Translational Oncology 14 (2021) 100969 Contents lists available at ScienceDirect Translational Oncology journal homepage: www.elsevier.com/locate/tranon Letters to the Editor Unmasking the expression of PD-L1 in Myeloid Derived explain the different response to targeted immunotherapy against PD- Suppressor Cells: A case study in lung cancer to discover L1, also suggesting that phenotypic characterization is not sufficient to new drugs with specific on-target efficacy identify MDSCs and an additional proof of the immunosuppressive func- tion is needed. PD-L1 was studied in a total of 5 samples, before and after PD-L1 expression on tumor cells and immune cells could predict re- immunotherapy (day 0, 1, 4, 6 and 8) showing differences in PD-L1 ex- sponse to immunotherapy [1]. However, tiny biopsy samples can result pression (Supplemental Fig. 3 and Supplemental Figure 4A) as well as in in false-negative PD-L1 evaluation [2]. Herein, we describe the accurate the relative number of MDSCs (Supplemental Figure 4B). Merging all to- identification of PD-L1 on peripheral blood myeloid derived suppressor gether into a single FCS file using a process called concatenation, periph- cells (MDSCs). eral blood samples were effectively compared (Fig. 1C and D). Dimen- A 72-year-old male, never-smoker, was diagnosed with recurrent sionality reduction was performed on the concatenated data set. After lung adenocarcinoma, PD-L1 negative (22C3), without actionable mu- PMA stimulation, PD-L1+ cells were displayed based on the expression tations. The patient was treated with nivolumab, and paclitaxel. Af- of 6 markers using t-SNE (t-distributed stochastic neighbor embedding) ter progression, he was re-treated with nivolumab plus glutaminase in- visualization. The axes show dimensionless values that were assigned hibitor until persistence of adrenal and distant lymph node metastasis to individual cells by the t-SNE algorithm, which places PD-L1+ MDSCs was observed. Atezolizumab plus T cell immunoglobulin mucin-domain that have similar expression profiles close to one another. Eliminating containing-3 (TIM3) inhibitor was administered as third line therapy. MDSCs by promoting PD-L1 stabilized unfolded states on both PMN- and The patient completed 11 cycles. Afterwards, the left adrenal gland was M-MDSCs could improve immunotherapy efficacy. High frequency of irradiated and a muscle metastasis in the left gluteus was resected. Tar- PMN-MDSCs [4] is an adverse prognostic factor in lung [5] and head and geted Next Generation Sequencing (NGS) in metastatic tissue and blood neck cancer [6,7]. This conformational change may be associated with were negative for actionable alterations. The patient received further a PD-L1 immunoregulatory mechanism that affects therapies targeting treatment with a novel anti-PD-L1 (PDR001) combination. However, the PD-1/PD-L1 checkpoint. Conformational changes, accumulation, further progression was identified with widespread bone and lymph expansion, and survival of MDSCs require research for PD-L1 unfolding. node metastases (More details in Supplementary Figure 1). Peripheral Monitoring peripheral blood MDSCs and identification of drugs with blood samples were serially collected before and after immunotherapy PMA-like effects is highly warranted. In view of these conformational for the assessment of MDSCs and PD-L1 expression by multi-color flow properties, analysis of accumulation, expansion and survival of patho- cytometry. logical immunosuppressive MDSCs could help to better understand and Prior to immunotherapy (day 0), peripheral blood smear analy- overcome the mechanisms of immunotherapy resistance, by developing sis showed mostly neutrophils and monocytes, and lymphocytes were new treatment strategies aimed at promoting PD-L1 stabilized unfolded clearly reduced (Supplementary Fig. 2A and B). Hematology analysis states. Altogether, our findings may indicate the clinical relevance of showed a decreased number of lymphocytes (5.5%), and a marked in- peripheral blood MDSCs assessment and immunosuppressive pattern, crease in monocytes (13%) and neutrophils (80.2%). At initial assess- as well as the need to find compounds that by themselves exhibit a ment, red-cell count, hemoglobin and hematocrit showed decreased con- PMA-like effect aimed at abrogating MDSCs. Limitations of this study centrations. White-cell analysis by multi-color flow cytometry showed include the enrollment of only one patient and a short follow–up period, that PD-L1 was dramatically increased after Phorbol 12-myristate 13- because exitus occurred after a few days of analysis. Important research acetate (PMA) stimulation. Peripheral blood cells were mostly PD-L1+ must be done to elucidate the mechanism by which PD-L1 is translo- MDSCs (81.50% polymorphonuclear PMN-MDSCs and 7.74% monocytic cated and the clinical relevance to those patients with higher PD- L1 M-MDSCs) (Fig. 1B). fold-change. Future experiments will attempt to further dissect to what Surprisingly, non-stimulated cells expressed undetectable levels of degree stimulation increases PD-L1 expression on MDSCs and how this PD-L1, as shown by the flow cytometry method when DMSO was used information can be translated into benefit for immunotherapy. The fea- (Fig. 1A). This suggests that PD-L1 displays some variation of spatial sibility of determining PD-L1 expression on MDSCs and its potential use conformation [3] in response to PMA stimulation, which may confer a as a biomarker to determine treatment response is promoting new ways critical enhancement in binding affinity. This variability may help to to study cancer, particularly in the promising area of immunotherapy. https://doi.org/10.1016/j.tranon.2020.100969 Received 29 July 2020; Received in revised form 13 November 2020; Accepted 20 November 2020 1936-5233/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Letters to the Editor Translational Oncology 14 (2021) 100969 Fig. 1. Evaluation of PD-L1 expression and clustering analysis in terms of cell number and antigenic density comparisons across immunotherapy. Evaluation of PD-L1 fold-increase of a 72-year-old male, a lifetime non-smoker, diagnosed with lung adenocarcinoma in 2014 the day before immunotherapy administration (day 0) and day 1, 4, 6 and 8 before immunotherapy. Panel A and B show a representative peripheral blood analysis on day 0stimulated for 5 min at 37 °C with PMA. Numbers in regions R5 to R7 indicate gated cell counts. Fold-change was calculated as the ratio between stimulated PD-L1+ MDSCs and non- stimulated PD-L1+ cells. PMN-MDSCs were identified by their expression of PD-L1 together with CD33+ CD11bhigh HLA-DR−/low and M-MDSCs were identified by their expression of PD-L1 together with CD33high CD11b+ HLA-DR−/low . The MDSCs PD-L1+ population was calculated over the total number of acquired leukocytes, identified in the DCV+ gate (R1), in the single cell gate (R2) and in the 7-AAD- gate (R3) (data not shown). Changes in PD-L1 expression after PMA treatment found a 191-fold increase after stimulation (Panel B), compared with non-stimulated cells (Panel A), suggesting that fold-change in PD-L1 appears to have an important regulatory factor that may relate to differences in underlying mechanisms within patients. Merging all measurements together into a single FCS file using a process called concatenation, peripheral blood samples obtained before and after immunotherapy treatment were effectively compared. Dimensionality reduction was performed on the concatenated data set. After PMA stimulation, PD-L1+ cells were displayed based on the expression of 6 markers using t-SNE (t-distributed stochastic neighbor embedding) visualization. The axes (t-SNE1 and t-SNE2) show dimensionless values that were assigned to individual cells by the t-SNE algorithm, which places PD-L1+ MDSCs that have similar expression profiles close to one another. In Panel C, the intensity of the coloring is relative to the cell density of PD-L1+ MDSCs. In Panel D, the intensity of the coloring is relative to the expression of PD-L1+ MDSCs. MDSCs from pre- and post-immunotherapy have similar cell density and expression patterns that identify them as well conserved entities across biologic therapy. Letters to the Editor Translational Oncology 14 (2021) 100969 Author contribution statement [2] S. Kitazono, Y. Fujiwara, K. Tsuta, H. Utsumi, S. Kanda, H. Horinouchi, H. Nokihara, N. Yamamoto, S. Sasada, S. Watanabe, H. Asamura, T. Tamura, et al., Reliability of small biopsy samples compared with resected specimens for the determination L.G.R., and J.P. performed the experiments. L.G.R., A.A.H, M.D.W., of programmed death-ligand 1 expression in non-small-cell lung cancer, Clin. Lung J.A.B., J.J., R.R. and J.P performed data interpretation, and drafted Cancer 16 (2015) 385–390. the manuscript. R.R. and J.P conceived the study, participated in its [3] R. Pascolutti, X. Sun, J. Kao, R.L. Maute, A.M. Ring, G.R. Bowman, A.C. Kruse, Struc- ture and dynamics of PD-L1 and an ultra-high-affinity PD-1 receptor mutant, Structure design, conducted most experiments, and contributed the manuscript 24 (2016) 1719–1728. equally (∗ ). [4] E. Tcyganov, J. Mastio, E. Chen, D.I. Gabrilovich, Plasticity of myeloid-derived sup- pressor cells in cancer, Curr. Opin. Immunol. 51 (2018) 76–82. [5] L. Barrera, E. Montes-Servin, J.M. Hernandez-Martinez, M. Orozco-Morales, CRediT author statement D. Michel-Tello, R.A. Morales-Flores, D. Flores-Estrada, O. Arrieta, Levels of pe- ripheral blood polymorphonuclear myeloid-derived suppressor cells and selected cy- Laura G. Rico: Methodology, Investigation, Formal analysis, Writing tokines are potentially prognostic of diseaseprogression for patients with non-small cell lung cancer, Cancer Immunol. Immunother. 67 (2018) 1393–1406. – Original draft. [6] S. Lang, K. Bruderek, C. Kaspar, B. Hoing, O. Kanaan, N. Dominas, T. Hussain, Andrés Aguilar Hernández: Formal analysis, Writing – Original F. Droege, C. Eyth, B. Hadaschik, S. Brandau, Clinical relevance and suppressive ca- draft. pacity of human myeloid-derived suppressor cell subsets, Clin. Cancer Res. 24 (2018) Michael D. Ward: Formal analysis, Writing – Original draft. 4834–4844. [7] H.T. Lee, S.H. Lee, Y.S. Heo, Molecular interactions of antibody drugs targeting PD-1, Jolene A. Bradford: Formal analysis, Writing – Original draft. PD-L1, and CTLA-4 in immuno-oncology, Molecules 24 (2019). Jordi Juncà: Formal analysis, Writing – Original draft. Rafael Rosell: Conceptualization, Methodology, Writing – Original draft, Writing - Review & Editing, Supervision. Laura G. Rico Jordi Petriz: Conceptualization, Methodology, Investigation, Writ- Functional Cytomics Group, Institut de Recerca contra la Leucèmia Josep ing – Original draft, Writing - Review & Editing, Supervision. Carreras, IJC, Campus ICO-Germans Trias i Pujol, Institut Germans Trias i Pujol (IGTP), Universitat Autònoma de Barcelona, UAB, Badalona, Declaration of Competing Interest Barcelona, Catalonia, 08916, Spain Andrés Aguilar Hernández M.D.W. and J.A.B. work for Thermo Fisher Scientific, which is in the Quirón Dexeus University Hospital, Dr Rosell Oncology Institute, business of selling flow cytometers and flow cytometry reagents. Barcelona, Spain Acknowledgements Michael D. Ward, Jolene A. Bradford Thermo Fisher Scientific, Eugene, OR, USA We thank CERCA Programme/Generalitat de Catalunya and The Jordi Juncà Josep Carreras Foundation for institutional support. The authors are also Institut Català d’Oncologia, Hospital Germans Trias i Pujol (HGTiP), very grateful for their advice and technical support to Sergio Ramón, Badalona, Spain Víctor Querol, Clara Streiff, Paola Paglia, and Lluís Sainz from Thermo Fisher for all his comments and discussions on earlier work in this re- Rafael Rosell∗∗ search field. Jordi Petriz also acknowledges the financial support from Quirón Dexeus University Hospital, Dr Rosell Oncology Institute, The Obra Social La Caixa and Thermo Fisher Scientific. Barcelona, Spain Institut Català d’Oncologia, Hospital Germans Trias i Pujol (HGTiP), Badalona, Spain Supplementary materials Jordi Petriz∗ Supplementary material associated with this article can be found, in Functional Cytomics Group, Institut de Recerca contra la Leucèmia Josep the online version, at doi:10.1016/j.tranon.2020.100969. Carreras, IJC, Campus ICO-Germans Trias i Pujol, Institut Germans Trias i Pujol (IGTP), Universitat Autònoma de Barcelona, UAB, Badalona, References ∗ Corresponding author. Barcelona, Catalonia, 08916, Spain ∗∗ Corresponding author at: Quirón Dexeus University Hospital, Dr Rosell Oncology Institute, Barcelona, Spain. [1] F.R. Hirsch, A. McElhinny, D. Stanforth, J. Ranger-Moore, M. Jansson, K. Kulangara, W. Richardson, P. Towne, D. Hanks, B. Vennapusa, A. Mistry, R. Kalamegham, et al., E-mail addresses: rrosell@iconcologia.net (R. Rosell), PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the jpetriz@carrerasresearch.org (J. Petriz) blueprint PD-L1 IHC assay comparison project, J. Thorac. Oncol. 12 (2017) 208–222. Revised 13 November 2020