Aligning tumor mutational burden (TMB) quantification across diagnostic platforms: phase II of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB) measurements aid in identifying patients who are likely to benefit from immunotherapy; however, there is empirical variability across panel assays and factors contributing to this variability have not been comprehensively investigated. Identifying sources of variability can help facilitate comparability across different panel assays, which may aid in broader adoption of panel assays and development of clinical applications. MATERIALS AND METHODS: Twenty-nine tumor samples and 10 human-derived cell lines were processed and distributed to 16 laboratories; each used their own bioinformatics pipelines to calculate TMB and compare to whole exome results. Additionally, theoretical positive percent agreement (PPA) and negative percent agreement (NPA) of TMB were estimated. The impact of filtering pathogenic and germline variants on TMB estimates was assessed. Calibration curves specific to each panel assay were developed to facilitate translation of panel TMB values to whole exome sequencing (WES) TMB values. RESULTS: Panel sizes >667 Kb are necessary to maintain adequate PPA and NPA for calling TMB high versus TMB low across the range of cut-offs used in practice. Failure to filter out pathogenic variants when estimating panel TMB resulted in overestimating TMB relative to WES for all assays. Filtering out potential germline variants at >0% population minor allele frequency resulted in the strongest correlation to WES TMB. Application of a calibration approach derived from The Cancer Genome Atlas data, tailored to each panel assay, reduced the spread of panel TMB values around the WES TMB as reflected in lower root mean squared error (RMSE) for 26/29 (90%) of the clinical samples. CONCLUSIONS: Estimation of TMB varies across different panels, with panel size, gene content, and bioinformatics pipelines contributing to empirical variability. Statistical calibration can achieve more consistent results across panels and allows for comparison of TMB values across various panel assays. To promote reproducibility and comparability across assays, a software tool was developed and made publicly available.

Inactivation of misselected CD8 T cells by CD8 gene methylation and cell death.

Misselected CD8 cells that express T cell receptors (TCRs) that do not recognize class I major histocompatibility complex (MHC) protein can emerge from thymic selection. A postthymic quality control mechanism that purges these cells from the repertoire is defined here. The failure of mature CD8 cells to simultaneously engage their TCR and CD8 coreceptor triggers an activation process that begins with inhibition of CD8 gene expression through remethylation and concludes with up-regulation of surface Fas and Fas ligand and cellular apoptosis. Thus, inhibition of a death signal through continued TCR-CD8 coengagement of MHC molecules is a key checkpoint for the continued survival of correctly selected T cells. Molecular defects that prevent delivery of the death signal to mistakenly selected T cells underlie the expansion of double-negative T cells, which is the cellular signature of a subset of systemic autoimmune diseases.

Suppression of immune responses by CD8 cells. I. Superantigen-activated CD8 cells induce unidirectional Fas-mediated apoptosis of antigen-activated CD4 cells.

Stimulation of mature CD4 cells through the TCR induces cellular activation and expansion that are often followed by clonal elimination by a form of apoptosis termed activation-induced cell death. This process of CD4 cell apoptosis is generally thought to reflect clonal suicide and to be independent of other cell types. Here we show that during the response to the superantigen Staphylococcal enterotoxin A, activated CD8 cells, but not activated CD4 cells, suppress the CD4 proliferative response. Suppression by CD8 cells reflects their ability to induce CD4 cell apoptosis via ligation of Fas. Moreover, although activated CD8 cells that express Fas ligand and Fas eliminate CD4 cells through a Fas-dependent mechanism, they are themselves resistant to Fas-dependent apoptosis. These findings indicate a fundamental difference between the two major T cell subsets with regard to sensitivity to Fas-dependent apoptosis, expression of Fas ligand, and mediation of suppressive activity following immunization with superantigen.

Phylogenetic and serological characterization of two Ugandan HIV-1 isolates.

HIV-1 isolates Ug06 and Ug23 were established in culture from peripheral blood mononuclear cells (PBMCs) of Ugandan subjects. The isolates were studied for phylogenetic and serological relationships with each other and with the laboratory strains, HTLV-IIIB and HIV-1MN. The results suggest that the Ugandan isolates are related to different subgroups of African viruses with 17.3% of genetic distance between UG06 and the U455 provirus (Uganda); and 12.6% of genetic distance between UG23 and the JY1 provirus (Zaire). Analysis of the predicted amino acid sequences for Ug06 and Ug23 showed marked sequence heterogeneity in the V3 region and CD4-binding site. A conserved amino acid sequence was identified in the C-terminal immunodominant region of the envelope glycoprotein gp120. The isolates were compared in virus-neutralization experiments with HTLV-IIIB and HIV-1MN stocks, using panels of Western blot-positive North American and Ugandan sera. The North American serum samples showed broad neutralizing activity against both of the Ugandan isolates. However, the Ugandan serum panel demonstrated strain-specific activity against either Ug06 or Ug23. Furthermore, the African serum specimens showed higher prevalence and titers of neutralizing activity against the HIV-1MN stock as compared with HTLV-IIIB.