Detection Of Mutations Research Articles

Peritoneal washing analysis in endometrial cancer: Does somatic mutation detection with panel sequencing build upon traditional cytologic analysis?

Peritoneal washing analysis in endometrial cancer: Does somatic mutation detection with panel sequencing build upon traditional cytologic analysis?

Development and preliminary assessment of the iFIND TBR: all-in- one molecular diagnostic assay for rapid detection of Mycobacterium tuberculosis and rifampicin resistance

IntroductionEarly and accurate diagnosis of tuberculosis (TB) is crucial for initiating timely treatment and preventing new infections. In this study, we introduced the iFIND TBR assay, an automated all-in-one tuberculosis detection approach that simultaneously detect Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance.MethodsThe limits of detection (LOD), sensitivity, specificity, and RIF-R rpoB mutation detection of the iFIND TBR were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M.tuberculosis H37Rv. Frozen clinical samples from patients suspected of having TB were also tested.ResultsThe LOD of the iFIND TBR for MTB detection were 13.34 CFU/ml (95% CI, 11.71-16.47), and for RIF resistance was 109.79CFU/mL (95% CI, 95-138.19). The iFIND TBR assay accurately distinguish MTB strains from non-tuberculous mycobacteria (NTM) without any cross reactivity. Testing on 157 clinical sputum samples, compared with the bacteriologically TB standard, the overall sensitivity and specificity of the iFIND TBR was 100% (95%CI, 94.64, 100) and 85.29% (95% CI, 74.61, 92.72), respectively. When assessing RIF susceptibility, the iFIND TBR achieved a sensitivity of 98.15% (95% CI, 90.11–99.95) and a specificity of 85.71% (95% CI, 67.33–95.97), compared with phenotypic drug susceptibility testing. Discordant RIF susceptibility results were more frequently observed in samples exhibiting heteroresistance.DiscussionThese findings demonstrate that iFIND TBR assay performs well in detecting TB and RIF resistance, and shows promise as a point-of-care tool in resource-limited areas.

Genetic and other omics-based information in the most-cited recent clinical trials

Abstract Introduction Since the genomic revolution, the utilization of genetic and omics technologies has undergone considerable expansion within clinical studies, employed in design, analysis, and interpretation. Methods A recent study of Siena et al. developed a database containing the 600 most cited clinical trials published from 2019 to 2022. Building on this database, the aim of our study is to assess how frequently genetic and other omics-based information has been used in the design, analysis, results or conclusion of these recent influential trials. Results 132 out 600 (22%) trials used genetic or other omics. These trials were more likely to be oncology trials (76%vs34%), having industry funding (59%vs47%), and having industry authors (75%vs54%) than others. Overall, genetics and genomics were present in 75% of the trials (100/132), and oncology was the predominant medical field (76%, n = 101). The prevailing use was detection of specific mutations (n = 99), often (n = 60) used as eligibility criterion. These mutations were mostly somatic (n = 85), while 14 were germline. Other applications included transcriptomics (20.4%), proteomics (10.6%), metabolomics (8.3%) and metagenomics (8.3%). 36 studies (27%) employed multiple applications. Genetics or other omics were used in 10% of the studies (14/132) for randomization stratification and in 63% (83/132) to conduct subgroup analysis. Among the latter, in 48% (40/83) the presence of a somatic mutation was significantly associated with the investigated health outcomes, while germline mutation or other omics only in the 12% (10/83). In the trials conclusions, authors addressed the relevance of the genetic or omics information in 62.1% (82/132) of the trials. Conclusions A sizeable proportion of the most-cited clinical trials use genetics or omics-based information, but the large majority pertains to cancer mutations in oncology trials. Use of genetics and omics needs to become more common in other clinical trial fields. Key messages • Genetic profiling and omics applications should be incorporated into trial design. • Genomic research is mostly oncology related and it needs to be expanded to other medical fields.

18-Years of single-centre DNA testing in over 7000 index cases with inherited retinal dystrophies and optic neuropathies

Inherited retinal dystrophies (IRDs) and inherited optic neuropathies (IONs) are characterized by distinct genetic causes and molecular mechanisms that can lead to varying degrees of visual impairment. The discovery of pathogenic variants in numerous genes associated with these conditions has deepened our understanding of the molecular pathways that influence both vision and disease manifestation and may ultimately lead to novel therapeutic approaches. Over the past 18 years, our DNA diagnostics unit has been performing genetic testing on patients suspected of having IRD or ION, using state-of-the-art mutation detection technologies that are continuously updated. This report presents a retrospective analysis of genetic data from 6237 IRD and 780 ION patients. Out of these, 3054 IRD patients (49.0%) and 211 ION patients (27.1%) received a definitive molecular diagnosis, with disease-causing variants identified in 139 different genes. The genes most implicated in disease pathologies are ABCA4, accounting for 23.8% of all IRD/ION index cases, followed by BEST1 (7.8%), USH2A (6.2%), PRPH2 (5.7%), RPGR (5.6%), RS1 (5.5%), OPA1 (4.3%), and RHO (3.1%). Our study has compiled the most extensive dataset in combined IRD/ION diagnostics to date and offers valuable insights into the frequencies of mutant alleles and the efficiency of mutation detection in various inherited retinal conditions.

Detection of PIK3CA hotspot mutations in canine mammary tumors using droplet digital PCR: tissue validation and liquid biopsy feasibility

Domestic dogs (Canis lupus familiaris) serve as valuable translational models for human cancer research due to their biological similarities. Canine mammary tumors (CMTs), frequently diagnosed in female dogs, share various characteristics with human breast cancers. This study investigates the PIK3CA (H1047R) mutation in CMTs using droplet digital PCR (ddPCR) and explores the potential of liquid biopsy for non-invasive detection. We analyzed 80 formalin-fixed, paraffin-embedded (FFPE) CMT tissue samples and compared ddPCR results with next-generation sequencing (NGS) data, achieving high concordance. Plasma and serum samples were also assessed for mutation concordance with tissue results. Our findings indicate a higher frequency of the PIK3CA (H1047R) mutations in benign and grade I malignant CMTs compared to more aggressive malignancies. The ddPCR assay demonstrated high sensitivity and specificity, with plasma testing showing 78.6% sensitivity and 87.5% specificity, and serum testing showing 66.7% sensitivity and 90.0% specificity. These results highlight the viability of liquid biopsy as a minimally invasive method for monitoring PIK3CA mutations in canine patients. The study suggests that liquid biopsy techniques hold significant promise for improving the early detection and monitoring of canine cancers, warranting further research to refine these methods and explore their applications in canine cancer diagnostics and treatment.

Tumor organoids improve mutation detection of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) presents challenges in detecting somatic mutations due to its complex cellular composition. This study investigated the utility of patient-derived organoids (PDOs) to overcome these obstacles and enhance somatic mutation identification. Surgically resected PDAC tumors and their paired PDOs from 21 patients were examined. Whole-exome sequencing (WES) of tumor tissue, organoids, and peripheral blood mononuclear cells was performed to identify somatic mutations. Our findings demonstrate that PDOs retained about 80% of the somatic mutations from the original tumors, showing high concordance in mutation types. PDOs exhibited increased tumor purity and uncovered key driver mutations, aiding in identifying clinically relevant genomic alterations. Moreover, eight cycles of FOLFIRINOX treatment did not significantly alter the mutational landscape at the DNA level, indicating the stability of the mutational profile after therapeutic pressure in patients. In conclusion, PDOs are potentially important tools for exploring the somatic mutational landscape of PDAC. While they can reveal mutations that may be challenging to detect through traditional biopsy sequencing due to the inherently low tumor purity of PDAC, it is important to note that PDOs may not always fully recapitulate all mutations found in primary tumors. Despite this limitation, PDOs can still offer critical insights into the genomic complexities of PDAC, which is crucial for the development of personalized vaccines and therapies for this disease.

Enrichment Strategies for Low-Abundant Single Nucleotide Mutations.

Over the past three decades, significant advancements have been made in mutation enrichment methods, driven by the increasing need for precise and efficient identification of rare genetic variants associated with diseases. Mutation-enrichment methods have emerged to boost sensitivity and enable easy detection of low-frequency mutations. These methods are crucial in genomics research and clinical diagnostics, allowing for the detection of low-frequency mutations within large genomic datasets. This review presents a summary of technological developments in rare mutation enrichment and emphasizes their mechanisms and applications in liquid biopsies.

Genome sequencing in the management of myelodysplastic syndromes and related disorders.

Myeloid neoplasms originate from the clonal proliferation of hematopoietic stem cells, which is driven by the acquisition of somatic genetic mutations. Within these disorders, myelodysplastic syndromes (MDS) are specifically characterized by morphologic abnormalities (dysplasia) and impaired maturation of myeloid precursors (ineffective hematopoiesis), resulting in peripheral blood cytopenia. Several studies have advanced the field of MDS, with a few landmark papers leading to a paradigm shift, opening new avenues of research and enabling a molecular revolution. These seminal papers include the first description of the 5q- syndrome, the identification of somatic mutations of TET2 in myeloid neoplasms, the detection of common pathway mutations in the splicing machinery, and the discovery of clonal hematopoiesis. The somatic genomic landscape of MDS is now well-defined. Genes that are recurrently mutated include epigenetic regulators, as well as genes of RNA splicing machinery, transcription regulation, DNA repair control, cohesin complex, and signal transduction. Furthermore, several disorders with a germline genetic predisposition to MDS have been identified, collectively accounting for up to 15% of all MDS cases. Genomic profiling can significantly improve the diagnostic approach to MDS, allowing the identification of distinct nosologic entities such as SF3B1-mutant or TP53-mutant MDS. The Molecular International Prognostic Scoring System for MDS (IPSS-M) has already proven to be a valuable tool for individualized risk assessment and treatment decisions. In addition, the recently developed molecular taxonomy of MDS will likely facilitate the implementation of precision medicine approaches for these disorders. This will necessitate the establishment of specialized infrastructures within public health systems, involving close collaboration between healthcare institutions, academia, and the life sciences industry.

An Optimized CRISPR/Cas12a Assay to Facilitate the BRAF V600E Mutation Detection.

Accurate detection of the BRAF V600E (1799T > A) mutation status can significantly contribute to selecting an optimal therapeutic strategy for diverse cancer types. CRISPR-based diagnostic platforms exhibit simple programming, cost-effectiveness, high sensitivity, and high specificity in detecting target sequences. The goal of this study is to develop a simple BRAF V600E mutation detection method. We combined the CRISPR/Cas12a system with recombinase polymerase amplification (RPA). Subsequently, several parameters related to CRISPR/Cas12a reaction efficiency were evaluated. Then, we conducted a comparative analysis of three distinct approaches toward identifying BRAF V600E mutations in the clinical samples. Our data suggest that CRISPR/Cas detection is considerably responsive to variations in buffer conditions. Magnesium acetate (MgOAc) demonstrated superior performance compared to all other examined additive salts. It was observed using 150 nM guide RNA (gRNA) in an optimized reaction buffer containing 14 mM MgOAc, coupled with a reduction in the volumes of PCR and RPA products to 1 μL and 3 μL, respectively, resulted in an enhanced sensitivity. Detection time was decreased to 75 min with a 2% limit of detection (LOD), as evidenced by the results obtained from the blue light illuminator. The CRISPR/Cas12a assay confirmed the real-time PCR results in 31 of 32 clinical samples to identify the BRAF V600E mutation status, while Sanger sequencing detected BRAF V600E mutations with lower sensitivity. We propose a potential diagnostic approach that is facile, fast, and affordable with high fidelity. This method can detect BRAF V600E mutation with a 2% LOD without the need for a thermocycler.

A novel dual probe-based method for mutation detection using isothermal amplification.

Cost efficient and rapid detection tools to detect mutations especially those linked to drug-resistance are important to address concerns of the rising multi-drug resistance infections. Here we integrated dual probes, namely a calibrator probe and an indicator probe, into isothermal amplification detection system. These two probes are designed to bind distinct regions on the same amplicon to determine the presence or absence of mutation. The calibrator probe signal is used as an internal signal calibrator for indicator probe which detects the presence or absence of the mutation. As an illustrative example, we evaluated the applicability of this dual probe method for detecting mutations associated with rifampicin (RIF) drug resistance at codons 516, 526 and 531 of the rpoB gene in Mycobacterium tuberculosis. In this assessment, we examined 127 artificial samples comprising wild types and mutants with single or multiple mutations. Our results demonstrated 100% accuracy for both wild types and mutants for mutations at codons 526 and 531. As regards to mutations at codon 516, the wild type was identified with 100% accuracy, while the mutants were identified with 95% accuracy. Moreover, when we extended our evaluation to include clinical MTB strains and the Zeptometrix MTB Verification panel, our method achieved 100% accuracy (5 out of 5) in identifying wild-type strains. Additionally, we successfully detected a RIF-resistant strain with mutations at codon 531 of the rpoB gene in Zeptometrix verification panel. Our isothermal mutation detection system, relying on dual probes exhibits a versatile approach. With the capability to identify mutations without prior knowledge of their specific mutation direction, our dual-probe method shows significant promise for applications in drug resistance nucleic acid testing, particularly in resource-limited settings.

Can molecular patterns help to classify overlapping entities in myeloid neoplasms?

Myeloid neoplasms include myeloproliferative and myelodysplastic neoplasms and acute myeloid leukaemia. Historically, these diseases have been diagnosed based on clinicopathological features with sometimes arbitrary thresholds that have persisted even as molecular features were gradually incorporated into their classification. As such, although current diagnostic approaches can classify the majority of myeloid neoplasms accurately using a combination of molecular and clinicopathological features, some areas of overlap persist and occasionally pose diagnostic challenges. These include overlap across BCR::ABL1-negative myeloproliferative neoplasms; between clonal cytopenia of undetermined significance and myelodysplastic neoplasms; myelodysplastic/myeloproliferative neoplasms; and, detection of KIT mutations in myeloid neoplasms other than mastocytosis, raising the prospect of systemic mastocytosis. Molecular testing has become state of the art in the diagnostic work-up of myeloid neoplasms, and molecular patterns can inherently help to classify overlapping entities if considered within a framework of haematological presentations. For future development, molecular testing will likely include whole genome and transcriptome sequencing, and primarily molecular classifications of myeloid neoplasms have already been suggested. As such, genetically defined groups should still constitute the basis for our understanding of disease development from early onset to progression, while clinicopathological features could then be used to describe the stage of the disease rather than the specific type of myeloid neoplasm.

Establishment of potential reference measurement procedure and reference materials for EML4-ALK fusion variants measurement

Echinoderm microtubule-associated protein 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene detection is of great significance in personalized tumor treatment. With the development of EML4-ALK fusion variants detection, it is necessary to establish traceability to ensure the consistency and comparability of its detection results in clinical practice. The establishment of traceability relies on SI traceable reference materials (RMs) and potential reference measurement procedures (RMPs). In this study, a potential RMP for the quantitative detection of V1 and V3 fusion mutations and the reference type (ALK-ref, including wild type, V1 and V3 mutant type) based on reverse transcription dPCR (RT-dPCR) and EML4-ALK fusion gene RMs were established. The proposed potential RMP has high specificity, good inter-laboratory reproducibility (CV < 7.3%) and good linear relationship (0.92 < slope < 1.06, R2 ≧ 0.99). The limit of detection (LoD) of V1, V3, and ALK-ref are 2 copies/reaction, 2 copies/reaction, and 1 copy/reaction, respectively. Interlaboratory studies using the EML4-ALK RMs and potential RMP showed that participating laboratories can produce consistent copy concentrations of fusion variant and ALK-ref as well as the ratio of EML4-ALK/ALK-ref. The established potential RMP with high specificity and accuracy can be used to characterize the EML4-ALK RMs, and the potential RMP and RM are useful to establish the traceability of EML4-ALK fusion measurement to improve the comparability and consistency in clinical tests.

Bioinformatics investigation of the prognostic value and mechanistic role of CD9 in glioma

In recent years, CD9 has been extensively studied as a potential biomarker for cancer. However, the biological role of CD9 in gliomas remains unclear. This study investigates the function of CD9 in gliomas and its molecular mechanisms. Utilizing pan-cancer analysis with TCGA, CGGA, and GEO databases, differential expression of CD9 was observed in 11 tumor types within the TCGA cohort, and it was associated with patient survival rates. Analysis of the CGGA glioma database revealed that patients with high CD9 expression had lower survival rates. The area under the ROC curve (AUC) for GSE16011 was greater than 0.7, indicating a high discriminative ability. Through gene set enrichment analysis (GSEA), immune-related analysis, and CD9 mutation detection, CD9 was found to have the strongest correlation with neutrophil involvement (cor = 0.30, P < 0.05), and the high CD9 expression group exhibited higher rejection responses and TIDE scores, suggesting a lower likelihood of successful immunotherapy. The high CD9 expression group was more sensitive to 81 drugs, indicating potential therapeutic effects for gliomas. Furthermore, overexpression of CD9 in gliomas may be associated with gene mutations. Down-regulation or up-regulation of CD9 expression in the glioblastoma cell line LN229 showed that CD9 could positively regulate the migratory ability of LN229 cells. Further, several marker genes, such as VEGFR-2, TGF-β1, CASP1 and PI3K, were down regulated in CD9 knockdown cell lines and up regulated in CD9 overexpression cell lines, compared with control cell line. This study preliminarily explores the role of CD9 in gliomas and its prognostic value, providing new insights for personalized treatment strategies in glioma therapy.

Delivery of functional Cas:DNA nucleoprotein complexes into recipient bacteria through a type IV secretion system

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells.

Improving the Utility of a Cancer Gene Mutation Panel for Multiple Myeloma Patients

Abstract Multiple myeloma is a plasma cell malignancy that accounts for 1-2% of all cancer diagnoses and 10-15% of hematopoietic neoplasms. Clonal rearrangement of the IG locus suggests a monoclonal model for pathogenesis. The presence of cytogenetic abnormalities in &amp;gt;90% of cases suggests transformation by chromosomal instability and rearrangement. Classification and consensus guidelines by The International Myeloma Working Group (IMWG) and others reflect this by categorizing myeloma based on ploidy, translocations, and chromosomal abnormalities. Interestingly, a portion of Emory’s test volume for cancer gene mutation profiling by second generation short-read sequencing is for multiple myeloma cases. This suggests our 75-gene myeloid neoplasm panel (MMP75) is being utilized for medical purposes that are not met by cytogenetic and microarray characterizations performed during the diagnostic workup of multiple myeloma. Inspired by this observation, this study investigates providers motivations for ordering MMP75 and assesses the adequacy of our current implementation for their stated purposes. Anecdotes from ordering physicians indicate interest in MMP75 results when informing prognosis, selecting treatments, and evaluating for treatment associated secondary neoplasms. A more formal survey is underway that will statistically capture how the results of MMP75 testing were ultimately used: to inform diagnosis, determine prognosis, guide treatment, explain progression, identify/rule-out secondary neoplasia, or no effect on management. We also explore the variants called by MMP75 when performed using bone marrow from patients who received a cytogenetic workup for multiple myeloma at some point. A retrospective analysis of the 5-year interval between Jan 2019 and Jan 2024 identified 156 cases meeting this criterion. The number of pathogenic variants per case ranged between 1 and 8, with mutations detected across 42 genes. The genes most frequently identified with pathogenic mutations were TP53 (22.4%), DNMT3A (19.2%), TET2 (14.7%), and ASXL1 (12.2%). All four have been associated with adverse outcomes in patients with multiple myeloma. Activating events in therapeutic targets included mutations in the RAS/RAF pathway (16.7%), JAK/STAT signaling (2.6%), and the kinase KIT (0.6%). While these findings support the use of MMP75 for detection of relevant mutations, the genetic variants identified are not unique to multiple myeloma. Mutations detected in DNMT3A, TET2, and ASXL1 are common in clonal hematopoiesis of indeterminant potential (CHIP) and myeloid neoplasms. Hotspot mutations in TP53 and activating events in the RAS/RAF pathway also occur in various hematologic malignancies. This test therefore detects events that occur in both the plasma cell compartment and other cells of origin. This encumbers distinguishing variants that inform on a patient’s multiple myeloma from events that suggest a secondary neoplasm. Future directions include determining whether the effect of plasma cell enrichment on a mutations variant allele frequency (VAF) in repeat testing can help distinguish true multiple myeloma variants from events in other cells.

Multi-gene mutations in ultrasound-guided fine-needle aspiration specimens of thyroid micronodules and diagnostic value in thyroid microcarcinomas using next-generation sequencing

Objective: To explore the detection of BRAF, RAS, TERT promoter, and TP53 gene mutations in solitary thyroid micronodule (TMN) specimens obtained by ultrasound-guided fine-needle aspiration (US-FNA) using next-generation sequencing (NGS) technology and assess its diagnostic value in thyroid microcarcinomas (TMC). Methods: On-site recruitment of 428 patients with single suspicious TMC who underwent thyroid ultrasound examination, US-FNA, and NGS from September 2018 to July 2021 at Beijing Tongren Hospital affiliated to Capital Medical University. A total of 147 patients were finally included. NGS was used to detect mutations in the BRAF, RAS, TERT promoter, and TP53 genes in the US-FNA specimens. Comparisons were made between patients with and without gene mutations in terms of age, gender, and the maximum diameter of nodules. The diagnostic efficiency of BRAF mutation for TMC was calculated using the receiver operating characteristic (ROC) curve, with postoperative pathology as the gold standard. Results: The age [M (Q1, Q3)] of the 147 patients was 43.0 (32.0, 51.0) years, and 37 were male (25.2%). Among the 147 US-FNA specimens, 97 (66.0%) were detected with BRAF gene mutations, all of which were p.V600E point mutation; 6 (4.1%) were detected with RAS gene mutation, and no TERT promoter or TP53 gene mutations were detected. Postoperative pathology confirmed that 136 cases were TMC, all of which were papillary thyroid microcarcinomas (PTMC); 11 cases (7.5%)were benign. Among 136 TMC samples, BRAF gene mutations were detected in 97 cases (71.3%). There were no statistically significant differences in age, gender, and maximum nodule diameter between patients with and without BRAF gene mutations (all P>0.05). The sensitivity and specificity of BRAF gene mutation in diagnosing TMC were 71.3% and 100.0%, respectively, with an area under the ROC curve (AUC) (95%CI) of 0.857 (0.789-0.925). For nodules classified as Bethesda Ⅲ-Ⅴ, the sensitivity and specificity were 63.0% and 100.0%, respectively, with an AUC (95%CI) of 0.815 (0.680-0.950). Conclusions: NGS technology can successfully detect multiple gene mutations in US-FNA specimens from TMN patients, especially BRAF gene mutation, and BRAF gene mutation has certain value in diagnosing TMC.

Uveal Effusion Syndrome Due to WNT10A Mutation.

Uveal effusion syndrome (UES) is an exudative detachment of ciliary body, retina and choroid. Various underlying causes leads to UES-like drugs (topiramate), inflammation and hypotony. Wnt gene involvement has never been associated with UES. We report a case of bilateral UES being misdiagnosed as Vogt-Koyanagi-Harada syndrome (VKH), with Wnt gene dysfunction as the underlying trigger. A 32-year-old male presented with diminution of vision in his left eye. He was found to have choroidal detachment with retinal detachment in his left eye. Choroidal detachment was noted in the right eye. Various ocular imaging including fundus fluorescein angiography and ocular coherence tomography was done. He was misdiagnosed as a case of VKH syndrome, for which he was treated with systemic immunomodulatory therapy. However, a subclinical response was made to revisit the diagnosis. Ultrasound biomicroscopy showed supraciliary effusion. Systemic and genetic evaluation led to the detection of Wnt10A pathway mutation. UES is an entity of diagnostic challenge. Careful and thorough systemic evaluation is required to clinch the diagnosis. We reported the first case of bilateral UES recalcitrant to corticosteroids, with Wnt10A gene mutation.

Rare Adult acute leukemia with Coexisting myeloblastic (basophilic differentiation) &amp; B-lymphoblastic subpopulations Case Study

Abstract Introduction/Objective Acute basophilic leukemia is a very rare subgroup of acute myeloid leukemia that is defined in the 2022 WHO classification by presence of at least 20% blast cells in peripheral blood or bone marrow with immature and maturing cells of basophil lineage. The simultaneous occurrence of two hematological malignancies in a middle- aged adult patient is quite a rare event as evidenced by the rare cases reported in the literature. To our knowledge, this is the first case to be reported with concomitant acute basophilic and B-lymphoblastic leukemia in adult patients. these findings necessitates the comprehensive systematic approach to attain accurate diagnosis and plan the appropriate treatment protocol. Methods/Case Report Bone marrow aspiration &amp; biopsy procedure was performed under local anesthesia and marrow samples were subjected to: lieshman staining of marrow films and direct examination. Immunophenotypic analysis of marrow aspirate by FACS Canto II flow cytometer. Cytogenetics studies were done as karyotyping and gene mutation detection by FISH. Results (if a Case Study enter NA) NA Conclusion Morphology of marrow films: 50% Blast cells, group of them are medium to larg-cells with high nucleocytoplasmic ratio and immature chromatin and the other group shows blast cells with sparse basophilic granulations, plus 20% immatue and maturing basophils. IPT shows two populations: the Myeloblast population (basophilic differentiation) is Positive for CD45dim, CD34het, CD13, CD33, CD117prt, CD25het, CD123het, cyt MPO part, HLA-DR partial. the B lineage lymphoblast population is Positive for CD45dim, CD34het, CD19, CD79a, TdT, CD20het, CD38. Cytogenetics studies: hyperploidy 56, (X, Y, 9, 10, 12, 13, 15, 18, 22, mar+ extra-chromosomes) FISH: +ve for FLT3-ITD mutation. the picture is by morphology &amp; IPT consistent with biclonal leukemia, myeloid clone (acute basophilic leukemia) &amp; ProB-ALL. The systematic laboratory approach from careful morphological examination of peripheral blood and bone marrow smears to confirmatory immunophenotypic analysis of marrow specimens by flow cytometry, and the comprehensive cytogenetics analysis, has a vital role in diagnosing such rare and aggressive phenomenon and provide a good chance for planning the appropriate treatment protocol with critical timesaving and cost effectiveness.

Development of molecular diagnostic protocols for simultaneous identification of common bed bugs (Cimex lectularius) and tropical bed bugs (Cimex hemipterus)

BackgroundThe resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species is crucial for implementing targeted capture traps and tailored pyrethroid resistance diagnosis, due to differences in genetic and physiological traits.MethodsTo develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species.ResultsBoth multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP suggest its potential for rapid and accurate diagnosis of bed bugs in the field. The diagnostic accuracy of these methods was validated through a blind test.ConclusionsThe multiplex PCR and LAMP protocols lay the foundation for rapid and accurate field identification of bed bug species, enabling the use of appropriate traps and the detection of species-specific pyrethroid resistance mutations. This approach ensures effective management tailored to the unique characteristics of each bed bug species.Graphical

Advancing Precision Oncology: Whole-Exome Sequencing in Endometrial Cancer Liquid-Based Cytology.

Diagnostic strategies for endometrial cancer have been evolving, with cytologic analysis being considered a key method in integrated oncologic diagnostics because of its less invasive nature and adaptability to various assessments. Liquid-based cytology (LBC) has emerged as a promising method for intact DNA preservation; it exhibits improved efficiency in advanced sequencing applications such as next-generation sequencing. However, despite the use of LBC in panel assays, its application in whole-exome sequencing (WES) for comprehensive genomic profiling remains underexplored. To investigate whether molecular classification is possible based on WES using DNA derived from LBC specimens. We combined WES with targeted gene panel analysis to compare genomic findings of LBC and traditional tissue samples obtained from 7 cases of endometrial cancer. We investigated pathogenic mutations, tumor mutational burden, and microsatellite instability, and achieved molecular classification with high accuracy. We found a substantial concordance between LBC and traditional tissue samples in terms of pathogenic mutation detection, with a 95% match in the LBC samples and 94% in the tissue samples. Notably, our results highlight the importance of combining WES with panel-based analysis in identifying the ultramutated status of a case that had been missed during panel analysis. Our findings emphasize the potential of LBC samples in the precise and noninvasive genomic analysis of cases of endometrial cancer and offer a new avenue for developing diagnostic and therapeutic strategies in precision oncology.