Automation of hybridization and capture based next generation sequencing library preparation requires reduction of on-deck bead binding and heated wash temperatures.

Capture-based library preparation for next generation sequencing (NGS) offers a balance between sequencing depth and bioinformatics cost of analysis. Liquid handling automation enhances the reliability of the library preparation process by reducing sample-to-sample variation and substantially enhances throughput, particularly when it can be employed in a 'walk-away' fashion with limited hands-on interaction. This requires complex series of mixing and heating steps like those utilized in capture chemistries to happen on the liquid handler. While developing liquid handling automation for Integrated DNA Technologies (IDT) xGen Exome, Illumina TruSight Oncology 500, and Personal Genome Diagnostics (PGDx) elio Plasma Resolve chemistries on the PerkinElmer Sciclone liquid handler, we found that applying the capture temperatures recommended for manual library preparation results in low yield on automation. To restore the final library yield, we reduced bead binding and/or heated wash temperatures of the Peltier heaters on the liquid handlers by about 10°C. Since this applied across three unique capture-based chemistries, we consider this a generalizable principle of automating capture on the Sciclone. We hypothesize that this is driven by the very different thermodynamic environments represented by a sealed plate on a thermal cycler and a plate with a lid on a Peltier heater. This phenomenon should be considered when automating NGS library preparation on PerkinElmer Sciclone instruments.

Preemptive Pharmacogenomic Testing for Precision Medicine: A Comprehensive Analysis of Five Actionable Pharmacogenomic Genes Using Next-Generation DNA Sequencing and a Customized CYP2D6 Genotyping Cascade.

Significant barriers, such as lack of professional guidelines, specialized training for interpretation of pharmacogenomics (PGx) data, and insufficient evidence to support clinical utility, prevent preemptive PGx testing from being widely clinically implemented. The current study, as a pilot project for the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol, was designed to evaluate the impact of preemptive PGx and to optimize the workflow in the clinic setting. We used an 84-gene next-generation sequencing panel that included SLCO1B1, CYP2C19, CYP2C9, and VKORC1 together with a custom-designed CYP2D6 testing cascade to genotype the 1013 subjects in laboratories approved by the Clinical Laboratory Improvement Act. Actionable PGx variants were placed in patient's electronic medical records where integrated clinical decision support rules alert providers when a relevant medication is ordered. The fraction of this cohort carrying actionable PGx variant(s) in individual genes ranged from 30% (SLCO1B1) to 79% (CYP2D6). When considering all five genes together, 99% of the subjects carried an actionable PGx variant(s) in at least one gene. Our study provides evidence in favor of preemptive PGx testing by identifying the risk of a variant being present in the population we studied.

Preemptive genotyping for personalized medicine: design of the right drug, right dose, right time-using genomic data to individualize treatment protocol.

OBJECTIVE: To report the design and implementation of the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment protocol that was developed to test the concept that prescribers can deliver genome-guided therapy at the point of care by using preemptive pharmacogenomics (PGx) data and clinical decision support (CDS) integrated into the electronic medical record (EMR). PATIENTS AND METHODS: We used a multivariate prediction model to identify patients with a high risk of initiating statin therapy within 3 years. The model was used to target a study cohort most likely to benefit from preemptive PGx testing among the Mayo Clinic Biobank participants, with a recruitment goal of 1000 patients. We used a Cox proportional hazards model with variables selected through the Lasso shrinkage method. An operational CDS model was adapted to implement PGx rules within the EMR. RESULTS: The prediction model included age, sex, race, and 6 chronic diseases categorized by the Clinical Classifications Software for International Classification of Diseases, Ninth Revision codes (dyslipidemia, diabetes, peripheral atherosclerosis, disease of the blood-forming organs, coronary atherosclerosis and other heart diseases, and hypertension). Of the 2000 Biobank participants invited, 1013 (51%) provided blood samples, 256 (13%) declined participation, 555 (28%) did not respond, and 176 (9%) consented but did not provide a blood sample within the recruitment window (October 4, 2012, through March 20, 2013). Preemptive PGx testing included CYP2D6 genotyping and targeted sequencing of 84 PGx genes. Synchronous real-time CDS was integrated into the EMR and flagged potential patient-specific drug-gene interactions and provided therapeutic guidance. CONCLUSION: This translational project provides an opportunity to begin to evaluate the impact of preemptive sequencing and EMR-driven genome-guided therapy. These interventions will improve understanding and implementation of genomic data in clinical practice.

Molecular characterization of endometrial cancer: a correlative study assessing microsatellite instability, MLH1 hypermethylation, DNA mismatch repair protein expression, and PTEN, PIK3CA, KRAS, and BRAF mutation analysis.

Endometrial cancer is associated with numeric and structural chromosomal abnormalities, microsatellite instability (MSI), and alterations that activate oncogenes and inactivate tumor suppressor genes. The aim of this study was to characterize a set of endometrial cancers using multiple molecular genetic and immunohistochemical techniques. Ninety-six cases were examined for genomic alterations by MSI, MLH1 promoter hypermethylation, p53 and mismatch repair protein expression (MLH1, MSH2, MSH6, PMS2), and PTEN, PIK3CA, KRAS, and BRAF mutation analysis. At least 1 alteration was identified in 48 of 87 (55%) specimens tested for PTEN, making it the most common abnormality in this study. A PIK3CA alteration was observed in 16 (17%) specimens. Twenty-nine of 94 (31%) MSI tested tumors exhibited an MSI-H phenotype. Of the 29 MSI-H cases, 24 (83%) were positive for methylation of the MLH1 promoter region. Twenty-three (82%) of the 28 MSI-H cases with immunohistochemistry results showed loss of expression of MLH1/PMS2 (n=19), MSH2/MSH6 (n=2), or MSH6 only (n=2). Of the 19 MSI-H cases with loss of MLH1/PMS2 on immunohistochemistry, 18 were positive, and 1 was equivocal for MLH1 promoter hypermethylation. Twelve of 94 cases (13%) analyzed for KRAS mutations were found to have a mutation. No BRAF V600E mutations were indentified. This study provides a comprehensive molecular genetic analysis of commonly analyzed targets in a large cohort of endometrial cancers.

Evaluation of oligonucleotide sequence capture arrays and comparison of next-generation sequencing platforms for use in molecular diagnostics.

BACKGROUND: Next-generation DNA sequencing (NGS) techniques have the potential to revolutionize molecular diagnostics; however, a thorough evaluation of these technologies is necessary to ensure their performance meets or exceeds that of current clinical sequencing methods. METHODS: We evaluated the NimbleGen Sequence Capture 385K Human Custom Arrays for enrichment of 22 genes. We sequenced each sample on both the Roche 454 Genome Sequencer FLX (GS-FLX) and the Illumina Genome Analyzer II (GAII) to compare platform performance. RESULTS: Although the sequence capture method allowed us to rapidly develop a large number of sequencing assays, we encountered difficulty enriching G+C-rich regions. Although a high proportion of reads consistently mapped outside of the targeted regions, >80% of targeted bases for the GAII and >30% of bases for the GS-FLX were covered by a read depth of > or =20, and > 90% of bases for the GAII and > 80% of bases for the GS-FLX were covered by a read depth of > or =5. We observed discrepancies among sequence variants identified by the different platforms. CONCLUSIONS: Although oligonucleotide arrays are quick and easy to develop, some problematic regions may evade capture, necessitating sequential redesigning for complete optimization. Neither sequencing technology was able to detect every variant identified by Sanger sequencing because of well-known drawbacks of the NGS technologies. The rapidly decreasing error rates and costs of these technologies, however, coupled with advancing bioinformatic capabilities, make them an attractive option for molecular diagnostics in the very near future.

Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non-small cell lung carcinomas.

Recently, the fusion gene EML4-ALK was identified in non-small cell lung carcinoma, which could be a potential therapeutic target. We investigated the prevalence of anaplastic lymphoma kinase protein expression in these tumors by immunohistochemistry and correlated the results with data from ALK molecular studies. Gene expression profiling was performed on 35 adenocarcinomas to identify cases with ALK gene up-regulation, which was correlated with protein overexpression by immunohistochemistry. Immunohistochemistry was also performed on an independent cohort consisting of 150 adenocarcinomas and 150 squamous cell carcinomas to evaluate the utility of anaplastic lymphoma kinase immunostaining as a screening tool. Florescence in situ hybridization for the ALK locus and reverse transcriptase-polymerase chain reaction for EML4-ALK were performed on tumors positive for anaplastic lymphoma kinase by immunohistochemistry. Transcriptional up-regulation of ALK was identified in 2 (6%) of 35 adenocarcinomas by gene expression profiling. These 2 cases were positive for anaplastic lymphoma kinase by immunohistochemistry, whereas the remaining 33 cases were completely negative. In the independent cohort, anaplastic lymphoma kinase immunostaining was positive in 1 of 150 squamous cell carcinomas and in 3 of 150 adenocarcinomas. The 6 cases positive for anaplastic lymphoma kinase by immunohistochemistry showed evidence of ALK locus rearrangement by florescence in situ hybridization but were negative for EGFR and KRAS mutation. The presence of EML4-ALK fusion transcript was confirmed in 2 cases by reverse transcriptase-polymerase chain reaction. In conclusion, anaplastic lymphoma kinase immunoreactivity in non-small cell lung carcinomas was associated with transcriptional up-regulation, ALK locus rearrangement, and the presence of EML4-ALK fusion transcript. Anaplastic lymphoma kinase immunohistochemistry may have utility as a screening tool or as a surrogate marker for the molecular techniques to detect the EML4-ALK fusion gene in these tumors.

Direct uterine sampling with the Tao brush sampler using a liquid-based preparation method for the detection of endometrial cancer and atypical hyperplasia: a feasibility study.

BACKGROUND: Endometrial cytology sampling devices for direct uterine sampling have been shown in previous studies to be a reliable and relatively painless method for detecting endometrial lesions. The purpose of the current study was to determine the performance characteristics of endometrial cytology for the detection of malignancy and atypical hyperplasia using liquid-based cytology specimens collected with the Tao brush sampler. METHODS: Brushings of the endometrial cavity were obtained from 139 hysterectomy specimens before routine histopathologic evaluation. Cytology specimens were fixed in PreservCyt and processed using ThinPrep technology. Cytology diagnoses were classified as nondiagnostic, negative, atypical, or positive for malignancy. Histopathologic findings were used as the gold standard for determining the performance characteristics of cytology. RESULTS: Histopathologic results from the 139 patients included 81 (58%) endometrial cancers, 7 (5%) complex hyperplasias with atypia, 2 (1%) complex hyperplasias without atypia, and 49 (35%) patients with benign histology. The number of specimens diagnosed cytologically as positive, atypical, negative, or nondiagnostic was 60 (43%), 40 (29%), 37 (27%), and 2 (1%) specimens, respectively. The overall sensitivity and specificity of cytology for detecting endometrial cancer and atypical hyperplasia were 95% and 66% when atypical cytology specimens were considered positive. CONCLUSIONS: The results of the current study indicate that direct endometrial sampling by liquid-based endometrial cytology collected with the Tao brush sampler produces specimens that contain cellular material that may be identified as endometrial cancer or atypical hyperplasia. Both atypical and positive cytology diagnoses are indicators for triage to more specific methods of diagnosis.

LOC387715/HTRA1 and complement factor H variants in patients with age-related macular degeneration seen at the mayo clinic.

PURPOSE: To confirm association of the complement factor H allelic variant (CFH Y402H) and the LOC387715/HTRA1 (LOC387715 A69S) risk alleles with age-related macular degeneration (AMD). STUDY POPULATION: Study of 89 Caucasian patients with neovascular (exudative) AMD and 232 Caucasian controls. METHODS: The Y402H variant of CFH gene and A69S variant of LOC387715/HTRA1 gene locus were examined. RESULTS: For CFH, the odds ratio for the homozygous variant was 4.97 (CI 2.52 to 9.79). For LOC387715/HTRA1 the odds ratio for the homozygous risk variant was 7.75 (CI 3.46 to 17.35). The odds ratio for heterozygous carriers was 3.35 (CI 1.91 to 5.90).

Relationship between age-related macular degeneration-associated variants of complement factor H and LOC387715 with coronary artery disease.

OBJECTIVE: To determine whether either of the gene variants associated with age-related macular degeneration is associated with coronary artery disease (CAD). PATIENTS AND METHODS: This study consisted of 493 patients who underwent clinically indicated coronary angiography between June 1, 1998, and January 1, 1999. The Y402H variant of the complement factor H (CFH) gene and the A69S variant of the LOC387715 gene locus were examined by restriction fragment length polymorphism. Multiple logistic regression models were used to assess the association of CFH and LOC gene variants with CAD. Covariates with well-established associations with CAD were also evaluated. RESULTS: Seventy patients (14%) were homozygous for the histidine variant (HH) of CFH, 237 (48%) were heterozygous for the histidine variant (HY), and 186 (38%) were homozygous for the tyrosine variant (YY). Three hundred eight patients (62%) were homozygous for the alanine allele of LOC387715, 170 (34%) were heterozygous for Ala and Ser alleles, and 15 (3%) were homozygous for the serine variant. The overall association of the CFH genotype with CAD was not statistically significant (P=.08). However, some evidence (P=-.046) suggested that CAD was increased for the HH genotype compared to the homozygous wild-type YY genotype (odds ratio, 1.95; 95% confidence interval, 1.01-3.76). Male sex, hypertension, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and age were other variables that demonstrated significant associations with CAD. The overall effect of the LOC genotype was not statistically significant (P=-.06). Heterozygosity for the serine variant was (P=-.02) associated with the absence of CAD vs the AS genotype (odds ratio, 0.59; confidence interval, 0.38-0.91; P=-.02). CONCLUSION: The CFH genotype may have an independent association with CAD, although our evidence did not show statistical significance. Controlling for known risk factors, the age-related macular degeneration-associated HH variant appears to be associated with CAD. The LOC387715 gene may also play a role in CAD.