Quantitative analysis of genomic DNA degradation in whole blood under various storage conditions for molecular diagnostic testing.

Proper storage of whole blood is crucial for isolating nucleic acids from leukocytes and to ensure adequate performance of downstream assays in the molecular diagnostic laboratory. Short-term and long-term storage recommendations are lacking for successful isolation of genomic DNA (gDNA). Container type (EDTA or heparin), temperature (4 °C and room temperature) and time (1-130 days) were assessed as criterion for sample acceptance policies. The percentage of integrated area (%Ti) between 150 and 10,000 bp from the 2200 TapeStation electropherogram was calculated to measure gDNA degradation. Refrigerated EDTA samples yielded gDNA with low %Ti (high quality). Heparinized samples stored at room temperature yielded gDNA of worst quality. Downstream analysis demonstrated that the quality of the gDNA correlated with the quality of the data; samples with high %Ti generated significantly lower levels of high molecular weight amplicons. Recommendations from these analyses include storing blood samples intended for nucleic acid isolation in EDTA tubes at 4 °C for long term storage (>10 days). gDNA should be extracted within 3 days when blood is stored at room temperature regardless of the container. Finally, refrigerated heparinized samples should not be stored longer than 9 days if expecting high quality gDNA isolates. Laboratories should consider many factors, in addition to the results obtained herein, to update their policies for sample acceptance for gDNA extraction intended for molecular genetic testing.

Correction: Molecular Subtypes in Head and Neck Cancer Exhibit Distinct Patterns of Chromosomal Gain and Loss of Canonical Cancer Genes.

[This corrects the article DOI: 10.1371/journal.pone.0056823.].

Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation.

Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples. DNAs retrieved from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are commonly degraded, and specimens such as core biopsies are sometimes too small to obtain enough DNA for NGS applications. Thus, it is important to measure both the DNA quantity and quality accurately from clinical samples. However, there is no standard method for DNA quantity and quality analyses for NGS library preparation. We tested four different methods (PicoGreen, Qubit® fluorometry, TaqMan and SYBR-Green-based qPCR assay) and compared each to RNase P TaqMan as a reference control. We found that SYBR-Green-based qPCR assay provides a consistent and accurate DNA quantification while keeping its cost relatively low and the throughput high. We designed a dual-probe SYBR-Green qPCR assay for DNA quantity and quality assessment for targeted NGS library preparation. This assay provides a Dscore (degradation score) of the interrogated DNA by analyzing two different sizes of amplicons. We show an example of a clinical sample with a very high Dscore (high degradation). With a regular DNA quantification, without considering the degradation status, no correct NGS libraries were obtained. However, after optimizing the library condition by considering its poor DNA quality, a reasonably good library and sequencing results were obtained. In summary, we developed and presented a new DNA quantity and quality analysis qPCR assay for the targeted NGS library preparation. This assay may be mostly efficient for the clinical samples with high degradation and poor DNA quality.

Comprehensive evaluation and validation of targeted next-generation sequencing performance in two clinical laboratories.

Next-generation sequencing (NGS) has led to breakthroughs for genetic and genomic analyses and personalized medicine approaches for many diseases. More and more clinical laboratories are using NGS as a genetic screening tool for providing mutation information that is used to select the best treatment regimens for cancer patients. However, several obstacles prevent the routine implementation of NGS technology into the clinical molecular diagnosis setting: the sophisticated sample preparation process, high cost, time-consuming data analyses, as well as the reproducibility and accuracy of interpretation. To systematically evaluate the performance and quality of targeted NGS cancer panel analyses in clinical laboratories, we performed three different tests: i) laboratory-to-laboratory accuracy test, ii) intra-laboratory precision validation, and iii) limit of detection test, using formalin-fixed, paraffin-embedded cancer tissue specimens, cell lines and mutation positive DNA. A laboratory-to-laboratory accuracy test performed using 51 samples showed 100% sensitivity and 99.97% specificity. For the intra-laboratory precision test, 100% reproducibility was observed. For the limit of detection test, KRAS mutations from samples diluted from 70 to 2% of mutant allele frequencies were detected correctly. We believe that the present study demonstrated the feasibility of clinical implementation of a targeted NGS cancer panel analysis for personalized medicine.

Molecular subtypes in head and neck cancer exhibit distinct patterns of chromosomal gain and loss of canonical cancer genes.

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal heterogeneous disease. Beyond the role of human papilloma virus (HPV), no validated molecular characterization of the disease has been established. Using an integrated genomic analysis and validation methodology we confirm four molecular classes of HNSCC (basal, mesenchymal, atypical, and classical) consistent with signatures established for squamous carcinoma of the lung, including deregulation of the KEAP1/NFE2L2 oxidative stress pathway, differential utilization of the lineage markers SOX2 and TP63, and preference for the oncogenes PIK3CA and EGFR. For potential clinical use the signatures are complimentary to classification by HPV infection status as well as the putative high risk marker CCND1 copy number gain. A molecular etiology for the subtypes is suggested by statistically significant chromosomal gains and losses and differential cell of origin expression patterns. Model systems representative of each of the four subtypes are also presented.

Crystal-storing histiocytosis as a cause of symptomatic cardiac mass.

Crystal-storing histiocytosis is a rare disorder that is typically associated with low-grade B-cell lymphomas and monoclonal gammopathy. We present a 64-year-old man with a prior history of weakness and weight loss and hematologic evaluation that had revealed immunoglobulin G kappa monoclonal light chains in the serum and negative bone marrow biopsy. He presented with supraventricular tachyarrhythmia and a right atrial mass seen on echocardiogram and excised surgically. Histologically, the tumor was composed of sheets of macrophages infiltrating the atrial myocardium. The histiocytes were filled with multiple needle-shaped, periodic acid-Schiff-negative crystals. These cells and associated plasma cells failed to show clonal light chain restriction by in situ hybridization or immunohistochemistry, and there was no area of lymphoma in the tumor. Ultrastructural examination showed numerous stick-like, trapezoidal, or polygonal dense crystals in the cytoplasm of histiocytes corroborating the diagnosis of crystal-storing histiocytosis. Although rare, crystal-storing histiocytosis should be included in the differential diagnosis of heart masses in patients with hematologic conditions associated with monoclonal gammopathy.

Primary histiocytic sarcoma arising in the head and neck with predominant spindle cell component.

This is the first case report of Histiocytic Sarcoma (HS) with predominant spindle cell component occurring in the head and neck region of a 41-year-old man. The tumor was composed of sheets of large round to oval cells with pleomorphic vesicular nuclei, prominent nucleoli and abundant eosinophilic cytoplasm. Multinucleated forms, numerous mitoses, and tumor necrosis were also noted. Sheets, fascicles, and whorls of spindle cells with spindled to ovoid vesicular nuclei, small to medium-sized distinct nucleoli, and eosinophilic cytoplasm were frequently observed. Immunohistochemical staining in the tumor cells was positive for CD163, CD68, lysozyme, CD45, and NSE. Focal expression of CD4 and S-100 was also noted. Electron microscopy demonstrated an abundance of lysosomes in the cytoplasm of tumor cells. Chromosome study revealed a 57-80 hyperdiploid [7]/46, XY [13] karyotype, including 3 to 4 copies of various chromosomes. The immunohistochemical and ultrastructural findings confirmed the diagnosis of HS.