Background Recent evidence suggests a role for the microbiome in pancreatic ductal adenocarcinoma (PDAC) aetiology and progression. Objective To explore the faecal and salivary microbiota as potential diagnostic biomarkers. Methods We applied shotgun metagenomic and 16S rRNA amplicon sequencing to samples from a Spanish case–control study (n=136), including 57 cases, 50 controls, and 29 patients with chronic pancreatitis in the discovery phase, and from a German case–control study (n=76), in the validation phase. Results Faecal metagenomic classifiers performed much better than saliva-based classifiers and identified patients with PDAC with an accuracy of up to 0.84 area under the receiver operating characteristic curve (AUROC) based on a set of 27 microbial species, with consistent accuracy across early and late disease stages. Performance further improved to up to 0.94 AUROC when we combined our microbiome-based predictions with serum levels of carbohydrate antigen (CA) 19–9, the only current non-invasive, Food and Drug Administration approved, low specificity PDAC diagnostic biomarker. Furthermore, a microbiota-based classification model confined to PDAC-enriched species was highly disease-specific when validated against 25 publicly available metagenomic study populations for various health conditions (n=5792). Both microbiome-based models had a high prediction accuracy on a German validation population (n=76). Several faecal PDAC marker species were detectable in pancreatic tumour and non-tumour tissue using 16S rRNA sequencing and fluorescence in situ hybridisation. Conclusion Taken together, our results indicate that non-invasive, robust and specific faecal microbiota-based screening for the early detection of PDAC is feasible.

In most clinical trials, human mesenchymal stem cells (hMSCs) are expanded in vitro before implantation. The genetic stability of human stem cells is critical for their clinical use. However, the relationship between stem-cell expansion and genetic stability is poorly understood. Here, we demonstrate that within the normal expansion period, hMSC cultures show a high percentage of aneuploid cells that progressively increases until senescence. Despite this accumulation, we show that in a heterogeneous culture the senescence-prone hMSC subpopulation has a lower proliferation potential and a higher incidence of aneuploidy than the non-senescent subpopulation. We further show that senescence is linked to a novel transcriptional signature that includes a set of genes implicated in ploidy control. Overexpression of the telomerase catalytic subunit (human telomerase reverse transcriptase, hTERT) inhibited senescence, markedly reducing the levels of aneuploidy and preventing the dysregulation of ploidy-controlling genes. hMSC-replicative senescence was accompanied by an increase in oxygen consumption rate (OCR) and oxidative stress, but in long-term cultures that overexpress hTERT, these parameters were maintained at basal levels, comparable to unmodified hMSCs at initial passages. We therefore propose that hTERT contributes to genetic stability through its classical telomere maintenance function and also by reducing the levels of oxidative stress, possibly, by controlling mitochondrial physiology. Finally, we propose that aneuploidy is a relevant factor in the induction of senescence and should be assessed in hMSCs before their clinical use.

PD-1/L1 and CTLA-4 blockade immunotherapies have been approved for 13 types of cancers and are being studied in diffuse large B-cell lymphoma (DLBCL), the most common aggressive B-cell lymphoma. However, whether both PD-1 and CTLA-4 checkpoints are active and clinically significant in DLBCL is unknown. Whether PD-1 ligands expressed by tumor cells or by the microenvironment of DLBCL are critical for the PD-1 immune checkpoint is unclear. We performed immunophenotypic profiling for 405 patients with de novo DLBCL using a MultiOmyx immunofluorescence platform and simultaneously quantitated expression/coexpression of 13 immune markers to identify prognostic determinants. In both training and validation cohorts, results demonstrated a central role of the tumor immune microenvironment, and when its functionality was impaired by deficiency in tumor-infiltrating T cells and/or natural killer cells, high PD-1 expression (but not CTLA-4) on CD8+ T cells, or PD-L1 expression on T cells and macrophages, patients had significantly poorer survival after rituximab–CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) immunochemotherapy. In contrast, tumor-cell PD-L2 expression was associated with superior survival, as well as PD-L1+CD20+ cells proximal (indicates interaction) to PD-1+CD8+ T cells in patients with low PD-1+ percentage of CD8+ T cells. Gene-expression profiling results suggested the reversibility of T-cell exhaustion in PD-1+/PD-L1+ patients with unfavorable prognosis and implication of LILRA/B, IDO1, CHI3L1, and SOD2 upregulation in the microenvironment dysfunction with PD-L1 expression. This study comprehensively characterized the DLBCL immune landscape, deciphered the differential roles of various checkpoint components in rituximab–CHOP resistance in DLBCL patients, and suggests targets for PD-1/PD-L1 blockade and combination immunotherapies.