Dichoroacetate (DCA) and trichloroacetate (TCA) were found to be hepatotoxic and hepatocarcinogenic in rodents. To investigate the role of oxidative stress in the long term hepatotoxicity of the compounds, groups of mice were administered 7.7, 77, 154 and 410 mg/kg/day, of either DCA or TCA, by gavage, for 4 weeks (4-W) and 13 weeks (13-W), and superoxide anion (SA), lipid peroxidation (LP) and DNA-single strand breaks (SSBs) were determined in the hepatic tissues. Significant increases in all of the biomarkers were observed in response to the tested doses of both compounds in the two tested periods, with significantly greater increases observed in the 13-W, as compared with the 4-W period. Hepatomegaly was only observed with a DCA dose of 410 mg/kg/day in the 13-W treatment period, and that was associated with significant declines in the biomarkers, when compared with the immediately lower dose. With the exception of LP production in the 13-W treatment period that was similarly induced by the two compounds, the DCA-induced increases in all of the biomarkers were significantly greater than those of TCA. Since those biomarkers were significantly induced by the compounds' doses that were shown to be carcinogenic but at earlier periods than those demonstrating hepatotoxicity/ haptocarcinogencity, they can be considered as initial events that may lead to later production of those long term effects. The results also suggest LP to be a more significant contributing mechanism than SA and DNA damage to the long term hepatotoxicity of TCA.

The induction of phagocytic activation in response to prolonged treatment with different doses of dichloroacetate (DCA) and trichloroacetate (TCA) has been investigated in mice. Groups of B6C3F1 male mice were administered 7.7, 77, 154 and 410 mg of DCA or TCA/ kg/day , post orally, for 4-and 13-weeks. Peritoneal lavage cells (PLCs) were isolated and assayed for the different biomarkers of phagocytyic activation, including superoxide anion (SA), tumor necrosis factor-alpha (TNF-α), and myeloperoxidase (MPO). In addition, the role of superoxide dismutase (SOD) in the SA production was also assessed. DCA and TCA produced significant and dose-dependent increases in SA and TNF-α production and in MPO activity but the increases in response to the high doses of the compounds (> 77 mg/kg/day) in the 13-week treatment period were less significant than those produced in the 4-week treatment period. Also, dose-dependent increases in SOD activity were observed in both periods of treatments. In general, the results demonstrate significant induction of the biomarkers of phagocytic activation by doses of DCA and TCA that were previously shown to be non carcinogenic, with significantly greater increases observed at the earlier period of exposure, as compared with later period. These findings may argue against the contribution of those mechanisms to the hepatotoxicity/hepatocarcinogenicity of the compounds and suggest them to be early adaptive/ protective mechanisms against their long term effects.

Background and aims: The MET exon 14 skipping (METex14) is an oncogenic driver mutation that provides a therapeutic opportunity in non-small cell lung cancer (NSCLCs) patients. This event often results from sequence changes at the MET canonical splicing sites. We characterize two novel non-canonical splicing site variants of MET that produce METex14. Materials and Methods: Two variants were identified in three advanced-stage NSCLC patients in a next-generation sequencing panel. The potential impact on splicing was predicted using in silico tools. METex14 mutation was confirmed using reverse transcription (RT)-PCR and a Sanger sequencing analysis on RNA extracted from stained cytology smears. Results: The interrogated MET (RefSeq ID NM_000245.3) variants include a single nucleotide substitution, c.3028+3A>T, in intron 14 and a deletion mutation, c.3012_3028del, in exon 14. The in silico prediction analysis exhibited reduced splicing strength in both variants compared with the MET normal transcript. The RT-PCR and subsequent Sanger sequencing analyses confirmed METex14 skipping in all three patients carrying these variants. Conclusion: This study reveals two non-canonical MET splice variants that cause exon 14 skipping, concurrently also proposes a clinical workflow for the classification of such non-canonical splicing site variants detected by routine DNA-based NGS test. It shows the usefulness of in silico prediction to identify potential METex14 driver mutation and exemplifies the opportunity of routine cytology slides for RNA-based testing.

The Molecular Pathology Section Q8 , Cleveland Clinic (Cleveland, OH Q9), has undergone enhancement of its testing portfolio and processes. An electronic Q10 -and paper-based data-management system was replaced with a commercially available laboratory information-management system (LIMS) software application, a separate bioinformatics platform, customized test-interpretation applications, a dedicated sampleaccessioning service, and a results-releasing software application. The customized LIMS solution manages complex workflows, large-scale data packets, and process automation. A customized approach was required because, in a survey of commercially available off-the-shelf Q11 software products, none met the diverse and complex needs of this molecular diagnostics service. The project utilized the expertise of clinical laboratorians, pathologists, genetics counselors, bioinformaticians, and systems analysts in partnering with software-engineering consultants to design and implement a solution. Concurrently, Agile software-building best practices were formulated, which may be emulated for scalable and costeffective laboratory-authored software.