Mucosal melanoma of the head and neck region: clinical-pathologic implications of tumor microenvironment evaluated with multiplex immunohistochemistry

Mucosal melanoma of the head and neck (MM-H&N) is an aggressive disease known for its frequent residual tumor/relapses (RT/R) at the surgical site, which encourages the need for additional therapeutical strategies. Our current understanding of the tumor microenvironment (TME) in MM-H&N and its correlation with clinical-pathologic, immunohistochemical, and molecular features, remains far less comprehensive than that of its cutaneous counterpart. Additionally, the TME composition of consecutive samples collected from single patients has not been investigated, which limits our ability to detail the prognosis and therapy of these patients. Thirty-eight MM-H&N specimens from 24 patients were analyzed using a four-label protocol of bright-field multiplex immunohistochemistry (IHC) with CD20 (purple), CD3 (DAB-brown), CD68 (green), and SOX10 (yellow). The cases were assessed using a previously validated score, dichotomized into low and high adopting the corresponding median values, and statistically correlated with clinical-pathological, immunohistochemical, and molecular features (χ² test; p-value < 0.05). CD3_high was associated with pT3 stage [p = 0.001; 9/9 (100%) pT4a/pT4b cases showed CD3_low]; CD20_high was associated with fused/mixed cytotype (p = 0.003) and lympho-vascular invasion (LVI) (p = 0.005); CD68_high was associated with LVI (p = 0.001) and pT4a/pT4b stage [p = 0.001; 9/9 (100%) pT4a/pT4b cases showed CD68_high]. No associations between mutational status and immune marker values were found. Additionally, 3/9 (33.3%) of patients with consecutive specimens showed different IHC results in the analyzed samples, without a specific correlation with pre-/inter-surgery systemic therapies and "molecular heterogeneity". Our study shows that variations in TME components (CD20/CD3/CD68) correlate with prognostically relevant pathological features in MM-H&N. Notably, a distinct "TME phenotype" (CD3_low/CD20_high/CD68_high) is linked with unfavorable features - such as LVI and advanced pT4a/pT4b staging - as well as the need for more aggressive therapies. We also observed discordant TME and molecular results in consecutive samples from a subset of patients, suggesting heterogeneity that may potentially affect prognosis and therapeutic strategies.