Publications by authors named "Enrico Munari"

73 Publications

Identification of neuroblastoma cell lines with uncommon TAZ/mesenchymal stromal cell phenotype with strong suppressive activity on natural killer cells.

J Immunother Cancer 2021 Jan;9(1)

Immunology Area, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy

Background: Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells.

Methods: We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset.

Results: Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105/CD90/CD73/CD29/CD146/GD2/TAZ). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105/CD73/TAZ). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome.Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells.

Conclusions: We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells.
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http://dx.doi.org/10.1136/jitc-2020-001313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7813384PMC
January 2021

Wilms' Tumor Primary Cells Display Potent Immunoregulatory Properties on NK Cells and Macrophages.

Cancers (Basel) 2021 Jan 9;13(2). Epub 2021 Jan 9.

Bambino Gesù Children's Hospital, IRCCS, 00165 Rome, Italy.

The immune response plays a crucial defensive role in cancer growth and metastasis and is a promising target in different tumors. The role of the immune system in Wilm's Tumor (WT), a common pediatric renal malignancy, is still to be explored. The characterization of the immune environment in WT could allow the identification of new therapeutic strategies for targeting possible inhibitory mechanisms and/or lowering toxicity of the current treatments. In this study, we stabilized four WT primary cultures expressing either a blastematous (CD56/CD133) or an epithelial (CD56/CD133) phenotype and investigated their interactions with innate immune cells, namely NK cells and monocytes. We show that cytokine-activated NK cells efficiently kill WT cells. However, after co-culture with WT primary cells, NK cells displayed an impaired cytotoxic activity, decreased production of IFNγ and expression of CD107a, DNAM-1 and NKp30. Analysis of the effects of the interaction between WT cells and monocytes revealed their polarization towards alternatively activated macrophages (M2) that, in turn, further impaired NK cell functions. In conclusion, we show that both WT blastematous and epithelial components may contribute directly and indirectly to a tumor immunosuppressive microenvironment that is likely to play a role in tumor progression.
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http://dx.doi.org/10.3390/cancers13020224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826641PMC
January 2021

HLA-G expression in melanomas.

Int Rev Immunol 2021 Jan 11:1-21. Epub 2021 Jan 11.

Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.

Objective: Human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule involved in inducing tolerance at the feto-maternal interface and in escape of immune response by tumor cells. The aim of the study is to review the published literature on the expression of HLA-G in malignant melanomas and its clinicopathological and prognostic correlates.

Methods: A systematic search was carried out in electronic databases. Studies dealing with HLA-G expression in surgically-removed human samples were retrieved and analyzed.

Results: Of 1737 retrieved articles, 16 were included. The main themes regarded HLA-G expression in malignant melanocytic lesions, assessed by immunohistochemistry (IHC), soluble or molecular techniques, and its relationship with clinicopathological features, such as tumor thickness and malignant behavior. Overall significant HLA-G expression was found in 460/843 tumors (55%), and specifically in 251/556 melanomas (45%) evaluated with IHC, in 208/250 cases (83%) examined with soluble methods and in 13/23 melanoma lesions (57%) tested with polymerase chain reaction. Despite the correlation with parameters indicating an aggressive behavior, no studies demonstrated any prognostic value of HLA-G expression. Furthermore, uveal melanomas were constantly negative for this biomarker.

Conclusion: Overall, published data indicate that while HLA-G is involved in the interactions between melanomas and the immune system, it is unlikely to be the only factor to play such a role, therefore making it difficult to designate it as a prognostically relevant molecule. Evidence further suggests that HLA-G is not implicated in the immunobiology of uveal melanomas.
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http://dx.doi.org/10.1080/08830185.2020.1869732DOI Listing
January 2021

Assessment of HER2 Protein Overexpression and Gene Amplification in Renal Collecting Duct Carcinoma: Therapeutic Implication.

Cancers (Basel) 2020 Nov 12;12(11). Epub 2020 Nov 12.

Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, via Orabona 4, 70126 Bari, Italy.

Collecting duct carcinoma (CDC) is rare and aggressive histology of kidney cancers. Although different therapeutic approaches have been tested, the 2-year survival remains very poor. Since CDC exhibits overlapping features with urothelial carcinoma, the analysis of shared molecular alterations could provide new insights into the understanding of this rare disease and also therapeutic options. We collected 26 CDC cases, and we assessed HER2 protein expression by immunohistochemistry (IHC) and gene amplification by fluorescence in-situ hybridization (FISH) according to 2018 ASCO/CAP HER2-testing recommendations. Six out of twenty-six (23%) tumors showed HER2 positive staining. In particular, 3+ score was present in 2/6 cases (33%), 2+ in 3/6 cases (50%) and 1+ in 1/6 cases (17%). The 6 HER2+ tumors were also analyzed by FISH to assess gene copy number. One out of six CDC with IHC 3+ was also HER2 amplified, showing an average HER2 copy number ≥4.0 (10.85) and a HER2/CEP17 ratio ≥ (5.63), while the 5/6 cases were HER2 negative. Based on the 2018 ASCO/CAP guidelines overall, 2/26 CDC cases (8%) were HER2+. The present study provides evidence for testing, in future studies, HER2 to assess its clinical value as a novel target for the treatment of this highly malignant cancer.
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http://dx.doi.org/10.3390/cancers12113345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697829PMC
November 2020

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

Cancers (Basel) 2020 Nov 6;12(11). Epub 2020 Nov 6.

Department of Immunology, IRCCS Bambino Gesù Children's Hospital, 00146 Rome, Italy.

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.
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http://dx.doi.org/10.3390/cancers12113285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694632PMC
November 2020

MDM2 gene amplification as selection tool for innovative targeted approaches in PD-L1 positive or negative muscle-invasive urothelial bladder carcinoma.

J Clin Pathol 2020 Nov 3. Epub 2020 Nov 3.

Division of Urology, University and Hospital Trust of Verona, Verona, Italy.

Aims: According to The Cancer Genome Atlas (TCGA), around 9% of bladder carcinomas usually show abnormalities of the murine double minute 2 (MDM2) gene, but a few studies have been investigated them. We profiled MDM2 gene amplification in a series of urothelial carcinomas (UC) considering the molecular subtypes and expression of programmed death ligand 1 (PD-L1).

Methods: 117 patients with muscle-invasive UC (pT2-3) without (N0) or with (N+) lymph-node metastases were revised. Only cases with availability of in toto specimens and follow-up were studied. Tissue microarray was built. p53, ER, RB1, GATA-3, CK20, CK5/6, CD44 and PD-L1 (clone sp263) immunoexpression was evaluated. Fluorescent in situ hybridisation was assessed by using the HER-2/neu, FGFR-3, CDKN2A and MDM2 probes. True (ratio 12q/CEP12 >2) MDM2 gene amplification was distinguished from polyploidy/gains (ratio <2, absolute copy number of MDM-2 >2). MDM2 and PD-L1 values were correlated to the TCGA molecular phenotypes. Statistical analysis was performed.

Results: 6/50 (12%) cases (5 N0 and 1 N+) were amplified for MDM2 without matching to molecular phenotypes. Of 50, 14 (37%) cases expressed PD-L1 at 1% cut-off; 3/50 (9%) at >50% cut-off; of these, 2 cases on side of neoplasia among inflammatory cells. Only one out of six (17%) cases amplified for MDM2 showed expression (>50% cut-off) of PD-L1. MDM2 amplification was independent to all documented profiles (k test=0.3) and was prevalent in recurrent UC.

Conclusion: MDM2 amplification has been seen in both PD-L1 positive and negative muscle-invasive bladder UC independently from the TCGA molecular phenotypes. MDM2 and PD-L1 might be assessed in order to predict a better response to combo/single targeted therapies.
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http://dx.doi.org/10.1136/jclinpath-2020-207089DOI Listing
November 2020

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

Front Immunol 2020 3;11:2156. Epub 2020 Sep 3.

Department of Immunology, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy.

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.
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http://dx.doi.org/10.3389/fimmu.2020.02156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494755PMC
September 2020

Concurrent Targeting of Potential Cancer Stem Cells Regulating Pathways Sensitizes Lung Adenocarcinoma to Standard Chemotherapy.

Mol Cancer Ther 2020 10 26;19(10):2175-2185. Epub 2020 Aug 26.

Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Cancer stem cells (CSC) are highly resistant to conventional chemotherapeutic drugs. YAP1 and STAT3 are the two transcription factors that facilitate the therapeutic resistance and expansion of CSCs. The objective of this study was to understand the cross-talk between YAP1 and STAT3 activities and to determine the therapeutic efficacy of targeting dual CSC-regulating pathways (YAP1 and STAT3) combined with chemotherapy in lung adenocarcinoma. Here, we showed that YAP1 contributes to CSC regulation and enhances tumor formation while suppressing apoptosis. Mechanistically, YAP1 promotes phosphorylation of STAT3 by upregulating IL6. In lung adenocarcinoma clinical specimens, expression correlated with that of ( < 0.01). More importantly, YAP1 and phosphorylated STAT3 (pSTAT3) protein expressions were significantly correlated ( < 0.0001) in primary lung adenocarcinoma as determined by IHC. Immunoblotting of 13 lung adenocarcinoma patient-derived xenografts (PDX) showed that all YAP1-expressing PDXs also exhibited pSTAT3. Additional investigations revealed that chemotherapy resistance and malignant stemness were influenced by upregulating NANOG, OCT4, and SOX2, and the expression of these targets significantly attenuated by genetically and pharmacologically hindering the activities of YAP1 and STAT3 and Therapeutically, the dual inhibition of YAP1 and STAT3 elicits a long-lasting therapeutic response by limiting CSC expansion following chemotherapy in cell line xenograft and PDX models of lung adenocarcinoma. Collectively, these findings provide a conceptual framework to target the YAP1 and STAT3 pathways concurrently with systemic chemotherapy to improve the clinical management of lung adenocarcinoma, based on evidence that these two pathways expand CSC populations that mediate resistance to chemotherapy.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541748PMC
October 2020

Prevalence of PD-L1 expression in head and neck squamous precancerous lesions: a systematic review and meta-analysis.

Head Neck 2020 10 22;42(10):3018-3030. Epub 2020 Jun 22.

Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.

Background: Studies concerning programmed death-ligand 1 (PD-L1) expression in precancerous lesions of head and neck (HN) region have shown variable results.

Methods: We systematically reviewed the published evidence on PD-L1 expression in HN precancerous lesions.

Results: Of 1058 original articles, 14 were included in systematic review and 9 in meta-analysis. The pooled estimate of PD-L1 expression was 48.25% (confidence interval [CI] 21.07-75.98, I 98%, tau2 0.18). PD-L1 expression appeared to be more frequent in precancerous lesions than in normal mucosa (risk ratio [RR] 1.65, CI 0.65-4.03, I 91%, tau2 0.82) and less frequent than in invasive squamous cell carcinoma (RR 0.68, CI 0.43-1.08, I 91%, tau2 0.22).

Conclusions: PD-L1 expression could reflect a point of balance between host immune response and cancer escape ability. High heterogeneity and moderate quality suggest that further studies with larger sample size and more rigorous case selection will allow more precise assessment of PD-L1 expression in HN precancerous lesions.
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http://dx.doi.org/10.1002/hed.26339DOI Listing
October 2020

Glucocorticoids and the cytokines IL-12, IL-15, and IL-18 present in the tumor microenvironment induce PD-1 expression on human natural killer cells.

J Allergy Clin Immunol 2021 Jan 14;147(1):349-360. Epub 2020 May 14.

Department of Immunology, Istituto di Ricovero e Cura a Carattere Scientifico Bambino Gesù Children's Hospital, Rome, Italy. Electronic address:

Background: Programmed cell death protein 1 (PD-1)-immune checkpoint blockade has provided significant clinical efficacy across various types of cancer by unleashing both T and natural killer (NK) cell-mediated antitumor responses. However, resistance to immunotherapy occurs for many patients, rendering the identification of the mechanisms that control PD-1 expression extremely important to increase the response to the therapy.

Objective: We sought to identify the stimuli and the molecular mechanisms that induce the de novo PD-1 expression on human NK cells in the tumor setting.

Methods: NK cells freshly isolated from peripheral blood of healthy donors were stimulated with different combinations of molecules, and PD-1 expression was studied at the mRNA and protein levels. Moreover, ex vivo analysis of tumor microenvironment and NK cell phenotype was performed.

Results: Glucocorticoids are indispensable for PD-1 induction on human NK cells, in cooperation with a combination of cytokines that are abundant at the tumor site. Mechanistically, glucocorticoids together with IL-12, IL-15, and IL-18 not only upregulate PDCD1 transcription, but also activate a previously unrecognized transcriptional program leading to enhanced mRNA translation and resulting in an increased PD-1 amount in NK cells.

Conclusions: These results provide evidence of a novel immune suppressive mechanism of glucocorticoids involving the transcriptional and translational control of an important immune checkpoint.
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http://dx.doi.org/10.1016/j.jaci.2020.04.044DOI Listing
January 2021

Characterization of Human NK Cell-Derived Exosomes: Role of DNAM1 Receptor In Exosome-Mediated Cytotoxicity Against Tumor.

Cancers (Basel) 2020 03 12;12(3). Epub 2020 Mar 12.

Immunology Research Area, IRCCS Bambino Gesù Pediatric Hospital, 00146 Rome, Italy.

Despite the pivotal role of natural killer (NK) cells in defenses against tumors, their exploitation in cancer treatment is still limited due to their reduced ability to reaching tumor sites and the inhibitory effects of tumor microenvironment (TME) on their function. In this study, we have characterized the exosomes from IL2- or IL15-cultured human NK cells. Both cytokines induced comparable amounts of exosomes with similar cargo composition. Analysis of molecules contained within or exposed at the exosome surface, allowed the identification of molecules playing important roles in the NK cell function including IFN-γ, Lymphocyte Function-Associated Antigen (LFA-1), DNAX Accessory Molecule-1 (DNAM1) and Programmed Cell Death Protein (PD-1). Importantly, we show that DNAM1 is involved in exosome-mediated cytotoxicity as revealed by experiments using blocking antibodies to DNAM1 or DNAM1 ligands. In addition, antibody-mediated inhibition of exosome cytotoxicity results in a delay in target cell apoptosis. We also provide evidence that NK-exosomes may exert their cytolytic activity after short time interval and even at low concentrations. Regarding their possible use in immunotherapy, NK exosomes, detectable in peripheral blood, can diffuse into tissues and exert their cytolytic effect at tumor sites. This property offers a clue to integrate cancer treatments with NK exosomes.
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http://dx.doi.org/10.3390/cancers12030661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140072PMC
March 2020

Comprehensive analysis of 34 MiT family translocation renal cell carcinomas and review of the literature: investigating prognostic markers and therapy targets.

Pathology 2020 Apr 24;52(3):297-309. Epub 2020 Feb 24.

Department of Diagnostic and Public Health, Section of Pathology, University of Verona, Italy; Department of Pathology, Pederzoli Hospital, Peschiera del Garda, Italy. Electronic address:

Recently cabozantinib, a tyrosine kinase inhibitor with activity against VEGF, MET, AXL, and downregulating cathepsin K in vitro, has been proposed for the treatment of advanced clear and non-clear renal cell carcinomas. Since it is well known that cathepsin K is expressed in the majority of MiT family translocation renal cell carcinomas, we investigated cathepsin K, MET, AXL, and VEGF in a large series of those tumours, looking for possible predictive markers. We collected the clinicopathological features of 34 genetically confirmed MiT family translocation renal cell carcinomas [26 Xp11 and 8 t(6;11) renal cell carcinomas] and studied them using an immunohistochemical panel including PAX8, cathepsin K, HMB45, Melan-A, CD68 (PG-M1), CK7, CA9, MET, AXL and by FISH for VEGFA and MET. Cathepsin K was expressed in 14 of 26, HMB45 in 8 of 25, and Melan-A in 4 of 23 Xp11 renal cell carcinomas, whereas labelling for CK7 and CA9 was minimal. In t(6;11) renal cell carcinoma, cathepsin K and melanogenesis markers were constantly positive, whereas CK7 and CA9 were negative. None of the 34 carcinomas showed CD68 (PG-M1) and AXL expression. One aggressive Xp11 renal cell carcinoma showed increased VEGFA gene copy number (4-5 copies) with concurrent gains of TFE3 and TFEB. None of the 34 carcinomas showed MET gene amplification, whereas staining for MET was found in 7 of 8 t(6;11) and in 16 of 24 Xp11 renal cell carcinomas, and in the latter cases, when the expression was >50%, correlated with aggressiveness (p=0.0049). In Xp11 renal cell carcinomas, the aggressiveness was also correlated with larger tumour size (p=0.0008) and the presence of necrosis (p=0.027) but not nucleolar grading (p=1). Interestingly, in patients with tumours exhibiting two of three parameters (necrosis, larger tumour size and MET immunolabelling >50%) an aggressive clinical behaviour was observed in 88% of cases. In conclusion, cathepsin K, CD68 (PG-M1), CK7, CA9, and PAX8 is a useful panel for the diagnosis. Larger tumour size, the presence of necrosis and MET immunohistochemical expression correlate with aggressive behaviour in Xp11 renal cell carcinomas, especially in combination. VEGF, MET, cathepsin K but not AXL may be potential predictive markers for targeted therapy in MiT family translocation renal cell carcinomas.
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http://dx.doi.org/10.1016/j.pathol.2019.11.006DOI Listing
April 2020

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

Front Immunol 2019 29;10:3140. Epub 2020 Jan 29.

Department of Immunology, IRCCS Bambino Gesù Children's Hospital, Rome, Italy.

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.
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http://dx.doi.org/10.3389/fimmu.2019.03140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000626PMC
November 2020

Exploiting Human NK Cells in Tumor Therapy.

Front Immunol 2019 17;10:3013. Epub 2020 Jan 17.

Immunology Research Area, IRCCS Bambino Gesù Pediatric Hospital, Rome, Italy.

NK cells play an important role in the innate defenses against tumor growth and metastases. Human NK cell activation and function are regulated by an array of HLA class I-specific inhibitory receptors and activating receptors recognizing ligands expressed on tumor or virus-infected cells. NK cells have been exploited in immunotherapy of cancer, including: (1) the infusion of IL-2 or IL-15, cytokines inducing activation and proliferation of NK cells that are frequently impaired in cancer patients. Nonetheless, the significant toxicity experienced, primarily with IL-2, limited their use except for combination therapies, e.g., IL-15 with checkpoint inhibitors; (2) the adoptive immunotherapy with cytokine-induced NK cells had effect on some melanoma metastases (lung), while other localizations were not affected; (3) a remarkable evolution of adoptive cell therapy is represented by NK cells engineered with CAR-targeting tumor antigens (CAR-NK). CAR-NK cells complement CAR-T cells as they do not cause GvHD and may be obtained from unrelated donors. Accordingly, CAR-NK cells may represent an "off-the-shelf" tool, readily available for effective tumor therapy; (4) the efficacy of adoptive cell therapy in cancer is also witnessed by the αβT cell- and B cell-depleted haploidentical HSC transplantation in which the infusion of donor NK cells and γδT cells, together with HSC, sharply reduces leukemia relapses and infections; (5) a true revolution in tumor therapy is the use of mAbs targeting checkpoint inhibitors including PD-1, CTLA-4, the HLA class I-specific KIR, and NKG2A. Since PD-1 is expressed not only by tumor-associated T cells but also by NK cells, its blocking might unleash NK cells playing a crucial effector role against HLA class I-deficient tumors that are undetectable by T cells.
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http://dx.doi.org/10.3389/fimmu.2019.03013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978749PMC
December 2020

Multiplex fluorescence in situ hybridisation to detect anaplastic lymphoma kinase and ROS proto-oncogene 1 receptor tyrosine kinase rearrangements in lung cancer cytological samples.

J Clin Pathol 2020 Feb 27;73(2):96-101. Epub 2019 Sep 27.

Department of Mental and Physic Health and Preventive Medicine, Pathology Unit, University of Campania Luigi Vanvitelli, Napoli, Italy

Aims: Several predictive biomarkers of response to specific inhibitors have become mandatory for the therapeutic choice in non-small-cell lung cancer (NSCLC). In most lung cancer patients, the biological materials available to morphological and molecular diagnosis are exclusively cytological samples and minimum tumour wastage is necessary. Multiplex fluorescence in situ hybridisation (mFISH) to detect simultaneously rearrangement and rearrangement on a single slide could be useful in clinical practice to save cytological samples for further molecular analysis. In this study, we aim to validate diagnostic performance of multiplex ALK/ROS1 fluorescence in situ hybridisation (FISH) approach in lung adenocarcinoma cytological series compared with classic single break apart probes.

Methods: We collected a series of 61 lung adenocarcinoma cytological specimens enriched in tumours harbouring rearrangement and rearrangement. and status were previously assessed by classic FISH test using single break apart probes and immunohistochemistry. Study population was composed of 6 -positive, 2 -positive and 53 -wild type. All specimens were analysed by multiplex FISH assay using FlexISH ALK/ROS1 DistinguISH Probe Zytovision.

Results: The dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests on two different slides. 6 -positive and 2 -positive were confirmed through multiplex FISH test, without false-positive and false-negative results. Multiplex ALK/ROS1 FISH test results agreed with immunohistochemistry assay staining results.

Conclusion: Multiplex ALK/ROS1 FISH probe test is a useful tool to detect simultaneously rearrangement and rearrangement on a single slide in cytological specimens with a small amount of biomaterial.
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http://dx.doi.org/10.1136/jclinpath-2019-206152DOI Listing
February 2020

"Interchangeability" of PD-L1 immunohistochemistry assays: a meta-analysis of diagnostic accuracy.

Mod Pathol 2020 01 5;33(1):4-17. Epub 2019 Aug 5.

University Health Network, Toronto, ON, Canada.

Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.
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http://dx.doi.org/10.1038/s41379-019-0327-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927905PMC
January 2020

MiT Family Translocation Renal Cell Carcinoma: from the Early Descriptions to the Current Knowledge.

Cancers (Basel) 2019 Aug 3;11(8). Epub 2019 Aug 3.

Department of Diagnostic and Public Health, Section of Pathology, University of Verona, Verona 37134, Italy.

The new category of MiT family translocation renal cell carcinoma has been included into the World Health Organization (WHO) classification in 2016. The MiT family translocation renal cell carcinoma comprises Xp11 translocation renal cell carcinoma harboring gene fusions and t(6;11) renal cell carcinoma harboring gene fusion. At the beginning, they were recognized in childhood; nevertheless, it has been demonstrated that these neoplasms can occur in adults as well. In the nineties, among Xp11 renal cell carcinoma, , , and () were the first genes recognized as partners in rearrangement. Recently, many other genes have been identified, and a wide spectrum of morphologies has been described. For this reason, the diagnosis may be challenging based on the histology, and the differential diagnosis includes the most common renal cell neoplasms and pure epithelioid PEComa/epithelioid angiomyolipoma of the kidney. During the last decades, many efforts have been made to identify immunohistochemical markers to reach the right diagnosis. To date, staining for PAX8, cathepsin K, and melanogenesis markers are the most useful identifiers. However, the diagnosis requires the demonstration of the chromosomal rearrangement, and fluorescent in situ hybridization (FISH) is considered the gold standard. The outcome of Xp11 translocation renal cell carcinoma is highly variable, with some patients surviving decades with indolent disease and others dying rapidly of progressive disease. Despite most instances of t(6;11) renal cell carcinoma having an indolent clinical course, a few published cases demonstrate aggressive behavior. Recently, renal cell carcinomas with amplification have been described in connection with t(6;11) renal cell carcinoma. Those tumors appear to be associated with a more aggressive clinical course. For the aggressive cases of MiT family translocation carcinoma, the optimal therapy remains to be determined; however, new target therapies seem to be promising, and the search for predictive markers is mandatory.
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http://dx.doi.org/10.3390/cancers11081110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721505PMC
August 2019

PD-L1 Expression in De Novo Metastatic Castration-sensitive Prostate Cancer.

J Immunother 2019 09;42(7):269-273

Oncologia Medica, Fondazione Policlinico Universitario Agostino Gemelli IRCCS.

De novo metastatic castration-sensitive prostate cancer (mCSPC) is small subgroup of prostate cancer associated with poor prognosis. The aim of our study was to assess the expression of programmed death-ligand 1 (PD-L1) in tumor cells of de novo mCSPC patients. Patients referred to our institution from January 2007 to October 2017 with de novo mCSPC and available diagnostic tissue were included. We tested the PD-L1 pharmaDx qualitative immunohistochemical assay, a monoclonal rabbit anti-PD-L1, clone 28-8 intended for use in the detection of PD-L1 protein in formalin-fixed paraffin-embedded. Kaplan-Meier method was used to estimate survivals according to the expression of PD-L1. The study population included 32 de novo mCSPC patients, analyzed for PD-L1 expression using 2 cut-off values (1% and 5%) to define the positivity. Total of 46.9% of cases had tumor PD-L1 expression ≥1%, and 31.3% had expression ≥5%. No differences were found between the PD-L1 expression ≥1% and the involvement of liver, lung, and number of bone metastases, and the disease volume based on CHAARTED classification. PD-L1 tumors had higher incidence of Gleason score ≥8 compared with PD-L1 tumors (P=0.037), while the incidence of lymph node metastases was higher in PD-L1 tumors (P=0.044). No difference in the median overall survival (mOS) was observed between the PD-L1 population and the PD-L1 patients (43.8 vs. 29.6 mo; P=0.88). The tumor PD-L1 expression cannot be considered a prognostic factor for de novo mCSPC, even if its prognostic and predictive significance have to be thoroughly investigated to better define the selected group of prostate cancer patients that might benefit from checkpoint blockade immunotherapy.
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http://dx.doi.org/10.1097/CJI.0000000000000287DOI Listing
September 2019

Killer Ig-Like Receptors (KIRs): Their Role in NK Cell Modulation and Developments Leading to Their Clinical Exploitation.

Front Immunol 2019 28;10:1179. Epub 2019 May 28.

Laboratory of Tumor Immunology, Department of Immunology, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy.

Natural killer (NK) cells contribute to the first line of defense against viruses and to the control of tumor growth and metastasis spread. The discovery of HLA class I specific inhibitory receptors, primarily of killer Ig-like receptors (KIRs), and of activating receptors has been fundamental to unravel NK cell function and the molecular mechanisms of tumor cell killing. Stemmed from the seminal discoveries in early '90s, in which Alessandro Moretta was the major actor, an extraordinary amount of research on KIR specificity, genetics, polymorphism, and repertoire has followed. These basic notions on NK cells and their receptors have been successfully translated to clinical applications, primarily to the haploidentical hematopoietic stem cell transplantation to cure otherwise fatal leukemia in patients with no HLA compatible donors. The finding that NK cells may express the PD-1 inhibitory checkpoint, particularly in cancer patients, may allow understanding how anti-PD-1 therapy could function also in case of HLA class I tumors, usually susceptible to NK-mediated killing. This, together with the synergy of therapeutic anti-checkpoint monoclonal antibodies, including those directed against NKG2A or KIRs, emerging in recent or ongoing studies, opened new solid perspectives in cancer therapy.
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http://dx.doi.org/10.3389/fimmu.2019.01179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558367PMC
August 2020

PD-L1 expression in non-small cell lung cancer: evaluation of the diagnostic accuracy of a laboratory-developed test using clone E1L3N in comparison with 22C3 and SP263 assays.

Hum Pathol 2019 08 21;90:54-59. Epub 2019 May 21.

Department of Pathology, IRCCS Sacro Cuore Don Calabria, 37024 Negrar (VR), Italy.

Different studies have evaluated the comparability of various immunohistochemical assays for PD-L1 expression evaluation, with contrasting results. Besides the important issues related to analytic performance and comparability of validated assays, not all platforms are available in all laboratories; moreover, standardized assays are very expensive, and funding for PD-L1 testing is hard to obtain, especially in the research setting. One of the most widely used and inexpensive PD-L1 clones is E1L3N (Cell Signaling Technology, Danvers, MA), which is labeled for research use only. In this work, we wanted to further study and validate in a larger cohort the analytical performance of E1L3N clone on Ventana platform (Ventana Medical Systems, Tucson, AZ) and its comparability with assays SP263 and 22C3 run onto their dedicated platforms. Serial sections of tissue microarrays built from 165 cases of resected lung cancer were stained for E1L3N onto Ventana platform following a previously reported protocol and for 22C3 and SP263 assays onto their respective platforms following manufacturer's instructions. Overall, we found very high concordance when comparing E1L3N with SP263 at both 1% and 50% cutoffs. Lower concordance was found between E1L3N and 22C3 at both cutoffs; however, 100% sensitivity was found for E1L3N compared with both SP263 and 22C3 at 50% cutoff. Given the 100% sensitivity at 50% cutoff demonstrated by E1L3N in comparison with both SP263 and 22C3 and therefore the lack of false-negative cases, we propose an algorithm for PD-L1 testing in NSCLC when considering pembrolizumab as first-line therapy.
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http://dx.doi.org/10.1016/j.humpath.2019.05.003DOI Listing
August 2019

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

Front Immunol 2019 26;10:910. Epub 2019 Apr 26.

Department of Immunology, IRCSS Bambino Gesù Children's Hospital, Rome, Italy.

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 CD4T 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.
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http://dx.doi.org/10.3389/fimmu.2019.00910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498986PMC
September 2020

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

Int J Cancer 2019 09 26;145(6):1660-1668. Epub 2019 Mar 26.

Department of Immunology, IRCCS Bambino Gesù Children's Hospital, Rome, Italy.

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.
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http://dx.doi.org/10.1002/ijc.32262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767381PMC
September 2019

Clinical implication of the mammalian target of rapamycin pathway in upper tract urothelial carcinoma with negative GATA binding protein 3 expression.

Int J Urol 2019 06 10;26(6):678-679. Epub 2019 Mar 10.

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

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http://dx.doi.org/10.1111/iju.13940DOI Listing
June 2019

Natural killer cells: From surface receptors to the cure of high-risk leukemia (Ceppellini Lecture).

HLA 2019 04;93(4):185-194

Department of Immunology, IRCCS Ospedale Pediatrico Bambino Gesù, Rome, Italy.

Natural killer (NK) cells are innate immune effector cells involved in the first line of defense against viral infections and malignancies. In the last three decades, the identification of HLA class I-specific inhibitory killer immunoglobulin-like receptors (KIR) and of the main activating receptors has strongly improved our understanding of the mechanisms regulating NK cell functions. The increased knowledge on how NK cells discriminate healthy cells from damaged cells has made it possible to transfer basic research notions to clinical applications. Of particular relevance is the strong NK-mediated anti-leukemia effect in haploidentical hematopoietic stem cell transplantation to cure high-risk leukemia.
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http://dx.doi.org/10.1111/tan.13509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767140PMC
April 2019

Human NK cells: surface receptors, inhibitory checkpoints, and translational applications.

Cell Mol Immunol 2019 05 18;16(5):430-441. Epub 2019 Feb 18.

Department of Immunology, IRCCS Bambino Gesù Children's Hospital, Rome, Italy.

NK cells play important roles in innate defenses against viruses and in the control of tumor growth and metastasis. The regulation/induction of NK cell function is mediated by an array of activating or inhibitory surface receptors. In humans, major activating receptors involved in target cell killing are the natural cytotoxicity receptors (NCRs) and NKG2D. Activating receptors recognize ligands that are overexpressed or expressed de novo upon cell stress, viral infection, or tumor transformation. The HLA-class I-specific inhibitory receptors, including KIRs recognizing HLA-class I allotypic determinants and CD94/NKG2A recognizing the class-Ib HLA-E, constitute a fail-safe mechanism to avoid unwanted NK-mediated damage to healthy cells. Other receptors such as PD-1, primarily expressed by activated T lymphocytes, are important inhibitory checkpoints of immune responses that ensure T-cell tolerance. PD-1 also may be expressed by NK cells in cancer patients. Since PD-1 ligand (PD-L1) may be expressed by different tumors, PD-1/PD-L1 interactions inactivate both T and NK cells. Thus, the reliable evaluation of PD-L1 expression in tumors has become a major issue to select patients who may benefit from therapy with mAbs disrupting PD-1/PD-L1 interactions. Recently, NKG2A was revealed to be an important checkpoint controlling both NK and T-cell activation. Since most tumors express HLA-E, mAbs targeting NKG2A has been used alone or in combination with other therapeutic mAbs targeting PD-1 or tumor antigens (e.g., EGFR), with encouraging results. The translational value of NK cells and their receptors is evidenced by the extraordinary therapeutic success of haploidentical HSCT to cure otherwise fatal high-risk leukemias.
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http://dx.doi.org/10.1038/s41423-019-0206-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474200PMC
May 2019

PD-1 is expressed by and regulates human group 3 innate lymphoid cells in human decidua.

Mucosal Immunol 2019 05 12;12(3):624-631. Epub 2019 Feb 12.

Department of Experimental Medicine (DIMES) and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Genoa, Italy.

Group 3 innate lymphoid cells (ILC3) have been detected in both murine and human decidual tissues where they are thought to play a relevant role in the induction and maintenance of pregnancy. However, limited information exists on the molecular mechanisms that regulate these cells, including immune checkpoints. Here, we show that ILC3 express the inhibitory checkpoints programmed cell death (PD-1) and T cell immunoglobulin and mucin domain containing protein 3 (TIM-3) during the first trimester of pregnancy and that these receptors could regulate production of cytokines, including IL-22, IL-8, and TNF-α, induced by IL-23. We also show that the intermediate extravillous trophoblast (iEVT) expresses high levels of the PD-1-ligand PD-L1, suggesting that PD-1/PD-L1 interaction may regulate ILC3 function at the feto-maternal interface. Our present data provide the first evidence that human decidual ILC3 express a functional PD-1. It is possible that an altered expression or function of PD-1 may break the immune-tolerance resulting in pregnancy failure.
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http://dx.doi.org/10.1038/s41385-019-0141-9DOI Listing
May 2019

PD-1 in human NK cells: evidence of cytoplasmic mRNA and protein expression.

Oncoimmunology 2019;8(3):1557030. Epub 2018 Dec 25.

Department of Immunology, IRCSS Bambino Gesù Children's Hospital, Rome, Italy.

Under physiological conditions, PD-1/PD-L1 interactions regulate unwanted over-reactions of immune cells and contribute to maintain peripheral tolerance. However, in tumor microenvironment, this interaction may greatly compromise the immune-mediated anti-tumor activity. PD-1 NK cells have been detected in high percentage in peripheral blood and ascitic fluid of ovarian carcinoma patients. To acquire information on PD-1 expression and physiology in human NK cells, we analyzed whether PD-1 mRNA and protein are present in resting, surface PD-1, NK cells from healthy donors. Both different splicing isoforms of PD-1 mRNA and a cytoplasmic pool of PD-1 protein were detected. Similar results were obtained also from both in vitro-activated and tumor-associated NK cells. PD-1 mRNA and protein were higher in CD56 than in CD56 NK cells. Confocal microscopy analyses revealed that PD-1 protein is present in virtually all NK cells analyzed. The present findings are compatible with a rapid surface expression of PD-1 in NK cells in response to appropriate, still undefined, stimuli.
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http://dx.doi.org/10.1080/2162402X.2018.1557030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350684PMC
December 2018

Expression of programmed cell death ligand 1 in non-small cell lung cancer: Comparison between cytologic smears, core biopsies, and whole sections using the SP263 assay.

Cancer Cytopathol 2019 02 30;127(1):52-61. Epub 2018 Nov 30.

Department of Pathology, Sacro Cuore Don Calabria Hospital, Negrar, Italy.

Background: Evaluation of programmed cell death ligand 1 (PD-L1) expression can be made on both resection specimens and diagnostic biopsies; however, more than 30% of patients with advanced non-small cell lung cancer (NSCLC) do not have adequate histologic material to perform PD-L1 assays and require additional biopsies. In addition, in our practice, more than 16% of cases have cytological smears as the only available material. Our aim was to validate the PD-L1 immunocytochemistry assay on cytological smears and compare its accuracy with the results obtained from tissue cores and whole tumor sections using the clinically relevant cutoff of 50%.

Method: We compared the PD-L1 staining results of cytological smears to those from tissue cores or whole sections in 50 and 53 NSCLC cases, respectively, using the SP263 assay after scanning hematoxylin and eosin slides.

Results: We found an overall agreement of 90.6% between cytological smears and whole sections; specifically, we found absolute concordance between smears with PD-L1 expressed in <10% and ≥50% of cells and whole sections with PD-L1 expressed in <50% and ≥50% of cells, respectively. In addition, slightly lower diagnostic accuracy was found for the cytological smears in comparison with the tissue cores, but the difference was not statistically significant. We found excellent intraobserver and good interobserver agreement in the evaluation of PD-L1 on smears.

Conclusion: Immunocytochemistry on cytological smears is a reliable method for determination of PD-L1 at the 50% cutoff when positive cells are <10% or ≥50%; for cases showing PD-L1 expression in 10% to 49% of cells, additional tissue sampling may be necessary.
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http://dx.doi.org/10.1002/cncy.22083DOI Listing
February 2019