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The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

TNF-alpha is an inducible cytokine with widely divergent effects on numerous target tissues. Secretion and response to TNF-alpha appear to be required in cellular immune function including that of classical cytotoxic T cell development, non-MHC restricted cytotoxicity, and the mixed lymphocyte reaction. In addition, production of TNF-alpha has been implicated in a number of pathophysiologic processes involving lymphocytes. In this report, we have investigated the regulation of TNF-alpha in normal lymphocytes stimulated with IL-2. Our results demonstrate that the level of TNF-alpha gene expression can be determined by the quantity of IL-2 present during lymphocyte activation. The increased steady-state levels of TNF-alpha mRNA were attributable to an enhanced rate of nuclear transcription. TNF-alpha secretion increased with escalating IL-2 dose in parallel to that of mRNA production. The modulation of TNF-alpha gene expression by IL-2 concentration has been previously unrecognized and may be an important mechanism in normal immunohomeostasis, cellular dysregulations involving TNF production, and the dose-dependent toxicities observed with high-dose IL-2 immunotherapy.

We investigated whether TNF-beta could induce or modulate TNF-alpha gene expression in human peripheral blood lymphocytes. For these experiments, lymphocytes were cultured in serum-free medium for 48 hours with human recombinant TNF-beta in the presence or absence of human recombinant IL-2. At the end of the culture period, total cellular RNA was isolated and probed for TNF-alpha mRNA using an S1 nuclease protection assay. TNF-beta alone was unable to induce lymphocyte TNF-alpha mRNA. However, when TNF-beta was used in combination with IL-2 an 8- to 12-fold increase in TNF-alpha mRNA compared to lymphocytes activated in IL-2 alone was observed. Secreted TNF-alpha was measured in the culture supernatants by TNF-alpha specific ELISA. Consistent with the results of mRNA analysis, TNF-beta alone did stimulate TNF-alpha secretion, but lymphocytes activated with TNF-beta/IL-2 demonstrated a 3- to 10-fold increase in secreted TNF-alpha over that induced by IL-2 alone. These experiments demonstrate that TNF-beta can participate in the upregulation of TNF-alpha gene expression in lymphocytes and suggest that cellular dysregulations involving autocrine TNF production may be exacerbated by this positive feedback pathway.

In order of decreasing potency, CdCl2 greater than HgCl2 greater than CoCl2 greater than CuSO4 greater than NiCl2 greater than ZnCl2 and PbSO4 slowed cell growth at concentrations ranging from 1 microM to 60 microM. Flow cytometry analysis of cell cycle position indicated that cell growth was selectively blocked in S phase by these concentrations. Water insoluble metals such as As, Ni, crystalline Ni3S2, crystalline NiS, crystalline Ni3Se2 and NiO also resulted in an S phase blockage of cells at concentrations of 1 to 10 micrograms/ml. The crystalline nickel sulfide and selenide compounds as well as As metal were the most potent of these. At higher concentrations blockage of cells in mitosis was also evident with a number of the water insoluble metal compounds. The potency of the metal compounds in blocking cells in S phase was related to their chemical reactivity and their uptake into cells. The S phase specific blockage produced by the metals examined was consistent with their genotoxic or carcinogenic activity since such activity indicated a selective interaction with DNA metabolism.