This translational research paper highlights the work of Florent Peyraud, MD, conducted during his PhD thesis at Explicyte under the supervision of Prof. Antoine Italiano (Bergonié & Gustave Roussy Comprehensive Cancer Centers) and Alban Bessede, PhD (Explicyte). This project was made possible thanks to a collaboration with the imCORE network (Roche/Genentech), and samples from the BIP trial (NCT02534649). The study aims to uncover why certain patients with non-small cell lung cancer (NSCLC) who exhibit mature tertiary lymphoid structures (mTLS)—a phenotype typically associated with a favorable response to immune checkpoint inhibitors (ICI)—do not respond to anti-PD1/PD-L1 immunotherapy.
Presence of mature TLS & clinical outcome in NSCLC
A comprehensive pathological analysis of 509 NSCLC patients treated with immune checkpoint inhibitors (ICI) was performed, confirming that the presence of mTLS predicts a better outcome in terms of response rate, overall survival, and median progression-free survival, independently of PD-L1 and genomic features.
Spatial transcriptomics in responding & non-responding mTLS-positive NSCLC patients
Specimens from 6 mTLS-positive patients with extreme ICI response (3 responders, 3 non-responders) were analyzed using the GeoMx Whole Transcriptome Atlas. While gene expression profiles did not differ within the TLS and tumor compartments, non-responders exhibited an enrichment in fibroblasts in the stromal compartment, with a pronounced expression of TGFβ signaling and epithelial-mesenchymal transition (EMT) pathways.
Multiplex immunohistofluorescence (mIHF) panel focusing on cancer-associated fibroblasts (CAFs) 77 m-TLS positive patient samples were then analyzed by digital pathology for the detection of tumor cells, cytotoxic CD8+ T cells, and FAP+αSMA+ CAF and MYH11+αSMA+ CAFs. Analysis revealed a higher stromal density of both CAF subsets in non-responders, correlating with poor clinical outcome.
Bulk transcriptomics to analyze interactions between the immune infiltrates & CAF status
Gene expression profiling of 40 mTLS-positive lung tumor samples identified that:
- FAP+αSMA+ CAF-High tumors were associated with inflammatory response and T-cell exhaustion, which may explain the limited efficacy of ICI.
- MYH11+αSMA+ CAF-High tumors were associated with an enrichment in regulatory T cell (Treg) gene signatures, which are known to participate in immunosuppression and be detrimental to ICI efficacy
Multiplex immunofluorescence panel focused on T cell exhaustion
Specimens of 64 mTLS positive NSCLC patients were then analyzed by digital pathology:
- A panel consisting of CD8, PD1, CD39, LAG3, TIGIT, TIM3, and DAPI revealed a high density of intratumoral CD8+PD1+ T cells in the FAP+αSMA+ CAF-High tumors, with a higher expression of exhaustion markers.
- A panel including CD4, CD8, CD20, FoxP3, ICOS, TIGIT and DAPI highlighted an increased presence of CD4+ T cells in the stroma of MYH11+αSMA+ CAF-High tumors, with an increased infiltration of regulatory CD4+FoxP3+ T cells expressing immunosuppressive markers.
Altogether, these findings suggest that the presence of FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs is associated with poor outcomes in mTLS-positive NSCLC patients undergoing ICI treatment. They could serve as biomarkers to stratify patients, and could constitute targets, in combination with strategies addressing T-cell exhaustion, to enhance ICI efficacy in mTLS-positive NSCLC patients.
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