Christina M. Lockwood

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.

Clinical next-generation sequencing in patients with non-small cell lung cancer.

A clinical assay was implemented to perform next‐generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non–small cell lung cancer (NSCLC).

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.

Circulating MicroRNA miR-323-3p as a Biomarker of Ectopic Pregnancy

BACKGROUND The use of serum human chorionic gonadotropin (hCG) and progesterone to identify patients with ectopic pregnancy (EP) has been shown to have poor clinical utility. Pregnancy-associated circulating microRNAs (miRNAs) have been proposed as potential biomarkers for the diagnosis of pregnancy-associated complications. This proof-of-concept study examined the diagnostic accuracy of various miRNAs to detect EP in an emergency department (ED) setting. METHODS This study was a retrospective case-control analysis of 89 women who presented to the ED with vaginal bleeding and/or abdominal pain/cramping and received a diagnosis of viable intrauterine pregnancy (VIP), spontaneous abortion (SA), or EP. Serum hCG and progesterone concentrations were measured by immunoassays. The serum concentrations of miRNAs miR-323-3p, miR-517a, miR-519d, and miR-525-3p were measured with TaqMan real-time PCR. Statistical analysis was performed to determine the clinical utility of these biomarkers, both as single markers and as multimarker panels for EP. RESULTS Concentrations of serum hCG, progesterone, miR-517a, miR-519d, and miR-525-3p were significantly lower in EP and SA cases than in VIP cases (P < 0.01). In contrast, the concentration of miR-323-3p was significantly increased in EP cases, compared with SA and VIP cases (P < 0.01). As a single marker, miR-323-3p had the highest sensitivity of 37.0% (at a fixed specificity of 90%). In comparison, the combined panel of hCG, progesterone, and miR-323-3p yielded the highest sensitivity (77.8%, at a fixed specificity of 90%). A stepwise analysis that used hCG first, added progesterone, and then added miR-323-3p yielded a 96.3% sensitivity and a 72.6% specificity. CONCLUSIONS Pregnancy-associated miRNAs, especially miR-323-3p, added substantial diagnostic accuracy to a panel including hCG and progesterone for the diagnosis of EP.

Design of targeted, capture-based, next generation sequencing tests for precision cancer therapy

In cancer medicine, next generation sequencing (NGS) has emerged as a practical method to generate patient- and tumor-specific genetic data for optimal selection of targeted therapies. Targeted sequencing allows clinical testing to focus on cancer-related genes, thus maximizing the tests sensitivity and specificity for actionable variants. In this review, we summarize the current regulatory environment surrounding clinical NGS, including regulations and professional opinions established by the College of American Pathologists, the Centers for Disease Control and Prevention, the Clinical Laboratory Improvement Amendments, the Clinical and Laboratory Standards Institute, the Association for Molecular Pathology, the New York State Department of Health, and the American College of Medical Genetics. We outline practical considerations for the design of targeted NGS assays, with an emphasis on capture-based methods. Finally, we discuss components of the validation process for clinical NGS assays as well as challenges that still remain for clinical NGS.

Serum Human Chorionic Gonadotropin Concentrations Greater than 400,000 IU/L Are Invariably Associated with Suppressed Serum Thyrotropin Concentrations

BACKGROUND During pregnancy, when human chorionic gonadotropin (hCG) concentrations are highest, there is a transient suppression of serum thyrotropin (TSH). In normal pregnancy, TSH concentrations generally remain within nonpregnant reference intervals; however, in some patients TSH is suppressed. Here we sought to extend previous studies to examine the relationship between very high serum concentrations of hCG (>200,000 IU/L) and the thyroid hormones TSH and free thyroxine (FT(4)). The objective of this study was to determine: 1) if there is an hCG concentration above which TSH concentrations are suppressed (< or =0.2 microIU/mL); 2) how thyroid hormone concentrations change in response to changes in hCG concentrations; and 3) the clinical symptoms in patients with such extremely elevated hCG concentrations. METHODS Residual specimens sent to the laboratories for physician-ordered hCG testing were utilized. Over 26 months, 15,597 physician-ordered hCG tests were performed. Sixty-nine specimens from 63 women with hCG concentrations >200,000 IU/L were identified, and TSH and FT(4) concentrations were measured. Medical records were reviewed for clinical information. RESULTS Thirty-seven percent of subjects had hyperemesis gravidarum (HG) and 19% had gestational trophoblastic disease (GTD). TSH was suppressed (< or =0.2 microIU/mL) in 67% of the specimens with hCG concentrations >200,000 IU/L and 100% of specimens with hCG concentrations >400,000 IU/L. FT(4) concentrations were elevated above the reference interval (1.8 ng/dL) in 32% of specimens with hCG concentrations >200,000 IU/L and in 80% of specimens with hCG concentrations >400,000 IU/L. Only four subjects had documented signs of hyperthyroidism. Women with GTD had a median hCG concentration twofold higher than women with HG and a median TSH concentration one half that of women with HG. CONCLUSIONS 1) At hCG concentrations >400,000 IU/L, TSH is consistently suppressed; 2) serum FT(4) and TSH respond to changes in serum hCG concentrations; and 3) most patients with hCG concentrations >200,000 IU/L lack overt hyperthyroid symptoms.

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.

Pre-analytical variables in miRNA analysis.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in the regulation of cellular processes and have been shown to be differentially expressed in neoplasia and other disease states. This renders miRNAs promising diagnostic and prognostic biomarkers in tissues and body fluids, especially in blood. However, numerous variables can influence the detection of miRNAs in the pre-analytical phase and lead to erroneous results. This is of particular concern when miRNA profiles are used clinically and alter diagnosis and patient treatment. Since miRNAs have been discovered relatively recently, systematic studies examining pre-analytical variables are rare. Therefore, this review comprehensively summarizes the current knowledge of pre-analytical variables that influence miRNA analysis in general, as well as pre-analytical variables that are specific to the detection of circulating miRNAs and tissue miRNAs.

Qualitative point-of-care and over-the-counter urine hCG devices differentially detect the hCG variants of early pregnancy

BACKGROUND Qualitative point-of-care (POC) tests for human chorionic gonadotropin (hCG) vary in their ability to detect purified hCG variants and there is data to suggest that over-the-counter (OTC) devices might also display similar variability. This could potentially influence the detection of urine hCG in early pregnancy. METHODS Six OTC devices were tested for their ability to detect 5 hCG variants. Ten early pregnancy urine specimens were selected for their diverse expression of hCG variants. The samples were tested with 6 brands of POC and 6 OTC devices. RESULTS OTC devices consistently recognized intact hCG, hCGn, and hCGbeta. hCGbetan was consistently recognized by 4 out of 6 brands. One brand inconsistently recognized hCGbetacf. OTC and POC devices varied greatly in their ability to detect hCG in early pregnancy urine, despite the fact that urine samples were adjusted to the same intact hCG concentration. Interestingly, we found that the OTC devices had better analytical sensitivity than the POC devices. Clinitest and First Response demonstrated the lowest hCG detection limits for POC and OTC devices, respectively. CONCLUSIONS Both OTC and POC devices are capable of detecting hCG concentrations in early pregnancy urine, and OTC devices demonstrated better analytical sensitivity relative to POC devices.

Circulating miR-122 as a potential biomarker of liver disease

AIM Expression profiles indicate that miR-122 is specifically and abundantly expressed in liver. This study sought to determine miR-122 plasma concentrations in 15 apparently healthy subjects and 30 patients with liver disease, and clarify whether plasma miR-122 correlates with ALT. MATERIALS & METHODS miR-122 was measured by quantitative PCR in healthy volunteers and patients with liver disease. RESULTS ALT was increased in two out of 15 (13%) apparently healthy subjects and 17 out of 30 (57%) liver disease patients. In healthy subjects, median miR-122 plasma concentration was 51.7 copies/20 pg RNA (range 16.0-312.0). In liver disease patients, median miR-122 was significantly elevated to 202.3 copies/20 pg RNA (range 20.9-1160.0; Mann-Whitney test between median concentrations; p = 0.0016). CONCLUSION This small proof-of-principle study suggests that miR-122 may be a potential plasma biomarker of liver damage.