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

[EN] 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.This work was supported by grant no. CB16/12/00350 from the Centro de Investigacion Biomedica en Red de Cancer (CIBEROnc) and Grant No. PI18/00266 from the Instituto de Salud Carlos III (ISCIII).Herreros-Pomares, A.; Doria, P.; Gallach, S.; Meri-Abad, M.; Guijarro, R.; Calabuig-Fariñas, S.; Camps, C.... (2022). A Sonic Hedgehog Pathway Score to Predict the Outcome of Resected Non-Small Cell Lung Cancer Patients. Annals of Surgical Oncology. 30(2):1225-1235. https://doi.org/10.1245/s10434-022-12565-21225123530

Interplay Between MicroRNAs and Oxidative Stress in Ovarian Conditions with a Focus on Ovarian Cancer and Endometriosis.

Ovarian cancer and endometriosis are two distinct gynaecological conditions that share many biological aspects incuding proliferation, invasion of surrounding tissue, inflammation, inhibition of apoptosis, deregulation of angiogenesis and the ability to spread at a distance. miRNAs are small non-coding RNAs (19-22 nt) that act as post-transcriptional modulators of gene expression and are involved in several of the aforementioned processes. In addition, a growing body of evidence supports the contribution of oxidative stress (OS) to these gynaecological diseases: increased peritoneal OS due to the decomposition of retrograde menstruation blood facilitates both endometriotic lesion development and fallopian tube malignant transformation leading to high-grade serous ovarian cancer (HGSOC). Furthermore, as HGSOC develops, increased OS levels are associated with chemoresistance. Finally, continued bleeding within ovarian endometrioma raises OS levels and contributes to the development of endometriosis-associated ovarian cancer (EAOC). Therefore, this review aims to address the need for a better understanding of the dialogue between miRNAs and oxidative stress in the pathophysiology of ovarian conditions: endometriosis, EAOC and HGSO

An integrated analysis of miRNA and gene copy numbers in xenografts of Ewing's sarcoma

<p>Abstract</p> <p>Background</p> <p>Xenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers.</p> <p>Method</p> <p>Microarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods.</p> <p>Results</p> <p>The most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, <it>EWSR1, FLI1 </it>and their fusion gene (<it>EWS-FLI1</it>). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers.</p> <p>Conclusion</p> <p>In the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, <it>FLI1, EWSR1</it>, and the <it>EWS-FLI1 </it>fusion genes.</p

CD5 and CD6 as immunoregulatory biomarkers in non-small cell lung cancer

Background: The study of immune surveillance in the tumour microenvironment is leading to the development of new biomarkers and therapies. The present research focuses on the expression of CD5 and CD6 - two lymphocyte surface markers involved in the fine tuning of TCR signaling - as potential prognostic biomarkers in resectable stages of non-small cell lung cancer (NSCLC). Methods: CD5 and CD6 gene expression was analysed by reverse transcription quantitative polymerase chain reaction (RTqPCR) in 186 paired fresh frozen tumour and normal tissue samples of resected NSCLC. Results: Patients with higher CD5 expression had significantly increased overall survival (OS, 49.63 vs. 99.90 months, p=0.013). CD5 expression levels were correlated to CD4 infiltration and expression levels, and survival analysis showed that patients with a higher CD5/CD4+ ratio had significantly improved prognosis. Multivariate analysis established CD5 expression as an independent prognostic biomarker for OS in early stages of NSCLC [HR=0.554; 95% CI, 0.360-0.853; p=0.007]. Further survival analysis of NSCLC cases (n=97) from The Cancer Genome Atlas (TCGA) database, confirmed the prognostic value of both CD5 and CD6 expression¸ although CD6 expression alone did not reach significant prognostic value in our NSCLC training cohort. Conclusions: Our data support further studies on CD5 and CD6 as novel prognostic markers in resectable NSCLC and other cancer types (i.e., melanoma), as well as a role for these receptors in immune surveillance

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

[EN] 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.This research was funded by Centro de Investigacion Biomedica en Red de Cancer, grant number CB16-12-00350, and Instituto de Salud Carlos III, grant number PI18/00266.Dong, N.; Moreno-Manuel, A.; Calabuig-Fariñas, S.; Gallach, S.; Zhang, F.; Blasco, A.; Aparisi, F.... (2021). Characterization of Circulating T Cell Receptor Repertoire Provides Information about Clinical Outcome after PD-1 Blockade in Advanced Non-Small Cell Lung Cancer Patients. Cancers. 13(12). https://doi.org/10.3390/cancers13122950131

Soluble galectin-3 as a microenvironment-relevant immunoregulator with prognostic and predictive value in lung adenocarcinoma

Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin-3 (GAL-3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non-small cell lung cancer (NSCLC) patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early-stage patients and commercial cell lines were cultured, using tumorsphere-forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL-3 using reverse transcription-quantitative real-time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry and immunoassay analysis. Our results using three-dimensional (3D) models of lung tumor cells revealed that soluble GAL-3 (sGAL-3) is highly expressed and secreted. To more accurately mimic the TME, a co-culture of tumorspheres and fibroblasts was used, revealing that GAL-3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (TREGS). In the translational phase, we confirmed that patients with high expression levels of GAL-3 had more TREGS, which suggests that tumors may be recruiting this population through GAL-3. Next, we evaluated levels of sGAL-3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL-3 could be used as an independent prognostic biomarker for overall survival and relapse-free survival in early-stage LUAD patients. Additionally, levels of sGAL-3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first-line pembrolizumab were evaluated, further supporting that sGAL-3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve (AUC) of 0.801 (p=0.011). Moreover, high levels might predict decreased progression-free survival and overall survival to anti-PD-1 therapy, with sGAL-3 being a prognosis-independent biomarker for advanced LUAD

Update on systemic treatment in early triple negative breast cancer

Triple negative breast cancer (TNBC) is a heterogeneous disease representing about 15% of all breast cancers. TNBC are usually high-grade histological tumors, and are generally more aggressive and difficult to treat due to the lack of targeted therapies available, and chemotherapy remains the standard treatment. There is a close relationship between pathological complete response after chemotherapy treatment and higher rates of disease-free survival and overall survival. In this review of systemic treatment in early triple negative breast cancer, our purpose is to analyze and compare different therapies, as well as to highlight the novelties of treatment in this breast cancer subtype.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors acknowledge grant CB16-12-00350 from CIBEROnc, the AMACMA foundation, and Lopez Trigo 2017.Medicin

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. Keywords: non-small cell lung cancer; liquid biopsy; exosomes; extracellular vesicles; cell cultures; adenocarcinoma; squamous cell carcinoma; biomarker; tumorsphere

Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells

Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.panishgovernment and the EU (FIS PI13/00089, FIS PI12/02838,FIS PI12/00956 and RD12/0036/0025), a grant from LaMaratóde TV3 Foundation (20132730), a grant from SEPAR(17/2014), Consejería de Educación de la Junta de Castilla yLeón (Project BU340U13), Ministerio de Economíaycompetitividad/Instituto de Salud Carlos III (SAF2014-55700-P), and ICREA Academia-201

Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients

MET alterations may provide a potential biomarker to evaluate patients who will benefit from treatment with MET inhibitors. Therefore, the purpose of the present study is to investigate the utility of a liquid biopsy-based strategy to assess MET alterations in cancer patients. We analyzed MET amplification in circulating free DNA (cfDNA) from 174 patients with cancer and 49 healthy controls and demonstrated the accuracy of the analysis to detect its alteration in patients. Importantly, a significant correlation between cfDNA concentration and MET copy number (CN) in cancer patients (r = 0.57, p <10−10) was determined. Furthermore, we evaluated two approaches to detect the presence of MET on circulating tumor cells (CTCs), using the CellSearch® and Parsortix systems and monitored patients under anti-EGFR treatment (n = 30) combining both cfDNA and CTCs analyses. This follow-up provides evidence for the potential of MET CN assessment when patients develop resistance to anti-EGFR therapy and a significant association between the presence of CTCs MET+ and the Overall Survival (OS) in head and neck cancer patients (P = 0.05; HR = 6.66). In conclusion, we develop specific and noninvasive assays to monitor MET status in cfDNA/CTCs and demonstrate the utility of plasma MET CN determination as a biomarker for monitoring the appearance of resistance to anti-EGFR therapyThis study was financed by all the donors who participated in the Liquid Biopsy Crowdfunding campaign in 2017. LMR is supported by Asociación Española Contra el Cancer (AECC). ADL is funded by a “Juan Rodés” contract (JR17/00016) from ISCIIIS