Mutant Allele Frequency Research Articles

Biased expression of mutant alleles in cancer-related genes in esophageal squamous cell carcinoma.

Recent progress of large-scale international studies has provided comprehensive catalogs of somatic mutations in cancers. Additionally, it has become evident that allelic imbalance in the abundance of somatic mutations between DNA and RNA were pervasive in various types of cancer. However, the allelic imbalance of the abundance of somatic mutations in esophageal squamous cell carcinoma (ESCC) has not been fully analyzed. We performed exome sequencing for 25 Japanese patients with ESCC to detect a comprehensive catalog of somatic mutations in ESCC. Additionally, we performed mRNA sequencing to evaluate the allelic imbalance of the identified somatic mutations at the transcriptional level by comparing the mutant allele frequencies between RNA and DNA. The exome sequencing showed that TP53 and ZNF750 were significantly mutated genes. The expression levels of TP53 and ZNF750 were different depending on the mutation status. In almost all the tumors with missense mutations in TP53 and ZNF750, the mutant allele frequencies were higher in the RNA sequencing than those in the exome sequencing, indicating that the mutant alleles were preferentially expressed. By examining the allelic imbalances for all the identified missense mutations, we demonstrated that genes showing preferential expressions of the mutant alleles were involved in the pathways including cell cycle, cell death, and chromatin modification. The results of this study suggest that the allelic imbalance of the abundance of somatic mutations plays important roles in the initiation and progression of ESCC by modulating cancer-related biological pathways.

Predictive values of mutational variant allele frequency in overall survival and leukemic progression of myelodysplastic syndromes.

The implication of mutational variant allelic frequency (VAF) has been increasingly considered in the prognostic interpretation of molecular data in myeloid malignancies. However, the impact of VAF on outcomes of myelodysplastic syndromes (MDS) has not been extensively explored. Targeted next-generation sequencing was performed in 350 newly diagnosed MDS cases. The associations of mutational VAF of each gene with overall survival (OS) and leukemia-free survival (LFS) were examined by multivariate Cox regression after univariate analysis. Shorter OS was independently associated with DNMT3A VAF (HR 1.020 per 1% VAF increase; 95% CI 1.005-1.035; p = 0.011) and TP53 VAF (HR 1.014 per 1% VAF increase; 95% CI 1.006-1.022; p = 0.001). LFS analyses revealed that TET2 VAF (HR 1.013 per 1% VAF increase; 95% CI 1.005-1.022; p = 0.003) and TP53 VAF (HR 1.012 per 1% VAF increase; 95% CI 1.004-1.021; p = 0.005) were independently associated with faster leukemic transformation. Furthermore, we established nomograms to predict OS and LFS, respectively,by integrating independent mutational predictors into the revised International Prognostic Scoring System. Our study highlights that VAF of certain genes should be incorporated into routine clinical prognostication of survival and leukemic transformationof MDS.

Polymorphism of coupled indels in porcine TNNC2 alters its transcript splicing and is associated with meat quality traits.

The TNNC2 gene encodes the fast-skeletal C subunit of the troponin complex that plays a vital role in the regulation of striated muscle contraction and could be a candidate gene for pork quality. Here, we identified coupled insertion/deletion (indel) variants, a 17-bp insertion and an 11-bp deletion, in porcine TNNC2. The coupled indel variants provide an alternative splicing donor site and cause a 42-bp truncation in the first exon of TNNC2-201, leading to increased expression of TNNC2-201. Polymorphism of the two indel variants is associated with the average backfat thickness (p=3.16×10-3 ), pH value 24h post-slaughter (p=4.31×10-4 ), intramuscular fat (IMF) content (p=1.54×10-2 ), and myofiber cross-sectional area (p=2.86×10-2 ) of longissimus dorsi in a population of 425 Duroc (♂)×Luchuan (♀) pigs. In an independent population of 1,304 commercial hybrid pigs, we further confirmed that it is associated with the IMF content (p=1.75×10-4 ), pH value 45min post-slaughter (p=6.34×10-3 ), and drip loss (p=2.88×10-2 ) of the longissimus dorsi muscle. An increased frequency of the mutant allele is linked to increased IMF content, smaller myofibers, and a relatively moderate pH value. Furthermore, we detected a mutant allele frequency of 96.67% in Luchuan pigs and 86.67% in Tongcheng pigs, whereas the frequency was 0.91% in Duroc pigs, 2.04% in Landrace pigs, and 0% in Yorkshire and Pietrain pigs, indicating its opposing distributions in lean-type and Chinese local pig breeds. The present results establish coupled indel variants of TNNC2 as a novel molecular marker for meat quality improvement.

Prevalence and molecular characterization of alpha and beta-Thalassemia mutations among Hakka people in southern China.

Our aim was to investigate molecular features of thalassemia for proper clinical consultation and prevention in Heyuan. In our research, a total of 25,437 positive screening subjects were further subjected to a genetic analysis of α-thalassemia (α-thal) and β-thalassemia (β-thal). The deletion of α-thal mutation was tested by Gap-PCR, while the non-deletion of α-thal and β-thal mutation were identified by the PCR-reverse dot blot (PCR-RDB) technique. Nested PCR detected Hkαα/-- SEA and Hkαα/αα. Among the 25,437 positive screening subjects, 44.09% (11216/25437) subjects were bearers of thalassemia variations, and 30.85% (7847/25437) subjects showed α-thal changes alone. Among the 23 genotypes with α-thal mutation alone, the three common genotypes were --SEA/αα(68.34%), -α3.7/αα(16.44%), and -α4.2/αα(6.38%). Of the 11.50% (2924/25437) subjects and 29 genotypes with β-thal mutation alone, the three common genotypes were βCD41-42/βN(36.22%), βIVS-II-654/βN(30.88%), and β-28/βN(13.47%). Additionally, of the 1.75% (445/25437) subjects and 55 genotypes showed both α- and β-thal mutations. We also identified 269 cases of Hb H and six patients of Hkαα. Furthermore, the common genotypes of α-thal and β-thal mutations were consistent with allele frequencies of mutations. Our study establishes molecular features of thalassemia among Hakka people in Heyuan. It will be useful for developing strategies to prevent thalassemia.

Effect of MTTP -493G/T, I128T, Q95H and Q244E polymorphisms on hepatic steatosis in patients with chronic hepatitis

BackgroundChronic hepatitis C is characterized by a progressive deterioration of liver function and is involved in metabolic complications, such as hepatic steatosis. ObjectiveThe aim of this study was to investigate the role of host and viral characteristics associated with -493G/T (rs1800591), I128T (rs3816873), Q95H (rs61733139), and Q244E (rs17599091) Single Nucleotide Polymorphisms (SNPs) in the Microsomal Triglyceride Transfer Protein (MTTP) gene on hepatic steatosis in chronic hepatitis C. MethodsSNPs were genotyped by PCR-RFLP and analyzed in combination with host and viral characteristics by multiple logistic regression in different genetic models of inheritance. ResultsThe authors analyzed 236 patients with chronic hepatitis C, and 53% had hepatic steatosis. The mutated allele frequencies were > 5%, and the genotypes were in Hardy-Weinberg equilibrium (p ≥ 0.05). It was observed that patients with HCV genotype 3 infection (OR = 2.74, 95% CI 1.24‒6.06, p = 0.013), female sex (OR = 2.28, 95% CI 1.21‒4.28, p = 0.011) and moderate- and high-intensity liver inflammatory activity (A2-A3) (OR = 3.61, 95% CI 1.86‒7.01, p < 0.001) alone exhibited a higher risk of steatosis. The results of multiple logistic regression analysis for interaction showed that for the -493G/T SNP, when the GT/TT genotype (dominant model) and the GT genotype (codominant model) were each combined with HCV genotype 3 infection, an 11.51-fold (95% CI 2.08‒63.59, p = 0.005) and a 15.69-fold (95% CI 2.46‒99.85, p = 0.004) increased risk of steatosis, respectively, was observed. For the I128T SNP, when both the IT/TT genotype (dominant model) and the IT genotype (codominant model) were combined with HCV genotype 3 infection, an 8.51-fold (95% CI 1.59‒45.54, p = 0.012) and an 8.40 fold (95% CI 1.51‒46.91, p = 0.015) increased risk of steatosis, respectively, was observed. ConclusionThe present study showed that the viral genotype combined with the -493G/T and I128T SNPs in the MTTP gene influences hepatic steatosis.

Mature plasmacytoid dendritic cells associated with acute myeloid leukemia show similar genetic mutations and expression profiles to leukemia cells.

Introduction:Mature plasmacytoid dendritic cells (pDCs) proliferation associated with myeloid neoplasms (MPDMN) are recognized as a neoplasm related to fully differentiated pDCs. Although it has been reported for many years, the genomic landscape of MPDMN is poorly understood.Methods:We reported two patients who developed acute myeloid leukemia (French-American-British M5 subtype) coexisted with immunophenotypically mature pDCs proliferation, which fit the diagnosis of MPDMN. We sorted pDCs from myeloid blasts by flow cytometry and performed whole-exome sequencing and RNA sequencing of the two cell populations, respectively.Results:The immunophenotypes of pDCs in both patients were positive for CD123bri, HLA-DR, CD4, CD303, CD304, and negative for CD56, CD34, CD117, and TdT. The variant allele frequency of gene mutations in myeloid blasts and pDCs were similar. The expression data showed myeloid blasts clustered tightly with hematopoietic stem cells, and pDCs from patients clustered tightly with granulocyte-monocyte progenitors/common myeloid progenitor, rather than with pDCs from the GEO platform.Conclusion:Our study suggested that pDCs derived from the leukemic clone, evidenced by a shared mutation profile and similar transcriptional signatures between pDCs and concurrent myeloid blasts.

Mitochondrial sirtuins genetic variations and gastric cancer risk: Evidence from retrospective observational study

The purpose of the present study was to analyze the role of selected polymorphisms of SIRT3 and SIRT5 in gastric carcinogenesis. For this study, 500 blood samples of GC patients and 500 blood samples of healthy individuals were collected. Six selected polymorphisms of mitochondrial sirtuins were analyzed for analysis using Tetra-Arms PCR followed by DNA sequencing. Mutant allele frequencies of selected polymorphisms [rs3782116 (p<0.0001), rs6598072 (p<0.0001) and rs11246020 (p<0.0001), rs938222 (p=0.0136), rs3757261 (p=0.0005) and rs2841511 (p=0.0015)] were observed significant higher in GC patients vs controls. Haplotype analysis was performed, and 51 haplotypes were generated using haploview software. Among these haplotypes, eleven haplotypes were found associated with a significantly increased risk of GC. Furthermore, SNP-SNP interaction showed a significant correlation between studied SNPs and GC risk. Kaplan Meier analysis showed that mutant allele frequencies of selected polymorphisms are linked with a significant decrease in survival of GC patients CONCLUSIONS: It can be concluded that selected SNPs may be associated with enhanced risk of GC and hence can be potential prognostic markers for prognosis and predisposition of GC.

An ancient positively selected BMPRIB missense variant increases litter size of Mongolian sheep populations following latitudinal gradient.

New gene mutation origination is a driving force for the evolution of organisms. The effect of FecB mutation in BMPRIB gene on the litter size of sheep has been well known for a long time, each copy of the mutant allele increases litter size by 0.4-0.5. However, the origin and adaptive evolution mechanism of FecB mutation are still unclear. Here we carried on the thorough analysis on evolutionary features of BMPRIB gene and found that 150 species as a whole is under purifying selection while sheep lineage shows evidence of positive selection. The results of allele age estimation revealed that the FecB mutation in Mongolian sheep of China originated in Mongolian Plateau at about 5000years ago. Due the two shape drops in temperature subsequently, Mongolian sheep migrated from north to south following the northern nomadic people. Accordingly, the FecB mutant allele frequency increased, with the lowest in sheep locating at Mongolian plateau (0.01) and the highest in sheep locating at Yangtze River valley (0.96). In conclusion, the FecB mutation in Mongolian sheep of China originated in Mongolian Plateau at about 5000years ago, and the differentiated litter size of Mongolian sheep might be the result of adaptation to various environments during the migration following latitudinal gradient. This study may well exemplify selection on an ancient variation triggered by drastic ecological shifts, and is also helpful to analyze the adaptive evolution mechanism of economic traits of domestic animals and identify major genes and molecular markers.

A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency.

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Miyazaki K, Sato Y, Satoh H, Hizawa N. A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency. Contemporary Oncology/Współczesna Onkologia. 2022;26(1):88-89. doi:10.5114/wo.2022.115447. APA Miyazaki, K., Sato, Y., Satoh, H., & Hizawa, N. (2022). A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency. Contemporary Oncology/Współczesna Onkologia, 26(1), 88-89. https://doi.org/10.5114/wo.2022.115447 Chicago Miyazaki, Kunihiko, Yoshiharu Sato, Hiroaki Satoh, and Nobuyuki Hizawa. 2022. "A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency". Contemporary Oncology/Współczesna Onkologia 26 (1): 88-89. doi:10.5114/wo.2022.115447. Harvard Miyazaki, K., Sato, Y., Satoh, H., and Hizawa, N. (2022). A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency. Contemporary Oncology/Współczesna Onkologia, 26(1), pp.88-89. https://doi.org/10.5114/wo.2022.115447 MLA Miyazaki, Kunihiko et al. "A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency." Contemporary Oncology/Współczesna Onkologia, vol. 26, no. 1, 2022, pp. 88-89. doi:10.5114/wo.2022.115447. Vancouver Miyazaki K, Sato Y, Satoh H, Hizawa N. A long-term responding epidermal growth factor receptor mutated non-small cell lung cancer patient with extremely high mutation allele frequency. Contemporary Oncology/Współczesna Onkologia. 2022;26(1):88-89. doi:10.5114/wo.2022.115447.

Using All Our Genomes: Blood-based Liquid Biopsies for the Early Detection of Cancer.

The pursuit of highly sensitive and specific cancer diagnostics based on cell-free (cf) nucleic acids isolated from minimally invasive liquid biopsies has been an area of intense research and commercial effort for at least two decades. Most of these tests detect cancer-specific mutations or epigenetic modifications on circulating DNA derived from tumor cells (ctDNA). Although recent FDA approvals of both single and multi-analyte liquid biopsy companion diagnostic assays are proof of the tremendous progress made in this domain, using ctDNA for the diagnosis of early-stage (stage I/II) cancers remains challenging due to several factors, such as low mutational allele frequency in circulation, overlapping profiles in genomic alterations among diverse cancers, and clonal hematopoiesis. This review discusses these analytical challenges, interim solutions, and the opportunity to complement ctDNA diagnostics with microbiome-aware analyses that may mitigate several existing ctDNA assay limitations.

CYP3A4*2 Gene Polymorphism and its association with Clinical Phenotyping (temperament) Concept of Unani Medicine Philosophy in Indian Population

Phenotypic heterogeneity is one of the major concerns in personalized medicine. Variations in drug metabolism rates vary in every individual and have been associated with drug metabolizing gene polymorphism. CYP3A4*2 gene polymorphism in drug metabolism has been reported to be associated with a range of diseases. In this regard, the present investigation is aimed to determine the possible association of Unani philosophy of temperaments (clinical phenotypes) with CYP3A4*2 gene polymorphism in patients and controls. In the present study, 800 subjects were analyzed for CYP3A4*2 gene polymorphism by PCR-RFLP technique in four different clinical phenotypes (Sanguine, Phlegmatic, Choleric and Melancholic) as per unani philosophy. Mutant allelic frequencies of patients and controls of four unani temperaments are sanguine (Damavi) (0.19 vs 0.12), phlegmatic (Balghami) (0.21 vs 0.11), bilious (Safravi) (0.23 vs 0.11) and melancholic (Saudavi) (0.16 vs 0.11) subjects. Safravi (p&lt;0.04) and Balghami (p&lt;0.01) results are statistically significant. Bilious (Safravi) and phlegmatic (Balghami) patients showed significant association with CYP3A4*2 gene polymorphism with 1.76 and 2.04 odds risk. Further such studies are essential in unani regime for providing better treatment for many diseases which require personalized medicine based on phenotype-genotype correlation.

Screening and Functional Analysis of TEK Mutations in Chinese Children With Primary Congenital Glaucoma.

Purposes: Recent studies have suggested that loss-of-function mutations of the tunica intima endothelial receptor tyrosine kinase (TEK) are responsible for approximately 5% of primary congenital glaucoma (PCG) cases in diverse populations. However, the causative role of TEK mutations has not been studied in Chinese PCG patients. Here, we report the mutation spectrum of TEK after screening a large cohort of PCG patients of Chinese Han origin and analyze the identified variants in functional assays. Methods: TEK-targeted next-generation sequencing (NGS) was performed in 200 PCG patients. Candidate variants were prioritized by mutation type and allele frequency in public datasets. Plasmids containing wild type and identified variants of TEK were constructed and used to assess protein expression, solubility, receptor auto-phosphorylation, and response to ligand stimulation in cell-based assays. Results: Ten missense and one nonsense heterozygous variants were detected by NGS in 11 families. The clinical features of TEK variants carriers were comparable to that of TEK-mutated patients identified in other populations and CYP1B1-mutated individuals from in-house database. Functional analysis confirmed four variants involving evolutionarily conserved residues to be loss-of-function, while one variant (p.R1003H) located in tyrosine kinase domain seemed to be an activating mutation. However, our results did not support the pathogenicity of the other five variants (p.H52R, p.M131I, p.M228V, p.H494Y, and p.L888P). Conclusion: We provide evidence for TEK variants to be causative in Chinese PCG patients for the first time. Attention needs to be paid to TEK mutations in future genetic testing.

The Impact of IL28B Gene Polymorphisms on Drug Responses

The Impact of IL28B Gene Polymorphisms on Drug Responses

Mutant alleles differentially shape fitness and other complex traits in cattle

Mutant alleles (MAs) that have been classically recognised have large effects on phenotype and tend to be deleterious to traits and fitness. Is this the case for mutations with small effects? We infer MAs for 8 million sequence variants in 113k cattle and quantify the effects of MA on 37 complex traits. Heterozygosity for variants at genomic sites conserved across 100 vertebrate species increase fertility, stature, and milk production, positively associating these traits with fitness. MAs decrease stature and fat and protein concentration in milk, but increase gestation length and somatic cell count in milk (the latter indicative of mastitis). However, the frequency of MAs decreasing stature and fat and protein concentration, increasing gestation length and somatic cell count were lower than the frequency of MAs with the opposite effect. These results suggest bias in the mutations direction of effect (e.g. towards reduced protein in milk), but selection operating to reduce the frequency of these MAs. Taken together, our results imply two classes of genomic sites subject to long-term selection: sites conserved across vertebrates show hybrid vigour while sites subject to less long-term selection show a bias in mutation towards undesirable alleles.

Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays.

BackgroundTumor heterogeneity may lead to false negative test results for tissue biopsy-based companion diagnostic tests. Real-time polymerase chain reaction (PCR) and digital PCR assays are used to detect rare alleles in cell-free circulating DNA for liquid biopsies; however, those tests lack strong sensitivity at low allele frequencies. We show here a novel real-time digital PCR instrument that utilizes cycle-based amplification curves to further improve the sensitivity and quantification accuracy of digital PCR.MethodsThe novel real-time digital PCR instrument was compared to an endpoint digital PCR system to determine the sensitivity and quantification accuracy of both instruments. Samples were all thermal cycled on the real-time digital PCR instrument but were analyzed on both endpoint and real-time digital PCR instruments to compare the performance without introducing other variables. Contrived samples for epidermal growth factor receptor (EGFR) exon 19 deletion, T790M, and L858R point mutations as well as human epidermal growth factor receptor 2 (HER2) amplification were tested. Different mutant allele frequencies and wildtype to mutant gene copy number ratios were tested for EGFR and HER2, respectively.ResultsBy removing false positive datapoints using real-time amplification curves, real-time digital PCR improved sensitivity by lowering the baseline for wildtype samples. For EGFR 19del assay, samples with 2 or more fluorescein amidite (FAM) labeled positive wells are determined positive by real-time digital PCR, while a minimum of 5 FAM positive datapoints is needed by endpoint digital PCR. Improved limit of detection for EGFR 19del mutation was also observed. Real-time digital PCR also had better quantification accuracy and sensitivity, resulting in the mutant allele frequencies being closer to the expected values for all EGFR mutations, especially at very low allele frequencies. However, at high allele frequencies or for gene amplification assays, real-time digital PCR is comparable with endpoint digital PCR.ConclusionsThis novel technology with improved sensitivity is important and needed because it addresses current issues with liquid biopsy tests. Due to limited amounts of circulating tumor DNA (ctDNA) obtained for liquid biopsy tests, few copies of mutant alleles are expected. With the lower baseline of real-time digital PCR, false negative test results from tissue biopsy would be more effectively reduced, leading to more patients receiving the targeted therapy they need for better survival.

Monitoring Circulating Tumor DNA During Surgical Treatment in Patients with Gastrointestinal Stromal Tumors.

The majority of patients diagnosed with advanced gastrointestinal stromal tumors (GISTs) are successfully treated with a combination of surgery and tyrosine kinase inhibitors (TKIs). However, it remains challenging to monitor treatment efficacy and identify relapse early. Here, we utilized a sequencing strategy based on molecular barcodes and developed a GIST-specific panel to monitor tumor-specific and TKI resistance mutations in cell-free DNA and applied the approach to patients undergoing surgical treatment. Thirty-two patients with GISTs were included, and 161 blood plasma samples were collected and analyzed at routine visits before and after surgery and at the beginning, during, and after surgery. Patients were included regardless of their risk category. Our GIST-specific sequencing approach allowed detection of tumor-specific mutations and TKI resistance mutations with mutant allele frequency < 0.1%. Circulating tumor DNA (ctDNA) was detected in at least one timepoint in nine of 32 patients, ranging from 0.04% to 93% in mutant allele frequency. High-risk patients were more often ctDNA positive than other risk groups (P < 0.05). Patients with detectable ctDNA also displayed higher tumor cell proliferation rates (P < 0.01) and larger tumor sizes (P < 0.01). All patients who were ctDNA positive during surgery became negative after surgery. Finally, in two patients who progressed on TKI treatment, we detected multiple resistance mutations. Our data show that ctDNA may become a clinically useful biomarker in monitoring treatment efficacy in patients with high-risk GISTs and can assist in treatment decision making.

Abstract CC07-01: Whole exome and whole genome methylation sequencing of low-input cfDNA to implement precision medicine in metastatic castration resistant prostate cancer

Abstract Purpose Liquid biopsy has become increasingly important in cancer diagnosis, personalized medicine, and disease progression monitoring. Conventional liquid biopsy relies on targeted cancer gene panels which often contain fewer than 500 genes. Despite the revolutionary impact of liquid biopsy on cancer research and patient care, targeted gene panels may miss key novel mutations involved in cancer development and drug response, and other novel genomic alternations underlining cancer development, such as whole genome structural or epigenetic changes. Additionally, current commonly used methodologies require relatively large amounts of cell free DNA (cfDNA) input material, thus limiting its application when only small amounts of cfDNA input is available. Experimental Design cfDNA was isolated from whole blood collected from commercially available prostate cancer patients or age-matched healthy donors and evaluated using Predicine’s platform for whole exome sequencing, plasma WES, and compared to the targeted 500 gene PredicineATLAS panel (cfDNA input 10 or 30 ng). Additionally, low-input cfDNA (cfDNA input 10 or 20 ng) was also evaluated for whole genome methylation sequencing using PredicineECM and compared with standard whole genome bisulfite sequencing (WGBS). Results Plasma WES profiling of 30 or 10 ng cfDNA detected variants with mutation allele frequency (MAF) of 1% and above in the entire exome region, which enabled more accurate genome wide copy number variation prediction. All mutations detected using the PredicineATLAS panel that were ≥1% allele frequency were also detected by plasma WES, although low input required greater depth (10 ng, 3,500x) compared with high input (30 ng, 2500x depth). Furthermore, plasma WES identified 1.5 to 15.3x additional mutations compared with the PredicineATLAS panel. PredicineECM methylation analysis was superior to WGBS in reducing DNA damage and GC bias, resulting in increased NGS read mapping rate and quality score. Profiling of 2.5, 5 and 10 ng cfDNA using PredicineECM showed higher mapping rate and quality compared with WGBS profiling of 10 ng cfDNA. Additionally, clustering analysis of CpG methylation using PredicineECM separated tumor samples from normal samples and may serve as additional markers for minimal residue disease (MRD) tracing or early cancer detection. Conclusion In conclusion, we have successfully developed a multi-omics analysis platform for low-input cfDNA that integrates whole exome mutation and whole genome methylation profiling to obtain a comprehensive picture of cancer genomics to enable precision medicine in prostate cancer. This technology can also be applied to other cancers. Citation Format: Raju Kandimalla, Binggang Xiang, Chao Dai, Amy Wang, Pan Du, Shujun Luo, Debbie Liao. Whole exome and whole genome methylation sequencing of low-input cfDNA to implement precision medicine in metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC07-01.

Characterization of Blood-Based Molecular Profiling in Pancreatic Adenocarcinoma.

Background: Molecular profiling is being explored in pancreatic adenocarcinoma (PDAC) as a tool to assist with early detection, prognosis, and patient selection in targeted therapy clinical trials. Due to the challenges and risks of traditional tissue biopsies in pancreatic adenocarcinoma, the utility of blood-based molecular profiling is now being explored more broadly. However, given its novelty, what value blood-based molecular profiling may provide to oncologists caring for individuals with PDAC remains unknown. Herein, we characterize the mutational landscape of metastatic PDAC using blood-based circulating tumor DNA (ctDNA) collected in patients with refractory, metastatic PDAC who were referred to an oncology drug development unit in Denver, Colorado, between August 2014 and May 2019. Methods: We retrospectively analyzed results of blood-based molecular profiling that was performed on 77 consecutive patients with metastatic PDAC who underwent Guardant-360 testing for whom results were available. Results: In our data set, 55% of patients (41/77) were men, median (SD) age was 66 (9.3) years (range, 44-83). Of 77 patients, 34 (44%) had 1 or more somatic alterations. Variants reported as being of unknown significance were not included in the analyses. The total number of alterations were 119 (nonunique) and 96 (unique). The median number of alterations per patient was 3 and the median mutant allele frequency was 0.5%. TP53 was the most commonly altered gene (29 unique alterations), followed by KRAS (27 unique alterations). Of the patients with any alteration, 34% had 1 or more actionable alterations that could be potentially targeted in a clinical trial. Conclusions: Detection of genomic alterations in ctDNA from patients with metastatic PDAC is feasible and reveals a wide range of genomic alterations, the actionability of which is being explored in clinical trials. Further investigation is needed to determine the extent to which blood-based molecular profiling can provide clinical utility in helping to select patients into clinical trials and determine its impact on survival.

Organ Specific Copy Number Variations in Visceral Metastases of Human Melanoma.

Simple SummaryMalignant melanoma is a highly metastatic disease disseminating to several distant sites. This potential is also of great clinical impact for patient survival and therapeutic success. Knowledge about melanoma genomics is mainly based on lymphatic or skin metastases derived data, whereas data from distant sites is limited. Therefore, an autopsy-based visceral metastasis biobank was established, and an array-based copy number variation (CNV) analysis was performed, focusing primarily on major organs (brain, lung, and liver) and completed partly by proteomic analysis. A unique picture emerged about organ-specific CNV-type distributions or gene alterations, including the frequent loss of DNA damage error genes in brain metastases, the presence of HGF/MET autocrine loop in brain and lung metastases, the traces of immunogenic mimicry exclusive for lung metastases or the correlation of BRAF copy number and mutant allele frequency, especially in lung metastases. All these above phenomena have a great influence on therapy efficacy or resistance.Malignant melanoma is one of the most aggressive skin cancers with high potential of visceral dissemination. Since the information about melanoma genomics is mainly based on primary tumors and lymphatic or skin metastases, an autopsy-based visceral metastasis biobank was established. We used copy number variation arrays (N = 38 samples) to reveal organ specific alterations. Results were partly completed by proteomic analysis. A significant increase of high-copy number gains was found in an organ-specific manner, whereas copy number losses were predominant in brain metastases, including the loss of numerous DNA damage response genes. Amplification of many immune genes was also observed, several of them are novel in melanoma, suggesting that their ectopic expression is possibly underestimated. This “immunogenic mimicry” was exclusive for lung metastasis. We also provided evidence for the possible autocrine activation of c-MET, especially in brain and lung metastases. Furthermore, frequent loss of 9p21 locus in brain metastases may predict higher metastatic potential to this organ. Finally, a significant correlation was observed between BRAF gene copy number and mutant allele frequency, mainly in lung metastases. All of these events may influence therapy efficacy in an organ specific manner, which knowledge may help in alleviating difficulties caused by resistance.

A Target Site Mutation Associated With Diamide Insecticide Resistance in the Diamondback Moth Plutella xylostella (Lepidoptera: Plutellidae) is Widespread in South Georgia and Florida Populations.

Laboratory colonies of diamondback moth (DBM) larvae were established from larvae collected from four sites in Georgia and Florida where diamide, specifically chlorantraniliprole, insecticide resistance was recently documented. Based on dose-response experiments, these colonies exhibited 109- to 4,298-fold resistance to chlorantraniliprole, compared to a commercially available susceptible control colony. Colonies exhibited 50- to 107-fold resistance to another diamide, cyantraniliprole, based on similar dose-response experiments. All colonies were screened for the presence of four known mutations in the ryanodine receptor (RyR), the target of diamide insecticides, previously associated with resistance in Asian DBM populations. One mutation, G4946E, was identified in colonies from all four field sites, but not the susceptible control colony. Three additional RyR target site mutations, E1338D, Q4594L, and I4790M, were not identified in any of the screened samples. The estimated allele frequency of the G4946E mutation in these colonies ranged from 32 to 90%. These data are consistent with recently reported chlorantraniliprole control failures in Georgia and Florida. It is likely that the G4946E mutation is currently an important contributing factor to chlorantraniliprole resistance in Georgia and Florida DBM populations.