McIntyre, C. A. | bioRxiv Science

bioRxiv2024

Genomic evolution of pancreatic cancer at single-cell resolution

We adapted a previously developed targeted single-nucleus DNA sequencing (snDNA-seq) method and constructed a new suite of computational analysis tools to study 137,491 single-nucleus DNA libraries from 24 pancreatic cancers collected under a variety of clinical scenarios including early and late diagnoses, different metastatic sites and before- and after-treatment. We refined the mutational landscape of pancreatic cancer by capturing events missed by bulk sequencing, and validated the evolution pattern of early fixation of driver single-nucleotide variants (SNVs) followed by generation of intratumoral heterogeneity for copy number variations (CNVs). Intertumoral convergent evolution was common, including subclonal inactivation of TGF-{beta} pathway by mutating various components of it; intratumoral convergence was rarely observed, likely due to strong selective force in pancreatic cancer development. Continuous evolution was frequently seen manifesting as CNVs. In the context of non-targeted treatments, no particular pattern was found across metastases or through treatment. In six pancreatic cancers with germline BRCA2 mutation, we discovered varied timing of biallelic inactivation of BRCA2, which sculpted different evolutionary trajectories and could presumably contribute to differential response to treatment. As the first large-scale application of targeted snDNA-seq on pancreatic cancer, this work provides a sample processing and computational analysis pipeline that warrants further clinical utility.

cancer biology↗

bioRxiv2023

Comprehensive Assessment of the Intrinsic Pancreatic Microbiome

BackgroundSmall studies in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) have suggested that intra-pancreatic microbial dysbiosis may drive malignant transformation. We sought to comprehensively profile tissue and cyst fluid in patients with benign, precancerous, and cancerous conditions of the pancreas to characterize the intrinsic pancreatic microbiome. MethodsPancreatic samples were collected at the time of resection from 109 patients. Samples included tumor tissue (control, n=20; IPMN, n=20; PDAC, n=19) and pancreatic cyst fluid (IPMN, n=30; SCA, n=10; MCN, n=10). Assessment of bacterial DNA by quantitative PCR and 16S ribosomal RNA gene sequencing was performed. Downstream analyses determined the relative abundances of individual taxa between groups and compared intergroup diversity. Whole-genome sequencing data from 140 patients with PDAC in the National Cancer Institutes Clinical Proteomic Tumor Analysis Consortium (CPTAC) were analyzed to validate findings. ResultsSequencing of pancreatic tissue yielded few microbial reads regardless of diagnosis, and analysis of pancreatic tissue showed no difference in the abundance and composition of bacterial taxa between normal pancreas, IPMN, or PDAC groups. Low-grade dysplasia (LGD) and high-grade dysplasia (HGD) IPMN were characterized by low bacterial abundances with no difference in tissue composition and a slight increase in Pseudomonas and Sediminibacterium in HGD cyst fluid. Decontamination analysis using the CPTAC database confirmed a low-biomass, low-diversity intrinsic pancreatic microbiome that did not differ by pathology. ConclusionsOur analysis of the pancreatic microbiome demonstrated very low intrinsic biomass that is relatively conserved across diverse neoplastic conditions and thus unlikely to drive malignant transformation. Significance of this studyO_ST_ABSWhat is already known on this subject?C_ST_ABSO_LIMicrobial colonization, infection, and dysbiosis have been implicated in the oncogenesis of various gastrointestinal tumors (e.g.: Helicobacter pylori in gastric cancer, hepatitis B and C in hepatocellular carcinoma, colon dysbiosis in colon cancer progression). C_LIO_LIFew studies have analyzed the intrinsic pancreatic microbiome, and these have produced conflicting results regarding microbial presence and alterations associated with malignant disease. C_LIO_LIIPMN is a precursor lesion to pancreatic cancer that represents a whole gland defect without an established driver event, and microbiome changes have been implicated as a possible etiology of cyst formation and dysplastic progression. C_LI What are the new findings?O_LIA low-biomass, low-diversity intrinsic pancreatic microbiome is present in both pancreatic tissue and cyst fluid. C_LIO_LIThis intrinsic pancreatic microbiome does not differ in terms of abundance, composition, or diversity between patients with PDAC, IPMN, or other benign conditions of the pancreas. C_LI How might it impact clinical practice in the foreseeable future?O_LIMicrobiome dysbiosis does not appear to be a driver of malignant degeneration of IPMN, and further research is needed to identify drivers of oncogenesis in order for possible chemoprevention strategies to be developed. C_LI

microbiology↗

bioRxiv2022

Application of high-throughput, high-depth, targeted single-nucleus DNA sequencing in pancreatic cancer

Despite insights gained by bulk DNA sequencing of cancer it remains challenging to resolve the admixture of normal and tumor cells, and/or of distinct tumor subclones; high throughput single-cell DNA sequencing circumvents these and brings cancer genomic studies to higher resolution. However, its application has been limited to liquid tumors or a small batch of solid tumors, mainly because of the lack of a scalable workflow to process solid tumor samples. Here we optimized a highly automated nuclei extraction workflow that achieved fast and reliable targeted single-nucleus DNA library preparation of 38 samples from 16 pancreatic adenocarcinoma (PDAC) patients, with an average library yield per sample of 2867 single nuclei. We demonstrate that this workflow not only performs well using low cellularity or low tumor purity samples but reveals novel genomic evolution patterns of PDAC as well.

cancer biology↗