Molecular Analysis Of Mutations Research Articles

EP.07D.05 Molecular Testing Care Gap Analysis of EGFR Mutation for Early-Stage NSCLC Patients in Community Practices

EP.07D.05 Molecular Testing Care Gap Analysis of EGFR Mutation for Early-Stage NSCLC Patients in Community Practices

Z/E configuration controlled by a Taxus sesquiterpene synthase facilitating the biosynthesis of (3Z,6E)-α-farnesene

Plant enzymes often present advantages in the synthesis of natural products with specific configurations. Farnesene is a pharmacologically active sesquiterpene with three natural Z/E configurations, among which the enzyme selectively responsible for the biosynthesis of (3Z,6E)-α-farnesene remains elusive. Herein, a sesquiterpene synthase TwSTPS1 biosynthesizing (3Z,6E)-α-farnesene as the major product was identified from Taxus wallichiana through genome mining. Utilizing molecular dynamics simulations and mutation analysis, the catalytic mechanism of TwSTPS1, especially Z/E configuration control, was explored. Moreover, the crucial residues associated with the specific catalytic activity of TwSTPS1 was elucidated through mutagenesis experiments. The findings contribute to our understanding of the Z/E configuration control by plant terpene synthases and also provide an alternative tool for manipulating (3Z,6E)-α-farnesene production using synthetic biology.

Functional identification of carbohydrate-binding module 13 and its application to quantification of hemicellulose in gramineous plants

Gramineous biomass is a type of lignocellulose commonly used as a renewable energy source. Accurate quantification of hemicellulose within this biomass is crucial for its efficient conversion. Carbohydrate-binding modules (CBMs) help catalytic domains anchor substrates, representing the potential to be developed as fluorescence probes for hemicellulose content determination. In this study, we discovered a CBM named EmCBM13, derived from the bifunctional enzyme Xyn10A/Fae1A within the bacterial consortium EMSD5. This CBM displays a remarkable ability to bind soluble and insoluble hemicellulose components of gramineous plants. Through molecular docking and mutational analysis, we pinpointed two essential tyrosine residues mediating ligand interaction of EmCBM13. Leveraging the binding characteristics of EmCBM13, we created a fluorescence probe called EmCBM13-GFP by fusing a GFP with EmCBM13. We observed a positive correlation between the fluorescence contents of EmCBM13-GFP bound to hemicellulose and the hemicellulose contents in various gramineous biomasses. Utilizing this correlation, we rapidly determined the hemicellulose content in different gramineous plants with 90–110 % accuracy. This probe shows promise in quickly evaluating the characteristics of gramineous biomass feedstock for research and production purposes.

Correlation of posterior segment lesions with anterior segment biometric parameters and FBN1 genotype in patients with Marfan syndrome

Objective: To investigate the characteristics of posterior segment lesions in Marfan syndrome (MFS) patients and their relationship with anterior segment biometric parameters and FBN1 genotype. Methods: A cross-sectional study was conducted. A total of 121 MFS patients, 76 males and 45 females, with an average age of (11.72±11.66) years, who visited the Department of Ophthalmology, Eye & ENT Hospital of Fudan University from January 2013 to March 2023 were included. The presence of posterior scleral staphyloma was observed using B-mode ultrasound, and macular lesions were identified and classified using the atrophy-traction-neovascularization system based on ultra-widefield fundus images, color fundus images, and optical coherence tomography scans. Anterior segment biometric parameters, including axial length of the eye, average corneal curvature, corneal astigmatism, horizontal corneal diameter, anterior chamber depth, and lens thickness, were collected, and the direction and extent of lens dislocation were observed. Molecular genetic analysis of FBN1 gene mutations in patients was performed using next-generation sequencing based on a panel of ocular genetic diseases, and the impact of the genotype and anterior segment biometric parameters on the posterior segment manifestations was analyzed. Results: Sixty patients exhibited posterior segment lesions, including retinal detachment (4 cases, 3.31%), macular lesions (47 cases, 38.84%), and posterior scleral staphyloma (54 cases, 44.63%). There was statistically significant difference in axial length of the eye between patients with and without posterior scleral staphyloma [23.09 (22.24, 24.43) and 27.04 (25.44, 28.88) mm], between patients with and without macular lesions [23.16 (22.24, 24.61) and 27.04 (25.74, 28.78) mm], and between patients with and without atrophic macular lesions [23.16 (22.24, 24.61) and 27.04 (25.74, 28.79) mm] (all P<0.001). There was statistically significant difference in anterior chamber depth between patients with and without macular lesions [3.11 (2.75, 3.30) and 3.34 (3.09, 3.60) mm] (P<0.05). There was also statistically significant difference in corneal astigmatism between patients with and without posterior scleral staphyloma [2.15 (1.20, 2.93) and 1.40 (1.00, 2.20) diopters] (P<0.05). The location and region of the FBN1 gene mutation not only showed statistically significant difference from the positive rates of posterior scleral staphyloma and macular lesions (all P<0.05), but also influenced the occurrence of atrophic macular lesions (both P<0.05). Patients with FBN1 mutations located in the transforming growth factor β regulatory sequence had the highest proportion of posterior scleral staphyloma and macular lesions (both 10/11). Conclusions: Posterior scleral staphyloma and macular lesions have a relatively high incidence in MFS patients and tend to progress to more severe grades. The age, axial length of the eye, anterior chamber depth, corneal astigmatism, and location and region of the FBN1 gene mutation are factors affecting the posterior segment lesions in MFS patients.

A Novel Ectodysplasin a Gene mutation of X-Linked Hypohidrotic Ectodermal Dysplasia.

Hypohidrotic ectodermal dysplasia (HED) is a genetic disorder that influences structures of ectodermal origin, such as teeth, hair, and sweat glands. Compared with autosomal recessive and dominant modes of inheritance, the X-linked HED (XLHED) characterized by Hypodontia/Oligodontia teeth, Absent/sparse hair, Anhidrosis/hypohidrosis, and characteristic facial features, is the most frequent and its primary cause is the mutation of ectodysplasin A (EDA) gene. This research aimed to expound the clinical and molecular features of a Chinese male with XLHED and to summarize and compare several previous findings. Genomic DNA was obtained from the peripheral blood of the proband and his family members, then Sanger sequencing was used to perform a mutational analysis of EDA. Real-time quantitative PCR and Western blotting were used to detect EDA expression. The transcriptional activity of NF-κB was detected using a luciferase assay. The probandwith XLHED was identified a novel EDA mutation, c.1119G>C(p.M373I), that affected the molecular analysis of transmembrane protein exon8 mutations, inherited from the mother. He showed a severe multiple-tooth loss, with over 20 permanent teeth missing and sparse hair and eyebrows, dry, thin, and itching skin. Furthermore, his sweating function was abnormal to a certain extent. The functional study showed that this novel mutant led to a significant decrease in the EDA expression level and transcriptional activity of NF-κB. Our findings extend the range of EDA mutations in XLHED patients, which provides the basis and idea for further exploring the pathogenesis of XLHED.

A slow but steady nanoLuc: R162A mutation results in a decreased, but stable, nanoLuc activity

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays, including gene expression analysis, protein-protein interaction, and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remain elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in living cells and in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in live cell and in vitro assays performed using lysates prepared from cells expressing the proteins and with the furimazine substrate. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity. SignificanceBioluminescence has been extensively utilized in developing a variety of biological and biomedical assays. In this regard, engineering of brighter bioluminescent proteins, i.e. luciferases, has played a significant role. This is acutely exemplified by the engineering of the NLuc luciferase, which is small in size and displays much enhanced bioluminescence and thermal stability compared to previously available luciferases. While enhanced bioluminescent activity is desirable in a multitude of biological and biomedical assays, it would also be useful to develop variants of the protein that display a prolonged bioluminescence activity. This is specifically relevant in designing assays that require bioluminescence for extended periods, such as in the case of biosensors designed for monitoring slow enzymatic or cellular signaling reactions, without necessitating multiple rounds of luciferase substrate addition or any specialized reagents that result in increased assay costs. In the current manuscript, we report a mutant NLuc that possesses a stable and prolonged bioluminescence activity, albeit lower than the wild-type NLuc, and envisage a wider application of the mutant NLuc in designing biosensors for monitoring slower biological and biomedical events.

Abstract 5184: Biomarker results from PEVENAZA, a randomized phase 2 study of venetoclax and azacitidine +/- pevonedistat in newly diagnosed AML patients unfit for intensive chemotherapy: Increased efficacy in a subset of patients with IDH1/2 mutations and other observations

Abstract Background: Many newly diagnosed patients with AML are ineligible for intensive chemotherapy due to pre-existing comorbidities and older age. VIALE-A, a phase-3 trial with venetoclax (ven) and azacitidine (aza) established this combination as a new standard of care (SOC) for this patient population. The addition of pevonedistat (pevo) to this combination was evaluated in the same patient population in the current trial (NCT04266795). Here we present results of biomarker analyses from baseline and on-treatment bone marrow samples. Methods: Molecular mutational analysis was performed using a next generation sequencing (NGS) panel on baseline bone marrow samples. Combining this data with cytogenetics allowed centralized assignment of risk category based on ELN2017 guidelines. Longitudinal bone marrow aspirate samples were also examined by multi-color flow cytometry for expression of select surface antigen expression and to identify AML leukemic stem cells (LSCs) from the blast population. Results: Eleven genes were selected for analyses based on prevalence and scientific interest, including DNMT3A, ASXL1, TET2, FLT3, and IDH1/2. In contrast to other genes and ELN risk categories, we observed a statistically significant increase (p-value 0.013) in CR+CRi rates for IDH1/2 mutant patients when treated with pevo + ven + aza (95%, n=21) compared to ven+aza control arm (62%, n=21). In addition, we evaluated the prevalence of CD33, CD123 and CLEC12a, targets of current AML developmental therapeutics, in both the blast and LSC populations at baseline. The median (n=120 pts) % blast cells expressing CD33, CD123, or CLEC12a were 89.8%, 63.2%, and 66.0%, respectively. The median (n=78 pts) % LSCs expressing CD33, CD123, or CLEC12a were 48.2%, 90.9%, and 32.0%, respectively. Importantly, we did not see any significant difference in median fluorescence intensity (MFI) or % expression in any of the three proteins after treatment with ven and aza (+/- pevo). Finally, no strong associations were observed between expression levels and either mutations or ELN risk categories. Conclusions: Analyses of biomarker samples as part of the PEVENAZA trial resulted in several insights for not only the pevonedistat program but also for other AML therapeutics in development. The observation that newly diagnosed patients with IDH1/2 mutations may benefit from the addition of pevo to ven+aza is certainly hypothesis-generating but would require further follow-up with higher patient numbers. In addition, baseline CD33, CD123, and CLEC12a expression levels were in line with published studies. Here we have also shown they remain similar after SOC treatment (ven+aza) supporting the development of agents that target these proteins in both first and second line settings for AML. Citation Format: Radha Ramesh, Xiang Fang, Shuli Li, Sharon Friedlander, Farhad Sedarati, Lionel Adès, Nicholas Short, Cristina Papayannidis, Tammie Yeh. Biomarker results from PEVENAZA, a randomized phase 2 study of venetoclax and azacitidine +/- pevonedistat in newly diagnosed AML patients unfit for intensive chemotherapy: Increased efficacy in a subset of patients with IDH1/2 mutations and other observations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5184.

Liquid biopsy guides successful molecular targeted therapy of an inoperable pediatric brainstem neoplasm

Midline CNS tumors are occasionally inaccessible for surgical biopsies. In these instances, cell-free DNA (cfDNA) may serve as a viable alternative for molecular analysis and identification of targetable mutations. Here, we report a young child with an inoperable brainstem tumor in whom a stereotactic biopsy was deemed unsafe. The tumor progressed on steroids and after radiotherapy the patient developed hydrocephalus and received a ventriculoperitoneal shunt. Droplet digital PCR analysis of cfDNA from an intraoperative cerebrospinal fluid liquid biopsy revealed a BRAF V600 mutation enabling targeted treatment with MEK and BRAF inhibitors. The patient, now on trametinib and dabrafenib for 1 year, has had substantial tumor volume regression and reduction of contrast enhancement on MRIs and is making remarkable clinical progress. This case highlights that in a subset of CNS tumors, access to liquid biopsy analysis may be crucial to identify actionable therapeutic targets that would otherwise go undiscovered.

Homozygous substitution of threonine 191 by proline in polymerase η causes Xeroderma pigmentosum variant

DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη function is associated with the human disease Xeroderma pigmentosum variant (XPV). We hereby report the case of a 60-year-old woman known for XPV and carrying a Polη Thr191Pro variant in homozygosity. We further characterize the variant in vitro and in vivo, providing molecular evidence that the substitution abrogates polymerase activity and results in UV sensitivity through deficient damage bypass. This is the first functional molecular characterization of a missense variant of Polη, whose reported pathogenic variants have thus far been loss of function truncation or frameshift mutations. Our work allows the upgrading of Polη Thr191Pro from ‘variant of uncertain significance’ to ‘likely pathogenic mutant’, bearing direct impact on molecular diagnosis and genetic counseling. Furthermore, we have established a robust experimental approach that will allow a precise molecular analysis of further missense mutations possibly linked to XPV. Finally, it provides insight into critical Polη residues that may be targeted to develop small molecule inhibitors for cancer therapeutics.

Molecular analysis of genetic mutations in non-small cell lung cancer in Morocco.

Non-small cell lung cancer (NSCLC) is a significant global health issue with diverse molecular profiles affecting treatment responses. Yet, NSCLC's molecular epidemiology in Morocco is largely unexplored. This study focuses on NSCLC genetic mutations, specifically in adenocarcinoma, among Moroccan patients to contribute to understanding NSCLC in this population. Ninety-four patients diagnosed with lung adenocarcinoma were analyzed. Formalin-fixed paraffin-embedded tissue samples were processed, and deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) was extracted using standardized protocols. Mutations were detected using the AmoyDx Pan Lung Cancer Polymerase Chain Reaction (PCR) Panel kit, and their frequencies were assessed through statistical analysis. Epidermal Growth Factor Receptor (EGFR) mutations were detected in 22.34% of patients, predominantly exon 19 deletions (66.66%) and exon 21 L858R mutations (23.80%). Anaplastic lymphoma kinase (ALK) gene fusion was observed in 3.19% of patients, and KRAS mutations in 1.06%. No mutations were found in other tested genes. A slightly higher mutation rate was noted in females (54.16%) compared to males (45.84%). The study reveals a distinct mutation profile in Moroccan NSCLC patients, with a notable prevalence of EGFR mutations, albeit lower than in some Asian populations. The significance of EGFR mutations in treatment response aligns with global findings, highlighting the importance of understanding regional molecular variations for personalized therapy. Despite limitations in sample size and clinical data, this study sheds light on the genetic landscape of NSCLC in Morocco. The observed mutation rates, particularly in EGFR, underscore the potential for targeted therapies in Moroccan NSCLC patients, emphasizing the need for further research to refine treatment strategies tailored to this population.

Circumscribed choroidal hemangioma and non-pigmented choroidal melanoma: clinical, instrumental and molecular genetic features

This study attempts to develop a method of non-invasive molecular genetic differential diagnostics of CCH and non-pigmented CM. Based on the results of clinical and instrumental examination methods, 60 patients (60 eyes) with CCH (n=30) and non-pigmented CM (n=30) were included in this prospective study. The control group consisted of 30 individuals without intraocular tumors. Mutations in the GNAQ/GNA11 genes were determined by real-time PCR using the analysis of genomic circulating tumor DNA isolated from peripheral blood plasma. The average follow-up period was 12.1±1.8 months. The study revealed a significant association of mutations in exons 4 and 5 of the GNAQ/GNA11 genes with the presence of non-pigmented CM (27/30; 90%). These mutations were not detected in the group of patients with CCH. Mutations in exons 4 and 5 of the GNAQ/GNA11 genes were also not detected in the control group of healthy individuals. This study proposes a method of non-invasive and low-cost differential diagnostics based on molecular genetic analysis and detection of mutations in exons 4 and 5 of the GNAQ and GNA11 genes, which are specific for CM (90%).

Comprehensive molecular analysis of driver mutations in non-small cell lung carcinomas and its correlation with PD-L1 expression, An Indian perspective

BackgroundThe understanding of molecular mechanisms involved in non-small cell lung carcinoma (NSCLC) has revolutionized significantly in the recent years. These have helped to develop personalized management strategies by identifying specific molecular alterations such as mutations in EGFR, ROS1, BRAF, ERBB2, MET, ALK, and KRAS genes. These mutations are targetable ensuring a better clinical outcome. Next-generation sequencing (NGS) methodology is the recommended technique for the identification of driver mutations in the five hot-spot genes (EGFR, ALK, ROS1, MET, and BRAF) involved in the NSCLC. NGS has numerous advantages including multiplexing, tissue conservation, identification of rare and novel variants, and reduced cost over the sequential single gene testing. Herein, we sought to demonstrate the mutational profile in NSCLC and their clinicopathologic correlation in a contemporary cohort of Indian NSCLC patients. Additionally, we studied the correlation of oncogenic driver mutations with PD-L1 status in these patients. Materials and MethodsFive fifty-two stage IV NSCLC patients (adenocarcinoma=490; squamous cell carcinoma=51; adenosquamous carcinoma=5; large cell carcinoma=2; sarcomatoid carcinoma=3; spindle cell carcinoma=1) underwent broad molecular profiling by a custom-made, targeted DNA- and RNA-based five hot-spot genes lung cancer panel (EGFR, ALK, ROS1, BRAF, and MET), compatible with the NGS Ion S5 system. The mutations were correlated with the clinicopathologic characteristics. Additionally, PD-L1 expression status, available on 252 tumors, was correlated with the oncogenic drivers. ResultsValidation of the 5 gene panel yielded the following results: a) specificity of 99.74%; b) sensitivity of 100% for single nucleotide variants (SNVs) (>5% variant allele frequency, VAF), indels (>10% VAF) and fusions; c) 100% intra- and inter-run reproducibility; d) 88% inter-laboratory agreement. Validated panel was then used to analyze clinical samples. Sixty percentage tumors harbored either one (54.71%) or multiple (3.26%) mutations. EGFR and BRAF V600E mutations, ALK and ROS1 rearrangements, and MET exon 14 skipping mutation were observed in 38.41% (n = 212) and 2.72% (n = 15) patients, 12.14% (n = 67) and 3.62% (n = 20) patients, and 1.09% (n = 6) patients, respectively. EGFR exon 19 deletion accounted for 52.83% of all mutations, followed by L858R (35.85%), T790M (5.19%), exon 20 insertions (6.6%), and other rare mutations (G719X, L861Q, S768I) (9.91%). Concurrent EGFR with ALK, EGFR with ROS1, EGFR with MET, and EGFR with BRAF were observed in 10, 4, 1, and 3 patients, respectively. PD-L1 was expressed in 134 patients (53.2%). Exon 19 deletion was more prevalent in PD-L1 negative tumors whereas exon 21 substitution (L858R) was seen more in PD-L1 positive tumors. ConclusionsThis is one of the largest cohorts of NSCLC for comprehensive targeted mutational profiling and correlation with the PD-L1 expression. The mutations are more prevalent in non-smoker females for all genes, except ALK (non-smoker males). MET and BRAF mutations are more common in elderly population whereas EGFR mutations, and ALK and ROS1 genes rearrangements are more prevalent in younger population. The most common histopathologic subtype/feature associated with various mutations was as follows: acinar with EGFR, solid with ALK, macronucleoli with ROS1, signet ring with MET, and micropapillary with BRAF.

Molecular Analysis of Dihydropteroate Synthase Gene Mutations in Pneumocystis jirovecii Isolates among Bulgarian Patients with Pneumocystis Pneumonia

Pneumocystis jirovecii pneumonia (PCP) is a significant cause of morbidity and mortality in immunocompromised people. The widespread use of trimethoprim-sulfamethoxazole (TMP-SMZ) for the treatment and prophylaxis of opportunistic infections (including PCP) has led to an increased selection of TMP-SMZ-resistant microorganisms. Sulfa/sulfone resistance has been demonstrated to result from specific point mutations in the DHPS gene. This study aims to investigate the presence of DHPS gene mutations among P. jirovecii isolates from Bulgarian patients with PCP. A total of 326 patients were examined via real-time PCR targeting the P. jirovecii mitochondrial large subunit rRNA gene and further at the DHPS locus. P. jirovecii DNA was detected in 50 (15.34%) specimens. A 370 bp DHPS locus fragment was successfully amplified in 21 samples from 19 PCP-positive patients, which was then purified, sequenced, and used for phylogenetic analysis. Based on the sequencing analysis, all (n = 21) P. jirovecii isolates showed DHPS genotype 1 (the wild type, with the nucleotide sequence ACA CGG CCT at codons 55, 56, and 57, respectively). In conclusion, infections caused by P. jirovecii mutants potentially resistant to sulfonamides are still rare events in Bulgaria. DHPS genotype 1 at codons 55 and 57 is the predominant P. jirovecii strain in the country.

Molecular Characterization and Mutational Analysis of Clarithromycin- and Levofloxacin-Resistance Genes in Helicobacter pylori from Gastric Biopsies in Southern Croatia.

Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic-protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.

Protein–lipid charge interactions control the folding of outer membrane proteins into asymmetric membranes

Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, each leaflet comprising a distinct lipid composition. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. In this study, we used cyclodextrin-mediated lipid exchange to generate liposomes with asymmetric bilayers and characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs), OmpA and BamA. We found that excess negative charge in the outer leaflet of a liposome impedes their insertion and folding, while excess negative charge in the inner leaflet accelerates their folding relative to symmetric liposomes with the same membrane composition. Using molecular dynamics, mutational analysis and bioinformatics, we identified a positively charged patch critical for folding and stability. These results rationalize the well-known ‘positive-outside’ rule of OMPs and suggest insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.

High frequency of WNT-activated medulloblastomas with CTNNB1 wild type suggests a higher proportion of hereditary cases in a Latin-Iberian population.

Medulloblastomas are the most common primary malignant brain tumors in children. They are divided into molecular subgroups: WNT-activated, SHH-Activated, TP53 mutant or wild type, and non-WNT/non-SHH (Groups 3 and 4). WNT-activated medulloblastomas are usually caused by mutations in the CTNNB1 gene (85%-90%), and most remaining cases of CTNNB1 wild type are thought to be caused by germline mutations in APC. So far, the frequencies of CTNNB1 have been reported mainly in North American and European populations. The aim of this study was to report the frequency of CTNNB1 mutations in WNT-activated medulloblastomas in a Latin-Iberian population and correlate with their clinicopathological characteristics. A total of 266 medulloblastomas from seven different institutions from Brazil (n=211), Portugal (n=38), and Argentina (n=17) were evaluated. Following RNA and DNA isolation from formalin-fixed, paraffin-embedded (FFPE) tumor tissues, the molecular classification and CTNNB1 mutation analysis were performed by nCounter and Sanger sequencing, respectively. WNT-activated medulloblastomas accounted for 15% (40/266) of the series. We observed that 73% of WNT-activated medulloblastomas harbored CTNNB1 mutations. CTNNB1 wild-type cases (27%) were more prevalent in female individuals and suggested to be associated with a worse outcome. Among the CTNNB1 wild-type cases, the available analysis of family history revealed two cases with familiar adenomatous polyposis, harboring APC germline variants. We observed a lower incidence of CTNNB1 mutations in WNT-activated medulloblastomas in our Latin-Iberian cohort compared to frequencies previously described in other populations. Considering that CTNNB1 wild-type cases may exhibit APC germline mutations, our study suggests a higher incidence (~30%) of hereditary WNT-activated medulloblastomas in the Latin-Iberian population.

Study of 5 Alpha Reductase Enzyme Gene Mutations in a Group of Patients with 46XY Disorders of Sex Development

Background Disorders of sex development (DSD) are a wide range of conditions with diverse features and pathophysiology that most often present in the newborn or the adolescent periods. Affected patients usually present with atypical genitalia like hypospadias, isolated micropenis and undescended testis. These clinical situations can often be difficult to manage, particularly in those cases where the sex rearing is uncertain. Aim of the Work Studying 5alpha reductase enzyme gene mutations (SRD5A2) in patients with 46XY DSD to detect its molecular characteristics. Patients and Methods This cross-sectional study was performed on 25 male patients with clinical, laboratory and genetic molecular analysis of SRD5A2 gene mutation fitting the diagnosis of 46XY DSD. To assess the degree of virilization external masculinization score (EMS) was used where EMS less than 7 is considered ambiguous. All parameters were evaluated at the pediatrics endocrinology clinic and genetics department, Ain Shams University from December 2019 to December 2020. Results Only 12 patients showed laboratory and molecular results compatible with the diagnosis of SRD5A2 gene mutation, The mean age of the patients was 3.64 ± 4.75 years. Results showed EMS of 4.3 denoting the high degree of genital ambiguity in our cases. Scrotum was bifid in 5 patients (41.6%), underdeveloped in 5 patients (41.6%) and fused in 2 patients (16.8%). Cryptorchidism was found in 4 patients only (33.3%). Four patients (33.3%) had normal urethral opening while hypospadias was present in 8 patients being perineal in 1 patient (8.3%), perineoscrotal in 5 patients (41.6%) and scrotal in 2 patients (16.6%). Micropenis was present in 7 patients (58.3%), four patients had 2 openings (33.3%) compared to a single opening in 8 patients (66.7%). Regarding type of SRD5A2 gene mutations 56.2% of mutations were Insertion deletion, 25% were Insertion, 12.5 % were Missense, 6.25% were Deletion. Conclusion This study demonstrated ten novel mutations of SRD5A2 gene were identified in association with 5alpha reductase enzyme deficiency. These mutations were reported among 12 patients with 46 XY DSD from total of 25 patients were fit for this study. No distinct relationships were observed between genotypic and phenotypic characteristics in the participants.

Pathology of Hepatocellular Carcinoma and Tumor-Bearing Liver Tissue in Association with hTERT Promoter Mutation.

The hTERT promoter mutation represents a common and early event in hepatocarcinogenesis, but its linkage to the morphological status of the underlying liver tissue is poorly understood. We analyzed the connection between the histopathological changes in tumor-bearing liver tissue and the occurrence of the hTERT promoter mutation in hepatocellular carcinoma (HCC), correlated with clinical data. The study cohort comprised 160 histologically confirmed HCC in patients with or without cirrhosis that were investigated for the hTERT promoter mutation. We evaluated the frequency of the hTERT promoter mutation in patients with HCC with or without cirrhosis and correlated it with potential clinical and histopathological drivers. In particular, we examined tumor-bearing noncirrhotic liver tissue regarding inflammation; the modified histological activity index (mHAI), fibrosis, and steatosis; and its correlation with the frequency of the hTERT promoter mutation in HCC. We evaluated overall survival with multivariate Cox regression. Furthermore, we compared hTERT antibody immunohistochemistry and molecular hTERT promoter mutation analysis of both HCC and background liver tissue. The hTERT promoter mutation was especially related to HCC in cirrhotic compared with noncirrhotic liver (p < 0.001) and independently of cirrhosis in patients ≥ 60 years (p = 0.005). Furthermore, the hTERT promoter mutation was associated with cirrhosis caused by alcohol toxicity and hepatitis C virus infection. In noncirrhotic liver tissue, the frequency of hTERT-promoter-mutated HCC increased with the degree of inflammation and fibrosis. Nevertheless, 25% of the hTERT-promoter-mutated HCC developed in normal liver tissue without HCC risk factors. Multivariate Cox regression analysis did not reveal an influence of the hTERT promoter mutation in HCC on overall survival at 3, 5, and 16 years. Immunohistochemical analysis with the hTERT antibodies LS-B95 and 2D8 in hTERT-promoter-mutated HCC and hTERT-wildtype HCC showed a mildly stronger immunoreaction compared with the tumor-bearing liver tissue (LS-B95: p < 0.01, 2D8: p < 0.01). Our study reveals a connection between pathological changes in tumor-bearing liver tissue and the hTERT promoter mutation in most HCC, even in noncirrhotic liver tissue. Immunohistochemical hTERT antibodies do not discriminate between hTERT-promoter-mutated and wildtype HCC.

Molecular analysis of rpoB gene mutation in MTB detected isolates in a tertiary care centre (AGMC) of North-East, India

BackgroundRifampicin (RIF), one of the first line drug in treatment of tuberculosis. It acts on rpoB gene which encodes RNA polymerase β subunit. In 95% of RIF resistant cases, mutations are present in rpoB gene. Most of them are within 81bp RIF-resistance determining region (RRDR).Xpert MTB/RIF assay has been tremendously revolutionalised the diagnosis of tuberculosis (TB).Also sequencially detect bacteria and resistance to rifampicin (rif).Approximately 96% of rif-resistant Mycobacterium tuberculosis (MTB) strains worldwide, showed mutations in a region at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene. Here evaluation is made about frequent regions of amplification and mutation in various codons of 81bp of rpoB gene in rif sensitive and rif resistant cases. MethodsA total of 4116 samples were received at Mycobacteriology laboratory, AGMC and processed in CBNAAT.Data of MTB detected samples were collected & statistically analysed to detect frequency of amplification & no amplification in various regions of 81bp of rpoB genes. ResultsOut of 4116 samples, MTB was detected in 1323 samples. Among them 1291 (97.58%) cases were Rif sensitive (RS) and 32 (2.41%) cases were rif resistance (RR).Most of the MTBC detected samples showed amplification in probe A then in probe C.78.12% rif resistant cases showed mutation in either of the probe, commonest is probe E. Study also showed low bacillary loads in most of the RR cases. ConclusionStudy highlighted variations in amplification of different regions of 81bp of rpoB gene in MTBC detected cases. North-east India, like other part of world, also showed highest frequency of mutation in probe E in rif resistant cases.

Clinical characteristics and outcome of PIK3CA mutated hormone receptor positive (HR+), human epidermal growth factor 2 negative (HER2-), advanced breast cancer (BC) treated with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i): Real world data.

e13078 Background: BC is the most common malignant disease and leading cancer-related cause of death in women. Despite significant achievements in the treatment, advanced BC (aBC) is still an incurable disease. HR+/HER2- subtype accounts for 70% of all BC. One of the most common mutations in BC is the PIK3CA gene mutation which can induce endocrine and chemo- resistance. In early BC, patients (pts) with PIK3CA mutations mostly have better, but in aBC worse prognosis. Standard first-line treatment for HR+/HER2- aBC is a combination of CDK4/6i and endocrine therapy. PIK3CA status is most often assessed in this group of pts and combination of alpelisib - PIK3CA inhibitor and fulvestrant is second-line treatment option in PIK3CA mutated pts. Methods: Medical records of HR+/HER2- aBC pts who started CDK4/6i therapy between 01.08.2018. and 01.10.2020. were analyzed with prior University Hospital Center Zagreb Ethics Committee approval. Demographics, tumor characteristics, clinical presentation and therapy information were collected. Molecular analysis of PIK3CA mutations was performed on tumor tissue samples. Time to treatment failure (TTF) was analyzed using the Kaplan-Meier method and the data cut-off was 31.12.2022. Results: Of the 103 pts with determined PIK3CA status, 45 (43.7%) had a detectable mutation. Median follow-up was 35 months. CDK4/6i were first line therapy in 31 (68.9%) pts with and 29 (50%) pts without PIK3CA mutation. Endocrine resistance, de novo metastatic disease and bone marrow infiltration were more common in PIK3CA mutated subgroup. The incidence of visceral metastases was similar in both groups, whereas liver metastases were more common in the unmutated group. Disease progression was observed in 69 (67%) pts, 28 (40.6%) of whom were PIK3CA mutated. Median TTF in mutated subgroup was 24 months (95% CI 21-33) and 25 months in unmutated subgroup (95% CI 19-36). During the follow-up period, 35 (34%) pts died, 14 (40%) of whom were PIK3CA mutated. Odds ratios in pts with a PIK3CA mutation. Conclusions: There was no relevant difference in TTF between two groups. However, PIK3CA mutated subgroup had more aggressive clinical features - more often presented with bone marrow infiltration and de novo aBC, statistically relevant OR 5.158 and 2.323 respectively, which is related to lower quality of life and highlights the importance of treatment improvement for this subgroup of pts. [Table: see text]