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.Ministerio de Ciencia e Innovación | Ref. PS09-00405Xunta de Galicia | Ref. INBIOMED 2012-273Xunta de Galicia | Ref. GRC2014/019Ministerio de Ciencia e Innovación | Ref. MTM2011-2320

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.Xunta de Galicia | Ref. 09CSA053905PRInstituto Carlos III | Ref. PI09 / 9038

Value of serum NEUROG1 methylation for the detection of advanced adenomas and colorectal cancer

Aberrant DNA methylation detected in liquid biopsies is a promising approach for colorectal cancer (CRC) detection, including premalignant advanced adenomas (AA). We evaluated the diagnostic capability of serum NEUROG1 methylation for the detection of AA and CRC. A CpG island in NEUROG1 promoter was assessed by bisulfite pyrosequencing in a case-control cohort to select optimal CpGs. Selected sites were evaluated through a nested methylation-specific qPCR custom assay in a screening cohort of 504 asymptomatic family-risk individuals. Individuals with no colorectal findings and benign pathologies showed low serum NEUROG1 methylation, similar to non-advanced adenomas. Contrarily, individuals bearing AA or CRC (advanced neoplasia—AN), exhibited increased NEUROG1 methylation. Using >1.3518% as NEUROG1 cut-off (90.60% specificity), 33.33% of AN and 32.08% of AA were identified, detecting 50% CRC cases. Nonetheless, the combination of NEUROG1 with fecal immunochemical test (FIT), together with age and gender through a multivariate logistic regression resulted in an AUC = 0.810 for AN, and 0.796 for AA, detecting all cancer cases and 35–47% AA (specificity 98–95%). The combination of NEUROG1 methylation with FIT, age and gender demonstrated a convenient performance for the detection of CRC and AA, providing a valuable tool for CRC screening programs in asymptomatic individuals.Instituto de Salud Carlos III | Ref. PI15/02007Xunta de Galicia | Ref. Centro Singular de Investigación de Galicia accreditation 2016-201

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.Instituto de Salud Carlos III | Ref. PI09/90385Instituto de Salud Carlos III | Ref. RETIC-FIS-ISCIII RD09/0076/00011Xunta de Galicia | Ref. 09CSA053905P

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.Fundação para a Ciência e a Tecnologia | Ref. UIDB / 50026/2020Fundação para a Ciência e a Tecnologia | Ref. UIDP / 50026/2020Fundação para a Ciência e a Tecnologia | Ref. 2020.03113.CEECINDXunta de Galicia | Ref. CT850A-2European Commission | Ref. NORTE-01-0145-FEDER-072555European Commission | Ref. NORTE-01-0145- FEDER-00003

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 FITpositive 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 addressedFundación Científica de la Asociación Española Contra el Cáncer | Ref. GCB13131592CASTXunta de Galicia | Ref. GRC2014/019Red Gallega de Investigacion sobre Cáncer Colorectal | Ref. REGICC, R2014/03

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

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGA detailed understanding of how and when 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 49 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 1-2 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.Xunta de Galicia | Ref. ED431C2018/54Banco SantanderConsejo Superior de Investigaciones CientíficasConferencia de Rectores de las Universidades EspañolasServizo Galego de Saúd

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.Xunta de Galicia | Ref. CT850A-2Banco Santander-CSIC-CRUE | Ref. EPICOVIGAL de FONDO SUPERA-COVID1