Paired primary and metastatic lesions of patients with ipilimumab-treated melanoma: high variation in lymphocyte infiltration and HLA-ABC expression whereas tumor mutational load is similar and correlates with clinical outcome

Abstract

BackgroundImmune checkpoint inhibitors (ICI) can lead to long-term responses in patients with metastatic melanoma. Still many patients with melanoma are intrinsically resistant or acquire secondary resistance. Previous studies have used primary or metastatic tumor tissue for biomarker assessment. Especially in melanoma, metastatic lesions are often present at different anatomical sites such as skin, lymph nodes, and visceral organs. The anatomical site may directly affect the tumor microenvironment (TME). To evaluate the impact of tumor evolution on the TME and on ICI treatment outcome, we directly compared paired primary and metastatic melanoma lesions for tumor mutational burden (TMB), HLA-ABC status, and tumor infiltrating lymphocytes (TILs) of patients that received ipilimumab.MethodsTMB was analyzed by sequencing primary and metastatic melanoma lesions using the TruSight Oncology 500 assay. Tumor tissues were subjected to multiplex immunohistochemistry to assess HLA-ABC status and for the detection of TIL subsets (B cells, cytotoxic T cells, helper T cells, and regulatory T cells), by using a machine-learning algorithm.ResultsWhile we observed a very good agreement between TMB of matched primary and metastatic melanoma lesions (intraclass coefficient=0.921), such association was absent for HLA-ABC status, TIL density, and subsets thereof. Interestingly, analyses of different metastatic melanoma lesions within a single patient revealed that TIL density and composition agreed remarkably well, rejecting the hypothesis that the TME of different anatomical sites affects TIL infiltration. Similarly, the HLA-ABC status between different metastatic lesions within patients was also comparable. Furthermore, high TMB, of either primary or metastatic melanoma tissue, directly correlated with response to ipilimumab, whereas lymphocyte density or composition did not. Loss of HLA-ABC in the metastatic lesion correlated to a shorter progression-free survival on ipilimumab.ConclusionsWe confirm the link between TMB and HLA-ABC status and the response to ipilimumab-based immunotherapy in melanoma, but no correlation was found for TIL density, neither in primary nor metastatic lesions. Our finding that TMB between paired primary and metastatic melanoma lesions is highly stable, demonstrates its independency of the time point and location of acquisition. TIL and HLA-ABC status in metastatic lesions of different anatomical sites are highly similar within an individual patient.

The immune microenvironment landscape shows treatment-specific differences in rectal cancer patients

“…In short, slides were sequentially stained using antibodies against CD8, CD20, CD3, Foxp3, CD11c, and pan-Cytokeratin AE 1/3 using the BOND RX automated research stainer (Leica Biosystems, Wetzlar, Germany). The anti-CD45RO used in a previous study ( 35 ) was exchanged for CD11c (ab52632, Abcam, Cambridge, UK, RRID: AB_2129793). Concentrations of antibodies, retrieval steps, and corresponding Opal dyes were adjusted (details available in Supplementary Table 1 ) to ensure the best signal intensity for each marker that would allow exposure times of 15-100 ms on multispectral regions.…”

“…Whole slide scans were acquired using x4 magnification and subsequently scanned at x20 magnification for the multispectral regions of interest ( Figure 1B ). Images were first processed using the inForm software (V.2.4.8, Akoya Biosciences, MA, USA, RRID: SCR_019155) for cell and tissue segmentation and then processed through an in-house AI pipeline to phenotype immune cells, which were thereafter analyzed in FlowJo™ (Ashland, OR, USA, RRID: SCR_008520) as previously described ( 35 , 36 ).…”

“…The multiplex IF staining protocol Opal ™ 7 Tumor Infiltrating Lymphocyte (TIL) Kit (OP7TL3001KT, Akoya Biosciences, MA, USA), was modified and optimized for rectal 4µm thick FFPE tissue sections (34). In short, slides were sequentially stained using antibodies against CD8, CD20, CD3, Foxp3, CD11c, and pan-Cytokeratin AE 1/3 using the BOND RX automated research stainer (Leica Biosystems, Wetzlar, Germany).…”

Immunological and Genomic Analysis Reveals Clinically Relevant Distinctions between Angiosarcoma Subgroups

“…An antibody against ERG, a transcription factor expressed in endothelial cells, served as tumor marker (clone EPR3864, Abcam, Cambridge, UK). Methods for panel optimization and validation were previously described [ 27 , 28 ]. Multispectral images were generated by scanning the slides with the Vectra ® Automated Quantitative Imaging System with software version 3.0.4 (PerkinElmer, Waltham, MA, USA).…”

CIMT 2022: Report on the 19th Annual Meeting of the Association for Cancer Immunotherapy

“…Given that lymphocyte infiltration, in contrast to high mutational burden, did not correlate with benefit from Ipilimumab treatment in melanoma patients, De Vries outlined that tumors with a high mutational load are good candidates for cancer immunotherapy. 3 One such cancer type is Lynch syndrome, for which De Vries presented peptides arising from frame-shift mutations in the genes encoding for TGFβRII and Caspase 5 as viable targets for dendritic cell (DC) vaccination approaches. In a clinical trial phase I/II of Lynch syndrome patients (NCT01885702), DC vaccination was well tolerated and induced neoantigen-specific T cells that recognized tumor cells expressing the mutant antigens ex vivo.…”