Abstract:
Introdcution: In-transit metastases (ITM) in melanoma is a type of locoregional metastasis found between the primary tumour site and the regional draining lymph node. Treating ITM can be a challenge; some recur after surgery alone and others are unresectable at presentation. Immunotherapy has been used to treat patients with ITM in the adjuvant or metastatic settings, but some tumours appear resistant to these treatments. Existing literatures have highlighted that multiomic profiles and spatial features of the tumour microenvironment can influence patient outcomes to immunotherapy. This study aims to characterise the distinct molecular and spatial cellular features of tumour microenvironment in patients with ITM.
Method: Forty-eight patient samples were collected from a cohort of patients with ITM receiving anti-PD-1-based therapies at pre-treatment and progression. Patients who achieved a best RECIST response of complete or partial response in the advanced setting or a recurrence-free survival of over 12 months (since treatment initiation) in the adjuvant setting were categorised as “responsive” to anti-PD-1-based therapies. Those not meeting these criteria were deemed “resistant”. To characterise the tumour immune ecosystem, single-cell sequencing and highplex 40-marker immunofluorescent imaging were performed on 18 matched tumour dissociates and formalin-fixed paraffin-embedded tissue samples from patients with ITM at pre-treatment (n=3 resistant and 5 responsive) and progression (n=10). Additionally, whole genome sequencing was performed on 12 fresh frozen samples (n=4 resistant and 8 responsive at pre-treatment) to characterise intrinsic variants that implicated biological processes in the tumour microenvironment. Computational analyses were performed using cellular neighbourhood characterisation, receptor-ligand interactions, unsupervised clustering and pathway enrichment.
Findings (Impact): Spatial neighbourhood analysis identified lymphoid aggregates consisting of B cells, CD4+ and CD8+ T cells and dendritic cells. The B and T cells are in close proximity with HLA-A+ melanoma at the tumour margin and interacted with tumour cells using ligand-receptor pairs including CD74/CD44 and CD74/CXCR4. Sequencing and imaging analyses revealed that resistant ITM samples exhibited various immune evasion phenotypes, including the upregulation of immune checkpoint receptors (LAG3, VISTA, IDO1), dysfunctional T cell signatures and reduced immune recruitment. Furthermore, pathway enrichment results demonstrated lowered T cell metabolism, due to repression of the phosphoinositide 3-kinase/protein kinase B pathway, was identified in patients resistant to immunotherapy.
Conclusion: Integrating spatial imaging with multiomics-based feature reveal both immune-responsive and immune-suppressive hubs within the tumour tissue architecture. This work provides a foundation for developing more effective immunotherapy strategies tailored to the unique immune landscape of ITM patients.
