Wenceslao Torre

A Prognostic DNA Methylation Signature for Stage I Non–Small-Cell Lung Cancer

PURPOSE Non-small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. PATIENTS AND METHODS A DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. RESULTS Unsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P = .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P = .001). CONCLUSION The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.

Altered patterns of expression of members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family in lung cancer.

hnRNP A2/B1 has been suggested as a useful early detection marker for lung carcinoma. hnRNP A2/B1 is a member of a large family of heterogeneous nuclear ribonucleoproteins (hnRNP proteins) involved in a variety of functions, including regulation of transcription, mRNA metabolism, and translation. In lung cancer, we have evaluated the expression and cellular localization of several members of the hnRNP family, hnRNP A1, A2, B1, C1, C2 and K. 16 cell lines (SCLC and NSCLC) and biopsies from 32 lung cancer patients were analyzed. Our results suggest that, besides hnRNP A2/B1, the expression of other members of the hnRNP family is altered both in SCLC and NSCLC. In the biopsies, negative or low expression of the hnRNP proteins analyzed was observed in normal epithelial cells whereas lung cancer cells showed highly intense nuclear or cytoplasmic immunolocalization. In all the lung cancer cell lines, the mRNA for all the hnRNP proteins was detected. In general, higher levels of hnRNP mRNAs were found in SCLC as compared with NSCLC. Our results also suggest that the expression and processing of each hnRNP protein in lung cancer is independently regulated and is not exclusively related to proliferation status. In SCLC cell lines, hnRNP A1 protein expression correlated with that of Bcl-x(L). In the lung cancer cell lines, hnRNP K protein localization varied with the cellular confluence.

Expression of Tumor-Derived Vascular Endothelial Growth Factor and Its Receptors Is Associated With Outcome in Early Squamous Cell Carcinoma of the Lung

PURPOSE Antiangiogenic therapies targeting the vascular endothelial growth factor (VEGF) pathway have yielded more modest clinical benefit to patients with non-small-cell lung cancer (NSCLC) than initially expected. Clinical data suggest a distinct biologic role of the VEGF pathway in the different histologic subtypes of lung cancer. To clarify the influence of histologic differentiation in the prognostic relevance of VEGF-mediated signaling in NSCLC, we performed a concomitant analysis of the expression of three key elements of the VEGF pathway in the earliest stages of the following two principal histologic subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (ADC). PATIENTS AND METHODS We evaluated tumor cell expression of VEGF, VEGF receptor (VEGFR) 1, and VEGFR2 using automatic immunostaining in a series of 298 patients with early-stage NSCLC recruited as part of the multicenter European Early Lung Cancer Detection Group project. A score measuring the VEGF signaling pathway was calculated by adding the tumor cell expression value of VEGF and its two receptors. The results were validated in two additional independent cohorts of patients with NSCLC. RESULTS The combination of high VEGF, VEGFR1, and VEGFR2 protein expression was associated with lower risk of disease progression in early SCC (univariate analysis, P = .008; multivariate analysis, hazard ratio, 0.62; 95% CI, 0.42 to 0.92; P = .02). The results were validated in two independent patient cohorts, confirming the favorable prognostic value of high VEGF signaling score in early lung SCC. CONCLUSION Our results clearly indicate that the combination of high expression of the three key elements in the VEGF pathway is associated with a good prognosis in patients with early SCC but not in patients with ADC.

Investigation of Complement Activation Product C4d as a Diagnostic and Prognostic Biomarker for Lung Cancer

BACKGROUND There is a medical need for diagnostic biomarkers in lung cancer. We evaluated the diagnostic performance of complement activation fragments. METHODS We assessed complement activation in four bronchial epithelial and seven lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; bronchoalveolar lavage supernatants from patients with lung cancer (n = 50) and nonmalignant respiratory diseases (n = 22); and plasma samples from advanced (n = 50) and early lung cancer patients (n = 84) subjects with inflammatory lung diseases (n = 133), and asymptomatic individuals enrolled in a lung cancer computed tomography screening program (n = 190). Two-sided P values were calculated by Mann-Whitney U test. RESULTS Lung cancer cells activated the classical complement pathway mediated by C1q binding that was inhibited by phosphomonoesters. Survival was decreased in patients with high C4d deposition in tumors (hazard ratio [HR] = 3.06; 95% confidence interval [CI] = 1.18 to 7.91). C4d levels were increased in bronchoalveolar lavage fluid from lung cancer patients compared with patients with nonmalignant respiratory diseases (0.61 ± 0.87 vs 0.16 ± 0.11 µg/mL; P < .001). C4d levels in plasma samples from lung cancer patients at both advanced and early stages were also increased compared with control subjects (4.13 ± 2.02 vs 1.86 ± 0.95 µg/mL, P < 0.001; 3.18 ± 3.20 vs 1.13 ± 0.69 µg/mL, P < .001, respectively). C4d plasma levels were associated with shorter survival in patients at advanced (HR = 1.59; 95% CI = 0.97 to 2.60) and early stages (HR = 5.57; 95% CI = 1.60 to 19.39). Plasma C4d levels were reduced after surgical removal of lung tumors (P < .001) and were associated with increased lung cancer risk in asymptomatic individuals with (n = 32) or without lung cancer (n = 158) (odds ratio = 4.38; 95% CI = 1.61 to 11.93). CONCLUSIONS Complement fragment C4d may serve as a biomarker for early diagnosis and prognosis of lung cancer.

The Oncoprotein SF2/ASF Promotes Non–Small Cell Lung Cancer Survival by Enhancing Survivin Expression

Purpose: SF2/ASF is a splicing factor recently described as an oncoprotein. In the present work, we examined the role of SF2/ASF in human non–small cell lung cancer (NSCLC) and analyzed the molecular mechanisms involved in SF2/ASF-related carcinogenesis. Experimental Design: SF2/ASF protein levels were analyzed in 81 NSCLC patients by immunohistochemistry. SF2/ASF downregulation cellular models were generated using small interfering RNAs, and the effects on proliferation and apoptosis were evaluated. Survivin and SF2/ASF expression in lung tumors was analyzed by Western blot and immunohistochemistry. Survival curves and log-rank test were used to identify the association between the expression of the proteins and time to progression. Results: Overexpression of SF2/ASF was found in most human primary NSCLC tumors. In vitro downregulation of SF2/ASF induced apoptosis in NSCLC cell lines. This effect was associated with a reduction in the expression of survivin, an antiapoptotic protein widely upregulated in cancer. In fact, SF2/ASF specifically bound survivin mRNA and enhanced its translation, via a mammalian target of rapamycin complex 1 (mTORC1) pathway-dependent mechanism, through the phosphorylation and inactivation of the translational repressor 4E-BP1. Moreover, SF2/ASF promoted the stability of survivin mRNA. A strong correlation was observed between the expression of SF2/ASF and survivin in tumor biopsies from NSCLC patients, supporting the concept that survivin expression levels are controlled by SF2/ASF. Furthermore, combined expression of these proteins was associated with prognosis. Conclusion: This study provides novel data on the mTORC1- and survivin-dependent mechanisms of SF2/ASF-related carcinogenic potential, and shows that SF2/ASF and survivin expression is involved in NSCLC progression. Clin Cancer Res; 16(16); 4113–25. ©2010 AACR.

Long-term results after resection for bone sarcoma pulmonary metastases

BACKGROUND Pulmonary metastases from bone sarcomas occur in approximately 40% of the cases. The combination of both chemotherapy and surgical resection is currently the standard treatment options for these patients. We aim to study the influence of different prognostic factors on long-term survival. METHODS We reviewed the prognostic factors and survival rate in 52 consecutive patients with pulmonary metastases from bone sarcomas. All of them were previously treated with chemotherapy and submitted to metastasectomy at our institution from 1996 to 2006. Clinical and demographic variables, related to the primary tumour as well as to the pulmonary metastases, and treatment procedures were registered. Univariate (log-rank) and multivariate (Cox regression) analysis were carried out to identify significant prognostic factors related to overall survival. Five-year survival rates were estimated using Kaplan-Meier methods. RESULTS Median follow-up was 28 months. Follow-up duration ranged 7-148 months; the median survival was 27 months. As many as 31% of the patients were alive without disease, 3% were alive with disease, 64% died of disease while 2% died from other causes. Complete resection was achieved in 49 cases (94%). The overall 3- and 5-year survival rates were 43% and 31%, respectively. Univariate analysis showed (1) disease-free interval between treatment of the primary bone tumour and first lung metastasectomy (DFI) and (2) disease-free interval between first and second lung surgery (DFI2) as prognostic factors. Gender, primary site, histology of primary tumour, surgical approach, number of lung nodules, type of lung resection and re-do lung surgery did not have a significant impact on survival. CONCLUSION The long-term survival after bone sarcoma lung metastasectomy is encouraging. In our series, DFI and DFI2 were identified as the only prognostic factors.

Molecular profiling of computed tomography screen-detected lung nodules shows multiple malignant features.

Rationale and Purpose: Low-dose spiral computerized axial tomography (spiral CT) is effective for the detection of small early lung cancers. Although published data seem promising, there has been a significant degree of discussion concerning the potential of overdiagnosis in the context of spiral CT–based screening. The objective of the current study was to analyze the phenotypic and genetic alterations in the small pulmonary malignancies resected after detection in the University of Navarra/International Early Lung Cancer Action Project spiral CT screening trial and to determine whether their malignant molecular features are similar to those of resected lung tumors diagnosed conventionally. Experimental Design: We analyzed 17 biomarkers of lung epithelial malignancy in a series of 11 tumors resected at our institution during the last 4 years (1,004 high-risk individuals screened), using immunohistochemistry and fluorescence in situ hybridization (FISH). A parallel series of 11 gender-, stage-, and histology-matched lung cancers diagnosed by other means except screening was used as control. Results: The molecular alterations and the frequency of phenotypic or genetic aberrations were very similar when screen-detected and nonscreen-detected lung cancers were compared. Furthermore, most of the alterations found in the screen-detected cancers from this study were concordant with what has been described previously for stage I-II lung cancer. Conclusions: Small early-stage lung cancers resected after detection in a spiral CT-based screening trial reveal malignant molecular features similar to those found in conventionally diagnosed lung cancers, suggesting that the screen-detected cancers are not overdiagnosed. (Cancer Epidemiol Biomarkers Prev 2006;15(2):373–80)

Cribado de cáncer de pulmón: catorce años de experiencia del Programa Internacional de Detección Precoz de Cáncer de Pulmón con TBDR de Pamplona (P-IELCAP)

INTRODUCTION AND OBJECTIVES European experience regarding lung cancer screening using low-dose chest CT (LDCT) is available. However, there is limited data on the Spanish experience in this matter. Our aim is to present the results from the longest ongoing screening program in Spain. METHODOLOGY The Pamplona International Early Lung Cancer Detection Program (P-IELCAP) is actively screening participants for lung cancer using LDCT since year 2000 following the IELCAP protocol, including spirometric assessments. Men and women, ≥40 years of age, current or former smokers with a tobacco history of ≥10 pack-years are included. Results are compared to those from other European trials. RESULTS A total of 2989 participants were screened until March 2014 (73% male). A median of 2 (IQR 1-3) annual screening rounds were performed. Sixty lung cancers were detected in 53 participants (73% in StageI). Adenocarcinoma was the most frequent. The lung cancer prevalence and incidence proportion was 1.0% and 1.4%, respectively, with an annual detection rate of 0.41. The estimated 10-year survival rate among individuals with lung cancer was 70%. Chronic obstructive pulmonary disease and emphysema are important lung cancer predictors. CONCLUSIONS The experience in Spains longest lung cancer screening program is comparable to what has been described in the rest of Europe, and confirms the feasibility and efficacy of lung cancer screening using LDCT.

Chest wall reconstruction using iliac bone allografts and muscle flaps.

Technically we can divide full-thickness thoracic reconstruction into 2 parts: providing a rigid support and ensuring well-vascularized coverage. Since 1986, the authors’ center has had ample experience with bone banks and the use of cryopreserved bone grafts, which led them to consider the possibility of using these grafts for full-thickness chest wall reconstruction. They describe 3 patients in whom resection of the tumor and reconstruction of the thorax were carried out using iliac bone allografts covered with muscle flaps (1 pectoralis major and 2 rectus abdominis). None of the patients experienced breathing difficulties, pain, or instability after 14 months, 18 months, and 11 years of follow-up. The result of the reconstruction was excellent in all 3 patients in terms of function and aesthetics. The advantage of allografts compared with synthetic materials is their potential integration; they can become part of the host patient’s living tissue.

Assessment of indeterminate pulmonary nodules detected in lung cancer screening: Diagnostic accuracy of FDG PET/CT

BACKGROUND A major drawback of lung cancer screening programs is the high frequency of false-positive findings on computed tomography (CT). We investigated the accuracy of selective 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (FDG) Positron Emission Tomography/Computed Tomography (PET/CT) scan in assessing radiologically indeterminate lung nodules detected in lung cancer screening. METHODS FDG PET/CT was performed to characterize 64 baseline lung nodules >10mm and 36 incidence nodules detected on low-dose CT screening in asymptomatic current or former smokers (83 men, age range 40-83 years) at high risk for lung cancer. CT images were acquired without intravenous contrast. Nodules were analyzed by size, density, and metabolic activity and visual scored on a 5-point scale for FDG uptake. Nodules were classified as negative for malignancy when no FDG uptake was observed, or positive when focal uptake was observed in the visual analysis, and the maximum standardized uptake value (SUVmax) was measured. Final diagnosis was based on histopathological evaluation or at least 24 months of follow-up. RESULTS A total of 100 nodules were included. The prevalence of lung cancer was 1%. The sensitivity, specificity, NPV and PPV of visual analysis to detect malignancy were 84%, 95%, 91%, and 91%, respectively, with an accuracy of 91% (AUC 0.893). FDG PET/CT accurately detected 31 malignant tumors (diameters 9-42mm, SUVmax range 0.6-14.2) and was falsely negative in 6 patients. With SUVmax thresholds for malignancy of 1.5, 2, and 2.5, specificity was 97% but sensitivity decreased to 65%, 49%, and 46% respectively, and accuracy decreased to 85%, 79%, and 78% respectively (AUC 0.872). CONCLUSIONS The visual analysis of FDG PET/CT scan is highly accurate in characterizing indeterminate pulmonary nodules detected in lung cancer screening with low-dose CT. Semi-quantitative analysis does not improve the accuracy of FDG PET/CT over that obtained with a qualitative method for lung nodule characterization.