Immunotherapy targeting inhibitory checkpoints: The role of NK and other innate lymphoid cells.

Monoclonal antibodies that target specific ligand-receptor signaling pathways and act as immune checkpoint inhibitors have been designed to remove the brakes in T cells and restore strong and long-term antitumor-immunity. Of note, many of these inhibitory receptors are also expressed by Innate Lymphoid Cells (ILCs), suggesting that also blockade of inhibitory pathways in innate lymphocytes has a role in the response to the treatment with checkpoint inhibitors. ILCs comprise cytotoxic NK cells and "helper" subsets and are important cellular components in the tumor microenvironment. In addition to killing tumor cells, ILCs release inflammatory cytokines, thus contributing to shape adaptive cell activation in the context of immunotherapy. Therefore, ILCs play both a direct and indirect role in the response to checkpoint blockade. Understanding the impact of ILC-mediated response on the treatment outcome would contribute to enhance immunotherapy efficacy, as still numerous patients resist or relapse.

Presence of innate lymphoid cells in pleural effusions of primary and metastatic tumors: Functional analysis and expression of PD-1 receptor.

The tumor microenvironment (TM) contains a wide variety of cell types and soluble factors capable of suppressing immune responses. While the presence of NK cells in pleural effusions (PE) has been documented, no information exists on the presence of other innate lymphoid cell (ILC) subsets and on the expression of programmed cell death-1 (PD-1) in NK and ILC. The presence of ILC was assessed in PE of 54 patients (n = 33 with mesothelioma, n = 15 with adenocarcinoma and n = 6 with inflammatory pleural diseases) by cell staining with suitable antibody combinations and cytofluorimetric analysis. The cytokine production of ILC isolated from both PE and autologous peripheral blood was analyzed upon cell stimulation and intracytoplasmic staining. We show that, in addition to NK cells, also ILC1, ILC2 and ILC3 are present in malignant PE and that the prevalent subset is ILC3. PE-ILC subsets produced their typical sets of cytokines upon activation. In addition, we analyzed the PD-1 expression on NK/ILC by multiparametric flow-cytometric analysis, while the expression of PD-1 ligand (PD-L1) was evaluated by immunohistochemical analysis. Both NK cells and ILC3 expressed functional PD-1, moreover, both tumor samples and malignant PE-derived tumor cell lines were PD-L1+ suggesting that the interaction between PD-1+ ILC and PD-L1+ tumor cells may hamper antitumor immune responses mediated by NK and ILC.

Yeti, an essential Drosophila melanogaster gene, encodes a protein required for chromatin organization.

The evolutionarily conserved family of Bucentaur (BCNT) proteins exhibits a widespread distribution in animal and plants, yet its biological role remains largely unknown. Using Drosophila melanogaster as a model organism, we investigated the in vivo role of the Drosophila BCNT member called YETI. We report that loss of YETI causes lethality before pupation and defects in higher-order chromatin organization, as evidenced by severe impairment in the association of histone H2A.V, nucleosomal histones and epigenetic marks with polytene chromosomes. We also find that YETI binds to polytene chromosomes through its conserved BCNT domain and interacts with the histone variant H2A.V, HP1a and Domino-A (DOM-A), the ATPase subunit of the DOM/Tip60 chromatin remodeling complex. Furthermore, we identify YETI as a downstream target of the Drosophila DOM-A. On the basis of these results, we propose that YETI interacts with H2A.V-exchanging machinery, as a chaperone or as a new subunit of the DOM/Tip60 remodeling complex, and acts to regulate the accumulation of H2A.V at chromatin sites. Overall, our findings suggest an unanticipated role of YETI protein in chromatin organization and provide, for the first time, mechanistic clues on how BCNT proteins control development in multicellular organisms.

Mitochondria dynamism: of shape, transport and cell migration.

Mitochondria are highly dynamic and functionally versatile organelles that continuously fragment and fuse in response to different physiological needs of the cell. The list of proteins that strictly regulate the morphology of these organelles is constantly growing, adding new players every day and new pieces to the comprehension and elucidation of this complex machinery. The structural complexity of mitochondria is only paralled by their functional versatility. Indeed, changes in mitochondria shape play critical roles in vertebrate development programmed cell death and in various processes of normal cell physiology, such as calcium signaling, reactive oxygen species production, and lifespan. Here, we present the latest findings on the regulation of mitochondrial dynamics and some of their physiological roles, focusing on cell migration. In cells where migration represents a crucial function in their physiology, such as T and tumoral metastatic cells, mitochondria need to be fragmented and recruited to specific subcellular regions to make movement possible. In depth analysis of this role of mitochondrial dynamics should help in identifying potential targeted therapy against cancer or in improving the immune system's efficiency.

The anti-inflammatory cytokine IL-37 improves the NK cell-mediated anti-tumor response.

IL-37 is a member of the IL-1 superfamily exerting anti-inflammatory functions in a number of diseases. Extracellular IL-37 triggers the inhibitory receptor IL-1R8 that is known to regulate different NK cell pathways and functional activities including their anti-tumor effect. However, the effect of IL-37 on human NK cell functions is still to be unveiled. This study aimed to investigate the functional effect of IL-37 in human NK cells activated with IL-15. We found that IL-37 enhanced both NK cell cytotoxic activity against different tumor cell lines and cytokines production. These effects were associated with increased phosphorylation of ERK and NF-Kb. The improved NK cell activity was also strictly related to a time-dependent GSK3ß-mediated degradation of IL-1R8. The enhanced activation profile of IL-37 treated NK cells possibly due to IL-1R8 degradation was confirmed by the results with IL-1R8-silenced NK cells. Lastly, in line with these data, through the analysis of the TNM plot database of a large group of patients, IL-37 mRNA expression was found to be significantly lower in colon and skin cancers than in normal tissues. Colon adenocarcinoma and neuroblastoma patients with higher IL-37 mRNA levels had significantly higher overall survival, suggesting that the presence of IL-37 might be considered an independent positive prognostic factor for this tumor. Our results provide novel information on the mechanisms regulating IL-1R8 function in human NK cells, highlighting the IL-37-IL-1R8 axis as a potential new target to improve the anti-tumor immune response.

TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells.

Background: In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s). Methods: SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs. Results: SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals. Conclusions: TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects.

Analysis of the mechanisms regulating soluble PD-1 production and function in human NK cells.

NK cells represent important effectors that play a major role in innate defences against pathogens and display potent cytolytic activity against tumor cells. An array of surface receptors finely regulate their function and inhibitory checkpoints, such as PD-1, can dampen the immune response inducing an immunosuppressive state. Indeed, PD-1 expression in human NK cells correlated with impaired effector function and tumor immune evasion. Importantly, blockade of the PD-1/PD-L1 axis has been shown to reverse NK cell exhaustion and increase their cytotoxicity. Recently, soluble counterparts of checkpoint receptors, such as soluble PD-1 (sPD-1), are rising high interest due to their biological activity and ability to modulate immune responses. It has been widely demonstrated that sPD-1 can modulate T cell effector functions and tumor growth. Tumor-infiltrating T cells are considered the main source of circulating sPD-1. In addition, recently, also stimulated macrophages have been demonstrated to release sPD-1. However, no data are present on the role of sPD-1 in the context of other innate immune cell subsets and therefore this study is aimed to unveil the effect of sPD-1 on human NK cell function. We produced the recombinant sPD-1 protein and demonstrated that it binds PD-L1 and that its presence results in increased NK cell cytotoxicity. Notably, we also identified a pathway regulating endogenous sPD-1 synthesis and release in human NK cells. Secreted endogenous sPD-1, retained its biological function and could modulate NK cell effector function. Overall, these data reveal a pivotal role of sPD-1 in regulating NK-mediated innate immune responses.

The roles of different forms of IL-15 in human melanoma progression.

Background: Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. Methods: The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Results: Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Conclusions: Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

IL-1R8 silencing improves the anti-tumor function of freshly isolated human NK cells.

The inhibitory receptor interleukin-1 receptor 8 (IL-1R8) has been recently recognized to be expressed also by human natural killer (NK) cells. This study was aimed to design and optimize IL-1R8 silencing conditions in human NK cells to precisely establish the activity of such receptor in these cells. Electroporation of freshly isolated or IL-2-cultured NK cells with small interfering RNA (siRNA), resulted in a marked, even though variable, IL-1R8-silencing. Although the expression profile revealed downregulation of most genes involved in several intracellular pathways, some genes related to proliferation, expression of some chemokine receptors, antibody-dependent cell cytotoxicity and cytotoxic activity were upregulated in IL-1R8-silenced NK cells. Furthermore, upon IL-15 activation, the majority of genes involved in NK cell function were upregulated in IL-1R8-siRNA-compared with control-siRNA-transfected NK cells. More importantly, in agreement with these findings, the reduction of IL-1R8 gene expression levels resulted in enhanced expression of NK cell activation markers, production of cytokines and chemokines, and cytotoxic activity against several NK cell targets with different susceptibility to NK-mediated lysis. Similar results were obtained following stimulation with IL-18. All together these data, deeply impacting on the main effector functions of human NK cells, can lead to a better understanding of IL-1R8-mediated regulation on these cells and to the design of new strategies for improving NK cell-mediated anti-tumor responses.

RNA polymerase I transcription silences noncoding RNAs at the ribosomal DNA locus in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the repeated units of the ribosomal locus, transcribed by RNA polymerase I (Pol I), are interrupted by nontranscribed spacers (NTSs). These NTS regions are transcribed by RNA polymerase III to synthesize 5S RNA and by RNA polymerase II (Pol II) to synthesize, at low levels, noncoding RNAs (ncRNAs). While transcription of both RNA polymerase I and III is highly characterized, at the ribosomal DNA (rDNA) locus only a few studies have been performed on Pol II, whose repression correlates with the SIR2-dependent silencing. The involvement of both chromatin organization and Pol I transcription has been proposed, and peculiar chromatin structures might justify "ribosomal" Pol II silencing. Reporter genes inserted within the rDNA units have been employed for these studies. We studied, in the natural context, yeast mutants differing in Pol I transcription in order to find whether correlations exist between Pol I transcription and Pol II ncRNA production. Here, we demonstrate that silencing at the rDNA locus represses ncRNAs with a strength inversely proportional to Pol I transcription. Moreover, localized regions of histone hyperacetylation appear in cryptic promoter elements when Pol II is active and in the coding region when Pol I is functional; in addition, DNA topoisomerase I site-specific activity follows RNA polymerase I transcription. The repression of ncRNAs at the rDNA locus, in response to RNA polymerase I transcription, could represent a physiological circuit control whose mechanism involves modification of histone acetylation.

The Immune Checkpoint PD-1 in Natural Killer Cells: Expression, Function and Targeting in Tumour Immunotherapy.

In the last years, immunotherapy with antibodies against programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has shown remarkable efficacy in the treatment of different types of tumours, representing a true revolution in oncology. While its efficacy has initially been attributed only to unleashing T cell responses, responsivity to PD-1/PD-L1 blockade was observed in some tumours with low Human Leukocyte Antigen (HLA) I expression and increasing evidence has revealed PD-1 surface expression and inhibitory function also in natural killer (NK) cells. Thus, the contribution of anti-PD-1/PD-L1 therapy to the recovery of NK cell anti-tumour response has recently been appreciated. Here, we summarize the studies investigating PD-1 expression and function in NK cells, together with the limitations and perspectives of immunotherapies. A better understanding of checkpoint biology is needed to design next-generation therapeutic strategies and to improve the clinical protocols of current therapies.

Inhibitory Receptors and Checkpoints in Human NK Cells, Implications for the Immunotherapy of Cancer.

The highly destructive mechanisms by which the immune system faces microbial infections is under the control of a series of inhibitory receptors. While most of these receptors prevent unwanted/excessive responses of individual effector cells, others play a more general role in immunity, acting as true inhibitory checkpoints controlling both innate and adaptive immunity. Regarding human NK cells, their function is finely regulated by HLA-class I-specific inhibitory receptors which allow discrimination between HLA-I+, healthy cells and tumor or virus-infected cells displaying loss or substantial alterations of HLA-I molecules, including allelic losses that are sensed by KIRs. A number of non-HLA-specific receptors have been identified which recognize cell surface or extracellular matrix ligands and may contribute to the physiologic control of immune responses and tolerance. Among these receptors, Siglec 7 (p75/AIRM-1), LAIR-1 and IRp60, recognize ligands including sialic acids, extracellular matrix/collagen or aminophospholipids, respectively. These ligands may be expressed at the surface of tumor cells, thus inhibiting NK cell function. Expression of the PD-1 checkpoint by NK cells requires particular cytokines (IL-15, IL-12, IL-18) together with cortisol, a combination that may occur in the microenvironment of different tumors. Blocking of single or combinations of inhibitory receptors unleashes NK cells and restore their anti-tumor activity, with obvious implications for tumor immunotherapy.

Interleukin-15 and cancer: some solved and many unsolved questions.

Soluble interleukin (IL)-15 exists under two forms: as monomer (sIL-15) or as heterodimeric complex in association with sIL-15Rα (sIL-15/IL-15Rα). Both forms have been successfully tested in experimental tumor murine models and are currently undergoing investigation in phase I/II clinical trials. Despite more than 20 years research on IL-15, some controversial issues remain to be addressed. A first point concerns the detection of the sIL-15/IL-15Rα in plasma of healthy donors or patients with cancer and its biological significance. The second and third unsolved question regards the protumorigenic role of the IL-15/IL-15Rα complex in human cancer and the detrimental immunological consequences associated to prolonged exposure of natural killer (NK) cells to both forms of soluble IL-15, respectively. Data suggest that in vivo prolonged or repeated exposure to monomeric sIL-15 or the soluble complex may lead to NK hypo-responsiveness through the expansion of the CD8+/CD44+ T cell subset that would suppress NK cell functions. In vitro experiments indicate that soluble complex and monomeric IL-15 may cause NK hyporesponsiveness through a direct effect caused by their prolonged stimulation, suggesting that this mechanism could also be effective in vivo. Therefore, a better knowledge of IL-15 and a more appropriate use of both its soluble forms, in terms of concentrations and time of exposure, are essential in order to improve their therapeutic use. In cancer, the overproduction of sIL-15/IL-15Rα could represent a novel mechanism of immune escape. The soluble complex may act as a decoy cytokine unable to efficiently foster NK cells, or could induce NK hyporesponsiveness through an excessive and prolonged stimulation depending on the type of IL-15Rα isoforms associated. All these unsolved questions are not merely limited to the knowledge of IL-15 pathophysiology, but are crucial also for the therapeutic use of this cytokine. Therefore, in this review, we will discuss key unanswered issues on the heterogeneity and biological significance of IL-15 isoforms, analyzing both their cancer-related biological functions and their therapeutic implications.

Innate Lymphoid Cells: Expression of PD-1 and Other Checkpoints in Normal and Pathological Conditions.

Innate lymphoid cells (ILCs) belong to a family of immune cells. Recently, ILCs have been classified into five different groups that mirror the function of adaptive T cell subsets counterparts. In particular, NK cells mirror CD8+ cytotoxic T cells while ILC1, ILC2, ILC3, and Lymphoid tissue inducer (LTi)-like cells reflect the function of CD4+T helper (Th) cells (Th1, Th2, and Th17 respectively). ILCs are involved in innate host defenses against pathogens and tumors, in lymphoid organogenesis, and in tissue remodeling/repair. In recent years, important molecular inducible checkpoints (PD-1, TIM3, and TIGIT) were shown to control/inactivate different immune cell types. The expression of many of these receptors has been detected on NK cells and subsets of tissue-resident ILCs in both physiological and pathological conditions, including cancer. In particular, it has been demonstrated that the interaction between PD-1+ immune cells and PD-L1/PD-L2+ tumor cells may compromise the anti-tumor effector function leading to tumor immune escape. However, while the effector function of NK cells in tumor is well-established, limited information exists on the other ILC subsets. We will summarize what is known to date on the expression and function of these checkpoint receptors on NK cells and ILCs, with a particular focus on the recent data that reveal an essential contribution of the blockade of PD-1 and TIGIT on NK cells to the immunotherapy of cancer. A better information regarding the presence and the function of different ILCs and of the inhibitory checkpoints in pathological conditions may offer important clues for the development of new immune therapeutic strategies.

Helper Innate Lymphoid Cells in Human Tumors: A Double-Edged Sword?

Innate lymphoid cells (ILCs) were found to be developmentally related to natural killer (NK) cells. In humans, they are mostly located in "barrier" tissues where they contribute to innate defenses against different pathogens. ILCs are heterogeneous and characterized by a high degree of plasticity. ILC1s are Tbet+, produce interferon gamma and tumor necrosis factor alpha, but, unlike NK cells, are non-cytolytic and are Eomes independent. ILC2 (GATA-3+) secrete type-2 cytokines, while ILC3s secrete interleukin-22 and interleukin-17. The cytokine signatures of ILC subsets mirror those of corresponding helper T-cell subsets. The ILC role in defenses against pathogens is well-documented, while their involvement in tumor defenses is still controversial. Different ILCs have been detected in tumors. In general, the conflicting data reported in different tumors on the role of ILC may reflect the heterogeneity and/or differences in tumor microenvironment. The remarkable plasticity of ILCs suggests new therapeutic approaches to induce differentiation/switch toward ILC subsets more favorable in tumor control.

Human CAR NK Cells: A New Non-viral Method Allowing High Efficient Transfection and Strong Tumor Cell Killing.

CAR-NK cells may represent a valuable tool, complementary to CAR-T cells, in adoptive immunotherapy of leukemia and solid tumors. However, gene transfer to human NK cells is a challenging task, particularly with non-virus-based techniques. Here, we describe a new procedure allowing efficient electroporation-based transfection of plasmid DNA, including CAR and CCR7 genes, in resting or cytokine-expanded human NK cell populations and NK-92 cell line. This procedure may offer a suitable platform for a safe and effective use of CAR-NK cells in adoptive immunotherapy of cancer.

Human natural killer cells and other innate lymphoid cells in cancer: Friends or foes?

Innate lymphoid cells (ILC) including NK cells (cytotoxic) and the recently identified "helper" ILC1, ILC2 and ILC3, play an important role in innate defenses against pathogens. Notably, they mirror analogous T cell subsets, regarding the pattern of cytokine produced, while the timing of their intervention is few hours vs days required for T cell-mediated adaptive responses. On the other hand, the effectiveness of ILC in anti-tumor defenses is controversial. The relevance of NK cells in the control of tumor growth and metastasis has been well documented and they have been exploited in the therapy of high risk leukemia in the haploidentical hematopoietic stem cell transplantation setting. In contrast, the actual involvement of helper ILCs remains contradictory. Thus, while certain functional capabilities of ILC1 and ILC3 may favor anti-tumor responses, other functions could rather favor tumor growth, neo-angiogenesis, epithelial-mesenchymal transition and metastasis. In addition, ILC2, by secreting type-2 cytokines, are thought to induce a prevalent pro-tumorigenic effect. Finally, the function of both NK cells and helper ILCs may be inhibited by the tumor microenvironment, thus adding further complexity to the interplay between ILC and tumors.

PD-L1 expression comparison between primary and relapsed non-small cell lung carcinoma using whole sections and clone SP263.

We assessed the concordance, in terms of PD-L1 expression, between primary and metastatic non-small cell lung carcinoma (NSCLC) of different histotypes using validated SP263 clone. A few samples of local recurrences have also been analyzed. Whole sections of consecutive cases of primary NSCLC and paired relapses undergone surgical resection have been stained with PD-L1 clone SP263; for scoring purposes, a three-tiered system was applied using the following thresholds: <1%, 1-49% and ≥50%. Eighty-four cases of paired primary and relapsed tumors from 83 patients were analyzed, including 75 metastases and 9 local recurrences. Regarding metastases, when considering a cutoff of 1%, discrepancy in PD-L1 expression occurred in 9/75 (12%) paired samples (kappa value = 0.75); at 50% cutoff, discrepancy in PD-L1 expression was detected in 7/75 (9.3%) of paired samples (kappa value = 0.61). Regarding recurrences, at 1% cutoff, the discrepancy in PD-L1 expression was seen in 3/9 (33%) paired samples and in all cases there was a gained PD-L1 expression; at 50% cutoff, 1/9 (11%) paired samples showed gained PD-L1 expression. Our data provide important information regarding the concordance between primary and relapsed NSCLC and the degree of reliability of metastatic sites in terms of PD-L1 expression evaluation.

PD-L1 Expression Heterogeneity in Non-Small Cell Lung Cancer: Defining Criteria for Harmonization between Biopsy Specimens and Whole Sections.

INTRODUCTION: Determination of programmed death ligand 1 (PD-L1) expression defines eligibility for treatment with pembrolizumab in patients with advanced NSCLC. This study was designed to better define which value across core biopsy specimens from the same case more closely reflects the PD-L1 expression status on whole sections and how many core biopsy specimens are needed for confident classification of tumors in terms of PD-L1 expression. METHODS: We built tissue microarrays as surrogates of biopsies collecting five cores per case from 268 cases and compared PD-L1 staining results obtained by using the validated clone SP263 with the results obtained by using whole tumor sections. RESULTS: We found an overall positivity in 39% of cases at a cutoff of 1% and in 10% of cases at a cutoff of 50%. The maximum value across cores was associated with high concordance between cores and whole sections and the lowest number of false-negative cases overall. To reach high concordance with whole sections, four and three cores are necessary at cutoffs of 1% and 50%, respectively. Importantly, with 20% as the cutoff for core biopsy specimens, fewer than three cores showed high sensitivity and specificity in identifying cases with 50% or more of tumor cells positive for PD-L1 on whole sections. Specifically, for PD-L1 expression values of 20% to 49% on cores, the probabilities of a tumor specimen expressing PD-L1 in at least 50% of cells on a whole section were 46% and 24% with one and two biopsy specimens, respectively. CONCLUSIONS: An accurate definition of the criteria to determine the PD-L1 status of a given tumor may greatly help in selecting those patients who could benefit from anti-programmed cell death 1/PD-L1 treatment.