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The presence of certain volatile biomarkers in the breath of patients with gastric cancer has been reported by several studies; however, the origin of these compounds remains controversial. In vitro studies, involving gastric cancer cells may address this problem and aid in revealing the biochemical pathways underlying the production and metabolism of gastric cancer volatile indicators. Gas chromatography with mass spectrometric detection, coupled with headspace needle trap extraction as the pre-concentration technique, has been applied to map the volatilomic footprints of human HGC-27 and CLS-145 gastric cancer cell lines and normal Human Stomach Epithelial Cells (HSEC). In total, 27 volatile compounds are found to be associated with metabolism occurring in HGC-27, CLS-145, and HSEC. Amongst these, the headspace concentrations of 12 volatiles were found to be reduced compared to those above just the cultivating medium, namely there was an observed uptake of eight aldehydes (2-methylpropanal, 2-methyl-2-propenal, 2-methylbutanal, 3-methylbutanal, hexanal, heptanal, nonanal, and benzaldehyde), three heterocyclic compounds (2-methyl-furan, 2-ethyl-furan, and 2-pentyl-furan), and one sulfur-containing compound (dimethyl disulphide). For the other 15 volatiles, the headspace concentrations above the healthy and cancerous cells were found to be higher than those found above the cultivating medium, namely the cells were found to release three esters (ethyl acetate, ethyl propanoate, and ethyl 2-methylbutyrate), seven ketones (2-pentanone, 2-heptanone, 2-nonanone, 2-undecanone, 2-tridecanone, 2-pentadecanone, and 2-heptadecanone), three alcohols (2-methyl-1-butanol, 3-methyl-1-butanol, and 2-ethyl-1-hexanol), one aromatic compound (toluene), and one sulfur containing compound [2-methyl-5-(methylthio) furan]. In comparison to HSEC, HGC-27 cancer cell lines were found to have significantly altered metabolism, manifested by an increased production of methyl ketones containing an odd number of carbons. Amongst these species, three volatiles were found exclusively to be produced by this cell line, namely 2-undecanone, 2-tridecanone, and 2-heptadecanone. Another interesting feature of the HGC-27 footprint is the lowered level of alcohols and esters. The CLS-145 cells exhibited less pronounced changes in their volatilomic pattern compared to HSEC. Their footprint was characterized by the upregulated production of esters and 2-ethyl-hexanol and downregulated production of other alcohols. We have therefore demonstrated that it is possible to differentiate between cancerous and healthy gastric cells using biochemical volatile signatures.

Tertiary lymphoid structures (TLS) are associated with favorable outcome in non-metastatic colorectal carcinoma (nmCRC), but the dynamics of TLS maturation and its association with effective anti-tumor immune surveillance in nmCRC are unclear. Here, we hypothesized that not only the number of TLS but also their composition harbors information on recurrence risk in nmCRC. In a comprehensive molecular, tissue, laboratory, and clinical analysis of 109 patients with stage II/III nmCRC, we assessed TLS numbers and degree of maturation in surgical specimens by multi-parameter immunofluorescence of follicular dendritic cell (FDC) and germinal center (GC) markers. TLS formed in most tumors and were significantly more prevalent in highly-microsatellite-instable (MSI-H) and/or BRAF-mutant nmCRC. We could distinguish three sequential TLS maturation stages which were characterized by increasing prevalence of FDCs and mature B-cells: [1] Early TLS, composed of dense lymphocytic aggregates without FDCs, [2] Primary follicle-like TLS, having FDCs but no GC reaction, and [3] Secondary follicle-like TLS, having an active GC reaction. A simple integrated TLS immunoscore reflecting these parameters identified a large subgroup of nmCRC patients with a very low risk of recurrence independently of clinical co-variables such as ECOG performance status, age, stage, and adjuvant chemotherapy. We conclude that (1) mismatch repair and BRAF mutation status are associated with the formation of TLS in nmCRC, (2) TLS formation in nmCRC follows sequential maturation steps, culminating in germinal center formation, and (3) this maturation process harbors important prognostic information on the risk of disease recurrence.

Coronary artery disease (CAD) has a complex etiology involving numerous environmental and genetic factors of disease risk. To date, the genetic 9p21 locus represents the most robust genetic finding for prevalent and incident CAD. However, limited information is available on the genetic background of the severity and distribution of CAD. CAD manifests itself as stable CAD or acute coronary syndrome. The Gensini score quantifies the extent CAD but requires coronary angiography. Here, we aimed to identify novel genetic variants associated with Gensini score severity and distribution of CAD. A two-stage approach including a discovery and a replication stage was used to assess genetic variants. In the discovery phase, a meta-analysis of genome-wide association data of 4,930 CAD-subjects assessed by the Gensini score was performed. Selected single nucleotide polymorphisms (SNPs) were replicated in 2,283 CAD-subjects by de novo genotyping. We identified genetic loci located on chromosome 2 and 9 to be associated with Gensini score severity and distribution of CAD in the discovery stage. Although the loci on chromosome 2 could not be replicated in the second stage, the known CAD-locus on chromosome 9p21, represented by rs133349, was identified and, thus, was confirmed as risk locus for CAD severity.