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. 2024;2(1):70.
doi: 10.1038/s44276-024-00096-0. Epub 2024 Sep 11.

Small extracellular vesicles as biomarkers of response in recurrent/metastatic HNSCC patients treated with immunotherapy

Dan P Zandberg  1 , Chang-Sook Hong  1   2 , Andrew Swartz  1 , Ronan Hsieh  1 , Jennifer Anderson  1 , Robert L Ferris  1 , Brenda Diergaarde  1   3 , Theresa L Whiteside  1   4
Affiliations

Small extracellular vesicles as biomarkers of response in recurrent/metastatic HNSCC patients treated with immunotherapy

Dan P Zandberg et al. BJC Rep. 2024.

Abstract

Background: Biomarkers that effectively predict response to anti-PD-1 mAb therapy in cancer patients are an unmet need. We evaluated the utility of small extracellular vesicles (sEV) as biomarkers of response to immunotherapy in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients.

Methods: Plasma sEV were isolated from 24 R/M HNSCC patients prior to immunotherapy initiation. sEV were separated by immune capture into T cell-derived CD3(+) and tumor-enriched CD3(-) subsets. Stimulatory and suppressive profiles of CD3(-) sEV were determined by on-bead flow cytometry. Differences were assessed using nonparametric tests. Multivariable Cox regression was used to evaluate the relationship with overall (OS) and progression free survival (PFS).

Results: CD3(-)CD44v3(+) sEV represented the majority of plasma sEV; the T-cell-derived CD3(+) fraction was significantly smaller. High CD3(+) sEV was associated with better OS and PFS. Total CD3(-)CD44v3(+) sEV was not associated with outcome. However, suppressive and stimulatory profiles were associated with OS; the suppressive/stimulatory ratio was associated with best response. Exploration of individual proteins on CD3(-) sEV showed that high PD-L1 and high CTLA-4 were associated with better outcomes.

Conclusions: Evaluation of the T cell-derived-CD3(+) and tumor-enriched CD3(-) plasma sEV subsets indicated their potential utility as biomarkers of response to immunotherapy.

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Conflict of interest statement

Competing interestsCompeting interests: None for C-SH, AS, RH, JA, BD and TLW. DPZ: Steering Committee (BICARA, Seagen Inc.); Consulting (Inhibrx, MacroGenics Inc.); Advisory Board (Merck, Prelude Therapeutics); Research support (institutional) for role as PI for clinical trials with Aduro Biotech Inc., Astra-Zeneca, Bicara Therapeutics Inc., Bristol-Myers Squibb, GlaxoSmithKline, MacroGenics Inc., Merck, Novasenta. RLF: Adagene Incorporated: Consulting; Adaptimmune: H&N Cancer Advisory Board; Aduro Biotech, Inc: Consulting; Astra-Zeneca/MedImmune: Clinical Trial, Research Funding; Bicara Therapeutics, Inc: Consultant; Bristol-Myers Squibb: Advisory Board, Clinical Trial, Research Funding; Brooklyn Immunotherapeutics LLC: Consultant; Catenion: Consultant; Coherus BioSciences, Inc.: Advisory Board; CureVac : H&N Advisory Board; CytoAgents: Board; Eisai Europe Limited: Advisory Board; EMD Serono: Consultant; Everest Clinical Research Corporation: Consultant; F. Hoffmann-La Roche Ltd: Consultant; Federation Bio, Inc: Consultant; Genocea Biosciences, Inc: Consultant; Genmab: Advisory Board; Hookipa Biotech GmbH: Advisory Board; Instil Bio, Inc: Advisory Board; Kowa Research Institute, Inc.: Consultant; Lifescience Dynamics Limited: Advisory Board; MacroGenics, Inc.: Advisory Board; MeiraGTx, LLC: Advisory Board; Merck: Advisory Board, Clinical Trial; Merus N.V: Advisory Board; Mirati Therapeutics, Inc: Consultant; Mirror Biologics Inc: Data Safety Monitoring Board; Nanobiotix: Consultant; Novartis Pharmaceutical Corporation: Consulting; Novasenta: Consulting, Stock, Research Funding; Numab Therapeutics AG: Advisory Board; OncoCyte Corporation: Advisory Board; Pfizer: Advisory Board; PPD Development, L.P.: Consultant; Rakuten Medical, Inc: Advisory Board; Regeneron: H&N Advisory Board; Sanofi: Consultant; Seagen, Inc: Advisory Board; SIRPant Immunotherapeutics, Inc: Advisory Board; Tesaro: Research Funding; Vir Biotechnology, Inc: Advisory Board; Zymeworks, Inc.: Consultant.

Figures

Fig. 1
Fig. 1. Absolute neutrophil counts (×103/uL; ANC), absolute lymphocyte counts (×103/uL; Abs Lymp) and the neutrophil/lymphocyte (NRL) ratio in all patients (PTs) and in the patients stratified based on best response to immunotherapy.
Wilcoxon–Mann–Whitney test was used to compare PR/SD with PD. PR partial response, SD stable disease; PD progressive disease. Each dot represents a subject. Mean with sd is shown. Note different scale of the y-axes.
Fig. 2
Fig. 2. TEP levels in HNSCC patients (PTs) and healthy donors (HDs) (left panel), and in the patients stratified based on best response to immunotherapy (right panel).
Wilcoxon–Mann–Whitney test was used to compare the groups. PR partial response, SD stable disease, PD progressive disease. Each dot represents a subject. Horizontal bars indicate median TEP level.
Fig. 3
Fig. 3. Quantitation of TCR/CD3(+) and CD3(−)CD44v3(+) sEV fractions from all 24 patients by on-bead flow cytometry.
Wilcoxon signed-rank test was used to compare the fractions. Each dot represents a subject. Horizontal bars indicate median RFV level. RFV relative fluorescence values.
Fig. 4
Fig. 4. CD3(−)CD44v3(+) sEV and TCR/CD3(+) sEV levels and patient outcomes.
Kaplan–Meier overall survival (OS) and progression free survival (PFS) estimates by CD3(−)CD44v3(+) sEV (a) and TCR/CD3(+) sEV (b) category (high/low based on the median). The reference group is “low”, adjusted hazard ratios (aHRs), corresponding 95% confidence intervals and P values are shown. Variables included in the Cox proportional hazards model: age at start immunotherapy, sex, race, HPV status (negative/not evaluated, positive), and smoking status (no, yes).
Fig. 5
Fig. 5. Levels of PD-L1 and CTLA-4 on TEX-enriched CD3(−) sEV and patient outcomes.
Kaplan–Meier overall survival (OS) and progression free survival (PFS) estimates by PD-L1 (a) and CTLA-4 (b) on TEX-enriched CD3(−) sEV category (high/low based on the median). The reference group is “low”, adjusted hazard ratios (aHRs), corresponding 95% confidence intervals and P values are shown. Variables included in the Cox proportional hazards model: age at start immunotherapy, sex, race, HPV status (negative/not evaluated, positive), and smoking status (no, yes). Additional adjustment with PD-1 (low, high) for PD-L1 and with PD-L1 (low, high) for CTLA-4 is indicated as a+HR.
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