobacco dependence in 6 counties of Lleida]. / Prevalencia de tabaquismo en 6 comarcas de Lleida.

OBJECTIVE: To find the prevalence of tobacco dependency in the population of six counties in Lleida and in patients with chronic diseases or vascular risk factors. DESIGN: Cross-sectional study. A descriptive analysis of the data. SETTING: Six counties in Lleida: 5 Pyrenean counties with a rural population and the Segrià county with an urban population. PARTICIPANTS: A personal questionnaire covering 900 people over 15 (95% CI, 4% accuracy), chosen by randomised sampling, stratified for urban or rural, from the Municipal Rolls. MEASUREMENTS AND MAIN RESULTS: 543 individuals took part. Prevalence of tobacco dependency was 19.7%, higher in men (27.7%) than in women (12%) (p < 0.05). However, among the young there were no difference between the sexes. CONCLUSIONS: We found a low, in comparison with other studies, percentage of smokers in our sample, with a high prevalence of ex-smokers among patients with ictus or Coronary Disease. However, the high prevalence of smokers among the individuals with some cardiovascular risk factor points to the need to redouble efforts in health education on tobacco.

Efectos del tabaquismo sobre los niveles plasmáticos de osteoprotegerina en adultos jóvenes sanos / No disponible

El tabaco es un conocido factor de riesgo de osteoporosis, pero los mecanismos por los que se produce esta asociación siguen siendo objeto de debate. Estudiosprevios han evaluado las variaciones de osteoprotegerina plasmática, una citoquina que regula la osteoclastogénesis y la resorción ósea, en fumadores, conresultados dispares. En este estudio se analizaron los niveles de osteoprotegerina plasmática en un grupo de 49 voluntarios jóvenes sanos, pero no existierondiferencias entre fumadores y no fumadores. Tampoco se encontraron diferencias tras ajustar por las diferencias basales existentes en estrona en varones y enedad e ingesta de calcio en mujeres. En conclusión, fumar no parece influir en los niveles plasmáticos de osteoprotegerina en voluntarios jóvenes sanos

While tobacco smoke is a known risk factor for osteoporosis, the mechanisms that produce this association must still elucidated. Several previous studies have evaluated the variations in serum levels of osteoprotegerin, a cytokine that regulatesosteoclastogenesis and bone resorption in smokers, however, the results have been inconclusive. This study, which makes an analysis of serum levels ofosteoprotegerin in a group of 49 healthy young volunteers, did not find any influence of smoking on this parameter. There were also no differences followingadjustment for baseline differences in estrone in men and for age and calcium intake in women. In conclusion, tobacco smoking does not seem to influenceosteoprotegerin serum levels in healthy young volunteers