Haley J. Abel

Validation of a Next-Generation Sequencing Assay for Clinical Molecular Oncology

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Detection of FLT3 Internal Tandem Duplication in Targeted, Short-Read-Length, Next-Generation Sequencing Data

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays.

Targeted next generation sequencing of clinically significant gene mutations and translocations in leukemia

Leukemias are currently subclassified based on the presence of recurrent cytogenetic abnormalities and gene mutations. These molecular findings are the basis for risk-adapted therapy; however, such data are generally obtained by disparate methods in the clinical laboratory, and often rely on low-resolution techniques such as fluorescent in situ hybridization. Using targeted next generation sequencing, we demonstrate that the full spectrum of prognostically significant gene mutations including translocations, single nucleotide variants (SNVs), and insertions/deletions (indels) can be identified simultaneously in multiplexed sequence data. As proof of concept, we performed hybrid capture using a panel of 20 genes implicated in leukemia prognosis (covering a total of 1 Mbp) from five leukemia cell lines including K562, NB4, OCI-AML3, kasumi-1, and MV4–11. Captured DNA was then sequenced in multiplex on an Illumina HiSeq. Using an analysis pipeline based on freely available software we correctly identified DNA-level translocations in three of the three cell lines where translocations were covered by our capture probes. Furthermore, we found all published gene mutations in commonly tested genes including NPM1, FLT3, and KIT. The same methodology was applied to DNA extracted from the bone marrow of a patient with acute myeloid leukemia, and identified a t(9;11) translocation with single base accuracy as well other gene mutations. These results indicate that targeted next generation sequencing can be successfully applied in the clinical laboratory to identify a full spectrum of DNA mutations ranging from SNVs and indels to translocations. Such methods have the potential to both greatly streamline and improve the accuracy of DNA-based diagnostics.

Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel-based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.

Retinoblastoma gene mutations detected by whole exome sequencing of Merkel cell carcinoma

Merkel cell carcinoma is a highly aggressive cutaneous neuroendocrine tumor that has been associated with Merkel cell polyomavirus in up to 80% of cases. Merkel cell polyomavirus is believed to influence pathogenesis, at least in part, through expression of the large T antigen, which includes a retinoblastoma protein-binding domain. However, there appears to be significant clinical and morphological overlap between polyomavirus-positive and polyomavirus-negative Merkel cell carcinoma cases. Although much of the recent focus of Merkel cell carcinoma pathogenesis has been on polyomavirus, the pathogenesis of polyomavirus-negative cases is still poorly understood. We hypothesized that there are underlying human somatic mutations that unify Merkel cell carcinoma pathogenesis across polyomavirus status, and to investigate we performed whole exome sequencing on five polyomavirus-positive cases and three polyomavirus-negative cases. We found that there were no significant differences in the overall number of single-nucleotide variations, copy number variations, insertion/deletions, and chromosomal rearrangements when comparing polyomavirus-positive to polyomavirus-negative cases. However, we did find that the retinoblastoma pathway genes harbored a high number of mutations in Merkel cell carcinoma. Furthermore, the retinoblastoma gene (RB1) was found to have nonsense truncating protein mutations in all three polyomavirus-negative cases; no such mutations were found in the polyomavirus-positive cases. In all eight cases, the retinoblastoma pathway dysregulation was confirmed by immunohistochemistry. Although polyomavirus-positive Merkel cell carcinoma is believed to undergo retinoblastoma dysregulation through viral large T antigen expression, our findings demonstrate that somatic mutations in polyomavirus-negative Merkel cell carcinoma lead to retinoblastoma dysregulation through an alternative pathway. This novel finding suggests that the retinoblastoma pathway dysregulation leads to an overlapping Merkel cell carcinoma phenotype and that oncogenesis occurs through either a polyomavirus-dependent (viral large T antigen expression) or polyomavirus-independent (host somatic mutation) mechanism.

T-cell clonality assessment by next-generation sequencing improves detection sensitivity in mycosis fungoides

BACKGROUND T-cell receptor (TCR) clonality assessment is a principal diagnostic test in the management of mycosis fungoides (MF). However, current polymerase chain reaction-based methods may produce ambiguous results, often because of low abundance of clonal T lymphocytes, resulting in weak clonal peaks that cannot be size-resolved by contemporary capillary electrophoresis (CE). OBJECTIVE We sought to determine if next-generation sequencing (NGS)-based detection has increased sensitivity for T-cell clonality over CE-based detection in MF. METHODS Clonality was determined by an NGS-based method in which the TCR-γ variable region was polymerase chain reaction amplified and the products sequenced to establish the identity of rearranged variable and joining regions. RESULTS Of the 35 MF cases tested, 29 (85%) showed a clonal T-cell rearrangement by NGS, compared with 15 (44%) by standard CE detection. Three patients with MF had follow-up testing that showed identical, clonal TCR sequences in subsequent skin biopsy specimens. LIMITATIONS Clonal T-cell populations have been described in benign conditions; evidence of clonality alone, by any method, is not sufficient for diagnosis. CONCLUSION TCR clonality assessment by NGS has superior sensitivity compared with CE-based detection. Further, NGS enables tracking of specific clones across multiple time points for more accurate identification of recurrent MF.

Whole Exome Sequencing Reveals the Order of Genetic Changes during Malignant Transformation and Metastasis in a Single Patient with NF1-plexiform Neurofibroma

Purpose: Malignant peripheral nerve sheath tumors (MPNST) occur at increased frequency in individuals with neurofibromatosis type 1 (NF1), where they likely arise from benign plexiform neurofibroma precursors. While previous studies have used a variety of discovery approaches to discover genes associated with MPNST pathogenesis, it is currently unclear what molecular events are associated with the evolution of MPNST from plexiform neurofibroma. Experimental Design: Whole-exome sequencing was performed on biopsy materials representing plexiform neurofibroma (n = 3), MPNST, and metastasis from a single individual with NF1 over a 14-year period. Additional validation cases were used to assess candidate genes involved in malignant progression, while a murine MPNST model was used for functional analysis. Results: There was an increasing proportion of cells with a somatic NF1 gene mutation as the tumors progressed from benign to malignant, suggesting a clonal process in MPNST development. Copy number variations, including loss of one copy of the TP53 gene, were identified in the primary tumor and the metastatic lesion, but not in benign precursor lesions. A limited number of genes with nonsynonymous somatic mutations (βIII-spectrin and ZNF208) were discovered, several of which were validated in additional primary and metastatic MPNST samples. Finally, increased βIII-spectrin expression was observed in the majority of MPNSTs, and shRNA-mediated knockdown reduced murine MPNST growth in vivo. Conclusions: Collectively, the ability to track the molecular evolution of MPNST in a single individual with NF1 offers new insights into the sequence of genetic events important for disease pathogenesis and progression for future mechanistic study. Clin Cancer Res; 21(18); 4201–11. ©2015 AACR.

Admixture Mapping Identifies a Quantitative Trait Locus Associated with FEV1/FVC in the COPDGene Study

African Americans are admixed with genetic contributions from European and African ancestral populations. Admixture mapping leverages this information to map genes influencing differential disease risk across populations. We performed admixture and association mapping in 3,300 African American current or former smokers from the COPDGene Study. We analyzed estimated local ancestry and SNP genotype information to identify regions associated with FEV1/FVC, the ratio of forced expiratory volume in one second to forced vital capacity, measured by spirometry performed after bronchodilator administration. Global African ancestry inversely associated with FEV1/FVC (P = 0.035). Genome‐wide admixture analysis, controlling for age, gender, body mass index, current smoking status, pack‐years smoked, and four principal components summarizing the genetic background of African Americans in the COPDGene Study, identified a region on chromosome 12q14.1 associated with FEV1/FVC (P = 2.1 × 10−6) when regressed on local ancestry. Allelic association in this region of chromosome 12 identified an intronic variant in FAM19A2 (rs348644) as associated with FEV1/FVC (P = 1.76 × 10−6). By combining admixture and association mapping, a marker on chromosome 12q14.1 was identified as being associated with reduced FEV1/FVC ratio among African Americans in the COPDGene Study.

Occult Specimen Contamination in Routine Clinical Next-Generation Sequencing Testing.

OBJECTIVES To evaluate the extent of human-to-human specimen contamination in clinical next-generation sequencing (NGS) data. METHODS Using haplotype analysis to detect specimen admixture, with orthogonal validation by short tandem repeat analysis, we determined the rate of clinically significant (>5%) DNA contamination in clinical NGS data from 296 consecutive cases. Haplotype analysis was performed using read haplotypes at common, closely spaced single-nucleotide polymorphisms in low linkage disequilibrium in the population, which were present in regions targeted by the clinical assay. Percent admixture was estimated based on frequencies of the read haplotypes at loci that showed evidence for contamination. RESULTS We identified nine (3%) cases with at least 5% DNA admixture. Three cases were bone marrow transplant patients known to be chimeric. Six admixed cases were incidents of contamination, and the rate of contamination was strongly correlated with DNA yield from the tissue specimen. CONCLUSIONS Human-human specimen contamination occurs in clinical NGS testing. Tools for detecting contamination in NGS sequence data should be integrated into clinical bioinformatics pipelines, especially as laboratories trend toward using smaller amounts of input DNA and reporting lower frequency variants. This study provides one estimate of the rate of clinically significant human-human specimen contamination in clinical NGS testing.

A Model Study of In Silico Proficiency Testing for Clinical Next-Generation Sequencing.

CONTEXT -Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample. OBJECTIVE -To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically. DESIGN -DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. RESULTS -Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. CONCLUSIONS -The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.