Comparison of an amplicon-based large panel next generation sequencing (NGS) assay with conventional testing methods for MET and HER2 amplification in lung and breast cancers.

The frequency of MET and HER2 amplification being detected by next generation sequencing (NGS) is increasing due to NGS being increasingly adopted for molecular profiling of cancers. However, the accuracy of NGS in detecting these gene amplifications remains uncertain due to conflicting reports in the scientific literature. We studied the accuracy of an amplicon-based large panel NGS assay in detecting MET and HER2 amplification in lung and breast cancers, respectively, by comparing it against conventional testing methods. Amongst 48 lung cancers, four of five cancers that were MET amplified on fluorescence in situ hybridisation (FISH) were classified as amplified on NGS while 42 of the remaining 43 non-amplified cancers were classified as non-amplified on NGS, giving a sensitivity of 80%, specificity of 97.7% and overall concordance of 95.8%. Of the 46 breast cancers tested, only six of the nine cancers that were HER2-positive on immunohistochemistry (IHC)/FISH were HER2-positive on NGS, while all the remaining HER2-negative cases were negative on NGS, giving a sensitivity of 66.7%, specificity of 100% and overall concordance of 93.5%. All the false-negative cases had low level gene amplification (MET:CEP7 or HER2:CEP17 FISH ratio of <3). The low sensitivity for HER2 amplification may be confounded by the small sample size and disproportionate number of cases with low level amplification. In summary, the NGS assay has good concordance with conventional testing methods but may be less sensitive in detecting low level gene amplification.

Cord blood-derived Vδ2+ and Vδ2- T cells acquire differential cell state compositions upon in vitro expansion.

Human cord blood-derived γδ T cells (CBγδ) display a highly diverse TCRγδ repertoire and have a unique subtype composition different from fetal or adult peripheral blood counterparts. We expanded CBγδ in vitro using an irradiated Epstein-Barr virus-transformed feeder cell-based modified rapid expansion protocol (REP). Single-cell RNA sequencing tracked progressive differentiation of naïve CBγδ into cells expressing neoantigen-reactive tumor-infiltrating lymphocyte as well as tissue-resident memory precursor-like and antigen-presenting cell-like gene signatures. TCRγδ clonal tracing revealed a bias toward cytotoxic effector differentiation in a much larger proportion of Vδ2- clones compared to Vδ2+ clones, resulting in the former being more cytotoxic at the population level. These clonotype-specific differentiation dynamics were not restricted to REP and were recapitulated upon secondary nonviral antigen stimulations. Thus, our data showed intrinsic cellular differences between major subtypes of human γδ T cells already in operation at early postnatal stage and highlighted key areas of consideration in optimizing cell manufacturing processes.