Detection of abnormalities in B-cell differentiation pattern is a useful tool to predict relapse in precursor-B-ALL

Immunophenotypic investigation of minimal residual disease (MRD) has traditionally been based on the investigation of phenotypic aberrants at diagnosis to be used later as a target for MRD detection. This approach has several shortcomings (it is only applicable to patients with aberrant phenotypes, requires a diagnostic sample, and is patient-specific) and therefore a search for simpler alternatives is warranted. The present study is based on the hypothesis that in precursor-B-ALL patients the persistence of residual leukaemic cells may induce abnormalities in the precursor-B-cell compartment in bone marrow (BM) and these could be used as a criteria to predict relapse. These abnormalities may include: (1) the presence of an increase in the frequencies of immature B cells (CD34+/CD19+ or CD20-/CD19+) or (2) the existence of an altered B-cell differentiation pathway due to a blockade or to the presence of B cells outside the normal pathway. A total of 180 BM samples from 45 consecutive precursor-B-ALL patients who achieved morphological complete remission (CR) were analysed by multiparametric flow cytometry. Our results show that a significant increase in immature B-cell subsets or an altered B-cell differentiation predicts a high relapse rate (P<0.01) and a shorter disease-free survival (P<0.01). Moreover, abnormalities in either of these two criteria detected at specific time points during follow-up (end of induction, maintenance, or after treatment) were associated with a significantly shorter disease-free survival (P<0.01). In summary, the investigation of abnormalities in B-cell differentiation is a relatively simple and cheap approach for predicting relapse in precursor-B-ALL patients.     

Versatile strategy for controlling the specificity and activity of engineered T cells

The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR–T-cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as “switch” molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a “universal” anti-FITC–directed CAR-T cell. Optimization of this CAR–switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR–T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy.Second-generation CD19-targeting chimeric antigen receptor (CAR) T cells engineered with costimulatory signaling domains have generated potent antileukemic responses in patients with refractory B-cell malignancies (1–3). In light of their clinical promise, there has been an explosion of interest in CAR-T cells for cancer therapy, especially for the treatment of relapsed, refractory malignancies. However, current CAR–T-cell therapy is associated with serious treatment-related toxicities resulting from the uncontrollable release of cytokines (cytokine release syndrome, CRS) from synchronously activated and rapidly proliferating CAR-T cells. CRS is currently managed with anti–IL-6 receptor antibodies or by suppressing CAR–T-cell activity with corticosteroids (4, 5). In addition, the “on-target, off-tumor” activity of CD19-specific CAR-T cells results in the long-term depletion of B-cells in patients, which can be managed with Ig replacement therapy (2, 6, 7). This latter example of normal tissue toxicity is more problematic when targeting solid tumor-associated antigens (TAAs), where antigen expression is generally less restricted than hematologic markers (5, 8–11).To minimize treatment-related toxicities yet retain potent antitumor activity, several strategies have been developed to regulate the persistence or enhance tumor selectivity of CAR–T-cell therapy. Suicide genes and transient mRNA CAR have been used to induce T-cell apoptosis and shorten the lifespan of these potent CAR-T cells, respectively (12, 13). To improve CAR–T-cell selectivity for tumor cells, dual targeting CARs that require the engagement of two TAAs to induce full activation and proliferation of CAR-T cells have been described (14). Another approach is the use of inhibitory CARs, which bind to antigens found on normal cells and result in the inhibition of CAR–T-cell function (15). Recently, Wu et al. described an approach using a small molecule to induce the assembly of a functional CAR complex, and thus control CAR–T-cell activity (16). Alternatively, it has been shown that CAR–T-cell activity can be controlled using soluble intermediary “switch” molecules. These switches are comprised of a tumor-targeting antibody or small-molecule ligand and a second moiety that selectively binds the CAR and not an endogenous receptor. CAR–T-cell activity is thus strictly dependent on the formation of a ternary complex between the CAR-T cell, switch, and tumor antigen. Therefore, titration or removal of the switch molecule can control or terminate CAR–T-cell response, respectively. Notably, unlike the suicide-gene approach described above, these switchable CAR-T cells are expected to remain in patients after termination of treatment, which may be beneficial in incidences that require further treatments. In addition, unlike the intracellular switch approach described by Wu et al., an intercellular switch approach enables the targeting of multiple TAAs with a “universal” CAR-T cell. Examples of switches used in this approach include TAA-specific monoclonal antibodies that elicit antitumor activity from Fc-specific CAR-T cells (17), and chemically or enzymatically modified antibody–hapten conjugates that redirect antihapten CAR-T cells (18, 19). Recently, we have demonstrated the redirection of anti-FITC CAR-T cells with a heterobifunctional small-molecule switch, folate-FITC, which selectively targets folate receptor-overexpressing cancers (20). Although these reports established the feasibility of eliciting an anticancer response with switchable CAR-T (sCAR-T) cells, it has yet to be shown whether the efficacy of sCAR-T cells is comparable to current CAR-T cell therapies in the clinic.Another limitation of conventional CAR–T-cell therapy is the inability of a single CAR-T cell to target multiple TAAs. For example, a second engineered CAR-T cell is required to target the outgrowth of resistant cancer cells lacking the original surface antigen target, or heterogeneous tumor populations expressing distinct TAAs. Moreover, recent studies have demonstrated the need to engineer the single-chain variable fragments (scFv) and the spacer region within the CAR structure for optimal efficacy (12, 21, 22). Therefore, generation of a universal CAR-T cell that can be readily endowed with multiple TAA specificities is highly desirable to minimize the risk for developing escape variants, and to simplify the clinical implementation of CAR–T-cell therapy.Herein, we describe the development of antibody-based switches with site-specific conjugation of an FITC tag that enable the rapid optimization of a productive pseudoimmunological synapse between anti-FITC CAR-T cells and a given TAA. We demonstrate potent antigen-specific and dose-dependent in vitro and in vivo efficacies of these sCAR-T cells against CD19-expressing cancer cell lines. More importantly, we demonstrate that the strict dependence of CAR–T-cell activity on switch concentration can be exploited to enhance the in vivo safety (severe CRS and long-term B-cell depletion) associated with CAR–T-cell therapies. Finally, using two clinically validated B-cell markers, CD19 and CD22, we establish that this approach can be used to rapidly optimize different antigen-specific switch intermediates, which should allow one to target distinct TAAs with a single CAR construct.     

Mechanisms of insulin release from pancreatic B-cells

Summary The following structures are mainly involved in the process of insulin secretion by pancreatic B-cells: the cell membrane, β-granules and elements of the pericapillary space. The authors have used high doses of glucose as stimulators. The B-cells have a wide range of secretory mechanisms: a) by means of active sectors; b) by emiocytosis; and c) by apocrine secretion. By ‘active releasing sector’ we mean a definite sector of the cell membrane in which microglobules of the β-granule core are concentrated: it is here that insulin secretion takes place. Traduzione a cura di G.U.     

Up-regulation of Bcl-2 during adipogenesis mediates apoptosis resistance in human adipocytes

Targeting apoptotic pathways in adipocytes has been suggested as a pharmacological approach to treat obesity. However, adipocyte apoptosis was identified as a cause for macrophage infiltration into adipose tissue. Previous studies suggest that mature adipocytes are less sensitive to apoptotic stimuli as compared to preadipocytes. Here, we aimed to identify proteins mediating apoptosis resistance in adipocytes.     

Prognostic Factors and Outcomes of Adults With Hemophagocytic Lymphohistiocytosis

ObjectiveTo describe the prognostic factors and outcomes of adults with hemophagocytic lymphohistiocytosis (HLH), a rare disorder caused by pathologic activation of the immune system.Patients and MethodsThe study population consisted of a consecutive cohort of adult (age ≥18 years) patients treated at Mayo Clinic in Rochester, Minnesota, from January 1, 1996, through December 31, 2011, in whom a diagnosis of HLH was suspected and subsequently confirmed by retrospective review using the HLH-04 diagnostic criteria.ResultsOf 250 adult patients suspected of having HLH, 62 met the HLH-04 diagnostic criteria and were included in the final analysis. The median age was 49 years (range, 18-87 years), and 42 (68%) were male. The underlying cause of HLH was malignant tumor in 32 patients (52%), infection in 21 patients (34%), autoimmune disorder in 5 patients (8%), and idiopathic disease in 4 patients (6%). After a median follow-up of 42 months, 41 patients (66%) had died. The median overall survival of the entire cohort was 2.1 months. The median overall survival of patients with tumor–associated HLH was 1.4 months compared with 22.8 months for patients with non-tumor–associated HLH (P=.01). The presence of a malignant tumor and hypoalbuminemia were significant predictors of inferior survival on multivariate analysis.ConclusionIn this large series of adults with secondary HLH treated at a single tertiary care center, patients with low serum albumin levels and tumor–associated HLH had a markedly worse survival. Hemophagocytic lymphohistiocytosis remains elusive and challenging to clinicians who must maintain a high index of suspicion. The recent discovery of several novel diagnostic and therapeutic modalities may improve outcomes of adult patients with HLH.     

Expression of B-Cell Surface Antigens in Subpopulations of Exosomes Released From B-Cell Lymphoma Cells

Purpose

Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10¹² exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81⁺ and CD63⁺ subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation.

Methods

Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81.

Findings

Magnetic bead–based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma–derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity.

Implications

Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use.     

同时发生myc和bcl-2/IgH或bcl-6易位的B细胞淋巴瘤临床病理特征

目的 观察同时发生myc和bcl-2/IgH或bcl-6易位的B细胞淋巴瘤(“双击”淋巴瘤)的临床病理学特点.方法 收集145例经活检诊断的侵袭性成熟B细胞淋巴瘤病例石蜡包埋组织,其中包括弥漫性大B细胞淋巴瘤(DLBCL) 129例,具有介于DLBCL和伯基特淋巴瘤(BL)之间特征无法分类的B细胞淋巴瘤(BCLU)5例,BL 7例和高级别滤泡淋巴瘤合并DLBCL4例,在组织芯片上进行myc、bcl-2/IgH和bcl-6系列探针的间期荧光原位杂交检测,记录基因易位情况、拷贝数变化和其他基因异常,同时收集病例的临床、病理学及随访资料.结果 共检测出同时发生myc和bcl-2或bcl-6基因易位(“双击”淋巴瘤)病例5例,检出率5/145(3.4％);其中myc伴bcl-2易位2例(原形态学诊断1例是DLBCL,1例是BCLU);myc伴bcl-6易位3例,均为DLBCL.Ki-67阳性指数60％ ～100％.另发现3例bcl-2伴bcl-6易位,均为DLBCL.其余DLBCL病例中出现单个基因易位或拷贝数增加的占51.2％ (66/129).随访资料显示,5例“双击”淋巴瘤患者中3例在诊断后2年内死亡,1例失访,1例随访36个月无病生存.结论 “双击”淋巴瘤属于罕见病例,主要根据基因检测手段来诊断,形态学上不完全等同于2008 WHO分类中的BCLU这一亚型,还可表现为其他的形态学谱系.大部分患者预后差.     

Richter's Syndrome manifested as diffuse large B-cell lymphoma of the mandible with lytic lesions and hypercalcemic crisis

Richter's Syndrome (RS) is a transformation from chronic lymphocytic leukemia (CLL) into more aggressive lymphoma. RS occurs in about 5% of patients with CLL and its clinical outcome is poor. Extranodal involvements of RS may be present in up to 40% of patients and central nervous system manifestation was found to be the most common. Mandibular localization of RS has not been reported so far. There is no established standard treatment for RS. The improvement of survival in RS patients is achievable with intensive anti-lymphoma chemotherapy and subsequent allogeneic stem cell transplantation (alloHSCT) performed in complete remission. Herein we report a female with RS of the right mandible and hypercalcemia was its first clinical manifestation.