Marta Usó

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.

miRNA detection methods and clinical implications in lung cancer

Lung cancer is the leading cause of cancer death worldwide. Therefore, advances in the diagnosis and treatment of the disease are urgently needed. miRNAs are a family of small, noncoding RNAs that regulate gene expression at the transcriptional level. miRNAs have been reported to be deregulated and to play a critical role in different types of cancer, including lung cancer. Thus, miRNA profiling in lung cancer patients has become the core of several investigations. To this end, the development of a multitude of platforms for miRNA profiling analysis has been essential. This article focuses on the different technologies available for assessing miRNAs and the most important results obtained to date in lung cancer.

MicroRNA profiling associated with non-small cell lung cancer: next generation sequencing detection, experimental validation, and prognostic value

BACKGROUND The average five-year survival index for non-small cell lung cancer (NSCLC) patients is approximately 15%. Emerging evidence indicates that microRNAs constitute a new class of gene regulators in humans that may play an important role in tumorigenesis. Hence, there is growing interest in studying their role as possible new biomarkers whose expression is aberrant in cancer. Therefore, in this study we identified deregulated miRNAs by next generation sequencing (NGS) and analyzed their prognostic value. METHODS Sequencing by oligo ligation detection technology was used to identify dysregulated miRNAs in a training cohort comprising paired tumor/normal tissue samples (N = 32). We validated 22 randomly selected differentially-expressed miRNAs by quantitative real time PCR in tumor and adjacent normal tissue samples (N = 178). Kaplan-Meier survival analysis and Cox regression were used in multivariate analysis to identify independent prognostic biomarkers. RESULTS NGS analysis revealed that 39 miRNAs were dysregulated in NSCLC: 28 were upregulated and 11 were downregulated. Twenty-two miRNAs were validated in an independent cohort. Interestingly, the group of patients with high expression of both miRNAs (miR-21high and miR-188high) showed shorter relapse-free survival (RFS) and overall survival (OS) times. Multivariate analysis showed that this miRNA combination is an independent prognostic marker for RFS and OS (p = 0.001 and p < 0.0001, respectively). CONCLUSIONS NGS technology can specifically identify dysregulated miRNA profiles in rsectable NSCLC samples. MiR-21 or miR-188 overexpression correlated with a negative prognosis, and their combined signature may represent a new independent prognostic biomarker for RFS and OS.Background The average five-year survival for non-small cell lung cancer (NSCLC) patients is approximately 15%. Emerging evidence indicates that microRNAs (miRNAs) constitute a new class of gene regulators in humans that may play an important role in tumorigenesis. Hence, there is growing interest in studying their role as possible new biomarkers whose expression is aberrant in cancer. Therefore, in this study we identified dysregulated miRNAs by next generation sequencing (NGS) and analyzed their prognostic value. Methods Sequencing by oligo ligation detection technology was used to identify dysregulated miRNAs in a training cohort comprising paired tumor/normal tissue samples (N = 32). We validated 22 randomly selected differentially-expressed miRNAs by quantitative real time PCR in tumor and adjacent normal tissue samples (N = 178). Kaplan-Meier survival analysis and Cox regression were used in multivariate analysis to identify independent prognostic biomarkers. Results NGS analysis revealed that 39 miRNAs were dysregulated in NSCLC: 28 were upregulated and 11 were downregulated. Twenty-two miRNAs were validated in an independent cohort. Interestingly, the group of patients with high expression of both miRNAs (miR-21high and miR-188high) showed shorter relapse-free survival (RFS) and overall survival (OS) times. Multivariate analysis confirmed that this combined signature is an independent prognostic marker for RFS and OS (p = 0.001 and p < 0.0001, respectively). Conclusions NGS technology can specifically identify dysregulated miRNA profiles in resectable NSCLC samples. MiR-21 or miR-188 overexpression correlated with a negative prognosis, and their combined signature may represent a new independent prognostic biomarker for RFS and OS.

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.

Analysis of the immune microenvironment in resected non-small cell lung cancer: the prognostic value of different T lymphocyte markers

The prognosis of non-small cell lung cancer (NSCLC) remains poor and heterogeneous and new biomarkers are needed. As the immune system plays a pivotal role in cancer, the study of immune-related markers may provide valuable prognostic information of NSCLC. In 122 formalin-fixed, paraffin-embedded tumor tissue samples from early-stage NSCLC, tumor and tumor-near stromal areas were microdissected and gene expression levels of conventional and regulatory T cell markers were assessed by quantitative polymerase chain reaction. Also, the presence of infiltrating CD4+, CD8+, and FOXP3+ cells in tumor samples was assessed by immunohistochemistry. The relative proportion of conventional and regulatory T cells present in the tumor environment was assessed and found to be key to understand the importance that the immune system analysis has in the prognostics of NSCLC patients. The presence of CD8+ cells in the tumor compartment was associated with better outcome, whereas the presence of FOXP3+ cells was associated with worse overall survival. The negative prognostic value of combined biomarkers, indicating high levels of FOXP3 in the stroma and low levels of CD4 or CD8 in tumors, was observed at mRNA level and was validated by immunohistochemistry.In conclusion, the proportion of T helper and cytotoxic cells vs. regulatory T cells in different locations of the tumor microenvironment have opposite prognostic impacts in resected NSCLC.

Analysis of the prognostic role of an immune checkpoint score in resected non-small cell lung cancer patients

ABSTRACT Tumors develop mechanisms to recruit tolerogenic immune cells and to induce the expression of molecules that act as immune checkpoints. This regulation of the immune microenvironment favors immune tolerance to the neoplastic cells. In this study, we have investigated the prognostic role of immune-checkpoint expression markers in a cohort of resectable non-small cell lung cancer (NSCLC) patients. RNA was isolated from fresh-frozen lung specimens (tumor and normal lung) (n = 178). RTqPCR was performed to analyze the relative expression of 20 immune-related genes that were normalized by the use of endogenous genes selected by GeNorm algorithm. Patients with higher expression levels of IL23A and LGALS2 presented better outcomes. In the clustering expression patterns, we observed that patients with higher expression of immunoregulatory genes had better survival rates. Additionally, these data were used to develop a gene expression score. Since CTLA4 and PD1 were associated with prognosis based on Cox regression analysis (Z-score > 1.5), a multivariate model including these two genes was created. Absolute regression coefficients from this analysis were used in order to calculate the immune-checkpoint score: (PD1×0.116) + (CTLA4×0.059) for each case. Kaplan–Meier survival analysis showed that patients with high immune-checkpoint score have longer overall survival (OS) [NR vs. 40.4 mo, p = 0.008] and longer relapse-free survival (RFS) [82.6 vs. 23 mo, p = 0.009]. Multivariate analysis in the entire cohort indicated that the immune-checkpoint score was an independent biomarker of prognosis for OS [HR: 0.308; 95% CI, 0.156–0.609; p = 0.001] and RFS [HR: 0.527; 95% CI, 0.298–0.933; p = 0.028] in early-stage NSCLC patients. In conclusion, this score provides relevant prognostic information for a better characterization of early stage NSCLS patients with strikingly different outcomes and who may be candidates for immune-based therapies.

Abstract 4330: Immune checkpoint expression score is an independent prognostic biomarker in resectable non-small cell lung cancer

BACKGROUND Immune checkpoints blockade, which activate antitumor immunity, has demonstrated promising clinical results in NSCLC. In this study we have investigated the prognostic role of immune checkpoint expression markers and its correlation with immune-cells infiltration and clinico-pathological characteristics in a cohort of resectable NSCLC patients. MATERIAL AND METHODS RNA was isolated from fresh-frozen lung specimens (tumor and normal lung) (n = 178). RTqPCR was performed to analyze the expression of CTLA-4, PD-1 and PD-L1 by the use of hydrolysis probes. Relative gene expression was assessed by Pfaffl formula and normalized by the use of CDKN1B, GUS and ACTB as endogenous genes (selected by GeNorm algorithm). These data was used to develop a gene expression score. Furthermore, the presence of CD4+, CD8+ and FOXP3+ lymphocytes was also assessed in FFPE samples from 63 of these patients by immunohistochemistry (IHC). All statistical analysis were considered significant at p RESULTS Patient9s median age was 65 years [26-85], 86.5% were male and 43.8% were adenocarcinomas (ADC). Since CTLA-4 and PD-1 were moderately associated with prognosis based on COX regression analysis (|Z-score| CONCLUSIONS The immune checkpoint score based on the expression levels of CTLA-4 and PD-1 correlates with the presence of CD8+ infiltrating lymphocytes in the tumor microenvironment. This score provides relevant prognostic information for a better characterization of early-stage NSCLC patients with strikingly different outcomes who may be candidates for immune-based therapies. Supported by grants PS09-01149 and RD12/0036/0025 from ISCIII. Citation Format: Marta Uso, Eloisa Jantus-Lewintre, Rafael Sirera, Silvia Calabuig, Enrique Pastor, Jeronimo Forteza, Carlos Camps. Immune checkpoint expression score is an independent prognostic biomarker in resectable non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4330. doi:10.1158/1538-7445.AM2015-4330

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.

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.

Analysis of the prognostic role of regulatory T-lymphocyte-associated marker expression in resectable NSCLC.

10643 Background: Regulatory T-lymphocytes (Tregs) play a critical role in immune tolerance to tumor cells. Several molecules, including CD4, CD25, CD127, CTLA-4, IL-10 and TGFβ1 have been reported markers of Tregs. Another 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 determine the expression of Treg-associated markers by RT-PCR and to correlate them with clinicopathological and prognostic variables in NSCLC. Methods: RNA was obtained from frozen lung specimens (tumor and normal lung) from resectable NSCLC patients (n = 135). RT-PCR was performed to analyze the expression of: CD4, CTLA-4, FoxP3, IL10, CD25, CD127 and TGFβ1. Relative expression was normalized by an endogenous gene (GUS) using the Pfaffl formulae. Statistical analyses were considered significant at p < 0.05. Results: Compared to normal lung tissue, tumor samples had significantly higher expression of CD25 (x2.1) an...