Multi-modal and longitudinal characterization of the tumor and immune microenvironment from primary to in-transit and distant metastasis in acral melanoma

e21518 Background: Patients with in-transit metastases (ITM) in melanoma are a heterogeneous subset of patients where melanoma recurrence is anatomically detected between the site of the primary tumor and the draining lymph node. Notably, a significant subset of those patients has locoregional recurrences with long-term survival without progression to distant sites. The underlying mechanisms responsible for these distinct phenotypes are unknown. Methods: To understand the evolution from primary melanoma to in-transit and distant metastasis, we characterized the tumor and immune microenvironment among 9 patients with acral lentiginous melanoma using whole exome sequencing (WES), bulk RNAseq, snRNAseq and high-plex Cyclic Immunofluorescence (CyCIF) imaging. We further profiled the dynamics and the evolution of the tumor and immune compartments using 28 tumor samples obtained longitudinally over 9 years from a single patient (pt222) with recurrent in-transit and distant metastases undergoing CDK4/6, MEK inhibition and immune checkpoint blockade. Results: Our cohort included 5 patients that progressed from ITM to distant disease and 4 patients with only local recurrence and without distant progression (Table). WES profiling identified enriched RAS mutations (5 out of 9), independent of progression and treatment response. Pt222 showed multiple morphological features related to aggressive tumor growth and high risk of recurrence such as clefting, ulceration detected within the primary tumor by the histopathological assessment of the H&E section. Phylogenetic analysis revealed co-evolution of 7 genetic lineages that shared 35 clonal mutations, including NRAS p.Q61H, with independent resistance-associated alterations. The metastases at distant sites emerged from a lineage featured with high aneuploidy and tumor mutational burden (TMB). The brain metastasis showed the highest TMB and aneuploidy. Bulk RNAseq deconvolution, snRNAseq, CyCIF and H&E revealed low and heterogeneous immune composition among different samples and lineages with macrophages and DCs as dominant populations. Interestingly, we did not observe the lineage characterized by the highest absolute immune infiltrates after ICB treatment. Another specific lineage that disappeared after the treatment with ICB showed angiotropic growth and the over-expression of a WNT-betacatenin transcriptional signature linked with T cell exclusion. An orthogonal validation using bulk RNAseq, snRNAseq and CyCIF confirmed these findings. Conclusions: This study helps define a high-resolution map of the evolutionary dynamics from primary to in-transit and distant metastasis. [Table: see text]