Malignant pleural effusion supernatant is an alternative liquid biopsy specimen for comprehensive mutational profiling.

BACKGROUND: The clinical utility of malignant pleural effusion (MPE) to detect mutation has been well documented; however, routine practice of the use of MPE involves collection of the cell pellet to detect mutation, and limited studies have interrogated the MPE supernatant as an alternative source of tumor-derived DNA for mutation profiling. In this study, we investigated the potential of MPE supernatant as a liquid biopsy specimen by comparing its mutation profile with that of matched MPE cell pellets, tissue, and plasma samples. METHODS: Sequencing data from 17 patients with matched lung tissue, plasma, and MPE samples were retrospectively analyzed. Capture-based targeted sequencing was performed on matched plasma and MPE supernatant samples obtained from 154 patients with advanced lung adenocarcinoma. RESULTS: MPE supernatants had significantly higher median maximum allelic fractions (maxAFs) than their corresponding cell pellets (P = 0.008) and plasma samples (P = 0.036), and a comparable maxAF value to that of tissue samples (P = 0.675). Comparison of MPE supernatant and matched plasma samples from the larger cohort (n = 154) revealed a comparable mutation detection rate; however, MPE supernatant had a significantly higher median maxAF than plasma (20.3% vs. 1.13%; P < 0.001). Furthermore, the concordance rates between MPE supernatant and plasma for single-nucleotide and copy number variations were 56% and 18%, respectively, suggesting that MPE supernatant reveals a more comprehensive mutation spectrum, particularly for copy number variations. CONCLUSION: Overall, our study shows that MPE supernatant is an optimal alternative source of tumor-derived DNA for comprehensive mutation profiling.

Hobnail variant of papillary thyroid carcinoma: molecular profiling and comparison to classical papillary thyroid carcinoma, poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma.

BACKGROUND: As a rare but aggressive papillary thyroid carcinoma (PTC) variant, the genetic changes of hobnail variant of PTC (HVPTC) are still unclear. RESULTS: The prevalence of HVPTC was 1.69% (18/1062) of all PTC diagnosed in our cohort. 73 samples from 55 patients (17 HVPTC, 26 CPTC, 7 PDTC and 5 ATC) were successfully analyzed using targeted NGS with an 18-gene panel. Thirty-seven mutation variant types were identified among 11 genes. BRAF V600E mutation was the most common mutation, which is present in almost all HVPTC samples (16/17, 94%), most CPTC samples (20/26, 77%), and none of the ATC and PDTC samples. TERT promoter mutation (C228T) was identified in 2 ATC and one HVPTC patient. RAS and TP53 mutation are almost exclusively present among ATC and PDTC samples although TP53 mutation was also observed in 3 HVPTC patients. Six different GNAS mutations were identified among 8 CPTC patients (31%) and none of the HVPTC patients. The only patient who died of disease progression harbored concomitant TERT C228T mutation, BRAF V600E mutation and TP53 mutation. METHODS: HVPTC cases were identified from a group of 1062 consecutive surgical specimens diagnosed as PTC between 2000 and 2010. Targeted next-generation sequencing (NGS) was applied to investigate the mutation spectrum of HVPTC, compared to classical PTC (CPTC), poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC). CONCLUSION: As an aggressive variant of PTC, HVPTC has relatively specific molecular features, which is somewhat different from both CPTC and ATC/PDTC and may underlie its relatively aggressive behavior.

Capture-Based Targeted Ultradeep Sequencing in Paired Tissue and Plasma Samples Demonstrates Differential Subclonal ctDNA-Releasing Capability in Advanced Lung Cancer.

INTRODUCTION: Circulating tumor DNA (ctDNA), which represents an unbiased way to assess tumor genetic profile noninvasively, facilitates studying intratumor heterogeneity. Although intratumor heterogeneity has been elucidated substantially in a few cancer types, including NSCLC, how it influences the ability of tumor cells harboring different genetic abnormalities in releasing their DNA remains elusive. We designed a capture-based panel targeting NSCLC to detect and quantify genetic alterations from plasma samples by using deep sequencing. By applying the panel to paired biopsy and plasma samples, we imputed and compared the ctDNA-releasing efficiency in subclones harboring distinct genetic variants. METHODS: We collected 40 pairs of matched biopsy and plasma samples from patients with advanced lung cancer and applied capture-based sequencing using our LungPlasma panel, which consists of critical exons and introns of 168 genes. We derived a normalized relative allelic fraction score (NRAFS) to reflect ctDNA-releasing efficiency. RESULTS: By using mutations detected in biopsy samples as a reference, we achieved 87.2% by-variant sensitivity, including for single-nucleotide variants, insertions or deletions, and gene fusions. Furthermore, the by-variant sensitivity for the seven most critical and actionable genes was 96.2%. The average NRAFS for subclones carrying mutations from seven actionable genes was 0.877; in contrast, the average NRAFS for other mutations was 0.658. Mutations from four genes involved in cell cycle pathways had a particularly low NRAFS (0.480) compared with the other two groups (p = 0.07). CONCLUSIONS: We have demonstrated that subclones carrying driver mutations are more prone to release DNA. We have also demonstrated the quantitative ability of capture-based sequencing, paving its way for routine utilization in clinical settings.

Sodium selenite-induced apoptosis mediated by ROS attack in human osteosarcoma U2OS cells.

Sodium selenite (Na(2)SeO(3), SSE) is an inorganic Se compound that is widely used in cancer chemoprevention studies. SSE has been shown to have anti-proliferative effects on several types of human cancer cells, but its effect on osteosarcoma cells has thus far not been reported. In this study, the cytotoxic effect of SSE on osteosarcoma cells U2OS was investigated in vitro and found to be higher than on comparable non-cancer cell lines 293 and L6. Treatment with SSE decreased cell growth in a dose- and time-dependent manner and altered cellular morphology. SSE also inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and accumulation of cells during the advanced phase of apoptosis. SSE-induced apoptosis correlated with the activation of CASP 3, downregulation of BCL-2, and upregulation of P53 and PTEN in U2OS cells. These results indicated that SSE induces apoptosis in U2OS cells mainly through an ROS-mediated caspase pathway. This is the first report to show a possible mechanism of the anti-proliferative effect of SSE for the prevention of osteosarcoma in cell culture models.