A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6⁺ lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility.