Harnessing machine learning to the immuohistochemical expression of AMBRA1 and Loricin to identify non-ulcerated AJCC Stage I/II melanomas at high-risk of metastasis

Background: Melanoma is one of the most common cancer types in the United States and accounts for 90% of the mortality due to skin cancers.Approximately 60% of melanoma patients develop resistance to immunotherapy, and roughly 82% to targeted therapies.The ErbB receptor family comprises the melanoma dedifferentiation marker EGFR, HER2, HER3, and HER4.These receptors play a crucial role in the onset and propagation of a variety of cancer entities.In a recent study we identified EGFR in therapy-nave metastatic tissue of stage III/IV melanoma patients as a negative predictor for response to adjuvant ICI [1].Here we investigate the ErbB receptor family as a whole in the context of melanoma.Methods: In a cohort of melanoma patients receiving adjuvant anti-PD-1 therapy (n=37) we assessed the ErbB receptor expression and dimerization patterns in FFPE tumors by IHC, IF and digital image analysis.We performed the same assessment in a second cohort of melanoma patients diagnosed with various stages of disease for which the primary and multiple metastatic tissues were available (n=18).In four metastatic melanoma cell lines (WM793, UACC257, MEWO, WM266-4) we assessed ErbB expression by IF, dimerization patterns by PLA and co-IP, and tested the effect of 8 FDA-approved TKIs.We assessed the effect of afatinib, neratinib, and osimertinib in a mouse melanoma model.In the A375, MEWO, and WM266-4 melanoma cell lines we assessed the production of ErbB ligands NRG-1, HB-EGF, and TGF-alpha.Results: In the melanoma cohorts we identify EGFR and HER3 as the major players of the ErbB receptor family, in both primary and metastatic tumors.We confirm the same to receptors as the only ones being expressed in metastatic melanoma cell lines.In both patient samples and in vitro we identify specific ErbB dimerization patterns.In melanoma cell lines we show that afatinib, neratinib, and osimertinib have the most significant effect on cell proliferation and apoptosis.In NSG mice injected with the MEWO cell line we show that treatment with afatinib leads to reduced tumor growth and a significant increase in survival compared to controls.We show that in melanoma EGFR-HER3 dependant pathways can be activated in an autocrine manner.Conclusions: Here we demonstrate the relevance of investigating the ErbB receptor family in melanoma as a whole, we provide insight into the mechanisms involving the ErbB receptors, and we provide a therapeutic option by targeting these receptors and their associated signalling pathways.