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Hypereosinophilic syndromes (HES) represent a group of rare dis-immune conditions characterized by blood hyper-eosinophilia and eosinophilic related burden. Especially the idiopathic subtype (I-HES) is particularly difficult to diagnose because of its heterogeneous clinical presentation, the lack of specific findings on physical exam, lab tools, and imaging informative enough to unequivocally confirm the diagnosis and the overlap with other entities, including eosinophilic organ-diseases or systemic dis-immune conditions other than I-HES (from atopy to eosinophilic granulomatosis with polyangiitis [EGPA], the last often extremely difficult to distinguish from HES). Taken together, all the features mentioned above account for an extremely difficult early recognition HES and on-time referral to a specialized centre. The referral itself is challenging due to a not univocal specialist identification, because of the variability of physicians managing HES in different settings (including allergist/clinical immunologist, haematologist, internal medicine doctors, pulmonologist, rheumatologist). Furthermore, the approach in terms of personalized treatment identification and follow-up plan (timing, organ assessment), is poorly standardized. Further translational and clinical research is needed to address the mentioned unmet needs, but on practical grounds increasing the overall clinicians’ awareness on HES and implementing healthcare pathways for HES patients represent a roadmap that every clinician might try to realize in his specific setting.The present review aims at providing an overview about the current challenges and unmet needs in the practical approach to HES and rare hypereosinophilic allergo-immunological diseases, including a proposal for an innovative multidisciplinary organizational model.

Abstract Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

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Hypereosinophilic syndrome (HES) encompasses a heterogeneous and complex group of different subtypes within the wider group of hypereosinophilic disorders. Despite increasing research interest, several unmet needs in terms of disease identification, pathobiology, phenotyping, and personalized treatment remain to be addressed. Also, the prospective burden of non-malignant HES and, more in general, HE disorders is currently unknown. On a practical note, shortening the diagnostic delay and the time to an appropriate treatment approach probably represents the most urgent issue, even in light of the great impact of HES on the quality of life of affected patients. The present document represents the first action that the Italian Society of Allergy, Asthma, and Clinical Immunology (SIAAIC) has finalized within a wider project aiming to establish a collaborative national network on HES (InHES—Italian Network on HES) for patients and physicians. The first step of the project could not but focus on defining a common language as well as sharing with all of the medical community an update on the most recent advances in the field. In fact, the existing literature has been carefully reviewed in order to critically integrate the different views on the topic and derive practical recommendations on disease identification and treatment approaches.

Abstract We analyzed BCR::ABL1 expression at stop and in the first month after discontinuation in 168 chronic myeloid leukemia patients who stopped imatinib or 2nd generation tyrosine kinase inhibitors (2G‐TKIs) while in sustained deep molecular response. Patients were divided among those who maintained response (group 1, n = 123) and those who lost major molecular response (group 2, n = 45). Mean BCR::ABL1 RNA levels 1 month after discontinuation were higher in group 2 than in group 1 (p = 0.0005) and the difference was more evident 2 months after stop (p

Membranous glomerulonephritis (MGN) is a rare extra-hematological autoimmune complication of chronic lymphocytic leukemia (CLL), clinically characterized by nephrotic-range proteinuria and, less frequently, renal failure. Because of the rarity of this condition, there is no standardized treatment. Chlorambucil and fludarabine-based regimens, possibly combined with rituximab, have been historically the most frequent therapeutic approaches, with renal response obtained in about two-third of the patients. However, responses are often transient and partial. Here we describe the first patient with rituximab-refractory, CLL-related MGN successfully treated with the Bcl-2 antagonist venetoclax. Nephrotic syndrome resolved as soon as three months after venetoclax initiation, with no unexpected toxicities. At the last follow-up, 17 months after venetoclax start, renal response persists, with proteinuria below 0.5 g/24 hours. This case suggests that targeted agents, particularly Bcl-2 antagonists, might be suitable options for patients with renal autoimmune disorders arising in the context of CLL.

Most patients with myelofibrosis (MF) discontinue ruxolitinib (JAK1/JAK2 inhibitor) in the first 5 years of therapy due to therapy failure. As the therapeutic possibilities of MF are expanding, it is critical to identify patients predisposed to early ruxolitinib monotherapy failure and worse outcomes. We investigated predictors of early ruxolitinib discontinuation and death on therapy in 889 patients included in the “RUX-MF” retrospective study. Overall, 172 patients were alive on ruxolitinib after ≥5 years (long-term ruxolitinib, LTR), 115 patients were alive but off ruxolitinib after ≥5 yrs (short-term RUX, STR), and 123 patients died while on ruxolitinib after p = 0.08). Overall survival (OS) was significantly longer in LTR pts (p = 0.002). In multivariate analysis, PLT < 100 × 109/L, Hb < 10 g/dL, primary MF, absence of spleen response at 3 months and ruxolitinib starting dose

Classical Hodgkin lymphoma (cHL) is a unique neoplastic ecosystem characterized by a heterogeneous immune infiltrate surrounding the rare malignant Hodgkin Reed-Sternberg cells. Though less abundant than T-cells, tumor-infiltrating macrophages play a pivotal role in supporting HRS survival through cell-to-cell and paracrine interactions. Traditional immunohistochemistry based upon the M1-M2 dichotomy yielded controversial results about the composition, functional role and prognostic impact of macrophages in cHL. More recent studies exploiting single-cell technologies and image analyses have highlighted the heterogeneity and the peculiar spatial arrangement of the macrophagic infiltrate, with the most immunosuppressive subpopulations lying in close proximity of HRS cells and the most tumor-hostile subsets kept far away from the neoplastic niches. High-throughput analysis of peripheral blood mononuclear cells in cHL patients have also identified a novel, potentially cytotoxic, subpopulation predicting better response to PD-1 blockade. This review examines the phenotypic profile, spatial localization and clinical impact of tumor-infiltrating macrophages and circulating monocytes in cHL, providing an up-do-date portrait of these innate immune cells with possible translational applications.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) offers a survival benefit to adult patients affected by acute lymphoblastic leukemia (ALL). However, to avoid an overt disease relapse, patients with pre or post transplant persistence or occurrence of measurable residual disease (MRD) may require cellular or pharmacological interventions with eventual side effects. While the significance of multiparametric flow cytometry (MFC) in the guidance of ALL treatment in both adult and pediatric patients is undebated, fewer data are available regarding the impact of MRD monitoring, as assessed by MFC analysis, in the allo-HSCT settings. Aim of this article is to summarize and discuss currently available information on the role of MFC detection of MRD in adult ALL patients undergoing allo-HSCT. The significance of MFC-based MRD according to sensitivity level, timing, and in relation to molecular techniques of MRD and chimerism assessment will be also discussed.

Background & Aims: Type 1 autoimmune pancreatitis (AIP) is an IgG4-related disease whose diagnosis is challenging. The aim of this study was to investigate the diagnostic value of circulating total and IgG4+ plasmablasts in differentiating this condition from the other main pancreatic diseases. Methods: Patients with type 1 AIP (n = 19) were prospectively enrolled in a tertiary center together with patients suffering from type 2 or not otherwise specified (NOS) AIP (n = 10), pancreatic adenocarcinoma (n = 17), chronic pancreatitis (n = 20), and intraductal papillary mucinous neoplasia or chronic asymptomatic pancreatic hyperenzymemia (n = 21) as control groups. Flow cytometry was used to measure the total plasmablast and IgG4+ plasmablast number by gating peripheral blood CD45+CD19+CD38hiCD20-CD24-CD27+ and CD45+CD19+CD38hiCD20-CD24-CD27+IgG4+ cells, respectively. In patients with AIP, these cell populations were also evaluated after 1 month of therapy, after 2–4 months from the end of treatment, and after 1 year from the enrollment. The study was approved by the local ethics committee (protocol number: 59133, 30/11/2017). Results: Total plasmablast quantification was capable of discriminating type 1 AIP from all the other pancreatic disorders with a sensitivity of 47% and a specificity of 81%, according to a cutoff of 4500 cells/mL (AUC = 0.738), whereas IgG4+ plasmablast count distinguished type 1 AIP from all the other pancreatic disorders with a sensitivity of 80% and a specificity of 97% when applying a cutoff of 210 IgG4+ cells/mL (AUC = 0.879). The basal IgG4+ plasmablast number was significantly higher (P = .0001) in type 1 AIP than in type 2/NOS AIP, decreased after steroid therapy, and increased at disease relapse. Conclusion: IgG4+ plasmablast count represents a potentially useful biomarker to differentiate type 1 from type 2/NOS AIP and from other pancreatic diseases.

In polycythemia vera (PV), the prognostic relevance of an ELN-defined complete response (CR) to hydroxyurea (HU), the predictors of response, and patients’ triggers for switching to ruxolitinib are uncertain. In a real-world analysis, we evaluated the predictors of response, their impact on the clinical outcomes of CR to HU, and the correlations between partial or no response (PR/NR) and a patient switching to ruxolitinib. Among 563 PV patients receiving HU for ≥12 months, 166 (29.5%) achieved CR, 264 achieved PR, and 133 achieved NR. In a multivariate analysis, the absence of splenomegaly (p = 0.03), pruritus (p = 0.002), and a median HU dose of ≥1 g/day (p < 0.001) remained associated with CR. Adverse events were more frequent with a median HU dose of ≥1 g/day. Overall, 283 PR/NR patients (71.3%) continued HU, and 114 switched to ruxolitinib. In the 449 patients receiving only HU, rates of thrombosis, hemorrhages, progression, and overall survival were comparable among the CR, PR, and NR groups. Many PV patients received underdosed HU, leading to lower CR and toxicity rates. In addition, many patients continued HU despite a PR/NR; however, splenomegaly and other symptoms were the main drivers of an early switch. Better HU management, standardization of the criteria for and timing of responses to HU, and adequate intervention in poor responders should be advised.

Abstract Background Patients with hematological malignancies are at an increased risk of SARS‐CoV‐2 disease (COVID‐19) and adverse outcome. However, a low mortality rate has been reported in patients with chronic myeloid leukemia (CML). Preclinical evidence suggests that tyrosine kinase inhibitors (TKIs) may have a protective role against severe COVID‐19. Methods We conducted a cross‐sectional study of 564 consecutive patients with CML who were tested for anti‐SARS‐CoV‐2 IgG/IgM antibodies at their first outpatient visit between May and early November 2020 in five hematologic centers representative of three Italian regions. Results The estimated serological prevalence of SARS‐CoV‐2 infection in patients with CML after the first pandemic wave was similar to that in the general population (about 2%), both at national and regional levels. CML patients with positive anti‐SARS‐CoV‐2 serology were more frequently male (p = 0.027) and active workers (p = 0.012), while there was no significant association with TKI treatment type. Only 3 out of 11 IgG‐positive patients had previously received a molecular diagnosis of COVID‐19, while the remainders were asymptomatic or with mild symptoms. Conclusions Our data confirm that the course of SARS‐CoV‐2 infection in patients with CML is generally mild and reassure about the safety of continuing TKIs during the COVID‐19 pandemic. Furthermore, we suggest that patients with CML succeed to mount an antibody response after exposure to SARS‐CoV‐2, similar to the general population.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the acquisition of t(9;22) generating the fusion tyrosine kinase BCR::ABL1. However, despite the crucial role of this protein in the dysregulation of numerous signal transduction pathways, a direct measure of BCR::ABL1 kinase activity in chronic phase (CP) CML was never accomplished due to intense degradative activity present in mature leukocytes. Therefore, we developed a procedure suitable to preserve BCR::ABL1 protein under non-denaturing, neutral pH conditions in primary, chronic phase (CP)-CML samples. As a result, specific kinase activity was detected utilizing a biotinylated peptide substrate highly selective for c-ABL1. Furthermore, through this approach, BCR::ABL1 kinase activity was barely detectable in CP-CML compared to Ph+ acute lymphoblastic leukemia primary samples, where kinase activity is comparable to those measured in Ph+ cell lines. These in vitro findings provide the first direct measure of BCR::ABL1 kinase activity in primary CP-CML and reveal the presence of a still uncharacterized inhibitory mechanism that maintains BCR::ABL1 in a low activity state in CP-CML despite its overexpression.

Multipotent stem cells persist within the stromal vascular fraction (SVF) of adipose tissue during adulthood. These cells, commonly referred to as adipose-derived stromal cells (ASC), have been extensively investigated over the past years as a promising therapeutic tool based on their regenerative and immunomodulatory properties. However, how ASC might mirror the age-related alteration of the fat they reside in remains unclear. Herein, we show that inguinal adipose tissue in mice turns from brown/beige- to white-like with age and resident ASC readily mirror these changes both at mRNA and microRNA transcriptional level. Mechanistically, our data suggest that these brown/age-related changes in ASC transcription rely on changes in the activity of E2F1 and NFkB transcription factors.

After more than a decade of controversy on the role of stromal cells in the tumor microenvironment, the emerging data shed light on pro-tumorigenic and potential anti-cancer factors, as well as on the roots of the discrepancies. We discuss the pro-tumorigenic effects of stromal cells, considering the effects of tumor drivers like hypoxia and tumor stiffness on these cells, as well as stromal cell-mediated adiposity and immunosuppression in the tumor microenvironment, and cancer initiating cells’ cellular senescence and adaptive metabolism. We summarize the emerging data supporting stromal cell therapeutic potential in cancer, discuss the possibility to reprogram stromal cells of the tumor microenvironment for anti-cancer effects, and explore some causes of discrepancies on the roles of stromal cells in cancer in the available literature.

Extracellular vesicles (EVs) are membrane-coated particles secreted by virtually all cell types in response to different stimuli, both in physiological and pathological conditions. Their content generally reflects their biological functions and includes a variety of molecules, such as nucleic acids, proteins and cellular components. The role of EVs as signaling vehicles has been widely demonstrated. In particular, they are actively involved in the pathogenesis of several hematological malignancies (HM), mainly interacting with a number of target cells and inducing functional and epigenetic changes. In this regard, by releasing their cargo, EVs play a pivotal role in the bilateral cross-talk between tumor microenvironment and cancer cells, thus facilitating mechanisms of immune escape and supporting tumor growth and progression. Recent advances in high-throughput technologies have allowed the deep characterization and functional interpretation of EV content. In this review, the current knowledge on the high-throughput technology-based characterization of EV cargo in HM is summarized.

Notch and Wnt signaling are highly conserved intercellular communication pathways involved in developmental processes, such as hematopoiesis. Even though data from literature support a role for these two pathways in both physiological hematopoiesis and leukemia, there are still many controversies concerning the nature of their contribution. Early studies, strengthened by findings from T-cell acute lymphoblastic leukemia (T-ALL), have focused their investigation on the mutations in genes encoding for components of the pathways, with limited results except for B-cell chronic lymphocytic leukemia (CLL); in because in other leukemia the two pathways could be hyper-expressed without genetic abnormalities. As normal and malignant hematopoiesis require close and complex interactions between hematopoietic cells and specialized bone marrow (BM) niche cells, recent studies have focused on the role of Notch and Wnt signaling in the context of normal crosstalk between hematopoietic/leukemia cells and stromal components. Amongst the latter, mesenchymal stromal/stem cells (MSCs) play a pivotal role as multipotent non-hematopoietic cells capable of giving rise to most of the BM niche stromal cells, including fibroblasts, adipocytes, and osteocytes. Indeed, MSCs express and secrete a broad pattern of bioactive molecules, including Notch and Wnt molecules, that support all the phases of the hematopoiesis, including self-renewal, proliferation and differentiation. Herein, we provide an overview on recent advances on the contribution of MSC-derived Notch and Wnt signaling to hematopoiesis and leukemia development.

The endothelin-1 (ET-1) receptors were recently found to mediate pro-survival functions in multiple myeloma (MM) cells in response to autocrine ET-1. This study investigated the effectiveness of macitentan, a dual ET-1 receptor antagonist, in MM treatment, and the mechanisms underlying its activities. Macitentan affected significantly MM cell (RPMI-8226, U266, KMS-12-PE) survival and pro-angiogenic cytokine release by down-modulating ET-1-activated MAPK/ERK and HIF-1α pathways, respectively. HIF-1α silencing abrogated the ET-1 mediated induction of genes encoding for pro-angiogenic cytokines such as VEGF-A, IL-8, Adrenomedullin, and ET-1 itself. Upon exposure to macitentan, MM cells cultured in the presence of the hypoxia-mimetic agent CoCl2, exogenous ET-1, or CoCl2 plus ET-1, down-regulated HIF-1α and the transcription and release of downstream pro-angiogenic cytokines. Consistently, macitentan limited significantly the basal pro-angiogenic activity of RPMI-8226 cells in chorioallantoic membrane assay. In xenograft mouse models, established by injecting NOG mice either via intra-caudal vein with U266 or subcutaneously with RPMI-8226 cells, macitentan reduced effectively the number of MM cells infiltrating bone marrow, and the size and microvascular density of subcutaneous MM tumors. ET-1 receptors targeting by macitentan represents an effective anti-proliferative and anti-angiogenic therapeutic approach in preclinical settings of MM.

In this study, we compared the overall gene and pathway expression profiles of HS-5 and HS-27A stromal cell lines with those of primary bone marrow MSCs to verify if they can be considered a reliable alternative tool for evaluating the contribution of MSCs in tumor development and immunomodulation. Indeed, due to their easier manipulation in vitro as compared to primary MSC cultures, several published studies took advantage of stromal cell lines to assess the biological mechanisms mediated by stromal cells in influencing tumor biology and immune responses. However, the process carried out to obtain immortalized cell lines could profoundly alter gene expression profile, and consequently their biological characteristics, leading to debatable results. Here, we evaluated the still undisclosed similarities and differences between HS-5, HS-27A cell lines and primary bone marrow MSCs in the context of tumor development and immunomodulation. Furthermore, we assessed by standardized immunological assays the capability of the cell lines to reproduce the general mechanisms of MSC immunoregulation. We found that only HS-5 cell line could be suitable to reproduce not only the MSC capacity to influence tumor biology, but also to evaluate the molecular mechanisms underlying tumor immune escape mediated by stroma cells. However, HS-5 pre-treatment with inflammatory cytokines, that normally enhances the immunosuppressive activity of primary MSCs, did not reproduce the same MSCs behavior, highlighting the necessity to accurately set up in vitro assays when HS-5 cell line is used instead of its primary counterpart.

Mesenchymal stem/stromal cells (MSCs) are multipotent cells residing in the stromal tissues of the body and capable of promoting tissue repair and attenuating inflammatory processes through their immunomodulatory properties. Preclinical and clinical observations revealed that not only direct intercellular communication mediates MSC properties; in fact, a pivotal role is also played by the release of soluble and bioactive factors, such as cytokines, growth factor and extracellular vesicles (EVs). EVs are membrane-coated vesicles containing a large variety of bioactive molecules, including lipids, proteins, and nucleic acids, such as RNA. EVs release their contents into target cells, thus influencing cell fate through the control of intracellular processes. In addition, MSC-derived EVs can mediate modulatory effects toward different effector cells belonging to both innate and adaptive immunity. In this review, we will discuss the literature data concerning MSC-derived EVs, including the current standardized methods for their isolation and characterization, the mechanisms supporting their immunoregulatory properties, and their potential clinical application as alternative to MSC-based therapy for inflammatory reactions, such as graft-versus-host disease (GvHD).

The role of mesenchymal stromal cells (MSCs) in the tumor microenvironment is well described. Available data support that MSCs display anticancer activities, and that their reprogramming by cancer cells in the tumor microenvironment induces their switch toward pro-tumorigenic activities. Here we discuss the recent evidence of pro-tumorigenic effects of stromal cells, in particular (i) MSC support to cancer cells through the metabolic reprogramming necessary to maintain their malignant behavior and stemness, and (ii) MSC role in cancer cell immunosenescence and in the establishment and maintenance of immunosuppression in the tumor microenvironment. We also discuss the mechanisms of tumor microenvironment mediated reprogramming of MSCs, including the effects of hypoxia, tumor stiffness, cancer-promoting cells, and tumor extracellular matrix. Finally, we summarize the emerging strategies for reprogramming tumor MSCs to reactivate anticancer functions of these stromal cells.

Management of chronic myeloid leukemia (CML) in advanced phases remains a challenge also in the era of tyrosine kinase inhibitors (TKIs) treatment. Cytogenetic clonal evolution and development of resistant mutations represent crucial events that limit the benefit of subsequent therapies in these patients. CML is diagnosed in accelerated (AP) or blast phase (BP) in

Abstract Background Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. Methods TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions.

Mesenchymal stromal cells (MSCs) are adult multipotent stem cells residing as pericytes in various tissues and organs where they can differentiate into specialized cells to replace dying cells and damaged tissues. These cells are commonly found at injury sites and in tumors that are known to behave like “wounds that do not heal.” In this article, we discuss the mechanisms of MSCs in migrating, homing, and repairing injured tissues. We also review a number of reports showing that tumor microenvironment triggers plasticity mechanisms in MSCs to induce malignant neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth. The antitumor properties and therapeutic potential of MSCs are also discussed.

Master developmental pathways, such as Notch, Wnt, and Hedgehog, are signaling systems that control proliferation, cell death, motility, migration, and stemness. These systems are not only commonly activated in many solid tumors, where they drive or contribute to cancer initiation, but also in primary and metastatic tumor development. The reactivation of developmental pathways in cancer stroma favors the development of cancer stem cells and allows their maintenance, indicating these signaling pathways as particularly attractive targets for efficient anticancer therapies, especially in advanced primary tumors and metastatic cancers. Metastasis is the worst feature of cancer development. This feature results from a cascade of events emerging from the hijacking of epithelial-mesenchymal transition, angiogenesis, migration, and invasion by transforming cells and is associated with poor survival, drug resistance, and tumor relapse. In the present review, we summarize and discuss experimental data suggesting pivotal roles for developmental pathways in cancer development and metastasis, considering the therapeutic potential. Emerging targeted antimetastatic therapies based on Notch, Wnt, and Hedgehog pathways are also discussed.

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches.

Classic Hodgkin lymphoma (cHL) is a unique lymphoid neoplasm characterized by extensive immune infiltrates surrounding rare malignant Hodgkin Reed–Sternberg (HRS) cells. Different subsets of T and NK cells have long been recognized in the cHL microenvironment, yet their distinct contribution to disease pathogenesis has remained enigmatic. Very recently, novel platforms for high dimensional analysis of immune cells, such as single-cell RNA sequencing and mass cytometry, have revealed unanticipated insights into the composition of T- and NK-cell compartments in cHL. Advances in imaging techniques have better defined specific T-helper subpopulations physically interacting with neoplastic cells. In addition, the identification of novel cytotoxic subsets with an exhausted phenotype, typically enriched in cHL milieu, is shedding light on previously unrecognized immune evasion mechanisms. This review examines the immunological features and the functional properties of T and NK subsets recently identified in the cHL microenvironment, highlighting their pathological interplay with HRS cells. We also discuss how this knowledge can be exploited to predict response to immunotherapy and to design novel strategies to improve PD-1 blockade efficacy.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma in adults. Despite the recognition of transcriptional subtypes with distinct functional characteristics, patient outcomes have not been substantially altered since the advent of chemoimmunotherapy (CIT) twenty years ago. Recently, a few pivotal studies added to the disease heterogeneity by describing several activating mutations, which have been associated with disease presentation, B-cell function and behavior, and final outcome. DLBCL arises from antigen exposed B-cells, with the B-cell receptor (BCR) playing a central role. BCR-activity related mutations, such as CD79B and MYD88, are responsible for chronic activation of the BCR in a substantial subset of patients. These mutations, often coexisting in the same patient, have been found in a substantial subset of patients with immune-privileged (IP) sites DLBCLs, and are drivers of lymphoma development conferring tissue-specific homing properties. Both mutations have been associated with disease behavior, including tumor response either to CIT or to BCR-targeted therapy. The recognition of CD79B and MYD88 mutations will contribute to the heterogeneity of the disease, both in recognizing the BCR as a potential therapeutic target and in providing genetic tools for personalized treatment.

Wnt/β-catenin signaling has been reported in Acute Myeloid leukemia, but little is known about its significance as a prognostic biomarker and drug target. In this study, we first evaluated the correlation between expression levels of Wnt molecules and clinical outcome. Then, we studied—in vitro and in vivo—the anti-leukemic value of combinatorial treatment between Wnt inhibitors and classic anti-leukemia drugs. Higher levels of β-catenin, Ser675-phospho-β-catenin and GSK-3α (total and Ser 9) were found in AML cells from intermediate or poor risk patients; nevertheless, patients presenting high activity of Wnt/β-catenin displayed shorter progression-free survival (PFS) according to univariate analysis. In vitro, many pharmacological inhibitors of Wnt signalling, i.e., LRP6 (Niclosamide), GSK-3 (LiCl, AR-A014418), and TCF/LEF (PNU-74654) but not Porcupine (IWP-2), significantly reduced proliferation and improved the drug sensitivity of AML cells cultured alone or in the presence of bone marrow stromal cells. In vivo, PNU-74654, Niclosamide and LiCl administration significantly reduced the bone marrow leukemic burden acting synergistically with Ara-C, thus improving mouse survival. Overall, our study demonstrates the antileukemic role of Wnt/β-catenin inhibition that may represent a potential new therapeutics strategy in AML.

Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. β-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between β-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene β-Catenin. PTPRG was found to be capable of dephosphorylating β-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of β-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and β-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and β-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells.

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygen-level response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

Death receptor (DR3) 3 is a member of the TNFR superfamily. Its ligand is TNF-like ligand 1A (TL1A), a member of the TNF superfamily. TL1A/DR3 interactions have been reported to modulate the functions of T cells, NK, and NKT cells and play a crucial role in driving inflammatory processes in several T-cell-dependent autoimmune diseases. However, TL1A expression and effects on B cells remain largely unknown. In this study, we described for the first time that B cells from human blood express significant amounts of DR3 in response to B cell receptor polyclonal stimulation. The relevance of these results has been confirmed by immunofluorescence analysis in tonsil and spleen tissue specimens, which showed the in situ expression of DR3 in antigen-stimulated B cells in vivo. Remarkably, we demonstrated that TL1A reduces B-cell proliferation induced by anti-IgM-antibodies and IL-2 but did not affect B-cell survival, suggesting that TL1A inhibits the signal(s) important for B-cell proliferation. These results revealed a novel function of TL1A in modulating B-cell proliferation in vitro and suggest that TL1A may contribute to homeostasis of effector B-cell functions in immune response and host defense, thus supporting the role of the TL1A/DR3 functional axis in modulating the adaptive immune response.

Mesenchymal stromal cells (MSCs) reside in many organs including lung, as shown by their isolation from fetal lung tissues, bronchial stromal compartment, bronchial-alveolar lavage and transplanted lung tissues. It is still controversial whether lung MSCs can undergo mesenchymal-to-epithelial-transition (MET) and possess immune regulatory properties. To this aim, we isolated, expanded and characterized MSCs from normal adult human lung (lung-hMSCs) and compared with human bone marrow-derived MSCs (BM-hMSCs). Our results show that lung-MSCs reside at the perivascular level and do not significantly differ from BM-hMSCs in terms of immunophenotype, stemness gene profile, mesodermal differentiation potential and modulation of T, B and NK cells. However, lung-hMSCs express higher basal level of the stemness-related marker nestin and show, following in vitro treatment with retinoic acid, higher epithelial cell polarization, which is anyway partial when compared to a control epithelial bronchial cell line. Although these results question the real capability of acquiring epithelial functions by MSCs and the feasibility of MSC-based therapeutic approaches to regenerate damaged lung tissues, the characterization of this lung-hMSC population may be useful to study the involvement of stromal cell compartment in lung diseases in which MET plays a role, such as in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

BACKGROUND: small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro. DESIGN AND METHOD: we established VR09 cell line with plasmacytic differentiation, obtained from a case of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization in the attempt at thoroughly characterizing the cell line. We showed VR09 tumorigenic potential in vivo, leading to the development of activated DLBCL with plasmacytic features. RESULTS: VR09 cells displayed plasmacytic appearance and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2(-/-) γ-chain(-/-) mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/- CD138+ cyclin D1- Ki67 80% IgM+ IgD+ MUM1+ MNDA+ CD10- CD22+ CD23+ CD43+ K+, λ- Bcl2+ Bcl6-) and they presented episomal EBV genome, chromosome 12 trisomy, lack of c-MYC rearrangement and Myd88 gene mutation, presence of somatic hypermutation in the VH region, and wild-type p53. CONCLUSION: This new EBV-positive cell line may be useful to further characterize in vivo activated DLBCL with plasmacytic features.

Background Cytokines released in the bone marrow and thymic microenvironments play a key role in the growth of T-cell acute lymphoblastic leukemia. Among such cytokines, interleukin-8 is highly expressed in T-cell acute lymphoblastic leukemia cells refractory to chemotherapy. In this study we explored whether bone marrow stromal cells can regulate IL-8 expression in T-cell acute lymphoblastic leukemia and investigated the role of the stromal CXCL12 chemokine in this event. We also investigated the roles of the nuclear factor-κB and Jun-N-terminal kinase (JNK)/activating protein (AP)-1 signaling pathways, which contribute to regulate interleukin-8 production in some cells.Design and Methods We analyzed the expression of interleukin-8 in primary cells from ten adult patients with T-cell acute lymphoblastic leukemia when these cells were cultured with bone marrow stromal cells or stimulated with exogenous CXCL12. Interleukin-8 mRNA was analyzed by a colorimetric assay. Cytokine production was assayed by cytometric antibody array and flow cytometry. Nuclear factor-κB and JNK/AP-1 activation was investigated by using specific inhibitors of these pathways, immunoblotting, electrophoretic mobility-shift assay and cell transfection assays.Results Bone marrow stromal cells upregulated interleukin-8 mRNA in T-cell acute lymphoblastic leukemia cells through the activity of CXCR4, the CXCL12 receptor, as assessed by the use of neutralizing antibodies. Exogenous CXCL12 induced a significant increase in the production of IL-8 mRNA and protein in all T-cell acute lymphoblastic leukemia cases. We showed that CXCL12 activates the nuclear factor-κB and JNK/AP-1 pathways, and that these events are required for increased expression of interleukin-8. Furthermore, the nuclear factor-κB and AP-1 elements of the interleukin-8 promoter are necessary for both constitutive and CXCL12-induced interleukin-8 expression.Conclusions Interleukin-8 is physiologically regulated by the CXCL12/CXCR4 axis and the nuclear factor-κB and JNK/AP-1 pathways are required for interleukin-8 expression in T-cell acute lymphoblastic leukemia. We propose that, by upregulating interleukin-8, the bone marrow microenvironment and the CXCL12/CXCR4 axis may play a role in the pathogenesis of T-cell acute lymphoblastic leukemia.

We explored the role of interleukin-7 (IL-7) in the bone marrow (BM) stroma-mediated survival of primary T-cell acute lymphoblastic leukemia (T-ALL) cells and normal thymocytes. We present evidence that IL-7 has a major role in the enhanced survival mediated by BM stroma both in T-ALL cells and thymocytes.