A Sonic Hedgehog Pathway Score to Predict the Outcome of Resected Non-Small Cell Lung Cancer Patients.

BACKGROUND: Mutations and deregulations in components of the Hedgehog (Hh) pathway have been associated with cancer onset and tumor growth in different malignancies, but their role in non-small cell lung cancer (NSCLC) remains unclear. This study aims to investigate the expression pattern of the main components of the Hh pathway in tumor and adjacent normal tissue biopsies of resected NSCLC patients. METHODS: The relative expression of GLI1, PTCH1, SHH, and SMO was analyzed by quantitative polymerase chain reaction (PCR) in a cohort of 245 NSCLC patients. Results were validated in an independent cohort of NSCLC patients from The Cancer Genome Atlas (TCGA). RESULTS: We found that SMO and GLI1 were overexpressed in the tumor compared with normal-paired tissue, whereas PTCH1 and SHH were underexpressed. In addition, patients with higher expression levels of PTCH1 presented better outcomes. A gene expression score, called the Hedgehog Score, was calculated using a multivariable model including analyzed components of the Hh signaling pathway. NSCLC patients with a high Hedgehog Score had significantly shorter relapse-free survival (RFS) and overall survival (OS) than patients with a low score, especially at stage I of the disease. Similarly, patients in the adenocarcinoma (ADC) subcohort had shorter RFS and OS. Multivariate Cox analysis exhibited that the Hedgehog Score is an independent prognostic biomarker for OS in both the entire training cohort and the ADC subcohort. The Hedgehog Score was validated in an independent cohort of NSCLC patients from TCGA, which confirmed its prognostic value. CONCLUSIONS: Our results provide relevant prognostic data for NSCLC patients and support further studies on the Hh pathway.

Circulating Immune Proteins: Improving the Diagnosis and Clinical Outcome in Advanced Non-Small Cell Lung Cancer.

Immunotherapy has been proven a viable treatment option for non-small cell lung cancer (NSCLC) treatment in patients. However, some patients still do not benefit. Finding new predictive biomarkers for immunocheckpoint inhibitor (ICI) response will improve treatment management in the clinical routine. In this regard, liquid biopsy is a useful and noninvasive alternative to surgical biopsies. In the present study, we evaluated the potential diagnostic, prognostic, and predictive value of seven different soluble mediators involved in immunoregulation. Fifty-two plasma samples from advanced NSCLC treated in first-line with pembrolizumab at baseline (PRE) and at first response assessment (FR) were analyzed. In terms of diagnostic value, our results revealed that sFGL1, sGAL-3, and sGAL-1 allowed for optimal diagnostic efficacy for cancer patients. Additionally, the combination of sFGL1 and sGAL-3 significantly improved diagnostic accuracy. Regarding the predictive value to assess patients' immune response, sCD276 levels at PRE were significantly higher in patients without tumor response (p = 0.035). Moreover, we observed that high levels of sMICB at PRE were associated with absence of clinical benefit (pembrolizumab treatment less than 6 months) (p = 0.049), and high levels of sMICB and sGAL-3 at FR are also related to a lack of clinical benefit (p = 0.027 and p = 0.03, respectively). Finally, in relation to prognosis significance, at PRE and FR, sMICB levels above the 75th percentile are related to poor progression-free survival (PFS) (p = 0.013 and p = 0.023, respectively) and overall survival (OS) (p = 0.001 and p = 0.011, respectively). An increase in sGAL3 levels at FR was associated with worse PFS (p = 0.037). Interestingly, high sGAL-3 at PRE was independently associated with PFS and OS with a hazard ratio (HR) of 2.45 (95% CI 1.14-5.25; p = 0.021) and 4.915 (95% CI 1.89-12.73; p = 0.001). In conclusion, plasma levels of sFGL1, sGAL-3, and sGAL-1 could serve as diagnostic indicators and sMICB, sCD276, and sGAL3 were linked to outcomes, suggesting their potential in assessing NSCLC under pembrolizumab treatment. Our results highlight the value of employing soluble immune biomarkers in advanced lung cancer patients treated with pembrolizumab at first-line.

A gene signature combining the tissue expression of three angiogenic factors is a prognostic marker in early-stage non-small cell lung cancer.

BACKGROUND: 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. RESULTS: Univariate 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. CONCLUSIONS: The 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 Exosomal Cargo Provides Accurate Clinical, Histologic and Mutational Information in Non-Small Cell Lung Cancer.

Lung cancer is a malignant disease with high mortality and poor prognosis, frequently diagnosed at advanced stages. Nowadays, immense progress in treatment has been achieved. However, the present scenario continues to be critical, and a full comprehension of tumor progression mechanisms is required, with exosomes being potentially relevant players. Exosomes are membranous vesicles that contain biological information, which can be transported cell-to-cell and modulate relevant processes in the hallmarks of cancer. The present research aims to characterize the exosomes' cargo and study their role in NSCLC to identify biomarkers. We analyzed exosomes secreted by primary cultures and cell lines, grown in monolayer and tumorsphere formations. Exosomal DNA content showed molecular alterations, whereas RNA high-throughput analysis resulted in a pattern of differentially expressed genes depending on histology. The most significant differences were found in XAGE1B, CABYR, NKX2-1, SEPP1, CAPRIN1, and RIOK3 genes when samples from two independent cohorts of resected NSCLC patients were analyzed. We identified and validated biomarkers for adenocarcinoma and squamous cell carcinoma. Our results could represent a relevant contribution concerning exosomes in clinical practice, allowing for the identification of biomarkers that provide information regarding tumor features, prognosis and clinical behavior of the disease.

Oral microbiome in Proliferative Verrucous Leukoplakia exhibits loss of diversity and enrichment of pathogens.

OBJECTIVES: Oral microbiome plays an important role in oral diseases. Among them, proliferative verrucous leucoplakia (PVL) is an uncommon form of progressive multifocal leukoplakia with a worryingly rate of malignant transformation. Here, we aimed to characterize the oral microbiome of PVL patients and compare it with those of healthy controls. MATERIAL AND METHODS: Oral biopsies from ten PVL patients and five healthy individuals were obtained and used to compare their microbial communities. The sequence of the V3-V4 region of 16S rRNA gene was used as the taxonomic basis to estimate and analyze the composition and diversity of bacterial populations present in the samples. RESULTS: Our results show that the oral microbial composition and diversity are significantly different among PVL patients and healthy donors. The average number of observed operational taxonomic units (OTUs) was higher for healthy donors than for PVL, proving a loss of diversity in PVL. Several OTUs were found to be more abundant in either group. Among those that were significantly enriched in PVL patients, potential protumorigenic pathogens like Oribacterium sp. oral taxon 108, Campylobacter jejuni, uncultured Eubacterium sp., Tannerella, and Porphyromonas were identified. CONCLUSION: Oral microbiome dysbiosis was found in patients suffering from PVL. To the best of our knowledge, this is the first study investigating the oral microbiome alterations in PVL and, due to the limited number of participants, additional studies are needed. Oral microbiota-based biomarkers may be helpful in predicting the risks for the development of PVL.

Characterization of Circulating T Cell Receptor Repertoire Provides Information about Clinical Outcome after PD-1 Blockade in Advanced Non-Small Cell Lung Cancer Patients.

Despite the success of immunotherapies in lung cancer, development of new biomarkers for patient selection is urgently needed. This study aims to explore minimally invasive approaches to characterize circulating T cell receptor beta chain (TCR-ß) repertoire in a cohort of advanced non-small cell lung cancer (NSCLC) patients treated with first-line pembrolizumab. Peripheral blood samples were obtained at two time points: i) pretreatment (PRE) and ii) first response assessment (FR). Next-generation sequencing (NGS) was used to analyze the hypervariable complementary determining region 3 (CDR3) of TCR-ß chain. Richness, evenness, convergence, and Jaccard similarity indexes plus variable (V) and joining (J)-gene usage were studied. Our results revealed that increased richness during treatment was associated with durable clinical benefit (DCB; p = 0.046), longer progression-free survival (PFS; p = 0.007) and overall survival (OS; p = 0.05). Patients with Jaccard similarity index ≥0.0605 between PRE and FR samples showed improved PFS (p = 0.021). Higher TRBV20-1 PRE usage was associated with DCB (p = 0.027). TRBV20-1 levels ≥9.14% in PRE and ≥9.02% in FR significantly increased PFS (p = 0.025 and p = 0.016) and OS (p = 0.035 and p = 0.018). Overall, analysis of circulating TCR-ß repertoire may provide information about the immune response in anti-PD-1 treated NSCLC patients; in this scenario, it can also offer important information about the clinical outcome.

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

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.

MicroRNAs: promising new antiangiogenic targets in cancer.

MicroRNAs are one class of small, endogenous, non-coding RNAs that are approximately 22 nucleotides in length; they are very numerous, have been phylogenetically conserved, and involved in biological processes such as development, differentiation, cell proliferation, and apoptosis. MicroRNAs contribute to modulating the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules and so they play a key role in both health and disease. Angiogenesis is the process of new blood vessel formation from preexisting ones, which is particularly relevant to cancer and its progression. Over the last few years, microRNAs have emerged as critical regulators of signalling pathways in multiple cell types including endothelial and perivascular cells. This review summarises the role of miRNAs in tumour angiogenesis and their potential implications as therapeutic targets in cancer.

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

AIM: Telomeres 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). METHODS: We 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. RESULTS: Peripheral 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. CONCLUSIONS: The 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.

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.

JunB breakdown in mid-/late G2 is required for down-regulation of cyclin A2 levels and proper mitosis.

JunB, a member of the AP-1 family of dimeric transcription factors, is best known as a cell proliferation inhibitor, a senescence inducer, and a tumor suppressor, although it also has been attributed a cell division-promoting activity. Its effects on the cell cycle have been studied mostly in G1 and S phases, whereas its role in G2 and M phases still is elusive. Using cell synchronization experiments, we show that JunB levels, which are high in S phase, drop during mid- to late G2 phase due to accelerated phosphorylation-dependent degradation by the proteasome. The forced expression of an ectopic JunB protein in late G2 phase indicates that JunB decay is necessary for the subsequent reduction of cyclin A2 levels in prometaphase, the latter event being essential for proper mitosis. Consistently, abnormal JunB expression in late G2 phase entails a variety of mitotic defects. As these aberrations may cause genetic instability, our findings contrast with the acknowledged tumor suppressor activity of JunB and reveal a mechanism by which the deregulation of JunB might contribute to tumorigenesis.