RESUMO
We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Gastrointestinais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pulmonares/genética , Melanoma/genética , Receptores ErbB/genética , Humanos , Hibridização in Situ Fluorescente , Limite de Detecção , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Mutação , Inclusão em Parafina , Controle de Qualidade , Receptor ErbB-2/genética , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/genéticaRESUMO
In this case we introduce Sporothrix pallida, a non-pathogenic environmental Sporothrix species as a cause of infectious keratitis in a corneal transplant recipient. Human infections caused by S. schenckii are well-known but human infection with Sporothrix pallida has not been previously reported.
