ISSUE HIGHLIGHTS - July 2020.

Abstract

Francesco Lanza [Color figure can be viewed at wileyonlinelibrary.com] It is a great pleasure to make some introductory remarks to this issue of Clinical Cytometry which includes one paper focusing on chimeric antigen receptor (CAR)-T, two manuscripts dealing with acute lymphoblastic leukemia (ALL), a study addressing the clinical significance of CD20 antigen in primary nasal peripheral T-cell lymphoma, and two further articles highlighting new markers expressed by B-lymphocytes during the B-lineage differentiation pathway, or the significance of HER2 expression by flow cytometry (FC) in urothelial carcinoma cells. With reference to the CAR-T study, it might be emphasized that CAR-T cells are a novel class of anti-cancer therapy in which autologous or allogeneic T cells are engineered to express a CAR targeting a membrane antigen (1-3). CAR-T cells are shaping a new dawn for haematologic disease treatment. Two FDA/EMA-approved autologous products, namely Tisagenlecleucel and Axicabtagene ciloleucel, have recently entered clinical practice worldwide (4, 5). In Europe, Tisagenlecleucel is approved for the treatment of refractory/relapsed B-cell ALL in children and young adults as well as relapsed/refractory diffuse large B-cell lymphoma, while Axicabtageneciloleucel is approved for the treatment of relapsed/refractory high-grade B-cell lymphoma and primary mediastinal B-cell lymphoma (2). The need to efficiently identify and monitoring in vivo CAR T-cell products with a reliable, reproducible and efficient technology would be of considerable importance and FC could be the ideal tool. In this issue of Cytometry, Maryamchik and colleagues explore the potential role of FC across all stages of CAR-T cells, from generation process to in vivo monitoring (1). Several reports have demonstrated that autologous CAR-T cells efficacy depends, at least partially, by the phenotypic characteristics of the cell product (6, 7). In fact, CAR-T cells derived from disparate T-cell subsets, exhibit peculiar proliferation and in vivo persistence properties (8). The utilization of specific T-cell types with high potential for longevity could allow long-term persistence of infused CAR-T cells (9). FC could help to identify specific T-cell populations within the apheresis product, that have such characteristics, in particular, lesser differentiated elements in T-cell ontogeny, like stem cell and central memory phenotypes (10, 11). Moreover, the selection of cell products with ideal metabolic fitness through FC identification of activation, exhaustion and degranulation markers on T-cells is equally important, in order to achieve high rates of responses and secure their durability. The correct recognition of CAR-T cells throughout FC assay is not an easy task. Fluorochrome-tagged antibodies against the extracellular portion of the CAR, anti-idiotype monoclonal antibodies for the antigen-binding domain, biotinylated protein L-based assays and soluble antigen-based detection methods are the most relevant methods employed; each one has its own benefits and pitfalls (3, 12-14). Labs at several Institutions are establishing and validating assays in order to have sensitive information for clinicians around the world. The utilization of high throughput FC protocols allow the synchronous identification of multiple CAR-T cell features from degree of activation (CD25) to exhaustion (CD44, LY6C, KLRG1, LAG3) and degranulation (CD107a), as well as the composition of the surrounding cytokines milieu (IFN-γ, TNF-α, IL-5). Tumor antigen expression represents arguably the single most important factor in ratifying CAR-T cell therapy success or failure (14). In order to deliver an effective and robust effect against the tumor, the targeted antigen must be expressed by tumor cell surface with adequate intensity and durability. Lower target antigen site densities decrease CAR-T cell efficacy, facilitating tumor immunologic escape (15). The ability of modulating antigen expression on malignant cells by exogenous agents, as well as combinatorial antigen targeting may be useful tools in order to reshape antitumor activity and overcome immunologic escape (16-18). FC could help physicians to understand how T-cell phenotype impacts on the efficacy and durability of response after CAR-T cell infusion. The field is a rapidly expanding area of translational research and therefore it could be of great interest to delineate some of these new directions for designing and implementing CAR-T related FC assays in the clinical and investigational settings (4, 19). In the paper by Gudapati et al., a novel immunological marker, neuropillin-1-CD304, was tested for the detection of minimal residual disease (MRD) in B-cell ALL (20). Recent data have clearly demonstrated that MRD assessment represents a predictive and independent risk factor and a useful tool in the clinical management of ALL (21, 22). Two methods coexist for studying MRD: polychromatic FC analysis of leukemia associated immunophenotypes (LAIP) and molecular biology studies such as polymerase chain reaction (PCR) amplification of antigen receptor rearrangements (21, 22). Based on these studies ALL status has emerged as the single, most powerful risk stratification criteria in pediatric BALL aiding in therapeutic decisions (21-23). In adults, the role played by MRD assessment is still an object of debate. Over the last few years, several authors have tested several new immunophenotypic markers such as CD123, CD73, CD146, and CD86 with the main aim of improving the applicability and accuracy of MRD by next generation flow in B-precursor cell-ALL (24-30). CD304/neuropilin-1 (NRP-1), is a non-tyrosine kinase transmembrane C-type lectin expressed on plasmacytoid dendritic cells, endothelial cells, and blastic plasmacytoid dendritic cells (20). In this paper, authors have provided evidence that CD304 is commonly expressed in leukemic-blasts of B-cell ALL and may play a role in distinguishing residual-disease from hematogones and is a valuable addition for enhancing the sensitivity and applicability of MRD assay in this setting. Interestingly, in almost half of B-precursors cells ALL patients, CD304 was usable for MRD detection studies. However, further investigations are needed to confirm the usefulness of this marker as well as to assess its reproducibility and inter-laboratory standardization; these characteristics are essential for evaluating the applicability of CD304 testing in routine laboratory. In the manuscript by Park et al., the expression profile of immune checkpoint receptors such as Programmed Cell Death Protein-1 (PD-1) on T cell subsets and ligands on B-lymphoblasts from ALL patients was assessed by multiparametric FC analysis (31). Results showed that PD-1 expression on T-cell subsets was found to be increased at diagnosis, and to a greater extent, at relapse, suggesting the potential usefulness of PD-1 blockade in the treatment of B-ALL, particularly at relapse. Taking into account the fact that 30–60% of adult B-ALL patients relapse, and only 25–40% achieve disease-free survival of three years or more, new treatment strategies are in high demand (32). Furthermore, chemotherapy regimens for refractory/relapsed B-ALL are associated with complete remission (CR) rates ranging from 31% to 44%. Novel immune-targeted therapies, such as blinatumomab and inotuzumab provide potential means of circumventing chemo-refractory B-ALL cells through novel mechanisms of action (33). Given the role of T-cell mediated immunity in the context allogeneic hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusions (DLI), strategies that enhance immune activation and improve immune dysfunction such as the occurrence of acute and chronic graft versus host disease (GVHD) represent potential therapeutic ways to ameliorate clinical outcomes in acute leukemia. Preliminary data suggest that immune dysfunction is a major contributor to acute leukemia progression and relapse. Increased expression of immune checkpoints such as PD-1 may cause acute myeloid leukemia (AML) immune evasion and eventually disease relapse. More recently various immune checkpoints which regulates immune activation and/or inhibition of immune effector function mediated by T cells, have been reported to be an important target of several checkpoint inhibitor antibody drugs for the treatment of various hematological malignancies. Since activation of co-inhibitory receptors inhibits function of effector T cells, the blockade of these receptors by antibodies such as PD-1 antibodies may increase T cell function by “jumping the immune checkpoint” and may enhance antitumor response induced by effector T cells (31). In the last few years, several clinical trials which used single-agent or combination of immune checkpoint inhibitors have demonstrated the clinical benefits of these therapeutic regimens in patients with Hodgkin and non-Hodgkin lymphomas, and in AML patients who underwent allogeneic HSCT. Immune checkpoint inhibitors in combination with chemotherapy and hypomethylating agents have been recently tested in AML, and, in a lesser extent, in B-cell ALL. However, further clinical trials are necessary to better elucidate the role played by anti-PD-1 antibodies in the management of acute leukemias. The biological and clinical significance of CD20 antigen expressed in primary nasal peripheral T-cell lymphoma (TCL) was explored by Li Li et al. (34). This is a very rare disease entity that is associated with the co-expression of a range of T cell lineage markers, such as, CD2, CD3, CD5, CD7, and/or CD20. CD43 and CD45RO are often positive. CD30 is present in 30% of the cases. It is an extranodal lymphoma characterized by vascular damage and destruction, prominent necrosis, cytotoxic phenotype, abnormality of chromosome 6 and frequent association with EBV (35-37). CD56, although a highly useful marker for natural killer (NK) cells, may be present in this entity. NK cells, the most important effectors of the innate lymphoid cells, play a key role in tumor immune-surveillance, defense against viruses and in innate immune responses. NK cell activation is mediated by several activating receptors and co-receptors able to recognize ligands on virus-infected or tumor cells (38-40). The functional implication related to CD56 expression in primary nasal peripheral TCL needs to be investigated in future studies. Furthermore, the biological and clinical significance of CD20 antigen expressed in TCL is still an object of debate. CD20 antigen, a transmembrane protein that functions as a calcium channel, has long been designated a pan-B antigen, and presents on all B cells, except the early pro-B cells and terminal plasma cells. However, expression of CD20 by TCL has also been reported, especially in peripheral TCL (PTCL) not otherwise specified (NOS). Authors reported a case of lymphoma cells expressing CD2, CD3, CD5, CD7, CD8 and CD20 antigens in PB, BM and lymph node samples. The overall findings favor PTCL-NOS, despite the expression of CD20. In previous publications CD20 positivity was detected in 1% of PTCL-NOS. The paper by Nini et al. has investigated the role of novel biomarkers in urothelial carcinoma (UC) using the circulating tumor cells (CTC) assay (41). Since the detection of CTC by techniques based on EpCAM appeared to be suboptimal in UC, authors have explored the utility of HER2 for CTC, since it is thought to be broadly expressed in UC (42). To reach this goal HER2 and EpCAM expression was analyzed in 18 UC cell lines (UCCs) by qRT-PCR, western blot and fluorescence-activated cell scanning (FACS). This study showed that HER2 is expressed in UCC lines independent of the cell lines’ phenotypes (epithelial vs. mesenchymal) but varies in intensity. Unfortunately, the relatively weak intensity of HER2+ cells may represent the most likely factor impairing detection by CellSearch analysis. Interestingly, EpCAM/HER2-double positive CTCs were detected in only 1 of the examined patients. In contrast, EpCAM expression was essentially restricted to UCCs with epithelioid phenotypes. Because of its widespread expression, particularly in patients with high risk of metastasis, detection of HER2 could improve identification of UC CTCs, which is why combined detection using antibodies for EpCAM and HER2 may be beneficial. The clinical implication related to the addition of an antibody directed against HER2 in bladder cancer should be evaluated in well-designed clinical trials. In the paper by Hasegawa et al., the expression profile of DNAM-1 on B-lineage cells has been investigated (43). Previous reports revealed that a considerable number of white blood cells including peripheral blood-derived B cells in the conjugated to platelet. Thus, the proportion of DNAM-1+ B-lineage cells determined by FC analysis in the previous reports might be overestimated (44). In the present study, authors have shown that DNAM-1 expression was detected on subpopulations of plasmoblasts, plasma cells and memory B cells, and upregulated by stimulation with some chemical compounds (43). Regarding the function of this marker, since IL-10 is known to enhance survival and differentiation of B cells and antibody production, and results from this analysis indicated that DNAM-1 on CD19+ B-lineage cells was clearly involved in production of IL-10, it was hypothesized that DNAM-1 could be involved in B-lineage cell-mediated adaptive immune responses (43-45).