Recent developments in deep (next-generation) sequencing technologies are significantly impacting medical research. The global analysis of protein coding regions in genomes of interest by whole exome sequencing is a widely used application. Many technologies for exome capture are commercially available; here we compare the performance of four of them: NimbleGen’s SeqCap EZ v3.0, Agilent’s SureSelect v4.0, Illumina’s TruSeq Exome, and Illumina’s Nextera Exome, all applied to the same human tumor DNA sample. Each capture technology was evaluated for its coverage of different exome databases, target coverage efficiency, GC bias, sensitivity in single nucleotide variant detection, sensitivity in small indel detection, and technical reproducibility. In general, all technologies performed well; however, our data demonstrated small, but consistent differences between the four capture technologies. Illumina technologies cover more bases in coding and untranslated regions. Furthermore, whereas most of the technologies provide reduced coverage in regions with low or high GC content, the Nextera technology tends to bias towards target regions with high GC content. We show key differences in performance between the four technologies. Our data should help researchers who are planning exome sequencing to select appropriate exome capture technology for their particular application.

Adoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome. We herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells. This is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells. These results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution. FUND: This work was supported by the Research Council of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the University of Lausanne.

Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs.

Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8.

Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of tumor-infiltrating Tregs in B-cell non-Hodgkin lymphomas (B-NHLs) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotypes and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Here, we applied single-cell RNA sequencing and T-cell receptor sequencing combined with high-dimensional cytometry to decipher the heterogeneity of intratumoral Tregs in diffuse large B-cell lymphoma and follicular lymphoma (FL), compared with that in nonmalignant tonsillar tissue. We identified 3 distinct transcriptional states of Tregs: resting, activated, and unconventional LAG3+FOXP3− Tregs. Activated Tregs were enriched in B-NHL tumors, coexpressed several checkpoint receptors, and had stronger immunosuppressive activity compared with resting Tregs. In FL, activated Tregs were found in closer proximity to CD4+ and CD8+ T cells than other cell types. Furthermore, we used a computational approach to develop unique gene signature matrices, which were used to enumerate each Treg subset in cohorts with bulk gene expression data. In 2 independent FL cohorts, activated Tregs was the major subset, and high abundance was associated with adverse outcome. This study demonstrates that Tregs infiltrating B-NHL tumors are transcriptionally and functionally diverse. Highly immunosuppressive activated Tregs were enriched in tumor tissue but absent in the peripheral blood. Our data suggest that a deeper understanding of Treg heterogeneity in B-NHL could open new paths for rational drug design, facilitating selective targeting to improve antitumor immunity.

Many cases of acute lymphoblastic leukemia (ALL) carry visible acquired chromosomal changes of pathogenetic, diagnostic, and prognostic importance. Nevertheless, from one-fourth to half of newly diagnosed ALL patients have no visible chromosomal changes detectable by G-banding analysis at diagnosis. The introduction of powerful molecular methodologies has shown that many karyotypically normal ALLs carry clinically important submicroscopic aberrations. We used fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), RNA sequencing, reverse transcription (RT) and genomic polymerase chain reaction (PCR), as well as Sanger sequencing to investigate a case of pediatric ALL with a normal karyotype. FISH with a commercial PDGFRB breakapart probe showed loss of the distal part of the probe suggesting a breakpoint within the PDGFRB locus. aCGH revealed submicroscopic deletions in chromosome bands 5q32q35.3 (about 30 Mb long, starting within PDGFRB and finishing in the CANX locus), 7q34 (within TCRB), 9p13 (PAX5), 10q26.13 (DMBT1), 14q11.2 (TRAC), and 14q32.33 (within the IGH locus). RNA sequencing detected an in-frame GTF2I–PDGFRB and an out-of-frame IKZF1–TYW1 fusion transcript. Both fusion transcripts were verified by RT-PCR together with Sanger sequencing and interphase FISH. The GTF2I–PDGFRB fusion was also verified by genomic PCR and FISH. The corresponding GTF2I–PDGFRB fusion protein would consist of almost the entire GTF2I and that part of PDGFRB which harbors the catalytic domain of the tyrosine kinase. It would therefore seem to lead to abnormal tyrosine kinase activity in a manner similar to what has been seen for other PDGFRB fusion proteins. The examined pediatric leukemia is a Ph-like ALL which carries novel GTF2I–PDGFRB and IKZF1–TYW1 fusion genes together with additional submicroscopic deletions. Because hematologic neoplasms with PDGFRB-fusion genes can be treated with tyrosine kinase inhibitors, the detection of such novel fusions may be clinically important. Since the GTF2I–PDGFRB could be detected only after molecular studies of the leukemic cells, further investigations of ALL-cases, perhaps especially but not exclusively with a normal karyotype, are needed in order to determine the frequency of GTF2I–PDGFRB in leukemia, and also to find out which clinical impact the fusion may have.