Mutant Allele Frequency Research Articles

The Profiling of Circulating Tumor DNA in Pediatric Mature B-Cell Non-Hodgkin Lymphoma（B-NHL）: A Multicenter and Prospective Clinical Study

BackgroundNext-generation sequencing (NGS) based on liquid biopsy has been an emerging technology to identify tumor-specific genetic aberrations in adult lymphoma. However, there were few studies on the genomic profiling of plasma circulating tumor-derived DNA (ctDNA) in pediatric mature B-NHL.MethodsParaffin-embedded tissue (FFPE) and plasma samples from the newly diagnosed patients were collected and sequenced by 475 genes panel before, during and post of treatment. Clinical stage system, risk stratification and treatment of pediatric mature B-NHL followed a the modified BFM-95 protocol.ResultsA total of 53 pediatric mature B-NHLs were enrolled，including 35 Burkitt lymphoma, 18 diffused large B cell lymphoma/high-grade B cell lymphoma (DLBCL/HGBCL). We collected 38 tissue and 124 plasma samples for somatic mutation testing. The number of somatic mutations and the TOP 5 genes detected in the 38 tissues and 31 baseline plasma samples, were 416 vs 496, and MYC(71%), DDX3X(45%)，ID3 (42%), TP53(40%), SMARCA4(29%) (Fig1. A)vs MYC(52%), DDX3X(45%)，ID3(42%), TP53(36%), GNA13(23%) (Fig1. B), respectively. The median allele frequency of mutations in plasma was 3%(ranged from 0.2% to 96.6%) and MYC, DDX3X, ID3, TP53, SMARCA4,ARID1A shows higher max somatic allele frequency (MSAF), indicated that was the early events in tumor genesis.The sensitivity of plasma ctDNA to detecting tissue mutations was 63.4% in the 19 matched samples (11 samples from BL and 8 samples from DLBCL/HGBCL) and the sensitivity in BL and DLBCL/HGBCL were 64.1% and 62%, respectively. All genomic alteration types, including single nucleotide variants (SNVs), indels, and gene fusions were detected in similar proportions in each sample type and the gene mutation rate of every gene detected in paired tissue and ctDNA samples.Among the 37 mature B-NHL patients, 6 patients were collected plasma samples after resection of tumor, of which 4 patients was not detected the somatic gene mutations in plasma (Fig. 2). The abundance of ctDNA in patients with stage IV (N=10) was significantly higher than that of stage I-II (N=6) (P=0.0002) and stage III patients(N=15)(P<0.0001)(Fig3. A). Similarly the abundance of ctDNA mutations in high risk patients(N =11)was significantly higher than that of low risk patients (N=8)(P<0.0001) and Medium risk patients (N=12) (P<0.0001) (Fig3. B). MSAF of ctDNA mutations was significantly correlated with LDH and the abundance of ctDNA mutations (P<0.0001) (Fig3. C,D).With a median follow-up of 182 Days, 33 patients have completed anti-tumor treatment, 27 patients completed post-treatment PET-CT, and 20 patients have done ctDNA testing synchronously. PET-CT showed tumor residue in 4 patients, of which 2 patients showed no tumor residue in pathology and ctDNA, 1 patient showed tumor residue in pathology but not in ctDNA and with tumor progression 6 months after treatment, 1 patient unable to take biopsy showed no tumor residue in ctDNA and was no tumor reccourence with regular follow-up. At the last follow-up, 1 patient was disease progression, and all of the 53 patients survived.ConclusionPlasma ctDNA testing by NGS was practicable in pediatric mature B-NHL. The abundance of ctDNA is significantly related to tumor burden. CtDNA testing may be more sensitive than PET-CT for residual disease assessment. Nevertheless, sample size expansion is required to verify such conclusions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Frequent PIK3CA mutations in eutopic endometrium of patients with ovarian clear cell carcinoma

Recent studies have reported cancer-associated mutations in normal endometrium. Mutations in eutopic endometrium may lead to endometriosis and endometriosis-associated ovarian cancer. We investigated PIK3CA mutations (PIK3CAm) for three hotspots (E542K, E545K, H1047R) in eutopic endometrium in patients with ovarian cancer and endometriosis from formalin-fixed paraffin-embedded specimens by laser-capture microdissection and droplet digital PCR. The presence of PIK3CAm in eutopic endometrial glands with mutant allele frequency ≥ 15% were as follows: ovarian clear cell carcinoma (OCCC) with PIK3CAm in tumors, 20/300 hotspots in 11/14 cases; OCCC without PIK3CAm, 42/78 hotspots in 11/12 cases; high-grade serous ovarian carcinoma, 8/45 hotspots in 3/5 cases; and endometriotic cysts, 5/63 hotspots in 5/6 cases. These rates were more frequent than in noncancer nonendometriosis controls (7/309 hotspots in 5/17 cases). In OCCC without PIK3CAm, 7/12 (58%) cases showed multiple hotspot mutations in the same eutopic endometrial glands. In 3/54 (5.6%) cases, PIK3CAm was found in eutopic endometrial stroma. Multisampling of the OCCC tumors with PIK3CAm showed intratumor heterogeneity in three of eight cases. In two cases, PIK3CAm was detected in the stromal component of the tumor. Homogenous PIK3CAm in the epithelial component of the tumor matched the mutation in eutopic endometrial glands in only one case. Eutopic endometrial glands in ovarian cancer and endometriosis show high frequency of PIK3CAm that is not consistent with tumors, and multiple hotspot mutations are often found in the same glands. While the mutations identified in eutopic endometrium may not be driver mutations in the patient's cancer, these are still driver mutations but this specific clone has not undergone the requisite steps for the development of cancer.

Association Between PON1 (L55M and Q192R) Genetic Polymorphism and Recurrent Pregnancy Loss in North Indian Women Exposed to Pesticides.

The aim of the present study was to examine the relation between the PON1 polymorphisms and recurrent pregnancy loss (RPL). In a cross-sectional study, blood samples were collected from 100 females. DNA was extracted and PON1 genotypes were determined by polymerase chain reaction (PCR) amplification. Regarding PON1 L55M, the mutated allele (M) frequency was found in 70.5% in RPL and in 53.5% in controls; the M allele was significantly associated with an increased risk of RPL (adjusted odds ratio [ORadj] = 2.07; 95% confidence interval [CI]; p < 0.001). However, regarding PON1 Q192R, the R mutated allele frequency was found in 28.5% in RPL and in 33% in controls. The R allele did not show any risk for RPL (ORadj 0.81; 95%CI; p = 0.329). The present study suggests that there is an effect of genetic polymorphism on RPL and provides additional evidence that combines with the growing information about the ways in which certain PON1 genotypes can affect the development of the fetus in the uterus.

KRAS A146 Mutations Are Associated With Distinct Clinical Behavior in Patients With Colorectal Liver Metastases.

PURPOSESomatic KRAS mutations occur in approximately half of the patients with metastatic colorectal cancer (mCRC). Biologic tumor characteristics differ on the basis of the KRAS mutation variant. KRAS mutations are known to influence patient prognosis and are used as predictive biomarker for treatment decisions. This study examined clinical features of patients with mCRC with a somatic mutation in KRAS G12, G13, Q61, K117, or A146.METHODSA total of 419 patients with colorectal cancer with initially unresectable liver-limited metastases, who participated in a multicenter prospective trial, were evaluated for tumor tissue KRAS mutation status. For the subgroup of patients who carried a KRAS mutation and were treated with bevacizumab and doublet or triplet chemotherapy (N = 156), pretreatment circulating tumor DNA levels were analyzed, and total tumor volume (TTV) was quantified on the pretreatment computed tomography images.RESULTSMost patients carried a KRAS G12 mutation (N = 112), followed by mutations in G13 (N = 15), A146 (N = 12), Q61 (N = 9), and K117 (N = 5). High plasma circulating tumor DNA levels were observed for patients carrying a KRAS A146 mutation versus those with a KRAS G12 mutation, with median mutant allele frequencies of 48% versus 19%, respectively. Radiologic TTV revealed this difference to be associated with a higher tumor load in patients harboring a KRAS A146 mutation (median TTV 672 cm3 [A146] v 74 cm3 [G12], P = .036). Moreover, KRAS A146 mutation carriers showed inferior overall survival compared with patients with mutations in KRAS G12 (median 10.7 v 26.4 months; hazard ratio = 2.5; P = .003).CONCLUSIONPatients with mCRC with a KRAS A146 mutation represent a distinct molecular subgroup of patients with higher tumor burden and worse clinical outcomes, who might benefit from more intensive treatments. These results highlight the importance of testing colorectal cancer for all KRAS mutations in routine clinical care.

Prevalence of the MDR1 gene mutation in herding dog breeds and Thai Ridgebacks in Thailand.

Background and Aim:A canine multi-drug resistance 1 (MDR1) nt230(del4) is a well-known inherited disorder that primarily affects collies and various herding breeds. The most recognized clinical implication for affected dogs is associated with an increased risk of multiple drug toxicity. To date, MDR1 gene mutations have been identified globally, especially in dogs from the USA and European countries. This study aimed to investigate the prevalence of MDR1 nt230(del4) in herding dog breeds and Thai Ridgebacks in Thailand.Materials and Methods:We clarified the prevalence of MDR1 nt230(del4) in 263 dogs of eight purebred dog breeds in Thailand using an allele-specific multiplex polymerase chain reaction method and direct DNA sequencing.Results:Rough Collies, Australian Shepherds, Shetland Sheepdogs, and Old English Sheepdogs were affected by the mutation with mutant allelic frequencies of 57.14%, 12.82%, 11.28%, and 8.33%, respectively. Among these populations, the prevalence of the MDR1 (+/–) genotype was 57.14% (12/21) for Rough Collies, 25.64% (10/39) for Australian Shepherds, 16.13% (15/93) for Shetland Sheepdogs, and 16.67% (2/12) for Old English Sheepdogs, whereas the MDR1 (–/–) mutation was only identified in Rough Collies and Shetland Sheepdogs, with prevalences of 28.57% (6/21) and 3.22% (3/93), respectively. However, the MDR1 nt230(del4) was not identified in Border Collies, German Shepherds, White Swiss Shepherds, or Thai Ridgebacks.Conclusion:This study provides the current situation regarding MDR1 nt230(del4) in herding dog breeds in Thailand. In this survey, we investigated for the first time the status of MDR1 genotype in Thai Ridgebacks. These results are helpful for veterinarians managing effective therapeutic plans for commonly affected dog breeds, and these results will encourage all breeders to improve their selective breeding programs based on the MDR1 nt230(del4) status.

The Effect of the Vitamin D Receptor on Bone Metabolism Disorders in Premature Infants Born Using In Vitro Fertilization

Aim. To study the effect of the vitamin D receptor on bone metabolism disorders in premature infants born using in vitro fertilization (IVF). Material and methods. A study was conducted on 189 premature infants, divided into 2 groups. The first group is premature babies born using the IVF method. The second group – children were born naturally. The VDR receptor gene was analyzed depending on the observation groups. Results. It was found that in children born with IVF, the signs of osteopenia were more pronounced. Genetic studies have shown that the frequency of mutant allele *B and homozygous mutant genotype B/B of the VDR gene is more common in premature babies born with IVF. Mutant homozygous genotype B/B occurs in the main group of children with a frequency of 35%, which is significantly higher than in the control group of children (17%). Changes in bone metabolism are one of the important causes of musculoskeletal system disorders, which is closely associated with the molecular genetic polymorphism of the VDR gene. Conclusion. The studies conducted led to the following conclusion: the frequency of the mutant allele *B and homozygous for the mutant allele genotype B/B, occurs more often in premature babies born using IVF. The frequency of the *B allele in the group of premature babies born using IVF is 50%, which is higher than in the group of premature babies born naturally (40%). However, the identified genetic differences are not associated with the IVF procedure. These are combinations of gene alleles inherited from the parents. Knowledge of hereditary risk factors for low vitamin D3 levels can be of great practical importance for predicting bone metabolism disorders in premature infants.

TP53 mutation in newly diagnosed acute myeloid leukemia and myelodysplastic syndrome

ObjectivesTP53 mutation is found frequently in therapy related acute myeloid leukemia (AML)/ myelodysplastic syndrome (MDS), AML and MDS patients with monosomy or complex karyotype. However, the prevalence and treatment outcome in TP53 mutated AML/MDS patients in Asian population are scarce. We therefore conducted this study to analyze the prevalence and the treatment outcomes of TP53 mutation in AML and MDS-EB patients.MethodsPatients with newly diagnosed AML and MDS-EB were recruited, extraction of deoxyribonucleic acid from bone marrow samples were done and then performing TP53 mutation analysis, using MassArray® System (Agena Bioscience, CA, USA).ResultsA total of 132 AML/MDS patients were recruited, patients with de novo AML, secondary AML, MDS-EB1, MDS-EB2 and T-AML/MDS were seen in 66, 13, 9, 9 and 3%, respectively. TP53 mutation was found in 14 patients (10.6%), and prevalence of TP53 mutation in T-AML/MDS, secondary AML, de novo AML and MDS-EB patients were 50, 17.6, 9.2 and 8%, respectively. Three patients had double heterozygous TP53 mutation. Mutated TP53 was significantly detected in patients with monosomy and complex chromosome. Common TP53 mutation were R290C, T220C, A249S and V31I which V31I mutation was reported only in Taiwanese patients. Most variant allele frequency (VAF) of TP53 mutation in the study were greater than 40%. Three year-overall survival (OS) in the whole population was 22%, 3y-OS in AML and MDS-EB patients were 22 and 27%, respectively. The 1y-OS in patients with TP53-mutant AML/MDS were shorter than that in TP53 wild-type patients, 14% versus 50%, P = 0.001. In multivariate analysis, factors affecting OS in 132 AML/MDS patients was mutant TP53 (P = 0.023, HR = 1.20–7.02), whereas, WBC count> 100,000/μL (P = 0.004, HR = 1.32–4.16) and complex karyotype (P = 0.038, HR = 1.07–9.78) were associated with shorter OS in AML patients.DiscussionIn this study, the prevalence of TP53 mutation in de novo AML and MDS-EB patients were low but it had impact on survival. Patients with monosomy or complex karyotype had more frequent TP53 mutation.

Genome Sequencing of SARS-CoV-2 Allows Monitoring of Variants of Concern through Wastewater

Monitoring SARS-CoV-2 in wastewater has shown to be an effective tool for epidemiological surveillance. More specifically, RNA levels determined with RT-qPCR have been shown to track with the infection dynamics within the population. However, the surveillance of individual lineages circulating in the population based on genomic sequencing of wastewater samples is challenging, as the genetic material constitutes a mixture of different viral haplotypes. Here, we identify specific signature mutations from individual SARS-CoV-2 lineages in wastewater samples to estimate lineages circulating in Luxembourg. We compare circulating lineages and mutations to those detected in clinical samples amongst infected individuals. We show that especially for dominant lineages, the allele frequencies of signature mutations correspond to the occurrence of particular lineages in the population. In addition, we provide evidence that regional clusters can also be discerned. We focused on the time period between November 2020 and March 2021 in which several variants of concern emerged and specifically traced the lineage B.1.1.7, which became dominant in Luxembourg during that time. During the subsequent time points, we were able to reconstruct short haplotypes, highlighting the co-occurrence of several signature mutations. Our results highlight the potential of genomic surveillance in wastewater samples based on amplicon short-read data. By extension, our work provides the basis for the early detection of novel SARS-CoV-2 variants.

Multiplex Epstein-Barr Virus Genotyping PCR Detects High-Risk Variants in Plasma for Population-Level Nasopharyngeal Carcinoma Screening

<h3>Purpose/Objective(s)</h3> Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is unusually geographically and ethnically restricted. Without screening, most patients present at an advanced stage. Population-level screening in high-risk populations can detect most cases at an early stage, but existing serologic or molecular diagnostics are limited by low positive predictive value secondary to benign EBV reactivation. In contrast to human genome-wide association studies, recent EBV genome-wide association studies have identified polymorphisms with much greater attributable risk for NPC. We hypothesized that a noninvasive molecular diagnostic could detect high-risk EBV haplotypes in plasma to triage patients who screen positive for plasma EBV DNA. <h3>Materials/Methods</h3> We designed a multiplex allele-specific real-time polymerase chain reaction (qPCR) genotyping assay to detect three non-synonymous polymorphisms within the EBV BALF2 gene (162215C > A, 162476T > C, 163364C > T). Two dsDNA gene fragments served as either the high-risk or low-risk variant controls. Supernatant from the EBV-infected B95-8 cell line served as an additional wild-type whole-virus control. Positive clinical specimens sent for routine plasma EBV EBNA-1 testing were collected from patients with a variety of benign and neoplastic EBV-associated disorders. Three sets of conserved primers flanked the polymorphisms, with one allele-specific hydrolysis probe for each of the three high-risk alleles. A fourth allele-specific probe (162215-mt) served as an internal control. The 95% lower limit of detection (LLOD) was assessed in replicates of 20; linearity was assessed from 0.0-6.0 log10 cp/µL. Mutant allele frequencies from 0-100% were assessed by mixing dsDNA controls. <h3>Results</h3> The assay's limit of quantitation and 95% LLOD were 50 and 5 copies per well, respectively. At 100 copies per well, the assay could detect mutant allele frequencies as low as 5%, below the intra-host heterozygosity threshold. Wild-type B95-8 DNA confirmed assay specificity. Forty EBV-positive plasma specimens were genotyped from patients with non-transplant-associated viremia, post-transplant lymphoproliferative disorders, EBV-associated lymphomas (diffuse large-cell, Hodgkin, NK/T-cell, peripheral T-cell), and EBV-positive NPC. High-risk haplotypes were more common among patients with NPC (64% vs. 10%, <i>P</i> = 0.002). Compared to screening with plasma BamHI-W PCR alone, triaging screen-detected EBV DNA with this genotyping assay modestly reduced screening sensitivity (97% to 91%) but could decrease false positives and unnecessary nasoendoscopies by 63% in a modeled high-risk population. <h3>Conclusion</h3> Triaging screen-detected plasma EBV DNA with a multiplex BALF2 genotyping assay has the potential to decrease false positives and resource utilization in high-risk populations screened for NPC. This may be a low-cost and accessible alternative to higher-complexity sequencing assays in low- and middle-income countries.

KRAS Mutation Allele Frequency Impacts Prognosis in Pancreatic Ductal Adenocarcinoma Using Next Generation Sequencing

Nauheim, David O. BA; Moskal, David BS; Renslo, Bryan BS; Jiang, Wei MD, PhD; Yeo, Charles J. MD, FACS; Nevler, Avinoam MD; Bowne, Wilbur B. MD, FACS; Lavu, Harish MD, FACS Author Information

BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics.

Simple SummaryThe mutational load of BRAFV600E in melanomas has been described as a possible prognostic biomarker but there is no information about the mutant allele frequency (MAF) variability of BRAFV600E within cutaneous melanomas and its potential prognostic implications. Our study suggests that the variation degree of BRAFV600E MAF within primary cutaneous melanoma could act as a determinant in the location of primary melanomas as well as their first metastases and could influence prognostic indicators such as Breslow and mitotic indexes. BRAFV600E MAF variation is also related to the neoplastic cell phenotype and tumor lymphocytic infiltrate of primary tumors. For all these reasons, detection of BRAFV600E MAF variation could be a useful prognostic biomarker. It is worth exploring the role of BRAFV600E MAF variation with regards to the response of melanoma to targeted therapies.Background: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. Methods: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. Results: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. Conclusions: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.

Serial circulating tumor DNA to predict early recurrence in patients with hepatocellular carcinoma: a prospective study.

We studied the value of circulating tumor DNA (ctDNA) in predicting early postoperative tumor recurrence and monitoring tumor burden in patients with hepatocellular carcinoma (HCC). Plasma‐free DNA, germline DNA, and tissue DNA were isolated from 41 patients with HCC. Serial ctDNAs were analyzed by next‐generation sequencing before and after operation. Whole‐exome sequencing was used to detect the DNA of HCC and adjacent tissues. In total, 47 gene mutations were identified in the ctDNA of the 41 patients analyzed before surgery. ctDNA was detected in 63.4% and 46% of the patient plasma pre‐ and postoperation, respectively. The preoperative ctDNA positivity rate was significantly lower in the nonrecurrence group than in the recurrence group. With a median follow‐up of 17.7 months, nine patients (22%) experienced tumor recurrence. ctDNA positivity at two time‐points was associated with significantly shorter recurrence‐free survival (RFS). Tumors with NRAS, NEF2L2, and MET mutations had significantly shorter times to recurrence than those without mutations and showed high recurrence prediction performance by machine learning. Multivariate analyses showed that the median variant allele frequency (VAF) of mutations in preoperative ctDNA was a strong independent predictor of RFS. ctDNA is a real‐time monitoring indicator that can accurately reflect tumor burden. The median VAF of baseline ctDNA is a strong independent predictor of RFS in individuals with HCC.

OA20.02 Pre-Treatment Levels of ctDNA for Long-Term Survival Prediction in Stage IIIA NSCLC Treated With Neoadjuvant Chemo-Immunotherapy

There are currently no predictive biomarkers for long-term survival after neoadjuvant chemoimmunotherapy. However, the identification of non-small lung cancer (NSCLC) patients who obtain long-term benefit from chemoimmunotherapy is essential to optimize therapies. Using samples from NADIM clinical trial (NCT03081689), in which resectable stage IIIA NSCLC patients were treated with neoadjuvant chemo-immunotherapy with nivolumab, we have evaluated the capacity of ctDNA levels before treatment initiation to predict overall survival (OS) and progression-free survival (PFS) by calculating Harrell’s C-statistic and we compare its predictive value with classical survival surrogates as the pathological response and clinical response assessed according to RECIST criteria v.1.1. The ctDNA was analyzed by NGS, using the Oncomine Pan-Cancer Cell-Free Assay™ (Thermo Fisher Scientific®). To explore the prognostic value of the amount of ctDNA at baseline, for each positive plasma sample, we calculated the sum of the mutant allele frequency (MAF) for all detected mutations. Patients who died from COVID19 were excluded from this analysis. In our study, clinical responses based on RECIST criteria were not predictive for OS or PFS. On the contrary, in the multivariate analysis, patients with low ctDNA levels (<1% MAF), in the baseline sample, had significantly improved PFS and OS than patients in whom the opposite situation occurred (adjusted HR: 0.22; 95%CI: 0.06-0.75; P=0.016 and adjusted HR: 0.04; 95%CI: 0.00-0.45; P=0.008 for PFS and OS, respectively). The adjusted C-statistic (c) to predict PFS for ctDNA was 0.68 (95%CI: 0.51-0.84), which was superior to that of RECIST criteria (c=0.61; 95%CI: 0.45-0.78) and similar to that of pathological response (c=0.68; 95%CI: 0.52-0.84). Similarly, baseline ctDNA levels predicted OS (c=0.85; 95%CI: 0.72-0.99) better than RECIST criteria (c=0.68; 95%CI: 0.44-0.93). Pre-treatment ctDNA levels predicted more accurately long-term survival than radiological assessments in NADIM study and might be useful for the design of new clinical trials.

A large-scale, multicentered trial evaluating the sensitivity and specificity of digital PCR versus ARMS-PCR for detecting ctDNA-based EGFR p.T790M in non-small-cell lung cancer patients.

BackgroundDeveloping liquid biopsy technology with higher sensitivity and specificity especially for low-frequency mutations remains crucial. This study demonstrated superior performance of the newly developed digital PCR (dPCR) kit for ctDNA-based EGFR p.T790M detection in metastatic non-small-cell lung cancer (NSCLC) against ARMS-PCR.MethodsThis large-scale, multi-centered diagnostic study recruited 1,045 patients including 1,029 patients diagnosed with advanced NSCLC and 16 patients with specific samples between April 1st 2018 and November 30th 2019. EGFR p.T790M in plasma samples from mNSCLC patients were tested using dPCR with ADx-ARMS PCR and Cobas® EGFR Mutation Test V2 as comparator assays to confirm cut-off value for dPCR and evaluate its performance against ARMS-PCR-based assays. Efficacy was evaluated for patients with EGFR p.T790M detected by dPCR or ARMS-PCR, who underwent Osimertinib treatment.ResultsThe sensitivity, specificity, and concordance of dPCR against ADx-ARMS PCR was 98.15%, 88.66% and 90.16%, respectively for 1,026 plasma samples. Additional 9.26% patients were detected positive by dPCR. The majority of those samples had a mutation allele frequency between 0.1% and 1%. In 45 paired tissue and plasma samples, the sensitivity improved from 30.77% to 53.85% by dPCR with the specificity over 90%. The response of Osimertinib in 74 EGFR p.T790M-positive patients detected by dPCR, including 26 determined as negative by ARMS-PCR, were evaluated to have an ORR of 44.59% and a DCR of 90.54%.ConclusionsdPCR is a sensitive and accurate tool for ctDNA-based EGFR p.T790M detection due to its significantly improved sensitivity without compromising specificity, and dPCR is equivalent to ARMS-PCR as a companion diagnostic tool while benefiting more patients under Osimertinib treatment.Trial RegistrationChinese Clinical Trial Registry identifier: ChiCTR2100043147.

A Sensitive PCR-Based Method for Somatic Mutations Enrichment and Screening.

BackgroundEGFR and KRAS are the most frequently mutated genes in lung cancers, occurring in about 60% of all cases. Mutation genes assay has emerged as a promising blood-based biomarker for monitoring cancer dynamics noninvasively. However, detection can be challenging in patients where plasma often contains low levels of tumor-derived DNA fragments.MethodsWe have developed a nuclease-based enrichment assay for detecting mutant alleles. The procedure is based on Surveyor endonuclease cleaves mismatched DNA molecules, and these DNA fragments were enriched for mutation screening. We screened lung cancer specimens for mutations in exons 18 and 21 of EGFR, and the majority of activating mutations in lung cancer occur in codons 12 (G12X) and 13 (G13X) of exon 2 of the KRAS gene. The method screened all mutant genes with the same pair primers and three relevant TaqMan probes.ResultsThe method can effectively remove wild-type sequences and enrich mutation DNA, and the sensitivity detectable mutant allele frequencies (MAF) achieved 0.001%. The method increases the sensitivity and efficiency of mutation DNA for cancers screening. This highlights the importance of complex DNA variation like mutations in exon 21 of EGFR and exon 2 of the KRAS gene detected by the same probe.ConclusionWe developed a simple and sensitive methodology for mutation gene screening. The method is a cost-effective and sensitive method for mutation DNA enrichment and detection.

Frequency of W24X Gene Polymorphism among the Students from Hearing Impaired Schools of Shimoga District

Introduction An important accompaniment of Non-syndromic sensorineural hearing loss is family history and consanguinity. One of the widely studied single nucleotide polymorphism is the gap junction protein beta-2 (GJB2) gene which encodes the protein connexin26. This study aims to detect the frequency of W24X mutation in a population with non-syndromic sensorineural hearing loss concerning the degree of consanguinity. Materials and Methods The study includes 76 subjects with Non-syndromic sensorineural hearing loss. These subjects had congenital sensorineural hearing loss and other causes for the same had been ruled out. The SNP rs 104894396 was identified by the PCR-RFLP method. Results The frequency of the wild allele was 0.84% and the mutant allele was 0.15%. The frequency of wild allele and mutant allele did not differ much between patients with and without consanguinity. The association between consanguineous marriage and allele frequency was not significant. Gene polymorphism was not present in 77 percent of our NSHL subjects, though 79 percent of our study population were a result of consanguineous marriage. Conclusion Though the role of consanguineous marriages in congenital sensorineural hearing loss is well established, the association between allele frequency and consanguineous marriage was not seen. We assume that other genes responsible for deafness may be involved in the population.

Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae.

Decisions on which pesticide to use in agriculture are expected to become more difficult, as the number of available chemicals is decreasing. For Tetranychus urticae (T. urticae), a major pest for which a number of candidate markers for pesticide resistance are in place, molecular diagnostics could support decision-making for the rational use of acaricides. A suite of 12 TaqMan qPCR assays [G314D (GluCl1), G326E, I321T (GluCl3), G119S, F331W (Ace-1), H92R (PSST), L1024V, F1538I (VGSC), I1017F (CHS1), G126S, S141F, P262T (cytb)], were validated against Sanger-sequencing, and subsequently adapted for use with the ddPCR technology. The concordance correlation coefficient between the actual and ddPCR measured mutant allelic frequencies was 0.995 (95% CI=0.991-0.998), and no systematic, proportional, or random differences were detected. The achieved Limit of Detection (LoD) was 0.1% (detection of one mutant in a background of 999 wild type mites). The ddPCR assay panel was then assessed in terms of agreement with phenotypic resistance, through a pilot application in field populations from Crete, with strong correlation and thus predictive and diagnostic value of the molecular assays in some cases (e.g., etoxazole and abamectin resistance). Molecular diagnostics were able to capture incipient resistance that was otherwise missed by phenotypic bioassays. The molecular and phenotypic resistance screening of T. urticae field populations from Crete, revealed both multi-resistant and susceptible populations. The highly sensitive T. urticae molecular diagnostic platforms developed in this study could prove a valuable tool for pesticide resistance management. © 2021 Society of Chemical Industry.

Genetic Profiling of Cell-Free DNA From Pleural Effusion in Advanced Lung Cancer as a Surrogate for Tumor Tissue and Revealed Additional Clinical Actionable Targets

BackgroundPleural effusion (PE) has been one of the promising sources of liquid biopsy in advanced lung cancer patients. However, its clinical utility is not widely accepted due to the lack of full estimation of its potential versus routine clinical samples. MethodA total of 164 advanced lung cancer patients were enrolled with 164 matched tumor tissue and PE-cfDNA, 153 accompanied plasma and 63 11PE cell sediment (PE-sDNA)PE-sDNA. ResultPE-cfDNA displayed significantly higher median mutant allele frequency and an overall mutation concordance rate of 65% to tissue, which was higher than PE-sDNA (43%) and plasma-cfDNA (43%). The discrepancies between PE-cfDNA and tumor tissue were high in several genes, including SMARCA4, PIK3CA, ERBB2, KM T2A, ALK and NF1. For clinically actionable mutations, the concordance rate between PE-cfDNA and tumor tissue is 87%. Eleven patients were identified with actionable mutations in PE-cfDNA and four patients benefited from PE-cfDNA-guided targeted. Meanwhile, PE-cfDNA recapitulated mutations of diverse tissue origins and provided more mutational information under the circumstance that tumor tissue or tumor tissue of different origins were unavailable. The combination of tumor tissue and PE-cfDNA profiling increased positive detection rates of patients compared to tumor tissue alone. Our finding highlighted the importance of PE-cfDNA in the optimal selection of patients for targeted therapy. ConclusionThe PE-cfDNA-based liquid biopsy displays better performance in the characterization of gene alterations than PE-sDNA and plasma-cfDNA. PE-cfDNA together with tumor tissue profiling optimizes comprehensively genomic profiling of lung cancer patients, which might be important for selecting patients for better treatment management.

High levels of pyrethroid resistance and super-kdr mutations in the populations of tropical bed bug, Cimex hemipterus, in Iran

BackgroundThe tropical bed bug, Cimex hemipterus, is an important ectoparasite causing various health problems. This species is mainly confined to tropical regions; however, insecticide resistance, global warming, and globalization have changed its distribution map. Molecular information on pyrethroid resistance, which is essential for the development of control programs, is unknown for C. hemipterus in expanded areas. The present study was designed to determine the permethrin resistance status, characterize the pyrethroid receptor sites in voltage-gated sodium channel (vgsc) gene, and identify the resistance-related mutations in the populations of tropical bed bug in Iran.MethodsLive bed bugs were collected, and adults of C. hemipterus were selected for bioassay and molecular surveys. Bioassay was performed by tarsal contact with permethrin 0.75% in mixed-sex of samples. Conventional and quantitative TaqMan and SYBR Green real-time PCR assays were conducted to characterize the vgsc gene and genotypes of studied populations.ResultsIn the bioassay tests, the mortality rates were in the range of 30.7–38.7% and 56.2–77.4% in 24 and 48 h, respectively. The knockdown rates of studied populations were in the range of 32.2–46.6% and 61.5–83.8% in the first and second days, respectively. The KT50 and KT90 values in the Cimex lectularius Kh1 strain were presented as 5.39 and 15.55 h, respectively. These values in the selected populations of C. hemipterus varied from 27.9 to 29.5 and from 82.8 to 104.4 h, respectively. Knockdown time ratios (KR50 and KR90) in these populations varied from 5.17 to 6.17-fold compared with those of the C. lectularius Kh1 strain. Fragments of vgsc gene with 355 bp and 812 bp were amplified. Analysis of sequences revealed the A468T substitution, kdr-associated D953G, and super-kdr M918I and L1014F mutations in all populations.ConclusionsThe specific/sensitive, safe, and rapid diagnostic assays developed in this study are recommended for detection of kdr/super-kdr mutations and frequency of mutant alleles. The presence of super-kdr mutations and high resistance to permethrin in all the populations necessitate the reconsideration of control approaches against C. hemipterus.Graphical

Metacommunities, fitness and gradual evolution

We analyze the evolution of a multidimensional quantitative trait in a class-structured focal species interacting with other species in a wider metacommunity. The evolutionary dynamics in the focal species as well as the ecological dynamics of the whole metacommunity is described as a continuous-time process with birth, physiological development, dispersal, and death given as rates that can depend on the state of the whole metacommunity. This can accommodate complex local community and global metacommunity environmental feedbacks owing to inter- and intra-specific interactions, as well as local environmental stochastic fluctuations. For the focal species, we derive a fitness measure for a mutant allele affecting class-specific trait expression. Using classical results from geometric singular perturbation theory, we provide a detailed proof that if the effect of the mutation on phenotypic expression is small (“weak selection”), the large system of dynamical equations needed to describe selection on the mutant allele in the metacommunity can be reduced to a single ordinary differential equation on the arithmetic mean mutant allele frequency that is of constant sign. This invariance on allele frequency entails the mutant either dies out or will out-compete the ancestral resident (or wild) type. Moreover, the directional selection coefficient driving arithmetic mean allele frequency can be expressed as an inclusive fitness effect calculated from the resident metacommunity alone, and depends, as expected, on individual fitness differentials, relatedness, and reproductive values. This formalizes the Darwinian process of gradual evolution driven by random mutation and natural selection in spatially and physiologically class-structured metacommunities.