DeltaMSI: artificial intelligence-based modeling of microsatellite instability scoring on next-generation sequencing data.

BACKGROUND: DNA mismatch repair deficiency (dMMR) testing is crucial for detection of microsatellite unstable (MSI) tumors. MSI is detected by aberrant indel length distributions of microsatellite markers, either by visual inspection of PCR-fragment length profiles or by automated bioinformatic scoring on next-generation sequencing (NGS) data. The former is time-consuming and low-throughput while the latter typically relies on simplified binary scoring of a single parameter of the indel distribution. The purpose of this study was to use machine learning to process the full complexity of indel distributions and integrate it into a robust script for screening of dMMR on small gene panel-based NGS data of clinical tumor samples without paired normal tissue. METHODS: Scikit-learn was used to train 7 models on normalized read depth data of 36 microsatellite loci in a cohort of 133 MMR proficient (pMMR) and 46 dMMR tumor samples, taking loss of MLH1/MSH2/PMS2/MSH6 protein expression as reference method. After selection of the optimal model and microsatellite panel the two top-performing models per locus (logistic regression and support vector machine) were integrated into a novel script (DeltaMSI) for combined prediction of MSI status on 28 marker loci at sample level. Diagnostic performance of DeltaMSI was compared to that of mSINGS, a widely used script for MSI detection on unpaired tumor samples. The robustness of DeltaMSI was evaluated on 1072 unselected, consecutive solid tumor samples in a real-world setting sequenced using capture chemistry, and 116 solid tumor samples sequenced by amplicon chemistry. Likelihood ratios were used to select result intervals with clinical validity. RESULTS: DeltaMSI achieved higher robustness at equal diagnostic power (AUC = 0.950; 95% CI 0.910-0.975) as compared to mSINGS (AUC = 0.876; 95% CI 0.823-0.918). Its sensitivity of 90% at 100% specificity indicated its clinical potential for high-throughput MSI screening in all tumor types. Clinical Trial Number/IRB B1172020000040, Ethical Committee, AZ Delta General Hospital.

Cov2MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein.

The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov2MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.

Diagnostic Performance of Chest CT for SARS-CoV-2 Infection in Individuals with or without COVID-19 Symptoms.

Background The use of chest CT for coronavirus disease 2019 (COVID-19) diagnosis or triage in health care settings with limited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) capacity is controversial. COVID-19 Reporting and Data System (CO-RADS) categorization of the level of COVID-19 suspicion might improve diagnostic performance. Purpose To investigate the value of chest CT with CO-RADS classification to screen for asymptomatic SARS-CoV-2 infections and to determine its diagnostic performance in individuals with COVID-19 symptoms during the exponential phase of viral spread. Materials and Methods In this secondary analysis of a prospective trial, from March 2020 to April 2020, parallel SARS-CoV-2 PCR and CT with categorization of COVID-19 suspicion was performed with CO-RADS for individuals with COVID-19 symptoms and control participants without COVID-19 symptoms admitted to the hospital for medical emergencies unrelated to COVID-19. CT with CO-RADS was categorized on a five-point scale from 1 (very low suspicion) to 5 (very high suspicion). Area under the receiver operating curve (AUC) was calculated in symptomatic versus asymptomatic individuals to predict positive SARS-CoV-2 PCR, and likelihood ratios for each CO-RADS score were used for rational selection of diagnostic thresholds. Results A total of 859 individuals (median age, 70 years; interquartile range, 52-81 years; 443 men) with COVID-19 symptoms and 1138 control participants (median age, 68 years; interquartile range, 52-81 years; 588 men) were evaluated. CT with CO-RADS had good diagnostic performance (P < .001) in both symptomatic (AUC, 0.89) and asymptomatic (AUC, 0.70) individuals. In symptomatic individuals (42% PCR positive), CO-RADS 3 or greater detected positive PCR with high sensitivity (89%, 319 of 358) and specificity of 73%. In asymptomatic individuals (5% PCR positive), a CO-RADS score of 3 or greater detected SARS-CoV-2 infection with low sensitivity (45%, 27 of 60) but high specificity (89%). Conclusion CT with Coronavirus Disease 2019 Reporting and Data System (CO-RADS) had good diagnostic performance in symptomatic individuals, supporting its application for triage. Sensitivity in asymptomatic individuals was insufficient to justify its use as a first-line screening approach. Incidental detection of CO-RADS 3 or greater in asymptomatic individuals should trigger testing for respiratory pathogens. © RSNA, 2020 Online supplemental material is available for this article.

Early treatment of a child with NAGS deficiency using N-carbamyl glutamate results in a normal neurological outcome.

Acute hyperammonemia has a variety of etiologies and clinical manifestations. If not treated early in neonates, it leads to irreversible brain damage or death. We present a 7-day-old female patient who was brought to the emergency department with drownsiness and vomiting. Metabolic work-up revealed a blood ammonia level of 290 µmol/L (normal <100 µmol/L in neonates) with a compensated respiratory alkalosis, normal glycaemia and lactate and absence of urinary ketones. Oral feeding was stopped, an infusion of 20 % glucose was started, and sodium benzoate and arginine hydrochloride were given. After a drop of ammonemia within 12 h of treatment, it started rising again. N-carbamylglutamate (NCG) was added resulting in a rapid normalisation of ammonemia. Feedings were progressively reintroduced, the ammonia levels remained low. The results of the metabolic work-up were compatible with carbamyl phosphate synthase 1 (CPS1) or N-acetyl glutamate synthase (NAGS) deficiency. Genetic analysis confirmed the latter diagnosis with a homozygous mutation c. 1450T > C (p.W484R) in exon 6 of the NAGS gene in the patient and a carrier state in both parents. At the age of 9 months, the child is growing well with normal neurological development, under treatment with NCG 100 mg/kg/day and a normal diet. Conclusion: This case highlights the importance of keeping a high index of suspicion and early testing for ammonia levels in neonates/children with unexplained encephalopathy. In neonates with congenital hyperammonemia, NCG should always be started together with the standard management of hyperammonemia until all laboratory investigations are complete or indicate another disease.

Acoustic ejection mass spectrometry empowers ultra-fast protein biomarker quantification.

The global scientific response to COVID 19 highlighted the urgent need for increased throughput and capacity in bioanalytical laboratories, especially for the precise quantification of proteins that pertain to health and disease. Acoustic ejection mass spectrometry (AEMS) represents a much-needed paradigm shift for ultra-fast biomarker screening. Here, a quantitative AEMS assays is presented, employing peptide immunocapture to enrich (i) 10 acute phase response (APR) protein markers from plasma, and (ii) SARS-CoV-2 NCAP peptides from nasopharyngeal swabs. The APR proteins were quantified in 267 plasma samples, in triplicate in 4.8 h, with %CV from 4.2% to 10.5%. SARS-CoV-2 peptides were quantified in triplicate from 145 viral swabs in 10 min. This assay represents a 15-fold speed improvement over LC-MS, with instrument stability demonstrated across 10,000 peptide measurements. The combination of speed from AEMS and selectivity from peptide immunocapture enables ultra-high throughput, reproducible quantitative biomarker screening in very large cohorts.

Homozygosity for aquaporin 7 G264V in three unrelated children with hyperglyceroluria and a mild platelet secretion defect.

PURPOSE: Aquaporin 7 (AQP7) belongs to the aquaglyceroporin family, which transports glycerol and water. AQP7-deficient mice develop obesity, insulin resistance, and hyperglyceroluria. However, AQP7's pathophysiologic role in humans is not yet known. METHODS: Three children with psychomotor retardation and hyperglyceroluria were screened for AQP7 mutations. The children were from unrelated families. Urine and plasma glycerol levels were measured using a three-step enzymatic approach. Platelet morphology and function were studied using electron microscopy, aggregations, and adenosine triphosphate (ATP) secretion tests. RESULTS: The index patients were homozygous for AQP7 G264V, which has previously been shown to inhibit transport of glycerol in Xenopus oocytes. We also detected a subclinical platelet secretion defect with reduced ATP secretion, and the absence of a secondary aggregation wave after epinephrine stimulation. Electron microscopy revealed round platelets with centrally located granules. Immunostaining showed AQP7 colocalization, with dense granules that seemed to be released after strong platelet activation. Healthy relatives of these patients, who were homozygous (not heterozygous) for G264V, also had hyperglyceroluria and platelet granule abnormalities. CONCLUSION: The discovery of an association between urine glycerol loss and a platelet secretion defect is a novel one, and our findings imply the involvement of AQPs in platelet secretion. Additional studies are needed to define whether AQP7 G264V is also a risk factor for mental disability.

PhIP-Seq characterization of autoantibodies from patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis.

Autoimmune disease results from a loss of tolerance to self-antigens in genetically susceptible individuals. Completely understanding this process requires that targeted antigens be identified, and so a number of techniques have been developed to determine immune receptor specificities. We previously reported the construction of a phage-displayed synthetic human peptidome and a proof-of-principle analysis of antibodies from three patients with neurological autoimmunity. Here we present data from a large-scale screen of 298 independent antibody repertoires, including those from 73 healthy sera, using phage immunoprecipitation sequencing. The resulting database of peptide-antibody interactions characterizes each individual's unique autoantibody fingerprint, and includes specificities found to occur frequently in the general population as well as those associated with disease. Screening type 1 diabetes (T1D) patients revealed a prematurely polyautoreactive phenotype compared with their matched controls. A collection of cerebrospinal fluids and sera from 63 multiple sclerosis patients uncovered novel, as well as previously reported antibody-peptide interactions. Finally, a screen of synovial fluids and sera from 64 rheumatoid arthritis patients revealed novel disease-associated antibody specificities that were independent of seropositivity status. This work demonstrates the utility of performing PhIP-Seq screens on large numbers of individuals and is another step toward defining the full complement of autoimmunoreactivities in health and disease.

Hereditary Tyrosinemia Type 1 Mice under Continuous Nitisinone Treatment Display Remnants of an Uncorrected Liver Disease Phenotype.

Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

Enzymatic pyruvate measurement by Cobas 6000 open channel assay.

BACKGROUND: Blood pyruvate measurement in conjunction with lactic acid is useful for differentiating pyruvate dehydrogenase deficiencies from primary or secondary disorders of mitochondrial electron transport. METHODS: We evaluated the analytical performance of pyruvate measurement by an enzymatic open channel assay on a Roche Cobas 6000. RESULTS: The assay was linear from 0.07 to 0.50 mmol/L pyruvate. Total imprecision ranged from 15.7% to 7.1% at pyruvate levels of 0.08 to 0.31 mmol/L, respectively. Functional sensitivity was 0.07 mmol/L. The assay showed no interference by lipids or bilirubin, whereas haemolysis influenced pyruvate concentrations in a hemoglobin concentration-independent manner. Method comparison with patient samples (n = 41) showed that the Cobas 6000 enzymatic method correlated well (r2 = 0.930) with a similar enzymatic assay on a Cobas Mira platform and showed better accuracy in external control schemes. CONCLUSIONS: Enzymatic pyruvate measurement by a Cobas 6000 open channel shows satisfactory analytical performance. The assay can be integrated in the automated laboratory workflow and is always ready for use thanks to its on-board reagents.

From Delta to Omicron SARS-CoV-2 variant: Switch to saliva sampling for higher detection rate.

Background: Real-time polymerase chain reaction (RT-PCR) testing on a nasopharyngeal swab is the current standard for SARS-CoV-2 virus detection. Since collection of this sample type is experienced uncomfortable by patients, saliva- and oropharyngeal swab collections should be considered as alternative specimens. Objectives: Evaluation of the relative performance of oropharyngeal swab, nasopharyngeal swab and saliva for the RT-PCR based SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variant detection. Study design: Nasopharyngeal swab, oropharyngeal swab and saliva were collected from 246 adult patients who presented for SARS-CoV-2 testing at the screening centre in Ypres (Belgium). RT-PCR SARS-CoV-2 detection was performed on all three sample types separately. Variant type was determined for each positive patient using whole genome sequencing or Allplex SARS-CoV-2 variants I and II Assay. Results and conclusions: Saliva is superior compared to nasopharyngeal swab for the detection of the Omicron variant. For the detection of the Delta variant, nasopharyngeal swab and saliva can be considered equivalent specimens. Oropharyngeal swab is the least sensitive sample type and shows little added value when collected in addition to a single nasopharyngeal swab.

Pitfalls of SARS-CoV-2 antigen testing at emergency department.

BACKGROUND: Current method for diagnosis of SARS-CoV-2 infection is an RT-PCR test on the nasopharyngeal or oropharyngeal swab. Rapid diagnosis is essential for containing viral spread and triage of symptomatic patients presenting to hospital ER departments. As a faster alternative to RT-PCR, we evaluated a SARS-Cov-2 Rapid Antigen test in symptomatic patients presenting to hospital ER departments. METHODS: We evaluated the diagnostic performance of the Roche SARS-CoV-2 Rapid Antigen test (SD Biosensor) for detection of SARS-CoV-2 compared to RT-PCR. RESULTS: Our study showed inferior performance of the SARS-CoV-2 Rapid Antigen test for detection of SARS-CoV-2. Firstly, because of the lack of specificity, which is potentially life-threatening due to the association of nosocomial-acquired SARS-CoV-2 infection. Secondly, with a sensitivity of 45.5%, it is impossible to rule out SARS-CoV-2 infection, resulting in reflex PCR-testing. Comparison of viral load in RT-PCR positive samples with corresponding antigen results showed a significant difference between antigen positive and negative samples. COVID-19 infection will not be detected in patients admitted to the hospital in an early or late phase, typically associated with low viral loads. Sensitivity increases when testing within 5-7 symptomatic days, but the implementation of this cut-off is impractical in ER settings. However, diagnostic performance is better to detect high viral load (> = 5 log10 copies/mL) linked with contagiousness. CONCLUSION: Our study showed inferior performance of the Roche SARS-CoV-2 Rapid Antigen test (SD Biosensor) for detection of SARS-CoV-2 which limits its use as a diagnostic gatekeeper in ER departments, but is able to differentiate contagious individuals.

Cycle Threshold Probability Score for Immediate and Sensitive Detection of B.1.351 SARS-CoV-2 Lineage.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern associated with immune escape is important to safeguard vaccination efficacy. We describe the potential of delayed N gene amplification in the Allplex SARS-CoV-2 Assay (Seegene) for screening of the B.1.351 (20H/501.V2, variant of concern 2 [VOC.V2], South African SARS-CoV-2 variant) lineage. METHODS: In a study cohort of 397 consecutive polymerase chain reaction-positive samples genotyped by whole-genome sequencing, amplification curves of E/N/S-RdRP targets indicated delayedN vs E gene amplification characteristic of B.1.351. Logistic regression was used to calculate a VOC.V2 probability score that was evaluated as a separate screening test in an independent validation cohort vs sequencing. RESULTS: B.1.351 showed a proportionally delayed amplification of the  N vs E gene. In logistic regression, only N and E gene cycle thresholds independently contributed to B.1.351 prediction, allowing calculation of a VOC.V2 probability score with an area under the curve of 0.94. At an optimal dichotomous cutoff point of 0.12, the VOC.V2 probability score achieved 98.7% sensitivity at 79.9% specificity, resulting in a negative predictive value (NPV) of 99.6% and a positive predictive value of 54.6%. The probability of B.1.351 increased with an increasing VOC.V2 probability score, achieving a likelihood ratio of 12.01 above 0.5. A near-maximal NPV was confirmed in 153 consecutive validation samples. CONCLUSIONS: Delayed N vs E gene amplification in the Allplex SARS-CoV-2 Assay can be used for fast and highly sensitive screening of B.1.351.

Three doses of BNT162b2 vaccine confer neutralising antibody capacity against the SARS-CoV-2 Omicron variant.

We report the levels of neutralising antibodies against Wuhan, Delta and Omicron variants in unimmunized infected (group 1), immunised and boosted (group 2) and infected immunised and boosted (group 3) adult individuals. Our observations support the rapid administration of a booster vaccine dose to prevent infection and disease caused by Omicron.

Two Years of Genomic Surveillance in Belgium during the SARS-CoV-2 Pandemic to Attain Country-Wide Coverage and Monitor the Introduction and Spread of Emerging Variants.

An adequate SARS-CoV-2 genomic surveillance strategy has proven to be essential for countries to obtain a thorough understanding of the variants and lineages being imported and successfully established within their borders. During 2020, genomic surveillance in Belgium was not structurally implemented but performed by individual research laboratories that had to acquire the necessary funds themselves to perform this important task. At the start of 2021, a nationwide genomic surveillance consortium was established in Belgium to markedly increase the country's genomic sequencing efforts (both in terms of intensity and representativeness), to perform quality control among participating laboratories, and to enable coordination and collaboration of research projects and publications. We here discuss the genomic surveillance efforts in Belgium before and after the establishment of its genomic sequencing consortium, provide an overview of the specifics of the consortium, and explore more details regarding the scientific studies that have been published as a result of the increased number of Belgian SARS-CoV-2 genomes that have become available.

Predictive model for BNT162b2 vaccine response in cancer patients based on blood cytokines and growth factors.

Background: Patients with cancer, especially hematological cancer, are at increased risk for breakthrough COVID-19 infection. So far, a predictive biomarker that can assess compromised vaccine-induced anti-SARS-CoV-2 immunity in cancer patients has not been proposed. Methods: We employed machine learning approaches to identify a biomarker signature based on blood cytokines, chemokines, and immune- and non-immune-related growth factors linked to vaccine immunogenicity in 199 cancer patients receiving the BNT162b2 vaccine. Results: C-reactive protein (general marker of inflammation), interleukin (IL)-15 (a pro-inflammatory cytokine), IL-18 (interferon-gamma inducing factor), and placental growth factor (an angiogenic cytokine) correctly classified patients with a diminished vaccine response assessed at day 49 with >80% accuracy. Amongst these, CRP showed the highest predictive value for poor response to vaccine administration. Importantly, this unique signature of vaccine response was present at different studied timepoints both before and after vaccination and was not majorly affected by different anti-cancer treatments. Conclusion: We propose a blood-based signature of cytokines and growth factors that can be employed in identifying cancer patients at persistent high risk of COVID-19 despite vaccination with BNT162b2. Our data also suggest that such a signature may reflect the inherent immunological constitution of some cancer patients who are refractive to immunotherapy.

Interleukin-6 regulates pancreatic alpha-cell mass expansion.

Interleukin-6 (IL-6) is systemically elevated in obesity and is a predictive factor to develop type 2 diabetes. Pancreatic islet pathology in type 2 diabetes is characterized by reduced beta-cell function and mass, an increased proportion of alpha-cells relative to beta-cells, and alpha-cell dysfunction. Here we show that the alpha cell is a primary target of IL-6 actions. Beginning with investigating the tissue-specific expression pattern of the IL-6 receptor (IL-6R) in both mice and rats, we find the highest expression of the IL-6R in the endocrine pancreas, with highest expression on the alpha-cell. The islet IL-6R is functional, and IL-6 acutely regulates both pro-glucagon mRNA and glucagon secretion in mouse and human islets, with no acute effect on insulin secretion. Furthermore, IL-6 stimulates alpha-cell proliferation, prevents apoptosis due to metabolic stress, and regulates alpha-cell mass in vivo. Using IL-6 KO mice fed a high-fat diet, we find that IL-6 is necessary for high-fat diet-induced increased alpha-cell mass, an effect that occurs early in response to diet change. Further, after high-fat diet feeding, IL-6 KO mice without expansion of alpha-cell mass display decreased fasting glucagon levels. However, despite these alpha-cell effects, high-fat feeding of IL-6 KO mice results in increased fed glycemia due to impaired insulin secretion, with unchanged insulin sensitivity and similar body weights. Thus, we conclude that IL-6 is necessary for the expansion of pancreatic alpha-cell mass in response to high-fat diet feeding, and we suggest that this expansion may be needed for functional beta-cell compensation to increased metabolic demand.

Serum 25(OH)D Level on Hospital Admission Associated With COVID-19 Stage and Mortality.

OBJECTIVES: Vitamin D deficiency was previously correlated with incidence and severity of coronavirus disease 2019 (COVID-19). We investigated the association between serum 25-hydroxyvitamin D (25(OH)D) level on admission and radiologic stage and outcome of COVID-19 pneumonia. METHODS: A retrospective observational trial was done on 186 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals hospitalized from March 1, 2020, to April 7, 2020, with combined chest computed tomography (CT) and 25(OH)D measurement on admission. Multivariate regression analysis was performed to study if vitamin D deficiency (25(OH)D <20 ng/mL) correlates with survival independently of confounding comorbidities. RESULTS: Of the patients with COVID-19, 59% were vitamin D deficient on admission: 47% of females and 67% of males. In particular, male patients with COVID-19 showed progressively lower 25(OH)D with advancing radiologic stage, with deficiency rates increasing from 55% in stage 1 to 74% in stage 3. Vitamin D deficiency on admission was not confounded by age, ethnicity, chronic lung disease, coronary artery disease/hypertension, or diabetes and was associated with mortality (odds ratio [OR], 3.87; 95% confidence interval [CI], 1.30-11.55), independent of age (OR, 1.09; 95% CI, 1.03-1.14), chronic lung disease (OR, 3.61; 95% CI, 1.18-11.09), and extent of lung damage expressed by chest CT severity score (OR, 1.12; 95% CI, 1.01-1.25). CONCLUSIONS: Low 25(OH)D levels on admission are associated with COVID-19 disease stage and mortality.

Comparison of microsatellite instability detection by immunohistochemistry and molecular techniques in colorectal and endometrial cancer.

DNA mismatch repair deficiency (dMMR) testing is crucial for diagnosing Lynch syndrome and detection of microsatellite unstable (MSI) tumors eligible for immunotherapy. The aim of this study was to compare the relative diagnostic performance of three molecular MSI assays: polymerase chain reaction (PCR), MSI testing by Idylla and next-generation-sequencing (NGS) on 49 tumor samples (28 colorectal and 21 endometrial adenocarcinomas) versus immunohistochemistry (IHC). Discrepancies were investigated by MLH1 methylation analysis and integrated with germline results if available. Overall, the molecular assays achieved equivalent diagnostic performance for MSI detection with area under the ROC curves (AUC) of respectively 0.91 for Idylla and PCR, and 0.93 for NGS. In colorectal cancers with tumor cell percentages ≥ 30% all three molecular assays achieved 100% sensitivity and specificity (AUC = 1) versus IHC. Also, in endometrial cancers, all three molecular assays showed equivalent diagnostic performance, albeit at a clearly lower sensitivity ranging from 58% for Idylla to 75% for NGS, corresponding to negative predictive values from 78 to 86%. PCR, Idylla and NGS show similar diagnostic performance for dMMR detection in colorectal and endometrial cancers. Molecular MSI analysis has lower sensitivity for dMMR detection in endometrial cancer indicating that combined use of both IHC and molecular methods is recommended.Clinical Trial Number/IRB: B1172020000040, Ethical Committee, AZ Delta General Hospital.

The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts.

Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. microRNA-375 (miR-375) ranks among the top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 h before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to the identification of eight microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome, and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.