Validation of a Molecular Diagnostic Test for Circulating Tumor DNA by Next-Gen Sequencing.

A modified version of the PGDx elioTM Plasma Resolve assay was validated as a laboratory-developed test (LDT) for clinical use in the Molecular Diagnostics Laboratory at Fox Chase Cancer Center. The test detects single nucleotide variants (SNVs) and small insertions and deletions (indels) in 33 target genes using fragmented genomic DNA extracted from plasma. The analytical performance of this assay was assessed with reference standard DNA and 29 samples from cancer patients and detected 66 SNVs and 23 indels. Using 50 ng of input DNA, the sensitivity was 95.5% to detect SNVs at 0.5% allele frequency, and the specificity was 92.3%. The sensitivity to detect indels at 1% allele frequency was 70.4%. A cutoff of 0.25% variant allele frequency (VAF) was set up for diagnostic reporting. An inter-laboratory study of concordance with an orthologous test resulted in a positive percent agreement (PPA) of 91.7%.

A genome-wide association study of early spontaneous preterm delivery.

Preterm birth is the leading cause of infant morbidity and mortality. Despite extensive research, the genetic contributions to spontaneous preterm birth (SPTB) are not well understood. Term controls were matched with cases by race/ethnicity, maternal age, and parity prior to recruitment. Genotyping was performed using Affymetrix SNP Array 6.0 assays. Statistical analyses utilized PLINK to compare allele occurrence rates between case and control groups, and incorporated quality control and multiple-testing adjustments. We analyzed DNA samples from mother-infant pairs from early SPTB cases (20(0/7)-33(6/7) weeks, 959 women and 979 neonates) and term delivery controls (39(0/7)-41(6/7) weeks, 960 women and 985 neonates). For validation purposes, we included an independent validation cohort consisting of early SPTB cases (293 mothers and 243 infants) and term controls (200 mothers and 149 infants). Clustering analysis revealed no population stratification. Multiple maternal SNPs were identified with association P-values between 10×10(-5) and 10×10(-6). The most significant maternal SNP was rs17053026 on chromosome 3 with an odds ratio (OR) 0.44 with a P-value of 1.0×10(-6). Two neonatal SNPs reached the genome-wide significance threshold, including rs17527054 on chromosome 6p22 with a P-value of 2.7×10(-12) and rs3777722 on chromosome 6q27 with a P-value of 1.4×10(-10). However, we could not replicate these findings after adjusting for multiple comparisons in a validation cohort. This is the first report of a genome-wide case-control study to identify single nucleotide polymorphisms (SNPs) that correlate with SPTB.

Acute myeloid leukemia with a novel AKAP9::PDGFRA fusion transformed from essential thrombocythemia: A case report and mini review.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy associated with various combinations of gene mutations, epigenetic abnormalities, and chromosome rearrangement-related gene fusions. Despite the significant degree of heterogeneity in its pathogenesis, many gene fusions and point mutations are recurrent in AML and have been employed in risk stratification over the last several decades. Gene fusions have long been recognized for understanding tumorigenesis and their proven roles in clinical diagnosis and targeted therapies. Advances in DNA sequencing technologies and computational biology have contributed significantly to the detection of known fusion genes as well as for the discovery of novel ones. Several recurring gene fusions in AML have been linked to prognosis, treatment response, and disease progression. In this report, we present a case with a long history of essential thrombocythemia and hallmark CALR mutation transforming to AML characterized by a previously unreported AKAP9::PDGFRA fusion gene. We propose mechanisms by which this fusion may contribute to the pathogenesis of AML and its potential as a molecular target for tyrosine kinase inhibitors.

Genomic Evolution of Oligometastatic Clear Cell Renal Cell Carcinoma Presenting Two Decades Following Radical Nephrectomy.

Metachronous oligometastatic clear cell renal cell carcinoma may take many years before becoming clinically apparent. Herein we report regional lymph node recurrence of clear cell renal cell carcinoma more than two decades following radical nephrectomy. Chromosomal microarray analysis demonstrated multiple chromosomal alterations, including 3pq deletion shared by the original and recurrent tumors, and 17p deletion containing the TP53 gene present only in the latter. Sequencing of 1550 genes revealed mutations of VHL in both the primary and metastasis and BAP1 only in the metastatic lesion. These findings genetically link the original and recurrent tumors and suggest that VHL, TP53, and BAP1 alterations played an evolutionary role in recurrence decades after initial resection.

Leukemic presentation and progressive genomic alterations of MCD/C5 diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of lymphoid malignancies. Based on gene expression profiling, it has been subdivided into germinal center (GC)-derived and activated B-cell (ABC) types. Advances in molecular methodologies have further refined the subclassification of DLBCL, based on recurrent genetic abnormalities. Here, we describe a distinct case of DLBCL that presented in leukemic form. DNA sequencing targeting 275 genes revealed pathogenically relevant mutations of CD79B, MyD88, TP53, TBL1XR1, and PIM1 genes, indicating that this lymphoma would be best classified as MCD/C5 DLBCL, an ABC subtype. Despite an initial good clinical response to BTK inhibitor ibrutinib, anti-CD20 antibody rituxan, alkylating agent bendamustine, and hematopoietic stem-cell transplant, the lymphoma relapsed, accompanied by morphologic and molecular evidence of disease progression. Specifically, the recurrent tumor developed loss of TP53 heterozygosity (LOH) and additional chromosomal changes central to ABC DLBCL pathogenesis, such as PRDM1 loss. Acquired resistance to ibrutinib and rituxan was indicated by the emergence of BTK and FOXO1 mutations, respectively, as well as apparent activation of alternative cell-activation pathways, through copy-number alterations (CNAs), detected by high-resolution chromosomal microarrays. In vitro, studies of relapsed lymphoma cells confirmed resistance to standard BTK inhibitors but sensitivity to vecabrutinib, a noncovalent inhibitor active against both wild-type as well as mutated BTK. In summary, we provide in-depth molecular characterization of a de novo leukemic DLBCL and discuss mechanisms that may have contributed to the lymphoma establishment, progression, and development of drug resistance.

Clinical Validation of an Alternative Specimen Collection Kit for SARS-CoV-2 Testing at Fox Chase Cancer Center.

Background: Supply chain disruptions during the COVID-19 pandemic have affected the availability of components for specimen collection kits to detect SARS-CoV-2. Plastic injection molding offers a rapid and cheap method for mass production of swabs for upper respiratory tract sampling. Local production of virus transport medium increases flexibility to assemble sample collection kits if the medium provides appropriate stability for SARS-CoV-2 detection. Methods: A locally produced virus transport medium and a novel injection molded plastic swab were validated for SARS-CoV-2 detection by reverse-transcription quantitative polymerase chain reaction. Both components were compared to standard counterparts using viral reference material and representative patient samples. Results: Clinical testing showed no significant differences between molded and flocked swabs. Commercial and in-house virus transport media provided stable test results for over 40 days of specimen storage and showed no differences in test results using patient samples. Conclusions: This collection kit provides new supply chain options for SARS-CoV-2 testing.

Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests.

Diagnosis of any infectious disease is vital for opportune treatment and to prevent dissemination. RT-qPCR tests for detection of SARS-CoV-2, the causative agent for COVID-19, are ideal in a hospital environment. However, mass testing requires cheaper and simpler tests, especially in settings that lack sophisticated machinery. The most common current diagnostic method is based on nasopharyngeal sample collection, RNA extraction, and RT-qPCR for amplification and detection of viral nucleic acids. Here, we show that samples obtained from nasopharyngeal swabs in VTM and in saliva can be used with or without RNA purification in an isothermal loop-mediated amplification (LAMP)-based assay, with 60-93% sensitivity for SARS-CoV-2 detection as compared to standard RT-qPCR tests. A series of simple modifications to standard RT-LAMP published methods to stabilize pH fluctuations due to salivary acidity resulted in a significant improvement in reliability, opening new avenues for efficient, low-cost testing of COVID-19 infection.

Performance assessment of DNA sequencing platforms in the ABRF Next-Generation Sequencing Study.

Assessing the reproducibility, accuracy and utility of massively parallel DNA sequencing platforms remains an ongoing challenge. Here the Association of Biomolecular Resource Facilities (ABRF) Next-Generation Sequencing Study benchmarks the performance of a set of sequencing instruments (HiSeq/NovaSeq/paired-end 2 × 250-bp chemistry, Ion S5/Proton, PacBio circular consensus sequencing (CCS), Oxford Nanopore Technologies PromethION/MinION, BGISEQ-500/MGISEQ-2000 and GS111) on human and bacterial reference DNA samples. Among short-read instruments, HiSeq 4000 and X10 provided the most consistent, highest genome coverage, while BGI/MGISEQ provided the lowest sequencing error rates. The long-read instrument PacBio CCS had the highest reference-based mapping rate and lowest non-mapping rate. The two long-read platforms PacBio CCS and PromethION/MinION showed the best sequence mapping in repeat-rich areas and across homopolymers. NovaSeq 6000 using 2 × 250-bp read chemistry was the most robust instrument for capturing known insertion/deletion events. This study serves as a benchmark for current genomics technologies, as well as a resource to inform experimental design and next-generation sequencing variant calling.

Evidence of a gene-environment interaction that predisposes to spontaneous preterm birth: a role for asymptomatic bacterial vaginosis and DNA variants in genes that control the inflammatory response.

OBJECTIVE: We determined whether an environmental exposure to bacterial vaginosis (BV) modified genetic susceptibilities for spontaneous preterm delivery within genes that regulate the inflammatory response. STUDY DESIGN: Maternal DNA samples and vaginal smears for Gram staining were collected from 743 women (68 preterm births). We used a 1536-single nucleotide polymorphism (SNP) custom chip to study associations between genotype distributions and preterm birth. RESULTS: For 8 SNPs in 3 genes (protein kinase C alpha, fms-like tyrosine kinase 1, and interleukin 6), the odds ratios for preterm birth ranged from 1.9-4.0 among women with susceptible genotypes who were BV positive. The odds ratios for preterm birth were 2.0-5.0 times greater among women who were BV positive than among women who were BV negative. The significance of these differences was demonstrated by logistic regression analyses for genotype/BV interaction. CONCLUSION: These results demonstrate that the risk of preterm delivery that is associated with tag SNPs in genes that regulate the inflammatory response is modified by an environmental exposure such as bacterial vaginosis.

Assessing the significance of conserved genomic aberrations using high resolution genomic microarrays.

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.

Differential expression of miRNAs in papillary thyroid carcinoma compared to multinodular goiter using formalin fixed paraffin embedded tissues.

microRNAs (miRNAs) are approximately 22 nt RNAs that negatively regulate target gene expression. Their dysregulation has been implicated in the pathogenesis of a number of human cancers, including papillary thyroid carcinoma (PTC). Whereas previous studies using microarray technologies have largely relied on the ability to procure fresh tissue at the time of surgery to characterize miRNA signatures in PTC, we exploited the ability to procure sufficient miRNA from formalin-fixed paraffin-embedded (FFPE) tissue to describe a series of miRNAs whose expression is dysregulated in PTC compared to benign proliferative multinodular goiter (MNG). We identified 13 miRNAs upregulated and 26 miRNAs downregulated in PTC versus MNG. These include miRNA-21, miRNA-31, miRNA-221, and miRNA-222. Their dysregulation was further validated by real time RT-PCR analysis in an independent set of FFPE tissues. Many of these have previously been described in fresh tissue studies as altered in PTC, confirming the utility of this approach. These results further highlight the applicability of miRNA expression patterns as potential markers of human cancer, and our results suggest that FFPE tissues are suitable resources for such miRNA expression analyses. The ability to utilize FFPE tissue in the molecular characterization of human malignancy will unlock a rich resource for future cancer studies.

Genomic Methods and Microbiological Technologies for Profiling Novel and Extreme Environments for the Extreme Microbiome Project (XMP).

The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.

Combined RT-qPCR of mRNA and microRNA Targets within One Fluidigm Integrated Fluidic Circuit.

The ability to profile expression levels of a large number of mRNAs and microRNAs (miRNAs) within the same sample, using a single assay method, would facilitate investigations of miRNA effects on mRNA abundance and streamline biomarker screening across multiple RNA classes. A protocol is described for reverse transcription of long RNA and miRNA targets, followed by preassay amplification of the pooled cDNAs and quantitative PCR (qPCR) detection for a mixed panel of candidate RNA biomarkers. The method provides flexibility for designing custom target panels, is robust over a range of input RNA amounts, and demonstrated a high assay success rate.

Genome wide nucleosome mapping for HSV-1 shows nucleosomes are deposited at preferred positions during lytic infection.

HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200 bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6 hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152 kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes.

Metagenomic assay for identification of microbial pathogens in tumor tissues.

UNLABELLED: Screening for thousands of viruses and other pathogenic microorganisms, including bacteria, fungi, and parasites, in human tumor tissues will provide a better understanding of the contributory role of the microbiome in the predisposition for, causes of, and therapeutic responses to the associated cancer. Metagenomic assays designed to perform these tasks will have to include rapid and economical processing of large numbers of samples, supported by straightforward data analysis pipeline and flexible sample preparation options for multiple input tissue types from individual patients, mammals, or environmental samples. To meet these requirements, the PathoChip platform was developed by targeting viral, prokaryotic, and eukaryotic genomes with multiple DNA probes in a microarray format that can be combined with a variety of upstream sample preparation protocols and downstream data analysis. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and deep sequencing methods. These studies demonstrate the use of the PathoChip technology combined with PCR and deep sequencing as a valuable strategy for detecting the presence of pathogens in human cancers and other diseases. IMPORTANCE: This work describes the design and testing of a PathoChip array containing probes with the ability to detect all known publicly available virus sequences as well as hundreds of pathogenic bacteria, fungi, parasites, and helminths. PathoChip provides wide coverage of microbial pathogens in an economical format. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and sequencing methods. These studies demonstrate that the PathoChip technology is a valuable strategy for detecting the presence of pathogens in human cancers and other diseases.

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study.

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.

Development of a genotyping microarray for studying the role of gene-environment interactions in risk for lung cancer.

A microarray (LungCaGxE), based on Illumina BeadChip technology, was developed for high-resolution genotyping of genes that are candidates for involvement in environmentally driven aspects of lung cancer oncogenesis and/or tumor growth. The iterative array design process illustrates techniques for managing large panels of candidate genes and optimizing marker selection, aided by a new bioinformatics pipeline component, Tagger Batch Assistant. The LungCaGxE platform targets 298 genes and the proximal genetic regions in which they are located, using ≈ 13,000 DNA single nucleotide polymorphisms (SNPs), which include haplotype linkage markers with a minimum allele frequency of 1% and additional specifically targeted SNPs, for which published reports have indicated functional consequences or associations with lung cancer or other smoking-related diseases. The overall assay conversion rate was 98.9%; 99.0% of markers with a minimum Illumina design score of 0.6 successfully generated allele calls using genomic DNA from a study population of 1873 lung-cancer patients and controls.

Global genomic analysis reveals rapid control of a robust innate response in SIV-infected sooty mangabeys.

Natural SIV infection of sooty mangabeys (SMs) is nonprogressive despite chronic virus replication. Strikingly, it is characterized by low levels of immune activation, while pathogenic SIV infection of rhesus macaques (RMs) is associated with chronic immune activation. To elucidate the mechanisms underlying this intriguing phenotype, we used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs) in blood and lymph nodes. While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of the lymphocyte inhibitory receptor LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV.