György Lang

Genome-Wide miRNA Expression Profiling Identifies miR-9-3 and miR-193a as Targets for DNA Methylation in Non–Small Cell Lung Cancers

Purpose: The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on miRNA silencing in non–small cell lung cancers (NSCLC). Experimental Design: We conducted microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. miRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding nonmalignant lung tissue samples of 101 patients with stage I–III NSCLC. Results: By comparing microarray data of untreated and drug-treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analyzed. Moreover, miR-9-3 and miR-193a were found to be tumor specifically methylated in patients with NSCLC. We observed a shorter disease-free survival of patients with miR-9-3 methylated lung squamous cell carcinoma (LSCC) than patients with miR-9-3 unmethylated LSCC by multivariate analysis [HR = 3.8; 95% confidence interval (CI), 1.3–11.2, P = 0.017] and a shorter overall survival of patients with miR-9-3 methylated LSCC than patients with miR-9-3 unmethylated LSCC by univariate analysis (P = 0.013). Conclusions: Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in patients with LSCC. Clin Cancer Res; 18(6); 1619–29. ©2012 AACR.

Clinical ex vivo lung perfusion--pushing the limits.

Ex vivo lung perfusion (EVLP) provides the ability to evaluate donor lungs before transplantation. Yet, limited prospective clinical data exist with regard to its potential to recondition unacceptable donor lungs. This paper summarizes the results of a prospective study of lung transplantation using only initially unacceptable donor lungs, which were improved by EVLP for 2–4 h. From March 2010–June 2011, 13 lungs were evaluated ex vivo. Median donor PaO2 at FiO21.0/PEEP5 was 216 mmHg (range 133–271). Four lungs, all with trauma history, showed no improvement and were discarded. Nine lungs improved to a ΔPO2 higher than 350 mmHg. Median PvO2 at final assessment in these lungs was 466 mmHg (range 434–525). These lungs were transplanted with a median total ischemic time of 577 min (range 486–678). None of the patients developed primary graft dysfunction grades 2 or 3 within 72 h after transplantation. One patient with secondary pulmonary hypertension was left on a planned prolonged extracorporeal membrane oxygenation postoperatively. Median intubation time was 2 days. Thirty‐day mortality was 0%. During the observation period, 119 patients received standard lung transplantation with comparable perioperative outcome. EVLP has a significant potential to improve the quality of otherwise unacceptable donor lungs.

Primary Lung Transplantation After Bridge With Extracorporeal Membrane Oxygenation: A Plea for a Shift in Our Paradigms for Indications

Background. The introduction of the lung allocation score has brought lung transplantation (LTX) of patients on extracorporeal membrane oxygenation (ECMO) bridge into the focus of interest. We reviewed our institutional experience with ECMO as a bridge to LTX. Methods. Between 1998 and 2011, 38 patients (median age 30.1 years, range 13–66 years) underwent ECMO support with intention to bridge to primary LTX. The underlying diagnosis was cystic fibrosis (n=17), pulmonary hypertension (n=4), idiopathic pulmonary fibrosis (n=9), adult respiratory distress syndrome (n=4), hemosiderosis (n=1), bronchiolitis obliterans (n=1), sarcoidosis (n=1), and bronchiectasis (n=1). The type of extracorporeal bridge was venovenous (n=18), venoarterial (n=15), interventional lung assist (n=1), or a stepwise combination of them (n=4). The median bridging time was 5.5 days (range 1–63) days. The type of transplantation was double LTX (n=7), size-reduced double LTX (n=8), lobar LTX (n=16), split LTX (n=2), and lobar LTX after ex vivo lung perfusion (n=1). Results. Four patients died before transplantation. Thirty-four patients underwent LTX, of them eight patients died in the hospital after a median stay of 24.5 days (range 1–180 days). Twenty-six patients left the hospital and returned to normal life (median hospital stay=47.5 days; range 21–90 days). The 1-, 3-, and 5-year survival for all transplanted patients was 60%, 60%, and 48%, respectively. The 1-, 3-, and 5-year survival conditional on 3-month survival for patients bridged with ECMO to LTX (78%, 78%, and 63%) was not worse than for other LTX patients within the same period of time (90%, 80%, and 72%, respectively, P=0.09, 0.505, and 0.344). Conclusion. Transplantation of patients bridged on ECMO to LTX is feasible and results in acceptable outcome.

Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas

TSLC1 (tumor suppressor in lung cancer-1, IGSF4) encodes a member of the immunoglobulin superfamily molecules, which is involved in cell–cell adhesion. TSLC1 is connected to the actin cytoskeleton by DAL-1 (differentially expressed in adenocarcinoma of the lung-1, EPB41L3) and it directly associates with MPP3, one of the human homologues of a Drosophila tumor suppressor gene, Discs large. Recent data suggest that aberrant promoter methylation is important for TSLC1 inactivation in lung carcinomas. However, little is known about the other two genes in this cascade, DAL-1 and MPP3. Thus, we investigated the expression and methylation patterns of these genes in lung cancer cell lines, primary lung carcinomas and nonmalignant lung tissue samples. By reverse transcription–polymerase chain reaction, loss of TSLC1 expression was observed in seven of 16 (44%) non-small-cell lung cancer (NSCLC) cell lines and in one of 11 (9%) small-cell lung cancer (SCLC) cell lines, while loss of DAL-1 expression was seen in 14 of 16 (87%) NSCLC cell lines and in four of 11 (36%) SCLC cell lines. By contrast, MPP3 expression was found in all tumor cell lines analysed. Similar results were obtained by microarray analysis. TSLC1 methylation was seen in 13 of 39 (33%) NSCLC cell lines, in one of 11 (9%) SCLC cell lines and in 100 of 268 (37%) primary NSCLCs. DAL-1 methylation was observed in 17 of 39 (44%) NSCLC cell lines, in three of 11 (27%) SCLC cell lines and in 147 of 268 (55%) primary NSCLCs. In tumors of NSCLC patients with stage II–III disease, DAL-1 methylation was seen at a statistically significant higher frequency compared to tumors of patients with stage I disease. A significant correlation between loss of expression and methylation of the genes in lung cancer cell lines was found. Overall, 65% of primary NSCLCs had either TSLC1 or DAL-1 methylated. Methylation of one of these genes was detected in 59% of NSCLC cell lines; however, in SCLC cell lines, methylation was much less frequently observed. The majority of nonmalignant lung tissue samples was not TSLC1 or DAL-1 methylated. Re-expression of TSLC1 and DAL-1 was seen after treatment of lung cancer cell lines with 5-aza-2′-deoxycytidine. Our results suggest that methylation of TSLC1 and/or DAL-1, leading to loss of their expression, is an important event in the pathogenesis of NSCLC.

Circulating endothelial cells, bone marrow-derived endothelial progenitor cells and proangiogenic hematopoietic cells in cancer: From biology to therapy

Vascularization, a hallmark of tumorigenesis, is classically thought to occur exclusively through angiogenesis (i.e. endothelial sprouting). However, there is a growing body of evidence that endothelial progenitor cells (EPCs) and proangiogenic hematopoietic cells (HCs) are able to support the vascularization of tumors and may therefore play a synergistic role with angiogenesis. An additional cell type being studied in the field of tumor vascularization is the circulating endothelial cell (CEC), whose presence in elevated numbers reflects vascular injury. Levels of EPCs and CECs are reported to correlate with tumor stage and have been evaluated as biomarkers of the efficacy of anticancer/antiangiogenic treatments. Furthermore, because EPCs and subtypes of proangiogenic HCs are actively participating in capillary growth, these cells are attractive potential vehicles for delivering therapeutic molecules. The current paper provides an update on the biology of CECs, EPCs and proangiogenic HCs, and explores the utility of these cell populations for clinical oncology.

Cytomegalovirus Prevention in High-risk Lung Transplant Recipients: Comparison of 3- vs 12-Month Valganciclovir Therapy

BACKGROUND Cytomegalovirus (CMV) infections are common after lung transplantation (LuTx) and have an influence on acute rejection rates and chronic organ dysfunction. The objective of this study was to determine the incidence of CMV infections by comparing a prolonged valganciclovir prophylaxis with a standard regimen in high-risk LuTx recipients. METHODS A retrospective, single-center study was performed comparing two different CMV prophylactic regimens in high-risk LuTx recipients (D(+)/R(-)). The study population received either 3 months (Group A, 15 patients) or 12 months (Group B, 17 patients) of oral valganciclovir 900 mg/day in combination with CMV hyperimmune globulin in four doses (Days 1, 7, 14 and 21 post-transplant). RESULTS CMV viremia was noted in 11 of 15 patients in Group A (75%) and 5 of 17 in Group B (33%) (p < 0.05) at 6 months after valganciclovir cessation. The incidence of symptomatic CMV disease/syndrome was 6 of 15 (44%) in Group A and 2 of 17 in Group B (13%) (p < 0.05). Histologically proven acute rejection episodes of ISHLT Grade > or =A2 were found in 4 patients in Group A and in 1 patient in Group B within the first year (p = 0.14). CONCLUSIONS A 12-month CMV prophylaxis with oral valganciclovir is effective in significantly reducing CMV viremia and CMV disease/syndrome in high-risk lung transplant recipients. In addition, a reduction in acute and recurrent rejection episodes was observed, possibly due to less CMV viremia and subsequent immunomodulatory effects.

Genome-wide CpG island methylation analyses in non-small cell lung cancer patients

DNA methylation is part of the epigenetic gene regulation complex, which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stages I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis. Overall, we identified 2414 genomic positions differentially methylated between tumor and non-malignant lung tissue samples. Ninety-seven percent of them were found to be tumor-specifically methylated. Annotation of these genomic positions resulted in the identification of 477 tumor-specifically methylated genes of which many are involved in regulation of gene transcription and cell adhesion. Tumor-specific methylation was confirmed by a gene-specific approach. In the majority of tumors, methylation of certain genes was associated with loss of their protein expression determined by immunohistochemistry. Treatment of NSCLC cells with epigenetically active drugs resulted in upregulated expression of many tumor-specifically methylated genes analyzed by gene expression microarrays suggesting that about one-third of these genes are transcriptionally regulated by methylation. Moreover, comparison of methylation results with certain clinicopathological characteristics of the patients suggests that methylation of HOXA2 and HOXA10 may be of prognostic relevance in squamous cell carcinoma (SCC) patients. In conclusion, we identified a large number of tumor-specifically methylated genes in NSCLC patients. Expression of many of them is regulated by methylation. Moreover, HOXA2 and HOXA10 methylation may serve as prognostic parameters in SCC patients. Overall, our findings emphasize the impact of methylation on the pathogenesis of NSCLCs.

High VEGFR-3-positive Circulating Lymphatic/Vascular Endothelial Progenitor Cell Level Is Associated with Poor Prognosis in Human Small Cell Lung Cancer

Purpose: The newly identified bone marrow–derived cell population, called lymphatic/vascular endothelial progenitor cells (LVEPC), has been shown to contribute to lymph capillary growth in experimental tumor systems. The clinical significance of these cells has not yet been investigated in a human malignancy. Our aim was to study whether peripheral blood circulating LVEPCs participate in the progression of human small cell lung cancer (SCLC). Experimental Design: A total of 88 patients with limited-stage SCLC and 32 tumor-free control subjects were included. Peripheral blood circulating LVEPC labeled with CD34 and vascular endothelial growth factor receptor-3 (VEGFR3) antibodies and the serum levels of the key lymphangiogenic molecule VEGF-C were measured by flow cytometry and ELISA, respectively. Results: CD34-positive/VEGFR3-positive LVEPC levels were significantly increased in patients (versus controls; P < 0.01), and there was also a significant relationship between LVEPC counts and lymph node metastasis (P < 0.01). High pretreatment circulating LVEPC numbers correlated with poor overall survival (P < 0.01). Although we observed significantly elevated VEGF-C concentrations in patients (versus controls; P < 0.01), there was no significant correlation between VEGF-C and LVEPC levels. Moreover, no significant differences in peripheral blood VEGF-C levels were seen between patients subgrouped by clinicopathologic variables including tumor and lymph node stages and survival. Conclusions: Peripheral blood levels of bone marrow–derived LVEPCs are significantly increased in patients with SCLC and correlate with lymphatic involvement and prognosis. This is the first study that shows evidence of increased numbers of circulating LVEPC in patients with a malignant tumor.

Expression of the candidate tumor suppressor gene hSRBC is frequently lost in primary lung cancers with and without DNA methylation.

Recently, the human SRBC (hSRBC) gene, a candidate tumor suppressor gene (TSG), has been mapped to the chromosomal region 11p15.5–p15.4 where frequent allele loss has been described in lung cancer. Aberrant methylation (referred to as methylation) of the promoter region of TSGs has been identified as an important mechanism for gene silencing. Loss of hSRBC protein expression occurs frequently in lung cancer cell lines and sodium bisulfite sequencing of the promoter region of hSRBC in several lung cancer cell lines suggested that methylation plays an important role in inactivating hSRBC. To determine the methylation status of hSRBC in a large collection of primary lung cancer samples, corresponding nonmalignant lung tissues and lung cancer cell lines (N=52), we designed primers for a methylation-specific PCR assay. Methylation was detected in 41% of primary non-small-cell lung cancers (NSCLC) (N=107) and in 80% of primary small-cell lung cancers (SCLC) (N=5), but was seen only in 4% of corresponding nonmalignant lung tissues (N=103). In all, 79% of lung cancer cell lines were methylated and the frequency of hSRBC methylation was significantly higher in SCLC (100%) than in NSCLC (58%) cell lines. Normal hSRBC protein expression was detected in only 18% of primary NSCLCs (N=93) by immunostaining and a significant association between loss of protein expression and methylation was found. hSRBC re-expression was observed after treatment of lung cancer cells with the demethylating agent 5-aza-2′-deoxycytidine. In addition, 45% of the 76 hSRBC immunostaining-negative NSCLCs did not have hSRBC promoter methylation, indicating that other mechanisms of hSRBC expression silencing also exist. Both hSRBC immunostaining and methylation results did not correlate with clinicopathological characteristics of these patients. Our findings suggest that hSRBC is a candidate TSG involved in lung cancer pathogenesis, where expression is frequently inactivated by methylation and other mechanisms.

Extracorporeal Membrane Oxygenation Support for Resection of Locally Advanced Thoracic Tumors

BACKGROUND The international experience with resection of advanced thoracic malignancies performed with extracorporeal membrane oxygenation (ECMO) support is limited. We examined our results to assess the risks and benefits of this approach. METHODS We retrospectively analyzed all patients with thoracic malignancies who underwent tumor resection with ECMO support in our department between 2001 and 2010. RESULTS Nine patients (aged 21 to 71 years; mean, 54.8±7.5 years) underwent complex tracheobronchial resections (n=6) or resections of greater thoracic vessels (n=3) under venoarterial (VA) ECMO support. In 7 patients the underlying pathologic condition was non-small cell lung cancer, in 1 patient it was carcinoid tumor, and in 1 patient it was synovial sarcoma. The indication for extracorporeal support was complex tracheobronchial reconstruction (n=5), resection of the descending aorta (n=2), and resection of the inferior vena cava (n=1). ECMO cannulation was central (n=4), peripheral (n=4), or combined (n=1). Mean time on bypass was 110±19 minutes (range 40 to 135 minutes). A complete resection (R0) was achieved in 8 patients (89%). One patient died perioperatively as a result of hepatic necrosis. Eight patients were discharged from the hospital after 7 to 42 days (median, 10 days). Median time in the intensive care unit was 1 day (range, 0 to 36 days). The only complication related to the use of ECMO was a lymphatic fistula in the groin. Mean follow-up time was 38±42 months (range, 9 to 111 months). The actuarial 3-month survival was 88.9%, and the 1-year, 3-year, and 5-year survival was 76.7%. CONCLUSIONS Based on this experience, we consider VA ECMO support to be a safe alternative to cardiopulmonary bypass (CPB) for advanced general thoracic operations.