Mutant Allele Frequency Research Articles

The significance of micro-EGFR T790M mutation on EGFR-TKI efficacy in patients with NSCLC: The WJOG13119L study.

e21177 Background: The significance of micro-pretreatment EGFR T790M mutation on EGFR-tyrosine kinase inhibitor (TKI) efficacy in patients with non-small cell lung cancer (NSCLC) remains uncertain. In addition, there has been no method to examine the micro-T790M mutation after excluding formalin-fixed and paraffin-embedded (FFPE)-derived artificial mutations. Therefore, we used a novel method to examine the variant allele frequency (VAF) of T790M mutation in the FFPE samples after excluding the artificial mutations. Subsequently, we retrospectively investigated the association between micro-T790M mutations and time to treatment failure (TTF) in patients treated with 1st, 2nd, or 3rd generation (1G, 2G, 3G) EGFR-TKIs. Methods: We enrolled patients with NSCLC who harbored EGFR exon 19 deletion (Del19) or L858R without T790M mutation using conventional methods. Patients who received 1G or 2G EGFR-TKIs until June 2018 or 3G EGFR-TKI until June 2019 from 31 institutions were included. Droplet digital PCR was performed using pretreatment FFPE tumor samples. We defined cases as micro-T790M positive when the VAF of T790M was higher than that of F795F, owing to the FFPE-derived artificial mutations. The ratio of micro-T790M mutation was calculated as follows: {(VAF of T790M) – (VAF of F795F)} /VAF of (Del19 or L858R). The lowest value was set as 0. Results: A total of 110, 102, and 103 patients received 1G, 2G, and 3G EGFR-TKIs, respectively. Of these patients, 48.1%, 47.0%, and 47.6% were classified as micro-T790M positive, respectively. The ratios of micro-T790M mutation, represented as median (%) (interquartile range), in 1G, 2G, and 3G EGFR-TKI groups were 0.27 (0.12-0.87), 0.28 (0.13-0.47), and 0.42 (0.20-0.68), respectively. The VAF of T790M or F795F in all patients, represented as median (%) (interquartile range), was 0.26 (0.19-0.35) or 0.25 (0.19-0.33), respectively. The TTF was not significantly different between the T790M-positive and negative groups in analysis based on each generation TKIs. In the Del19 group, the TTF of the 1G EGFR-TKI was significantly shorter, while that of the 3G EGFR-TKI was significantly longer in the T790M-positive group than in the negative group ( p= 0.02, 0.04, respectively). In the T790M-positive group, the TTF of the 2G or 3G EGFR-TKI group was significantly longer than that of the 1G EGFR-TKI group ( p= 0.04, 0.01, respectively). This result was observed in the Del19 group but not in the L858 group. Furthermore, TTF did not significantly differ between the 1G, 2G, and 3G EGFR-TKIs in the T790M-negative group. Conclusions: This study showed that micro- EGFR T790M mutations influence the EGFR-TKI efficacy. Detection of micro-T790M mutations will facilitate selection of the optimal EGFR-TKI for patients with EGFR mutated NSCLC.

Variation in liquid biopsy cfDNA yield predicted by somatic mutation and clinical phenotypes across primary cancers.

e13553 Background: Liquid biopsy provides a non-invasive alternative to tissue biopsy by profiling the genetic mutations and biomarker characteristics of cell-free DNA (cfDNA) derived from primary and metastatic tumors found in patients’ peripheral blood. Previous studies have demonstrated significant variation in cfDNA and circulating tumor DNA (ctDNA) yield based on patient age, sex, primary tumor type, disease stage, and treatment regimen. Somatic mutations are a primary determinant of tumor phenotype, characterizing therapeutic sensitivity and resistance, as well as growth and metastasis rates. However, there has not been extensive investigation as to how presence of somatic mutations affect downstream cfDNA yield and ctDNA fraction. Methods: We screened 140,000 samples analyzed using Guardant360 liquid biopsy test for patient age, diagnosis, treatment, mutations, and associated cfDNA yield (from assay input of 3-10ml peripheral blood, 1 streck tube). We selected and grouped somatic mutations based on prevalence across the cohort. Estimated ctDNA fractions of cfDNA yields were made using the maximum mutant allele frequency (MAF) from among somatic mutations. Samples were organized by phenotype and cfDNA yield and ctDNA fraction was assessed across groups using the Wilcoxon and Kruskal-Wallis for bi-variate and multivariate comparisons, respectively. To understand impact of cfDNA yield variation on assay performance, group comparisons were further evaluated on samples binned by input cfDNA (< 5ng, 5-15ng, 15-30ng, > 30ng) representing cfDNA input requirement ranges current commercial liquid biopsy tests advertise. Results: We found significant yield variation in sample cfDNA and estimated ctDNA fraction between primary disease types ( Myield diff = 16.35ng, p < 0.0001), therapy histories ( Myield diff = 1.49ng, p < 0.001), and patient age groups ( Myield diff = 2.24ng, p < 0.0001), although this finding did not extend to comparisons within input yield bins. We found significant differences in cfDNA and ctDNA fraction across several common somatic mutations, adjusting for test multiplicity, with the greatest difference between TERT splice + and - patients ( Myield diff = 8.57ng, p < 0.0001). Further, we found significant and strong correlations (𝝆 > 0.8, p < 0.001) between EGFR exon 19 deletions and EGFR L858R MAF and estimated ctDNA fraction, and moderate correlation between MAF and cfDNA (𝝆 > 0.25, p < 0.001). Conclusions: Our results indicate that input cfDNA and ctDNA fraction varies by patient age, diagnosis and treatment, as well as with the presence of several common somatic mutations. The demonstrated correlation between somatic mutation presence and cfDNA yield offers a potential explanation for the broad variation of cfDNA yields within tumor and patient phenotypes, and may help inform blood collection strategies for patients with known somatic variants or diagnoses.

Potential indicators in circulating cell-free DNA for monitoring PARP inhibitor resistance in high-grade serous ovarian cancer.

5564 Background: Although the clinical application of PARP inhibitors (PARPis) has brought great survival benefits to patients with high-grade serous ovarian carcinoma (HGSOC), its resistance has gradually become a major challenge for clinicians with the widespread use of PARPis. Unfortunately, there are no effective, non-invasive means for monitoring PARPis resistance in time during maintain therapy. Methods: We collected peripheral blood samples (n = 37) from 37 healthy subjects and a series of longitudinal peripheral blood samples (n = 61) of 25 platinum-sensitive HGSOC patients undergoing Olaparib maintenance therapy. The genome of white blood cell and cfDNA in plasma was extracted for germline and somatic mutation detection by Circular Ligation Amplification and sequencing (CLAmp-seq) based on a targeted 42-gene panel, respectively. Before cfDNA mutation analysis, background noise introduced by random NGS error was removed and clonal hematopoietic mutations were filtered. Variant-supporting reads > 2 and without germline mutations were defined as the criterion for somatic mutations. Progression-free survival (PFS) was collected through regular follow-up. We analyzed the dynamic changes of cfDNA mutation profiles, the correlation between cfDNA mutations and the prognosis of patients, and screened specific mutation sites that closely associated with Olaparib resistance. Results: The elevation of maximum mutant allele frequency (Max MAF) in cfDNA during Olaparib maintenance therapy predicted a poor prognosis of patients ( P = 0.0043). Pathogenic germline mutations in BRCA1/2 or RAD51 were strongly associated with longer PFS ( P = 0.0229) and acquired new MRE11A mutations significantly shortened the PFS in patients ( P = 0.0005). Dynamic fluctuations of somatic mutation sites in CHEK2:p.K373E ( P = 0.0091) and CHEK2:p.R406H ( P = 0.0002) can be used to evaluate the therapeutic efficacy of patients. Remarkably, MRE11A:p.K464R may be a vital driving factor of Olaparib resistance and all patients who acquired new MRE11A:p.K464R in post-treatment cfDNA developed resistance to Olaparib and had significantly shorter PFS than those without it ( P = 0.0005). Besides, the combination of CHEK2:p.R406H and acquired new MRE11A:p.K464R in post-treatment improved the predictive efficiency of patients’ prognosis compared with them alone ( P < 0.0001). Conclusions: Olaparib resistance was robustly associated with the mutation load of tumor cells, and analysis of mutation profiles in cfDNA can be accurately monitor the status of Olaparib resistance in patients with HGSOC. Acquired new MRE11A:p.K464R may be a vital driver of Olaparib resistance and is expected to be a target for anti-tumor drug development.

Early detection of cancer using cell-free DNA (cfDNA) size analysis on a multiplexed amplicon-based next-generation sequencing (NGS) platform.

3041 Background: Tumor-derived cfDNA fragments are observed to be shorter in length than normal cfDNA. This size (length) difference can be analyzed as a tumor-specific signal. Whole genome or probe hybridization-based NGS methods can capture cfDNA fragments of native sizes. However, amplicon-based NGS assays are not directly amenable to cfDNA size analysis due to predetermination of amplicon sizes by design. Here we present a method to extract relative distribution of cfDNA fragment lengths from a targeted amplicon-based assay and show its utility in cancer detection. Methods: The LiquidHALLMARK cfDNA assay is an amplicon-based NGS test for the sensitive detection of genomic alterations in 80 genes. Although panel design is optimized for cfDNA with average amplicon length of ̃150 bp, the consecutive tiling design of amplicons for genes with contiguously targeted regions, e.g. BRCA permits the formation of longer amplicons (> 150 bp) from physically subsequent primer pairs, provided longer template cfDNA molecules are present. Cancer samples (n = 281), clinically tested by LiquidHALLMARK during Sep 2020-Sep 2021, and healthy samples (n = 28) were included for analysis. For each sample, fragment lengths were inferred from sequencing alignment files, and binned into “short” (0-150 bp) and “long” (151-500 bp) groups. A relative “size ratio” of the total number of short vs. long fragments per sample was calculated, and examined with clinical features, plasma cfDNA concentration (cfDNA/ml) and highest mutation allele frequency (AF%), in a model to predict cancer. Results: Calculated size ratios (relative abundance of short fragments) were higher in cancer than normal samples (median 47.6 vs. 31.2, p < 0.001). Cholangiocarcinoma and colorectal cancer samples had the highest size ratios (medians: 62.9 and 60.9, respectively) in agreement with a genome-wide NGS study that profiled cfDNA sizes. Size ratios were higher in metastatic (n = 143) compared to early stage (n = 30) lung cancers (p = 0.0039), indicating a stage-dependent accumulation of shorter cfDNA fragments. Size ratio was correlated with cfDNA/ml ( r= 0.63, p < 0.01) and AF% ( r= 0.42, p < 0.01). In 20-fold cross-validation of a logistic regression model trained to predict cancer, average area under curve (AUC) was 0.82 using size ratio, 0.86 using cfDNA/ml, and increased to 0.95 with the two features combined. Conclusions: Our analysis shows that it is feasible to derive meaningful cfDNA fragment size information from amplicon-based NGS data. Importantly, relative fragment size distributions observed in cancer and healthy plasma samples by this method are concordant with alternate target capture methods. Fragment size ratios derived from relatively small, targeted amplicon panels are a novel feature that, combined with other molecular and clinical features, can enhance non-invasive methods of cancer detection.

Clinical and genomic distinction of class 1/2/3 BRAF-mutant colorectal cancer and differential prognosis.

3533 Background: Mutations occurring at the V600 amino acid of BRAF is the most common BRAF mutations in colorectal cancer (CRC), which lead to RAS-independent active monomers (Class 1) and are the targets of BRAF inhibitors. BRAF non-V600 mutants can be further classified as RAS-independent active dimers (Class 2) and RAS-dependent impaired kinase (Class 3). The clinical and genetic distinction of CRC patients carrying different subtypes of BRAF mutations remain to be revealed. Methods: A multicenter retrospective study investigated the mutational profiles of 2,118 CRC patients whose baseline tumor samples were analyzed between June 2015 and June 2020 using capture-based hybrid NGS targeting 400+ cancer-related genes. Clinical characteristics including age, sex, stage, and tumor location were analyzed. A public cBioPortal cohort of 471 metastatic CRC patients was used for survival analysis. Results: A total of 473 patients were identified to contain BRAF mutations and can be sub-grouped into Class 1 (N = 246), Class 2 (N = 29), Class 3 (N = 53), and others (N = 145). All Class 1 BRAF mutations were V600E, while the Class 2 and Class 3 mutations were predominantly G469 (52%) and D594 (56%), respectively. No difference in patient’s age, sex, and stage was observed among BRAF Class 1-3 subgroups, but the anatomical location of the tumor differed among subgroups, particularly between Class 1 and Class 3 BRAF-mutant patients significantly (p = 0.027). Specifically, 39% of tumors carrying Class 1 mutations occurred at the right colon, while the most frequent location of tumors with Class 3 mutations was rectum (58%). Mutational profiling revealed that KRAS and APC mutations were enriched in Class 2/3 compared to Class 1 BRAF-mutant patients, while RNF43 and SMAD4 mutations were more frequently observed in Class 1 patients. Mutations causing the activation of the Wnt or RTK/RAS signaling pathways were also more common in Class 2/3 subgroups comparing to Class 1 patients. Furthermore, based on the analysis of mutation allele frequency, Class 1 BRAF mutations tended to be drivers while Class 2/3 BRAF mutations were more likely to be passengers. In addition, mutational signature profiling showed that the NER signature was enriched in Class 1 BRAF-mutant patients but the APOBEC signature in Class2/3 classes. The tumor mutational burden of Class 2 BRAF tumors was higher than the other two subgroups (median: 10.4 vs 7.6 and 8.4), and microsatellite instability-high tumors were more common in Class 1 (11% vs 7% and 4%). In the cBioPortal cohort, Class 1 BRAF-mutant patients had the worst overall survival whereas Class 3 patients demonstrated the best prognosis. Conclusions: The clinical and genetic features of CRC patients harboring Class 2 and 3 BRAF mutations were different from those carrying Class 1 BRAF mutations in aspects including tumor location, concurrent mutations, mutational signatures, as well as survival outcomes.

TP53-altered chronic lymphocytic leukemia treated with firstline Bruton's tyrosine kinase inhibitor-based therapy: A retrospective analysis.

Long-term follow up of prospective studies has shown that continuous Bruton's tyrosine kinase inhibitor (BTKi) therapy leads to durable remissions in previously untreated patients with TP53-altered chronic lymphocytic leukemia (CLL); however, it is unknown how variant allele frequency (VAF) of TP53 mutation (TP53-m) or percentage of cells with deletion of chromosome 17p [del(17p)] influences efficacy of firstline BTKi. We performed a retrospective analysis of 130 patients with CLL with baseline del(17p) and/or TP53-m treated with BTKi with or without the BCL2 inhibitor venetoclax (VEN) and with or without CD20 antibody in the firstline setting. A total of 104/130 (80%) patients had del(17p). TP53-m was noted in 89/110 (81%) patients tested; there were 101 unique TP53-m with an available VAF. The 4-year progression-free survival (PFS) and overall survival (OS) rates were 72.9% and 83.6%. No baseline characteristics including IGHV mutation status and number of TP53 alterations were associated with significant differences in PFS or OS, though a trend toward shorter PFS with increasing karyotypic complexity (hazard ratio 1.08, p=.066) was observed. Del(17p) was identified in <25% of cells in 26/104 (25%) of patients, and 28/101 (28%) of TP53-m were low-burden with a VAF of <10%; outcomes of these patients were similar to those with high-burden lesions. This study suggests that low-burden TP53 alterations should not be ignored when assessing genomic risk in CLL in the era of targeted therapy.

Osimertinib for lung cancer cells harboring low-frequency EGFR T790M mutation

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, shows significant benefit among patients with EGFR T790M mutation at disease progression. We analyzed the whole exome sequence of 48 samples obtained from 16 lung cancer patients with a longitudinal follow-up: treatment-naïve-baseline primary tumors positive for EGFR activating-mutations, paired re-biopsies upon disease progression but negative for EGFR T790M mutation based on qPCR, and their matched normal blood samples. Our Next generation sequencing (NGS) analysis identified an additional set of 25% re-biopsy samples to harbor EGFR T790M mutation occurring at a low-allele frequency of 5% or less, undetectable by conventional qPCR-based assays. Notably, the clinical utility of osimertinib among patients harboring low-allele frequency of EGFR T790M in tissue biopsy upon disease progression remains less explored. We established erlotinib-resistant PC-9R cells and twenty single-cell sub-clones from erlotinib-sensitive lung cancer PC-9 cells using in vitro drug-escalation protocol. NGS and allele-specific PCR confirmed the low-allele frequency of EGFR T790M present at 5% with a 100-fold higher resistance to erlotinib in the PC-9R cells and its sub-clones. Additionally, luciferase tagged PC-9, and PC-9R cells were orthotopically injected through the intercostal muscle into NOD-SCID mice. The orthotopic lung tumors formed were observed by non-invasive bioluminescence imaging. Consistent with in vitro data, osimertinib, but not erlotinib, caused tumor regression in mice injected with PC-9R cells, while both osimertinib and erlotinib inhibited tumors in mice injected with PC-9 cells. Taken together, our findings could extend the benefit of osimertinib treatment to patients with low EGFR T790M mutation allele frequency on disease progression.

Gene alternation of cerebrospinal fluid in patients with leptomeningeal metastases of lung adenocarcinoma using next-generation sequencing

BackgroundEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) provide a better prognosis in EGFR-mutant non-small cell lung cancer (NSCLC). Nevertheless, the outcome of leptomeningeal metastasis (LM) remains poor. In addition, due to limited access to intracranial tumour tissue, gene alterations associated with leptomeningeal metastasis from lung adenocarcinoma (LM-LUAD) are unclear.MethodsForty-five patients with LM-LUAD from May 2019 to June 2021 in Guangdong Sanjiu Brain Hospital were enrolled in this study. Seventy-five percent (34/45) of patients with LM harbored EGFR mutations, and patients with progressive disease (PD) of LM had 3rd-generation EGFR-TKI therapy and were defined as Cohort 1; those without 3rd-generation EGFR-TKI therapy were defined as Cohort 2. Next-generation targeted panel sequencing (NGS) was performed in each cerebrospinal fluid (CSF) sample of the two cohorts, and 9/45 LM-LUAD patients had matched plasma (PLA).ResultsThe common gene alterations discovered in the CSF of LM-LUAD were EGFR mutation (34/45, 75%), TP53 (25/45, 56%), CDKN2A (9/45, 20%), ALK (7/45, 16%), CTNNB1 (6/45, 13%), MET (5/45, 11%), APC (4/45, 9%), FGF4 (4/45, 9%), FGF3 (4/45, 9%), ERBB2 (4/45, 9%), and PIK3CG (4/45, 9%). Cooccurring mutations of TP53 and EGFR were found in 49% (22/45) of patients and correlated with poor prognosis. CDKN2A was identified in 20% (9/45) of patients and presented slightly shorter overall survival (OS) than those without (7.1 versus 8.8 months, p = 0.2). Cohort 1 had more genes associated with poor prognosis, consisting of CDK4, CDKN2A, PIK3CG, or PIK3CA, and YES1 and MET were more likely to be detected in cohort 2. The alteration of EGFR was comparable between CSF and matched PLA. Incidences of gene alterations such as CDK4, CDKN2A, MET, SOX2, JAK2, BRAF, and PIK3CG were more likely to be identified in CSF. All mutant allele frequencies (MAF) were much higher in CSF than in matched PLA.ConclusionsCSF could be a potential candidate for the genetic profiling of LM-LUAD, demonstrating the genetic characteristics of LM in EGFR-mutated lung adenocarcinoma on diverse EGFR-TKI therapies.

Analysis of mutation frequencies of the BRCA1 5382insC and BRCA2 6174delT genes in the population of Ufa city

BACKGROUND: Currently, there is an acute problem of assessing the real genetic danger and the impact of environmental pollution on human health. One of the cities that is affected by the whole complex of anthropogenic mutagenic factors is Ufa (Republic of Bashkortostan).&#x0D; AIM: The aim of the study was to analyze the frequency of pathogenic mutations in the BRCA1 5382insC and BRCA2 6174delT genes in healthy residents of an industrial city.&#x0D; MATERIALS AND METHODS: The mutations were determined by real-time PCR using the TaqMan probe technology on the Bio-Rad CFX96 device (Bio-Rad, USA) in accordance with protocol in 379 DNA samples of healthy residents of average age (18.32 0.45 years).&#x0D; RESULTS: The total mutations frequency in the BRCA1 and BRCA2 genes reached 4.22% (n = 16). The highest frequency of mutant alleles in the BRCA1 and BRCA2 genes of 6.18% (n = 11) was present in residents of the 1 observation area, where petrochemical production enterprises are concentrated. The mutant homozygous genotype of the BRCA2 gene was detected in 3.37% of cases only among residents of the 1 observation area (n = 6, p = 0.0002). The risk of formation of mutations in the BRCA2 gene is 6 times higher in residents of the 1 observation area (2 = 7.75; df 1; p = 0.006; OR 6.31; 95% CI 1.4427.67).&#x0D; CONCLUSIONS: The frequency of the BRCA family genes varies depending on the studied region, the degree of environmental pollution and the studied group.

Use of the Biocartis Idylla™ Platform for the Detection of Epidermal Growth Factor Receptor, BRAF and KRAS Proto-Oncogene Mutations in Liquid-Based Cytology Specimens from Patients with Non-Small Cell Lung Carcinoma and Pancreatic Adenocarcinoma

The study aimed to demonstrate rapid and effective molecular testing on liquid-based cytology (LBC) samples for EGFR, KRAS and BRAF mutations using the Biocartis Idylla™. Rapid on-site evaluation (ROSE) LBC samples for patients with non-small cell lung carcinoma (NSCLC) or pancreatic ductal adenocarcinoma (PDAC) were tested for EGFR, KRAS and BRAF mutations based on the relevance to tumour subtype. The quantification values (Cq values) and mutation detection status were compared between LBC samples and routine formalin-fixed paraffin-embedded (FFPE) clot samples. ROSE LBC samples (n = 54) showed a higher yield of well-preserved tumour and wild type (WT) DNA, demonstrated by lower quantification cycles, no false positives or false negatives, and a higher sensitivity for low allele frequency mutations when compared with FFPE clot samples. The Biocartis Idylla™ provides highly sensitive, reliable and rapid testing for LBC samples for the detection of EFGR and KRAS mutations. BRAF mutations were not detected in the participant cohort; however, all LBC WT BRAF results correlated with the results from the FFPE clot samples. Access to rapid molecular testing using LBC samples can detect the most frequent driver mutations closer to the time of diagnosis, enabling the selection of the most effective first-line targeted therapy sooner, reducing delays or side effects from suboptimal treatments, patient anxiety and costs to healthcare systems, whilst improving patient outcomes.

Carrier Rate and Mutant Allele Frequency of GM1 Gangliosidosis in Miniature Shiba Inus (Mame Shiba): Population Screening of Breeding Dogs in Japan.

Simple SummaryGM1 gangliosidosis, a progressive neurodegenerative lysosomal storage disease, occurs in the Shiba Inu breed due to the GLB1:c.1649delC (p.P550Rfs*50) mutation. Previous surveys performed of the Shiba Inu population in Japan showed that the carrier rate was 1.02–2.94%. Currently, a miniature type of the Shiba Inu called “Mame Shiba”, bred via artificial selection to yield smaller individuals, is becoming more popular than the standard type. A GM1 gangliosidosis mutation survey has yet to be carried out in the Mame Shiba population. Therefore, this study aimed to determine the frequency of the mutant allele and carrier rate of GM1 gangliosidosis in the Mame Shiba breed. We surveyed 1832 adult Mame Shiba Inus used for breeding across 143 Japanese kennels using a genotyping assay. As a result, nine Mame Shiba Inus were found to be carriers, indicating that the carrier rate was 0.49% (corresponding mutant allele frequency = 0.00246). This study demonstrates that there are certain Mame Shiba Inus carrying the mutation for GM1 gangliosidosis and that their breeding should be prevented or controlled.GM1 gangliosidosis is a progressive, recessive, autosomal, neurodegenerative, lysosomal storage disorder that affects the brain and multiple systemic organs due to an acid β-galactosidase deficiency encoded by the GLB1 gene. This disease occurs in the Shiba Inu breed, which is one of the most popular traditional breeds in Japan, due to the GLB1:c.1649delC (p.P550Rfs*50) mutation. Previous surveys performed of the Shiba Inu population in Japan found a carrier rate of 1.02–2.94%. Currently, a miniature type of the Shiba Inu called “Mame Shiba”, bred via artificial selection to yield smaller individuals, is becoming more popular than the standard Shiba Inu and it is now one of the most popular breeds in Japan and China. The GM1 gangliosidosis mutation has yet to be surveyed in the Mame Shiba population. This study aimed to determine the frequency of the mutant allele and carrier rate of GM1 gangliosidosis in the Mame Shiba breed. Blood samples were collected from 1832 clinically healthy adult Mame Shiba Inus used for breeding across 143 Japanese kennels. The genotyping was performed using a real-time PCR assay. The survey found nine carriers among the Mame Shibas, indicating that the carrier rate and mutant allele frequency were 0.49% and 0.00246, respectively. This study demonstrated that the mutant allele has already been inherited by the Mame Shiba population. There is a risk of GM1 gangliosidosis occurrence in the Mame Shiba breed if breeders use carriers for mating. Further genotyping surveys are necessary for breeding Mame Shibas to prevent the inheritance of this disease.

Genetic changes during leukemic transformation to secondary acute myeloid leukemia from myeloproliferative neoplasms

Leukemic transformation (LT) is the main cause of death for patients with myeloproliferative neoplasms (MPNs). To study genetic changes associated with the LT, we performed targeted sequencing in 26 MPN patients including 21 with paired samples. We observed that, besides three driver genes, IDH2 (19%) and ASXL1 (14%) were also frequently mutated at MPN diagnosis. Although variant allele frequencies (VAFs) of mutations in DNA methylation and spliceosome did not expand during LT, they were enriched in patients with LT (the LT group). At follow-up, we also observed acquisition of mutations, mostly in the LT group. When considering dynamics of VAF from diagnosis to follow-up, VAFs in the LT group expanded (median VAF, 36.7–43.7%, p = 0.045). In contrast, mutations in patients with no clinical progression was stable (median VAF, 36.3–35.7%, p = 0.739). Overall, the present study demonstrates genetic changes during LT and provides the potential for prognostic application.

Screening and Carrier Rate of Neuronal Ceroid Lipofuscinosis in Chihuahua Dogs in Japan.

Simple SummaryNeuronal ceroid lipofuscinosis (NCL) is a rare group of lethal neurodegenerative lysosomal storage diseases, in which a homozygous single base-pair deletion (c.846delT) in the canine CLN7/MFSD8 gene has been identified as a causative mutation in Chihuahuas. In this study, we aimed to determine the carrier rate of NCL in Chihuahuas in Japan based on the screening of puppies using a newly designed real-time PCR assay. A survey conducted among 1007 Chihuahua puppies indicated that the carrier rate was 1.29%. Given the lethality of this disease, the mutant allele frequency (0.00645) is sufficiently high to warrant measures for disease control and prevention using the genotyping assay.Neuronal ceroid lipofuscinosis (NCL) is a group of rare lethal neurodegenerative lysosomal storage diseases that occur in a range of dog breeds, including Chihuahuas. Recently, a homozygous single base-pair deletion (c.846delT), which causes a frame shift generating a premature stop codon (p.Phe282Leufs13*) in the canine CLN7/MFSD8 gene, has been identified as a causative mutation for NCL in Chihuahuas. The objective of this study was to determine the frequency of the mutant allele and/or carrier rate of NCL in Chihuahuas in Japan using a newly designed real-time PCR assay. Samples of saliva were randomly collected from 1007 Chihuahua puppies during physical examinations prior to the transportation to pet shops. Screening results revealed a carrier rate of 1.29%, indicating a mutant allele frequency (0.00645) that is considered sufficiently high to warrant measures for the control and prevention of this lethal disease. The genotyping assay designed in this study could make a valuable contribution to the control and prevention of NCL.

Spatial heterogeneity of knockdown resistance mutations in the dengue vector Aedesalbopictus in Guangzhou, China

BackgroundThe city of Guangzhou has been the epicenter of dengue fever in China since the 1990s, with Aedesalbopictus being the primary vector. The main method used to control vectors and prevent dengue fever has been the application of chemical insecticides; however, this control strategy has resulted in the development of resistance to these insecticides in mosquitoes. Here we report our investigation of the patterns of knockdown resistance (kdr) mutations in 15 field populations of Ae.albopictus collected from 11 districts in Guangzhou.ResultsFour mutant alleles (V1016G, F1534S, F1534C, F1534L) were detected in domain II and III of the voltage-gated sodium channel (VGSC) gene. Various allele frequencies of kdr mutations were observed (3.1–25.9% for V1016G, 22.6–85.5% for F1534S, 0–29.0% for F1534L, 0.6–54.2% for F1534C). Seven kdr haplotypes (VF, VS, VL, VC, GF, GC, GS) were identified; the highest frequency of haplotypes was found for the single mutant haplotype VS (50.8%), followed by the wild-type VF haplotype (21.7%) and the single mutant haplotype VC (11.9%). Of the three double mutant haplotypes, GF was the most frequent (8.8%), followed by GC (1.2%) and GS (0.8%). Aedesalbopictus showed spatial heterogeneity in deltamethrin resistance in populations collected in Guangzhou. We also observed significant differences in haplotype frequency. The frequency of the VC haplotype was significantly higher in high-risk dengue areas than in low-risk ones.ConclusionsThe kdr allele V1016G was discovered for the first time in Guangzhou. Genetic isolation in mosquito populations and long-term insecticide selection seem to be responsible for the persistent, patchy distribution of kdr mutant alleles. The small-scale spatial heterogeneity in the distribution and frequency of kdr mutations may have important implications for vector control operations and insecticide resistance management strategies.Graphical

A Common Missense Variant Causing Factor XI Deficiency and Increased Bleeding Tendency in Maine Coon Cats.

Hereditary factor XI (FXI) deficiency is characterized as an autosomal mild to moderate coagulopathy in humans and domestic animals. Coagulation testing revealed FXI deficiency in a core family of Maine Coon cats (MCCs) in the United States. Factor XI-deficient MCCs were homozygous for a guanine to adenine transition resulting in a methionine substitution for the highly conserved valine-516 in the FXI catalytic domain. Immunoblots detected FXI of normal size and quantity in plasmas of MCCs homozygous for V516M. Some FXI-deficient MCCs experienced excessive post-operative/traumatic bleeding. Screening of 263 MCCs in Europe revealed a mutant allele frequency of 0.232 (23.2%). However, V516M was not found among 100 cats of other breeds. Recombinant feline FXI-M516 (fFXI-M516) expressed ~4% of the activity of wild-type fFXI-V516 in plasma clotting assays. Furthermore, fFXIa-M516 cleaved the chromogenic substrate S-2366 with ~4.3-fold lower catalytic efficacy (kcat/Km) than fFXIa-V516, supporting a conformational alteration of the protease active site. The rate of FIX activation by fFXIa-M516 was reduced >3-fold compared with fFXIa-V516. The common missense variant FXI-V516M causes a cross-reactive material positive FXI deficiency in MCCs that is associated with mild-moderate bleeding tendencies. Given the prevalence of the variant in MCCs, genotyping is recommended prior to invasive procedures or breeding.

Molecular Epidemiology of Na+-Taurocholate Cotransporting Polypeptide Deficiency in Guangdong Province, China: A Pilot Study by Screening for Four Prevalent Variants of the Causative Gene SLC10A1.

Na+-taurocholate cotransporting polypeptide deficiency (NTCPD) is an autosomal recessive disorder arising from biallelic SLC10A1 mutations. As a newly-described inborn error of bile acid metabolism, the epidemiology of this condition remains largely unclear in Chinese population so far. In this study, a total of 2,828 peripheral blood samples were collected from 12 cities in Guangdong, a province with the largest population in China, and the four prevalent SLC10A1 variants c.800C > T (p.Ser267Phe), c.263T > C (p.Ile88Thr), c.595A > C (p.Ser199Arg) and c.665T > C (p.Leu222Ser) were screened for by using polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP). As a result, 663 mutated SLC10A1 alleles were detected, and the mutated allele frequency was calculated to be 11.72% (663/5,656), with a carrier frequency 20.69% (1/5) and a theoretical morbidity rate 1.37% (1/73) of NTCPD in Guangdong province. The variant c.800C > T (p.Ser267Phe) exhibited highest allele frequency among the four prevalent variants (χ2 = 1501.27, p < 0.0001) as well as higher allele frequency in the peripheral region than that within the Pearl River Delta (χ2 = 4.834, p < 0.05). The results suggested that NTCPD might be a disorder rather common in Guangdong province. The findings depicted the molecular epidemiologic features of NTCPD, providing preliminary but significant laboratory evidences for the subsequent NTCPD diagnosis and management in Guangdong population.

Anchored Multiplex PCR Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich TERT promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the TERT promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on BRAF/NRAS/TERT promoter mutations, was 14/21 (67%) patient samples which included a TERT C250T mutation in one BRAF and NRAS mutation negative sample. A BRAF or NRAS mutation was detected in the ctDNA of 13/21 (62%) patients while TERT promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 BRAF/NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on BRAF, NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an NRAS/BRAF/TERT promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.

Next-generation T cell-activating vaccination increases influenza virus mutation prevalence.

To determine the potential for viral adaptation to T cell responses, we probed the full influenza virus genome by next-generation sequencing directly ex vivo from infected mice, in the context of an experimental T cell–based vaccine, an H5N1-based viral vectored vaccinia vaccine Wyeth/IL-15/5Flu, versus the current standard-of-care, seasonal inactivated influenza vaccine (IIV) and unvaccinated conditions. Wyeth/IL-15/5Flu vaccination was coincident with increased mutation incidence and frequency across the influenza genome; however, mutations were not enriched within T cell epitope regions, but high allele frequency mutations within conserved hemagglutinin stem regions and PB2 mammalian adaptive mutations arose. Depletion of CD4+ and CD8+ T cell subsets led to reduced frequency of mutants in vaccinated mice; therefore, vaccine-mediated T cell responses were important drivers of virus diversification. Our findings suggest that Wyeth/IL-15/5Flu does not generate T cell escape mutants but increases stochastic events for virus adaptation by stringent bottlenecks.

The role of clonal hematopoiesis as driver of therapy-related myeloid neoplasms after autologous stem cell transplantation.

Therapy-related myeloid neoplasm (t-MN) is a threatening complication of autologous stem cell transplantation (ASCT). Detecting clonal hematopoiesis (CH) mutations in cryopreserved cells before ASCT has been associated with a higher risk of t-MN, but the evolution of molecular abnormalities from pre-ASCT to t-MN, within the same patient, remains to be elucidated. We evaluated the mutational profile of 19 lymphoma/myeloma patients, at both pre-ASCT and t-MN diagnosis, using a targeted NGS approach; 26 non-developing t-MN control patients were also studied pre-ASCT. At ASCT, we found a higher frequency of CH in patients developing t-MN (58%) than in those who did not (23%) (P = 0.029); mutations in epigenetic (DNMT3A, TET2, and ASXL1) and DNA repair genes (PPM1D, RAD21, TP53, and STAG2) were the most represented. At t-MN, CH increased to 82% of patients. Cumulative mutational burden and variant allele frequency (VAF) also increased at t-MN. CH clones detected at ASCT were found at t-MN in eight out of 16 patients, mainly with stable VAF. Among the new driver mutations appeared at t-MN, TP53 increased from one to 13 mutations, in nine patients; being associated with complex karyotype. Mutations in transcription factor (RUNX1, CEBPA) and intracellular signaling genes (FLT3, RAS genes) also increased from three to 17 mutations in eight patients, presenting with a normal karyotype. Overall, we found that preexisting CH at ASCT rarely causes t-MN directly, but may rather facilitate the appearance of new mutations, especially those involving TP53, RUNX1, and RAS, that can drive the evolution to t-MN of at least two distinct types.

Droplet digital PCR-based analyses for robust, rapid, and sensitive molecular diagnostics of gliomas

Classification of gliomas involves the combination of histological features with molecular biomarkers to establish an integrated histomolecular diagnosis. Here, we report on the application and validation of a set of molecular assays for glioma diagnostics based on digital PCR technology using the QX200™ Droplet Digital™ PCR (ddPCR) system. The investigated ddPCR-based assays enable the detection of diagnostically relevant glioma-associated mutations in the IDH1, IDH2, H3-3A, BRAF, and PRKCA genes, as well as in the TERT promoter. In addition, ddPCR-based assays assessing diagnostically relevant copy number alterations were studied, including 1p/19q codeletion, gain of chromosome 7 and loss of chromosome 10 (+ 7/-10), EGFR amplification, duplication of the BRAF locus, and CDKN2A homozygous deletion. Results obtained by ddPCR were validated by other methods, including immunohistochemistry, Sanger sequencing, pyrosequencing, microsatellite analyses for loss of heterozygosity, as well as real-time PCR- or microarray-based copy number assays. Particular strengths of the ddPCR approach are (1) its high analytical sensitivity allowing for reliable detection of mutations even with low mutant allele frequencies, (2) its quantitative determination of mutant allele frequencies and copy number changes, and (3) its rapid generation of results within a single day. Thus, in line with other recent studies our findings support ddPCR analysis as a valuable approach for molecular glioma diagnostics in a fast, quantitative and highly sensitive manner.