Spike-Seq: An amplicon-based high-throughput sequencing approach for the sensitive detection and characterization of SARS-CoV-2 genetic variations in environmental samples.

Ever since the outbreak of COVID-19 disease in Wuhan, China, different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been identified. Wastewater-based epidemiology (WBE), an approach that has been successfully applied in numerous case studies worldwide, offers a cost-effective and rapid way for monitoring trends of SARS-Cov-2 in the community level without selection bias. Despite being a gold-standard procedure, WBE is a challenging approach due to the sample instability and the moderate efficiency of SARS-CoV-2 concentration in wastewater. In the present study, we introduce Spike-Seq, a custom amplicon-based approach for the S gene sequencing of SARS-CoV-2 in wastewater samples, which enables not only the accurate identification of the existing Spike-related genetic markers, but also the estimation of their frequency in the investigated samples. The implementation of Spike-Seq involves the combination of nested PCR-based assays that efficiently amplify the entire nucleotide sequence of the S gene and next-generation sequencing, which enables the variant detection and the estimation of their frequency. In the framework of the current work, Spike-Seq was performed to investigate the mutational profile of SARS-CoV-2 in samples from the Wastewater Treatment Plant (WWTP) of Athens, Greece, which originated from multiple timepoints, ranging from March 2021 until July 2022. Our findings demonstrate that Spike-Seq efficiently detected major genetic markers of B.1.1.7 (Alpha), B.1.617.2 (Delta) as well as B.1.1.529 (Omicron) variants in wastewater samples and provided their frequency levels, showing similar variant distributions with the published clinical data from the National Public Health organization. The presented approach can prove to be a useful tool for the detection of SARS-CoV-2 in challenging wastewater samples and the identification of the existing genetic variants of S gene.

Development and Validation of a Novel Dual-Drop-off ddPCR Assay for the Simultaneous Detection of Ten Hotspots PIK3CA Mutations.

Breast cancer is the leading cause of cancer-related deaths in women worldwide. Approximately 40% of patients with hormone receptor-positive, human epidermal growth factor receptor-2-negative breast cancer have activating mutations in the PIK3CA gene. We developed a highly sensitive, specific, cost-effective, and reproducible dual-drop-off droplet digital polymerase chain reaction (PCR) assay for the simultaneous detection of ten hotspots of PIK3CA mutations in plasma cell-free (cf) DNA. We first evaluated the analytical specificity, sensitivity, limit of blank, repeatability, and reproducibility of the assay, which simultaneously detects seven mutations in exon9 and three in exon20. We further applied this assay in 11 gDNA and 18 plasma cfDNA samples from healthy donors and 35 plasma cfDNA samples from metastatic breast cancer patients. The assay is highly sensitive, specific, and applicable for clinical samples containing at least 1-5% mutant DNA. We detected PIK3CA mutations in 9/35(26%) plasma cfDNA samples in exon 9 and in 9/35(26%) in exon 20. Direct comparison of the developed assay with amplification refractory mutation system-based PCR (using plasma samples) and with the Food and Drug Administration-approved cobas PIK3CA mutation assay (using formalin fixed paraffin embedded samples) showed high concordance of our developed assay with the cobas PIK3CA assay. The developed assay is cost-effective and can reliably and simultaneously detect ten hotspot PIK3CA mutations in plasma cfDNA. The clinical performance of the assay will be further evaluated in liquid biopsy samples.

Wastewater surveillance of the most common circulating respiratory viruses in Athens: The impact of COVID-19 on their seasonality.

Due to governments' actions to contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the activity of common circulating respiratory viruses was significantly disrupted after the COVID-19 pandemic and thorough surveillance of respiratory pathogens was considered essential worldwide. Wastewater-based epidemiology has proven to be a valuable tool, that provides complementary information on disease outbreaks and is increasingly used to study the infection dynamics of other viruses, apart from SARS-CoV-2. The aims of the present study were the detection of four commonly circulating respiratory viruses: SARS-CoV-2, influenza A, B and Human Respiratory Syncytial Virus (RSV), the evaluation of the COVID-19 pandemic impact on their seasonality and the determination of the possible common trends in the viral load of these viruses in the wastewater of the Attica region. A standardized and validated concentration and extraction protocol was used, generic for all four viruses, followed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays. The study proved that there was a prolonged period when all four viruses circulated in the population and an early outbreak of seasonal influenza and RSV in 2022-2023, compared to data from the pre-COVID-19 period. SARS-CoV-2, influenza A and RSV concentrations showed peak levels during December, followed by a slight decline in influenza A concentrations, followed by steady increase of influenza B concentrations in January 2023. SARS-CoV-2 was the dominant virus throughout the whole study period. This is the first study in Greece that investigated the most common circulating viruses simultaneously and in one of the largest timelines, providing crucial information about their infection dynamics during a period when an outbreak of respiratory diseases was declared by the National Public Health Organization. Presented results highlight the establishment of environmental surveillance as a non-invasive and complementary virus outbreak monitoring tool and the importance of influenza A, B and RSV integration into a wastewater-based surveillance system to help in disease management.

Highly Sensitive Detection Method of CXCR4 Tumor Hotspot Mutations by Drop-Off Droplet Digital PCR in Patients with IgM Monoclonal Gammopathies.

CXCR4 mutations impact disease presentation and treatment outcomes in Waldenström macroglobulinemia. Current techniques used for CXCR4 mutation detection have a number of limitations. The aim of the present study was to develop and analytically validate a novel droplet digital PCR (ddPCR) assay for the simultaneous detection of five of the most common CXCR4 mutations in bone marrow (BM). In silico novel primers and probes designed for simultaneous detection of five hotspot mutations of CXCR4 were first performed. Experimental conditions were optimized, and the assay was analytically validated. The developed assay was further applied in 95 BM samples from patients with IgM gammopathy, 7 BM samples from patients with non-IgM gammopathy and 12 PBMCs from healthy donors, whereas a direct comparison study of Sanger sequencing and allele-specific PCR was performed by using 95 and 39 identical patient tumor DNA samples, respectively. The drop-off ddPCR assay is a robust, cost-effective, highly sensitive, and highly specific screening tool for CXCR4 mutations. Of 95 patients with IgM gammopathy samples, 27 had at least one CXCR4 mutation in their BM samples. With Sanger sequencing, 12 of the 95 samples tested positive, whereas the direct comparison of the developed assay with allele-specific PCR revealed substantial agreement. The clinical performance of the developed assay will be prospectively evaluated in a large number of patients, and the applicability of this assay will be further evaluated.

Detection and Molecular Characterization of Circulating Tumour Cells: Challenges for the Clinical Setting.

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of circulating tumour cells (CTCs) and circulating tumour-derived material, such as circulating tumour DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTC analysis has already had an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients. Numerous clinical trials nowadays include a liquid biopsy arm. CTC analysis is now an exponentially expanding field in almost all types of solid cancers. Functional studies, mainly based on CTC-derived cell-lines and CTC-derived explants (CDx), provide important insights into the metastatic process. The purpose of this review is to summarize the latest findings on the clinical significance of CTCs for the management of cancer patients, covering the last four years. This review focuses on providing a comprehensive overview of CTC analysis in breast, prostate and non-small-cell lung cancer. The unique potential of CTC single-cell analysis for understanding metastasis biology, and the importance of quality control and standardization of methodologies used in this field, is also discussed.

International multicenter survey on screening and confirmatory testing in primary aldosteronism.

OBJECTIVE: Primary aldosteronism (PA) is one of the most frequent causes of secondary hypertension. Although clinical practice guidelines recommend a diagnostic process, details of the steps remain incompletely standardized. DESIGN: In the present SCOT-PA survey, we have investigated the diversity of approaches utilized for each diagnostic step in different expert centers through a survey using Google questionnaires. A total of 33 centers from 3 continents participated. RESULTS: We demonstrated a prominent diversity in the conditions of blood sampling, assay methods for aldosterone and renin, and the methods and diagnostic cutoff for screening and confirmatory tests. The most standard measures were modification of antihypertensive medication and sitting posture for blood sampling, measurement of plasma aldosterone concentration (PAC) and active renin concentration by chemiluminescence enzyme immunoassay, a combination of aldosterone-to-renin ratio with PAC as an index for screening, and saline infusion test in a seated position for confirmatory testing. The cutoff values for screening and confirmatory testing showed significant variation among centers. CONCLUSIONS: Diversity of the diagnostic steps may lead to an inconsistent diagnosis of PA among centers and limit comparison of evidence for PA between different centers. We expect the impact of this diversity to be most prominent in patients with mild PA. The survey raises 2 issues: the need for standardization of the diagnostic process and revisiting the concept of mild PA. Further standardization of the diagnostic process/criteria will improve the quality of evidence and management of patients with PA.

Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer.

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients.

Delta SARS-CoV-2 variant is entirely substituted by the omicron variant during the fifth COVID-19 wave in Attica region.

Wastewater analysis is the most attractive alternative way for the quantification and variant profiling of SARS-CoV-2. Infection dynamics can be monitored by RT-qPCR assays while NGS can provide evidence for the presence of existing or new emerging SARS-CoV-2 variants. Herein, apart from the infection dynamic in Attica since June 1st, 2021, the monitoring of 9 mutations of the omicron and 4 mutations of the delta SARS-CoV-2 variants, utilizing both novel Nested-Seq and RT-PCR, is reported and the substitution of the delta variant (B.1.617.2) by the omicron variant (B.1.1.529) in Attica, Greece within approximately one month is highlighted. The key difference between the two methodologies is discovery power. RT-PCR can only detect known sequences cost-effectively, while NGS is a hypothesis-free approach that does not require prior knowledge to detect novel genes. Overall, the potential of wastewater genomic surveillance for the early discovery and monitoring of variants important for disease management at the community level is underlined. This is the first study, reporting the SARS-CoV-2 infection dynamic for an extended time period and the first attempt to monitor two of the most severe variants with two different methodologies in Greece.

Pre- and peri-operative characteristics, complications and outcomes of patients with biochemically silent pheochromocytomas; a case series.

PURPOSE: Pheochromocytomas are rare tumors and biochemically silent ones with normal catecholamine levels are even rarer. Up to date, biochemically inactive pheochromocytomas are poorly investigated. We aimed to systematically assess the pre- and peri-operative characteristics and the outcomes of patients with these tumors who had been treated and followed-up in 2 tertiary centers. METHODS: Clinical, laboratory and imaging data, treatment outcomes and follow-up of biochemically silent pheochromocytoma patients were recorded. RESULTS: Ten patients (5 men) [median age at diagnosis 52.5 years (24-72)] were included. Adrenal masses were incidentally discovered in all patients except from one who presented with pheochromocytoma-related manifestations. Twenty-four-hour urine metanephrine and normetanephrine levels were in the low-normal, normal and high-normal range in 4, 4 and 2 patients and in 1, 6 and 3 patients, respectively. Tumors were unilateral [median size 46 mm (17-125)] and high density on pre-contrast CT imaging or high signal intensity on T2-weighted MRI scans were found in all cases. Pre-operatively, 5 patients were treated with phenoxybenzamine [median total daily dose 70 mg (20-100)]. Intra-operatively, 4 patients developed hypertension requiring vasodilator administration and 8 developed hypotension; vasoconstrictors were required in 5 cases. One patient, not pre-operatively treated with phenoxybenzamine, developed Takotsubo cardiomyopathy. During a median 24-month (12-88) follow-up period, one patient had disease progression. CONCLUSIONS: The majority (90%) of patients with biochemically silent pheochromocytomas developed hemodynamic instability during adrenal surgery. In patients with biochemically silent adrenal lesions and a high suspicion index for pheochromocytoma based on tumor imaging characteristics, pre-operative alpha-blockade treatment may be advisable.

High Prevalence of Primary Aldosteronism in Patients with Type 2 Diabetes Mellitus and Hypertension.

Primary aldosteronism (PA) is the most common cause of endocrine hypertension. The prevalence of hypertension is higher in patients with diabetes mellitus-2 (DM-2). Following the limited existing data, we prospectively investigated the prevalence of aldosterone excess either as autonomous secretion (PA) or as a hyper-response to stress in hypertensive patients with DM-2 (HDM-2). A total of 137 HDM-2 patients and 61 non-diabetics with essential hypertension who served as controls (EH-C) underwent a combined, overnight diagnostic test, the Dexamethasone-captopril-valsartan test (DCVT) used for the diagnosis of PA and an ultralow dose (0.3 µg) ACTH stimulation test to identify an exaggerated aldosterone response to ACTH stimulation. Twenty-three normotensive individuals served as controls (NC) to define the normal response of aldosterone (ALD) and aldosterone-to-renin ratio (ARR) to the ultralow dose ACTH test. Using post-DCVTALD and ARR from the EH-C, and post-ACTH peak ALD and ARR from the NC, 47 (34.3%) HDM-2 patients were found to have PA, whereas 6 (10.4%) HDM-2 patients without PA (DCVT-negative) exhibited an exaggerated aldosterone response to stress-a prevalence much higher than ever reported. Treatment with mineralocorticoid receptor antagonists (MRAs) induced a significant and permanent reduction of BP in all HDM-2 patients. Early diagnosis and targeted treatment of PA is crucial to prevent any aggravating effect on chronic diabetic complications.

Detection of ESR1 Mutations in Primary Tumors and Plasma Cell-Free DNA in High-Grade Serous Ovarian Carcinoma Patients.

ESR1 mutations have been recently associated with resistance to endocrine therapy in metastatic breast cancer and their detection has led to the development and current evaluation of novel, highly promising therapeutic strategies. In ovarian cancer there have been just a few reports on the presence of ESR1 mutations. The aim of our study was to evaluate the frequency and the clinical relevance of ESR1 mutations in high-grade serous ovarian cancer (HGSOC). Drop-off droplet digital PCR (ddPCR) was first used to screen for ESR1 mutations in 60 primary tumors (FFPEs) from HGSOC patients and in 80 plasma cell-free DNA (cfDNA) samples from advanced and metastatic ovarian cancer patients. We further used our recently developed ESR1-NAPA assay to identify individual ESR1 mutations in drop-off ddPCR-positive samples. We report for the first time the presence of ESR1 mutations in 15% of FFPEs and in 13.8% of plasma cfDNA samples from advanced and metastatic ovarian cancer patients. To define the clinical significance of this finding, our results should be further validated in a large and well-defined cohort of ovarian cancer patients.

Genetic screening of hypertensive patients with aldosterone hypersecretion under conditions of stress.

PURPOSE: Although ACTH is considered a secondary regulator of aldosterone production, patients with apparent essential hypertension have been treated with mineralocorticoid receptor antagonists (MRAs). In this study, we aimed to identify potentially damaging variants that might be implicated in the phenotype of a well-characterized cohort of 21 hypertensive patients without PA but with stress-induced aldosterone hypersecretion. The patients' blood pressure was normalized though MRA administration. METHODS: Genetic screening was performed through whole-exome sequencing (WES), and variants in PA-associated or in ion-channels of aldosterone-regulating genes were prioritized. Variants with population frequency < 0.01, predicted to alter protein structure and classified as likely pathogenic by in silico tools, were retained. RESULTS: Qualifying variants were identified in nine of the 21 patients screened. Seven patients were carriers of six potentially damaging variants in six genes associated with PA (KCNK9, KCNK5, ATP13A3, SLC26A2, CACNA1H, and CACNA1D). A novel variant in the KCNK9 gene (p.V221M) is reported. Our analysis revealed two variants in two novel susceptibility genes for aldosterone hypersecretion, namely, KCNK16 (p.P255H) and CACNA2D3 (p.V557I). CONCLUSION: WES revealed potentially damaging germline variants in genes participating in aldosterone synthesis/regulating pathways in 9/21 patients of our cohort. The variants identified might play a role in aldosterone hypersecretion under conditions of stress. The potential pathogenicity of these variants should be examined in future functional studies.

Evaluation of viral concentration and extraction methods for SARS-CoV-2 recovery from wastewater using droplet digital and quantitative RT-PCR.

The ongoing pandemic caused by the emergence of SARS-CoV-2 has resulted in millions of deaths worldwide despite the various measures announced by the authorities. Wastewater-based epidemiology has the ability to provide a day-to-day estimation of the number of infected people in a fast and cost-effective manner. However, owing to the complex nature of wastewater, wastewater monitoring for viral genome copies is affected by the extensive viral fragmentation that takes place all the way to the sewage and the analytical lab. The aim of this study was to evaluate different methodologies for the concentration and extraction of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. We compare 5 different concentration methods and 4 commercially available kits for the RNA extraction. To evaluate the performance and the recovery of these, SARS-CoV-2 isolated from patients was used as a spike control. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and N3). Using spiked samples, recoveries were estimated 2.1-37.6% using different extraction kits and 0.1-2.1% using different concentration kits. It was found that a direct capture-based method, evaluated against a variety of concentration methods, is the best in terms of recovery, time and cost. Interestingly, we noticed a good agreement between the results provided by RT-qPCR and RT-ddPCR in terms of recovery. This evaluation can serve as a guide for laboratories establishing a protocol to perform wastewater monitoring of SARS-CoV-2. Overall, data presented here reinforces the validity of WBE for SARS-CoV-2 surveillance, uncovers potential caveats in the selection of concentration and extraction protocols and points towards optimal solutions to maximize its potential.

Enhanced performance of a modified diagnostic test of primary aldosteronism in patients with adrenal adenomas.

OBJECTIVE: Primary aldosteronism (PA) is the commonest cause of endocrine hypertension ranging from 4.6 to 16.6% according to the diagnostic tests employed. The aim of this study was to compare the traditional saline infusion test (SIT) with the modified post-dexamethasone saline infusion test (DSIT) by applying both tests on the same subjects. METHODS: We studied 68 patients (72% hypertensives) with single adrenal adenoma and 55 normotensive controls with normal adrenal imaging. Serum cortisol, aldosterone, and plasma renin concentration (PRC) were measured and the aldosterone-to-renin ratio (ARR) was calculated. Using the mean ± 2 s.d. values from the controls, we defined the upper normal limits for cortisol, aldosterone, and PRC for both the SIT and DSIT. RESULTS: In the controls, the post-DSIT aldosterone levels and the ARR were approximately two-fold and three-fold lower, respectively, than the corresponding post-SIT values (all P = 0.001) leading to lower cut-offs of aldosterone suppression. Applying these cut-offs to patients with adrenal adenomas, the prevalence of PA was 13.2% following the SIT and 29.4% following the DSIT, respectively. In addition, 54.5% of patients with PA had concomitant autonomous cortisol secretion (ACS). Targeted treatment of PA resulted in resolution of hypertension and restoration of normal secretory aldosterone dynamics. CONCLUSIONS: The DSIT improves the diagnostic accuracy of PA, allowing for the detection of milder forms of PA in patients with adrenal adenomas. This is of particular importance as such patients may be at an increased risk of developing cardiovascular and renal morbidity that could be enhanced in the presence of concomitant ACS.

Determination of MYD88L265P mutation fraction in IgM monoclonal gammopathies.

We describe a novel method for the detection of MYD88L265P mutation using a competitive allele-specific polymerase chain reaction (Cast-PCR) assay. This assay has a sensitivity of 1 × 10-3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg), and allowed the detection of MYD88L265P somatic mutation in both tumor-derived DNA (tDNA) and cell-free DNA (cfDNA). In addition, using the Cast-PCR assay, we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with immunoglobulin M monoclonal gammopathy of undetermined significance and 110 with Waldenström's macroglobulinemia [WM], of whom 54 were asymptomatic and 56 were symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared with asymptomatic WM and in those with asymptomatic WM compared with those with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance. In addition, the evaluation of sequential samples showed that MAF decreased after treatment, whereas it increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tDNA and cfDNA samples, that also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

SARS-CoV-2 wastewater surveillance data can predict hospitalizations and ICU admissions.

We measured SARS-CoV-2 RNA load in raw wastewater in Attica, Greece, by RT-qPCR for the environmental surveillance of COVID-19 for 6 months. The lag between RNA load and pandemic indicators (COVID-19 hospital and intensive care unit (ICU) admissions) was calculated using a grid search. Our results showed that RNA load in raw wastewater is a leading indicator of positive COVID-19 cases, new hospitalization and admission into ICUs by 5, 8 and 9 days, respectively. Modelling techniques based on distributed/fixed lag modelling, linear regression and artificial neural networks were utilized to build relationships between SARS-CoV-2 RNA load in wastewater and pandemic health indicators. SARS-CoV-2 mutation analysis in wastewater during the third pandemic wave revealed that the alpha-variant was dominant. Our results demonstrate that clinical and environmental surveillance data can be combined to create robust models to study the on-going COVID-19 infection dynamics and provide an early warning for increased hospital admissions.

Prevalence of Primary Aldosteronism Across the Stages of Hypertension Based on a New Combined Overnight Test.

Primary aldosteronism (PA) is the most common endocrine cause of arterial hypertension. Despite the increasing incidence of hypertension worldwide, the true prevalence of PA in hypertension was only recently recognized. The objective of the work was to estimate the prevalence of PA in patients at different stages of hypertension based on a newly developed screening-diagnostic overnight test. This is a prospective study with hypertensive patients (n=265) at stage I (n=100), II (n=88), and III (n=77) of hypertension. A group of 103 patients with essential hypertension without PA was used as controls. PA diagnosis was based on a combined screening-diagnostic overnight test, the Dexamethasone-Captopril-Valsartan Test (DCVT) that evaluates aldosterone secretion after pharmaceutical blockade of angiotensin-II and adrenocorticotropic hormone. DCVT was performed in all participants independently of the basal aldosterone to renin ratio (ARR). The calculated upper normal limits for post-DCVT aldosterone levels [3 ng/dl (85 pmol/l)] and post-DCVT ARR [0.32 ng/dl/µU/ml (9 pmol/IU)] from controls, were applied together to establish PA diagnosis. Using these criteria PA was confirmed in 80 of 265 (30%) hypertensives. The prevalence of PA was: 21% (21/100) in stage I, 33% (29/88) in stage II, and 39% (30/77) in stage III. Serum K+ levels were negatively correlated and urinary K+ was positively correlated in PA patients with post-DCVT ARR (r=-0.349, p <0.01, and r=0.27, p <0.05 respectively). In conclusion, DCVT revealed that PA is a highly prevalent cause of hypertension. DCVT could be employed as a diagnostic tool in all subjects with arterial hypertension of unknown cause.

The Effect of Genomic DNA Contamination on the Detection of Circulating Long Non-Coding RNAs: The Paradigm of MALAT1.

The presence of contaminating gDNA in RNA preparations is a frequent cause of false positives in RT-PCR-based analysis. However, in some cases, this cannot be avoided, especially when there are no exons-intron junctions in the lncRNA sequences. Due to the lack of exons in few of long noncoding RNAs (lncRNAs) and the lack of DNAse treatment step in most studies reported so far, serious questions are raised about the specificity of lncRNA detection and the potential of reporting false-positive results. We hypothesized that minute amounts of gDNA usually co-extracted with RNA could give false-positive signals since primers would specifically bind to gDNA due to the lack of junction. In the current study, we evaluated the effect of gDNA and other forms of DNA like extrachromosomal circular DNAs (eccDNAs) contamination and the importance of including a DNAse treatment step on lncRNAsexpression.As a model, we have chosen as one of the most widely studied lncRNAs in cancer namely MALAT1, which lacks exons. When we tested this hypothesis in plasma and primary tissue samples from NSCLC patients, our findings clearly indicated that results on MALAT1 expression are highly affected by the presence of DNA contamination and that the DNAse treatment step is absolutely necessary to avoid false positive results.

Generation of Non-Small Cell Lung Cancer Patient-Derived Xenografts to Study Intratumor Heterogeneity.

Recent advances in sequencing technologies have allowed the in-depth molecular study of tumors, even at the single cell level. Sequencing efforts have uncovered a previously unappreciated heterogeneity among tumor cells, which has been postulated to be the driving force of tumor evolution and to facilitate recurrence, metastasis, and drug resistance. In the current study, focused on early-stage operable non-small cell lung cancer, we used tumor growth in patient-derived xenograft (PDX) models in mice as a fast-forward tumor evolution process to investigate the molecular characteristics of tumor cells that grow in mice, as well as the parameters that affect the grafting efficiency. We found that squamous cell carcinomas grafted significantly more efficiently compared with adenocarcinomas. Advanced stage, patient age and primary tumor size were positively correlated with grafting. Additionally, we isolated and characterized circulating tumor cells (CTC) from patients' peripheral blood and found that the presence of CTCs expressing epithelial-to-mesenchymal (EMT) markers correlated with the grafting potential. Interestingly, exome sequencing of the PDX tumor identified genetic alterations in DNA repair and genome integrity genes that were under-represented in the human primary counterpart. In conclusion, through the generation of a PDX biobank of NSCLC, we identified the clinical and molecular properties of tumors that affected growth in mice.