The increasing accessibility of next-generation sequencing has empowered researchers to investigate somatic mutations in cancer. The complexity of variant analysis pipelines, terminology, and tool selection remains a major barrier, especially for those new to the field or working in translational settings. To address this challenge, we present a practical framework that guides researchers through the critical steps of variant interrogation in tumor samples. This guide is broken into four phases: Planning —laying the foundation for thoughtful experimental design and a clear understanding of sequencing outputs; Gathering Resources —assembling the tools, reference data, and variant annotation sets required for analysis; Filtering and Validation —executing a systematic approach to prioritize meaningful variants; and Dissemination and Storage —ensuring findings are reproducible and accessible through transparent reporting and data sharing. Developed with an emphasis on accessibility, reproducibility, and clinical relevance, this framework equips researchers with the guidance to navigate variant analysis with confidence and rigor.

Background: At diagnosis, ~80% of pancreatic ductal adenocarcinomas (PDAC) have metastasized. Relapse is thus common even among patients who undergo surgical resection, the only curative option. PDAC progresses rapidly, and existing immunotherapies have been ineffective. We hypothesized that natural killer (NK) cell immunotherapies could be effective against PDAC because they recognize conserved and heterogeneous features associated with cellular stress and transformation and can seek out metastases distal to the primary tumor site. Here, we aim to define the key features of NK cells as effective agents for PDAC immunotherapy. Methods: We used The Cancer Genome Atlas Program (TCGA) PDAC Firehose data and flow cytometry to predict and measure the most common activating or inhibitory ligands available on PDAC for NK cell activation. To ascertain how the tumor might alter expression of these ligands during treatment, inflammation or immune pressure, we measured expression of NK ligands at rest, or after exposure to immune cells or inflammation. To test and rank the functional importance of these dynamic ligands in the recognition, killing and control of PDAC we used co-culture, antibody-blocking and an NK-competent humanized mouse model. Results: Leveraging the known sequential acquisition of mutations as a surrogate for disease progression, we observed a progressive loss of transcript expression for activating NK cell ligands and chemoattractants. Exposure of PDAC to NK cells or interferon-γ, an inflammatory stimulus, drove dynamic changes in expression of both activating and inhibitory ligands. In vitro co-culture assays revealed a redundancy in the activating receptors engaged in NK:PDAC interactions, but that human leukocyte antigens (HLA)-killer immunoglobulin-like receptors (KIR) signaling dominantly interrupted anti-PDAC activity. In NK-competent humanized mice, adoptively transferred, unselected, unmodified NK cells slowed tumor growth in a dose-dependent manner, but NK cells selected to avoid HLA I-driven inhibition were the most competent effectors for PDAC control. Conclusions: Although there is redundancy among activating ligand:receptor pairs for recognizing PDAC tumors, interactions between KIR and HLA define the extent to which antitumor activity can proceed. During tumor progression and in response to immunotherapy, NK:tumor interactions drive upregulation of HLA I molecules. Thus, educated NK cells from HLA I-disparate donors may be the more effective allogeneic NK immunotherapy for PDAC.

Pancreatic cancer is a complex malignancy associated with poor prognosis and high symptom burden. Optimal patient care relies on the integration of various sectors in the healthcare field as well as innovation through research. The Canadian National Pancreas Conference (NPC) was co-organized and hosted by Craig’s Cause Pancreatic Cancer Society and The Royal College of Physicians and Surgeons in November 2023 in Montreal, Canada. The conference sought to bridge the gap between Canadian healthcare providers and researchers who share the common goal of improving the prognosis, quality of life, and survival for patients with pancreatic cancer. The accredited event featured discussion topics including diagnosis and screening, value-based and palliative care, pancreatic enzyme replacement therapy, cancer-reducing treatment, and an overview of the current management landscape. The present article reviews the NPC sessions and discusses the presented content with respect to the current literature.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers, and has limited options for treatment. Low immune infiltration, desmoplastic stroma, and poor tumour immunogenicity are all expected to contribute to PDAC’s rapid progression and limited response to existing immunotherapies. PDAC tumours are mosaics of different sub-tumour microenvironments, including some that do contain immune cells. We hypothesized that increasing frequency of lymphocyte:tumour interactions would correlate with lengthened survival for patients with PDAC. Methods: Using multiplex immunofluorescence, digital pathology, and computational analyses, we profiled the spatial distribution, co-localization, and neighbourhood architecture of immune cells in tumours from 73 patients with PDAC. Results: Higher densities of CD3+CD8– (CD4+) T cells were associated with improved five-year overall survival, particularly when enriched at the tumour-stroma boundary (i.e. peritumoural). CD3+CD8-T cells, CD8+ T cells (CD3+CD8+), and B cells (CD20+) frequently co-infiltrated and co-localized, forming distinct immune neighbourhoods indicative of organized adaptive immunity. We defined three common immune neighbourhoods within PDAC (1) Macrophage-dominant; (2) T cell dominant; and (3) Disorganized. With greater tumour and peri-tumoural area represented by the T cell dominant neighbourhood, overall survival was increased. The other neighbourhoods were not significantly associated with outcomes. Conclusion: Immune cells self-assemble in predictable patterns in PDAC. The presence of T cell dominant neighbourhoods, which we interpret as supporting ongoing immune activation, best predict lengthened survival. Spatially organized immune interactions may serve as prognostic indicators and inform future studies for immunotherapies in PDAC.