Search

Abstract Background B‐cell chronic lymphocytic leukemia (B‐CLL) is characterized by the expansion of CD5+ malignant B lymphocytes. Recent discoveries have shown that double‐negative T (DNT) cells, double‐positive T (DPT) cells, and natural killer T (NKT)‐cells may be involved in tumor surveillance. Methods A detailed immunophenotypic analysis of the peripheral blood T‐cell compartment of 50 patients with B‐CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain‐lyse‐no wash technique and a comprehensive six‐color antibody panels. Results Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B‐CLL, as already reported. In particular, DNT, DPT, and NKT‐like percentages were significantly lower than in the controls, except for NKT‐like in the low‐risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low‐risk prognostic group of NKT‐like cells was found. A significant correlation of the absolute values of NKT‐like cells in the intermediate‐risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3+ T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T‐cell subset plays a key role in the immune T response in B‐CLL. Conclusion These early results supported that DNT, DPT, and NKT‐like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations.

Lymphoproliferative diseases are a heterogeneous set of malignant clonal proliferations of lymphocytes. Despite well-established diagnostic criteria, the diagnosis remains difficult due to their variety in clinical presentation and immunophenotypic profile. Lymphoid T-cell disorders are less common than B-cell entities, and the lack of a clear immunophenotypic characteristic makes their identification hard. Flow cytometry turned out to be a useful tool in diagnosing T-cell disorders and to resolve complicated cases, especially if the number of analyzable neoplastic cells is small. We present a case of a 55-year-old man with simultaneous lymphoproliferative neoplastic T-cell clones, one αβ and the other γδ, identified and characterized by flow cytometry (FC), exploiting the variable expression intensity of specific markers. However, the patient’s rapid decline made it impossible to define a differential diagnosis in order to confirm the identity of the γδ clone, which remains uncertain. This case is added to the few other cases already documented in the literature, characterized by the co-existence of T-large granular lymphocytic leukemia (T-LGLL)-αβ and T-LGLL-γδ/Hepatosplenic T-cell lymphoma (HSTCL). Our case underlines the key role of sensitive diagnostic tools in the assessment of potential relationship between the diagnosis, prognosis, and treatment in the two pathologies.

Measurable residual disease (MRD) is defined as the presence of residual cancer cells after treatment in patients with clinically undetectable disease, who would otherwise be considered in complete remission. It is a highly sensitive parameter which indicates the disease burden and predicts survival in this setting of patients. In recent years, MRD has gained a role in many hematological malignancies as a surrogate endpoint for clinical trials: undetectable MRD has been correlated to longer progression free survival (PFS) and overall survival (OS). New drugs and combinations have been developed with the aim to achieve MRD negativity, which would indicate favorable prognosis. Different methods to measure MRD have also been devised, which include flow cytometry, polymerase chain reaction (PCR) and next generation sequencing (NGS), with different sensitivity and accuracy in evaluating deep remission after treatment. In this review, we will analyze the current recommendations for the detection of MRD, with particular focus on its role in Chronic Lymphocytic Leukemia (CLL), as well as the different detection methods. Moreover, we will discuss the results of clinical trials and the role of MRD in new therapeutic schemes with inhibitors and monoclonal antibodies. MRD is not currently used in the clinical practice to evaluate response to treatment, due to technical and economical limitations, but it’s gaining more and more interest in trials settings, especially since the introduction of venetoclax. The use of MRD in trials will likely be followed by a broader practical application in the future. The aim of this work is to provide a reader-friendly summary of the state of art in the field, as MRD will soon become an accessible tool to evaluate our patients, predict their survival and guide physician’s therapeutic choices and preferences.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory checkpoint receptor that negatively regulates Tcell responses. CD226 competes with TIGIT for binding to the CD155 ligand, delivering a positive signal to the T cell. Here we studied the expression of TIGIT and CD226 in a cohort of 115 patients with chronic lymphocytic leukemia (CLL) and report expression of TIGIT and CD226 by leukemic cells. By devising a TIGIT/CD226 ratio, we showed that CLL cells favoring TIGIT over CD226 are typical of a more indolent disease, while those favoring CD226 are characterized by a shorter time to first treatment and shorter progression-free survival after first treatment. TIGIT expression was inversely correlated to the B-cell receptor (BCR) signaling capacity, as determined by studying BTK phosphorylation, cell proliferation and interleukin- 10 production. In CLL cells treated with ibrutinib, in which surface IgM and BCR signaling capacity are temporarily increased, TIGIT expression was downmodulated, in line with data indicating transient recovery from anergy. Lastly, cells from patients with Richter syndrome were characterized by high levels of CD226, with low to undetectable TIGIT, in keeping with their high proliferative drive. Together, these data suggest that TIGIT contributes to CLL anergy by downregulating BCR signaling, identifying novel and actionable molecular circuits regulating anergy and modulating CLL cell functions.

A case of concomitant hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) in a 50- year-old man was reported. Flow cytometry and droplet digital PCR (ddPCR) were used to detect the B-Raf proto-oncogene (BRAF) V600E mutation. The HCL population was the predominant component. The patient was first treated with cladribine and then with rituximab and achieved HCL partial remission. Importantly, the high sensitivity of our flow cytometric approach allowed the detection of a small population “P3,” in addition to the typical HCL and CLL clones. The P3 clone changed over time, from an HCL-like to a CLL-like immunophenotype. This case is added to the few other cases of synchronous HCL and CLL already reported in the literature and underlines the importance of analyzing chronic lymphoproliferative disorders by highly sensitive diagnostic techniques, like the multicolor flow cytometry and ddPCR, to evaluate the possible association between HCL and CLL at diagnosis.

Breast cancer is the most common malignancy among women worldwide. Several studies indicate that, in addition to established risk factors for breast cancer, other factors such as cortisol release related to psychological stress and drug treatment with high levels of glucocorticoids may also contribute significantly to the initiation of breast cancer. There are several possible mechanisms by which glucocorticoids might promote neoplastic transformation of breast tissue. Among these, the least known and studied is the inhibition of the nuclear erythroid factor 2-related (Nrf2)-antioxidant/electrophile response element (ARE/EpRE) pathway by high levels of glucocorticoids. Specifically, Nrf2 is a potent transcriptional activator that plays a central role in the basal and inducible expression of many cytoprotective genes that effectively protect mammalian cells from various forms of stress and reduce the propensity of tissues and organisms to develop disease or malignancy including breast cancer. Consequently, a loss of Nrf2 in response to high levels of gluco-corticoids may lead to a decrease in cellular defense against oxidative stress, which plays an important role in the initiation of human mammary carcinogenesis. In the present review, we provide a comprehensive overview of the current state of knowledge of the cellular mechanisms by which both glucocorticoid pharmacotherapy and endogenous GCs (cortisol in humans and corticosterone in rodents) may contribute to breast cancer development through inhibition of the Nrf2-ARE/EpRE pathway and the protective role of melatonin against glucocorticoid-induced apoptosis in the immune system.

CD200, a transmembrane type Ia glycoprotein belonging to the immunoglobulin protein superfamily, is broadly expressed on a wide variety of cell types, such as B lymphocytes, a subset of T lymphocytes, dendritic cells, endothelial and neuronal cells. It delivers immunosuppressive signals through its receptor CD200R, which is expressed on monocytes/myeloid cells and T lymphocytes. Moreover, interaction of CD200 with CD200R has also been reported to play a role in the regulation of tumor immunity. Overexpression of CD200 has been reported in chronic lymphocytic leukemia (CLL) and hairy cell leukemia but not in mantle cell lymphoma, thus helping to better discriminate between these different B cell malignancies with different prognosis. In this review, we focus on the role of CD200 expression in the differential diagnosis of mature B-cell neoplasms and on the prognostic significance of CD200 expression in CLL, where conflicting results have been published so far. Of interest, increasing evidences indicate that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing malignancies, such as CLL.

Chronic lymphocytic leukemia (CLL) is characterized by low CD20 expression, in part explained by an epigenetic-driven downregulation triggered by mutations of the NOTCH1 gene. In the present study, by taking advantage of a wide and well-characterized CLL cohort (n=537), we demonstrate that CD20 expression is downregulated in SF3B1-mutated CLL to an extent similar to NOTCH1-mutated CLL. In fact, SF3B1-mutated CLL cells show common features with NOTCH1- mutated CLL cells, including a gene expression profile enriched in NOTCH1-related gene sets and elevated expression of the active intracytoplasmic NOTCH1. Activation of the NOTCH1 signaling and downregulation of surface CD20 in SF3B1-mutated CLL cells correlate with overexpression of an alternatively spliced form of DVL2, a component of the Wnt pathway and negative regulator of the NOTCH1 pathway. These findings were confirmed by separately analyzing the CD20dim and CD20bright cell fractions from SF3B1-mutated cases as well as by DVL2 knockout experiments in CLL-like cell models. Together, the clinical and biological features that characterize NOTCH1-mutated CLL may also be recapitulated in SF3B1-mutated CLL, contributing to explain the poor prognosis of this CLL subset and providing the rationale for expanding therapies based on novel agents to SF3B1-mutated CLL.

We present a laboratory-based prognostic calculator (designated CRO score) to risk stratify treatment-free survival in early stage (Rai 0) chronic lymphocytic leukemia (CLL) developed using a training-validation model in a series of 1,879 cases from Italy, the United Kingdom and the United States. By means of regression analysis, we identified five prognostic variables with weighting as follows: deletion of the short arm of chromosome 17 and unmutated immunoglobulin heavy chain gene status, 2 points; deletion of the long arm of chromosome 11, trisomy of chromosome 12, and white blood cell count >32.0x103/microliter, 1 point. Low-, intermediate- and high-risk categories were established by recursive partitioning in a training cohort of 478 cases, and then validated in four independent cohorts of 144 / 395 / 540 / 322 cases, as well as in the composite validation cohort. Concordance indices were 0.75 in the training cohort and ranged from 0.63 to 0.74 in the four validation cohorts (0.69 in the composite validation cohort). These findings advocate potential application of our novel prognostic calculator to better stratify early-stage CLL, and aid case selection in risk-adapted treatment for early disease. Furthermore, they support immunocytogenetic analysis in Rai 0 CLL being performed at the time of diagnosis to aid prognosis and treatment, particularly in today’s chemofree era.

Background and objectives: Nonagenarians and centenarians (NCs) are an extremely fragile population, particularly in regard to their physical and cognitive function. The aim of this study was to define the neurocognitive profiles among 29 NCs and their 49 younger cohabitants aged 50–75 years from The Cilento Initiative on Aging Outcomes (CIAO) Pilot study in the South of Italy that had provided initial hypotheses regarding positive psychological traits related to exceptional longevity. Materials and Methods: During the home visits, lifestyle information with specific questionnaires, functional autonomy and the neuropsychological Mini Mental Scale Examination (MMSE), and the Alzheimer’s Disease Assessment Scale-Cognitive (ADAS-Cog) scale were obtained by qualified study personnel. The total blood oxidative capacity was also determined by testing the reactive derivative of oxygen metabolites (d-ROM) and by the Biological Antioxidant Potential (BAP). In all individuals, the APOE genotype determination was also performed. Results: All the subjects in both groups showed high adherence to the Mediterranean Diet. None of the NCs had severe cognitive impairment, and a very low incidence of dementia was found. The data obtained on the Activities ed Instrumental Activities of Daily Living (ADL-IADL) scale showed that the majority of NCs (16/29) were autonomous in daily life activities. The comparative assessment of NCs and cohabitants showed no significant differences in the laboratory assessment of oxidative stress and APOE genotype. Conclusions: In the Cilento Region of Southern Italy, NCs seemed to have good cognitive status when compared to younger cohabitants aging 50–65 years without significant differences in oxidative stress markers or APOE genotype. These results might be related to optimal adherence to the Mediterranean diet, although other lifestyle factors and positive personality traits may also contribute to their healthy aging. Further studies on a larger population should be performed to confirm the results of this pilot study.

Regulatory T cells (Tregs) have a fundamental function in monitoring the immune homeostasis in healthy individuals. In cancer and, in particular, in hematological malignancies, Tregs exert a major immunosuppressive activity, thus playing a critical role in tumor cell growth, proliferation, and survival. Here, we summarize published data on the prognostic significance of Tregs in hematological malignancies and show that they are highly conflicting. The heterogeneity of the experimental approaches that were used explains—at least in part—the discordant results reported by different groups that have investigated the role of Tregs in cancer. In fact, different tissues have been studied (i.e., peripheral blood, bone marrow, and lymph node), applying different methods (i.e., flow cytometry versus immunohistochemistry, whole blood versus isolated peripheral blood mononuclear cells versus depletion of CD25+ cells, various panels of monoclonal antibodies, techniques of fixation and permeabilization, and gating strategies). This is of relevance in order to stress the need to apply standardized approaches in the study of Tregs in hematological malignancies and in cancer in general.

The frequency and function of regulatory T-cells (Tregs) in multiple myeloma (MM) are still matter of debate. The percentage and absolute number of circulating Tregs (CD4+CD25+high densityCD127-/low density) from 39 patients with untreated MM and 44 patients with monoclonal gammopathies of uncertain significance (MGUS) were tested and compared with 20 healthy subjects as controls. The mean percentage number of circulating Tregs was 2.1% ± 1.0 (range 0.75–6.1%) in MM patients; 2.1% ± 0.9 (range 0.3–4.4%) in MGUS; and 1.5% ± 0.4 (range 0.9–2.1%) in controls (p ns). Mean absolute number of Tregs was 36.3/μL ± 23.7 (range 6.7–149/μL) in MM; 38.8/μL ± 19.1 (range 4.3–87/μL) in MGUS; and 39.4/μL ± 12.5 (range 18–63/μL) in controls (p ns). After a median follow-up of 38 months, 5 MGUS and 2 smoldering MM (SMM) transformed into overt MM; however Tregs number did not predict this evolution. With respect to MM patients and after a median follow-up of 33 months, Tregs did not show any significant correlation with main clinical and laboratory characteristics. Finally, from a functional point of view, Tregs displayed an effective suppressor function, irrespective of disease status. This study indicates that the number of circulating Tregs does not differ in different monoclonal gammopathies and normal subjects and do not correlate with clinical features of MM.

In mammals, a master clock is located within the suprachiasmatic nucleus (SCN) of the hypothalamus, a region that receives input from the retina that is transmitted by the retinohypothalamic tract. The SCN controls the nocturnal synthesis of melatonin by the pineal gland that can influence the activity of the clock’s genes and be involved in the inhibition of cancer development. On the other hand, in the literature, some papers highlight that artificial light exposure at night (LAN)-induced circadian disruptions promote cancer. In the present review, we summarize the potential mechanisms by which LAN-evoked disruption of the nocturnal increase in melatonin synthesis counteracts its preventive action on human cancer development and progression. In detail, we discuss: (i) the Warburg effect related to tumor metabolism modification; (ii) genomic instability associated with L1 activity; and (iii) regulation of immunity, including regulatory T cell (Treg) regulation and activity. A better understanding of these processes could significantly contribute to new treatment and prevention strategies against hormone-related cancer types.

Acquired hemophilia A (AHA) is a rare bleeding disorder due to the development of specific autoantibodies against factor VIII. The anti-CD20 monoclonal antibody Rituximab has been proven to be effective in obtaining a long-term suppression of inhibitors of AHA, besides other immunosuppressive standard treatments. Here we describe a case of idiopathic AHA in a 60-year old man successfully treated with rituximab. He showed a complete clinical response with a normalization of clotting parameters after 5 weekly courses of rituximab given at a dose of 375 mg/sqm. , but after stopping rituximab, an initial worsening of coagulation parameters induced the addition of 3 further courses. At present, the patient is in complete clinical and hematological remission after 200 days. This case confirms that Rituximab may be a safe and useful tool to treat AHA and, a prolonged administration can overcome the initial resistance. However, the precise position of this drug in the therapeutic strategy (first or second-line, alone or in combination with other drugs) remains to be established and warrants further investigation.

Infection with hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC) in developed countries. Epidemiological reports indicate that the incidence of HBV-related HCC is higher in males and postmenopausal females than other females. Increasing evidence suggests that sex hormones such as androgens and estrogens play an important role in the progression of an HBV infection and in the development of HBV-related HCC. While androgen is supposed to stimulate the androgen signaling pathway and cooperate to the increased transcription and replication of HBV genes, estrogen may play a protecting role against the progression of HBV infections and in the development of HBV-related HCC through decreasing HBV RNA transcription and inflammatory cytokines levels. Additionally, sex hormones can also affect HBV-related hepatocarcinogenesis by inducing epigenetic changes such as the regulation of mRNA levels by microRNAs (miRNAs), DNA methylation, and histone modification in liver tissue. This review describes the molecular mechanisms underlying the gender disparity in HBV-related HCC with the aim of improving the understanding of key factors underneath the sex disparity often observed in HBV infections. Furthermore, the review will propose more effective prevention strategies and treatments of HBV-derived diseases.

Regulatory T-cells (Tregs) constitute a small subset of cells that are actively involved in maintaining self-tolerance, in immune homeostasis and in antitumor immunity. They are thought to play a significant role in the progression of cancer and are generally increased in patient with chronic lymphocytic leukemia (CLL). Their number correlates with more aggressive disease status and is predictive of the time to treatment, as well. Moreover, it is now clear that dysregulation in Tregs cell frequency and/or function may result in a plethora of autoimmune diseases, including multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, systemic lupus erythematosis, autoimmune lymphoproliferative disorders, rheumatoid arthritis, and psoriasis. Efforts are made aiming to develop approaches to deplete Tregs or inhibit their function in either cancer and autoimmune disorders.

This work investigates the possibility that HLA-G, a molecule modulating innate and adaptive immunity, is part of an immune escape strategy of chronic lymphocytic leukemia cells. A 14 base pair insertion/deletion polymorphism (rs66554220) in the 3′-untranslated region of HLA-G influences mRNA stability and protein expression. The analysis of a cohort of patients with chronic lymphocytic leukemia confirmed that del/del individuals are characterized by higher levels of surface and soluble HLA-G than subjects with the other two genotypes. In line with its role in immunomodulation, the percentage of regulatory T lymphocytes is higher in del/del patients than in patients with the other genotypes and correlates with the amounts of surface or soluble HLA-G. Furthermore, addition of sHLA-G-rich plasma from patients with chronic lymphocytic leukemia induces natural killer cell apoptosis and impairs natural killer cell lysis, with effects proportional to the amount of soluble HLA-G added. Lastly, the presence of an HLA-G 14 base pair polymorphism is of prognostic value, with del/del patients showing reduced overall survival, as compared to those with other genotypes. These results suggest that: (i) the HLA-G 14 base pair polymorphism influences the levels of surface and soluble HLA-G expression, and (ii) the over-expression of HLA-G molecules contributes to creating tolerogenic conditions.

Fournier’s gangrene (FG) is a rare but severe necrotizing fasciitis of the external genitalia that may complicate the clinical course of hematologic malignancies and sometimes may be the first sign of the disease. The clinical course of FG is very aggressive and the mortality is still high despite the improvement in its management. Early recognition of FG and prompt appropriate treatment with surgical debridement and administration of antibiotics are the cornerstone of the management of this very severe disease.

Chronic lymphocytic leukemia is marked by profound defects in T-cell function. Programmed death-1 is a receptor involved in tumor-mediated immunosuppression through binding of the PD-L1 ligand. Multiparametric flow cytometry and immunohistochemistry were used to study PD-1/PD-L1 expression. Functional assays were used to determine the involvement of the PD-1/PD-L1 axis in T-cell responses. PD-1 expression by CD4+ and CD8+ T lymphocytes was significantly higher in 117 chronic lymphocytic leukemia patients than in 33 donors of a comparable age. CD4+ and CD8+ T lymphocytes from chronic lymphocytic leukemia patients displayed increased numbers of effector memory and terminally differentiated cells, respectively, when compared to controls. The number of effector memory CD4+ and terminally differentiated CD8+ lymphocytes positively associated with a more advanced stage of disease, treatment requirements and unfavorable genomic aberrations. Furthermore, leukemic lymphocytes expressed higher levels of PD-L1 than circulating B lymphocytes from normal donors. PD-1 and PD-L1 surface expression spiked in proliferating T and B lymphocytes, suggesting that this interaction works efficiently in activated environments. Within chronic lymphocytic leukemia proliferation centers in the lymph node, CD4+/PD-1+ T lymphocytes were found to be in close contact with PD-L1+ chronic lymphocytic leukemia cells. Lastly, functional experiments using recombinant soluble PD-L1 and blocking antibodies indicated that this axis contributes to the inhibition of IFN-γ production by CD8+ T cells. These observations suggest that pharmacological manipulation of the PD-1/PD-L1 axis may contribute to restoring T-cell functions in the chronic lymphocytic leukemia microenvironment.

We report a patient with chronic lymphocytic leukemia (CLL) in whom a leukemic involvement of prostate and penis occurred in the advanced phase of his disease. Obstructive urinary symptoms were indicative of prostatic CLL infiltration, followed by the occurrence of an ulcerative lesion on the glans. Histologic examination confirmed the neoplastic B-cell infiltration. Both localizations responded to conventional treatments. A review of the literature confirms that leukemic involvement of the genito-urinary system is uncommon in CLL patients. However, such an involvement should be considered in CLL patients with urologic symptoms and a long history of the disease.

The clinical course of chronic lymphocytic leukemia (CLL) may be complicated at any time by autoimmune phenomena.The most common ones are hematologic disorders, such as autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP). Pure red cell aplasia (PRCA) and autoimmune agranulocytosis (AG) are, indeed, more rarely seen. However, they are probably underestimated due to the possible misleading presence of cytopenias secondary to leukemic bone marrow involvement or to chemotherapy cytotoxicity. The source of autoantibodies is still uncertain, despite the most convincing data are in favor of the involvement of resting normal B-cells. In general, excluding the specific treatment of underlying CLL, the managementof these complications is not different from that of idiopathic autoimmune cytopenias or of those associated to other causes. Among different therapeutic approaches, monoclonal antibody rituximab, given alone or in combination, has shown to be very effective.

Regulatory T-cells (Tregs) constitute a small subset of cells that are actively involved in maintaining self-tolerance, in immune homeostasis and in antitumor immunity. They are thought to play a significant role in the progression of cancer and are generally increased in patient with chronic lymphocytic leukemia (CLL). Their number correlates with more aggressive disease status and is predictive of the time to treatment, as well. Moreover, it is now clear that dysregulation in Tregs cell frequency and/or function may result in a plethora of autoimmune diseases, including multiple sclerosis, type 1 diabetes mellitus, myasthenia gravis, systemic lupus erythematosis, autoimmune lymphoproliferative disorders, rheumatoid arthritis, and psoriasis. Efforts are made aiming to develop approaches to deplete Tregs or inhibit their function in either cancer and autoimmune disorders.