Elena Sanmartín

Circulating DNA is a useful prognostic factor in patients with advanced non-small cell lung cancer

Background: Circulating DNA is observed at higher concentrations in patients with lung cancer than in controls. Qualitative and quantitative analysis of circulating DNA is a promising noninvasive tool. Our aim was to prospectively study the association between the catalytic subunit of telomerase (human telomerase reverse transcriptase [hTERT]) in plasma and clinical variables and survival in a large-scale non-small cell lung cancer (NSCLC) study. Methods: Four hundred forty-six patients with stages IIIB and IV NSCLC with a median follow-up of 9.7 months (range, 0.5–45) were analyzed. Blood samples were collected before therapy start (cisplatin/docetaxel). Quantification of baseline circulating DNA was determined as the amount of free hTERT in plasma, by using real-time quantitative polymerase chain reaction. Results: Patients with hTERT ≤49.8 ng/ml (median value) had a median time to progression (TTP) of 6.3 months compared with 4.9 for hTERT more than 49.8 ng/ml (p = 0.001). Overall survival (OS) was significantly higher (10.9 versus 9.3 months) at lower hTERT levels (p = 0.012). When calculations were done using hTERT as continuous variable, we did not observe independent significant differences. Thus, there is an apparent discrepancy in p values when hTERT is considered as a continuous versus dichotomized variable. There was a tendency to differentiate median hTERT levels with respect to response rates (complete response + partial response: 33.1 versus stable disease + progressive disease: 50.7 ng/ml, p = 0.12), but other clinical variables such as age, gender, performance status, stage, histology, and number of metastatic locations were not associated with hTERT. In multivariate analysis, hTERT was an independent prognostic variable for both TTP (hazard ratio: 1.44, p < 0.001) and OS (hazard ratio: 1.33, p = 0.007). Conclusions: In advanced NSCLC, high pretreatment circulating hTERT level is an independent poor prognostic marker for TTP and OS. Circulating DNA is a noninvasive marker, which may help to improve the prognostic profile of these patients.

Combined VEGF-A and VEGFR-2 concentrations in plasma: Diagnostic and prognostic implications in patients with advanced NSCLC

INTRODUCTION The vascular endothelial growth factor (VEGF) family of ligands and receptors (VEGFR) play an important role in tumor angiogenesis. Increased expression of angiogenic factors in tumors or in blood is associated with poor prognosis. The aim of this study was to investigate the role of VEGF-A and soluble VEGFR-2 (sVEGFR-2) as biomarkers in advanced non-small-cell lung cancer (NSCLC). METHODS We studied 432 patients with advanced NSCLC (stages IIIB-IV) treated with cisplatin and docetaxel and 89 healthy age-matched controls. Blood samples were collected before chemotherapy, and VEGF-A and sVEGFR-2 levels were determined by ELISA. RESULTS VEGF-A and sVEGFR-2 levels were higher in NSCLC patients than in the controls, but VEGF-A behaves as a better diagnostic biomarker. There were no significant associations between VEGF-A and sVEGFR-2 concentrations and clinical characteristics, such as ECOG-PS, gender, stage, histology, metastases, and treatment response. A patient subgroup characterized by a combination of high VEGF-A and low sVEGFR-2 levels exhibited the worst patient prognoses in terms of TTP and OS. CONCLUSIONS VEGF-A and sVEGFR-2 levels were significantly higher in patients than in the controls. A combination of VEGF-A and sVEGFR-2 can be used as an independent prognostic biomarker in advanced NSCLC.

The identification of KRAS mutations at codon 12 in plasma DNA is not a prognostic factor in advanced non-small cell lung cancer patients.

BACKGROUND Qualitative analysis of circulating DNA in the blood is a promising non-invasive diagnostic and prognostic tool. Our aim was to study the association between the presence of KRAS mutations at codon 12 and several clinical variables in advanced non-small cell lung cancer (NSCLC) patients. METHODS We examined 308 stage IIIB and IV NSCLC patients who were treated with cisplatin and docetaxel. Blood samples were collected before chemotherapy, and circulating DNA was extracted from the plasma using commercial adsorption columns. The KRAS mutational status was determined by an RT-PCR method that is based on allelic discrimination. RESULTS The median age of the patients was 60 years [31-80], 84% were male, 98% had a performance status of 0-1 and 84% of the patients were in stage IV. The histological subtypes were as follows: 30% squamous cell carcinoma (SCC), 51% adenocarcinoma (ADC) and 19% others. Of the 277 response-evaluated patients, 1% achieved a complete response (CR), 26% achieved a partial response (PR), 34% had stable disease (SD) and 39% had progressive disease (PD). Additionally, 27 (8.8%) patients had KRAS mutations; 26 had a KRAS codon 12 TGT mutation, and 1 had a codon 12 GTT mutation. Plasmatic KRAS mutations were found in patients presenting SCC or ADC. Patients with KRAS mutations in plasma DNA had a median progression free survival (PFS) of 5.77 months [3.39-8.14], whereas for patients with wild-type (wt) KRAS, the PFS was 5.43 months [4.65-6.22] (p=0.277). The median overall survival (OS) in KRAS-mutated patients was 9.07 months [4.43-13.70] vs 10.03 months [8.80-11.26] in wt patients (p=0.514). CONCLUSIONS In advanced NSCLC patients, there were no significant differences between patients with or without KRAS mutations in plasma-free DNA with respect to the baseline characteristics, response rates, PFS or OS.

Update on biomarkers for the detection of lung cancer

Patients at risk for lung cancer may have subclinical disease for years before presentation. The diagnosis of this disease is primarily based on symptoms, and detection often occurs after curative intervention is no longer possible. At present, no lung cancer early-detection biomarker is clinically available. This study reviews the most recent advances in early detection and molecular diagnostic biomarkers for the detection of lung cancer. This review includes an overview of the various biological specimens and matrices in which these biomarkers could be analyzed, as well as the diverse strategies and approaches for identifying new biomarkers that are currently being explored. Several novel and attractive biomarker candidates for the early detection of lung cancer exist. A remarkable shift is taking place from research based on single markers to analyzing signatures that are more complex in order to take advantage of new high-throughput technologies. However, it is still necessary to validate the most promising markers and the standardization of procedures that will lead to specific clinical applications.

The prognostic value of hTERT expression levels in advanced-stage colorectal cancer patients: a comparison between tissue and serum expression.

AimTelomeres are regions of highly repetitive, non-coding DNA located at the termini of chromosomes whose principal function is to maintain the structural stability of these ends. In 90% of human tumours, telomere length is maintained by the expression and activation of telomerase reverse transcriptase. Various studies have demonstrated an increase in telomerase activity in tumour tissue, which suggests its possible prognostic value. The main objective of our study was to study the prognostic value of the expression level of telomerase catalytic component (hTERT) in patients with colorectal cancer (CRC).MethodsWe analysed the prognostic value of the ratio of telomerase expression in tumour tissue to telomerase expression in the adjacent healthy mucosa and the prognostic value of the expression level of hTERT in the serum of patients diagnosed with CRC. As secondary objectives of the study, we (1) analysed the correlation between telomerase expression in the serum and that in the tumour tissue and (2) analysed the relationship between telomerase expression and different clinical parameters.ResultsPeripheral blood and tissue samples taken from 48 patients with CRC were analysed. No significant differences were observed in disease-free survival (DFS) or overall survival time (OST) between the groups of patients categorised based on the ratio of telomerase expression between tumour tissue and healthy tissue. The correlation index (Pearson’s coefficient) between telomerase levels in the serum and those in tissue was 0.32. Our study of the relationship between telomerase levels in the serum and different clinical variables, such as tumour size, ganglion affectation, preoperative carcinoembryonic antigen levels and stage, revealed a higher telomerase expression level in patients with stage IV CRC. There was no significant association between telomerase expression in tumour tissue and the clinical parameters analysed.ConclusionsThe results obtained in our study do not allow us to propose that the level of telomerase expression be used as a prognostic factor in colorectal cancer. Thus, we cannot consider telomerase expression in the serum as a surrogate marker of its expression in tumour tissue.

A Gene Signature Combining the Tissue Expression of Three Angiogenic Factors is a Prognostic Marker in Early-stage Non-small Cell Lung Cancer

AbstractBackground Angiogenesis and lymphangiogenesis are key mechanisms for tumor growth and dissemination. They are mainly regulated by the vascular endothelial growth factor (VEGF) family of ligands and receptors. The aim of this study was to analyze relative expression levels of angiogenic markers in resectable non-small cell lung cancer patients in order to asses a prognostic signature that could improve characterization of patients with worse clinical outcomes. Methods RNA was obtained from tumor and normal lung specimens from 175 patients. Quantitative polymerase chain reaction was performed to analyze the relative expression of HIF1A, PlGF, VEGFA, VEGFA165b, VEGFB, VEGFC, VEGFD, VEGFR1, VEGFR2, VEGFR3, NRP1 and NRP2.ResultsUnivariate analysis showed that tumor size and ECOG-PS are prognostic factors for time to progression (TTP) and overall survival (OS). This analysis in the case of angiogenic factors also revealed that PlGF, VEGFA, VEGFB and VEGFD distinguish patients with different outcomes. Taking into account the complex interplay between the different ligands of the VEGF family and to more precisely predict the outcome of the patients, we considered a new analysis combining several VEGF ligands. In order to find independent prognostic variables, we performed a multivariate Cox analysis, which showed that the subgroup of patients with higher relative expression of VEGFA plus lower VEGFB and VEGFD presented the poorest outcome for both TTP and OS.ConclusionsThe relative expression of these three genes can be considered as an angiogenic gene signature whose applicability for the selection of candidates for targeted therapies needs to be further validated.

Association of Foxp3 expression with prognosis in resected non-small cell lung cancer (NSCLC).

e21114 Background: Regulatory T cells (Tregs) play a critical role in suppressing T-cell-mediated immunity in patients with cancer. A highly specific marker for Tregs is Foxp3, a transcription factor which appears to be a master control gene for their development and function. The aim of this study was to evaluate the role of Foxp3 as a prognostic marker in resectable NSCLC patients. METHODS Foxp3 expression was assessed by RT-PCR in frozen samples (tumor and normal lung) from 150 NSCLC patients. Foxp3 relative expression was normalized by an endogenous gene (GUSB) and compared with clinicopathological variables. Statistical analyses were considered significant at p<0.05. RESULTS RT-PCR analysis showed that Foxp3 was over-expressed (>2.0X) in 41/150 tumor lung tissues. No significant associations were found between Foxp3 expression and gender, ECOG-PS, stage or histology. Patients with Foxp3 > 1.48X (median value) showed a shorter TTP (median 22.1 vs 66.6 months, p= 0.017) and OS (median 26.8 vs 49.7 months, p= 0.036) than others. CONCLUSIONS Foxp3 is a transcription factor necessary and sufficient for induction of the immunosuppressive functions in Tregs. In concordance, our results indicate that high expression of Foxp3 in tumor samples is a poor prognostic marker for TTP and OS in resectable NSCLC. It could be inferred that overcoming Treg activity, may be beneficial for the treatment of this type of cancer. Supported by grants PS09-01149 and RD06/0020/1024 from ISCIII.

Prognostic implications of lymphangiogenic markers in early-stage NSCLC.

e21108 Background: The lymphatic system constitutes one of the most important pathways for tumor cell dissemination. The process of lymphangiogenesis is mainly regulated by VEGF family members. Specifically, the activation of VEGFR-3 by the binding of VEGF-C and VEGF-D is the initial signal stimulating the proliferation and migration of lymphatic endothelial cells. The aim of the present study was to analyze the expression of these genes in a cohort of resectable NSCLC patients and to correlate them with clinico-pathological variables and prognosis. METHODS RNA was obtained from tumor and normal lung specimens from 150 resectable NSCLC patients. RT-PCR was performed to assess the expression of VEGF-C, VEGF-D and VEGFR-3. Relative expression was normalized by an endogenous gene (GUS) using the Pfaffl formulae. Differences were considered statistically significant at p<0.05. RESULTS We found that tumor samples had a significant lower expression of VEGF-D compared to normal tissue (0.030X). In regard to the smoking status, the group of active smoking patients showed higher expressions levels of VEGF-C and VEGFR-3 genes (p=0.012 and p=0.014, respectively). There was s trend correlating lower expression of VEGF-D with lymph node metastasis. The survival analysis revealed that the group of patients with values of VEGF-D below the median had a significantly reduced OS rate (p=0.002). CONCLUSIONS VEGF-D is a master control gene of the lymphangiogenic process and may have a crucial role in the development of metastasis, therefore the lower expression of this gene found in tumor samples compared with normal lung tissue needs to be further investigated. Moreover, the expression of VEGF-D below the median is a poor prognostic marker for OS in our cohort of early-stage NSCLC patients. Supported by grants PS09-01149 and RD06/0020/1024 from ISCIII.

The surfactant protein B (SFTPB) as a surrogate of circulating tumor cells (CTC) with prognostic value in advanced-stage NSCLC.

7596 Background: The detection of CTCs in the peripheral blood of cancer patients is of great interest in oncology but remains technically challenging. Molecular methodologies such as QRT-PCR are highly sensitive, specific and reproducible, but the use of appropriate mRNA markers for CTCs is required. The aim of the present study, in a cohort of advanced NSCLC, was to analyze eight CTCs associated markers using QRT-PCR pre- and post-chemotherapy, and to correlate them with clinico-pathological variables and prognosis. METHODS RNA was obtained from peripheral blood samples preserved in PAXgene tubes (whole blood) of 69 NSCLC patients and 59 healthy age-, gender-matched controls. Patients samples were obtained before and after 3 cycles of chemotherapy with cisplatin and docetaxel. Eight genes surrogate of CTCs were selected in silico (CEA, CK19, CK20, CK7, SFTPB, AGR2, S100A13 and hTERT), mRNA was reverse transcribed and QRT-PCR was performed using TaqMan technology, GUS-B as endogenous control and a cell line as a calibrator. Differences were considered significant at p ≤0.05. RESULTS Median age 59.2 [37.1-75.5]; 84.1% males; 73.9 % stage IV; 30.4% squamous cell, 50.7% adenocarcinomas and 18.8% other histologies. We found significant differences in the expression of SFTPB between controls and patients (14.6 vs 25.6 respectively, p=0.05). Splitting the patients into two groups according to median gene expression levels, it could be observed that overall survival was significantly higher in patients with lower levels of SFTPB, 14.6 vs 10.8 months (p= 0.049). CONCLUSIONS Quantification of CTCs associated markers by QRT-PCR is technically possible in whole blood. SFTPB levels are significantly higher in NSCLC patients than controls. Patients with higher levels of this gene presented poorer overall survival. Validation with an independent group of patients is necessary in order to assess their role as diagnostic and/or prognostic biomarker. Supported by RD06/0020/1024 grant by ISCIII.

Expression of angiogenic genes in tumor and stroma as biomarkers of prognosis in NSCLC.

e21013 Background: In tumor angiogenesis there is a complex interplay between endothelial, stromal, and tumor cells. The VEGF and PDGF families of ligands and receptors play important roles in this process. We investigate the correlations between the expression of ligands and receptors of both families in tumor and stromal cells in patients with resectable NSCLC and their possible role as prognostic biomarkers. Methods: Primary tumor tissues (n = 84) from NSCLC patients (I-IIIA) were used in this study. Representative areas of tumor, stroma and normal tissue were carefully selected. RNA was extracted from 2 sampled cores (0.6 mm) and RT-PCR was performed using TaqMan probes. Relative quantification was calculated using actin-β as endogenous control. Results were normalized against a calibrator sample (NCI-H23 cell line). Statistical analysis were considered significant at p < 0.05. Results: Significant differences between tumor and stroma expression were found for VEGFR-1, -3, neuropilin-1 (NRP-1), PDGF-B...