Fragment Analysis Research Articles

Molecular networking-assisted isolation of chlorophenolic glycosides from the rhizomes of Curculigo orchioides and their inhibitory effect on α-glucosidase

Molecular networking analysis and in silico tools, such as Network Annotation Propagation (NAP) and MolNetEnhancer, were applied to explore bioactive constituents present in the ethyl acetate-soluble fraction of the rhizomes of Curculigo orchioides. Among the molecular networks, the most abundant cluster was classified as a phenolic glycoside using the ClassyFire module of MolNetEnhancer. Further, the major node in this cluster was accurately predicted as curculigine A using the in silico fragment analysis tool, NAP. Six undescribed chlorophenolic glycosides (1–6) and 11 known phenolic glycosides were isolated, using molecular networking-assisted isolation methods, and their structures were elucidated using 1D, 2D-NMR and HRESIMS. In particular, the structures of the isolated chlorophenolic glycosides, which have non-protonated aromatic rings, were determined using various NMR experiments, such as 1D-selective NOE, ROESY, and LR-HMBC, and acid hydrolysis. All isolated compounds were examined to determine their inhibitory effects on α-glucosidase and compounds 3, 8, 10, 11, 13, 14, and 16 revealed the IC50 values ranging from 19.6 to 35.5 μM. Their structure-activity relationships were also evaluated based on the analysis of their inhibitory effects and performance of molecular docking simulations.

The role of social media in sustainable development and strengthening the image of a political leader: Kazakhstan experience

In this article, based on the analysis of domestic and foreign studies, the concepts of "image", "individual image", "political image", as well as the features and tech-nologies of forming and strengthening the image of a political leader in the infor-mation space of Kazakhstan are disclosed. Having determined the theoretical and methodological foundations of the study, the team of authors conducted a con-tent analysis of social media texts that formed the public image of the President of the Republic of Kazakhstan (RK) Kasim-Jomart Kemelovich Tokayev. Within the framework of this work, the political image was studied as a set of me-dia texts in social media that provide public communication between a politician and the audience in order to create an image that has characteristics that are con-gruent with the desires and moods of the masses. At the same time, a hypothesis was put forward, according to which, whatever the image of a political leader, his personal qualities, character traits influence his important political decisions and the results of his activities. Thus, the correlation of psychological qualities and the media image of a political leader, the topic of the direct influence of personality traits and character of a political leader on his image in the media and mass con-sciousness will always remain one of the most relevant. The hypothesis put forward was tested by a complete analysis of the political situation: general monitoring and selective content analysis of the materials of public speeches of the President of the Republic of Kazakhstan K. K. Tokayev, covering the domestic and foreign policy of the Republic of Kazakhstan from March 2019 to March 2021. Subsequently, with the help of the Polpred program, a fragmented situational analysis was performed: monitoring and content analysis of the image information of the President of the Republic of Kazakhstan K.K. The choice of this time period was due to the fact that the month of January 2021 in Kazakhstan was politically saturated, i.e., elections were held to the lower house of the Parliament (Mazhilis and Maslikhats), there was a change in the Government of the Republic of Kazakhstan. The method of content analysis of social media texts, which was used in the work, helped to identify the role of modern media in managing the image of a political leader in conditions of social tension, as well as the specifics of working with them in the context of solving this problem. Based on the results of the study of media texts, a model for managing the image of a political leader was developed, consisting of the following elements: factors affecting the image of a political leader; effective methods, technologies and tools for managing the image of a political leader; communication aspect in the use of selected methods, technologies and tools for optimizing public capital. In conclusion, conclusions are formulated about possible mechanisms for strengthening the image of the political leader of the Republic of Kazakhstan in the mass consciousness of Kazakhstanis.

A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples.

CYP2D6 is one of the most polymorphic drug-metabolizing enzymes in the liver. While genetic CYP2D6 variants serve as clinical biomarkers to predict CYP2D6 activity, large inter-person variability in CYP2D6 expression remains unaccounted for. Previous results suggest that there is variable expression of a CYP2D6 splice isoform with an in-frame deletion of exon 3 (CYP2D6ΔE3) encoding a protein lacking numerous active site residues. Here, using fragment analysis and RT-qPCR, we revealed that rs1058164G (MAF = 27%-43%) is associated with increased formation of CYP2D6∆E3 in human liver samples (1.4-2.5-fold) and transfected cells. Furthermore, western blots showed that rs1058164G was associated with a 50% decrease in full-length hepatic CYP2D6 protein expression. In addition, by studying a larger liver cohort, we confirmed our previous results that rs16947 (CYP2D6*2) reduces full-length CYP2D6 mRNA by increasing the production of an unstable splice isoform lacking exon 6 (CYP2D6ΔE6) and that the impact of CYP2D6ΔE6 is offset in carriers of the downstream enhancer variant rs5758550. The three frequent SNPs (rs1058164, rs16947, and rs5758550) form various 3-SNP-haplotypes, each with distinct CYP2D6 expression characteristics. Using an expression score (ES) system, we tested the impact of the 3-SNP-haplotype on improving the standard model to predict hepatic CYP2D6 protein expression based on genotype. A model that incorporates the 3-SNP-haplotype provided the best fit for CYP2D6 expression and also accounted for more variability in CYP2D6 protein levels (59%) than a model based on the accepted standard (36%) or one that only adds rs16947 and rs5758550 (42%). Clinical studies are needed to determine whether including the 3-SNP-haplotype alongside current standard CYP2D6 models improves the predictive value of CYP2D6 panels.

PROFASA-a web-based protein fragment and structure analysis workstation.

Introduction: In the field of bioinformatics and computational biology, protein structure modelling and analysis is a crucial aspect. However, most existing tools require a high degree of technical expertise and lack a user-friendly interface. To address this problem, we developed a protein workstation called PROFASA. Methods: PROFASA is an innovative protein workstation that combines state-of-the-art protein structure visualisation techniques with cutting-edge tools and algorithms for protein analysis. Our goal is to provide users with a comprehensive platform for all protein sequence and structure analyses. PROFASA is designed with the idea of simplifying complex protein analysis workflows into one-click operations, while providing powerful customisation options to meet the needs of professional users. Results: PROFASA provides a one-stop solution that enables users to perform protein structure evaluation, parametric analysis and protein visualisation. Users can use I-TASSER or AlphaFold2 to construct protein models with one click, generate new protein sequences, models, and calculate protein parameters. In addition, PROFASA offers features such as real-time collaboration, note sharing, and shared projects, making it an ideal tool for researchers and teaching professionals. Discussion: PROFASA's innovation lies in its user-friendly interface and one-stop solution. It not only lowers the barrier to entry for protein computation, analysis and visualisation tools, but also opens up new possibilities for protein research and education. We expect PROFASA to advance the study of protein design and engineering and open up new research areas.

Genetic diversity and population structure of indigenous guinea fowl (Numida meleagris) in Benin using simple sequence repeat markers.

This study aimed to assess the genetic diversity and population structure of 12 guinea fowl phenotypes from three climatic zones (Guinean, Sudano-Guinean and Sudanian) of Benin. A total of 96 adult guinea fowl, aged at least 6months, were selected for blood sampling. Fragment analysis was carried out using 17 polymorphic microsatellite markers. The informative marker combinations revealed a total of 83alleles across all loci, with an average of 5alleles per locus and a mean polymorphic information content (PIC) of0.793. This study showed an observed heterozygosity of0.492. The inbreeding coefficient values ranged from0.086 in white phenotype to0.226 in cinnamon, showing a deficit of heterozygotes, suggesting a moderate inbreeding level. A relatively low population differentiation was observed, with a mean fixation index(Fst) value of0.033. The short genetic distances between phenotypes, unlike the strong genetic identities, revealed high genetic proximity between the 12 phenotypes of indigenous guinea fowl in Benin. These data indicate the existence of a single indigenous guinea fowl population with high intra-population genetic diversity with respect to climatic zones or phenotypes. This study will help in the selection of parental breeding stock for genetic improvement programs, as well as in the conservation for biodiversity maintenance and sustainable use of the indigenous guinea fowl in the study zones in Benin.

Performance of Liquid Biopsy for Diagnosis and Surveillance of Human Papillomavirus–Associated Oropharyngeal Cancer

There is growing interest in the use of circulating plasma tumor human papillomavirus (HPV) DNA for diagnosis and surveillance of patients with HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Recent advances in the assays, combining the identification of circulating HPV tumor DNA and tumor DNA fragment analysis (tumor tissue-modified viral [TTMV]-HPV DNA), have been shown to be highly accurate. However, use of these newer techniques has been limited to small cohort studies and clinical trials. To establish the clinical efficacy of plasma TTMV-HPV DNA testing in the diagnosis and surveillance of HPV-associated OPSCC in a contemporary clinical setting. This retrospective observational cohort study included patients with OPSCC who underwent TTMV-HPV DNA testing between April 2020 and September 2022 during the course of routine clinical care. For the diagnosis cohort, patients with at least 1 TTMV-HPV DNA measurement prior to initiation of primary therapy were included. Patients were included in the surveillance cohort if they had at least 1 TTMV-HPV DNA test performed after completion of definitive or salvage therapy. Per-test performance metrics, including sensitivity, specificity, positive predictive value, and negative predictive value, for TTMV-HPV DNA testing. Of 399 patients included in the analysis, 163 were in the diagnostic cohort (median [IQR] age, 63 [56-68.5] years; 142 [87.1%] male), and 290 were in the surveillance cohort (median [IQR] age, 63 [57-70] years; 237 [81.7%] male). Of the 163 patients in the diagnostic cohort, 152 (93.3%) had HPV-associated OPSCC while 11 (6.7%) had HPV-negative OPSCC. The TTMV-HPV DNA sensitivity in pretreatment diagnosis was 91.5% (95% CI, 85.8%-95.4% [139 of 152 tests]), and the specificity was 100% (95% CI, 71.5%-100% [11 of 11 tests]). In the surveillance cohort, 591 tests conducted in 290 patients were evaluated. A total of 23 patients had molecularly confirmed pathologic recurrences. The TTMV-HPV DNA test demonstrated sensitivity of 88.4% (95% CI, 74.9%-96.1% [38 of 43 tests]) and specificity of 100% (95% CI, 99.3%-100% [548 of 548 tests]) in detecting the recurrences. Positive predictive value was 100% (95% CI, 90.7%-100% [38 of 38 tests]), and negative predictive value was 99.1% (95% CI, 97.9%-99.7% [548 of 553 tests]). The median (range) lead time from positive TTMV-HPV DNA test to pathologic confirmation was 47 (0-507) days. This cohort study demonstrated that when evaluated in a clinical setting, the TTMV-HPV DNA assay demonstrated 100% specificity in both diagnosis and surveillance. However, the sensitivity was 91.5% for the diagnosis cohort and 88.4% for the surveillance cohort, signifying that nearly 1 in 10 negative tests among patients with HPV-associated OPSCC was a false negative. Additional research is required to validate the assay's performance and, if validated, then further research into the implementation of this assay into standard clinical practice guidelines will be required.

Qualitative and quantitative analysis of the chemical components in Yuquan capsules by using ultra-performance liquid chromatography-mass spectrometry.

The Yuquan capsules is a commonly used traditional Chinese Patent Medicine used for the treatment of diabetes mellitus. In this study, a high-throughput analytical method for identifying the chemical composition of Yuquan capsules was established for the first time by using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry. The data obtained were subjected to fragment analysis and this was combined with UNIFI processing of natural products. One-hundred sixteen compounds were characterized from Yuquan capsules. Twelve of the bioactive compounds were quantitatively analyzed by ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry. This study was undertaken to obtain a comprehensive chemical profile analysis as well as to evaluate the overall quality of Yuquan capsules. The results will provide a reference for the quality evaluation of different Yuquan preparations. In addition, the data will enable basic pharmacodynamic research into these extensively used capsules.

Effects of mobile-supervised question-driven collaborative dialogues on EFL learners' communication strategy use and academic oral English performance.

This study investigated the effects of mobile-supervised question-driven collaborative dialogues (QDCDs) on reducing lower-intermediate-level English as a foreign language (EFL) participants' tendency of their first language (L1) use in academic collaborative dialogues and on improving their academic foreign language (L2) oral performance. Throughout a whole semester, one group (n = 20) was involved in a mobile-supervised QDCDs intervention and a control group (n = 26) was involved in QDCDs with no supervision. Three semi-open-ended and three closed-ended academic questions were used to elicit pre-and post-study oral performance data from the participants. Independent-samples t-tests showed that after the intervention, the mobile-supervised group outperformed its control counterpart in a statistically significant manner in terms of Non-repeated L2 word production (NRW), T-unit count (TC), and Mean Length of Run after pruning (MLRP). The intervention group also significantly reduced their dependence on their L1-based speaker compensatory communication strategies (SC-CSs) in QDCDs. These results suggest that the intervention group outperformed the control group in their L2 academic oral performance and their language use tendency moves toward the L2 during QDCD. Based on the findings, we conclude that, even though L1 oral output may temporally enhance the quality of lower-intermediate-level EFL learners' tasks, it may inhibit their academic oral proficiency development in the long run. Methods for fragmental bilingual oral output analysis are introduced. Pedagogical implications of the findings for MALL are also discussed.

English

Knowledge of the genetic variability of plantain cultivars in the Democratic Republic of the Congo (DRC) is limited. This study assessed genetic diversity and relationships in three plantain accessions differing in pseudostem color at seven microsatellite loci using SSR markers. DNA was extracted from banana leaves, and PCR was performed with unlabeled and fluorescent-labeled SSR primers and eventual capillary sequencing. Different population genetic analysis software was used to interpret the fragment analysis data. There was a high allelic power with a mean polymorphic information content of 0.83. A high average genetic diversity was indicated by Nei's Gene Diversity Index (h: 1.38) and the Shannon Information Index (I: 2.22). Pairwise genetic similarity between plantain genotypes averaged 0.82. A dendrogram grouped the 60 genotypes into three clusters. AMOVA showed high genetic variation within the population (98%) compared to the variation between populations, with no significant difference between the three cultivars regarding the color of the pseudostem. In contrast, these cultivars were closely related to each other. No correlation between the morphological and molecular characterization of cultivars was observed. We recommend a follow-up study with other plantain cultivars from other regions and large sample size for more representative data.   Key words: Characterization, microsatellites, genetic diversity, plantain cultivars.

Association of Low Serum Bilirubin Concentrations and Promoter Variations in the UGT1A1 and HMOX1 Genes with Type 2 Diabetes Mellitus in the Czech Population.

Bilirubin has potent biological beneficial effects, protecting against atherosclerosis, obesity, and metabolic syndrome. The aim of this study was to assess serum bilirubin concentrations and (TA)n and (GT)n microsatellite variations in the promoter regions of the UGT1A1 and HMOX1 genes, respectively, in patients with type 2 diabetes mellitus (T2DM). The study was carried out in 220 patients with T2DM and 231 healthy control subjects, in whom standard biochemical tests were performed. The (TA)n and (GT)n dinucleotide variations were determined by means of fragment (size-based) analysis using an automated capillary DNA sequencer. Compared to controls, both male and female patients with T2DM had lower serum bilirubin concentrations (9.9 vs. 12.9 μmol/L, and 9.0 vs. 10.6 μmol/L, in men and women, respectively, p < 0.001). Phenotypic Gilbert syndrome was much less prevalent in T2DM patients, as was the frequency of the (TA)7/7UGT1A1 genotype in male T2DM patients. (GT)nHMOX1 genetic variations did not differ between diabetic patients and controls. Our results demonstrate that the manifestation of T2DM is associated with lower serum bilirubin concentrations. Consumption of bilirubin due to increased oxidative stress associated with T2DM seems to be the main explanation, although (TA)n repeat variations in UGT1A1 partially contribute to this phenomenon.

Comparative study of next generation sequencing and immunohistochemistry on molecular classification of endometrial carcinoma

Objective: To investigate the differences in molecular classification of endometrial carcinoma (EC) between various technical methods and to explore molecular classification schemes suitable for Chinese population. Methods: The study used a comprehensive scheme of next generation sequencing (NGS) and immunohistochemistry for molecular classification of 254 EC cases that were obtained at Department of Pathology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China from April 2021 to March 2022. According to the recommended threshold of Sanger sequencing which was approximate-20% variant allele fraction (VAF), NGS data were extracted to simulate the results of Sanger sequencing. Results: The 254 EC patients had a mean age of 51 years (range, 24 to 89 years). Combination of POLE (9-14 exons), TP53 total exons and microsatellite instability (MSI) detection was a better single scheme than NGS alone, while combination of MSI fragment analysis and conventional immunohistochemistry was the best solution and seemed best aligned with TCGA data and recent studies. POLE ultramuted type, mismatch repair defect type, TP53 mutant type and non-specific molecular characteristic type accounted for 11.4% (29/254), 31.5% (80/254), 22.4% (57/254) and 34.6% (88/254) of the cases, respectively. If Sanger sequencing was adopted for POLE and TP53 detection, the frequencies of these EC types were 9.1% (23/254), 31.5% (80/254), 12.9% (33/254) and 46.6% (118/254), respectively, with greatly increasing non-specific molecular characteristics cases. If POLE was detected by Sanger sequencing and others by immunohistochemistry, they were 9.1% (23/254), 42.2% (92/218), 13.8% (35/254) and 40.9% (105/254), respectively, with increasing the false positive rates of the mismatch repair defect group. Conclusions: Small and medium-sized NGS panels with MSI detection is a better solution than NGS alone. Sanger sequencing is currently available for POLE mutation detection, which is not sensitive enough for TP53 mutation detection, and seems equivalent to the efficiency of TP53 by immunohistochemistry. Further optimization of small and medium-sized NGS panels covering MSI detection and POLE and TP53 full exons may be the best choice for the future to meet national conditions.

HLA-G susceptibility to hepatitis B infection and related hepatocellular carcinoma in the Japanese population

AimsHuman leukocyte antigen (HLA)-G plays a role in various physiological immunomodulatory functions. Aberrant HLA-G expression is observed in various disease states, including tumors, autoimmune disorders, and viral infections. The present study investigated the association between HLA-G functional gene polymorphisms (rs1736933 [-486 C > A], rs1049033 [+2018 C > T], 14 bp Insertion [Ins]/Deletion [Del] [+2961 Del > Ins], and rs1063320 [+3142 C > G]) and disease susceptibility, hepatocellular carcinoma (HCC) development, and hepatitis B surface antigen (HBsAg) clearance. MethodsAllele discrimination of the 3 SNPs (-486 C > A, +2018 C > T, +3142 C > G) was determined by a TaqMan 5′ exonuclease assay, while the 14 bp Ins/Del polymorphism was typed by fragment analysis using Genetic Analyzer and GeneMapper software. The above polymorphisms were analyzed for 325 Japanese hepatitis B virus (HBV) patients, 355 Japanese healthy subjects (Controls) as healthy controls, and 799 Japanese hepatitis C virus (HCV) patients as disease controls, respectively. ResultsThe 14 bp Insertion allele was significantly more frequent in HBV patients than Controls (27.1 % vs 20.6 %, odds ratio [OR] 1.43, P = 0.005) but did not differ between HCV patients and Controls. Similar results were found for the rs1063320 G allele (38.9 % vs 26.3 %, OR 1.78, P < 0.001) and the rs1736933 T allele (32.2 % vs 26.9 %, OR 1.29, P = 0.034) between HBV and Controls. The rs1049033 T allele showed a weak but significant association with HCC development in the dominant model (OR 1.95, P = 0.04). Regarding HBsAg clearance, the A allele at rs1736933 was significantly correlated in the recessive model (OR 3.23, P = 0.003). ConclusionsThis study revealed significant associations of HLA-G gene polymorphisms with disease susceptibility, HCC development, and HBsAg clearance in HBV patients.

Introducing the New Editorial Team

Introducing the New Editorial Team

Studying the Genetic Diversity of the Rice Break Cause &lt;i&gt;Pyricularia oryzae&lt;/i&gt; Cav. on the Complex of Molecular and Morphological Features

On the basis of molecular genetic approaches, the study of the biodiversity of the phytopathogenic fungus Pyricularia oryzae Cav. in the south of Russia and a classification of the morphological and cultural characteristics of the pathogen was carried out. For 2020–2022 monitoring was carried out and 24 strains of the pathogen were isolated from the affected herbarium material collected from the fields of five ecological rice growing zones of the Krasnodar Territory (Krasnoarmeisky, Kalininsky, Abinsky, Slavyansky districts, Krasnodar). Using the multiplex PCR system developed by us based on fragment analysis, five genotypes were identified among the studied Pyricularia oryzae Cav. strains over the entire period of the project, each of which is characterized by a unique genetic profile. Compiled their “DNA passport”. The studied isolates of the causative agent of blast were characterized by morphological and cultural characteristics. Six morphotypes of the pathogen have been identified.

Genomic landscapes to characterize mismatch-repair deficiency (dMMR)/microsatellite instability-high (MSI-H) gastrointestinal (GI) cancers stratified by tumor mutation burden (TMB).

2618 Background: TMB is a predictive biomarker for immune checkpoint inhibitor treatment in dMMR/MSI-H cancers. We aimed to characterize specific molecular features of a large cohort of dMMR/MSI-H GI tumors with a range of TMB levels. Methods: A total of 2272 GI tumors tested as dMMR/MSI-H were analyzed using Next-generation sequencing (NGS) and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). MMR/MSI status was evaluated by a combination of IHC, Fragment Analysis and NGS. TMB-H were defined using differing TMB cutoffs (10, 20, 50 mutations/Mb). Molecular features were compared in four groups (TMB &lt; 10 vs 10-20 vs 20-50 vs ≥50mutations/Mb) using Fisher-Exact or Chi-square and adjusted for multiple comparison by Benjamini-Hochberg. The impact of TMB on tumor immune environment was analyzed using a TCGA cohort. Significance was determined by adjusted (adj) P &lt; 0.05. Results: Tumors with TMB over 10, 20, 50 mutations/Mb were observed in 95.38%, 86.05% and 14.47% respectively in the dMMR/MSI-H GI cohort. Compared to other groups, dMMR/MSI-H GI tumors with TMB over 50 mutations/Mb exhibited the highest mutations rates in RNF43, MSH6, KMT2D, BRCA2 and ASXL1 (Top 5, all Padj&lt; 0.0001). Among the four MMR genes, only MLH1 mutation had no association with higher TMB. The rates of CNNE1 amplification (i.e. the Cycle E1 gene) and HER2 overexpression were the highest in tumors with TMB below 10 mutations/Mb (all Padj&lt; 0.05). The association between PD-L1 positivity and TMB levels was only observed in gastroesophageal cancers ( P= 0.03). The lymphocyte infiltration signature score, IFNγ response and M1 macrophage infiltration were significantly lower, while fraction of genome alterations was significantly increased in dMMR/MSI-H tumors with TMB below 10 mutations/Mb, compared to other subgroups (all P&lt; 0.05). Neoantigens were significantly enriched in those with TMB over 50 mutations/Mb, compared to other subgroups ( P&lt; 0.0001). Conclusions: This is the largest study to investigate the distinct molecular landscape of dMMR/MSI-H GI cancers with different degrees (magnitude) of TMB. These data may inform our understanding of the efficacy of ICB in dMMR/MSI-H GI tumors.

Microsatellite instability in sarcomas: Which tests are ideal for diagnosis, and a systematic review.

e23519 Background: There is a lack of clinically relevant biomarkers to inform immunotherapy for patients with advanced sarcoma. High levels of microsatellite instability (MSI-H) is a histology-agnostic biomarker of response to pembrolizumab. However, the frequency of MSI-H in sarcoma is low - ranging from &lt; 0.5% to 4%. MSI status can be assessed through Next Generation Sequencing (NGS), immunohistochemistry (IHC), or PCR followed by fragment analysis with capillary electrophoresis (PCR). To date, both the College of American Pathologists and the European Society of Medical Oncology have not determined the best method to evaluate MSI in sarcomas. We performed a systematic review to evaluate the methods to diagnose MSI-H in sarcomas and to evaluate the impact of this biomarker in treatment with pembrolizumab. Methods: We performed a literature search using the PubMed database to identify all English full-text available articles between January 2020 and December 2022, with the terms “sarcoma and microsatellite instability” and “sarcoma and pembrolizumab”. One independent reviewer screened the studies, two independent reviewers analyzed the selected studies, and disparities were resolved with a fourth reviewer. Results: Concerning the diagnosis of MSI-H in sarcomas, 187 abstracts were found, and 31 articles were identified for full analysis. MSI status was reported for 7958 patients - 7762 with a single test and 196 with more than one test. NGS was used for 6649, IHC for 1222, and PCR for 285. MSI-H tumors were 25 (0.4%), 38 (3.1%), and 21 (7.33%) for each technique, respectively. Two investigators performed the three available tests to evaluate concordance, with one concordant case and one discordant case. 194 cases were evaluated for both IHC and PCR, with 4 discordant results. No article that described NGS results performed a second test. To analyze the results of pembrolizumab therapy in MSI-H sarcomas, we analyzed 199 abstracts of which we selected 13 prospective and 31 retrospective papers for full analysis. The K cohort of Keynote-158 is the only prospective trial concerning therapies guided by MSI-H in sarcoma (n = 14 patients, ORR = 33%). Regarding retrospective data, we identified 2 case reports of the use of pembrolizumab in patients with advanced sarcoma – both achieved a response to therapy. Conclusions: There is limited data to establish the best diagnostic test for MSI-H in patients with sarcoma. The concordance between NGS, IHC, and PCR seems to be high, but discrepancies have been described and need to be further evaluated. There is only one prospective trial that enrolled patients with sarcomas utilizing MSI-H as a biomarker. Albeit only a small number of patients were enrolled (n = 14), efficacy is consistent with that seen in other histologies, and scarce retrospective data corroborate the potential role of MSI-H as a predictive biomarker of pembrolizumab efficacy in sarcoma.

Mutational and DNA fragment analysis may help in the triage of FIT+ patients enrolled in colorectal cancer screening programs.

e15546 * F. Pepe made an equal contribution Background: Mutational and DNA fragment analysis have already been investigated for a possible role in the detection of colorectal cancer. This pilot study, conducted at ASL NA 3 SUD (Naples, Italy) with the University of Naples Federico II, attempts to understand their possible role in the detection of Advanced Adenomas+ (AA+) and their potential ability to triage subjects with a positive FIT test for referral to colonoscopy. Methods: 160 FIT+ subjects of both genders, aged 50-74, participating in a colorectal cancer screening program were enrolled in a clinical trial focusing on a novel biomarker for colorectal cancer diagnostics. Of these, 14 subjects had disease (12 AAs and 2 colorectal cancers, from now on “CRC”). After ethical committee’s approval and release of informed consents, a 20 ml whole-blood sample was collected from each subject. Samples were centrifuged following standard procedures and stored at -80°C. DNA extraction was later performed using a Minielute kit (Qiagen). Mutational and DNA fragment analysis were performed using a qPCR-based technology: the ColoScape™ 2.5 kit (DiaCarta Inc, Pleasanton, CA). A logistic regression was run to determine the probability of disease in subjects showing positivity for predictors. Estimates of sensitivity, specificity, PPV, NPV (with 95% C.I.), PLR, NLR and Youden’s Index were also obtained. Results: The logistic regression showed statistical significance, with a p-value &lt; 0.0001, that is, the logistic regression model with provides a better fit than the model without the independent variable. ColoScape identified 12 subjects with disease out of 14, for a sensitivity of 86% (67%,100%); both CRCs were picked up by ColoScape together with 10 AAs. 2 AAs were missed. Out of 146 healthy subjects, 107 were successfully identified as negatives, for a specificity of 73% (66%, 80%). PPV was 24% (12%, 35%). NPV was 98% (96%, 100%). PLR, NLR and Youden’s Index were 3.21, 0.19 and 0.59 respectively. Conclusions: This pilot study was intended to provide performance indicators to design a larger population study that is currently underway. These preliminary findings suggest that liquid biopsy-based mutational and DNA fragment analysis can play a role in selecting subjects with a higher risk of returning a positive colonoscopy, thus potentially improving the quality of colorectal cancer screening programs and patients’ experience.

Possible False Results With cobas® EGFR Mutation Test v2 and Oncomine Dx Target Test for EGFR Mutation.

Disparities in the results of next-generation sequencing-based multiplex gene panel tests and those of single-gene tests when detecting epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) have been reported. However, the possible underlying causes have not been investigated. The aim of this study was to explore the possibilities and causes of false results obtained using cobas® EGFR Mutation Test v2 (cobas® EGFR) and Oncomine Dx Target Test (ODxTT). The data of patients with NSCLC who underwent gene assessment using both cobas® EGFR and ODxTT between April 2021 and May 2022 were retrospectively reviewed. Disparate results of EGFR mutation analyses were then reviewed. One hundred and sixteen patients were included in the analysis. The results of six samples were inconsistent. In four samples, exon 20 insertion mutations were detected using cobas® EGFR, but not identified using ODxTT. A fragment analysis was performed on three of the four samples, and all showed negative results for exon 20 insertion. Furthermore, one false negative result was obtained in the ODxTT for both exon 19 deletion and L858R mutations. For exon 19 deletion mutation, a single nucleotide variant from adenine to thymine was identified close to the mutation site. False positives for exon 20 insertion may occur when using cobas® EGFR, and false negatives for exon 19 deletion and L858R mutations may occur when using ODxTT.

UBTF tandem duplications are rare but recurrent alterations in adult AML and associated with younger age, myelodysplasia, and inferior outcome

Tandem-duplication mutations of the UBTF gene (UBTF-TDs) coding for the upstream binding transcription factor have recently been described in pediatric patients with acute myeloid leukemia (AML) and were found to be associated with particular genetics (trisomy 8 (+8), FLT3-internal tandem duplications (FLT3-ITD), WT1-mutations) and inferior outcome. Due to limited knowledge on UBTF-TDs in adult AML, we screened 4247 newly diagnosed adult AML and higher-risk myelodysplastic syndrome (MDS) patients using high-resolution fragment analysis. UBTF-TDs were overall rare (n = 52/4247; 1.2%), but significantly enriched in younger patients (median age 41 years) and associated with MDS-related morphology as well as significantly lower hemoglobin and platelet levels. Patients with UBTF-TDs had significantly higher rates of +8 (34% vs. 9%), WT1 (52% vs. 7%) and FLT3-ITD (50% vs. 20.8%) co-mutations, whereas UBTF-TDs were mutually exclusive with several class-defining lesions such as mutant NPM1, in-frame CEBPAbZIP mutations as well as t(8;21). Based on the high-variant allele frequency found and the fact that all relapsed patients analyzed (n = 5) retained the UBTF-TD mutation, UBTF-TDs represent early clonal events and are stable over the disease course. In univariate analysis, UBTF-TDs did not represent a significant factor for overall or relapse-free survival in the entire cohort. However, in patients under 50 years of age, who represent the majority of UBTF-mutant patients, UBTF-TDs were an independent prognostic factor for inferior event-free (EFS), relapse-free (RFS) and overall survival (OS), which was confirmed by multivariable analyses including established risk factors such as age and ELN2022 genetic risk groups (EFS [HR: 2.20; 95% CI 1.52–3.17, p < 0.001], RFS [HR: 1.59; 95% CI 1.02–2.46, p = 0.039] and OS [HR: 1.64; 95% CI 1.08–2.49, p = 0.020]). In summary, UBTF-TDs appear to represent a novel class-defining lesion not only in pediatric AML but also younger adults and are associated with myelodysplasia and inferior outcome in these patients.

Oxidative stress and genotoxicity in oral epithelial cells from subjects undergoing orthodontic treatment with fixed appliances.

The objective of this work was to evaluate the impact of fixed orthodontic appliances on oxidative stress (OS) and genotoxicity from oral epithelial cells. Samples of oral epithelial cells were obtained from fifty-one healthy voluntary subjects who had an indication for orthodontic treatment. The samples were obtained before treatment and after 6 and 9months of treatment. OS was evaluated by quantitating 8-hydroxy-2'deoxyguanosine (8-OHdG) and by performing relative gene expression with antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). DNA degradation and instability were evaluated by multiplex polymerase chain reaction (PCR) and fragment analysis for human identification. The quantitation results showed that 8-OHdG increased during treatment, although this increase was not statistically significant. SOD increased by 2.5- and 2.6-fold after 6 and 9months of treatment, respectively. CAT increased by threefold after 6months of treatment, while after 9months of treatment, the expression level decreased to a level similar to that before treatment. DNA degradation was found in 8% and 12% of DNA samples after 6 and 9months of treatment, respectively, while DNA instability was detected in only 2% and 8% of DNA samples after 6 and 9months of treatment, respectively. The results showed that OS and genotoxicity slightly changed after treatment with a fixed orthodontic appliance; in addition, a biological adaptation response to the treatment may occur after 6months. OS and genotoxicity in the buccal cavity are risk factors for oral and systemic diseases. This risk may be reduced through antioxidant supplementation, by using thermoplastic materials, or by reducing the orthodontic treatment time.