Primary head and neck cancer cell cultures are susceptible to proliferation of Epstein-Barr virus infected lymphocytes.

BACKGROUND: New concepts for a more effective anti-cancer therapy are urgently needed. Experimental flaws represent a major counter player of this development and lead to inaccurate and unreproducible data as well as unsuccessful translation of research approaches into clinics. In a previous study we have created epithelial cell cultures from head and neck squamous cell carcinoma (HNSCC) tissue. METHODS: We characterize primary cell populations isolated from human papillomavirus positive HNSCC tissue for their marker expression by RT-qPCR, flow cytometry, and immunofluorescence staining. Their sensitivity to MDM2-inhibition was measured using cell viability assays. RESULTS: Primary HNSCC cell cultures showed the delayed formation of spheroids at higher passages. These spheroids mimicked the morphology and growth characteristics of other established HNSCC spheroid models. However, expression of epithelial and mesenchymal markers could not be detected in these cells despite the presence of the HNSCC stem cell marker aldehyde dehydrogenase 1 family member A1. Instead, strong expression of B- and T-lymphocytes markers was observed. Flow cytometry analysis revealed a heterogeneous mixture of CD3 + /CD25 + T-lymphocytes and CD19 + B-lymphocytes at a ratio of 4:1 at passage 5 and transformed lymphocytes at late passages (≥ passage 12) with CD45 + CD19 + CD20 + , of which around 10 to 20% were CD3 + CD25 + CD56 + . Interestingly, the whole population was FOXP3-positive indicative of regulatory B-cells (Bregs). Expression of transcripts specific for the Epstein-Barr-virus (EBV) was detected to increase in these spheroid cells along late passages, and this population was vulnerable to MDM2 inhibition. HPV + HNSCC cells but not EBV + lymphocytes were detected to engraft into immunodeficient mice. CONCLUSIONS: In this study we present a primary cell culture of EBV-infected tumor-infiltrating B-lymphocytes, which could be used to study the role of these cells in tumor biology in future research projects. Moreover, by describing the detailed characteristics of these cells, we aim to caution other researchers in the HNSCC field to test for EBV-infected lymphocyte contaminations in primary cell cultures ahead of further experiments. Especially researchers who are interested in TIL-based adopted immunotherapy should exclude these cells in their primary tumor models, e.g. by MDM2-inhibitor treatment. BI-12-derived xenograft tumors represent a suitable model for in vivo targeting studies.

Classification of three corynebacterial strains isolated from a small paddock in North Rhine-Westphalia: proposal of Corynebacterium kalinowskii sp. nov., Corynebacterium comes sp. nov. and Corynebacterium occultum sp. nov.

Three novel corynebacterial species were isolated from soil sampled at a paddock in Vilsendorf, North Rhine-Westphalia, Germany. The strains were coccoid or irregular rod-shaped, catalase-positive and pale white to yellow-orange in colour. By whole genome sequencing and comparison of the 16S rRNA genes as well as the whole genome structure, it was shown that all three strains represent novel species of the family Corynebacteriaceae, order Corynebacteriales, class Actinobacteria. This project describes the isolation, identification, sequencing, and phenotypic characterization of the three novel Corynebacterium species. We propose the names Corynebacterium kalinowskii sp. nov. (DSM 110639T=LMG 31801T), Corynebacterium comes sp. nov. (DSM 110640T=LMG 31802T), and Corynebacterium occultum sp. nov. (DSM 110642T=LMG 31803T).

Molecular pathogenesis of cutaneous lymphoma-Future directions.

The pathogenesis of cutaneous T-cell lymphomas is not clear. In recent years, the genetic changes in CTCL were explored. The detected mutations showed a great deal of heterogeneity between individual patients. The studies documented various copy number variations (CNV) and single nucleotide variations (SNV) in multiple genes involved in multiple signalling pathways. Recurrently mutated signalling pathways include JAK-STAT, MAPK, T-cell receptor, TNF receptor and NFκB signalling. In the period between 2018 and today, additional studies towards the genetic changes in CTCL were carried out. Genetic changes in gamma delta T-cell lymphoma are also shown in genes of the JAK-STAT, MAPK, MYC and chromatin signalling pathways. These studies might indicate a shift away from targeted sequencing approaches towards whole-genome sequencing. This approach demands additional resources in terms of funding but has the advantage of finding mutations in non-coding regions. These mutations were neglected for a long time, but as shown in contemporary research these regions harbour highly recurrent mutations affecting gene expression and regulation. Nevertheless, the detection of specific molecular changes in known pathways enables considerations for targeted therapies.

Long-read sequencing and structural variant characterization in 1,019 samples from the 1000 Genomes Project.

Structural variants (SVs) contribute significantly to human genetic diversity and disease 1-4 . Previously, SVs have remained incompletely resolved by population genomics, with short-read sequencing facing limitations in capturing the whole spectrum of SVs at nucleotide resolution 5-7 . Here we leveraged nanopore sequencing 8 to construct an intermediate coverage resource of 1,019 long-read genomes sampled within 26 human populations from the 1000 Genomes Project. By integrating linear and graph-based approaches for SV analysis via pangenome graph-augmentation, we uncover 167,291 sequence-resolved SVs in these samples, considerably advancing SV characterization compared to population-wide short-read sequencing studies 3,4 . Our analysis details diverse SV classes-deletions, duplications, insertions, and inversions-at population-scale. LINE-1 and SVA retrotransposition activities frequently mediate transductions 9,10 of unique sequences, with both mobile element classes transducing sequences at either the 3'- or 5'-end, depending on the source element locus. Furthermore, analyses of SV breakpoint junctions suggest a continuum of homology-mediated rearrangement processes are integral to SV formation, and highlight evidence for SV recurrence involving repeat sequences. Our open-access dataset underscores the transformative impact of long-read sequencing in advancing the characterisation of polymorphic genomic architectures, and provides a resource for guiding variant prioritisation in future long-read sequencing-based disease studies.

[New insights into the pathogenesis and molecular understanding of cutaneous T-cell lymphomas]. / Neues zu Pathogenese und molekularem Verständnis bei kutanen T-Zell-Lymphomen.

The pathogenesis of cutaneous T­cell lymphomas (CTCL) is still an enigma. Therefore, extensive translational research efforts have been undertaken in recent years to gain further clinical and molecular insights. There is increasing evidence that the different clinical appearance of the CTCL subtypes derives from the assumption that they develop from different skin subpopulations of T cells. Detection and quantification of the malignant T­cell clones is crucial for the diagnosis and prognosis of CTCL. Numerous recurrent mutant cellular signalling pathways have been found in recent years. This includes the JAK-STAT, NFκB, T­cell receptor and MAP kinase signalling pathways, as well as cell cycle control and epigenetics. The most recent analyses imply a tumour evolution model with initial copy number variation, like amplification or deletions of specific DNA fragments (CNVs) and only subsequent later single nucleotide variations (SNVs). The crucial question, however, is which CNVs are sufficient to initiate general tumourigenesis? The challenge is to identify possible driver genes. Increasing molecular understanding in CTCL will include new breakthrough therapeutic options in the near future.

Unraveling the Structural Variations of Early-Stage Mycosis Fungoides-CD3 Based Purification and Third Generation Sequencing as Novel Tools for the Genomic Landscape in CTCL.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL). At present, knowledge of genetic changes in early-stage MF is insufficient. Additionally, low tumor cell fraction renders calling of copy-number variations as the predominant mutations in MF challenging, thereby impeding further investigations. We show that enrichment of T cells from a biopsy of a stage I MF patient greatly increases tumor fraction. This improvement enables accurate calling of recurrent MF copy-number variants such as ARID1A and CDKN2A deletion and STAT5 amplification, undetected in the unprocessed biopsy. Furthermore, we demonstrate that application of long-read nanopore sequencing is especially useful for the structural variant rich CTCL. We detect the structural variants underlying recurrent MF copy-number variants and show phasing of multiple breakpoints into complex structural variant haplotypes. Additionally, we record multiple occurrences of templated insertion structural variants in this sample. Taken together, this study suggests a workflow to make the early stages of MF accessible for genetic analysis, and indicates long-read sequencing as a major tool for genetic analysis for MF.

The Desmin Mutation DES-c.735G>C Causes Severe Restrictive Cardiomyopathy by Inducing In-Frame Skipping of Exon-3.

Currently, little is known about the genetic background of restrictive cardiomyopathy (RCM). Herein, we screened an index patient with RCM in combination with atrial fibrillation using a next generation sequencing (NGS) approach and identified the heterozygous mutation DES-c.735G>C. As DES-c.735G>C affects the last base pair of exon-3, it is unknown whether putative missense or splice site mutations are caused. Therefore, we applied nanopore amplicon sequencing revealing the expression of a transcript without exon-3 in the explanted myocardial tissue of the index patient. Western blot analysis verified this finding at the protein level. In addition, we performed cell culture experiments revealing an abnormal cytoplasmic aggregation of the truncated desmin form (p.D214-E245del) but not of the missense variant (p.E245D). In conclusion, we show that DES-c.735G>C causes a splicing defect leading to exon-3 skipping of the DES gene. DES-c.735G>C can be classified as a pathogenic mutation associated with RCM and atrial fibrillation. In the future, this finding might have relevance for the genetic understanding of similar cases.