Antonio Torres

Epigenetic Silencing of the Tumor Suppressor MicroRNA Hsa-miR-124a Regulates CDK6 Expression and Confers a Poor Prognosis in Acute Lymphoblastic Leukemia

Whereas transcriptional silencing of genes due to epigenetic mechanisms is one of the most important alterations in acute lymphoblastic leukemia (ALL), some recent studies indicate that DNA methylation contributes to down-regulation of miRNAs during tumorigenesis. To explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL. Expression of miR-124a was down-regulated in ALL by hypermethylation of the promoter and histone modifications including decreased levels of 3mk4H3 and AcH3 and increased levels of 2mK9H3, 3mK9H3, and 3mK27H3. Epigenetic down-regulation of miR-124a induced an up-regulation of its target, CDK6, and phosphorylation of retinoblastoma (Rb) and contributed to the abnormal proliferation of ALL cells both in vitro and in vivo. Cyclin-dependent kinase 6 (CDK6) inhibition by sodium butyrate or PD-0332991 decreased ALL cell growth in vitro, whereas overexpression of pre-miR124a led to decreased tumorigenicity in a xenogeneic in vivo Rag2(-/-)gammac(-/-) mouse model. The clinical implications of these findings were analyzed in a group of 353 patients diagnosed with ALL. Methylation of hsa-miR-124a was observed in 59% of the patients, which correlated with down-regulation of miR-124a (P < 0.001). Furthermore, hypermethylation of hsa-miR-124a was associated with higher relapse rate (P = 0.001) and mortality rate (P < 0.001), being an independent prognostic factor for disease-free survival (P < 0.001) and overall survival (P = 0.005) in the multivariate analysis. These results provide the grounds for new therapeutic strategies in ALL either targeting the epigenetic regulation of microRNAs and/or directly targeting the CDK6-Rb pathway.

Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia

Aberrant genome-wide hypomethylation is thought to be related to tumorigenesis by promoting genomic instability. Since DNA methylation is considered an important mechanism for the silencing of retroelements, hypomethylation in human tumors may lead to their reactivation. However, the role of DNA hypomethylation in chronic myeloid leukemia (CML) remains to be elucidated. In this study, the methylation status of the LINE-1 (L1) retrotransposon promoter was analysed in CML samples from the chronic-phase (CP, n=140) and the blast crisis (BC, n=47). L1 hypomethylation was significantly more frequent in BC (74.5%) than in CP (38%) (P<0.0001). Furthermore, L1 hypomethylation led to activation of both ORF1 sense transcription (P<0.0001) and c-MET gene antisense transcription (P<0.0001), and was significantly associated with high levels of BCR–ABL (P=0.02) and DNMT3b4 (P=0.001) transcripts. Interestingly, in CP-CML, extensive L1 hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (P=0.004) or imatinib (P=0.034) and progression-free survival (P=0.005). The above results strongly suggest that activation of both sense and antisense transcriptions by aberrant promoter hypomethylation of the L1 elements plays a role in the progression and clinical behavior of the CML.

Preoperative Chemotherapy Plus Surgery Versus Surgery Plus Adjuvant Chemotherapy Versus Surgery Alone in Early-Stage Non–Small-Cell Lung Cancer

PURPOSE To address whether preoperative chemotherapy plus surgery or surgery plus adjuvant chemotherapy prolongs disease-free survival compared with surgery alone among patients with resectable non-small-cell lung cancer. PATIENTS AND METHODS In this phase III trial, 624 patients with stage IA (tumor size > 2 cm), IB, II, or T3N1 were randomly assigned to surgery alone (212 patients), three cycles of preoperative paclitaxel-carboplatin followed by surgery (201 patients), or surgery followed by three cycles of adjuvant paclitaxel-carboplatin (211 patients). The primary end point was disease-free survival. RESULTS In the preoperative arm, 97% of patients started the planned chemotherapy, and radiologic response rate was 53.3%. In the adjuvant arm, 66.2% started the planned chemotherapy. Ninety-four percent of patients underwent surgery; surgical procedures and postoperative mortality were similar across the three arms. Patients in the preoperative arm had a nonsignificant trend toward longer disease-free survival than those assigned to surgery alone (5-year disease-free survival 38.3% v 34.1%; hazard ratio [HR] for progression or death, 0.92; P = .176). Five-year disease-free survival rates were 36.6% in the adjuvant arm versus 34.1% in the surgery arm (HR 0.96; P = .74). CONCLUSION In early-stage patients, no statistically significant differences in disease-free survival were found with the addition of preoperative or adjuvant chemotherapy to surgery. In this trial, in which the treatment decision was made before surgery, more patients were able to receive preoperative than adjuvant treatment.

Lack of CpG Island Methylator Phenotype Defines a Clinical Subtype of T-Cell Acute Lymphoblastic Leukemia Associated With Good Prognosis

PURPOSE To examine cancer genes undergoing epigenetic inactivation in a set of T-cell acute lymphoblastic leukemias (T-ALLs) to obtain the CpG island methylator phenotype (CIMP) in the disease and its possible correlation with clinical features and outcome of the patients. PATIENTS AND METHODS Methylation-specific polymerase chain reaction was used to analyze methylation of the ADAMTS-1, ADAMTS-5, APAF-1, ASPP-1, CDH1, CDH13, DAPK, DIABLO, DKK-3, LATS-1, LATS-2, NES-1, p14, p15, p16, p57, p73, PARK-2, PTEN, sFRP1/2/4/5, SHP-1, SYK, TMS-1, and WIF-1 genes in samples from 50 consecutive T-ALL patients (19 children and 31 adults). Results were compared with results obtained in 286 B-cell acute lymphoblastic leukemias (B-ALLs). RESULTS A total of 88% of the T-ALL samples had at least one gene methylated. According to the number of methylated genes observed in each individual sample, 12 patients (24%) were included in the CIMP- group (zero to two methylated genes), and 38 patients (76%) were included in the CIMP+ group (> two methylated genes). Clinical features and remission rate did not differ significantly among both groups of patients. Estimated disease-free survival (DFS) rate at 12 years and overall survival (OS) rate at 13 years were 100% and 91% for the CIMP- group and 20% and 17% for the CIMP+ group, respectively (P = .0006 and P = .003, respectively). Multivariate analysis demonstrated that methylation profile was an independent prognostic factor in predicting DFS (P = .05) and OS (P = .02). A group of five genes (SYK-1, ASPP-1, sFRP-2, sFRP-5, and WIF-1) showed specificity for T-ALL compared with B-ALL. CONCLUSION Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in T-ALL.

Prospective Comparison of Severity Scores for Predicting Clinically Relevant Outcomes for Patients Hospitalized With Community-Acquired Pneumonia

BACKGROUND The comparative accuracy and discriminatory power of three validated rules for predicting clinically relevant outcomes other than mortality in patients hospitalized with community-acquired pneumonia (CAP) are unknown. METHODS We prospectively compared the newly developed severe community-acquired pneumonia (SCAP) score, pneumonia severity index (PSI), and the British Thoracic Society confusion, urea > 7 mmol/L, respiratory rate > or = 30 breaths/min, BP < 90 mm Hg systolic or < 60 mm Hg diastolic, age > or = 65 years (CURB-65) rule in an internal validation cohort of 1,189 consecutive adult inpatients with CAP from one hospital and an external validation cohort of 671 consecutive adult inpatients from three other hospitals. Major adverse outcomes were admission to ICU, need for mechanical ventilation, progression to severe sepsis, or treatment failure. Mean hospital length of stay (LOS) was also evaluated. The rules were compared based on sensitivity, specificity, and area under the curve (AUC) of the receiver operating characteristic. RESULTS The rate of all adverse outcomes and hospital LOS increased directly with increasing SCAP, PSI, or CURB-65 scores (p < 0.001) in both cohorts. Patients classified as high risk by the SCAP score showed higher rates of adverse outcomes (ICU admission, 35.8%; mechanical ventilation, 16.4%; severe sepsis, 98.5%; treatment failure, 22.4%) than PSI and CURB-65 high-risk classes. The discriminatory power of SCAP, as measured by AUC, was 0.75 for ICU admission, 0.76 for mechanical ventilation, 0.79 for severe sepsis, and 0.61 for treatment failure in the external validation cohort. AUC differences with PSI or CURB-65 were found. CONCLUSIONS The SCAP score is as accurate or better than other current scoring systems in predicting adverse outcomes in patients hospitalized with CAP while helping classify patients into different categories of increasing risk for potentially closer monitoring.

Cadherin-13, a Mediator of Calcium-Dependent Cell-Cell Adhesion, Is Silenced by Methylation in Chronic Myeloid Leukemia and Correlates With Pretreatment Risk Profile and Cytogenetic Response to Interferon Alfa

PURPOSE Cadherin-13 (CDH13) is a newly characterized cadherin molecule responsible for selective cell recognition and adhesion, the expression of which is decreased by methylation in a variety of human cancers, indicating that the CDH13 gene functions as a tumor suppressor gene. Although defective progenitor-stromal adhesion is a well-recognized feature of chronic myeloid leukemia (CML), the role of CDH13 abnormalities has not been evaluated in this disease. PATIENTS AND METHODS We examined the methylation status of the CDH13 promoter in 179 chronic phase (CP)-CML patients and in 52 advanced-phase samples and correlated it with mRNA expression using methylation-specific polymerase chain reaction (PCR) and reverse transcriptase PCR. RESULTS Aberrant de novo methylation of the CDH13 promoter region was observed in 99 (55%) of 179 of CP-CML patients, and 90 of the patients failed to express CDH13 mRNA (P <.0001). Advanced-stage samples (n = 52) showed concordant methylation results with their corresponding CP tumors, indicating that CDH13 methylation was not acquired during the course of the disease. Nevertheless, absence of CDH13 expression was more frequently observed among Sokal high-risk patients (P =.01) and was also independently associated with a shorter median progression-free survival time (P =.03) and poor cytogenetic response to interferon alfa treatment (P =.0001). CONCLUSION Our data indicate that the silencing of CDH13 expression by aberrant promoter methylation occurs at an early stage in CML pathogenesis and probably influences the clinical behavior of the disease.

Downregulation of the large tumor suppressor 2 (LATS2/KPM) gene is associated with poor prognosis in acute lymphoblastic leukemia

Downregulation of the large tumor suppressor 2 (LATS2/KPM) gene is associated with poor prognosis in acute lymphoblastic leukemia

Clinical value of immunological monitoring of minimal residual disease in acute lymphoblastic leukaemia after allogeneic transplantation

Summary. In this study, we used multiparameter flow cytometry to quantify minimal residual disease (MRD) in 165 serial bone marrow samples from 40 patients diagnosed with acute lymphoblastic leukaemia (ALL) who underwent allogeneic stem cell transplantation (allo‐SCT) from siblings (n = 34) or unrelated donors (n = 6). Samples were prospectively taken from 24 patients before starting the conditioning regimen, at days +30, +60 and +90 and subsequently every 2–3 months. Samples from 16 patients in complete remission (CR) after allo‐SCT were taken at least twice. Six of 24 patients harboured MRD (0·2–10% of mononuclear cells) at transplant and 18 were negative. Estimated disease‐free survival for the MRD+ and MRD– groups at transplant was 33·3% and 73·5% respectively (P = 0·03). During follow‐up, increasing MRD levels were detected in nine patients, a finding that preceded marrow relapse by 1–6 months. Two patients with stable low MRD levels remained in CR. When we used flow cytometry to test the effect of donor leucocyte infusions (DLI) in six patients, we observed that the only sustained remission was achieved when DLI was applied prior to overt relapse. We conclude that MRD by flow cytometry can rapidly assess tumoral burden before transplant to predict outcome, and can be clinically useful for the timing of DLI for increasing levels of leukaemia after transplant.

Kinetic of regulatory CD25high and activated CD134+ (OX40) T lymphocytes during acute and chronic graft-versus-host disease after allogeneic bone marrow transplantation

Graft‐versus‐host disease (GVHD) is still a major complication after allogeneic stem cell transplantation. In murine models, freshly isolated or ex vivo expanded CD4+CD25high regulatory T cells (Treg) are able to ameliorate GVHD while maintaining graft‐versus‐leukaemia reactions. However, in the human setting, prospective studies of this population and its interaction with activated non‐regulatory CD134+ (OX40) lymphocytes during post‐transplant follow‐up are lacking. In this study, we prospectively quantified CD4+CD25high and activated CD134+ lymphocytes in 119 peripheral blood samples from 35 consecutive patients who underwent allogeneic bone marrow transplantation (BMT). Fifty‐five samples obtained less than 100 d after allogeneic BMT, were not statistically different regarding CD4+CD25high Treg or CD134+ lymphocytes compared with those obtained from patients with (n = 35) or without (n = 20) acute GVHD. Chronic GVHD was associated with a small, but not statistically significant, increase in the number of Treg (9·9 vs. 6·7 × 106/L). However, the CD134/CD25high ratio was significantly higher during chronic GVHD (cGHVD) when compared with either patients without cGVHD (67·7 ± 40·3 vs. 4·0 ± 0·9, P < 0·01) or cGVHD after treatment (67·7 ± 40·3 vs. 3·7 ± 0·8, P < 0·01). Our findings suggest that the suppressive activity of CD4+CD25high Treg could be abrogated in vivo during cGVHD by CD134 expression in a much higher number of activated donor T lymphocytes. In addition to CD4+CD25highex vivo expansion protocols, OX40 blocking might be crucial to optimize the use of Treg to prevent GVHD.

Disparity for the minor histocompatibility antigen HA-1 is associated with an increased risk of acute graft-versus-host disease (GvHD) but it does not affect chronic GvHD incidence, disease-free survival or overall survival after allogeneic human leucocyte antigen-identical sibling donor transplantation

Disparity for the minor histocompatibility antigen HA‐1 between patient and donor has been associated with an increased risk of acute graft‐versus‐host disease (GvHD) after allogeneic human leucocyte antigen (HLA)‐identical sibling donor stem cell transplantation (SCT). However, no data concerning the impact of such disparity on chronic GvHD, relapse or overall survival are available. A retrospective multicentre study was performed on 215 HLA‐A2‐positive patients who received an HLA‐identical sibling SCT, in order to determine the differences in acute and chronic GvHD incidence on the basis of the presence or absence of the HA‐1 antigen mismatch. Disease‐free survival and overall survival were also analysed. We detected 34 patient–donor pairs mismatched for HA‐1 antigen (15·8%). Grades II–IV acute GvHD occurred in 51·6% of the HA‐1‐mismatched pairs compared with 37·1% of the non‐mismatched. The multivariate logistic regression model showed statistical significance (P: 0·035, OR: 2·96, 95% CI: 1·07–8·14). No differences were observed between the two groups for grades III–IV acute GvHD, chronic GvHD, disease‐free survival or overall survival. These results confirmed the association between HA‐1 mismatch and risk of mild acute GvHD, but HA‐1 mismatch was not associated with an increased incidence of chronic GvHD and did not affect relapse or overall survival.