Rare variants in Toll-like receptor 7 results in functional impairment and downregulation of cytokine-mediated signaling in COVID-19 patients.

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity.

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management.

A pilot study of next generation sequencing-liquid biopsy on cell-free DNA as a novel non-invasive diagnostic tool for Klippel-Trenaunay syndrome.

OBJECTIVES: Somatic mosaicism of PIK3CA gene is currently recognized as the molecular driver of Klippel-Trenaunay syndrome. However, given the limitation of the current technologies, PIK3CA somatic mutations are detected only in a limited proportion of Klippel-Trenaunay syndrome cases and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next generation sequencing liquid biopsy using cell-free DNA has emerged as an innovative non-invasive approach for early detection and monitoring of cancer. This approach, overcoming the space-time profile constraint of tissue biopsies, opens a new scenario also for others diseases caused by somatic mutations. METHODS: In the present study, we performed a comprehensive analysis of seven patients (four females and three males) with Klippel-Trenaunay syndrome. Blood samples from both peripheral and efferent vein from malformation were collected and cell-free DNA was extracted from plasma. Tissue biopsies from vascular lesions were also collected when available. Cell-free DNA libraries were performed using Oncomine™ Pan-Cancer Cell-Free Assay. Ion Proton for sequencing and Ion Reporter Software for analysis were used (Life Technologies, Carlsbad, CA, USA). RESULTS: Cell-free circulating DNA analysis revealed pathogenic mutations in PIK3CA gene in all patients. The mutational load was higher in plasma obtained from the efferent vein at lesional site (0.81%) than in the peripheral vein (0.64%) leading to conclude for a causative role of the identified variants. Tissue analysis, available for one amputated patient, confirmed the presence of the mutation at the malformation site at a high molecular frequency (14-25%), confirming its causative role. CONCLUSIONS: Our data prove for the first time that the cell-free DNA-next generation sequencing-liquid biopsy, which is currently used exclusively in an oncologic setting, is indeed the most effective tool for Klippel-Trenaunay syndrome diagnosis and tailored personalized treatment.

C9orf72 Intermediate Repeats Confer Genetic Risk for Severe COVID-19 Pneumonia Independently of Age.

A cytokine storm, autoimmune features and dysfunctions of myeloid cells significantly contribute to severe coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Genetic background of the host seems to be partly responsible for severe phenotype and genes related to innate immune response seem critical host determinants. The C9orf72 gene has a role in vesicular trafficking, autophagy regulation and lysosome functions, is highly expressed in myeloid cells and is involved in immune functions, regulating the lysosomal degradation of mediators of innate immunity. A large non-coding hexanucleotide repeat expansion (HRE) in this gene is the main genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), both characterized by neuroinflammation and high systemic levels of proinflammatory cytokines, while HREs of intermediate length, although rare, are more frequent in autoimmune disorders. C9orf72 full mutation results in haploinsufficiency and intermediate HREs seem to modulate gene expression as well and impair autophagy. Herein, we sought to explore whether intermediate HREs in C9orf72 may be a risk factor for severe COVID-19. Although we found intermediate HREs in only a small portion of 240 patients with severe COVID-19 pneumonia, the magnitude of risk for requiring non-invasive or mechanical ventilation conferred by harboring intermediate repeats >10 units in at least one C9orf72 allele was more than twice respect to having shorter expansions, when adjusted for age (odds ratio (OR) 2.36; 95% confidence interval (CI) 1.04-5.37, p = 0.040). The association between intermediate repeats >10 units and more severe clinical outcome (p = 0.025) was also validated in an independent cohort of 201 SARS-CoV-2 infected patients. These data suggest that C9orf72 HREs >10 units may influence the pathogenic process driving more severe COVID-19 phenotypes.

PIK3CA-CDKN2A clonal evolution in metastatic breast cancer and multiple points cell-free DNA analysis.

BACKGROUND: Daily experience tells us that breast cancer can be controlled using standard protocols up to the advent of a relapse. Now new frontiers in precision medicine like liquid biopsy of cell free DNA (cfDNA) give us the possibility to understand cancer evolution and pick up the key mutation on specific cancer driver gene. However, tight schedule of standardized protocol may impair the use of personalized experimental drugs in a timely therapeutic window. MAIN BODY: Here, using a combination of deep next generation sequencing and cfDNA liquid biopsy, we demonstrated that it is possible to monitor cancer relapse over time. We showed for the first time the exact correspondence from the increasing clonal expansion and clinical worsening of metastatic breast cancer. CONCLUSION: Thanks to liquid biopsy may be possible to introduce new experimental drugs in the correct therapeutic window which would lead in the near future to an effective treatment which otherwise remains challenging.

iPSC-derived neurons profiling reveals GABAergic circuit disruption and acetylated α-tubulin defect which improves after iHDAC6 treatment in Rett syndrome.

Mutations in MECP2 gene have been identified in more than 95% of patients with classic Rett syndrome, one of the most common neurodevelopmental disorders in females. Taking advantage of the breakthrough technology of genetic reprogramming, we investigated transcriptome changes in neurons differentiated from induced Pluripotent Stem Cells (iPSCs) derived from patients with different mutations. Profiling by RNA-seq in terminally differentiated neurons revealed a prominent GABAergic circuit disruption along with a perturbation of cytoskeleton dynamics. In particular, in mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with selective inhibitors of HDAC6, the main α-tubulin deacetylase. These findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the treatment of Rett-associated epileptic behavior as well as for the definition of new therapeutic strategies for Rett syndrome.

Urine-derived podocytes-lineage cells: A promising tool for precision medicine in Alport Syndrome.

Alport Syndrome (ATS) is a rare genetic disorder caused by collagen IV genes mutations, leading to glomerular basement membrane damage up to end-stage renal disease. Podocytes, the main component of the glomerular structure, are the only cells able to produce all the three collagens IV alpha chains associated with ATS and thus, they are key players in ATS pathogenesis. However, podocytes-targeted therapeutic strategies have been hampered by the difficulty of non-invasively isolating them and transcripts-based diagnostic approaches are complicated by the inaccessibility of other COL4 chains-expressing cells. We firstly isolated podocyte-lineage cells from ATS patients' urine samples, in a non-invasive way. RT-PCR analysis revealed COL4A3, COL4A4, and COL4A5 expression. Transcripts analysis on RNA extracted from patient's urine derived podocyte-lineage cells allowed defining the pathogenic role of intronic variants, namely one mutation in COL4A3 (c.3882+5G>A), three mutations in COL4A4 (c.1623+2T>A, c.3699_3706+1del, c.2545+143T>A), and one mutation in COL4A5 (c.3454+2T>C). Therefore, our cellular model represents a novel tool, essential to unequivocally prove the effect of spliceogenic intronic variants on transcripts expressed exclusively at a glomerular level. This process is a key step for providing the patient with a definite molecular diagnosis and with a proper recurrence risk. The established system also opens up the possibility of testing personalized therapeutic approaches on disease-relevant cells.

Mouse pulmonary adenoma susceptibility 1 locus is an expression QTL modulating Kras-4A.

Pulmonary adenoma susceptibility 1 (Pas1) is the major locus responsible for lung tumor susceptibility in mice; among the six genes mapping in this locus, Kras is considered the best candidate for Pas1 function although how it determines tumor susceptibility remains unknown. In an (A/J × C57BL/6)F4 intercross population treated with urethane to induce lung tumors, Pas1 not only modulated tumor susceptibility (LOD score = 48, 69% of phenotypic variance explained) but also acted, in lung tumor tissue, as an expression quantitative trait locus (QTL) for Kras-4A, one of two alternatively spliced Kras transcripts, but not Kras-4B. Additionally, Kras-4A showed differential allelic expression in lung tumor tissue of (A/J × C57BL/6)F4 heterozygous mice, with significantly higher expression from the A/J-derived allele; these results suggest that cis-acting elements control Kras-4A expression. In normal lung tissue from untreated mice of the same cross, Kras-4A levels were also highly linked to the Pas1 locus (LOD score = 23.2, 62% of phenotypic variance explained) and preferentially generated from the A/J-derived allele, indicating that Pas1 is an expression QTL in normal lung tissue as well. Overall, the present findings shed new light on the genetic mechanism by which Pas1 modulates the susceptibility to lung tumorigenesis, through the fine control of Kras isoform levels.

Exome sequencing coupled with mRNA analysis identifies NDUFAF6 as a Leigh gene.

We report here the case of a young male who started to show verbal fluency disturbance, clumsiness and gait anomalies at the age of 3.5years and presented bilateral striatal necrosis. Clinically, the diagnosis was compatible with Leigh syndrome but the underlying molecular defect remained elusive even after exome analysis using autosomal/X-linked recessive or de novo models. Dosage of respiratory chain activity on fibroblasts, but not in muscle, underlined a deficit in complex I. Re-analysis of heterozygous probably pathogenic variants, inherited from one healthy parent, identified the p.Ala178Pro in NDUFAF6, a complex I assembly factor. RNA analysis showed an almost mono-allelic expression of the mutated allele in blood and fibroblasts and puromycin treatment on cultured fibroblasts did not lead to the rescue of the maternal allele expression, not supporting the involvement of nonsense-mediated RNA decay mechanism. Complementation assay underlined a recovery of complex I activity after transduction of the wild-type gene. Since the second mutation was not detected and promoter methylation analysis resulted normal, we hypothesized a non-exonic event in the maternal allele affecting a regulatory element that, in conjunction with the paternal mutation, leads to the autosomal recessive disorder and the different allele expression in various tissues. This paper confirms NDUFAF6 as a genuine morbid gene and proposes the coupling of exome sequencing with mRNA analysis as a method useful for enhancing the exome sequencing detection rate when the simple application of classical inheritance models fails.

Potentially Treatable Disorder Diagnosed Post Mortem by Exome Analysis in a Boy with Respiratory Distress.

We highlight the importance of exome sequencing in solving a clinical case of a child who died at 14 months after a series of respiratory crises. He was the half-brother of a girl diagnosed at 7 years with the early-onset seizure variant of Rett syndrome due to CDKL5 mutation. We performed a test for CDKL5 in the boy, which came back negative. Driven by the mother's compelling need for a diagnosis, we moved forward performing whole exome sequencing analysis. Surprisingly, two missense mutations in compound heterozygosity were identified in the RAPSN gene encoding a receptor-associated protein with a key role in clustering and anchoring nicotinic acetylcholine receptors at synaptic sites. This gene is responsible for a congenital form of myasthenic syndrome, a disease potentially treatable with cholinesterase inhibitors. Therefore, an earlier diagnosis in this boy would have led to a better clinical management and prognosis. Our study supports the key role of exome sequencing in achieving a definite diagnosis in severe perinatal diseases, an essential step especially when a specific therapy is available.

Germline polymorphisms and survival of lung adenocarcinoma patients: a genome-wide study in two European patient series.

In lung cancer, the survival of patients with the same clinical stage varies widely for unknown reasons. In this two-phase study, we examined the hypothesis that germline variations influence the survival of patients with lung adenocarcinoma. First, we analyzed existing genotype and clinical data from 289 UK-resident patients with lung adenocarcinoma, identifying 86 single nucleotide polymorphisms (SNPs) that associated with survival (p < 0.01). We then genotyped these candidate SNPs in a validation series of 748 patients from Italy that resulted genetically compatible with the UK series based on principal component analysis. In a Cox proportional hazard model adjusted for age, sex and clinical stage, four SNPs were confirmed on the basis of their having a hazard ratio (HR) indicating the same direction of effect in the two series and p < 0.05. The strongest association was provided by rs2107561, an intronic SNP of PTPRG, protein tyrosine phosphatase, receptor type, G; the C allele was associated with poorer survival in both patient series (pooled analysis loge HR = 0.31; 95% CI: 0.15-0.46, p = 8.5 × 10(-5) ). PTPRG mRNA levels in 43 samples of lung adenocarcinoma were 40% of those observed in noninvolved lung tissue from the same patients. PTPRG overexpression significantly inhibited the clonogenicity of A549 lung carcinoma cells and the anchorage-independent growth of the NCI-H460 large cell lung cancer line. These four germline variants represent promising candidates that, with further study, may help predict clinical outcome. In addition, the PTPRG locus may have a role in tumor progression.

Interstitial 22q13 deletions not involving SHANK3 gene: a new contiguous gene syndrome.

Phelan-McDermid syndrome (22q13.3 deletion syndrome) is a contiguous gene disorder resulting from the deletion of the distal long arm of chromosome 22. SHANK3, a gene within the minimal critical region, is a candidate gene for the major neurological features of this syndrome. We report clinical and molecular data from a study of nine patients with overlapping interstitial deletions in 22q13 not involving SHANK3. All of these deletions overlap with the largest, but not with the smallest deletion associated with Phelan-McDermid syndrome. The deletion sizes and breakpoints varied considerably among our patients, with the largest deletion spanning 6.9 Mb and the smallest deletion spanning 2.7 Mb. Eight out of nine patients had a de novo deletion, while in one patient the origin of deletion was unknown. These patients shared clinical features common to Phelan-McDermid syndrome: developmental delay (11/12), speech delay (11/12), hypotonia (9/12), and feeding difficulties (7/12). Moreover, the majority of patients (8/12) exhibited macrocephaly. In the minimal deleted region, we identified two candidate genes, SULT4A1 and PARVB (associated with the PTEN pathway), which could be associated in our cohort with neurological features and macrocephaly/hypotonia, respectively. This study suggests that the haploinsufficiency of genes in the 22q13 region beside SHANK3 contributes to cognitive and speech development, and that these genes are involved in the phenotype associated with the larger Phelan-McDermid syndrome 22q13 deletions. Moreover, because the deletions in our patients do not involve the SHANK3 gene, we posit the existence of a new contiguous gene syndrome proximal to the smallest terminal deletions in the 22q13 region.

Differential gene expression analysis pipelines and bioinformatic tools for the identification of specific biomarkers: A review.

In recent years, the role of bioinformatics and computational biology together with omics techniques and transcriptomics has gained tremendous importance in biomedicine and healthcare, particularly for the identification of biomarkers for precision medicine and drug discovery. Differential gene expression (DGE) analysis is one of the most used techniques for RNA-sequencing (RNA-seq) data analysis. This tool, which is typically used in various RNA-seq data processing applications, allows the identification of differentially expressed genes across two or more sample sets. Functional enrichment analyses can then be performed to annotate and contextualize the resulting gene lists. These studies provide valuable information about disease-causing biological processes and can help in identifying molecular targets for novel therapies. This review focuses on differential gene expression (DGE) analysis pipelines and bioinformatic techniques commonly used to identify specific biomarkers and discuss the advantages and disadvantages of these techniques.

Gene expression signature of non-involved lung tissue associated with survival in lung adenocarcinoma patients.

Lung adenocarcinoma patients of similar clinical stage and undergoing the same treatments often have marked interindividual variations in prognosis. These clinical discrepancies may be due to the genetic background modulating an individual's predisposition to fighting cancer. Herein, we hypothesized that the lung microenvironment, as reflected by its expression profile, may affect lung adenocarcinoma patients' survival. The transcriptome of non-involved lung tissue, excised from a discovery series of 204 lung adenocarcinoma patients, was evaluated using whole-genome expression microarrays (with probes corresponding to 28 688 well-annotated coding sequences). Genes associated with survival status at 60 months were identified by Cox regression analysis (adjusted for gender, age and clinical stage) and retested in a validation series of 78 additional cases. RNA-Seq analysis from non-involved lung tissue of 12 patients was performed to characterize the different isoforms of candidate genes. Ten genes for which the loge-transformed hazard ratios expressed the same direction of effect in the discovery (P < 1.0 × 10(-3)) and validation series comprised the gene expression signature associated with survival: CNTNAP1, PKNOX1, FAM156A, FRMD8, GALNTL1, TXNDC12, SNTB1, PPP3R1, SNX10 and SERPINH1. RNA sequencing highlighted the complex expression pattern of these genes in non-involved lung tissue from different patients and permitted the detection of a read-through gene fusion between PPP3R1 and the flanking gene (CNRIP1) as well as a novel isoform of CNTNAP1. Our findings support the hypothesis that individual genetic characteristics, evidenced by the expression pattern of non-involved tissue, influence the outcome of lung adenocarcinoma patients.

Different Liquid Biopsies for the Management of Non-Small Cell Lung Cancer in the Mutational Oncology Era.

In the last ten years, liquid biopsy has been slowly joining the traditional invasive techniques for the diagnosis and monitoring of tumors. Liquid biopsies allow easy repeated sampling of blood, reflect the tumor scenario, and make personalized therapy real for the patient. Liquid biopsies isolate and utilize different substrates present in patients' body fluids such as circulating tumor cells, circulating tumor DNA, tumor extracellular vesicles, etc. One of the most-used solid cancers in the development of the non-invasive liquid biopsy approach that has benefited from scientific advances is non-small cell lung cancer (NSCLC). Using liquid biopsy, it is possible to have more details on NSCLC staging, progression, heterogeneity, gene mutations and clonal evolution, etc., basing the treatment on precision medicine as well as on the screening of markers for therapeutic resistance. With this review, the authors propose a complete and current overview of all different liquid biopsies available to date, to understand how much has been carried out and how much remains to be completed for a better characterization of NSCLC.

Modeling PCDH19 clustering epilepsy by Neurogenin 2 induction of patient-derived induced pluripotent stem cells.

BACKGROUND: Loss of function mutations in PCDH19 gene causes an X-linked, infant-onset clustering epilepsy, associated with intellectual disability and autistic features. The unique pattern of inheritance includes random X-chromosome inactivation, which leads to pathological tissue mosaicism. Females carrying PCDH19 mutations are affected, while males have a normal phenotype. No cure is presently available for this disease. METHODS: Fibroblasts from a female patient carrying frameshift mutation were reprogrammed into human induced pluripotent stem cells (hiPSCs). To create a cell model of PCDH19-clustering epilepsy (PCDH19-CE) where both cell populations co-exist, we created mosaic neurons by mixing wild-type (WT) and mutated (mut) hiPSC clones, and differentiated them into mature neurons with overexpression of the transcriptional factor Neurogenin 2. RESULTS: We generated functional neurons from patient-derived iPSC using a rapid and efficient method of differentiation through overexpression of Neurogenin 2. Was revealed an accelerated maturation and higher arborisation in the mutated neurons, while the mosaic neurons showed the highest frequency of action potential firing and hyperexcitability features, compared to mutated and WT neurons. CONCLUSIONS: Our findings provide evidence that PCDH19 c.2133delG mutation affects proper metaphases with increased numbers of centrosomes in stem cells and accelerates neuronal maturation in premature cells. PCDH19 mosaic neurons showed elevated excitability, representing the situation in PCDH19-CE brain. We suggest Ngn2 hiPSC-derived PCDH19 neurons as an informative experimental tool for understanding the pathogenesis of PCDH19-CE and a suitable approach for use in targeted drug screening strategies.

Case report: PIK3CA somatic mutation leading to Klippel Trenaunay Syndrome and multiple tumors.

We report a case of Klippel Trenaunay Syndrome that was monitored both clinically and molecularly over a period of 9 years. A somatic mosaic mutation of PIK3CA (p(E545G)) was identified using both cfDNA NGS liquid biopsy and tissue biopsy. At the age of 56, due to intervening clonal mutations in PIK3CA background, she developed a squamous cell carcinoma in the right affected leg which was treated surgically. Nine years later, lung bilateral adenocarcinoma arose on PIK3CA mutated tissues supported by different clonal mutations. One year later, the patient died from metastases led by a new FGFR3 clone unresponsive to standard-of-care, immunotherapy-based. Our results highlight the presence of a molecular hallmark underlying neoplastic transformation that occurs upon an angiodysplastic process and support the view that PIK3CA mutated tissues must be treated as precancerous lesions. Importantly, they remark the effectiveness of combining cfDNA NGS liquid and tissue biopsies to monitor disease evolution as well as to identify aggressive clones targetable by tailored therapy, which is more efficient than conventional protocols.

Association of lung adenocarcinoma clinical stage with gene expression pattern in noninvolved lung tissue.

Associations between clinical outcome of cancer patients and the gene expression signature in primary tumors at time of diagnosis have been reported. To test whether gene expression patterns in noninvolved lung tissue might correlate with clinical stage in lung adenocarcinoma (ADCA) patients, we compared the transcriptome of noninvolved lung samples from 60 ADCA smoker patients of clinical stage I versus 60 patients with stage>I. Quantitative PCR of 10 genes with the most significant differential expression confirmed the statistical association with clinical stage in eight genes, six of which were downregulated in high-stage patients. Five of these six genes were also downregulated in lung ADCA tissue as compared to noninvolved tissue. Studies in vitro indicated that four of the genes (SLC14A1, SMAD6, TMEM100 and TXNIP) inhibited colony formation of lung cancer cell lines transfected to overexpress the genes, suggesting their potential tumor-suppressor activity. Our findings suggest that individual variations in the transcriptional profile of noninvolved lung tissue may reflect the lung ADCA patient's predisposition to tumor aggressiveness.

Diagnostic accuracy of circulating free DNA testing for the detection of KRAS mutations in non-small cell lung cancer: A systematic review and meta-analysis.

Kirsten rat sarcoma viral oncogene homolog (KRAS) gene encodes a GTPase that acts as a molecular switch for intracellular signal transduction, promoting cell growth and proliferation. Mutations in the KRAS gene represent important biomarkers for NSCLC targeted therapy. However, detection of KRAS mutations in tissues has shown some limitations. During the last years, analyses of circulating free DNA (cfDNA) has emerged as an alternative and minimally invasive, approach to investigate tumor molecular changes. Here, we assessed the diagnostic performance of cfDNA analysis, compared to tissues through a meta-analysis and systematic review of existing literature. From 561 candidate papers, we finally identified 40 studies, including 2,805 NSCLC patients. We extracted values relating to the number of true-positive, false-positive, false-negative, and true-negative. Pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio, each with 95% CI, were calculated. A summary receiver operating characteristic curve and the area under curve (AUC) were used to evaluate the overall diagnostic performance. The pooled sensitivity was 0.71 (95% CI 0.68-0.74) and the specificity was 0.93 (95% CI 0.92-0.94). The diagnostic odds ratio was 35.24 (95% CI 24.88-49.91) and the area under the curve was 0.92 (SE = 0.094). These results provide evidence that detection of KRAS mutation using cfDNA testing is of adequate diagnostic accuracy thus offering to the clinicians a new promising screening test for NSCLC patients.