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Abstract Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2− γδ T cells. In the context of γδ T–cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T–cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti–PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T–cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions. See related Spotlight on p. 600

Plain Language SummaryWhat is this summary about?ENZAMET is a large international clinical trial involving people with metastatic hormone sensitive prostate cancer (mHSPC). Prior to the trial, standard treatment included testosterone suppression and sometimes, chemotherapy. However, the researchers thought enzalutamide, an anti-androgen (hormone) medicine, which helps to block the effects of testosterone, might be helpful if added to testosterone suppression.What are the key takeaways?ENZAMET showed that the addition of enzalutamide to standard testosterone suppression treatment for mHSPC improved survival, resulting in an increase in the percent of participants alive at 5 years from 57% to 67%. Enzalutamide extended the time until the cancer grew. Side effects were in keeping with what was already known about enzalutamide and these risks were outweighed by improved cancer control in the long-term.What are the main conclusions reported by the researchers?The combination of testosterone suppression plus enzalutamide is a very effective first-line treatment for mHSPC.This is an abstract of the Plain Language Summary of Publication article.View the full Plain Language Summary PDF of this article to read the full-textThis article is related to: AcknowledgmentsAustralian and New Zealand Urogenital and Prostate Cancer Trials Group (ANZUP); National Health and Medical Research Council Clinical Trials Centre, University of Sydney; Cancer Trials Ireland; the Canadian Cancer Trials Group; The Dana Farber Cancer Institute.We thank the trial participants, principal investigators, co-investigators, and study coordinators at all participating centres for their commitment to this trial.Disclosure statementThe authors of this PLSP include; consumers and consumer-members from the ANZUP consumer advisory panel, as well as several original authors from the ENZAMET publication(s). Ciara Conduit – reports honoraria from Astra Zeneca, Bristol-Myers Squibb and Janssen; travel/conference support from Merck and Bayer.Andrisha-Jade Inderjeeth – Speaker fee honoraria from; BMS, Pfizer and Astellas. Eibhlin Mulroe – works for Cancer Trials Ireland. Robert R. Zielinski – Honoraria from Astra Zeneca and Pfizer, and Speaker fees from Pierre Fabre and Janssen. Alastair Thomson – Speaker fees: Novartis (Jan 24), Roche, Exact Sciences (Mar 24), Gilead(Sep 24), Accord (Sep 24), Pfizer (Sep 24), Advisory Boards: MSD, Exact Sciences, Novartis (Oct 23), Amgen, Seagen (Nov 23), Gilead (Dec 23), Support for attending conferences: BMS, MSD, Astellas, Ipsen, EUSA, Lilly, Gilead (Dec 23), Novartis (Oct 23), Menarini Stemline Oncology (Sep 24) and Educational meetings support: Roche Thean Hsiang Tan – Honoraria from Astra Zeneca. Shahneen K. Sandhu – reports institutional research grants or contracts from Novartis, AstraZeneca, Merck Sharp & Dohme, Roche/Genentech, Pfizer, Senhwa Biosciences, AMGEN, Merck Healthcare and support for institution from Merck Sharp Dohme, Bristol Myers Squibb,AstraZeneca, Janssen, Novartis, Roche/Genentech, SkylineDx, Abbvie for participation in advisory boards or consulting. M. Neil Reaume – Advisory Board fees from Pfizer, Ipsen, EMD serono, Merck and Bayer. David W. Pook – Institution grants from Medivation, Bristol-Myers Squibb, SymVivo, Roche, Amgen, Exelixis, Pfizer, Bayer,Astellas Pharma and Merck Sharp & Dohme, Consulting fees from Pfizer & Merck Sharp & Dohme (Institution), and personal: Bristol-Myers Squibb, Pfizer, Merck Sharp & Dohme, Merck, Merck/Pfizer, Bristol Myers Squibb Foundation, Cipla and Astellas Pharma, honoraria from Merck/Pfizer and Bayer, support for attending meetings from Bristol-Myers Squibb, Astellas Pharma, Pfizer, Amgen, Merck/Pfizer and Janssen. Scott A. North – reports honoraria from AAA, Astellas, AstraZeneca,BMS, Eisai, EMD Serono, Ipsen, Janssen, Merck, Pfizer. Anthony Joshua – reports stock ownership Pricilium Therapeutics and Opthea, Consulting or Advisory Role(from his institution): Janssen Oncology, Ipsen, AstraZeneca, Sanofi, Pfizer, Novartis, Merck Serono, IDEAYA Biosciences, IQvia, Bayer, Astellas Pharma, Grey Wolf Therapeutics, Medison, Starpharma, Eisai, Mayne Pharma and MSD Oncology, Patents, Royalties, Other Intellectual Property – Cancer therapeutic methods, Research Funding (through his institution): Bristol-Myers Squibb, Janssen Oncology, Merck Sharp & Dohme, Mayne Pharma, Roche/Genentech, Bayer, Lilly, Pfizer, AstraZeneca,Corvus Pharmaceuticals, Genentech, BeiGene, Myeloid Therapeutics and Immunocore. Lisa Horvath – Honoraria for talks given in 2020, 2022 & 2023 from Astellas Pharma (all honoraria donated to Chirs O'Brien Lifehouse), Support for attending meetings from Astellas Pharma. Ray McDermott - reports honoraria from Janssen,Astellas, BMS, MSD and Ipsen, and travel/conference support from Novartis, Pfizer and Bayer. Simon Chowdhury – Consulting fees from Astellas Pharma, Novartis,Bayer, Pfizer, BeiGene and Janssen-Cilag, Honoraria from Novartis & Clovis Oncology, Speakers bureau from Janssen-Cilag and Sanofi/Aventis and stock from Clovis Oncology. Kim N. Chi – Honoraria/Grants: Astellas, AstraZeneca, Bayer, BMS, Janssen, Merck, Novartis, Pfizer, Point Biopharma and Roche. Alison Y. Zhang – Grants to institution from Astellas, Amgen, Astra Zeneca, Bionomics, Bristol-Myers Squibb, Celgene, Medivation, Merck Sharp & Dohme, Pfizer, Roche, Sanofi and Tilray,Consulting fees from Merck Sharp & Dohme, Honoraria from Merck Sharpe Dohme, Astellas, Bayer, Pfizer, Merck, Mundipharma, Janssen and Astra Zeneca, Advisory Board from Merck, Merck Sharpe Dohme, Astellas and Bayer. Martin R. Stockler – reports Grants to his institution from: Astellas, Amgen, Astra Zeneca, Bionomics,Bristol-Myers Squibb, Celgene, Medivation, Merck Sharp & Dohme, Pfizer, Roche, Sanofi and Tilray. Ian D. Davis – ANZUP Cancer Trials Group, Member of Board of Directors, Director and Chair; unremunerated, Co-Chair of the ENZAMET trial. Christopher J. Sweeney – Consulting or Advisory Role: Janssen, Astellas Pharma,Bayer, Genentech, Pfizer, Lilly, MDS, Point Biopharma; Advancell, CellCentric, Amphista Research Funding: Janssen Biotech (Inst), Astellas Pharma (Inst), Sanofi (Inst),Bayer (Inst), Patents, Royalties, Other Intellectual Property: Parthenolide (Indiana University): dimethylaminoparthenolide (Leuchemix); Exelixis: Abiraterone plus cabozantinib combination; FRAS1 SNP and tristetraprolin and KDM5D as biomarkers of lethal prostate cancer. Stock or Other Ownership: Leuchemix; AdvanCell. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.Patient reviewers on this PLSP have received honorarium from Future Oncology for their review work but have no other relevant financial relationships to disclose.FundingStudy drug and costs of the trial were provided by the manufacturer of enzalutamide (Astellas). The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

The neurotrophic factor ARTEMIN (ARTN) has been reported to possess a role in mammary carcinoma progression and metastasis. Herein, we report that ARTN modulates endothelial cell behaviour and promotes angiogenesis in ER-mammary carcinoma (ER-MC). Human microvascular endothelial cells (HMEC-1) do not express ARTN but respond to exogenously added, and paracrine ARTN secreted by ER-MC cells. ARTN promoted endothelial cell proliferation, migration, invasion and 3D matrigel tube formation. Angiogenic behaviour promoted by ARTN secreted by ER-MC cells was mediated by AKT with resultant increased TWIST1 and subsequently VEGF-A expression. In a patient cohort of ER-MC, ARTN positively correlated with VEGF-A expression as measured by Spearman's rank correlation analysis. In xenograft experiments, ER-MC cells with forced expression of ARTN produced tumors with increased VEGF-A expression and increased microvessel density (CD31 and CD34) compared to tumors formed by control cells. Functional inhibition of ARTN by siRNA decreased the angiogenic effects of ER-MC cells. Bevacizumab (a humanized monoclonal anti-VEGF-A antibody) partially inhibited the ARTN mediated angiogenic effects of ER-MC cells and combined inhibition of ARTN and VEGF-A by the same resulted in further significant decrease in the angiogenic effects of ER-MC cells. Thus, ARTN stimulates de novo tumor angiogenesis mediated in part by VEGF-A. ARTN therefore co-ordinately regulates multiple aspects of tumor growth and metastasis.

Background Vaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8 + Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate with longer survival in metastatic cutaneous melanoma. However, their capacity to respond to VM and their neoantigens is not known. Methods Using longitudinal samples, we explored the evolution of VM mutations by whole-exome sequencing and RNAseq, we also defined the immune context using multiplex immunohistochemistry and nanostring pan cancer immune profile. Then using fresh single cell suspensions of the metastatic samples, we explored VM T cells via mass cytometry and single cell RNAseq and T cell receptor sequencing (TCRseq). Finally, we investigated TRM, pre-TRM and exhausted T cell function against melanoma neo-antigens and melanoma differentiation antigens in vitro. Results Primary VM was non-inflamed and devoid of CD8 + TRM cells. In contrast, both metastases showed proliferating CD8 + TRM were clustered at the tumor margin, with increased numbers in the second ICB-refractory metastasis. The first metastasis showed dense infiltration of CD8 + T cells, the second showed immune exclusion with loss of melanoma cell Major histocompatibility complex (MHC)-I expression associated with downregulation of antigen presentation pathway gene expression. CD8 + TRM from both metastases responded to autologous melanoma cells more robustly than all other CD8 + T cell subsets. In addition, CD8 + TRM shared TCR clones across metastases, suggesting a response to common antigens, which was supported by recognition of the same neoantigen by expanded tumor infiltrating lymphocytes. Conclusions In this study, we identified TRM clusters in VM metastases from a patient, but not primary disease. We showed TRM location at the tumor margin, and their superior functional response to autologous tumor cells, predicted neoantigens and melanoma differentiation antigens. These CD8 + TRM exhibited the highest tumor-responsive potential and shared their TCR with tumor-infiltrating effector memory T cells. This suggests VM metastases from this patient retain strong antitumor T cell functional responses; however, this response is suppressed in vivo. The loss of VG MHC-I expression is a common immune escape mechanism which was not addressed by anti-PD-1 monotherapy; rather an additional targeted approach to upregulate MHC-I expression is required.

Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca 2+ mobilization and was inhibited by Cl − channel blockers. Calcium wave propagation also was reduced by purinergic receptor antagonists and by Cl − channel blockers but insensitive to gap junction inhibitors. These observations suggest that cell-to-cell signaling associated with connexin expression results from enhanced ATP release and not, as previously believed, from an increase in intercellular coupling.

Additional file 3: Table S7. Decontamination criteria outcome for ASVs that passed the in silico decontamination strategy applied to plasma samples of the extension cohort.

Supplementary Figure from Tumor Genomic Testing for >4,000 Men with Metastatic Castration-resistant Prostate Cancer in the Phase III Trial PROfound (Olaparib)

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