Catherine E. Cottrell

Outcome modeling with CRLF2, IKZF1, JAK, and minimal residual disease in pediatric acute lymphoblastic leukemia: a Children's Oncology Group Study

As controversy exists regarding the prognostic significance of genomic rearrangements of CRLF2 in pediatric B-precursor acute lymphoblastic leukemia (ALL) classified as standard/intermediate-risk (SR) or high-risk (HR), we assessed the prognostic significance of CRLF2 mRNA expression, CRLF2 genomic lesions (IGH@-CRLF2, P2RY8-CRLF2, CRLF2 F232C), deletion/mutation in genes frequently associated with high CRLF2 expression (IKZF1, JAK, IL7R), and minimal residual disease (MRD) in 1061 pediatric ALL patients (499 HR and 562 SR) on COG Trials P9905/P9906. Whereas very high CRLF2 expression was found in 17.5% of cases, only 51.4% of high CRLF2 expressors had CRLF2 genomic lesions. The mechanism underlying elevated CRLF2 expression in cases lacking known genomic lesions remains to be determined. All CRLF2 genomic lesions and virtually all JAK mutations were found in high CRLF2 expressors, whereas IKZF1 deletions/mutations were distributed across the full cohort. In multivariate analyses, NCI risk group, MRD, high CRLF2 expression, and IKZF1 lesions were associated with relapse-free survival. Within HR ALL, only MRD and CRLF2 expression predicted a poorer relapse-free survival; no difference was seen between cases with or without CRLF2 genomic lesions. Thus, high CRLF2 expression is associated with a very poor outcome in high-risk, but not standard-risk, ALL. This study is registered at www.clinicaltrials.gov as NCT00005596 and NCT00005603.

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.

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.

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.

Contactin 4 as an Autism Susceptibility Locus

Structural and sequence variation have been described in several members of the contactin (CNTN) and contactin‐associated protein (CNTNAP) gene families in association with neurodevelopmental disorders, including autism. Using array comparative genome hybridization (CGH), we identified a maternally inherited ∼535 kb deletion at 3p26.3 encompassing the 5′ end of the contactin 4 gene (CNTN4) in a patient with autism. Based on this finding and previous reports implicating genomic rearrangements of CNTN4 in autism spectrum disorders (ASDs) and 3p− microdeletion syndrome, we undertook sequencing of the coding regions of the gene in a local ASD cohort in comparison with a set of controls. Unique missense variants were identified in 4 of 75 unrelated individuals with ASD, as well as in 1 of 107 controls. All of the amino acid substitutions were nonsynonomous, occurred at evolutionarily conserved positions, and were, thus, felt likely to be deleterious. However, these data did not reach statistical significance, nor did the variants segregate with disease within all of the ASD families. Finally, there was no detectable difference in binding of two of the variants to the interacting protein PTPRG in vitro. Thus, additional larger studies will be necessary to determine whether CNTN4 functions as an autism susceptibility locus in combination with other genetic and/or environmental factors.Autism Res 2011,4:189–199.

Identification of medically actionable secondary findings in the 1000 genomes

The American College of Medical Genetics and Genomics (ACMG) recommends that clinical sequencing laboratories return secondary findings in 56 genes associated with medically actionable conditions. Our goal was to apply a systematic, stringent approach consistent with clinical standards to estimate the prevalence of pathogenic variants associated with such conditions using a diverse sequencing reference sample. Candidate variants in the 56 ACMG genes were selected from Phase 1 of the 1000 Genomes dataset, which contains sequencing information on 1,092 unrelated individuals from across the world. These variants were filtered using the Human Gene Mutation Database (HGMD) Professional version and defined parameters, appraised through literature review, and examined by a clinical laboratory specialist and expert physician. Over 70,000 genetic variants were extracted from the 56 genes, and filtering identified 237 variants annotated as disease causing by HGMD Professional. Literature review and expert evaluation determined that 7 of these variants were pathogenic or likely pathogenic. Furthermore, 5 additional truncating variants not listed as disease causing in HGMD Professional were identified as likely pathogenic. These 12 secondary findings are associated with diseases that could inform medical follow-up, including cancer predisposition syndromes, cardiac conditions, and familial hypercholesterolemia. The majority of the identified medically actionable findings were in individuals from the European (5/379) and Americas (4/181) ancestry groups, with fewer findings in Asian (2/286) and African (1/246) ancestry groups. Our results suggest that medically relevant secondary findings can be identified in approximately 1% (12/1092) of individuals in a diverse reference sample. As clinical sequencing laboratories continue to implement the ACMG recommendations, our results highlight that at least a small number of potentially important secondary findings can be selected for return. Our results also confirm that understudied populations will not reap proportionate benefits of genomic medicine, highlighting the need for continued research efforts on genetic diseases in these populations.

Diagnostic utility of targeted next-generation sequencing in problematic cases.

Targeted next-generation sequencing (NGS) provides predictive and prognostic information in the routine care of patients with cancer. However, with increasing knowledge of the biological basis of cancer, NGS of the same gene sets can also provide diagnostic information in challenging cases, on the basis of identification of both known and novel variants, including single-nucleotide variants, insertions and deletions, copy number alterations, and translocations. Here, we present 3 clinical cases in which targeted NGS of hybrid-capture–enriched DNA from formalin-fixed, paraffin-embedded tumor samples provided unique and clinically important diagnostic and/or staging information in 3 different challenging clinical scenarios. In the first patient, NGS played a key role in both diagnosis and staging in a patient with multiple tumors of the same histologic type. The second case demonstrates the ability of NGS to clarify the tumor tissue type in a single mass involving multiple organs, and thereby guide appropriate chemotherapy. The third case illustrates that information regarding susceptibility to targeted therapeutics can also clarify the original histologic diagnosis.

Utility of clinical high-depth next generation sequencing for somatic variant detection in the PIK3CA-related overgrowth spectrum

Next‐generation sequencing (NGS) has revolutionized the approach of studying sequence variation, and has been well described in the clinical laboratory setting for the detection of constitutional alterations, as well as somatic tumor‐associated variants. It is increasingly recognized that post‐zygotic somatic alteration can be associated with congenital phenotypic abnormalities. Variation within the PI3K/AKT/mTOR pathway, including PIK3CA, has been described in somatic overgrowth syndromes and vascular malformations. Detection of PIK3CA somatic alteration is challenging because of low variant allele frequency (VAF) along with the need to assay involved tissue, thus necessitating a highly sensitive methodology. Here we describe the utility of target hybrid capture coupled with NGS for the identification of somatic variation in the PIK3CA‐related overgrowth spectrum (PROS) among 14 patients submitted for clinical testing. Assay detection of low allelic fraction variation is coverage dependent with >90% sensitivity at 400× unique read depth for VAF of 10%, and approaching 100% at 1000×. Average read depth among the patient dataset across PIK3CA coding regions was 788.4. The diagnostic yield among this cohort was 71%, including the detection of two PIK3CA alterations novel in the setting of PROS. This report expands the mutational scope and phenotypic attributes of PROS disorders.

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.

Maternal uniparental disomy of chromosome 4 in a patient with limb-girdle muscular dystrophy 2E confirmed by SNP array technology.

Cottrell CE, Mendell J, Hart‐Kothari M, Ell D, Thrush DL, Astbury C, Pastore M, Gastier‐Foster JM, Pyatt RE. Maternal uniparental disomy of chromosome 4 in a patient with limb‐girdle muscular dystrophy 2E confirmed by SNP array technology.