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. 2023 Mar 8;12(6):832.
doi: 10.3390/cells12060832.

Prognostic Role of Soluble and Extracellular Vesicle-Associated PD-L1, B7-H3 and B7-H4 in Non-Small Cell Lung Cancer Patients Treated with Immune Checkpoint Inhibitors

Carlo Genova  1   2 , Roberta Tasso  3   4 , Alessandra Rosa  5 , Giovanni Rossi  6   7 , Daniele Reverberi  8 , Vincenzo Fontana  5 , Silvia Marconi  9 , Michela Croce  10 , Maria Giovanna Dal Bello  11 , Chiara Dellepiane  6 , Marco Tagliamento  2 , Maria Chiara Ciferri  3 , Lodovica Zullo  6 , Alessandro Fedeli  12 , Angela Alama  9 , Katia Cortese  3 , Chiara Gentili  3   4 , Eugenia Cella  6 , Giorgia Anselmi  13 , Marco Mora  13 , Giulia Barletta  6 , Erika Rijavec  14 , Francesco Grossi  14 , Paolo Pronzato  6 , Simona Coco  9
Affiliations

Prognostic Role of Soluble and Extracellular Vesicle-Associated PD-L1, B7-H3 and B7-H4 in Non-Small Cell Lung Cancer Patients Treated with Immune Checkpoint Inhibitors

Carlo Genova et al. Cells. .

Abstract

The treatment of non-small cell lung cancer (NSCLC) has changed dramatically with the advent of immune checkpoint inhibitors (ICIs). Despite encouraging results, their efficacy remains limited to a subgroup of patients. Circulating immune checkpoints in soluble (s) form and associated with extracellular vesicles (EVs) represent promising markers, especially in ICI-based therapeutic settings. We evaluated the prognostic role of PD-L1 and of two B7 family members (B7-H3, B7-H4), both soluble and EV-associated, in a cohort of advanced NSCLC patients treated with first- (n = 56) or second-line (n = 126) ICIs. In treatment-naïve patients, high baseline concentrations of sPD-L1 (>24.2 pg/mL) were linked to worse survival, whereas high levels of sB7-H3 (>0.5 ng/mL) and sB7-H4 (>63.9 pg/mL) were associated with better outcomes. EV characterization confirmed the presence of EVs positive for PD-L1 and B7-H3, while only a small portion of EVs expressed B7-H4. The comparison between biomarker levels at the baseline and in the first radiological assessment under ICI-based treatment showed a significant decrease in EV-PD-L1 and an increase in EV-B7H3 in patients in the disease response to ICIs. Our study shows that sPD-L1, sB7-H3 and sB7-H4 levels are emerging prognostic markers in patients with advanced NSCLC treated with ICIs and suggests potential EV involvement in the disease response to ICIs.

Keywords: B7-H3; B7-H4; NSCLC; PD-L1; extracellular vesicle; immune checkpoint inhibitors; platelets; prognosis; soluble protein.

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Conflict of interest statement

C.G. (Carlo Genova) declares honoraria from Amgen, AstraZeneca, Bristol-Myers-Squibb, Eli-Lilly, Merck Sharp and Dohme, Novartis, Roche, Sanofi, Takeda and ThermoFisher. Research grants from: Bristol-Myers Squibb and Italian Ministry of Health. G.R. declares honoraria from AstraZeneca, Bristol-Myers Squibb, Roche, MSD and Janssen. C.D. declares honoraria from Bristol-Myers Squibb, Roche and Astra Zeneca. M.T. declares honoraria for travel, accommodation, expenses from Roche, Bristol-Myers Squibb, AstraZeneca, Takeda and Eli Lilly. Honoraria as medical writers: Novartis, Amgen and MSD. G.B. declares honoraria from AstraZeneca, Roche, Pierre Fabre and GSK. E.R. declares a travel grant from: daichii sankyo and honoraria from BMS, SANOFI, ASTRA ZENECA, MSD and ROCHE. F.G. declares his advisory role in ad hoc advisory boards/consultations (last 3 years); honoraria from Eli Lilly, Roche, Boehringer Ingelheim, AstraZeneca, Pierre Fabre, BMS, MSD, Novartis, Merck, Otsuka, Novartis and Takeda; Honoraria for seminars/talks to the industry (last 3 years): Eli Lilly, Roche, Boehringer Ingelheim, AstraZeneca, Pierre Fabre, AMGEN, Celgene, BMS and MSD; Research Funding (last 3 years): AstraZeneca, BMS and MSD. The other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Study design: (a) explore the role of sPD-L1, sB7-H3 and sB7-H4 as prognostic markers in patients with advanced NSCLC treated with nivolumab or pembrolizumab; (b) assess the involvement of EVs expressing PD-L1 and B7-H3 in the mechanisms of the response to ICIs.
Figure 2
Figure 2
Overall (a–c) and progression-free (d–f) survival probabilities estimated through multivariable Cox regression analysis in 56 patients of Pembro cohort stratified according to biomarker median values: sPD-L1 = 24.2 pg/mL (a,d); sB7-H3 = 0.5 ng/mL (b,e); sB7-H4 = 63.9 pg/mL (c,f). Legend—HR: hazard rate ratio adjusted for gender, age, cycles of therapy, ECOG-PS and histotype; 95% CL: 95% confidence limits for HR.
Figure 3
Figure 3
Comparisons between prognostic effects of study biomarkers in Pembro (PC) and Nivo (NC) cohorts on mortality (OS), progression (PFS) and non-response (NR) rates estimated through multivariable modified Poisson (NR) and Cox (OS and PFS) regressions. Legend—relative risk: (black points) ratio between adverse outcome rates for patients with higher vs. lower biomarker levels; 95% CL: (gray whiskers) 95% confidence limits for relative risk. Note—all relative risks were adjusted for gender, age, cycles, ECOG and histotype; relative risk = 1: (vertical dashed line) equal rates in both biomarker categories; relative risk >1: greater rates in higher categories; relative risk <1: lower rates in higher categories.
Figure 4
Figure 4
(a) Representative flow cytometry analysis of EVs derived from plasma of NR (#146) and CR (#174) patients, at both T0 and T1. Areas under blue and pink lines indicate EVs reacting with PD-L1 (upper panels) or B7-H3 (bottom panels) at T0 and T1, respectively. Areas under the grey lines indicate the interactions of vesicles with corresponding non-reactive immunoglobulin of the same isotype. (b,c) Histograms representing the percentage of PD-L1- (b) or B7-H3-positive EVs (c) derived from NR and CR/PR patients, at both T0 and T1. *: p < 0.05, **: p < 0.01, ****: p < 0.0001 (one-way ANOVA). (d) Western blot analysis of Flotillin-1, PD-L1 and B7-H3 on EVs derived from plasma of NR (#146) and CR (#174) patients, at both T0 and T1.
Figure 5
Figure 5
EV surface proteins profiled by multiplex bead-based flow cytometry assay. Captured EVs were counterstained with APC-labelled detection antibodies (mixture of anti-CD9, -CD63 and -CD81 antibodies). (a,d,g) Dot charts of beads corresponding to each of the 37 proteins. Red dots indicate: (a) PD-L1-expressing EVs identified above detection threshold in any of the patients, after normalizing their expression against CD9/CD63/CD81 MFI; (d) B7-H3Bright EVs identified above detection threshold after normalizing their expression against CD9 MFI; (g) B7-H3Dim EVs identified above detection threshold after normalizing their expression against CD81 MFI. Blue dots correspond to tetraspanin marker whereas black dots correspond to negative markers. (b,e,h) Histograms represent: (b) 9 out of 37 proteins that were found to be positive in PD-L1-expressing EVs isolated from any of the patients; (e) 7 out of 37 proteins that were found to be positive in B7-H3Bright EVs isolated from any of the patients; (h) 4 out of 37 proteins that were found to be positive in B7-H3Dim EVs isolated from any patients. (c,f) Histograms represent: percentage of B7-H3Bright (c) B7-H3Dim (f) and EVs isolated from NR and CR/PR patients at both T0 and T1. Error bars in graphs represent mean ± SD. ** p < 0.01 (Ordinary one-way ANOVA).
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