FNA smears as a potential source of DNA for targeted next-generation sequencing of lung adenocarcinomas.

BACKGROUND: Diff-Quik-stained fine-needle aspiration (FNA) smears and touch preparations from biopsies represent alternative specimens for molecular testing when cell block or biopsy material is insufficient. This study describes the use of these samples for targeted next-generation sequencing (NGS) of primary and metastatic lung adenocarcinoma and reports the DNA quality and success rates of FNA smears versus other specimens from 1 year of clinical use. METHODS: A validation set of 10 slides from 9 patients with prior clinical epidermal growth factor receptor (EGFR) Sanger sequencing and KRAS pyrosequencing (5 KRAS-positive/EGFR-negative and 4 KRAS-negative/EGFR-negative) underwent DNA extraction, quality assessment, and targeted NGS. Subsequently, lung adenocarcinoma specimens submitted for NGS solid tumor mutation panel testing in 1 calendar year (60 biopsies, 57 resections, 33 FNA cell blocks, 12 FNA smears, and 10 body fluid cell blocks) were reviewed for specimen adequacy, sequencing success, and DNA quality. RESULTS: All 10 validation samples met the DNA quality threshold (delta Ct threshold < 8; range, -2.2 to 4.9) and yielded 0.5 to 22 µg of DNA. The KRAS and EGFR mutation status from FNA smears according to NGS was concordant with previous clinical testing for all 10 samples. In the 1-year review, FNA smears were 100% successful, and this suggested a performance equivalent to or better than the performance of established specimen types, including FNA cell blocks. DNA quality according to ΔCt was significantly better with FNA smears versus biopsies, resections, and FNA cell blocks. CONCLUSIONS: FNA smears of lung adenocarcinomas are high-quality alternative specimens for a targeted NGS panel with a high success rate in clinical practice. Cancer Cytopathol 2016;124:406-14. © 2016 American Cancer Society.

UGT1A1 promoter genotype is not strongly associated with severity of coronary artery disease.

Atherosclerosis is a leading cause of morbidity and mortality. Oxidative stress is thought to play a role in its pathogenesis. Bilirubin is an endogenous antioxidant that is mildly elevated in people with Gilbert syndrome. Homozygosity for a A(TA)7TAA variant of the UDP-glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) promoter is necessary for expression of the Gilbert phenotype. We studied the relationship between coronary artery disease (CAD) and the Gilbert genotype. Decedents who underwent autopsy were categorized into none/mild, moderate, and severe CAD groups based on autopsy findings. Known CAD risk factors were evaluated for each decedent in the severe CAD group (n=35), and for an age, race, and sex-matched control group with none/mild CAD (n=45). Formalin-fixed paraffin-embedded (FFPE) tissue was tested for UGT1A1 promoter variants by polymerase chain reaction and capillary electrophoresis. To our knowledge, this is the first study to successfully apply UGT1A1 promoter genotyping to formalin-fixed paraffin-embedded tissue, which may facilitate more thorough examination of clinicopathologic correlations. The frequency of the Gilbert genotype was compared between the none/mild cohort and the severe cohort. UGT1A1 promoter genotype data were obtained for 76/80 cases. The overall frequency of the Gilbert genotype compared well with previously reported frequencies at 16%, with a frequency of 16% in the none/mild CAD group and 15% in the severe CAD group. These findings suggest that UGT1A1 promoter genotype is not a major factor contributing to risk of CAD.

Diagnosis of human congenital cytomegalovirus infection by amplification of viral DNA from dried blood spots on perinatal cards.

Congenital human cytomegalovirus (HCMV) infection affects 1% of children and is the most common infectious cause of sensorineural hearing loss. Due to the difficulty of diagnosing deafness and other neurological disorders in infants, affected individuals may not be recognized until much later when active infection has resolved and culture is no longer informative. To overcome this problem, congenital HCMV infection was diagnosed retrospectively by testing residual blood samples collected from newborns and dried on perinatal cards as part of the North Carolina Newborn Screening Program. We modified the Qiagen method for purifying DNA from dried blood spots to increase the sample size and recovery of the lysate. A multiplex, real-time TaqMan polymerase chain reaction assay on an ABI 7900 instrument measured a highly conserved segment of the HCMV polymerase gene and the APOB human control gene. HCMV DNA was detected in blood dried on perinatal cards from all seven infants with culture-proven congenital infection, and all 24 negative control cases lacked detectable HCMV DNA. Our findings suggest that it is possible to diagnose congenital HCMV infection using dried blood collected up to 20 months earlier. Further studies are warranted on patients with hearing loss or other neurological deficits to determine the percentage that is attributable to congenital HCMV infection.