Dispersal history of SARS-CoV-2 in Galicia, Spain.

The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Serum DNA methylome of the colorectal cancer serrated pathway enables non-invasive detection.

The clinical relevance of the colorectal cancer serrated pathway is evident, but the screening of serrated lesions remains challenging. We aimed to characterize the serum methylome of the serrated pathway and to evaluate circulating cell-free DNA (cfDNA) methylomes as a potential source of biomarkers for the non-invasive detection of serrated lesions. We collected serum samples from individuals with serrated adenocarcinoma (SAC), traditional serrated adenomas, sessile serrated lesions, hyperplastic polyps and individuals with no colorectal findings. First, we quantified cfDNA methylation with the MethylationEPIC array. Then, we compared the methylation profiles with tissue and serum datasets. Finally, we evaluated the utility of serum cfDNA methylation biomarkers. We identified a differential methylation profile able to distinguish high-risk serrated lesions from no serrated neoplasia, showing concordance with tissue methylation from SAC and sessile serrated lesions. Serum methylation profiles are pathway-specific, clearly separating serrated lesions from conventional adenomas. The combination of ninjurin 2 (NINJ2) and glutamate-rich 1 (ERICH1) methylation discriminated high-risk serrated lesions and SAC with 91.4% sensitivity (64.4% specificity), while zinc finger protein 718 (ZNF718) methylation reported 100% sensitivity for the detection of SAC (96% specificity). This is the first study exploring the serum methylome of serrated lesions. Differential methylation of cfDNA can be used for the non-invasive detection of colorectal serrated lesions.

Serum methylation of GALNT9, UPF3A, WARS, and LDB2 as noninvasive biomarkers for the early detection of colorectal cancer and advanced adenomas.

BACKGROUND: Early detection has proven to be the most effective strategy to reduce the incidence and mortality of colorectal cancer (CRC). Nevertheless, most current screening programs suffer from low participation rates. A blood test may improve both the adherence to screening and the selection to colonoscopy. In this study, we conducted a serum-based discovery and validation of cfDNA methylation biomarkers for CRC screening in a multicenter cohort of 433 serum samples including healthy controls, benign pathologies, advanced adenomas (AA), and CRC. RESULTS: First, we performed an epigenome-wide methylation analysis with the MethylationEPIC array using a sample pooling approach, followed by a robust prioritization of candidate biomarkers for the detection of advanced neoplasia (AN: AA and CRC). Then, candidate biomarkers were validated by pyrosequencing in independent individual cfDNA samples. We report GALNT9, UPF3A, WARS, and LDB2 as new noninvasive biomarkers for the early detection of AN. The combination of GALNT9/UPF3A by logistic regression discriminated AN with 78.8% sensitivity and 100% specificity, outperforming the commonly used fecal immunochemical test and the methylated SEPT9 blood test. CONCLUSIONS: Overall, this study highlights the utility of cfDNA methylation for CRC screening. Our results suggest that the combination methylated GALNT9/UPF3A has the potential to serve as a highly specific and sensitive blood-based test for screening and early detection of CRC.

Evaluation of Blood Soluble CD26 as a Complementary Biomarker for Colorectal Cancer Screening Programs.

Fecal hemoglobin immunodetection (FIT) in combination with endoscopy has been implemented to reduce mortality from colorectal cancer (CRC), although there are issues that can be improved in relation to participation rates. We studied whether the blood biomarker soluble-CD26 (sCD26), related at least in part to the immune system and inflammation, and/or its dipeptidyl peptidase enzyme activity (DPP4), could help reduce false positives. In a cohort of 1703 individuals who underwent colonoscopy and had a serum sample, sCD26 and DPP4 activity showed statistically significant differences regarding sex and age. According to the colonoscopy findings, sCD26 and DPP4 activity progressively decreased in advanced adenomas and CRC, with statistically significant differences, even between both groups; 918 of them had a FIT result (n = 596 positive cases) with approximately 70% of these (n = 412) false positives. With cut-offs of 440 ng/mL for sCD26, 42 mU/mL for DPP4, and 11 ng/mU for their ratio, the combined information of the three biomarkers (at least positive for one biomarker) identified almost all advanced adenomas and CRC cases in the FIT cohort with approximately half of the false positives compared to FIT. A sequential testing strategy with FIT and our blood biomarker test is proposed.

Limited genomic reconstruction of SARS-CoV-2 transmission history within local epidemiological clusters.

A detailed understanding of how and when severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission occurs is crucial for designing effective prevention measures. Other than contact tracing, genome sequencing provides information to help infer who infected whom. However, the effectiveness of the genomic approach in this context depends on both (high enough) mutation and (low enough) transmission rates. Today, the level of resolution that we can obtain when describing SARS-CoV-2 outbreaks using just genomic information alone remains unclear. In order to answer this question, we sequenced forty-nine SARS-CoV-2 patient samples from ten local clusters in NW Spain for which partial epidemiological information was available and inferred transmission history using genomic variants. Importantly, we obtained high-quality genomic data, sequencing each sample twice and using unique barcodes to exclude cross-sample contamination. Phylogenetic and cluster analyses showed that consensus genomes were generally sufficient to discriminate among independent transmission clusters. However, levels of intrahost variation were low, which prevented in most cases the unambiguous identification of direct transmission events. After filtering out recurrent variants across clusters, the genomic data were generally compatible with the epidemiological information but did not support specific transmission events over possible alternatives. We estimated the effective transmission bottleneck size to be one to two viral particles for sample pairs whose donor-recipient relationship was likely. Our analyses suggest that intrahost genomic variation in SARS-CoV-2 might be generally limited and that homoplasy and recurrent errors complicate identifying shared intrahost variants. Reliable reconstruction of direct SARS-CoV-2 transmission based solely on genomic data seems hindered by a slow mutation rate, potential convergent events, and technical artifacts. Detailed contact tracing seems essential in most cases to study SARS-CoV-2 transmission at high resolution.

SARS-CoV-2 Evolution and Spike-Specific CD4+ T-Cell Response in Persistent COVID-19 with Severe HIV Immune Suppression.

Intra-host evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported in cases with persistent coronavirus disease 2019 (COVID-19). In this study, we describe a severely immunosuppressed individual with HIV-1/SARS-CoV-2 coinfection with a long-term course of SARS-CoV-2 infection. A 28-year-old man was diagnosed with HIV-1 infection (CD4+ count: 3 cells/µL nd 563000 HIV-1 RNA copies/mL) and simultaneous Pneumocystis jirovecii pneumonia, disseminated Mycobacterium avium complex infection and SARS-CoV-2 infection. SARS-CoV-2 real-time reverse transcription polymerase chain reaction positivity from nasopharyngeal samples was prolonged for 15 weeks. SARS-CoV-2 was identified as variant Alpha (PANGO lineage B.1.1.7) with mutation S:E484K. Spike-specific T-cell response was similar to HIV-negative controls although enriched in IL-2, and showed disproportionately increased immunological exhaustion marker levels. Despite persistent SARS-CoV-2 infection, adaptive intra-host SARS-CoV-2 evolution, was not identified. Spike-specific T-cell response protected against a severe COVID-19 outcome and the increased immunological exhaustion marker levels might have favoured SARS-CoV-2 persistence.

OmniSARS2: A Highly Sensitive and Specific RT-qPCR-Based COVID-19 Diagnostic Method Designed to Withstand SARS-CoV-2 Lineage Evolution.

Extensive transmission of SARS-CoV-2 during the COVID-19 pandemic allowed the generation of thousands of mutations within its genome. While several of these become rare, others largely increase in prevalence, potentially jeopardizing the sensitivity of PCR-based diagnostics. Taking advantage of SARS-CoV-2 genomic knowledge, we designed a one-step probe-based multiplex RT-qPCR (OmniSARS2) to simultaneously detect short fragments of the SARS-CoV-2 genome in ORF1ab, E gene and S gene. Comparative genomics of the most common SARS-CoV-2 lineages, other human betacoronavirus and alphacoronavirus, was the basis for this design, targeting both highly conserved regions across SARS-CoV-2 lineages and variable or absent in other Coronaviridae viruses. The highest analytical sensitivity of this method for SARS-CoV-2 detection was 94.2 copies/mL at 95% detection probability (~1 copy per total reaction volume) for the S gene assay, matching the most sensitive available methods. In vitro specificity tests, performed using reference strains, showed no cross-reactivity with other human coronavirus or common pathogens. The method was compared with commercially available methods and detected the virus in clinical samples encompassing different SARS-CoV-2 lineages, including B.1, B.1.1, B.1.177 or B.1.1.7 and rarer lineages. OmniSARS2 revealed a sensitive and specific viral detection method that is less likely to be affected by lineage evolution oligonucleotide-sample mismatch, of relevance to ensure the accuracy of COVID-19 molecular diagnostic methods.

Colorectal cancer screening and diagnosis: omics-based technologies for development of a non-invasive blood-based method.

Introduction: Colorectal cancer (CRC) is one of the most important health problems in the Western world. In order to reduce the burden of the disease, two strategies are proposed: screening and prompt detection in symptomatic patients. Although diagnosis and prevention are mainly based on colonoscopy, fecal hemoglobin detection has been widely implemented as a noninvasive strategy. Various studies aiming to discover blood-based biomarkers have recently emerged.Areas covered: The burgeoning omics field provides diverse high-throughput approaches for CRC blood-based biomarker discovery. In this review, we appraise the most robust and commonly used technologies within the fields of genomics, transcriptomics, epigenomics, proteomics, and metabolomics, together with their targeted validation approaches. We summarize the transference process from the discovery phase until clinical translation. Finally, we review the best candidate biomarkers and their potential clinical applicability.Expert opinion: Some available biomarkers are promising, especially in the field of epigenomics: DNA methylation and microRNA. Transference requires the joint collaboration of basic researchers, intellectual property experts, technology transfer officers and clinicians. Blood-based biomarkers will be selected not only based on their diagnostic accuracy and cost but also on their reliability, applicability to clinical analysis laboratories and their acceptance by the population.

Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment.

Different methodological approaches are available to assess DNA methylation biomarkers. In this study, we evaluated two sodium bisulfite conversion-dependent methods, namely pyrosequencing and methylation-specific qPCR (MS-qPCR), with the aim of measuring the closeness of agreement of methylation values between these two methods and its effect when setting a cut-off. Methylation of tumor suppressor gene p16/INK4A was evaluated in 80 lung cancer patients from which cytological lymph node samples were obtained. Cluster analyses were used to establish methylated and unmethylated groups for each method. Agreement and concordance between pyrosequencing and MS-qPCR was evaluated with Pearson's correlation, Bland-Altman, Cohen's kappa index and ROC curve analyses. Based on these analyses, cut-offs were derived for MS-qPCR. An acceptable correlation (Pearson's R2 = 0.738) was found between pyrosequencing (PYRmean) and MS-qPCR (NMP; normalized methylation percentage), providing similar clinical results when categorizing data as binary using cluster analysis. Compared to pyrosequencing, MS-qPCR tended to underestimate methylation for values between 0 and 15%, while for methylation >30% overestimation was observed. The estimated cut-off for MS-qPCR data based on cluster analysis, kappa-index agreement and ROC curve analysis were much lower than that derived from pyrosequencing. In conclusion, our results indicate that independently of the approach used for estimating the cut-off, the methylation percentage obtained through MS-qPCR is lower than that calculated for pyrosequencing. These differences in data and therefore in the cut-off should be examined when using methylation biomarkers in the clinical practice.

CD26-Related Serum Biomarkers: sCD26 Protein, DPP4 Activity, and Anti-CD26 Isotype Levels in a Colorectal Cancer-Screening Context.

Current screening trials are showing reduction in colorectal cancer incidence and mortality. However, participation rates are often low, and blood-based tests could complement existing screening strategies. CD26 protein (sCD26) and its dipeptidyl peptidase IV (DPP4) enzymatic activity in circulation have been proposed as biomarkers for colorectal cancer and other diseases. However, changes in sCD26 and DPP4 levels show complex degrees of correlation, and their physiological or pathophysiological role is unclear. The aim of this study was to analyse if anti-CD26 autoantibodies are related to sCD26 and DPP4 and to determine their relevance in a context of colorectal cancer screening for complementing the value of sCD26 and DPP4 as biomarkers. These biomarkers were measured in a large prospective cohort (n = 497, except the anti-CD26 antibodies, evaluated in 125 samples) that included a subgroup of individuals that were positive for the faecal immunological occult blood test (FIT) (n = 86) and underwent a colonoscopy (n = 47). We confirmed for the first time higher DPP4 activity in men compared to women (Student's t test, p = 0.002), though this difference between sexes was not seen for serum sCD26 protein. These biomarkers correlated (R = 0.246, p = 0.003) only in women. Correlations were found between anti-CD26 isotypes but not with DPP4 activity or sCD26 concentration, except for a negative correlation only in men between anti-CD26 IgA isotype and sCD26 (R = -0.232, p = 0.044), and an almost significant negative correlation between anti-CD26 IgG and sCD26 limited to FIT-positive men. Interestingly, patients with advanced adenomas displayed the most elevated mean levels of anti-CD26 IgA, IgM, and particularly IgG (Mann-Whitney U test, p = 0.030) in comparison with the other FIT positives without adenomas, and these levels did not correlate with sCD26 or its DPP4 activity. Our preliminary results suggest that the combination of these measures using sex as confounder could perhaps be used as biomarkers for colorectal disease. It also suggests that events affecting the gut influence the levels of anti-CD26 antibodies, which show little or no effect in antigen clearance. These findings should be confirmed in a larger cohort of individuals with colonoscopy. The physiological origin of the sex differences observed should be further addressed.

Methylation Assessment for the Prediction of Malignancy in Mediastinal Adenopathies Obtained by Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration in Patients with Lung Cancer.

The evaluation of mediastinal lymph nodes is critical for the correct staging of patients with lung cancer (LC). Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique for mediastinal staging, though unfortunately lymph node micrometastasis is often missed by cytological analysis. The aim of this study was to evaluate the predictive capacity of methylation biomarkers and provide a classification rule for predicting malignancy in false negative EBUS-TBNA samples. The study included 112 patients with a new or suspected diagnosis of LC that were referred to EBUS-TBNA. Methylation of p16/INK4a, MGMT, SHOX2, E-cadherin, DLEC1, and RASSF1A was quantified by nested methylation-specific qPCR in 218 EBUS-TBNA lymph node samples. Cross-validated linear regression models were evaluated to predict malignancy. According to EBUS-TBNA and final diagnosis, 90 samples were true positives for malignancy, 110 were true negatives, and 18 were false negatives. MGMT, SHOX2, and E-cadherin were the methylation markers that better predicted malignancy. The model including sex, age, short axis diameter and standard uptake value of adenopathy, and SHOX2 showed 82.7% cross-validated sensitivity and 82.4% specificity for the detection of malignant lymphadenopathies among negative cytology samples. Our results suggest that the predictive model approach proposed can complement EBUS-TBNA for mediastinal staging.

A new approach to epigenome-wide discovery of non-invasive methylation biomarkers for colorectal cancer screening in circulating cell-free DNA using pooled samples.

Background: Colorectal cancer is the fourth cause of cancer-related deaths worldwide, though detection at early stages associates with good prognosis. Thus, there is a clear demand for novel non-invasive tests for the early detection of colorectal cancer and premalignant advanced adenomas, to be used in population-wide screening programs. Aberrant DNA methylation detected in liquid biopsies, such as serum circulating cell-free DNA (cfDNA), is a promising source of non-invasive biomarkers. This study aimed to assess the feasibility of using cfDNA pooled samples to identify potential serum methylation biomarkers for the detection of advanced colorectal neoplasia (colorectal cancer or advanced adenomas) using microarray-based technology. Results: cfDNA was extracted from serum samples from 20 individuals with no colorectal findings, 20 patients with advanced adenomas, and 20 patients with colorectal cancer (stages I and II). Two pooled samples were prepared for each pathological group using equal amounts of cfDNA from 10 individuals, sex-, age-, and recruitment hospital-matched. We measured the methylation levels of 866,836 CpG positions across the genome using the MethylationEPIC array. Pooled serum cfDNA methylation data meets the quality requirements. The proportion of detected CpG in all pools (> 99% with detection p value < 0.01) exceeded Illumina Infinium methylation data quality metrics of the number of sites detected. The differential methylation analysis revealed 1384 CpG sites (5% false discovery rate) with at least 10% difference in the methylation level between no colorectal findings controls and advanced neoplasia, the majority of which were hypomethylated. Unsupervised clustering showed that cfDNA methylation patterns can distinguish advanced neoplasia from healthy controls, as well as separate tumor tissue from healthy mucosa in an independent dataset. We also observed that advanced adenomas and stage I/II colorectal cancer methylation profiles, grouped as advanced neoplasia, are largely homogenous and clustered close together. Conclusions: This preliminary study shows the viability of microarray-based methylation biomarker discovery using pooled serum cfDNA samples as an alternative approach to tissue specimens. Our strategy sets an open door for deciphering new non-invasive biomarkers not only for colorectal cancer detection, but also for other types of cancers.

Proteomic Profiling for Colorectal Cancer Biomarker Discovery.

Nowadays, the ideal biomarker for colorectal cancer (CRC) has not been found. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) are suitable techniques for searching new biomarkers. In this chapter, we describe methodology for biomarker discovery based on a proteomic approach. In addition, special attention is given to the sample preparation, including protein extraction, fractionation, and cleanup, as we consider this a critical step. Comparing the proteomic profile of tumor and mucosa, we identified the nucleoside diphosphate kinase A (NDKA) protein as a candidate biomarker for CRC. Finally, we validated NDKA with an ELISA kit using serum samples from individuals of a screening cohort. Our results suggest that serum NDKA is a potential biomarker for screening of CRC and premalignant advanced adenomas (AA).

Relevance of matrix metalloproteases in non-small cell lung cancer diagnosis.

BACKGROUND: The need for novel biomarkers that could aid in non-small cell lung cancer (NSCLC) detection, together with the relevance of Matrix Metalloproteases (MMPs) -1, -2, -7, -9 and -10 in lung tumorigenesis, prompted us to assess the diagnostic usefulness of these MMPs and the Tissue Inhibitor of Metalloproteinase (TIMP) -1 in NSCLC patients. METHODS: Markers were evaluated in an initial study cohort (19 NSCLC cases and 19 healthy controls). Those that better performed were analyzed in a larger sample including patients with benign lung diseases. Serum MMPs and TIMP-1 were determined by multiplexed immunoassays. Logistic regression was employed for multivariate analysis of biomarker combinations. RESULTS: MMPs and TIMP-1 were elevated in the serum of NSCLC patients compared to healthy controls. MMP-1, -7 and -9 performed at best and were further evaluated in the sample including benign pathologies, corroborating the superiority of MMP-9 in NSCLC discrimination, also at early-stage NSCLC. The optimal diagnostic value was obtained with the model including MMP-9, gender, age and smoking history, that demonstrated an AUC of 0.787, 85.54% sensitivity and 64.89% specificity. CONCLUSION: Our results suggest that MMP-9 is a potential biomarker for NSCLC diagnosis and its combined measurement with other biomarkers could improve NSCLC detection.

Highly Sensitive Marker Panel for Guidance in Lung Cancer Rapid Diagnostic Units.

While evidence for lung cancer screening implementation in Europe is awaited, Rapid Diagnostic Units have been established in many hospitals to accelerate the early diagnosis of lung cancer. We seek to develop an algorithm to detect lung cancer in a symptomatic population attending such unit, based on a sensitive serum marker panel. Serum concentrations of Epidermal Growth Factor, sCD26, Calprotectin, Matrix Metalloproteinases -1, -7, -9, CEA and CYFRA 21.1 were determined in 140 patients with respiratory symptoms (lung cancer and controls with/without benign pathology). Logistic Lasso regression was performed to derive a lung cancer prediction model, and the resulting algorithm was tested in a validation set. A classification rule based on EGF, sCD26, Calprotectin and CEA was established, able to reasonably discriminate lung cancer with 97% sensitivity and 43% specificity in the training set, and 91.7% sensitivity and 45.4% specificity in the validation set. Overall, the panel identified with high sensitivity stage I non-small cell lung cancer (94.7%) and 100% small-cell lung cancers. Our study provides a sensitive 4-marker classification algorithm for lung cancer detection to aid in the management of suspicious lung cancer patients in the context of Rapid Diagnostic Units.

Evaluation of serum nucleoside diphosphate kinase A for the detection of colorectal cancer.

We previously described the over-expression of nucleoside diphosphate kinase A (NDKA) in tumours and serum from colorectal cancer (CRC) patients, suggesting its use as biomarker. In this study we evaluated the diagnostic accuracy of serum NDKA to detect advanced neoplasia (CRC or advanced adenomas). Furthermore, the performance of NDKA was compared with the faecal immunochemical test (FIT). The study population included a case-control cohort and a screening cohort (511 asymptomatic first-degree relatives of CRC patients that underwent a colonoscopy and a FIT). Serum NDKA was elevated in CRC patients in the case-control cohort (p = 0.002). In the screening cohort, NDKA levels were higher for advanced adenomas (p = 0.010) and advanced neoplasia (p = 0.006) compared to no neoplasia. Moreover, elevated NDKA was associated with severe characteristics of adenomas (≥3 lesions, size ≥ 1 cm or villous component). Setting specificity to 85%, NDKA showed a sensitivity of 30.19% and 29.82% for advanced adenomas and advanced neoplasia, respectively. NDKA combined with FIT (100 ng/mL cut-off) detected advanced adenomas and advanced neoplasia with 45.28% and 49.12% sensitivity, with specificity close to 90%. The combination of serum NDKA and FIT can improve the detection of advanced neoplasia, mainly for lesions located on the proximal colon, in asymptomatic individuals with CRC family-risk.

Serum matrix metalloproteinase-9 in colorectal cancer family-risk population screening.

Matrix metalloproteinase-9 (MMP-9) is related to tumour development and progression in colorectal cancer (CRC) and its utility as biomarker has been suggested. The aim of our study was to measure serum MMP-9 in asymptomatic first-degree relatives of CRC patients, and to analyse its diagnostic accuracy for the detection of advanced neoplasia (AN: advanced adenomas and CRC). Additionally, we compared its diagnostic capability with the most used non-invasive faecal immunochemical test (FIT). Serum MMP-9 was quantified by ELISA in 516 asymptomatic individuals that underwent a colonoscopy and a FIT. MMP-9 levels were significantly related to age and gender and therefore the concentration was corrected by these confounders. Corrected MMP-9 (cMMP-9) levels were higher in individuals with advanced adenomas (AA; p-value = 0.029) and AN (p-value = 0.056) compared to individuals with no neoplasia. Moreover, elevated cMMP-9 concentration was associated with more severe characteristics of adenomas (number of lesions, size and histology). Nevertheless, the diagnostic accuracy of cMMP-9 was considerably lower than that of FIT for identifying AA (22.64% vs. 47.17% sensitivity, 90% specificity) or AN (19.30% vs. 52.63% sensitivity, 90% specificity). According to our results, serum MMP-9 cannot be considered of utility for the diagnosis of AN in CRC family-risk population screening.

Postoperative serum levels of sCD26 for surveillance in colorectal cancer patients.

One of the main aims of the follow-up after curative resection of colorectal cancer is the early detection and treatment of tumor recurrence. We previously demonstrated decreased preoperative soluble CD26 (sCD26) levels in serum from colorectal cancer patients. We extended now the study to investigate if sCD26 levels in postoperative serum serve as marker of recurrence of the disease during surveillance. Soluble sCD26 was measured in pre- and postoperative serum samples of 43 patients with primary colorectal cancer. Carcinoembryonic antigen, carbohydrate antigen 19.9 and 72.4 levels were also measured during surveillance. The average follow-up period was 41.8 ± 20.8 months. sCD26 levels during follow-up showed well-defined patterns in patients without disease (n = 28), and in patients with tumor persistence (n = 2), local recurrence (n = 3) or distant metastasis (n = 10). Disease-free patients showed stable levels between 460-850 ng/mL during follow-up, while high (over 850 ng/mL) and unstable sCD26 levels were found before recurrence was diagnosed. The mean maximum/minimum sCD26 ratios during surveillance were 1.52, 2.12 and 2.63 for patients with no recurrence, local recurrence and metastasis, respectively (p = 0.005). From the cut-off obtained from a receiver operator characteristics (ROC) curve built with the maximum/minimum sCD26 ratios and the upper and lower cut-offs of sCD26, we were able to discriminate patients with and without recurrent disease. We propose that the measurement of serum sCD26 during the follow-up of patients diagnosed of colorectal cancer could be valuable for the early detection of local and distant recurrence. A large, randomized, prospective trial should be performed to confirm our findings.

Viability of lymph node samples obtained by echobronchoscopy in the study of epigenetic alterations in patients with lung cancer.

INTRODUCTION: The diagnosis of microscopic lymph node metastasis in lung cancer is challenging despite the constant advances in tumor staging. The analysis of the methylation status of certain genes in lymph node samples could improve the diagnostic capability of conventional cyto-histological methods. The aim of this study was to demonstrate the feasibility of methylation studies using cytological lymph node samples. METHODS: Prospective study including 88 patients with a diagnosis or strong suspicion of non-small cell lung cancer, in which an echobronchoscopy was performed on mediastinal or hilar lymph nodes for diagnostic and/or staging. DNA was extracted from cytological lymph node samples and sodium bisulfite modification was performed. Methylation studies for p16/INK4a and SHOX2 were accomplished by MS-qPCR and pyrosequencing. RESULTS: The methodology used in our study yielded optimal/good DNA quality in 90% of the cases. No differences in DNA concentration were observed with respect to the lymph node biopsied and final diagnosis. Methylation analyses using MS-qPCR and pyrosequencing were not possible in a small number of samples mainly due to low DNA concentration, inadequate purity, fragmentation and/or degradation as a consequence of bisulfite conversion. CONCLUSION: Methylation quantification using MS-qPCR and pyrosequencing of cytological lymph node samples obtained using echobronchoscopy is feasible if an appropriate DNA concentration is obtained, notably contributing to the identification of epigenetic biomarkers capable of improving decision-making for the benefit of potentially curable lung cancer patients.

Prognostic value of aberrant hypermethylation in pleural effusion of lung adenocarcinoma.

Lung adenocarcinoma is one of the most frequent causes of malignant pleural effusions (MPE). The presence of MPE bears a poor prognosis. Although epigenetic changes are commonly related to human neoplasia, scarce date is available on patients with MPE. We aimed to estimate the prognostic value of DNA methylation of tumor suppressor genes from pleural fluid. Thirty patients with MPE due to lung adenocarcinoma were prospectively included. Methylation-specific (MS) PCR was used to study the methylation status of the promoter region of tumor suppressor genes p16/INK4a, MGMT, BRCA1 and RARß in pleural fluid. Clinical data and survival were collected. Survival analysis was performed using Kaplan-Meier plots and Cox regression. Hypermethylation in at least one gene was detected in 25 patients (83.3%). On multivariate analysis factors significantly associated with shorter survival were the lack of hypermethylation in any of the studied genes (hazard ratio = 9.3; p = 0.001), Charlson index ≥ 3 (hazard ratio = 9.6, p = 0.002) and no oncological treatment (hazard ratio = 11.1; p < 0.001). Analysis of aberrant promoter hypermethylation of tumor suppressor genes may be useful in predicting prognosis, but further studies are needed to validate our findings.