Allele Frequencies In Samples Research Articles

Rare germline genetic variation in PAX8 transcription factor binding sites and susceptibility to epithelial ovarian cancer.

Common genetic variation throughout the genome together with rare coding variants identified to date explain about a half of the inherited genetic component of epithelial ovarian cancer risk. It is likely that rare variation in the non-coding genome will explain some of the unexplained heritability, but identifying such variants is challenging. The primary problem is lack of statistical power to identifying individual risk variants by association as power is a function of sample size, effect size and allele frequency. Power can be increased by using burden tests which test for association of carriers of any variant in a specified genomic region. This has the effect of increasing the putative effect allele frequency. PAX8 is a transcription factor that plays a critical role in tumour progression, migration and invasion. Furthermore, regulatory elements proximal to target genes of PAX8 are enriched for common ovarian cancer risk variants. We hypothesised that rare variation in PAX8 binding sites are also associated with ovarian cancer risk, but unlikely to be associated with risk of breast, colorectal or endometrial cancer. We have used publicly available, whole-genome sequencing data from the UK 100,000 Genomes Project to evaluate the burden of rare variation in PAX8 binding sites across the genome. Data were available for 522 ovarian cancers, 2,984 breast cancers, 2,696 colorectal cancers, 836 endometrial cancers and 2253 non-cancer controls. Active binding sites were defined using data from multiple PAX8 and H3K27 ChIPseq experiments. We found no association between the burden of rare variation in PAX8 binding sites (defined in several ways) and risk of ovarian, breast or endometrial cancer. An apparent association with colorectal cancer was likely to be a technical artefact as a similar association was also detected for rare variation in random regions of the genome. Despite the null result this study provides a proof-of -principle for using burden testing to identify rare, non-coding germline genetic variation associated with disease. Larger sample sizes available from large-scale sequencing projects together with improved understanding of the function of the non-coding genome will increase the potential of similar studies in the future.

The expected sample allele frequencies from populations of changing size via orthogonal polynomials

In this article, discrete and stochastic changes in (effective) population size are incorporated into the spectral representation of a biallelic diffusion process for drift and small mutation rates. A forward algorithm inspired by Hidden-Markov-Model (HMM) literature is used to compute exact sample allele frequency spectra for three demographic scenarios: single changes in (effective) population size, boom-bust dynamics, and stochastic fluctuations in (effective) population size. An approach for fully agnostic demographic inference from these sample allele spectra is explored, and sufficient statistics for stepwise changes in population size are found. Further, convergence behaviours of the polymorphic sample spectra for population size changes on different time scales are examined and discussed within the context of inference of the effective population size. Joint visual assessment of the sample spectra and the temporal coefficients of the spectral decomposition of the forward diffusion process is found to be important in determining departure from equilibrium. Stochastic changes in (effective) population size are shown to shape sample spectra particularly strongly.

Abstract 5033: Sub-0.01% KRAS G12C dPCR variant detection by mutation specific blocker (MSB) cassettes

Abstract Purpose: Kirsten rat sarcoma viral oncogene homologue (KRAS) is one of the most well-known oncogenes and is found commonly mutated in numerous cancer subtypes. Despite its prevalence as a driver of disease state, until recently KRAS-targeting therapeutics have proven difficult to develop due to lack of specificity (Huang 2021). Now, several treatments have been approved for KRAS mutated cancers that are driven specifically by the G12C variant, and additional compounds are under investigation. Enrollment on these therapies is conditional on the G12C mutation presence, and thus accurate, sensitive detection of G12C is critical. Herein, we describe the development of KRAS G12C-targeting MSB dPCR assays capable of detecting G12C variants down to less than 0.01% variant allele frequency in whole blood, FFPE, and cfDNA samples. Methods: Multiple KRAS G12C MSB cassettes were developed, composed of four components: a mutation-specific forward primer with an engineered penultimate nucleotide mismatch, a non-extendable blocking oligo for the wild-type (WT) KRAS G12 sequence, a TaqMan probe, and a standard reverse primer. Initial cassette performance was assessed using synthetic KRAS G12C and KRAS G12 WT sequences inserted into an IDT plasmid backbone and tested directly or spiked into whole blood or extracted genomic DNA obtained from healthy donors prior to testing. Data obtained with the MSB cassettes across multiple matrices was compared to a BioRad off-the-shelf KRAS G12C droplet digital PCR (ddPCR) assay for accuracy. Cassette designs achieving the desired specificity and sensitivity were subsequently run on cell-free DNA samples possessing KRAS G12C, KRAS G12D, and WT sequences to evaluate performance in patient material. Results: The enhanced WT suppression design of the assay allowed the lower limit of detection and limit of blank to remain at 6 copies/reaction (cps/rxn) and 3 cps/rxn, respectively, across 30 ng/rxn to 350 ng/rxn gDNA background. At the theoretical maximum input of 200,000 cps/rxn, the assay can achieve 0.003% frequency detection in a single well, with robust detection down to 5 ng/rxn input, making it ideal for samples with limited availability. The assay demonstrated high accuracy, with a correlation coefficient ≥ 0.95 to known values and the BioRad reference assay, and high precision, with less than 10% coefficient of variation among allele frequencies of the same sample. Conclusion: With the emergence of targeted therapies for KRAS G12C driven cancers, detection of this mutation has become crucial in the diagnosis. While multiple methods are available for variant identification, MSB cassettes represent a powerful approach to achieve a sensitivity of molecular detection not possible with other techniques. This enhanced sensitivity directly impacts patient access to potentially life-saving therapies, ensuring all possible options are available to diagnose the patients sensitively and early. Citation Format: Ashley Beams, Kevin Lee, Samantha Addison, Nathan Riccitelli. Sub-0.01% KRAS G12C dPCR variant detection by mutation specific blocker (MSB) cassettes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5033.

Correlation between Peripheral Blood and Bone Marrow Somatic Mutations Among Patients with Suspected or Established Myelodysplastic Syndromes from the National MDS Study

INTRODUCTION: Myelodysplastic Syndromes (MDS) are caused by progressive clonal dominance of mutated hematopoietic stem cells. While assessment of mutations in peripheral blood (PB) is an established standard for monitoring patients with some diseases such as chronic myeloid leukemia, the sensitivity and precision of PB screening and monitoring for somatic mutations in patients with MDS and related conditions such as clonal cytopenias of undetermined significance (CCUS) is less certain. Multiple published guidelines require bone marrow (BM) evaluations for the above, an invasive procedure not often performed outside of clinical trials. We compared somatic mutations and variant allele frequencies (VAFs) in paired PB and BM samples to assess the utility of PB results as a surrogate for diagnosis and monitoring. METHODS: The National MDS Natural History Study (National MDS Study) is an observational longitudinal cohort study of cytopenic patients suspected of having a diagnosis of MDS. All patients have PB and BM sampled when entering the study, prior to formal diagnosis. For this study, paired PB and BM baseline DNA samples from 36 patients were assessed for somatic mutations. Only subjects with minimum of one variant detected in their BM were included in this study. DNA was extracted from PB using an automated FlexSTAR (AutoGen) and from BM cell pellets using a QIAamp DNA Mini Kit (QIAGEN). Targeted exon sequencing of 96 genes was performed using a NovaSeq 6000 at a mean coverage of >1,200X and a mean breadth (bases covered at ≥100X) of >99.9%. Reads were aligned against a patched version of the build GRCh38 using BWA-MEM. VarScan2 was used to detect single nucleotide variants and short insertions/deletions with a minimum VAF of 2% and 5%, respectively. Resulting variants from 53 genes were manually reviewed to retain likely disease-causing variants. Correlation analysis between PB and BM VAFs was performed using Pearson correlation and linear regression. To confirm paired samples were sourced from the same subject, the Somalier software using a set of highly polymorphic SNPs across different ancestries was utilized to ensure a high level of relatedness. RESULTS: The 36 patients included 10 (28%) with MDS, 2 (6%) with MDS/MPN, 1 (3%) with AML (<30% blasts), and 23 (64%) with CCUS. The median age was 73 years (Table 1). Five of 36 patients had circulating PB blasts. Among the 191 mutations identified, the most common were TET2 (27%), SRSF2 (13%), DNMT3A (9%), SF3B1 (8%), and ASXL1 (8%). Correlation analysis of mutations (n=180) shared between paired PB and BM samples indicated a linear relationship between VAFs detected in PB and BM (Pearson correlation r=0.95, Figure 1), with a slight increase in variance observed with increasing VAFs. The deviation from the perfect (grey line) to the observed (yellow line) linear fit indicated that PB VAFs underestimated VAFs in BM by approximately 23% overall (Figure 1). For example, a BM VAF of 0.20 translated to a mean PB VAF of 0.16 based on the regression fit. The lower sensitivity was confirmed when looking at the number of total missed variants (present in BM, absent in PB): 9 of 101 (9%). Two mutations were detected in PB, but not BM. These mutations and diagnoses were 1) ETNK1 (VAF 0.026) in a patient with MDS-EB-1; and 2) CBL (VAF 0.021) in a patient with MDS with isolated del5q. Next, we assessed the relationship between percent of detected mutations and minimum VAF that was detected using linear regression and found that the higher the minimum VAF, the lower the percent of missed mutations. For example, for a 0.1, 0.2, and 0.3 minimum VAF, 11%, 9%, and 8% of BM mutations were missed, respectively. There was no difference in concordance between MDS and CCUS. SUMMARY & CONCLUSIONS: PB can be used to reliably identify somatic mutations in patients with suspected or established MDS and related conditions. Linear correlation between PB and BM VAFs showed, unsurprisingly, that BM mutations with higher VAFs are more likely to be detected. If PB is positive, this method of variant detection may be used potentially to diagnose and monitor. However, if negative (or if VAF is less than 0.10), bone marrow genomics are still necessary to exclude detectable clonal mutations. For quantitative predictive tools that use VAFs, PB VAFs would need to be corrected to improve predictive results; our estimated linear regression equation could be used to guide this in a larger prospective study.

PoolHelper: An R package to help in designing Pool‐Seq studies

Abstract Next‐generation sequencing of pooled samples (Pool‐seq) is an important tool in population genomics and molecular ecology. In Pool‐seq, the relative number of reads with an allele reflects the allele frequencies in the sample. However, unequal individual contributions to the pool and sequencing errors can lead to inaccurate allele frequency estimates, influencing downstream analysis. When designing Pool‐seq studies, researchers need to decide the pool size (number of individuals) and average depth of coverage (sequencing effort). An efficient sampling design should maximise the accuracy of allele frequency estimates while minimising the sequencing effort. We describe a novel tool to simulate single nucleotide polymorphism (SNP) data using coalescent theory and account for sources of uncertainty in Pool‐seq. We introduce an R package, poolHelper, enabling users to simulate Pool‐seq data under different combinations of average depth of coverage and pool size, accounting for unequal individual contributions and sequencing errors, modelled by adjustable parameters. The mean absolute error is computed by comparing the sample allele frequencies obtained based on individual genotypes with the frequency estimates obtained with Pool‐seq. poolHelper enables users to simulate multiple combinations of pooling errors, average depth of coverage, pool sizes and number of pools to assess how they influence the error of sample allele frequencies and expected heterozygosity. Using simulations under a single population model, we illustrate that increasing the depth of coverage does not necessarily lead to more accurate estimates, reinforcing that finding the best Pool‐seq study design is not straightforward. Moreover, we show that simulations can be used to identify different combinations of parameters with similarly low mean absolute errors. This can help users to define an effective sampling design by using those combinations of parameters that minimise the sequencing effort. The poolHelper package provides tools for performing simulations with different combinations of parameters (e.g. pool size, depth of coverage, unequal individual contribution) before sampling and generating data, allowing users to define sampling schemes based on simulations. This allows researchers to focus on the best sampling scheme to answer their research questions. poolHelper is comprehensively documented with examples to guide effective use.

CtDNA for clonal evolution surveillance and therapeutic efficacy prediction in unresectable locally advanced non-small cell lung cancer under radiotherapy.

e20555 Background: Therapeutic efficacy differs across unresectable locally advanced non-small cell lung cancer (LA-NSCLC) patients receiving definitive radiotherapy/chemoradiotherapy (RT/CRT), but the underlying resistance mechanism, clonal evolution and molecular prognostic factors remain poorly understood. Methods: Unresectable LA-NSCLC patients treated with definitive RT/CRT were consecutively enrolled between May 2018 and November 2020. Next-generation sequencing covering 474 cancer-relevant genes was performed on plasma ctDNA collected at pre-treatment (TP0), 4-week post-RT (TP1), 1-month post-RT (TP2), 3-month post-RT (TP3), and progression (TP4). Main study endpoints included disease progression, progression-free survival (PFS), death from any cause, overall survival (OS), distant metastasis and distant metastasis free survival. Fisher’s exact test and two-sample t-tests were performed to compare the frequencies and means of independent groups, respectively. The median follow-up time was estimated using the reverse Kaplan-Meier method. Hazard ratios (HR) with 95% confidence intervals (CI) were estimated by Cox proportional hazards models. Results: A total of 46 patients were included (median age: 63 [range: 40–80], males: 87%, clinical stage IIIB: 52%, squamous cell carcinoma: 54%, TP0 ctDNA shedding: 63%, consolidation immunotherapy: 33%). Twenty-nine (63%) patients developed disease progression by the end of follow-up (median follow-up: 23.7 months), with median PFS and OS being 15.5 and 25.4 months, respectively. The polymerase epsilon ( POLE) mutational signature at TP0 indicated inferior PFS (HR: 5.32, 95% CI: 1.83–15.45) and worse OS (HR: 20.68, 95% CI: 3.94–108.62). Notably, newly-discovered/lost mutations were rarely observed at TP4 when compared to TP0. Patients with a high ratio of mean over max variant allele frequency (m/mVAF) in TP0 samples, indicative of a clonal predominant tumor with a low subclone fraction ( P< 0.01), had relatively poor OS, compared to patients with low m/mVAF (HR: 2.92, 95% CI: 0.82–10.46) or undetectable ctDNA (HR: 3.39, 95% CI: 0.98–11.75). The prognostic value was validated in an independent publicly available dataset of 431 LA-NSCLC patients receiving definitive RT/CRT, showing patients with high m/mVAF demonstrated worse OS than those with low m/mVAF (HR: 1.43, 95% CI: 1.08–1.89). Additionally, increased m/mVAF between TP0 and TP1 indicated an elevated risk for disease progression ( vs. non-increase, HR: 3.49, 95% CI: 1.20–10.16), especially for distant metastasis ( vs. non-increase, HR: 2.85, 95% CI: 0.84–9.69). Conclusions: Our findings demonstrated the clinical value of plasma ctDNA and its derived molecular features in delineating clonal evolution, monitoring therapeutic resistance, and predicting prognosis of LA-NSCLC under definitive RT.

The size and composition of haplotype reference panels impact the accuracy of imputation from low-pass sequencing in cattle

BackgroundLow-pass sequencing followed by sequence variant genotype imputation is an alternative to the routine microarray-based genotyping in cattle. However, the impact of haplotype reference panels and their interplay with the coverage of low-pass whole-genome sequencing data have not been sufficiently explored in typical livestock settings where only a small number of reference samples is available.MethodsSequence variant genotyping accuracy was compared between two variant callers, GATK and DeepVariant, in 50 Brown Swiss cattle with sequencing coverages ranging from 4- to 63-fold. Haplotype reference panels of varying sizes and composition were built with DeepVariant based on 501 individuals from nine breeds. High-coverage sequence data for 24 Brown Swiss cattle were downsampled to between 0.01- and 4-fold to mimic low-pass sequencing. GLIMPSE was used to infer sequence variant genotypes from the low-pass sequencing data using different haplotype reference panels. The accuracy of the sequence variant genotypes that were inferred from low-pass sequencing data was compared with sequence variant genotypes called from high-coverage data.ResultsDeepVariant was used to establish bovine haplotype reference panels because it outperformed GATK in all evaluations. Within-breed haplotype reference panels were more accurate and efficient to impute sequence variant genotypes from low-pass sequencing than equally-sized multibreed haplotype reference panels for all target sample coverages and allele frequencies. F1 scores greater than 0.9, which indicate high harmonic means of recall and precision of called genotypes, were achieved with 0.25-fold sequencing coverage when large breed-specific haplotype reference panels (n = 150) were used. In absence of such large within-breed haplotype panels, variant genotyping accuracy from low-pass sequencing could be increased either by adding non-related samples to the haplotype reference panel or by increasing the coverage of the low-pass sequencing data. Sequence variant genotyping from low-pass sequencing was substantially less accurate when the reference panel lacked individuals from the target breed.ConclusionsVariant genotyping is more accurate with DeepVariant than GATK. DeepVariant is therefore suitable to establish bovine haplotype reference panels. Medium-sized breed-specific haplotype reference panels and large multibreed haplotype reference panels enable accurate imputation of low-pass sequencing data in a typical cattle breed.

Abstract PD17-01: Genomic analysis of circulating tumor DNA (ctDNA) from patients with HR+, HER2-mutant metastatic breast cancer (MBC) enrolled in SUMMIT: mechanisms of acquired resistance to neratinib + fulvestrant + trastuzumab (N+F+T)

Abstract Background: In the SUMMIT trial, original cohorts of patients with HR+, HER2-negative (locally assessed), HER2-mutant MBC received N alone or N+F, with promising clinical response rates but abbreviated duration. Clinical progression coincided with emergence of additional HER2 mutations and/or amplification of the mutant allele [Smyth et al. Cancer Discov 2020;10:198–213]. Addition of T to the combination was postulated to prolong response; the combination of N+F+T in heavily pretreated patients with HR+, HER2-mutant MBC who had received CDK4/6 inhibitors (n=51) yielded a confirmed overall response rate (ORR) of 35.3%, median duration of response (DOR) of 14.3 months, clinical benefit rate (CBR) of 47.1%, and median progression-free survival (PFS) of 8.2 months [Jhaveri et al. J Clin Oncol 2022;40:1028]. Seven of these 51 patients were part of a randomized (1:1:1) cohort who received N+F+T, F+T, or F alone. Patients randomized to F+T or F could crossover to N+F+T upon progression. No patients responded to F or F+T; however, one of four patients who crossed over to N+F+T upon progression on F+T responded to the triplet, as did two of six who crossed over upon progression on F. We undertook ctDNA sequencing in patients who responded to N+F+T upfront and after crossover. Methods: Patients with HR+, HER2-negative MBC with activating HER2 mutation(s) and prior CDK4/6i received N+F+T (oral N 240 mg/d with loperamide prophylaxis, im F 500 mg d1, d15, and d29 of cycle 1 then q4w, iv T 8 mg/kg initially then 6 mg/kg q3w) or F+T or F alone. Efficacy endpoints included investigator-assessed ORR and CBR (RECIST v1.1), DOR, and best overall response. ctDNA was collected at baseline, every third cycle during treatment, and at the end of treatment and analyzed by next-generation sequencing. Samples were analyzed using the TEMPUS xF assay. Results: Sequencing results from ctDNA analysis are pending at the time of this abstract submission. Baseline HER2 mutations and co-alterations will be reported and compared with those found in tissue samples. Genomic spectrum and variant allele frequencies in samples taken at baseline, on treatment, and at the end of treatment from patients who experienced complete or partial response to N+F+T and then progressed (n=25) will be sequenced and mechanisms of acquired resistance will be posited. ctDNA genomic profiles of serial time points from patients randomized to F or F+T before and after crossover to N+F+T (n=10) will also be evaluated. Conclusions: Similarities and differences between the mechanisms of acquired resistance to N+F+T, and those previously reported to be associated with progression on N or N+F, will be discussed. Citation Format: Cynthia Ma, James Waisman, Adam M. Brufsky, Eddy S. Yang, Hans Wildiers, John P. Crown, Sarina A. Piha-Paul, Jennifer M. Suga, José Ángel García-Sáenz, Valentina Gambardella, Angel Guerrero, Salomon Stemmer, Ron Bose, Tonya Novara-Demgen, Daniel DiPrimeo, Lisa D. Eli, Komal Jhaveri. Genomic analysis of circulating tumor DNA (ctDNA) from patients with HR+, HER2-mutant metastatic breast cancer (MBC) enrolled in SUMMIT: mechanisms of acquired resistance to neratinib + fulvestrant + trastuzumab (N+F+T) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-01.

Association of GCK (rs1799884), GCKR (rs780094), and G6PC2 (rs560887) Gene Polymorphisms with Type 2 Diabetes among Malay Ethnics.

Background Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder, and the underlying causes remain unknown and have not been fully elucidated. Several candidate genes have been associated with T2DM in various populations with conflicting results. The variations found in glucokinase ( GCK ), glucokinase regulatory protein ( GCKR ), and glucose-6-phosphatase 2 ( G6PC2 ) genes were not well studied, particularly among Asians. Aims The main objective of this study was to determine the candidate genetic polymorphisms of GCK (rs1799884), GCKR (rs780094), and G6PC2 (rs560887) genes in T2DM among Malay ethnics. Methods In this candidate gene association study, a total of 180 T2DM subjects and 180 control subjects were recruited to determine the genotypes using polymerase chain reaction-restriction fragment length polymorphism and Taqman probe assay methods. Genotype and allele frequencies in case and control samples were compared using the chi-squared test to determine a significant difference. Results The body mass index, fasting blood glucose, hemoglobin A1c, systolic and diastolic blood pressure, and total cholesterol were significantly different ( p < 0.05) between T2DM and control subjects. The genotypic and allelic frequencies of GCK (rs1799884), GCKR (rs780094), and G6PC2 (rs560887) gene polymorphisms were significantly different between T2DM and controls ( p < 0.05). Conclusion Hence, rs1799884 of GCK gene and rs780094 of GCKR gene and rs560887 of the G6PC2 gene are possible genetic biomarkers in T2DM development among Malay ethnics in Malaysia.

Assessment of tissue and blood tumor mutational burden in patients with melanoma using a 523-gene clinical assay.

e21570 Background: High tumor mutation burden (TMB) predicts improved efficacy of immune checkpoint inhibitors (ICIs) across a range of malignancies, including melanoma. Circulating tumor DNA (ctDNA) has emerged as a powerful complement to tumor biopsies for the diagnosis and clinical management of cancer patients. The TruSight Oncology 500 (TSO500) clinical assay interrogates 523 cancer-related genes and has shown good concordance between tissue and ctDNA using a high throughput sequencer such as NovaSeq. There are no studies looking at concordance and utility of this assay using more affordable mid throughput platforms. Here we present a pilot study comparing TSO500 sequencing results from matched tumor and plasma specimens and concordance of selected actionable gene variants investigated by ctDNA digital PCR. Methods: Samples from 18 metastatic cutaneous melanoma patients with matched formalin-fixed, paraffin-embedded (FFPE) tumor tissue and plasma collected in Streck tubes had DNA extracted for library preparation using the Illumina TSO500 NGS assay with inputs of ≥40ng of FFPE DNA and ≥10ng of ctDNA. Libraries were sequenced on a NextSeq2000 P3 flow cell to achieve recommended sequencing depth of 800 million reads per sample. A TMB cut off ≥10 mut/Mb was used as high TMB. Droplet digital PCR (ddPCR) was performed for orthogonal validation of selected clinically significant somatic mutations. Results: Sequencing on the NextSeq2000 achieved an average of 797 million pass filter reads per sample and somatic variant allele frequency (VAF) sensitivity down to 0.12%, which exceeded vendor recommendations. There was 75% concordance on the TMB classification (low and high values, Cohen’s k = 0.714) based on tumor and plasma (r = 0.86, p &lt; 0.01) on 8 paired samples (extended analysis will be presented). Some high TMB cases carried POLE or POLD1 variants. Actionable variants identified by ctTSO500 in 15 cases were confirmed by ddPCR, with an outstanding concordance of their VAF (R2= 0.99, p &lt; 0.0001). Diagnosis of tier I/II actionable variants (21 variants; 81% accuracy) was achieved in 7 matched tumor tissue and blood samples. Some variants found in tumor tissue were not detected in the blood. Conclusions: This pilot study demonstrates the clinical utility of the TSO500 ctDNA panel using an affordable mid throughput sequencing platform to identify actionable variants and TMB from blood specimens with high sensitivity and specificity. Testing of a larger cohort of matched tumor and plasma samples will serve to evaluate the clinical utility of the TSO500 ctDNA panel as a complement or alternative strategy to tissue biopsy for the genomic profiling of melanoma patients.

Genomic Alteration Spectrum of Non-Small Cell Lung Cancer Patients in East-China Characterized by Tumor Tissue DNA and Cell-Free DNA.

IntroductionFrom an oncologic perspective, genetic detection is becoming a frontline clinical test, used to identify actionable alterations for targeted therapy, monitor molecular clonal tumor evolution, indicate disease progression and prognosis, and predict medication efficacy and resistance. From analysis of both tumor tissue and cell-free DNA from a large cohort of non-small cell lung cancer patients in East-China, we characterized the full spectrum of genomic alterations.MethodsThe study comprised 3000 unpaired samples including 1351 tumor tissue DNA (tDNA) and 1649 cell-free circulating tumor DNA (cfDNA) samples, from which 67 cancer-related genes were sequenced and the genetic alteration profiles were depicted. Integrative molecular analyses identified the frequently mutated genes, uncovered co-occurring somatic alterations, described the distribution of hotspot variants, analyzed the frequency of variant alleles, and notably distinguished actionable, novel variants.ResultsThe most commonly affected genes were EGFR, TP53, KRAS, CDKN2A, and PIK3CA in both tDNA and cfDNA samples. EGFR and CTNNB1, PIK3CA and PTEN, ERBB2 and SMO were found to have frequent co-occurring alterations in tDNA samples, while EGFR and SMO, KRAS and PDGFRA, PIK3CA and KDR were in cfDNA samples. A large number of primary druggable variants were identified in tDNA samples, while numerous drug-resistance variants, rare actionable variants, and non-EGFR actionable variants were detected in cfDNA samples. Novel variants were enriched in KDR, KIT, TP53, ABL1, FGFR1 in tDNA samples while the majority of novel variants were distributed in PDGFRA, EGFR, KIT, ROS1, BRCA2 in cfDNA samples. Variant allele frequency in tDNA samples was significantly (P < 0.001) higher than that in cfDNA samples.ConclusionThe results revealed considerable differences in the alteration characteristics between the two kinds of specimens. To date, this study represents the largest real-world investigation of its kind, derived from the largest number of patients in East-China. It reinforced and expanded the mechanism of molecular analysis of neoplastic genetic profiles.

Freqpcr: Estimation of population allele frequency using qPCR ΔΔCq measures from bulk samples.

PCR techniques, both quantitative (qPCR) and nonquantitative, have been used to estimate the frequency of a specific allele in a population. However, the labour required to sample numerous individuals and subsequently handle each sample renders the quantification of rare mutations (e.g., pesticide resistance gene mutations at the early stages of resistance development) challenging. Meanwhile, pooling DNA from multiple individuals as a “bulk sample” combined with qPCR may reduce handling costs. The qPCR output for a bulk sample, however, contains uncertainty owing to variations in DNA yields from each individual, in addition to measurement errors. In this study, we have developed a statistical model to estimate the frequency of the specific allele and its confidence interval when the sample allele frequencies are obtained in the form of ΔΔCq in the qPCR analyses on multiple bulk samples collected from a population. We assumed a gamma distribution as the individual DNA yield and developed an R package for parameter estimation, which was verified using real DNA samples from acaricide‐resistant spider mites, as well as a numerical simulation. Our model resulted in unbiased point estimates of the allele frequency compared with simple averaging of the ΔΔCq values. The confidence intervals suggest that dividing the bulk samples into more parts will improve precision if the total number of individuals is equal; however, if the cost of PCR analysis is higher than that of sampling, increasing the total number and pooling them into a few bulk samples may also yield comparable precision.

Efficient approaches for large-scale GWAS with genotype uncertainty.

Association studies using genetic data from SNP-chip-based imputation or low-depth sequencing data provide a cost-efficient design for large-scale association studies. We explore methods for performing association studies applicable to such genetic data and investigate how using different priors when estimating genotype probabilities affects the association results. Our proposed method, ANGSD-asso’s latent model, models the unobserved genotype as a latent variable in a generalized linear model framework. The software is implemented in C/C++ and can be run multi-threaded. ANGSD-asso is based on genotype probabilities, which can be estimated using either the sample allele frequency or the individual allele frequencies as a prior. We explore through simulations how genotype probability-based methods compare with using genetic dosages. Our simulations show that in a structured population using the individual allele frequency prior has better power than the sample allele frequency. In scenarios with sequencing depth and phenotype correlation ANGSD-asso’s latent model has higher statistical power and less bias than using dosages. Adding additional covariates to the linear model of ANGSD-asso’s latent model has higher statistical power and less bias than other methods that accommodate genotype uncertainty, while also being much faster. This is shown with imputed data from UK Biobank and simulations.

Rank-invariant estimation of inbreeding coefficients

The two alleles an individual carries at a locus are identical by descent (ibd) if they have descended from a single ancestral allele in a reference population, and the probability of such identity is the inbreeding coefficient of the individual. Inbreeding coefficients can be predicted from pedigrees with founders constituting the reference population, but estimation from genetic data is not possible without data from the reference population. Most inbreeding estimators that make explicit use of sample allele frequencies as estimates of allele probabilities in the reference population are confounded by average kinships with other individuals. This means that the ranking of those estimates depends on the scope of the study sample and we show the variation in rankings for common estimators applied to different subdivisions of 1000 Genomes data. Allele-sharing estimators of within-population inbreeding relative to average kinship in a study sample, however, do have invariant rankings across all studies including those individuals. They are unbiased with a large number of SNPs. We discuss how allele sharing estimates are the relevant quantities for a range of empirical applications.

Clinical outcomes of patients taking first-generation EGFR-TKIs may predict the benefits afforded by osimertinib in EGFR T790M-mutant NSCLC patients.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are widely used to treat patients with EGFR-mutated non-small cell lung cancers (NSCLCs). The association between the clinical outcomes of patients on first-line EGFR-TKIs and the efficacy of osimertinib as second-line treatment has not been previously assessed. This is our topic here. We retrospectively analysed 67 patients with EGFR mutations on osimertinib after treatment with first-generation EGFR-TKIs. We evaluated patient characteristics, the EGFR T790M allele frequency in plasma samples and clinical outcomes. When osimertinib was given as second-line treatment, the median progression-free survival (PFS) was 6.0months, and the response rate and disease control rate were 32.8% and 91.0%, respectively. Correlation analysis showed that the female sex and isolated (not multiple) progression on first-line EGFR-TKIs were correlated with a superior response to osimertinib. Kaplan-Meier analysis showed that patients exhibiting a partial response, isolated progression, and longer PFS on first-line EGFR-TKIs experienced prolonged PFS on osimertinib. Univariate analysis indicated that the treatment response, PFS and progression when on first-line EGFR-TKIs affected the PFS on osimertinib. Multivariate analysis showed that progression when on first-line EGFR-TKIs was independently prognostic of a response to osimertinib. The median PFS of patients with isolated progressive disease PD alone who were receiving brain radiotherapy was significantly longer than that of patients with isolated progressive disease alone who did not receive brain radiotherapy as well as patients exhibiting multiple progression. A low frequency of the EGFR T790M allele in plasma tended to predict an inferior efficacy of osimertinib and shorter PFS. We found that patients who benefited from first-line EGFR-TKIs may experience prolonged PFS and a higher response rate when subsequently given osimertinib. A low plasma frequency of the EGFR T790M allele may predict poor osimertinib efficacy and shorter PFS.

Association between single nucleotide polymorphisms of nLvALF1 and PEN2-1 genes and resistance to Vibrio parahaemolyticus in the Pacific white shrimp Litopenaeusvannamei

Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease (AHPND) in penaeid shrimps. In the Pacific white shrimp farming industry, one possible strategy to reduce economic loss due to AHPND is the development of a shrimp line resistant to the disease. In this study, we identified single nucleotide polymorphisms (SNPs) in the Litopenaeus vannamei anti-lipopolysaccharide factor 1 (nLvALF1) and penaeidin 2-1 (PEN2-1) genes, and we analyzed the associations between these SNPs and resistance/susceptibility to V. parahaemolyticus infection in the Pacific white shrimp. Postlarvae (PL20) shrimp from a local hatchery in Prachuap Khiri Khan Province were challenged with an isolate of VPAHPND and mortality was observed for 14 days. DNA was extracted from susceptible (died within 6 days) and resistant (survived the challenge) shrimp (45 individuals/group) and used for PCR amplifications of nLvALF1 (397 bp) and PEN2-1 (637 bp) gene fragments. PCR products were sequenced by direct sequencing and SNPs were identified from sequencing chromatograms. Nine and seven SNPs were identified in nLvALF1 and PEN2-1 gene fragments, respectively. Analyses of allele frequencies in susceptible and resistant samples using Chi-square tests revealed that four and six SNPs in nLvALF1 and PEN2-1, respectively, were associated with resistance/susceptibility to V. parahaemolyticus infection (P < 0.05). The SNPs in the candidate genes identified here are potential DNA markers for breeding V. parahaemolyticus-resistant Pacific white shrimp in the study population; however, further validation will be required if these SNPs are to be used across populations.

Liquid Biopsy for the Identification of Intravascular Large B-Cell Lymphoma

[Backgrounds] Intravascular large B cell lymphoma (IVLBCL) is a rare subtype of extranodal diffuse large B-cell lymphoma (DLBCL) that features lymphoma cells lodged in lumina of small vessels. Because of the lack of mass formation, diagnosis by tumor biopsy is often difficult, and thus, cell-free DNA (cfDNA) analysis might be useful for assisting diagnosis of IVLBCL. In this study, we first aimed at investigating the prevalence of mutations in genes involved in the B-cell receptor, Toll-like receptor/interleukin-1 receptor, and nuclear factor kappa B pathways. We then evaluated the liquid biopsies for detection of hot-spot mutations by droplet digital polymerase chain reaction (ddPCR).[Patients and Methods] Twenty-seven IVLBCL patients whose archived samples were available were included in this study. The patients were diagnosed as IVLBCL from January 2005 to May 2017 in University of Tsukuba Hospital, JA Toride Medical Center, and Kameda Medical Center. The study was approved by the review board in each institution, and conducted according to the Declaration of Helsinki. Tissue-derived DNA (tdDNA) was extracted from formalin-fixed paraffin-embedded specimens and/or cryo-preserved bone marrow samples, in which the lymphoma cell infiltration had been pathologically confirmed. cfDNA was extracted from 1 ml of serum or plasma obtained prior to chemotherapy. We performed targeted sequence for 8 genes (B2M, BTG2, CARD11, CD79B, MYD88, PIM1, PRDM1, and TNFAIP3) in paired tdDNA and cfDNA from 9 IVLBCL subjects by Ion Torrent Personal Genome Machine (Thermo Fisher Scientific). MYD88 L265P (c.794T>C) mutational analysis was performed by ddPCR assays (BioRad) for 46 samples in total collected from 27 subjects. Statistical analysis was performed with EZR version 1.3.2. Paired t-test was used to compare variant allele frequencies (VAFs) in paired samples.[Results] All cases were categorized as the non-GCB by Hans classification. Random skin biopsies (RSB) and bone marrow biopsies (BMB) were performed in 21 and 27 patients. The diagnostic yield of RSB was 81.0% (17 of 21). Initial BMB detected lymphoma cells in 48.1% (13/27) patients, but repeated BMB increased successful detection up to 66.7% (18/27). tdDNA was available in 26 out of 27 subjects, and in 9 of which paired cfDNA was also available (Figure 1). In one subject, only cfDNA but not tdDNA was available. Targeted sequencing for 8 genes demonstrated at least one mutation in CD79B, MYD88, PIM1, or PRDM1 in both tdDNA and cfDNA, or only in cfDNA, in 8 of 9 subjects (Figure 2). We identified 3 missense CD79B mutations in 6 (66.7%; Y196C in 2, Y196H in 3, and L199P in 1). All MYD88 mutations were L265P, which were found in 5 of 9 (55.6%). Together, either Y196 CD79B or L265P MYD88 mutation was found in 7 of 9 (77.8%). PIM1 mutations and PRDM1 mutations were seen each in 3 (33.3%). VAFs were higher in cfDNA than in tdDNA (mean, 45.6% vs. 3.7%; P=0.0006, Figure 3). Four mutations in 3 patients were detected only in cfDNA. On the other hand, all variants detected in tdDNA were confirmed in cfDNA. ddPCR assay for the L265P MYD88 mutation with all 46 samples including tdDNA and cfDNA revealed the L265P MYD88 mutation in 24/46 (52.2%) samples from 16/27 (59.3%) IVLBCL patients. Furthermore, sequential analysis of cfDNA was performed in one of the MYD88 -mutated patients. The L265P MYD88 mutation was detected (VAF=30%) in cfDNA collected 17 weeks before the diagnosis of IVLBCL, whereas the mutation was not detected in DNA derived from the bone marrow sample that was collected at the same timing and resulted in the negative pathological finding.[Conclusion] Mutations in MYD88 and CD79B are frequent in IVLBCL . Targeted sequencing suggested that tumor cell-derived DNA is abundant in serum/plasma. Liquid biopsy to detect L265P MYD88 and Y196 CD79B may provide a powerful tool for approaching diagnosis of IVLBCL. [Display omitted] DisclosuresChiba:Nippon Shinyaku: Honoraria, Research Funding.

Serial circulating tumor DNA (ctDNA) monitoring in metastatic colorectal cancer (mCRC) reveals dynamic profile of actionable alterations.

3572 Background: Serial ctDNA can measure dynamic changes in disease burden over time, however utility of serial profiling to detect changes in actionable alterations remains unclear. Methods: We evaluated 501 patients with ≥3 serial Guardant360 assays performed between 09/2016 and 11/2020 and compared MSI, fusion, amplification and single nucleotide variant (SNV) detection over time. This comprised 2147 assays with a median of 4 assays per patient (min 3, max 18) occurring an average of 163 days apart (+/- SD of 147 days). Maximum detected variant allele frequency in samples (maxVAF) was assessed for relation to changes in detected alterations as a surrogate for tumor volume. Results: Among 406 patients with assays assessable for MSI-status, 17 (4.2%) had MSI detected. New MSI detection on a subsequent assay always occurred with a rising maxVAF (3/3) that was also ≥0.7%, while loss of detectable MSI between assays always associated with falling maxVAFs (7/7) with 6/7 occurring when maxVAF fell below 0.4%. Fusions were noted in 9/501 (2%) patients. Among 3 patients who lost a detectable fusion, maxVAF decreased in 1 patient and changed ≤0.2% between assays in 2, while 2/3 patients with new fusions had rising maxVAFs and 1 patient had a falling maxVAF. Amplifications were detected in 242/501 patients (48%). While most genes had highly variable amplification detection between assays (9% serially detected), ERBB2 amplifications were more consistent and serially detected in 39% of detected cases (P &lt; 0.0001). New detection of amplifications occurred more commonly in cases with rising maxVAF (OR 11.70, 95% CI 7.61-18.00, P &lt; 0.0001) and loss of detectable amplifications occurred more between samples with falling maxVAF (OR 12.37, 95% CI 8.35-18.66, P &lt; 0.0001). Change in maxVAF correlated with change in number of detected amplifications (r = 0.62, P &lt; 0.0001), but only partially explained changes seen (R2= 0.39). Between serial assays, SNVs changed a median of 0 variants (IQR -1 to 1), however some patients had significant changes (max gain 21/max loss 18). Among 1646 serial time points, 454 (28%) had no change in SNVs, 674 (41%) gained SNVs, and 518 (31%) lost SNVs on subsequent assays. Gains were more common in samples with rising maxVAF (OR 7.76, 95% CI 6.18-9.73, P &lt; 0.0001) while losses were more common when maxVAF fell (OR 6.90, 95% CI 5.47-8.66, P &lt; 0.0001). The correlation between maxVAF change and SNV change was significant (r = 0.29, P &lt; 0.0001), but minimally explained SNV changes (R2= 0.086) and was a much weaker association than noted for amplification changes. Conclusions: We noted significant differences in detection of actionable alterations across serial ctDNA assays. Increased ctDNA volume (higher maxVAF) due to tumor progression may explain some variation over time, but variability also occurs outside these changes, likely reflecting clonal evolution following therapy.

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Prevalence of the Brazilian TP53 Founder c.1010G>A (p.Arg337His) in Lung Adenocarcinoma: Is Genotyping Warranted in All Brazilian Patients?

In Southern and Southeastern Brazil, there is a germline pathogenic variant with incomplete penetrance located in the oligomerization domain of TP53, c.1010G>A (p.Arg337His). Due to a founder effect, the variant is present in 0.3% of the general population of the region. Recently, this variant was identified in 4.4 and 8.9% of two apparently unselected, single center case series of Brazilian lung adenocarcinoma (LUAD) patients from the Southeastern and Central regions of the country, respectively. In the present study, our aim was to examine TP53 c.1010G>A allele and genotype frequencies in LUAD samples obtained from patients diagnosed in Southern Brazil. A total of 586 LUAD samples (tumor DNA) recruited from multiple centers in the region were tested, and the mutant allele was identified using TaqMan® assays in seven cases (7/586, 1.2%) which were submitted to next generation sequencing analyses for confirmation. Somatic EGFR mutations were more frequent in TP53 c.1010G>A carriers than in non-carriers (57.1 vs. 17.6%, respectively). Further studies are needed to confirm if TP53 c.1010G>A is a driver in LUAD carcinogenesis and to verify if there is a combined effect of EGFR and germline TP53 c.1010G>A. Although variant frequency was higher than observed in the general population, it is less than previously reported in LUAD patients from other Brazilian regions. Additional data, producing regional allele frequency information in larger series of patients and including cost-effectiveness analyses, are necessary to determine if TP53 c.1010G>A screening in all Brazilian LUAD patients is justified.