SUMMARY Increasing evidence links the gut microbiota with colorectal cancer. Metagenomic analyses indicate that commensal Fusobacterium spp. are associated with human colorectal carcinoma but whether this is an indirect or causal link remains unclear. We find that Fusobacterium spp. are enriched in human colonic adenomas relative to surrounding tissues and in stool samples from colorectal adenoma and carcinoma patients compared to healthy subjects. Additionally, in the ApcMin/+ mouse model of intestinal tumorigenesis, Fusobacterium nucleatum increases tumor multiplicity and selectively recruits tumor-infiltrating myeloid cells, which can promote tumor progression. Tumors from ApcMin/+ mice exposed to F. nucleatum exhibit a pro-inflammatory expression signature that is shared with human fusobacteria-positive colorectal carcinomas. However, unlike other bacteria linked to colorectal carcinoma, F. nucleatum does not exacerbate colitis, enteritis or inflammation-associated intestinal carcinogenesis. Collectively, these data suggest that, through recruitment of tumor-infiltrating immune cells, fusobacteria, generate a pro-inflammatory microenvironment that is conducive for colorectal neoplasia progression.

Colorectal cancers comprise a complex mixture of malignant cells, non-transformed cells, and microorganisms. Fusobacterium nucleatum is among the most prevalent bacterial species in colorectal cancer tissues. Here we show that colonization of human colorectal cancers with Fusobacterium and its associated microbiome, —including Bacteroides, Selenomonas, and Prevotella species, —is maintained in distal metastases, demonstrating microbiome stability between paired primary and -metastatic tumors. In situ hybridization analysis revealed that Fusobacterium is predominantly associated with cancer cells in the metastatic lesions. Mouse xenografts of human primary colorectal adenocarcinomas were found to retain viable Fusobacterium and its associated microbiome through successive passages. Treatment of mice bearing a colon cancer xenograft with the antibiotic metronidazole reduced Fusobacterium load, cancer cell proliferation, and overall tumor growth. These observations argue for further investigation of antimicrobial interventions as a potential treatment for patients with Fusobacterium-associated colorectal cancer.

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity, using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two prostate cancer patients including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were observed in matched tissue. Moreover, we identified 10 early-trunk and 56 metastatic-trunk mutations in the non-CTC tumor samples and found 90% and 73% of these, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

Highlights d A scRNA-seq study reveals shared and distinct features of human MMRd and MMRp CRC d Co-variation of single-cell transcriptional programs across specimens predicts immune hubs d A myeloid-rich inflammatory hub is identified below the colonic lumen in human CRC d CXCR3-ligand+ cells form foci with activated T cells in human MMRd CRC

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC. Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. .

SUMMARY Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo , we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo -relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

Importance Although patients with resected pancreatic adenocarcinoma are at high risk for disease recurrence, few markers are available to inform patient outcomes. Objective To evaluate alterations of the four main driver genes for pancreatic adenocarcinoma and patient outcomes after cancer resection. Design, Setting, and Participants We analyzed protein expression and DNA alterations for KRAS, CDKN2A, SMAD4, and TP53 by immunohistochemistry and next-generation sequencing in formalin-fixed, paraffin-embedded tumors from 356 patients with resected pancreatic adenocarcinoma evaluated at three U.S. centers. Associations of driver gene alterations with disease-free survival (DFS) and overall survival (OS) were evaluated using Cox proportional hazards regression with estimation of hazard ratios (HR) and 95% confidence intervals (CI) and adjustment for age, sex, tumor characteristics, institution, and peri-operative treatment. Main Outcomes DFS and OS among patients with resected pancreatic adenocarcinoma Results Patients with KRAS mutant tumors had worse DFS and OS compared to patients with KRAS wild-type tumors, with median OS of 20.3 versus 38.6 months and 5-year OS of 13.0% versus 30.2%, respectively. Particularly poor outcomes were identified in patients with KRAS G12D-mutant tumors, who had median OS of 15.3 months. Patients whose tumors lacked CDKN2A expression had worse DFS and OS compared to patients whose tumors retained CDKN2A, with median OS of 19.7 versus 24.6 months and 5-year OS of 11.9% versus 19.5%, respectively. SMAD4 status was not associated with DFS or OS, while TP53 status was associated only with DFS (P=0.04). Patients had worse DFS and OS with greater number of altered driver genes. Compared to patients with 0-2 altered genes, those with 4 altered genes had HR for DFS of 1.79 (1.24-2.59; P<0.01) and OS of 1.38 (0.98-1.94; P=0.06). Five-year OS was 18.4% for patients with 0-2 gene alterations, 14.1% for 3 alterations and 8.2% for 4 alterations. Alterations in the four driver genes were not significantly associated with local recurrence as the first site of disease recurrence. Conclusions and Relevance Patient outcomes are associated with alterations of the four main driver genes in resected pancreatic adenocarcinoma.

Nearly 10% of PDAC patients harbor germline variants, although the majority lack somatic second hits, the therapeutic significance of which warrants further study.