Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and insights into both disease etiology and targeted intervention are needed. A total of 109 micro-dissected PDA cases were subjected to whole-exome sequencing. Microdissection enriches tumour cellularity and enhances mutation calling. Here we show that environmental stress and alterations in DNA repair genes associate with distinct mutation spectra. Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype. We identify multiple novel mutated genes in PDA, with select genes harbouring prognostic significance. RBM10 mutations associate with longer survival in spite of histological features of aggressive disease. KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival. Oncogenic BRAF mutations are mutually exclusive with KRAS and define sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signalling, chromatin remodelling, Hedgehog signalling, DNA repair and cell cycle processes are observed. Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.

Introduction: Monocarboxylate transporter 1 (MCT1) is an importer of monocarboxylates such as lactate and pyruvate and a marker of mitochondrial metabolism. MCT1 is highly expressed in a subgroup of cancer cells to allow for catabolite uptake from the tumor microenvironment to support mitochondrial metabolism. We studied the protein expression of MCT1 in a broad group of breast invasive ductal carcinoma specimens to determine its association with breast cancer subtypes and outcomes.Methods: MCT1 expression was evaluated by immunohistochemistry on tissue micro-arrays (TMA) obtained through our tumor bank. Two hundred and fifty-seven cases were analyzed: 180 cases were estrogen receptor and/or progesterone receptor positive (ER+ and/or PR+), 62 cases were human epidermal growth factor receptor 2 positive (HER2+), and 56 cases were triple negative breast cancers (TNBC). MCT1 expression was quantified by digital pathology with Aperio software. The intensity of the staining was measured on a continuous scale (0-black to 255-bright white) using a co-localization algorithm. Statistical analysis was performed using a linear mixed model.Results: High MCT1 expression was more commonly found in TNBC compared to ER+ and/or PR+ and compared to HER-2+ (p < 0.001). Tumors with an in-situ component were less likely to stain strongly for MCT1 (p < 0.05). High nuclear grade was associated with higher MCT1 staining (p < 0.01). Higher T stage tumors were noted to have a higher expression of MCT1 (p < 0.05). High MCT1 staining in cancer cells was associated with shorter progression free survival, increased risk of recurrence, and larger size independent of TNBC status (p < 0.05).Conclusion: MCT1 expression, which is a marker of high catabolite uptake and mitochondrial metabolism, is associated with recurrence in breast invasive ductal carcinoma. MCT1 expression as quantified with digital image analysis may be useful as a prognostic biomarker and to design clinical trials using MCT1 inhibitors.

Cancer biology is influenced by the tumor microenvironment that impacts on disease prognosis and therapeutic intervention. The inter-relationship of tumor infiltrating lymphocytes, immune response regulators, and a glycolytic tumor environment was evaluated in a cohort of 183 largely consecutive patients with triple negative breast cancer diagnosis. High levels of tumor infiltrating lymphocytes were associated with improved survival of triple negative breast cancer cases. However, elevated levels of PD-L1, CD163, and FOXP3 were individually associated with significantly decreased overall survival. These three determinants were significantly correlated, and could serve to differentiate the prognostic significance of tumor infiltrating lymphocytes. Interestingly, a glycolytic tumor environment, as determined by the expression of MCT4 in the tumor stroma, was associated with the immune evasive environment and poor prognosis. Clustering of all markers defined four distinct triple negative breast cancer subtypes that harbored prognostic significance in multi-variate analysis. Immune and metabolic markers stratify triple negative breast cancer into subtypes that have prognostic significance and implications for therapies targeting immune checkpoints and tumor metabolism.

Objective The tumor microenvironment frequently displays abnormal cellular metabolism, which contributes to aggressive behavior. Metformin inhibits mitochondrial oxidative phosphorylation, altering metabolism. Though the mechanism is unclear, epidemiologic studies show an association between metformin use and improved outcomes in HNSCC. We sought to determine if metformin alters metabolism and apoptosis in HNSCC tumors. Study Design Window of opportunity trial of metformin between diagnostic biopsy and resection. Participants were patients with newly diagnosed HNSCC. 50 patients were enrolled, and 39 completed a full treatment course. Metformin was titrated to standard diabetic dose (2000 mg/day) for a course of 9 or more days prior to surgery. Level of Evidence 4 Methods Immunohistochemistry (IHC) for the metabolic markers caveolin-1 (CAV1), B-galactosidase (GALB) and monocarboxylate transporter 4 (MCT4) as well as the TUNEL apoptosis assay and Ki-67 IHC were performed in pre- and post-metformin specimens. Exploratory mass spectroscopy imaging (MSI) to assess lactate levels was also performed in three subjects. Results Metformin was well tolerated. The average treatment course was 13.6 days. Post-treatment specimens showed a significant increase in stromal CAV1 (p<0.001) and GALB (p<0.005) and tumor cell apoptosis by TUNEL assay (p<0.001). There was no significant change in stromal MCT4 expression or proliferation measured by Ki67. Lactate levels in carcinoma cells were increased 2.4-fold post-metformin (p<0.05) as measured by MSI. Conclusions Metformin increases markers of reduced catabolism and increased senescence in stromal cells and increased carcinoma cell apoptosis. This study demonstrates that metformin modulates metabolism in the HNSCC microenvironment. Trial Registration ClinicalTrials.gov Identifier NCT02083692.

Highlights d VISTA is heterogeneously expressed on melanoma cells d VISTA expression promotes tumor onset and alters the immune microenvironment d FOXD3 suppresses the expression of VISTA d BRAF/MEK inhibitor treatment reduces levels of VISTA