Detection Of Point Mutations Research Articles

High-resolution and quantitative spatial analysis reveal intra-ductal phenotypic and functional diversification in pancreatic cancer.

A 'classical' and a 'basal-like' subtype of pancreatic cancer have been reported, with differential expression of GATA6 and different dosages of mutant KRAS. We established in situ detection of KRAS point mutations and mRNA panels for the consensus subtypes aiming to project these findings to paraffin-embedded clinical tumour samples for spatial quantitative analysis. We unveiled that, next to inter-patient and intra-patient inter-ductal heterogeneity, intraductal spatial phenotypes exist with anti-correlating expression levels of GATA6 and KRASG12D . The basal-like mRNA panel better captured the basal-like cell states than widely used protein markers. The panels corroborated the co-existence of the classical and basal-like cell states in a single tumour duct with functional diversification, i.e. proliferation and epithelial-to-mesenchymal transition respectively. Mutant KRASG12D detection ascertained an epithelial origin of vimentin-positive cells in the tumour. Uneven spatial distribution of cancer-associated fibroblasts could recreate similar intra-organoid diversification. This extensive heterogeneity with functional cooperation of plastic tumour cells poses extra challenges to therapeutic approaches. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Recombinase polymerase amplification in combination with electrochemical readout for sensitive and specific detection of PIK3CA point mutations

As part of the ongoing evolution towards personalized anticancer therapy, mutation screening is becoming increasingly important and, therefore, also alternative detection strategies that allow for fast genetic diagnostics at the point of care. In the case of breast cancer, detecting cancer-associated point mutations in the PIK3CA gene is of particular importance for treatment decisions. We developed a recombinase polymerase amplification assay combined with an enzyme-linked electrochemical assay on multi-channel screen-printed gold sensors for specific and highly sensitive detection of three PIK3CA point mutations (H1047R, E545K, and E542K). Recombinase polymerase amplification (RPA) of the target sequences was optimized and characterized with a real-time RPA assay. Comparison with real-time PCR reveals that RPA is slightly inferior in terms of efficiency and sensitivity. However, the desired target DNA is successfully amplified at initial concentrations down to 100 copies μL−1. For electrochemical readout, biotinylated dCTP is used to label the target DNA during RPA. Single-stranded target DNA is produced with either asymmetric RPA or symmetric RPA followed by lambda exonuclease digestion. Characterization of the two different approaches in terms of sensitivity results in comparable detection limits (229 copies μL−1 and 224 copies μL−1, respectively), though RPA followed by lambda exonuclease digestion yields significantly higher currents. Finally, this method, together with a designed wild-type blocking oligo that inhibits binding of the wild-type target DNA during probe-target hybridization, allows for detecting the PIK3CA point mutations H1047R, E545K, and E542K in the presence of wild-type target DNA when the proportion of mutant target DNA is >20%.

Comprehensive application of multiple molecular diagnostic techniques in pre-implantation genetic testing for monogenic.

Pre-implantation genetic testing for monogenic disorders (PGT-M) is an effective approach to reducing the incidence of birth defects by preventing the transmission of inherited diseases to offspring. However, there are still controversies regarding the detection methods and transplantation of embryos. This paper aims to evaluate the effectiveness of different detection technologies applied to PGT-M through a retrospective analysis of clinical detection data. The carrier status of pathogenic mutations and chromosomal copy number variants (CNVs) in 892 embryos was characterized using next-generation sequencing (NGS), single-nucleotide polymorphism (SNP) array, and PCR-based detection technologies. Clinical data from PGT-M cases were retrospectively analyzed to assess the effectiveness of these detection methods in identifying genetic abnormalities in embryos. A total of 829 embryos were analyzed, with 63 being unsuccessful. Our study revealed that the success rate of detecting deletional mutations using Gap-PCR 84.9%, which is lower than that of SNP array (98.7%) and NGS (92.5%). However, no significant difference was observed when detecting point mutations using any of the methods. These findings suggest that, when detecting deletional mutations, SNP array and NGS are more suitable choices compared to Gap-PCR. While SNP array may have a lower resolution and success rate (80.5%) in analyzing CNVs compared to NGS (95.5%), it may still be useful for revealing certain abnormal types. In conclusion, this study found that SNP analysis is advantageous for identifying polygenic and deletional mutations, whereas NGS is more cost-efficient for detecting common monogenic diseases. Additionally, SNP-based haplotyping and PCR-based direct detection of mutations can be used together to enhance the accuracy and success rates of PGT-M. Our findings offer valuable insights for PGT technicians in choosing suitable detection methods for patients.

Response to insecticides and underlying mechanisms of resistance in the field populations of Aedes aegypti Linnaeus (Diptera: Culicidae) in Puducherry, India.

Mosquito-borne diseases are major threats to human health worldwide. Successful control of vector mosquitoes requires periodic updates on their response to the insecticides that are in use. Different classes of neurotoxic insecticides have been used in vector control programs. Ae. aegypti and Ae. albopictus are the primary vectors of dengue and have developed resistance to organophosphates and synthetic pyrethroids that are used in vector control programs. Monitoring insecticide pressure and studying the underlying mechanisms of resistance in the field populations of Aedes aegypti are important to formulate resistant management strategies for their control programs. Aedes aegypti were collected from study sites Lawspet and Abishegapakkam and F1 progeny was subject to biochemical assays to determine the enzyme activity. Insecticide susceptibility tests were conducted to determine vector susceptibility/resistance to malathion and deltamethrin. Adult dried mosquitoes were subjected to multiplex PCR to detect point mutation in the VGSC gene. Insecticide susceptibility test results revealed that Aedes aegypti is resistant to malathion and incipient resistance to deltamethrin has emerged. It was observed that β-esterase and monoxygense activity were significantly higher in Lawspet sample than the laboratory strain, whereas it was comparatively lower in Abishegapakkam sample than laboratory strain. Multiplex PCR assays showed no kdr mutation in all Ae. aegypti strains. Monitoring insecticide resistance in Ae. aegypti would help the local health authorities to implement a rationalized approach for insecticide use in vector control.

Rapid Classification of Sarcomas Using Methylation Fingerprint: A Pilot Study.

Sarcoma classification is challenging and can lead to treatment delays. Previous studies used DNA aberrations and machine-learning classifiers based on methylation profiles for diagnosis. We aimed to classify sarcomas by analyzing methylation signatures obtained from low-coverage whole-genome sequencing, which also identifies copy-number alterations. DNA was extracted from 23 suspected sarcoma samples and sequenced on an Oxford Nanopore sequencer. The methylation-based classifier, applied in the nanoDx pipeline, was customized using a reference set based on processed Illumina-based methylation data. Classification analysis utilized the Random Forest algorithm and t-distributed stochastic neighbor embedding, while copy-number alterations were detected using a designated R package. Out of the 23 samples encompassing a restricted range of sarcoma types, 20 were successfully sequenced, but two did not contain tumor tissue, according to the pathologist. Among the 18 tumor samples, 14 were classified as reported in the pathology results. Four classifications were discordant with the pathological report, with one compatible and three showing discrepancies. Improving tissue handling, DNA extraction methods, and detecting point mutations and translocations could enhance accuracy. We envision that rapid, accurate, point-of-care sarcoma classification using nanopore sequencing could be achieved through additional validation in a diverse tumor cohort and the integration of methylation-based classification and other DNA aberrations.

Linked-read based analysis of the medulloblastoma genome.

Medulloblastoma is the most common type of malignant pediatric brain tumor with group 4 medulloblastomas (G4 MBs) accounting for 40% of cases. However, the molecular mechanisms that underlie this subgroup are still poorly understood. Point mutations are detected in a large number of genes at low incidence per gene while the detection of complex structural variants in recurrently affected genes typically requires the application of long-read technologies. Here, we applied linked-read sequencing, which combines the long-range genome information of long-read sequencing with the high base pair accuracy of short read sequencing and very low sample input requirements. We demonstrate the detection of complex structural variants and point mutations in these tumors, and, for the first time, the detection of extrachromosomal DNA (ecDNA) with linked-reads. We provide further evidence for the high heterogeneity of somatic mutations in G4 MBs and add new complex events associated with it. We detected several enhancer-hijacking events, an ecDNA containing the MYCN gene, and rare structural rearrangements, such a chromothripsis in a G4 medulloblastoma, chromoplexy involving 8 different chromosomes, a TERT gene rearrangement, and a PRDM6 duplication.

Commercial next generation sequencing in a real-world cohort of patients with metastatic breast cancer.

e13082 Background: Clinicians increasingly order next generation sequencing (NGS) of tissue or liquid (ctDNA) biopsies from patients with metastatic breast cancer. Although some genetic information is clinically actionable, some, particularly non-ERBB2 gene amplifications, is not. We reviewed NGS data from a real-world cohort of metastatic breast cancer patients to evaluate the prevalence of somatic abnormalities using commercial NGS technology. Methods: We evaluated Foundation One NGS data from 44 tissue and 17 liquid biopsies from patients seen in a medical oncology practice between 2016 and 2022. We used KM Plotter to measure overall survival in early-stage patients by relative gene expression. Results: 76% of patients had ductal and 24% had lobular carcinomas. 59% of patients had luminal (ER-positive) and 32% of patients had basal breast cancer (ER-negative). Only 9% of patients had HER2-positive disease. 48% of the tumors had a p53 abnormality, either a point (59%) or frameshift mutation (36%). 50% tumors had a somatic mutation in a tumor suppressor gene such as PTEN, RB or APC. One patient had a germline BRCA1 mutation and 5 patients had germline BRCA2 mutations. 39% of patients carried a somatic mutation in PIK3CA, predominantly H1047R (45%) in the kinase domain or E545X (20%) in the helicase domain. KM plotter showed worse OS in early-stage patients with low p53 gene expression or high PIK3CA expression. 10/11 patients with lobular carcinoma had somatic mutations in CDH1, 70% of which were frameshift mutations. 8/27 patients with luminal cancer had an ESR1 mutation, predominantly Y537 or D538. 9 patients, predominantly luminal, had amplifications of the 11q13 (CCND1). 7 patients had amplifications of 8q24 (MYC). 20q13 (AURKA) amplifications were only seen in luminal cancers. 12p13 (CCND2) amplifications were only seen in basal cancers. 27% of lobular cancers (vs. 6-17% of ductal cancers) demonstrated amplification of the 11q13, 8q24 and/or 8p11 amplicons. 20q13 and 12p13 amplifications were only seen in ductal cancers. Liquid biopsies were more likely to detect point mutations than tissue biopsies. p53 mutations were seen in 65% of liquid vs. 48% of tissue biopsies. 47% of liquid biopsies showed two p53 mutations. PIK3CA mutations were seen in 71% of liquid vs. 39% of tissue biopsies. 18% of liquid biopsies showed two PIK3CA mutations. 93% of basal cancers had at least one p53 mutation. 44% of luminal cancers had a PIK3CA mutation. ESR1, GATA3 and AKT1 mutations were only seen in luminal cancers. While ESR1 mutations were seen in ductal and lobular cancers, GATA3 and AKT1 mutations were only seen in ductal cancers. Conclusions: Commercial NGS testing of a real-world cohort yields actionable mutations that can impact clinical management and outcome of metastatic breast cancer patients. NGS testing demonstrates the range of gene amplifications and corresponds closely with what has been reported in the published literature.

The utilisation of digital droplet PCR to enhance the diagnosis of bladder and pancreaticobiliary tumours in cytology specimens.

Digital droplet PCR (ddPCR) is a relatively new technique used to detect molecular alterations with unprecedented precision and accuracy. It is particularly useful for detecting point mutations and copy number variation (CNV) in samples with small amounts of target DNA. This proof of principle study was conducted to see if ddPCR technology could be applied to cytology specimens to detect molecular alterations which may influence diagnostic decision making. A range of cytological specimens derived from bladder or pancreaticobiliary origin, with varying diagnoses but with an emphasis on abnormality, underwent ddPCR testing. DNA was manually extracted from Thinprep vials, cell blocks or direct fine needle aspiration smears. ddPCR was performed using two somatic point mutations TP53 R248W and TP53 R273H assays for both urine and pancreaticobiliary cytology specimens. Three CNV assays; CDKN2A, E2F3 and YWHAZ were applied to urine samples. SMAD4 and CDKN2A CNVs were applied to the pancreaticobiliary samples. 16 of 21 urine specimens showed molecular alterations using ddPCR testing. 12 of those 16 cases were associated with malignant outcomes. The pancreaticobiliary specimens showed 14 of 37 specimens with molecular alterations, all of which were associated with carcinoma. We demonstrated an increasing percentage of molecular aberrations associated with increasing severity of cytological results. Our results have shown that ddPCR can identify both mutations and CNVs in urine and pancreaticobiliary cytology derived samples. Being able to detect these molecular alterations may reduce the number of equivocal results leading to more timely and informed patient management decisions.

Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis.

The World Health Organization (WHO) Global tuberculosis Report 2021 stated that rifampicin-resistant tuberculosis (RR-TB) remains a major public health threat. However, the in-practice diagnostic techniques for RR-TB have a variety of limitations including longer time, lack of sensitivity, and undetectable low proportion of heterogeneous drug resistance. Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison. The sensitivity of the MLP-RAP assay could reach 5 copies/μl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/μl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%. MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.

Detection of Cytb Point Mutation (G143A) that Confers High-Level Resistance to Pyraclostrobin in Glomerella cingulata Using LAMP Method.

Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emerging disease that results in severe defoliation and fruit spots in apples. In China, the compound of pyraclostrobin and tebuconazole was registered to control GLS in 2018 and has achieved excellent control efficiency. In this study, we showed that the high-level resistant isolates of G. cingulata to pyraclostrobin, caused by the point mutation at codon 143 (GGT→GCT, G143A) in the cytochrome b gene, has appeared in apple orchards in Shandong Province in 2020, and the resistance frequency was 4.8%. Based on the genotype of the resistant isolates, we developed a loop-mediated isothermal amplification (LAMP) assay for detection of the pyraclostrobin resistance. The LAMP assay was demonstrated to have good specificity, sensitivity, and repeatability, and it exhibited high accuracy in detecting pyraclostrobin resistance in the field. This study reported the resistance status of GLS to pyraclostrobin in Shandong Province and developed a molecular tool for the detection of pyraclostrobin resistance, which is of practical significance for the scientific control of GLS.

A molecular beacon-assisted nanotheranostic agent for real-time detection and effective combined management of acquired resistance to EGFR-TKIs

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is inevitably developed in non-small cell lung cancer (NSCLC) and the major cause of resistance to first-generation EGFR-TKIs is the T790M secondary mutation. Real-time detection of the acquired T790M mutation to avoid futile treatment and timely adjust the therapeutic interventions remains a great challenge. Molecular beacon (MB) could achieve real-time detection of point mutation and the fluorophores located at the terminal of MB could be activated for photodynamic therapy (PDT). Herein, a multifunctional nanoplatform CQEM based on erlotinib and tertiary amine dual-fabricated chitosan (CQE) loaded with cyanine dyes-labeled MB was prepared for simultaneous detection of the degree of T790M mutation in real-time and reversal of the resistance by PDT. CQE could protect MB against DNase I digestion and CQEM could achieve real-time sensing of T790M mutation during the course of EGFR-TKI treatments. With 808 nm laser irradiation, CQEM showed substantial therapeutic efficiency and could overcome the acquired resistance by the synergism of targeted molecular therapy and dual-cyanine dyes-mediated PDT. Thus, the CQEM nanoplatform could be an auspicious tool for individualized accurate therapy of NSCLC by real-time detection and management of the efficacy of EGFR-TKIs.

Analysis of cell-free DNA concentration, fragmentation patterns and TP53 gene expression in mammary tumor-bearing dogs: A pilot study.

Liquid biopsy based on the analysis of circulating cell-free DNA (cfDNA), as well as on detection of point mutations by digital droplet PCR (ddPCR), has revolutionized the research in oncology. In recent years, this technique has been pioneering in veterinary medicine since it is a minimally invasive approach with very promising results for characterization of tumors. The aim of this study was, firstly, to analyze the concentration and the fragmentation pattern of cfDNA of dogs with mammary tumors (n = 36) and healthy dogs (n = 5) and its correlation with clinicopathological data. Secondly, analysis of TP53 gene expression and the point mutation in the codon 245 were performed in cfDNA and in tumor tissues to assess their potential as plasma biomarkers. Our results highlighted that those dogs with worse clinicopathological characteristics (simple or undifferentiated carcinomas, higher histological grade and presence of peritumoral inflammation) shown higher cfDNA concentration and higher concentrations of short-fragments (<190 bp) than healthy dogs. In addition, although no detection of the point mutation in codon 245 of TP53 gene could be detected neither in plasma nor tumor tissue, an increased TP53 expression was detected in animals with tumors bearing malignant characteristics. Finally, a high concordance with TP53 gene expression in plasma and tumor tissue and cfDNA concentration was also found. The results derived from this work confirm the valuable potential of cfDNA and its fragments, as well as the analysis of TP53 expression in plasma as useful liquid biomarkers for clinical application in veterinary oncology.

EtcPRSMut as a molecular marker of halofuginone resistance in Eimeria tenella and Toxoplasma gondii

The control of coccidiosis, causing huge economic losses in the poultry industry, is facing the stagnation of the development of new drugs and the emergence of drug resistance. Thus, the priority for coccidiosis control is to decipher the effect mechanisms and resistance mechanisms of anticoccidial drugs. In this study, we mined and validated a molecular marker for halofuginone resistance in Eimeria tenella through forward and reverse genetic approaches. We screened whole-genome sequencing data and detected point mutations in the ETH2_1020900 gene (encoding prolyl-tRNA synthetase, PRS). Then, we introduced this mutated gene into E.tenella and Toxoplasma gondii and validated that overexpression of this mutated gene confers resistance to halofuginone invivo and invitro. These results together show that mutations A1852G and A1854G on the ETH2_1020900 gene are pivotal to halofuginone resistance in E.tenella, encouraging the exploration of mechanisms of drug resistance against other anticoccidial drugs in eimerian parasites.

Non-invasive Monitoring of Soft-tissue Sarcoma Response to Neoadjuvant Radiotherapy by Quantification of Circulating Tumor DNA (ctDNA) and Multiparametric MRI (mpMRI)

Introduction: Soft-tissue sarcomas (STS) are a heterogenous group of rare, yet malignant tumors. Wide resection remains the cornerstone of treatment for localized STS. Current S3 guidelines recommend neoadjuvant radiotherapy for primary, high-grade tumors; however, therapy response for different histological subtypes and gradings has not been systematically investigated. This ongoing prospective study evaluates the response of various STS during the course of neoadjuvant radiotherapy using innovative, non-invasive methods. Through quantification of ctDNA, tumor activity and thus therapy response will be evaluated in correlation with imaging data. Materials and Methods: Liquid biopsies: Detection of circulating tumor DNA (ctDNA) in patient blood samples through targeted next-generation sequencing (NGS). Patient-specific exome sequencing allows the detection of individual structural variants (SVs) and point mutations. Circulating free DNA (cfDNA) is isolated from peritherapeutically collected patient plasma samples and ctDNA quantified therein. Bioinformatic analysis is performed using the MIRACUM Pipeline. Multiparametric MRI (mpMRI): Combination of conventional MRI sequences with diffusion-weighted imaging (DWI) and dynamic contrast enhancement (DCE). Multiparametric MRI is performed before, during, and after neoadjuvant radiotherapy. Accurate co-registration and correlation of mpMRI with the macroscopic, histologic, and immunohistochemical resected specimen allow an analysis of morphological changes during therapy. Results: Quantification of ctDNA mirrors the clinical course and tumor activity. Furthermore, we present imaging markers that allow the characterization of tumor masses for heterogenous STS and the evaluation of therapy response. Conclusion: Liquid biopsies can be used to detect the primary tumor and metastases, and to monitor the course of therapy. Quantification of ctDNA in combination with tumor mass characterization through co-registration of mpMRI and histopathology enables the evaluation of STS response to neoadjuvant radiotherapy. The participation and support of a wider group of oncologic surgeons are needed to validate these findings on a larger patient cohort to create future tumor- and patient-specific therapy concepts.

MLST genotypes and quinolone resistance profiles of Campylobacter jejuni isolates from various sources in Turkey

This study was conducted to determine the overall genetic diversity, as well as prevalence and mechanisms of resistance to quinolone antibiotics of 178 Campylobacter jejuni isolated from humans, cattle, dogs, and chickens in Turkey. Multilocus sequence typing (MLST) and E-test were performed for genotyping and antimicrobial susceptibility testing, respectively. Mismatch Amplification Mutation Assay, Polymerase Chain Reaction (MAMA-PCR) was used to detect point mutations associated with quinolone resistance. Of the 178 isolates tested, 151 were included in 21 clonal complexes (CCs); the remaining 27 isolates did not belong to any existing CCs. CC21, CC353, CC206, and CC257 were the predominant clones, representing 38 % of all C. jejuni isolates tested. The isolates were assigned to 78 different sequence types (STs), three of which were novel (ST 8082, ST 8083, and ST 8084). Resistance to quinolones was found in 73 (41 %) of the isolates (42.85 %, 2.85 %, 20.58 %, and 43.75 % in human, cattle, dog, and chicken isolates, respectively). All of the resistant isolates had Thr-86-Ile mutation in the gyrA gene. The highest Sorensen coefficient index was detected for human/chicken meat and human/dog C. jejuni isolates (Ss = 0.71), suggesting a strong link between the isolates from respective sources. The Simpson diversity index of C. jejuni isolates analyzed was detected between 0.92 and 0.98.The study provides detailed information on the quinolone resistance and MLST-based genetic relatedness of C. jejuni isolates from humans, cattle, dog, and broiler meat in Turkey for the first time, enabling a better understanding of the transmission pathways of C. jejuni in this country. Our results suggest that broiler meat and dogs may be the most important sources of human campylobacteriosis in Turkey.

Extracellular Vesicles' Genetic Cargo as Noninvasive Biomarkers in Cancer: A Pilot Study Using ExoGAG Technology.

The two most developed biomarkers in liquid biopsy (LB)-circulating tumor cells and circulating tumor DNA-have been joined by the analysis of extracellular vesicles (EVs). EVs are lipid-bilayer enclosed structures released by all cell types containing a variety of molecules, including DNA, mRNA and miRNA. However, fast, efficient and a high degree of purity isolation technologies are necessary for their clinical routine implementation. In this work, the use of ExoGAG, a new easy-to-use EV isolation technology, was validated for the isolation of EVs from plasma and urine samples. After demonstrating its efficiency, an analysis of the genetic material contained in the EVs was carried out. Firstly, the sensitivity of the detection of point mutations in DNA from plasma EVs isolated by ExoGAG was analyzed. Then, a pilot study of mRNA expression using the nCounter NanoString platform in EV-mRNA from a healthy donor, a benign prostate hyperplasia patient and metastatic prostate cancer patient plasma and urine samples was performed, identifying the prostate cancer pathway as one of the main ones. This work provides evidence for the value of using ExoGAG for the isolation of EVs from plasma and urine samples, enabling downstream applications of the analysis of their genetic cargo.

Comprehensive ctDNA Measurements Improve Prediction of Clinical Outcomes and Enable Dynamic Tracking of Disease Progression in Advanced Pancreatic Cancer.

Circulating tumor DNA (ctDNA) has emerged as a promising tumor-specific biomarker in pancreatic cancer, but current evidence of the clinical potential of ctDNA is limited. In this study, we used comprehensive detection methodology to explore the utility of longitudinal ctDNA measurements in patients with advanced pancreatic cancer. A targeted eight-gene next-generation sequencing panel was used to detect point mutations and copy-number aberrations (CNA) in ctDNA from 324 pre-treatment and longitudinal plasma samples obtained from 56 patients with advanced pancreatic cancer. The benefit of ctDNA measurements to predict clinical outcome and track disease progression was assessed. We detected ctDNA in 35/56 (63%) patients at baseline and found that it was an independent predictor of shorter progression-free survival (PFS) and overall survival (OS). After initiation of treatment, ctDNA levels decreased significantly before significantly increasing by the time of progression. In some patients, ctDNA persistence was observed after the first chemotherapy cycles, and it was associated with rapid disease progression and shorter OS. Longitudinal monitoring of ctDNA levels in 27 patients for whom multiple samples were available detected progression in 19 (70%) patients. The median lead time of ctDNA measurements on radiologically determined progression/time of death was 19 days (P = 0.002), compared with 6 days (P = 0.007) using carbohydrate antigen 19-9. ctDNA is an independent prognostic marker that can be used to detect treatment failure and disease progression in patients with advanced pancreatic cancer.

Genetic analyses and detection of point mutations in the acetylcholinesterase-1 gene associated with organophosphate insecticide resistance in fall armyworm (Spodoptera frugiperda) populations from Uganda

BackgroundThe fall armyworm (FAW), Spodoptera frugiperda; J.E. Smith (Lepidoptera: Noctuidae), is now an economically important pest that causes huge losses to maize productivity in sub-Saharan Africa. Variations in sub-population genetics and the processes of rapid adaptation underpinning the invasion remain unclear. For this, the genetic identity and diversity of FAW populations in Uganda were revealed by sequencing 87 samples (collected across the country). Based on the partial mitochondrial cytochrome oxidase I (COI) gene polymorphisms, we further examined the mitochondrial haplotype configuration and compared the FAW in Uganda with sequences from other parts of the world. The molecular target for organophosphate and carbamate resistance, acetylcholinesterase, was also investigated.ResultsAnalysis of the partial COI gene sequences showed the presence of both rice (predominant) and corn strain haplotypes, with a haplotype diversity of 0.382. Based on the COI marker, pairwise difference distribution analyses, and neutrality tests, showed that the FAW populations in Uganda and the rest of Africa are evolving neutrally, but those in America and Asia are undergoing expansion. Our findings support observations that invasive FAW populations throughout the rest of Africa and Asia share a common origin. Sequencing of the S. frugiperda ace-1 gene revealed four amino acid substitutions, two of which (A201S and F290V) were previously shown to confer organophosphate resistance in both S. frugiperda and several other insect species. The other two previously reported new variations in positions g-396 and g-768, are presumed to be related to the development of insecticide resistance.ConclusionsThis research has increased our knowledge of the genetics of FAW in Uganda, which is critical for pest surveillance and the detection of resistance. However, due to the low gene polymorphism of COI, more evolutionary studies incorporating the Spodoptera frugiperda whole-genome sequence are required to precisely understand the FAW population dynamics, introduction paths, origin, and subsequent spread.

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3'-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

An innovative single-base extension method for synchronous detection of point mutations and MSI status in colorectal cancer.

An accurate genotyping analysis is one of the critical prerequisites for patients with colorectal cancer receiving matched therapies. Conventional genotyping analysis is currently used to detect either gene mutations or MSI status, delaying the detection of critical tumor biomarkers and thus the optimal time for treatment. An assay that analyzes both biomarkers in a streamlined process is eagerly needed. We developed an assay combining Multiplex PCR Amplification, Single-base Extension and capillary electrophoresis (CE) analysis (MASE-CE) for synchronous detection of KRAS/NRAS/BRAF mutations and MSI status. In a 190 colorectal cancer cohort, we identified seven somatic mutations in KRAS, NRAS and BRAF as well as five MSI loci (D2S123/D5S346/D17S250/BAT-25/BAT-26) simultaneously. KRAS/NRAS/BRAF mutations were detected by NGS and MASE-CE, and MSI status were detected by PCR-CE and MASE-CE methods. The MASE-CE method showed high consistency with NGS for mutation detection (Kappa value ≥0.8) and PCR-CE (Kappa value=0.79). In addition, the limits of detection (LOD) of MASE-CE assay for MSI and somatic mutation were 5% and 2%, respectively. In somatic mutation detection and MSI detection, the LOD of MASE-CE assay was superior to that of qPCR and NGS. MASE-CE assay is a highly sensitive, time-saving and specimen-saving method, which can greatly avoid the cumbersome testing process and provide clinical decision for doctors in time.