Applications

Tumor- and circulating-free DNA methylation identifies clinically relevant small cell lung cancer subtypes. Simon Heeke, Carl M. Gay, Marcos R. Estecio, et al. Cancer Cell   January 25, 2024

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy composed of distinct transcriptional subtypes, but implementing subtyping in the clinic has remained challenging, particularly due to limited tissue availability. Given the known epigenetic regulation of critical SCLC transcriptional programs, we hypothesized that subtype-specific patterns of DNA methylation could be detected in tumor or blood from SCLC patients. Using genomic-wide reduced-representation bisulfite sequencing (RRBS) in two cohorts totaling 179 SCLC patients and using machine learning approaches, we report a highly accurate DNA methylation-based classifier (SCLC-DMC) that can distinguish SCLC subtypes. We further adjust the classifier for circulating-free DNA (cfDNA) to subtype SCLC from plasma. Using the cfDNA classifier (cfDMC), we demonstrate that SCLC phenotypes can evolve during disease progression, highlighting the need for longitudinal tracking of SCLC during clinical treatment. These data establish that tumor and cfDNA methylation can be used to identify SCLC subtypes and might guide precision SCLC therapy.

(Methods section)  

Critical commercial assays

Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit Apostle Bio A17622-250

Nucleic acid extraction

cfDNA was extracted using the Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit (Apostle Inc). 

Terminal modifications independent cell-free RNA sequencing enables sensitive early cancer detection and classification. Jun Wang, Jinyong Huang, Yunlong Hu, et al. Nature Communications   15, Article number: 156 (2024) 

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Cell-free RNAs (cfRNAs) offer an opportunity to detect diseases from a transcriptomic perspective, however, existing techniques have fallen short in generating a comprehensive cell-free transcriptome profile. We develop a sensitive library preparation method that is robust down to 100 µl input plasma to analyze cfRNAs independent of their 5’-end modifications. We show that it outperforms adapter ligation-based method in detecting a greater number of cfRNA species. We perform transcriptome-wide characterizations in 165 lung cancer, 30 breast cancer, 37 colorectal cancer, 55 gastric cancer, 15 liver cancer, and 133 cancer-free participants and demonstrate its ability to identify transcriptomic changes occurring in early-stage tumors. We also leverage machine learning analyses on the differentially expressed cfRNA signatures and reveal their robust performance in cancer detection and classification. Our work sets the stage for in-depth study of the cfRNA repertoire and highlights the value of cfRNAs as cancer biomarkers in clinical applications.

(Methods section)  cfRNA extraction

Frozen plasma samples were thawed on ice prior to cfRNA extraction. 200 μl of plasma samples were subjected to cfRNA extraction using the Apostle MiniMax High-Efficiency cfRNA Isolation Kit (Apostle), following the manufacturer’s protocol with minor modifications.

Integrative modeling of tumor genomes and epigenomes for enhanced cancer diagnosis by cell-free DNA. 

Mingyun Bae, Gyuhee Kim, Tae-Rim Lee, et al. Nature Communications  14, Article number: 2017 (2023)

(Note: Apostle MiniMax technology is used in this study.) 

Abstract Multi-cancer early detection remains a key challenge in cell-free DNA (cfDNA)-based liquid biopsy. Here, we perform cfDNA whole-genome sequencing to generate two test datasets covering 2125 patient samples of 9 cancer types and 1241 normal control samples, and also a reference dataset for background variant filtering based on 20,529 low-depth healthy samples. An external cfDNA dataset consisting of 208 cancer and 214 normal control samples is used for additional evaluation. Accuracy for cancer detection and tissue-of-origin localization is achieved using our algorithm, which incorporates cancer type-specific profiles of mutation distribution and chromatin organization in tumor tissues as model references. Our integrative model detects early-stage cancers, including those of pancreatic origin, with high sensitivity that is comparable to that of late-stage detection. Model interpretation reveals the contribution of cancer type-specific genomic and epigenomic features. Our methodologies may lay the groundwork for accurate cfDNA-based cancer diagnosis, especially at early stages..

(Methods section)  cfDNA was extracted from 0.4 mL plasma ... and eluted in a final volume of 22 μL, using an Apostle MiniMax High Efficiency cfDNA Isolation Kit (Apostle, US) according to the manufacturer’s instructions. 

Individualized, heterologous chimpanzee adenovirus and self-amplifying mRNA neoantigen vaccine for advanced metastatic solid tumors: phase 1 trial interim results.

 Christine D. Palmer, Amy R. Rappaport, Matthew J. Davis, et al. Nature Medicine  volume 28, pages 1619–1629 (2022)

Abstract Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 1012 viral particles (VP) ChAd68 and 30 µg samRNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manufacturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circulating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analyses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies.

(Methods section)  cfDNA was extracted from the entire plasma volume of a single draw using the Apostle MiniMax cfDNA Isolation kit (ApostleBio) 

(Methods section) Enriched libraries were sequenced on an Illumina NovaSeq to a minimum mean raw depth of 80,000... Variant allele frequency was converted to mutated haploid genomic equivalents per milliliter plasma (hGE ml1 ) using the extracted plasma volume and total cfDNA yield from the extraction. Percent change in ctDNA was calculated as the change of the median mutated hGE ml1 from the baseline sample.

Simultaneous analysis of mutations and methylations in circulating cell-free DNA for hepatocellular carcinoma detection.

Pei Wang, Qianqian Song, Jie Ren, Weilong Zhang, Yuting Wang, Lin Zhou, Dongmei Wang, Kun Chen, Liping Jiang, Bochao Zhang, Wanqing Chen, Chunfeng Qu, Hong Zhao, Yuchen Jiao. National Cancer Center of China. Science Translational Medicine 14, eabp8704 (2022) 23 November 2022

Abstract Cell-free DNA (cfDNA)–based liquid biopsy is a promising approach for the early detection of cancer. A major hurdle is the limited yield of cfDNA from one blood draw, limiting the use of most samples to one test of either mutation or methylation. Here, we develop a technology, Mutation Capsule Plus (MCP), which enables multiplex profiling of one cfDNA sample, including simultaneous detection of genetic and epigenetic alterations and genome-wide discovery of methylation markers. With this technology, we performed de novo screening of methylation markers on cfDNA samples from 30 hepatocellular carcinoma (HCC) cases and 30 non-HCC controls. The methylation markers enriched in HCC cfDNA were further profiled in parallel with a panel of mutations on a training cohort of 60 HCC and 60 non-HCC cases, resulting in an HCC detection model. We validated the model in an independent retrospective cohort with 58 HCC and 198 non-HCC cases and got 90% sensitivity with 94% specificity. Furthermore, we applied the model to a prospective cohort of 311 asymptomatic hepatitis B virus carriers with normal liver ultrasonography and serum AFP concentration. The model detected four of the five HCC cases in the cohort, showing 80% sensitivity and 94% specificity. These findings demonstrate that the MCP technology has potential for the discovery and validation of multiomics biomarkers for the noninvasive detection of cancer. This study also provides a comprehensive database of genetic and epigenetic alterations in the cfDNA of a large cohort of HCC cases and high-risk non-HCC individuals.

(Methods Section) cfDNA was extracted from the plasma samples using the Apostle MiniMax cfDNA isolation kit (C43468, Apostle). 

Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy.

Jin et al. PNAS February 2, 2021 118 (5) e2017421118; https://doi.org/10.1073/pnas.2017421118 

Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery. 

Plasma DNA extraction was performed using 2 to 5 mL of plasma with the Apostle MiniMax High-Efficiency cfDNA Isolation Kit, according to the product manual. 

Reliability of Cell-Free DNA and Targeted NGS in Predicting Chromosomal Abnormalities of Patients With Myeloid Neoplasms.

Andrew Ip et al. Frontiers in Oncology June 14, 2022; https://doi.org/10.3389/fonc.2022.923809

Introduction: Cytogenetic analysis is important for stratifying patients with various neoplasms. We explored the use of targeted next generation sequencing (NGS) in detecting chromosomal structural abnormalities or copy number variations (CNVs) in patients with myeloid neoplasms.

Methods: Plasma cell-free DNA (cfDNA) from 2821 myeloid or lymphoid neoplasm patients were collected. cfDNA was sequenced using a 275 gene panel. CNVkit software was used for analyzing and visualizing CNVs. Cytogenetic data from corresponding bone marrow (BM) samples was available on 89 myeloid samples.

Results: Of the 2821 samples, 1539 (54.5%) showed evidence of mutations consistent with the presence of neoplastic clones in circulation. Of these 1539 samples, 906 (59%) showed abnormalities associated with myeloid neoplasms and 633 (41%) with lymphoid neoplasms. Chromosomal structural abnormalities in cfDNA were detected in 146 (16%) myeloid samples and 76 (12%) lymphoid samples. Upon comparison of the myeloid samples with 89 BM patients, NGS testing was able to reliably detect chromosomal gain or loss, except for fusion abnormalities. When cytogenetic abnormalities were classified according to prognostic classes, there was a complete (100%) concordance between cfDNA NGS data and cytogenetic data.

Conclusions: This data shows that liquid biopsy using targeted NGS is reliable in detecting chromosomal structural abnormalities in myeloid neoplasms. In specific circumstances, targeted NGS may be reliable and efficient to provide adequate information without the need for BM biopsy considering broad mutation profiling can be obtained through adequate sequencing within the same test. Overall, this study supports the use of liquid biopsy for early diagnosis and monitoring of patients with myeloid neoplasms.

We extracted cfDNA from 2821 peripheral blood samples using the Apostle MiniMax High Efficiency cfDNA Isolation Kit (San Jose, CA). 

Cell-free chromatin immunoprecipitation can determine tumor gene expression in lung cancer patients. Christoffer Trier Maansson, Peter Meldgaard, Magnus Stougaard, Anders Lade Nielsen, Boe Sandahl Sorensen.  Molecular Oncology.  February 24, 2023. DOI: 10.1002/1878-0261.13394.

(Note: Apostle MiniMax technology is used in this study.)

Cell-free DNA (cfDNA) in blood plasma can be bound to nucleosomes that contain post-translational modifications representing the epigenetic profile of the cell of origin. This includes histone H3 lysine 36 trimethylation (H3K36me3), a marker of active transcription. We hypothesized that cell-free chromatin immunoprecipitation (cfChIP) of H3K36me3-modified nucleosomes present in blood plasma can delineate tumor gene expression levels. H3K36me3 cfChIP followed by targeted NGS (cfChIP-seq) was performed on blood plasma samples from non-small cell lung cancer patients (NSCLC, n = 8), small cell lung cancer patients (SCLC, n = 4) and healthy controls (n = 4). H3K36me3 cfChIP-seq demonstrated increased enrichment of mutated alleles compared to normal alleles in plasma from patients with known somatic cancer mutations. Additionally, genes identified to be differentially expressed in SCLC and NSCLC tumors had concordant H3K36me3 cfChIP enrichment profiles in NSCLC (sensitivity = 0.80) and SCLC blood plasma (sensitivity = 0.86). Findings here expand the utility of cfDNA in liquid biopsies to characterize treatment resistance, cancer subtyping, and disease progression.

(Material and Methods section) -Both the input as well as the cfChIP samples were purified using Apostle MiniMax High Efficiency cfDNA Isolation Kit (Beckman Coulter, Indianapolis, IN, USA) according to manufacturer’s instructions. 

Response prediction and risk stratification of patients with rectal cancer after neoadjuvant therapy through an analysis of circulating tumour DNA.

Liu W, Li Y, et al. EBioMedicine March 17, 2022; https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(22)00129-3/fulltext

Background - Multiple approaches based on cell-free DNA (cfDNA) have been applied to detect minimal residual disease (MRD) and to predict prognosis or recurrence. However, a comparison of the approaches used in different cohorts and studies is difficult. We aimed to compare multiple approaches for MRD analysis after neoadjuvant therapy (NAT) in patients with locally advanced rectal cancer (LARC).

Methods - Sixty patients with LARC from a multicentre, phase II/III randomized trial were included, with tissue and blood samples collected. For each cfDNA sample, we profiled MRD using 3 approaches: personalized assay targeting tumour-informed mutations, universal panel of genes frequently mutated in colorectal cancer (CRC), and low depth sequencing for copy number alterations (CNAs).

Findings - Positive MRD based on post-NAT personalized assay was significantly associated with an increased risk of recurrence (HR = 27.38; log-rank P < 0.0001). MRD analysis based on universal panel (HR = 5.18; log-rank P = 0.00086) and CNAs analysis (HR = 9.24; log-rank P = 0.00017) showed a compromised performance in predicting recurrence. Both the personalized assay and universal panel showed complementary pattern to CNAs analysis in detecting cases with recurrence and the combination of the two types of biomarkers may lead to better performance.

Interpretation - The combination of mutation profiling and CNA profiling can improve the detection of MRD, which may help optimize the treatment strategies for patients with LARC.

(Methods Section) cfDNA was extracted from 1.5-4.5 mL of plasma with the Apostle MiniMax cfDNA isolation kit (C40605, Apostle; San Jose, CA, USA). 

Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study. 

Yang et al. Targeted Oncology. 2019;14:719-728. Published: 06 November 2019. 

Background  Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations.  

Objective  We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC).  Methods  Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples.  

Results  We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression.  

Conclusions We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC.

Trial Registration Clinical Trials.gov identifier: NCT02804100. 

cfDNA was extracted from 2–6 ml of plasma using an Apostle MiniMax High Efficiency cfDNA Isolation Kit (standard edition) (Apostle, Silicon Valley, CA, USA) according to the manufacturer’s instructions.

A method for early diagnosis of lung cancer from tumor originated DNA fragments using plasma cfDNA methylome and fragmentome profiles.

 Yeo Jin Kim, Hahyeon Jeona, Sungwon Jeon, et al.  Molecular and Cellular Probes Volume 66, December 2022

Abstract Early detection is critical for minimizing mortality from cancer. Plasma cell-free DNA (cfDNA) contains the signatures of tumor DNA, allowing us to quantify the signature and diagnose early-stage tumors. Here, we report a novel tumor fragment quantification method, TOF (Tumor Originated Fragment) for the diagnosis of lung cancer by quantifying and analyzing both the plasma cfDNA methylation patterns and fragmentomic signatures. TOF utilizes the amount of ctDNA predicted from the methylation density information of each cfDNA read mapped on 6243 lung-tumor-specific CpG markers. The 6243 tumor-specific markers were derived from lung tumor tissues by comparing them with corresponding normal tissues and healthy blood from public methylation data. TOF also utilizes two cfDNA fragmentomic signatures: 1) the short fragment ratio, and 2) the 5′ end-motif profile. We used 298 plasma samples to analyze cfDNA signatures using enzymatic methyl-sequencing data from 201 lung cancer patients and 97 healthy controls. The TOF score showed 0.98 of the area under the curve in correctly classifying lung cancer from normal samples. The TOF score resolution was high enough to clearly differentiate even the early-stage non-small cell lung cancer patients from the healthy controls. The same was true for small cell lung cancer patients.

(Methods Section) Cell-free DNA was extracted from 3 to 5 ml of plasma using XXX method or Apostle MiniMax High Efficiency Isolation Kit (Beckman Coulter Life Sciences, C40603), according to the manufacturers’ procedures. 

BGI’s Three Complementary Kits of Colorectal Cancer Testing Have Been CE Marked.

BGI.  July 13, 2021

BGI Genomics announces that its Stool Sample Collection Kit, DNA Isolation Kit together with Sample Pretreatment Kit for Methylation Detection have been CE marked.

All three kits are used in conjunction with the previously CE marked Colorectal Cancer Testing Product which can detect the methylation of SDC2, ADHFE1 and PPP2R5C genes in human fecal samples.

According to the Global Cancer 2020 (GLOBOCAN) statistics, there are about 19.3 million new cases of colorectal cancer each year, accounting for 10 percent of all new cancer cases. About 935,000 colorectal cancer deaths occur each year, accounting for 9.4 percent of all cancer deaths.

So far, BGI has obtained CE mark for all four products used in the colorectal cancer detection workflow, from sample collection, DNA extraction, DNA pre-treatment to methylation detection, providing customers with reliable and standardized reagents and services.

(Note: Apostle is the Original Equipment Manufacturer or OEM for the Stool DNA Isolation Kit mentioned in this news. Apostle 's branded product is called Apostle MiniGenomics Stool Fast Kit.)

A systematic evaluation of stool DNA preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers.

Jin et al. Clinical Chemistry and Laboratory Medicine (CCLM). 2021; 59(1): 91–99

Objectives - Colorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays.

Methods - We systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol.

Results - The yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively.

Conclusions - Through the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.

(Methods section) For magnetic beads-based method, Apostle Stool gDNA Isolation Kit (APOSTLE) was used according to the manufacturer’s instructions. Either 0.2 g pellets or 0.2 mL supernatant from homogenized stool was mixed with 1 mL lysis buffer (APOSTLE) for DNA extractions.

Nicotine dose-dependent epigenomic-wide DNA methylation changes in the mice with long-term electronic cigarette exposure. 

Gang Peng,* Yibo Xi,* Chiara Bellini, Kien Pham, Zhen W Zhuang, Qin Yan, Man Jia, Guilin Wang, Lingeng Lu, Moon-Shong Tang, Hongyu Zhao, and He Wang American Journal of Cancer Research. Volume 12, Issue 8, August 2022, Pages 3679–3692. 

Abstract

Epigenomic-wide DNA methylation profiling holds the potential to reflect both electronic cigarette exposure-associated risks and individual poor health outcomes. However, a systemic study in animals or humans is still lacking. Using the Infinium Mouse Methylation BeadChip, we examined the DNA methylation status of white blood cells in male ApoE-/- mice after 14 weeks of electronic cigarette exposure with the InExpose system (2 hr/day, 5 days/week, 50% PG and 50% VG) with low (6 mg/ml) and high (36 mg/ml) nicotine concentrations. Our results indicate that electronic cigarette aerosol inhalation induces significant alteration of 8,985 CpGs in a dose-dependent manner (FDR<0.05); 7,389 (82.2%) of the CpG sites are annotated with known genes. Among the top 6 significant CpG sites (P-value<1e-8), 4 CpG sites are located in the known genes, and most (3/5) of these genes have been related to cigarette smoking. The other two CpGs are close to/associated with the Phc2 gene that was recently linked to smoking in a transcriptome-wide associations study. Furthermore, the gene set enrichment analysis highlights the activation of MAPK and 4 cardiomyocyte/cardiomyopathy-related signaling pathways (including adrenergic signaling in cardiomyocytes and arrhythmogenic right ventricular cardiomyopathy) following repeated electronic cigarette use. The MAPK pathway activation correlates well with our finding of increased cytokine mRNA expression after electronic cigarette exposure in the same batch of mice. Interestingly, two pathways related to mitochondrial activities, namely mitochondrial gene expression and mitochondrial translation, are also activated after electronic cigarette exposure. Elucidating the relationship between these pathways and the increased circulating mitochondrial DNA observed here will provide further insight into the cell-damaging effects of prolonged inhalation of e-cigarette aerosols.

(Methods section) Plasma cfDNA and mtDNA/nDNA ratio and oxidization of cell-free DNA

For measurement of in vivo plasma mtDNA/nDNA ratio, mice plasma was first subjected to cell-free DNA (cfDNA) extraction using Apostle minimax cfDNA extraction kit (ApostleBio, CA, US). 

Noninvasive Prenatal Screening for Common Fetal Aneuploidies Using Single-Molecule Sequencing.  Yeqing Qian, Yongfeng Liu, Kai Yan, et al.  Laboratory Investigation Volume 103, Issue 4, April 2023, 100043

(Note: Apostle MiniMax technology is used in this study.)

Amplification biases caused by next-generation sequencing (NGS) for noninvasive prenatal screening (NIPS) may be reduced using single-molecule sequencing (SMS), during which PCR is omitted. Therefore, the performance of SMS-based NIPS was evaluated. We used SMS-based NIPS to screen for common fetal aneuploidies in 477 pregnant women. The sensitivity, specificity, positive predictive value, and negative predictive value were estimated. The GC-induced bias was compared between the SMS- and NGS-based NIPS methods. Notably, a sensitivity of 100% was achieved for fetal trisomy 13 (T13), trisomy 18 (T18), and trisomy 21 (T21). The positive predictive value was 46.15% for T13, 96.77% for T18, and 99.07% for T21. The overall specificity was 100% (334/334). Compared with NGS, SMS (without PCR) had less GC bias, a better distinction between T21 or T18 and euploidies, and better diagnostic performance. Overall, our results suggest that SMS improves the performance of NIPS for common fetal aneuploidies by reducing the GC bias introduced during library preparation and sequencing.

(Materials and Methods section) - cfDNA was extracted from 0.6 mL of maternal plasma using Apostle MiniMaxTM High-Efficiency cfDNA Isolation Kit (ref. number: A17622-50; Apostle) according to the manufacturer’s instructions. 

Segmental duplication as potential biomarkers for non-invasive prenatal testing of aneuploidies.

Chen et al. EBioMedicine August 11, 2021; https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(21)00328-5/fulltext

We developed a computational program whereby available SD regions can be processed and analyzed efficiently for their potential use as biomarkers of the aneuploidy of interest. For the five common aneuploidies, i.e., trisomy 13, 18, 21, and two sex chromosome aneuploidies, a total of 21,772 candidate SD biomarker sequences together with their corresponding primer/probe sets were generated. The primer/probe sets were tested using a real-time PCR-based multicolour melting curve analysis for simultaneous detection of the five common aneuploidies, and yielded 100% clinical sensitivity and 99.64% specificity when subjected to a clinical evaluation. Following the observations that the SD biomarkers for aneuploidy could be better detected by digital PCR with improved accuracy, we established a noninvasive prenatal testing protocol for trisomy 21 and attained 100% concordance with next generation sequencing.

Our study confirmed that SD regions are preferred biomarkers for aneuploidy detection and in particular SD-based digital PCR could find potential use for NIPT of trisomy. A similar strategy can be applied to other chromosomal abnormality and genetic disorders.

The cfDNA was isolated from 1 mL of each plasma sample by the Apostle MiniMax™ High Efficiency cfDNA Isolation Kit (Apostle Inc, San Jose, CA) in line with the manufacturer's instructions.

The SneakPeek Early Gender DNA Test

SneakPeek by Gateway Genomics was founded with the goal to make DNA-based prenatal and pediatric information accessible and affordable for parents everywhere.

The SneakPeek Early Gender DNA Test is designed to specifically focus on fetal sex and return just one answer – male or female. This enables the test to be run on magnitudes smaller volumes of blood than the typical non-invasive prenatal test. When maternal blood samples arrive at SneakPeek Labs, extracted cell-free fetal DNA is run through real-time quantitative PCR to detect with a sensitivity down to a single Y chromosome. If Y chromosomes are found, the result is a boy. If they are absent, the baby is a girl. The test is run in 3 hours with 99.9% accuracy¹.

The SneakPeek Early Traits DNA Test lets parents find out what their infant or child will look like as an adult, predicted nutrition levels and sleep behavior. The DNA collection process is simple with a rub of the inner cheek using a swab, no blood samples are required for this test making it easy for parents. When the DNA samples arrive at SneakPeek Labs, genotyping method is used to analyze the differences in DNA and determine which traits an individual may have as a result.

(Note: Apostle is a technological product provider to SneakPeek by Gateway Genomics). 

¹In a recent large-scale study, SneakPeek accurately determined fetal sex in 99.9% of 1,029 pregnant women between 7-37 weeks gestational age. In a separate clinical study run in 2021, SneakPeek accurately determined fetal sex in 75 out of 75 pregnant women at 7 weeks into pregnancy. 

High-resolution DNA size enrichment using a magnetic nano-platform and application in non-invasive prenatal testing.  

Zhang et al. Analyst.  July 2020, 145, 5733-5739  (PDF)

Precise DNA sizing can boost sequencing efficiency, reduce cost, improve data quality, and even allow sequencing of low-input samples, while current pervasive DNA sizing approaches are incapable of differentiating DNA fragments under 200 bp with high resolution (<20 bp). In non-invasive prenatal testing (NIPT), the size distribution of cell-free fetal DNA in maternal plasma (main peak at 143 bp) is significantly different from that of maternal cell-free DNA (main peak at 166 bp). The current pervasive workflow of NIPT and DNA sizing is unable to take advantage of this 20 bp difference, resulting in sample rejection, test inaccuracy, and restricted clinical utility. Here we report a simple, automatable, high-resolution DNA size enrichment workflow, named MiniEnrich, on a magnetic nano-platform to exploit this 20 bp size difference and to enrich fetal DNA fragments from maternal blood. Two types of magnetic nanoparticles were developed, with one able to filter high-molecular-weight DNA with high resolution and the other able to recover the remaining DNA fragments under the size threshold of interest with >95% yield. Using this method, the average fetal fraction was increased from 13% to 20% after the enrichment, as measured by plasma DNA sequencing. This approach provides a new tool for high-resolution DNA size enrichment under 200 bp, which may improve NIPT accuracy by rescuing rejected non-reportable clinical samples, and enable NIPT earlier in pregnancy. It also has the potential to improve non-invasive screening for fetal monogenic disorders, differentiate tumor-related DNA in liquid biopsy and find more applications in autoimmune disease diagnosis.

Quantifying Fetal DNA in Maternal Blood Plasma by ddPCR Using DNA Methylation.

E. Hall, T. Riel, M. Ramesh, M. Gencoglu, O. Mikhaylichenko, R. Dannebaum, S. Margeridon, M. Herrera. Bio-Rad Laboratories, Pleasanton, CA. Association for Molecular Pathology 2022 Annual Meeting Abstracts. J Mol Diagn 2022, 24:S1 Abstract G072.

Introduction: The proportion of cell-free DNA (cfDNA) circulating in maternal blood that originates from the fetus, the fetal fraction, is an important quality control metric when performing tests on fetal-derived cfDNA. Epigenetic differences produce dissimilar DNA methylation patterns, allowing for leveraging regions of high methylation contrast using methylation-sensitive restriction enzyme (MSRE) digestion to quantify fetal and maternal DNA via droplet digital PCR (ddPCR). This advancement positions ddPCR as a faster and less expensive alternative to next-generation sequencing (NGS) for fetal fraction estimation.

Methods: Assays were designed to target MSRE- compatible regions with high methylation contrast between maternal and fetal cfDNA. Fetal assays targeted sites hypermethylated in fetal cfDNA and maternal assays targeted sites hypermethylated in maternal cfDNA. The assay multiplex was tested against contrived and clinical samples using an in-droplet MSRE-ddPCR workflow. The reaction mix was dropletized to create about 20,000 droplets per 24-μL reaction, thermocycled, and analyzed in a QX ONE instrument. The thermocycling profile included a 45-minute MSRE incubation step prior to PCR amplification. Contrived samples were constructed by spiking DNA-free plasma with micrococcal nuclease-digested DNA from an amniotic fluid cell line ("fetal" component) and a B-lymphocyte cell line ("maternal" component). Clinical samples were remnant diagnostic samples with existing NGS non-invasive prenatal testing results attached. DNA was extracted from all samples with the Apostle MiniMax kit on the KingFisher Flex.

Results: From an initial set of 15 assays, a final five-assay multiplex was produced following amplicon sequencing with NGS and ddPCR screening. Although amplicon sequencing did not completely predict ddPCR performance and non- specific interactions, it was valuable for guiding the final ddPCR screen. The five-assay multiplex, consisting of three fetal assays and two maternal assays, produced an excellent linear response against contrived samples from 0% to 25% fetal fraction (R2 >0.99). Similarly, a high correlation was observed between ddPCR-estimated fetal fraction and NGS fetal fraction for a set of clinical samples (n=6, plus two non- pregnant controls, R2 >0.94).

Conclusions: As an epigenetic trait, DNA methylation is a useful way to discriminate between otherwise highly similar DNA sequences in an efficient and effective manner. Leveraging DNA methylation may be done with minimal impact to the standard ddPCR workflow. The high sensitivity, speed, and direct quantification of ddPCR make it an attractive alternative to NGS for fetal fraction estimation.

Next-Generation Digital Polymerase Chain Reaction: High-Dynamic-Range Single-Molecule DNA Counting via Ultrapartitioning.

 Eleen Y. Shum, Janice H. Lai, Sixing Li, Haeun G. Lee, Jesse Soliman, Vedant K. Raol, Cavina K. Lee, Stephen P.A. Fodor, H. Christina Fan Anal. Chem. 2022, Publication Date:December 12, 2022 https://doi.org/10.1021/acs.analchem.2c03649

(Note: Apostle MiniMax technology is used in this study.) 

ABSTRACT: Digital PCR (dPCR) was first conceived for single-molecule quantitation. However, current dPCR systems often require DNA templates to share partitions due to limited partitioning capacities. Here, we introduce UltraPCR, a next-generation dPCR system where DNA counting is performed in a single-molecule regimen through a 6-log dynamic range using a swift and parallelized workflow. Each UltraPCR reaction is divided into >30 million partitions without microfluidics to achieve single template occupancy. Combined with a unique emulsion chemistry, partitions are optically clear, enabling the use of a three-dimensional imaging technique to rapidly detect DNA-positive partitions. Single-molecule occupancy also allows for more straightforward multiplex assay development due to the absence of partition-specific competition. As a proof of concept, we developed a 222-plex UltraPCR assay and demonstrated its potential use as a rapid, low-cost screening assay for noninvasive prenatal testing for as low as 4% trisomy fraction samples with high precision, accuracy, and reproducibility.

(Methods Section) Cell-free plasma was isolated using the Apostle MiniMax kit (Beckman Coulter) according to the manufacturer’s protocol with an elution volume of 60 μL.

Safety and tolerability of AAV8 delivery of a broadly neutralizing antibody in adults living with HIV: a phase 1, dose-escalation trial.

Casazza et al. Nature Medicine. April 11, 2022 

Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial (NCT03374202). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1–3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml−1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.

...

AAV8-VRC07 vector DNA quantitation. Plasma AAV8-VRC07 plasmid DNA was measured by extracting DNA from plasma, concentrating and then using a real-time PCR assay to measure a 103 base sequence spanning the junction of the IgG heavy chain sequence and F2A insert. DNA was extracted from serum using an Apostle MiniMax High Efficiency cfDNA Isolation Kit, following the manufacturer’s protocol with slight modification.

Apostle COVID-19 RNA Extraction System Applied in the Effective Detection of SARS-CoV-2

Ed. Horner S. Application Note.

The current coronavirus disease 2019 (COVID-19) pandemic started in late 2019. COVID-19 is the result of severe acute respiratory syndrome 2 (SARS-CoV-2) virus contraction. COVID-19 is often accompanied by a wide range of symptoms including fever, cough, and shortness of breath. The SARS-CoV-2 virus consists of a ~30 kb RNA genome encoding for 15 proteins, including the spike protein that enables the virus to enter host cells. The current gold standard qualitative detection method, qRT-PCR, reverse transcribes the viral RNA into cDNA, which is subsequently amplified and quantitated.

This application note illustrates the effective detection of SARS-CoV-2 using the Apostle COVID-19 Viral RNA Isolation Automation System and qRT-PCR in clinical lab settings. This system uses efficient MiniGenomics magnetic nanoparticle technology for fast extraction and purification of viral nucleic acids from various types of biological samples collected in transport media. The proficient and consistent systems provide reliable test results to individuals that contribute to COVID-19 pandemic relief.

To date, our clients have processed more than 20 million swabs in various CAP/CLIA clinical labs in the United States. 

“Apostle COVID-19 RNA Extraction System is a fast and reliable solution for SARS-CoV-2 viral RNA extraction. We look forward to continuing the collaboration with Apostle and providing high quality COVID-19 tests for our community.” commented by Harry Gao, MD, PhD, DABMG, FACMG, Lab Director and Chief Scientific Officer of Fulgent Genetics.

Included in: FDA EUA Summary: Fulgent COVID-19 by RT-PCR TEST(FULGENT THERAPEUTICS).  For In vitro Diagnostic Use. Rx Only. For use under Emergency Use Authorization (EUA) only. US FDA. April 12, 2021. 

Detecting SARS-CoV-2 variants in wastewater and their correlation with circulating variants in the communities. Lin Li, Timsy Uppal, Paul D. Hartley, Andrew Gorzalski, Mark Pandori, Michael A. Picker, Subhash C. Verma & Krishna Pagilla.  Scientific Reports volume 12, Article number: 16141 (2022) , September 2022

Abstract

Detection of SARS-CoV-2 viral load in wastewater has been highly informative in estimating the approximate number of infected individuals in the surrounding communities. Recent developments in wastewater monitoring to determine community prevalence of COVID-19 further extends into identifying SARS-CoV-2 variants, including those being monitored for having enhanced transmissibility. We sequenced genomic RNA derived from wastewater to determine the variants of coronaviruses circulating in the communities. Wastewater samples were collected from Truckee Meadows Water Reclamation Facility (TMWRF) from November 2020 to June 2021. SARS-CoV-2 variants resulting from wastewater were compared with the variants detected in infected individuals' clinical specimens (nasal/nasopharyngeal swabs) during the same period and found conclusively in agreement. Therefore, wastewater monitoring for SARS-CoV-2 variants in the community is a feasible strategy as a complementary tool to clinical specimen testing in the latter's absence.

(Methods Section) The NP swabs received at the Nevada State Public Health Laboratory (NSPHL) from the Reno-Sparks metropolitan for SARS-CoV-2 diagnostic testing were subjected for RNA extraction using Virus Total NA Isolation Fast Kit (Apostle MiniGenomics, San Jose, CA, USA), followed by RT-PCR for the detection of SARS-CoV-2 genome using US Federal Drug Administration Emergency Use Authorization (FDA-EUA) diagnostic kits, as described previously.

Interview with Lauryn Massic, Association of Public Health Laboratories infectious disease fellow at the Nevada State Public Health Laboratory

Full interview

Lauryn Massic, Association of Public Health Laboratories infectious diseases fellow, shares how breakthrough technologies from Ceres Nanosciences and Apostle enables SARS-CoV-2 wastewater surveillance results that are efficient and accurate.

Please share with us more about your workflow and your metrics for success.

LM: Our goal in setting up our workflow was to have a turnaround time of less than one day for detection and quantification of the virus in wastewater samples. We wanted to be able to test wastewater from each community facility at least three times per week and to test the campus dorms every day of the week.

The workflow we follow in the lab starts with the use of Ceres Nanosciences’ Nanotrap Magnetic Virus Particles on the Apostle MagTouch 2000 for virus concentration from the wastewater. This is followed by RNA extraction using ThermoFisher and Apostle Bio reagents on the MagTouch 1000. We are analyzing the RNA using the Promega SARS-CoV -2 Wastewater RT-qPCR kit and are sequencing extracted RNA using Illumina short read sequencing on a MiniSeq.

How has the Apostle automation accelerated your testing capabilities?

LM: With Apostle automation, my team and I have been able to develop a method to detect and quantify SARS-CoV-2 in wastewater samples, while also having the ability to sequence viral RNA from the wastewater samples. We can accomplish the detection and quantification portion all in the span of a day, and the hands- free time of the automated process gives us the ability to complete other wastewater-related tasks.

Rapid repeat infection of SARS-CoV-2 by two highly distinct delta-lineage viruses.

Andrew J. Gorzalski , Christina Boyles, Victoria Sepcic, Subhash Verma, Joel Sevinsky, Kevin Libuit, Stephanie Van Hooser, Mark W. Pandori. Diagnostic Microbiology and Infectious Disease. Volume 104, Issue 1, September 2022, 115747; https://doi.org/10.1016/j.diagmicrobio.2022.115747

An instance of sequential infection of an individual with, firstly, the Delta variant and secondly a Delta-sub-lineage has been identified. The individual was found positive for the AY.26 lineage 22 days after being found positive for the Delta [B.1.617.2] variant. The viruses associated with the cases showed dramatic genomic difference, including 31 changes that resulted in deletions or amino acid substitutions. Seven of these differences were observed in the Spike protein. The patient in question was between 30 and 35 years old and had no underlying health conditions. Though singular, this case illustrates the possibility that infection with the Delta variant may not itself be fully protective against  a population of SARS-CoV-2 variants that are becoming increasingly diverse.

Nucleic acid extractions were performed by Apostle MagTouch Nucleic Acid Extraction Automation Systems [Apostle Inc, San Jose, CA]. 

Electronic Cigarettes Induce Mitochondrial DNA Damage and Trigger TLR9 (Toll-Like Receptor 9)-Mediated Atherosclerosis. 

Li et al.  Arteriosclerosis, Thrombosis, and Vascular Biology. 2021;41:839–853

Objective:

Electronic cigarette (e-cig) use has recently been implicated in promoting atherosclerosis. In this study, we aimed to investigate the mechanism of e-cig exposure accelerated atherosclerotic lesion development.

Approach and Results:

Eight-week-old ApoE−/− mice fed normal laboratory diet were exposed to e-cig vapor (ECV) for 2 hours/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining and greatly upregulated TLR9 (toll-like receptor 9) expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by enhanced TLR9 expression in human femoral artery atherosclerotic plaques from e-cig smokers. Intriguingly, we found a significant increase of oxidative mitochondria DNA lesion in the plasma of ECV-exposed mice. Administration of TLR9 antagonist before ECV exposure not only alleviated atherosclerosis and the upregulation of TLR9 in plaques but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the plaque accumulation of lipid and macrophages, and decreased the frequency of blood CCR2+ (C-C chemokine receptor type 2) classical monocytes. Surprisingly, we found that cytoplasmic mitochondrial DNA isolated from ECV extract-treated macrophages can enhance TLR9 activation in reporter cells and the induction of inflammatory cytokine could be suppressed by TLR9 inhibitor in macrophages.

Conclusions:

E-cig increases level of damaged mitochondrial DNA in circulating blood and induces the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacological target of ECV-related inflammation and cardiovascular diseases.

Mice plasma was first centrifuged at 16 000×g for 10 minutes at 4 °C, and the supernatant was subjected to cell-free DNA (cfDNA) extraction using Apostle minimax cfDNA extraction kit (ApostleBio, CA).