Abstract
In melanoma, the lymphocytic infiltrate is a prognostic parameter classified morphologically into 'brisk', 'non-brisk' and 'absent' entailing a functional association that has never been proved. Recently, it has been shown that lymphocytic populations can be very heterogeneous, and that anti-PD-1 immunotherapy supports activated T cells. Here, we characterize the immune landscape in primary melanoma by high-dimensional single-cell multiplex analysis in tissue sections (MILAN technique) followed by image analysis, RT-PCR and shotgun proteomics. We observed that the brisk and non-brisk patterns are heterogeneous functional categories that can be further sub-classified into active, transitional or exhausted. The classification of primary melanomas based on the functional paradigm also shows correlation with spontaneous regression, and an improved prognostic value when compared to that of the brisk classification. Finally, the main inflammatory cell subpopulations that are present in the microenvironment associated with activation and exhaustion and their spatial relationships are described using neighbourhood analysis.
Highlights
The lymphocytic infiltrate in melanoma is a prognostic parameter reported by the pathologist as patterns of tumour-infiltrating lymphocytes (TILs)
Repeating the survival analysis in the SKCM TCGA data set confirmed that the patients in the ‘Active’ group had better prognosis than the patients in the ‘Exhausted’ group validating the results obtained with our dataset (Figure 2—figure supplement 1)
Since immunotherapy is generally administered only if the patient develops metastasis, to confirm the existence of the same functional subcategories in metastatic melanoma samples, we perform qPCR followed by proteomics on microdissected TILs, comparing melanoma metastasis that we classified as brisk and non-brisk, using an ‘absent’ case and a ‘tumoral melanosis’ case as controls
Summary
The lymphocytic infiltrate in melanoma is a prognostic parameter reported by the pathologist as patterns of tumour-infiltrating lymphocytes (TILs). We characterize the immune landscape at single cell-level in primary melanoma based on a panel of 39 immune markers applied on one single tissue section through a high-dimensional multiplexing method (Cattoretti et al, 2001; Bolognesi et al, 2017), a RT-PCR expression evaluation and a shotgun proteomic analysis This approach allowed us (a) to further categorize the brisk and non-brisk morphological patterns of TILs into three functional categories; (b) to define the correlation between T-cell activation and spontaneous melanoma regression; (c) to investigate the most important inflammatory subpopulations involved in TILs exhaustion (Figure 1)
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