Resectable Non-Small Cell Lung Cancer Heterogeneity and Recurrence Assessed by Tissue Next-Generation Sequencing Genotyping and Circulating Tumor Cell EZH2 Characterization.

INTRODUCTION: Non-small cell lung cancer (NSCLC) is the most common type of lung neoplasm. Despite surgical resection, it has a high relapse rate, accounting for 30-55% of all cases. Next-generation sequencing (NGS) based on a customized gene panel and the analysis of circulating tumor cells (CTCs) can help identify heterogeneity, stratify high-risk patients, and guide treatment decisions. In this descriptive study involving a small prospective cohort, we focus on the phenotypic characterization of CTCs, particularly concerning EZH2 expression (a member of the Polycomb Repression Complex 2), as well as on the mutation profiles of the tissue using a customized gene panel and their association with poor outcomes in NSCLC. METHODS: Isolation and characterization of EZH2 on CTCs were evaluated before surgical resection (CTC1) and one month after surgery (CTC2) in resectable NSCLC patients. Targeted NGS was performed using a customized 50-gene panel on tissue samples from a subset of patients. RESULTS: 76 patients with resectable NSCLC were recruited. The top mutated genes in the cohort included TP53, FLT1, MUC5AC, EGFR, and NLRP3. Pair of genes that had mutually exclusive mutations was TP53-RIN3, and pairs of genes with co-occurring mutations were CD163-TLR4, FGF10-FOXP2, ADAMTSL3-FLT1, ADAMTSL3-MUC5AC and MUC5AC-NLRP3. CTCs decreased significantly between the two time points CTC1 and CTC2 (p<0.0001), and CTCs+ patients with high EZH2 expression had an 87% increased risk of death (p=0.018). CONCLUSIONS: Integrating molecular profiling of tumors and CTC characterization can provide valuable insights into tumor heterogeneity and improve patient stratification for resectable NSCLC.

Unveiling the impact of circulating tumor cells: Two decades of discovery and clinical advancements in solid tumors.

Circulating tumor cells (CTCs) enumeration and molecular profiling hold promise in revolutionizing the management of solid tumors. Their understanding has evolved significantly over the past two decades, encompassing pivotal biological discoveries and clinical studies across various malignancies. While for some tumor types, such as breast, prostate, and colorectal cancer, CTCs are ready to enter clinical practice, for others, additional research is required. CTCs serve as versatile biomarkers, offering insights into tumor biology, metastatic progression, and treatment response. This review summarizes the latest advancements in CTC research and highlights future directions of investigation. Special attention is given to concurrent evaluations of CTCs and other circulating biomarkers, particularly circulating tumor DNA. Multi-analyte assessment holds the potential to unlock the full clinical capabilities of liquid biopsy. In conclusion, CTCs represent a transformative biomarker in precision oncology, offering extraordinary opportunities to translate scientific discoveries into tangible improvements in patient care.

Baseline extracellular vesicle miRNA-30c and autophagic CTCs predict chemoradiotherapy resistance and outcomes in patients with lung cancer.

Concurrent chemoradiotherapy (cCRT) is the mainstay of treatment for patients diagnosed with locally advanced non-small cell lung cancer (NSCLC). One significant challenge in the effectiveness of this therapy is the potential development of resistance mechanisms, where autophagy up-regulation has been proposed as a key contributing factor. However, there is a lack of reliable biomarkers to predict outcomes on these patients. Interestingly, for addressing this gap, extracellular vesicles (EVs) and circulating tumor cells (CTCs) have emerged as potential sources of such biomarkers. In this study, we investigated EV-associated miRNAs and presence of autophagic CTCs in prospectively collected serial samples from 38 patients with stage III NSCLC undergoing cCRT. Our findings revealed that non-responders exhibited low levels of baseline EV miR-375, miR-200c, and miR-30c. In particular, EV miR-30c showed high predictive value with an area under the curve of 87.2%. Low EV miR-30c and the presence of autophagic-activated CTCs emerged as independent predictive biomarkers for shorter relapse-free survival and overall survival. Furthermore, in experimental models simulating the effects of chemo- and radiotherapy, the administration of miR-30c, either through direct transfection or encapsulation into human EVs, led to the inhibition of autophagy in these cells. This is the first report demonstrating that EV miR-30c inhibits tumor autophagy and its quantification, together with autophagic-activated CTCs, could be used as biomarkers for the stratification and monitoring of patients with NSCLC undergoing cCRT, and they may hold promising potential for guiding subsequent consolidation treatment with immunotherapy or other novel therapies based on autophagy inhibitors.

Circulating tumor cells in cancer-risk populations as a cancer interception tool.

Cancer interception (CI) is a new approach to cancer prevention and treatment in a cancer-risk population that aims to detect and treat pre-tumoral stages. It has several potential advantages over traditional cancer diagnosis and monitoring methods because it is non-invasive, making it less painful and risky than conventional biopsy procedures. The circulating tumor cells (CTCs), liquid biopsy family members, are essential for the CI approach; then, the liquid biopsy (LB) is used as a CI tool. LB can be performed frequently because of its easy sampling and early pathological stages, which allow repeated non-invasive monitoring of cancer progression and response to treatment. CTCs have been found in the bloodstream of several types of cancer patients, including in early-stage cancer and premalignant lesions, suggesting a tumor development role in cancer's early stages. This chapter will present foundational scientific studies addressing CI and the clinical impact of CTC screening in a population at risk for cancer.

Los Servicios de Salud Públicos de la Región Metropolitana (RM) cuentan con 9 camas psiquiátricas de corta estadía por 100.000 habitantes mayores de 18 años, insuficiente para la necesidad actual / The Public Health Services at the Metropolitan Region (MR) of Chile have nine acute psychiatric beds per 100,000 over 18 years old inhabitants, insufficient for current need Alfonso González1 2

INTRODUCCIÓN: Los Servicios de Salud Públicos de la Región Metropolitana (RM) cuentan con 9 camas psiquiátricas de corta estadía por 100.000 habitantes adultos, por debajo de las recomendaciones internacionales. OBJETIVO: El presente estudio evaluará la capacidad de resolución del principal Servicio de Urgencias Psiquiátricas de la RM lo que puede ser de utilidad para evaluar el impacto de la disponibilidad de camas de corta estadía en la RM. MATERIALES Y MÉTODO: Se realizó un estudio observacional retrospectivo de todas las atenciones realizadas en el Servicio de Urgencias del Instituto Psiquiátrico "Dr. José Horwitz B." entre los años 2017 y 2020 y las indicaciones de hospitalización y su resolución. Se obtuvieron Razones de Tasas de Incidencia crudas y ajustadas para la indicación de hospitalización, las efectuadas y aquellas rechazadas por falta de vacantes. RESULTADOS: Se realizaron 90.464 atenciones a 41.541 usuarios y se indicó la hospitalización al 12,5% de ellas. La hospitalización se efectúa en el 59,5% de las atenciones y 35,9% no se pueden realizar por falta de vacantes. Al comparar las Tasas de Incidencia ajustadas se observó solamente una mayor tasa de hospitalización efectuada para los usuarios de regiones (IRR = 1,27; IC95%: 1,11-1,44; valor-p < 0,001) y durante el primer semestre de 2020 (IRR = 1,49; IC95%: 1,35-1,65; valor-p < 0,001). CONCLUSIONES: La evidente demanda por las hospitalizaciones psiquiátricas y la baja disponibilidad de camas de corta estadía en la Región Metropolitana probablemente tiene consecuencias insospechadas. Su abordaje es un desafío que requiere de una planificación multinivel entre todos los actores involucrados.

BACKGROUND: The Public Health Services at the Metropolitan Region (MR) of Chile have nine acute psychiatric beds per 100,000 inhabitants, under international recommendations. AIM: The present study will evaluate the resolution capacity of the main MR Psychiatric Emergency Room (PER), which may help assess the impact of the availability of acute beds in the MR. MATERIAL AND METHODS: A retrospective observational study of electronic patient records for all adult patients attending PER of the Psychiatric Institute "Dr. José Horwitz B." between 2017 and 2020 was analyzed. Crude and adjusted Incidence Rate Ratios were obtained for the indication of hospitalization, admissions, and those rejected due to lack of acute psychiatric beds. RESULTS: 90,464 attendances were evaluated on 41,541 patients, and hospitalization was indicated for 12.5% of them. Admissions were carried out in 59.5%, and 35.9% did not occur due to a lack of acute psychiatric beds. When comparing the adjusted Incidence Rates, only a higher hospitalization rate was observed for users from regions (IRR = 1,267; 95% CI: 1,11-1,44; p-value < 0.001) and during the first half of 2020 (IRR = 1.49; CI95%: 1.35-1.65; p-value < 0.001). CONCLUSIONS: The demand for psychiatric hospitalizations and the low availability of acute psychiatric beds in the MR probably have unsuspected consequences. The solution requires multilevel planning among all the actors involved.

microRNAs signature in relapse metastasis and de novo metastasis of breast cancer. A systematic review.

miRNAs have been widely identified as important players in cancer development and progression. Metastasis in breast cancer can occur as relapse of a treated primary tumour or at the time of diagnosis of the tumour. The aim of this review is to show if both metastasis are different molecular entities characterised by different miRNA signatures that could be studied as specific biomarkers for each entity. For this, we systematically searched the PubMed, Scopus and Web of Science databases. After searching and reviewing the literature, a total of 30 records were included in this review. Results showed a genetic signature including a total of 5 upregulated miRNAs in metastasis compared with early stages. Of them, miR-23b and miR-200c were exclusively present in relapse metastasis. Finally, we proposed a molecular signature for future studies that can be used as a complementary tool at clinical trials for the diagnosis and characterization of metastasis.

Quantification and Characterization of CTCs and Clusters in Pancreatic Cancer by Means of the Hough Transform Algorithm.

Circulating Tumor Cells (CTCs) are considered a prognostic marker in pancreatic cancer. In this study we present a new approach for counting CTCs and CTC clusters in patients with pancreatic cancer using the IsofluxTM System with the Hough transform algorithm (Hough-IsofluxTM). The Hough-IsofluxTM approach is based on the counting of an array of pixels with a nucleus and cytokeratin expression excluding the CD45 signal. Total CTCs including free and CTC clusters were evaluated in healthy donor samples mixed with pancreatic cancer cells (PCCs) and in samples from patients with pancreatic ductal adenocarcinoma (PDAC). The IsofluxTM System with manual counting was used in a blinded manner by three technicians who used Manual-IsofluxTM as a reference. The accuracy of the Hough-IsofluxTM approach for detecting PCC based on counted events was 91.00% [84.50, 93.50] with a PCC recovery rate of 80.75 ± 16.41%. A high correlation between the Hough-IsofluxTM and Manual-IsofluxTM was observed for both free CTCs and for clusters in experimental PCC (R2 = 0.993 and R2 = 0.902 respectively). However, the correlation rate was better for free CTCs than for clusters in PDAC patient samples (R2 = 0.974 and R2 = 0.790 respectively). In conclusion, the Hough-IsofluxTM approach showed high accuracy for the detection of circulating pancreatic cancer cells. A better correlation rate was observed between Hough-IsofluxTM approach and with the Manual-IsofluxTM for isolated CTCs than for clusters in PDAC patient samples.

Baseline extracellular vesicle TGF-ß is a predictive biomarker for response to immune checkpoint inhibitors and survival in non-small cell lung cancer.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) are an effective therapeutic strategy, improving the survival of patients with lung cancer compared with conventional treatments. However, novel predictive biomarkers are needed to stratify which patients derive clinical benefit because the currently used and highly heterogenic histological PD-L1 has shown low accuracy. Liquid biopsy is the analysis of biomarkers in body fluids and represents a minimally invasive tool that can be used to monitor tumor evolution and treatment effects, potentially reducing biases associated with tumor heterogeneity associated with tissue biopsies. In this context, cytokines, such as transforming growth factor-ß (TGF-ß), can be found free in circulation in the blood and packaged into extracellular vesicles (EVs), which have a specific delivery tropism and can affect in tumor/immune system interaction. TGF-ß is an immunosuppressive cytokine that plays a crucial role in tumor immune escape, treatment resistance, and metastasis. Thus, we aimed to evaluate the predictive value of circulating and EV TGF-ß in patients with non-small-cell lung cancer receiving ICIs. METHODS: Plasma samples were collected in 33 patients with advanced non-small-cell lung cancer before and during treatment with ICIs. EV were isolated from plasma by serial ultracentrifugation methods and circulating and EV TGF-ß expression levels were evaluated by enzyme-linked immunosorbent assay. RESULTS: Baseline high expression of TGF-ß in EVs was associated with nonresponse to ICIs as well as shorter progression-free survival and overall survival, outperforming circulating TGF-ß levels and tissue PD-L1 as a predictive biomarker. CONCLUSION: If validated, EV TGF-ß could be used to improve patient stratification, increasing the effectiveness of treatment with ICIs and potentially informing combinatory treatments with TGF-ß blockade. PLAIN LANGUAGE SUMMARY: Treatment with immune-checkpoint inhibitors (ICIs) has improved the survival of some patients with lung cancer. However, the majority of patients do not benefit from this treatment, making it essential to develop more reliable biomarkers to identify patients most likely to benefit. In this pilot study, the expression of transforming growth factor-ß (TGF-ß) in blood circulation and in extracellular vesicles was analyzed. The levels of extracellular vesicle TGF-ß before treatment were able to determine which patients would benefit from treatment with ICIs and have a longer survival with higher accuracy than circulating TGF-ß and tissue PD-L1, which is the currently used biomarker in clinical practice.

Microfluidic amperometric immunosensor based on porous nanomaterial towards claudin7 determination for colorectal cancer diagnosis.

In this work, we present a microfluidic amperometric immunosensor for cancer biomarker claudin7 (CLD7) determination in circulating extracellular vesicles (EVs) as well as its validation in colorectal cancer (CC) patients. The device is based on synthetized nanosized MIL-125-NH2 particles, covalently anchored to the central channel of the microfluidic immunosensor. This nanomaterial was employed as efficient platform for anti-CLD7 monoclonal antibodies immobilization for specifically recognize and capture CLD7 in EVs samples. Afterwards, the amount of this trapped CLD7 was quantified by HRP-conjugated anti-CLD7-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude was directly proportional to the level of CLD7 in the sample. This immunosensor, under optimum conditions, gave the limit of detection for CLD7 of 0.1 pg mL-1, with a wide linear range from 2 to 1000 pg mL-1. The results reported herein open up the use of porous open framework platforms for sensing applications for biomedicine and diagnosis.

Hepatocellular carcinoma risk-stratification based on ASGR1 in circulating epithelial cells for cancer interception.

Purpose: Lack of diagnostic and prognostic biomarkers in hepatocellular carcinoma impedes stratifying patients based on their risk of developing cancer. The aim of this study was to evaluate phenotypic and genetic heterogeneity of circulating epithelial cells (CECs) based on asialoglycoprotein receptor 1 (ASGR1) and miR-122-5p expression as potential diagnostic and prognostic tools in patients with hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Methods: Peripheral blood samples were extracted from LC and HCC patients at different disease stages. CECs were isolated using positive immunomagnetic selection. Genetic and phenotypic characterization was validated by double immunocytochemistry for cytokeratin (CK) and ASGR1 or by in situ hybridization with miR-122-5p and CECs were visualized by confocal microscopy. Results: The presence of CECs increased HCC risk by 2.58-fold, however, this was only significant for patients with previous LC (p = 0.028) and not for those without prior LC (p = 0.23). Furthermore, the number of CECs lacking ASGR1 expression correlated significantly with HCC incidence and absence of miR-122-5p expression (p = 0.014; r = 0.23). Finally, overall survival was significantly greater for patients at earlier cancer stages (p = 0.018), but this difference was only maintained in the group with the presence of CECs (p = 0.021) whereas progression-free survival was influenced by the absence of ASGR1 expression. Conclusion: Identification and characterization of CECs by ASGR1 and/or miR-122-5p expression may be used as a risk-stratification tool in LC patients, as it was shown to be an independent prognostic and risk-stratification marker in LC and early disease stage HCC patients.

A Novel, Quick, and Reliable Smartphone-Based Method for Serum PSA Quantification: Original Design of a Portable Microfluidic Immunosensor-Based System.

We describe a versatile, portable, and simple platform that includes a microfluidic electrochemical immunosensor for prostate-specific antigen (PSA) detection. It is based on the covalent immobilization of the anti-PSA monoclonal antibody on magnetic microbeads retained in the central channel of a microfluidic device. Image flow cytometry and scanning electron microscopy were used to characterize the magnetic microbeads. A direct sandwich immunoassay (with horseradish peroxidase-conjugated PSA antibody) served to quantify the cancer biomarker in serum samples. The enzymatic product was detected at -100 mV by amperometry on sputtered thin-film electrodes. Electrochemical reaction produced a current proportional to the PSA level, with a linear range from 10 pg mL-1 to 1500 pg mL-1. The sensitivity was demonstrated by a detection limit of 2 pg mL-1 and the reproducibility by a coefficient of variation of 6.16%. The clinical performance of this platform was tested in serum samples from patients with prostate cancer (PCa), observing high specificity and full correlation with gold standard determinations. In conclusion, this analytical platform is a promising tool for measuring PSA levels in patients with PCa, offering a high sensitivity and reduced variability. The small platform size and low cost of this quantitative methodology support its suitability for the fast and sensitive analysis of PSA and other circulating biomarkers in patients. Further research is warranted to verify these findings and explore its potential application at all healthcare levels.

Variability of Reaction Time as a Marker of Executive Function Impairments in Fibromyalgia.

In addition to chronic widespread pain and depression and anxiety symptoms, patients with fibromyalgia frequently experience cognitive problems. This study investigated executive functions in fibromyalgia via a Go/No-Go task. To obtain comprehensive information about performance, traditional and ex-Gaussian parameters of reaction time (RT) variability were used, in addition to speed and accuracy indices. Ex-Gaussian parameters show an excellent fit to empirical RT distributions. Fifty-two female fibromyalgia patients and twenty-eight healthy controls participated. The task included 60 visual stimuli, which participants had to respond to (Go stimuli) or withhold the response to (No-Go stimuli). After 30 trials, the task rule changed, such that previous No-Go stimuli had to be responded to. Performance was indexed by the hit rate, false alarm rate, and mean (M) and intraindividual standard deviation (SD) of RT and the ex-Gaussian parameters mu, sigma, and tau. Mu and sigma indicate the M and SD of the Gaussian distribution; tau reflects the M and SD of the exponential function. Patients exhibited a lower hit rate, higher M RT, and higher tau than controls. Moreover, patients showed greater decrease of the hit rate after the change of task rule. In the entire sample, SD, sigma, and tau were inversely associated with the hit rate and positively associated with the false alarm rate. While the greater decline in hit rate after the change in task rule indicates deficient cognitive flexibility, the lack of any difference in false alarm rate suggests intact response inhibition. Higher M RT reflects reduced cognitive or motor speed. Increased tau in fibromyalgia indicates greater fluctuations in executive control and more frequent temporary lapses of attention. For the first time, this study demonstrated that indices of RT variability, in particular those derived from the ex-Gaussian function, may complement speed and accuracy parameters in the assessment of executive function impairments in fibromyalgia. Optimized assessment may facilitate the personalization of therapies aimed at improving the cognitive function of those with the disorder.

Fibromyalgia Syndrome and Cognitive Decline: The Role of Body Mass Index and Clinical Symptoms.

The high prevalence of obesity and overweight in fibromyalgia (FM) may be an important factor in the well-known cognitive deficits seen in the disorder. This study analyzed the influence of body mass index (BMI) and primary clinical symptoms of FM (pain, fatigue, insomnia, anxiety, and depression) on attention, memory, and processing speed in FM. Fifty-two FM patients and thirty-two healthy participants completed cognitive tasks assessing selective, sustained, and divided attention; visuospatial and verbal memory; and information processing speed. Furthermore, they were evaluated in terms of the main clinical symptoms of the disorder. FM patients showed a marked reduction of cognitive performance in terms of selective, sustained, and divided attention; visuospatial memory; and processing speed, but no group differences were observed in verbal memory. BMI negatively affects sustained and selective attention, verbal memory, and processing speed and is the main predictor of performance in these basic cognitive domains. Our findings confirm the presence of cognitive deficits with respect to attention and visual memory, as well as slower processing speed, in FM. Moreover, the results support a role of BMI in the observed cognitive deficits. Interventions increasing physical activity and promoting cognitive stimulation could be useful for strengthening cognitive function in FM patients.

Extracellular vesicle PD-L1 dynamics predict durable response to immune-checkpoint inhibitors and survival in patients with non-small cell lung cancer.

BACKGROUND: Immune-checkpoint inhibitors (ICIs) changed the therapeutic landscape of patients with lung cancer. However, only a subset of them derived clinical benefit and evidenced the need to identify reliable predictive biomarkers. Liquid biopsy is the non-invasive and repeatable analysis of biological material in body fluids and a promising tool for cancer biomarkers discovery. In particular, there is growing evidence that extracellular vesicles (EVs) play an important role in tumor progression and in tumor-immune interactions. Thus, we evaluated whether extracellular vesicle PD-L1 expression could be used as a biomarker for prediction of durable treatment response and survival in patients with non-small cell lung cancer (NSCLC) undergoing treatment with ICIs. METHODS: Dynamic changes in EV PD-L1 were analyzed in plasma samples collected before and at 9 ± 1 weeks during treatment in a retrospective and a prospective independent cohorts of 33 and 39 patients, respectively. RESULTS: As a result, an increase in EV PD-L1 was observed in non-responders in comparison to responders and was an independent biomarker for shorter progression-free survival and overall survival. To the contrary, tissue PD-L1 expression, the commonly used biomarker, was not predictive neither for durable response nor survival. CONCLUSION: These findings indicate that EV PD-L1 dynamics could be used to stratify patients with advanced NSCLC who would experience durable benefit from ICIs.

Circulating Tumor Cells Enumeration from the Portal Vein for Risk Stratification in Early Pancreatic Cancer Patients.

BACKGROUND: Effective biomarkers are needed to enable personalized medicine for pancreatic cancer patients. This study analyzes the prognostic value, in early pancreatic cancer, of single circulating tumor cell (CTC) and CTC clusters from the central venous catheter (CVC) and portal blood (PV). METHODS: In total, 7 mL of PV and CVC blood from 35 patients with early pancreatic cancer were analyzed. CTC were isolated using a positive immunomagnetic selection. The detection and identification of CTC were performed by immunocytochemistry (ICC) and were analyzed by Epi-fluorescence and confocal microscopy. RESULTS: CTC and the clusters were detected both in PV and CVC. In both samples, the CTC number per cluster was higher in patients with grade three or poorly differentiated tumors (G3) than in patients with well (G1) or moderately (G2) differentiated. Patients with fewer than 185 CTC in PV exhibited a longer OS than patients with more than 185 CTC (24.5 vs. 10.0 months; p = 0.018). Similarly, patients with fewer than 15 clusters in PV showed a longer OS than patients with more than 15 clusters (19 vs. 10 months; p = 0.004). These significant correlations were not observed in CVC analyses. CONCLUSIONS: CTC presence in PV could be an important prognostic factor to predict poor prognosis in early pancreatic cancer. In addition, the number of clustered-CTC correlate to a tumor negative differentiation degree and, therefore, could be used as a diagnostic biomarker for pancreatic cancer.

Liquid biopsy from research to clinical practice: focus on non-small cell lung cancer.

INTRODUCTION: In the current era of personalized medicine, liquid biopsy has acquired a relevant importance in patient management of advanced stage non-small cell lung cancer (NSCLC). As a matter of fact, liquid biopsy may supplant the problem of inadequate tissue for molecular testing. The term 'liquid biopsy' refers to a number of different biological fluids, but is most clearly associated with plasma-related platforms. It must be taken into account that pre-analytical processing and the selection of the appropriate technology according to the clinical context may condition the results obtained. In addition, novel clinical applications beyond the evaluation of the molecular status of predictive biomarkers are currently under investigation. AREAS COVERED: This review summarizes the available evidence on pre-analytical issues and different clinical applications of liquid biopsies in NSCLC patients. EXPERT OPINION: Liquid biopsy should be considered not only as a valid alternative but as complementary to tissue-based molecular approaches. Careful attention should be paid to the optimization and standardization of all phases of liquid biopsy samples management in order to determine a significant improvement in either sensitivity or specificity, while significant reducing the number of 'false negative' or 'false positive' molecular results.

Next generation sequencing for liquid biopsy based testing in non-small cell lung cancer in 2021.

Lung cancer is the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) representing its most commonly diagnosed sub-type. Despite the significant improvements in lung cancer biomarkers knowledge, accompanied by substantial technological advances in molecular tumor profiling, a considerable fraction (up to 30 %) of advanced NSCLC patient presents with major testing challenges or tissue unavailability for molecular analysis. In this context, liquid biopsy is on the rise, currently gaining considerable interest within the molecular pathology and oncology community. Molecular profiling of liquid biopsy specimens using next generation molecular biology methodologies is a rapidly evolving field with promising applications not exclusively limited to advanced stages but also more recently expanding to early stages cancer patients. Here, we offer an overview of some of the most consolidated and emerging applications of next generation sequencing technologies for liquid biopsy testing in NSCLC.