Gary J. Latham

Targeted, High-Depth, Next-Generation Sequencing of Cancer Genes in Formalin-Fixed, Paraffin-Embedded and Fine-Needle Aspiration Tumor Specimens

Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.

A Novel FMR1 PCR Method for the Routine Detection of Low Abundance Expanded Alleles and Full Mutations in Fragile X Syndrome

BACKGROUNDnFragile X syndrome (FXS) is a trinucleotide-repeat disease caused by the expansion of CGG sequences in the 5 untranslated region of the FMR1 (fragile X mental retardation 1) gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on 2 tests, PCR and Southern blotting; however, performance or throughput limitations of these methods currently constrain routine testing.nnnMETHODSnWe evaluated a novel FMR1 gene-specific PCR technology with DNA templates from 20 cell lines and 146 blinded clinical samples. The CGG repeat number was determined by fragment sizing of PCR amplicons with capillary electrophoresis, and results were compared with those for FMR1 Southern blotting analyses with the same samples.nnnRESULTSnThe FMR1 PCR accurately detected full-mutation alleles up to at least 1300 CGG repeats and consisting of >99% GC character. All categories of alleles detected by Southern blotting, including 66 samples with full mutations, were also identified by the FMR1 PCR for each of the 146 clinical samples. Because all full mutation alleles in samples from heterozygous females were detected by the PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940-CGG allele in a background of 99% 23-CGG allele-a roughly 5- fold greater sensitivity than obtained with Southern blotting.nnnCONCLUSIONSnThe novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify; reproducibly detect low abundance full mutation alleles; and correctly infer homozygosity in female samples, thus greatly reducing the need for sample reflexing to Southern blotting.

An Information-Rich CGG Repeat Primed PCR That Detects the Full Range of Fragile X Expanded Alleles and Minimizes the Need for Southern Blot Analysis

(CGG)(n) repeat expansion in the FMR1 gene is associated with fragile X syndrome and other disorders. Current methods for FMR1 molecular testing rely on Southern blot analysis to detect expanded alleles too large to be PCR-amplified and to identify female homozygous alleles that often confound interpretations of PCR data. A novel, single-tube CGG repeat primed FMR1 PCR technology was designed with two gene-specific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG)(n) repeat. This PCR was evaluated with 171 unique DNA samples, including a blinded set of 146 clinical specimens. The method detected all alleles reported by Southern blot analysis, including full mutations in 66 clinical samples and comprised up to 1300 CGG. Furthermore, a blinded cohort of 42 female homozygous and heterozygous specimens, including 21 with full mutation alleles, was resolved with 100% accuracy. Last, AGG interrupter sequences, which may influence the risk of (CGG)(n) expansion in the children of some carriers, were each correctly identified in 14 male and female clinical samples as referenced to DNA sequencing. As a result, this PCR provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis and producing more comprehensive FMR1 genotyping data than other methods.

Activation of the NOTCH pathway in Head and Neck Cancer

NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently.

Fragile X AGG analysis provides new risk predictions for 45-69 repeat alleles.

We investigated the effect of AGG interruptions on fragile X repeat instability upon transmission of fragile X intermediate and small premutation alleles with 45–69 CGG repeats. The FMR1 repeat structure was determined for 375 mothers, 48 fathers, and 538 offspring (457 maternal and 81 paternal transmissions) using a novel PCR assay to determine repeat length and AGG interruptions. The number of AGG interruptions and the length of uninterrupted CGG repeats at the 3′ end were correlated with repeat instability on transmission. Maternal alleles with no AGGs conferred the greatest risk for unstable transmissions. All nine full mutation expansions were inherited from maternal alleles with no AGGs. Furthermore, the magnitude of repeat expansion was larger for alleles lacking AGG interruptions. Transmissions from paternal alleles with no AGGs also exhibited greater instability than those with one or more AGGs. Our results demonstrate that characterization of the AGG structure within the FMR1 repeat allows more accurate risk estimates of repeat instability and expansion to full mutations for intermediate and small premutation alleles.

High-resolution methylation polymerase chain reaction for fragile X analysis: Evidence for novel FMR1 methylation patterns undetected in Southern blot analyses

Purpose: Fragile X syndrome is associated with the expansion of CGG trinucleotide repeats and subsequent methylation of the FMR1 gene. Molecular diagnosis of fragile X currently requires Southern blot analysis to assess methylation. This study describes the evaluation of a polymerase chain reaction-only workflow for the determination of methylation status across a broad range of FMR1 genotypes in male and female specimens.Methods: We evaluated a novel method that combines allele-specific methylation polymerase chain reaction and capillary electrophoresis with eight cell line and 80 clinical samples, including 39 full mutations. Methylation status was determined using a three-step workflow: (1) differential treatment of genomic DNA using a methylation-sensitive restriction enzyme; (2) polymerase chain reaction with two sets of dye-tagged primers; and (3) amplicon sizing by capillary electrophoresis. All samples were analyzed by both methylation polymerase chain reaction and Southern blot analysis.Results: FMR1 methylation status and CGG repeat sizing were accurately and reproducibly determined in a set of methylation controls and genomic DNA samples representing a spectrum of CGG repeat lengths and methylation states. Moreover, methylation polymerase chain reaction revealed allele-specific methylation patterns in premutation alleles that were unobtainable using Southern blot analysis.Conclusions: Methylation polymerase chain reaction enabled high throughput, high resolution, and semiquantitative methylation assessments of FMR1 alleles, as well as determinations of CGG repeat length. Results for all samples were concordant with corresponding Southern blot analyses. As a result, this study presents a polymerase chain reaction-based method for comprehensive FMR1 analysis. In addition, the identification of novel methylation mosaic patterns revealed after polymerase chain reaction and capillary electrophoresis may be relevant to several FMR1 disorders.

AGG interruptions and maternal age affect FMR1 CGG repeat allele stability during transmission

BackgroundThe presence of AGG interruptions in the CGG repeat locus of the fragile X mental retardation 1 (FMR1) gene decreases the instability of the allele during transmission from parent to child, and decreases the risk of expansion of a premutation allele to a full mutation allele (the predominant cause of fragile X syndrome) during maternal transmission.MethodsTo strengthen recent findings on the utility of AGG interruptions in predicting instability or expansion to a full mutation of FMR1 CGG repeat alleles, we assessed the outcomes of 108 intermediate (also named gray zone) and 710 premutation alleles that were transmitted from parent to child, and collected from four international clinical sites. We have used the results to revise our initial model that predicted the risk of a maternal premutation allele expanding to a full mutation during transmission and to test the effect of AGG interruptions on the magnitude of expanded allele instability of intermediate or premutation alleles that did not expand to a full mutation.ResultsConsistent with previous studies, the number of AGG triplets that interrupts the CGG repeat locus was found to influence the risk of allele instability, including expansion to a full mutation. The total length of the CGG repeat allele remains the best predictor of instability or expansion to a full mutation, but the number of AGG interruptions and, to a much lesser degree, maternal age are also factors when considering the risk of transmission of the premutation allele to a full mutation.ConclusionsOur findings demonstrate that a model with total CGG length, number of AGG interruptions, and maternal age is recommended for calculating the risk of expansion to a full mutation during maternal transmission. Taken together, the results of this study provide relevant information for the genetic counseling of female premutation carriers, and improve the current predictive models which calculate risk of expansion to a full mutation using only total CGG repeat length.

Novel insight into mutational landscape of head and neck squamous cell carcinoma

Development of head and neck squamous cell carcinoma (HNSCC) is characterized by accumulation of mutations in several oncogenes and tumor suppressor genes. We have formerly described the mutation pattern of HNSCC and described NOTCH signaling pathway alterations. Given the complexity of the HNSCC, here we extend the previous study to understand the overall HNSCC mutation context and to discover additional genetic alterations. We performed high depth targeted exon sequencing of 51 highly actionable cancer-related genes with a high frequency of mutation across many cancer types, including head and neck. DNA from primary tumor tissues and matched normal tissues was analyzed for 37 HNSCC patients. We identified 26 non-synonymous or stop-gained mutations targeting 11 of 51 selected genes. These genes were mutated in 17 out of 37 (46%) studied HNSCC patients. Smokers harbored 3.2-fold more mutations than non-smokers. Importantly, TP53 was mutated in 30%, NOTCH1 in 8% and FGFR3 in 5% of HNSCC. HPV negative patients harbored 4-fold more TP53 mutations than HPV positive patients. These data confirm prior reports of the HNSCC mutational profile. Additionally, we detected mutations in two new genes, CEBPA and FES, which have not been previously reported in HNSCC. These data extend the spectrum of HNSCC mutations and define novel mutation targets in HNSCC carcinogenesis, especially for smokers and HNSCC without HPV infection.

CGG allele size somatic mosaicism and methylation in FMR1 premutation alleles

Background Greater than 200 CGG repeats in the 5′UTR of the FMR1 gene lead to epigenetic silencing and lack of the FMR1 protein, causing fragile X Syndrome. Individual carriers of a premutation (PM) allele with 55–200 CGG repeats are typically unmethylated and can present with clinical features defined as FMR1-associated conditions. Methods Blood samples from 17 male PM carriers were assessed clinically and molecularly by Southern blot, western blot, PCR and QRT-PCR. Blood and brain tissue from an additional 18 PM males were also similarly examined. Continuous outcomes were modelled using linear regression and binary outcomes were modelled using logistic regression. Results Methylated alleles were detected in different fractions of blood cells in all PM cases (n=17). CGG repeat numbers correlated with percent of methylation and mRNA levels and, especially in the upper PM range, with greater number of clinical involvements. Inter-tissue/intra-tissue somatic instability and differences in percent methylation were observed between blood and fibroblasts (n=4) and also observed between blood and different brain regions in three of the 18 PM cases examined. CGG repeat lengths in lymphocytes remained unchanged over a period of time ranging from 2 to 6u2005years, three cases for whom multiple samples were available. Conclusions In addition to CGG size instability, individuals with a PM expanded allele can exhibit methylation and display more clinical features likely due to RNA toxicity and/or FMR1 silencing. The observed association between CGG repeat length and percent of methylation with the severity of the clinical phenotypes underscores the potential value of methylation in affected PM to further understand penetrance, inform diagnosis and expand treatment options.

Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel.

BackgroundImprovements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures.MethodsThe evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance.ResultsWe observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250u2009ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value <10-16), and the incorporation of orthogonal sequencing strategies increased the harmonic mean of sensitivity and positive predictive value of mutation detection by 41%. In one cohort of FFPE tumor samples, coverage and inter-platform concordance were positively correlated with the QFI, emphasizing the need for pre-analytical sample quality control to reduce the risk of false positives and negatives. In a separate cohort of FFPE samples, the 51-gene panel achieved 78% sensitivity (95% CIu2009=u200956.3, 92.5) with 100% PPV (95% CIu2009=u200981.5, 100.0) based on known mutations at 7.9% median abundance. By sequencing specimens using an orthogonal NGS technology, sensitivity was improved to 87.0% (95% CIu2009=u200966.4,97.2) while maintaining PPV.ConclusionsThe results highlight the value of process integration in a comprehensive targeted NGS system, enabling both discovery and diagnostic applications, particularly when sequencing low-quality cancer specimens.