Candida Vitale

IGHV unmutated CLL B cells are more prone to spontaneous apoptosis and subject to environmental prosurvival signals than mutated CLL B cells

Tumor cells in chronic lymphocytic leukemia (CLL) are more prone to apoptosis when cultured ex vivo, because they lack prosurvival signals furnished in vivo via B-cell receptor (BCR)-dependent and -independent pathways. This study compared the susceptibility of unmutated (UM) and mutated (M) CLL B cells to spontaneous apoptosis and prosurvival signals. UM CLL B cells showed a significantly higher rate of spontaneous apoptosis than M CLL B cells. Nuclear factor-kB (NF-kB) was rapidly inactivated, and B-cell leukemia/lymphoma 2 (Bcl-2) expression progressively down-regulated in the UM CLL B cells. CD40-Ligand, interleukin-4 and stromal cells significantly improved their viability and partially recovered Bcl-2, but not NF-kB expression. Peripheral blood mononuclear cells also offered protection of UM CLL B cells, and recovered both NF-kB and Bcl-2 expression. T cells, rather than nurse-like cells, were responsible for protecting UM CLL B cells by means of cell-to-cell contact and soluble factors. Despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions.

Vγ9Vδ2 T cell-based immunotherapy in hematological malignancies: from bench to bedside

Many hematological malignancies consist of tumor cells that are spontaneously recognized and killed by Vγ9Vδ2 T cells. These tumor cells generate high amounts of intracellular phosphorylated metabolites mimicking the natural ligands and display a wide range of stress-induced self-ligands that are recognized by Vγ9Vδ2 T cells via TCR-dependent and TCR-independent mechanisms. The intrinsic features of Vγ9Vδ2 T cells and that of tumor cells of hematological origin constitute an ideal combination from which to develop Vγ9Vδ2 T cell-based immune interventions. In this review, we will discuss the rationale, preclinical and clinical data in favor of this therapeutic strategy and the future perspectives of its development.

Dysfunctional Vγ9Vδ2 T cells are negative prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells

The role of Vγ9Vδ2 T cells in chronic lymphocytic leukemia (CLL) is unexplored, although these cells have a natural inclination to react against B-cell malignancies. Proliferation induced by zoledronic acid was used as a surrogate of γδ TCR-dependent stimulation to functionally interrogate Vγ9Vδ2 T cells in 106 untreated CLL patients. This assay permitted the identification of responder and low-responder (LR) patients. The LR status was associated with greater baseline counts of Vγ9Vδ2 T cells and to the expansion of the effector memory and terminally differentiated effector memory subsets. The tumor immunoglobulin heavy chain variable region was more frequently unmutated in CLL cells of LR patients, and the mevalonate pathway, which generates Vγ9Vδ2 TCR ligands, was more active in unmutated CLL cells. In addition, greater numbers of circulating regulatory T cells were detected in LR patients. In multivariate analysis, the LR condition was an independent predictor of shorter time-to-first treatment. Accordingly, the time-to-first treatment was significantly shorter in patients with greater baseline numbers of total Vγ9Vδ2 T cells and effector memory and terminally differentiated effector memory subpopulations. These results unveil a clinically relevant in vivo relationship between the mevalonate pathway activity of CLL cells and dys-functional Vγ9Vδ2 T cells.

Comprehensive assessment of the TCRBV repertoire in small T-cell samples by means of an improved and convenient multiplex PCR method

OBJECTIVE Overall diversity of the T-cell receptor (TCR) repertoire can be regarded as a recapitulatory signature of a hosts immunocompetence status. We aimed to establish a time- and cost-saving multiplex polymerase chain reaction (PCR) method for determining the TCR repertoire of conventional alphabeta T cells in small T-cell samples. MATERIALS AND METHODS The method estimates the length distribution of the complementarity-determining regions 3 (CDR3) of beta variable (BV) gene segments (TCRBV repertoire) by multiplex PCR, followed by fluorescent run-off reactions to visualize BV-BC and/or BV-BJ rearrangements. Run-off products are separated on a capillary sequencer and subsequently analyzed with GeneScan or Genotyper programs. Detection-limit studies with normal T cells, KMS27 cells, and regulatory T cells were carried out to evaluate sensitivity and reproducibility. RESULTS Head-to-head comparison of the method with conventional immunoscope assay has shown that it is a time- and cost-saving approach to characterize TCRBV and TCRBJ repertoires, including the presence of oligoclonal T cells in samples containing as few as 1 x 10(5) T cells. CONCLUSION We have developed a multiplex PCR method that allows comprehensive assessment of the TCRBV repertoire at the BV-BC and BV-BJ levels, and saves a considerable amount of time, reagents, and cell input.

The enzymatic activities of CD38 enhance CLL growth and trafficking: implications for therapeutic targeting

The ecto-enzyme CD38 is gaining momentum as a novel therapeutic target for patients with hematological malignancies, with several anti-CD38 monoclonal antibodies in clinical trials with promising results. In chronic lymphocytic leukemia (CLL) CD38 is a marker of unfavorable prognosis and a central factor in the pathogenetic network underlying the disease: activation of CD38 regulates genetic pathways involved in proliferation and movement. Here we show that CD38 is enzymatically active in primary CLL cells and that its forced expression increases disease aggressiveness in a xenograft model. The effect is completely lost when using an enzyme-deficient version of CD38 with a single amino-acid mutation. Through the enzymatic conversion of NAD into ADPR (ADP-ribose) and cADPR (cyclic ADP-ribose), CD38 increases cytoplasmic Ca2+ concentrations, positively influencing proliferation and signaling mediated via chemokine receptors or integrins. Consistently, inhibition of the enzymatic activities of CD38 using the flavonoid kuromanin blocks CLL chemotaxis, adhesion and in vivo homing. In a short-term xenograft model using primary cells, kuromanin treatment traps CLL cells in the blood, thereby increasing responses to chemotherapy. These results suggest that monoclonal antibodies that block the enzymatic activities of CD38 or enzyme inhibitors may prove therapeutically useful.

Anergic bone marrow Vγ9Vδ2 T cells as early and long-lasting markers of PD-1-targetable microenvironment-induced immune suppression in human myeloma

Vγ9Vδ2 T cells have a natural inclination to recognize malignant B cells in vitro via receptors for stress-induced self-ligands and TCR-dependent recognition of phosphoantigens (pAgs) generated in the mevalonate (Mev) pathway. This inclination is continuously challenged in vivo by the immune suppression operated by tumor cells. Multiple myeloma (MM) is a prototypic B-cell malignancy in which myeloma cells subvert the local microenvironment to reshape antitumor immune responses. In this study, we have investigated the immune competence of bone marrow (BM) Vγ9Vδ2 T cells in a large series of MM patients. We have found that the BM microenvironment significantly hampers the pAg-reactivity of BM Vγ9Vδ2 T cells, which become largely PD-1+ and are surrounded by PD-L1+ myeloma cells and increased numbers of PD-L1+ myeloid-derived suppressor cells (MDSC). Vγ9Vδ2 T-cell dysfunction is an early event that can be already detected in individuals with monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even when MM patients achieve clinical remission. Anti-PD-1 treatment increases the cytotoxic potential of Vγ9Vδ2 T cells by almost 5-fold after pAg stimulation, and appears to be a promising strategy for effective immune interventions in MM.

Simvastatin and downstream inhibitors circumvent constitutive and stromal cell-induced resistance to doxorubicin in IGHV unmutated CLL cells

The immunoglobulin heavy-chain variable region (IGHV) mutational status is a strong determinant of remission duration in chronic lymphocytic leukemia (CLL). The aim of this work was to compare the multidrug resistance (MDR) signature of IGHV mutated and unmutated CLL cells, identifying biochemical and molecular targets potentially amenable to therapeutic intervention. We found that the mevalonate pathway-dependent Ras/ERK1–2 and RhoA/RhoA kinase signaling cascades, and the downstream HIF-1α/P-glycoprotein axis were more active in IGHV unmutated than in mutated cells, leading to a constitutive protection from doxorubicin-induced cytotoxicity. The constitutive MDR phenotype of IGHV unmutated cells was partially dependent on B cell receptor signaling, as shown by the inhibitory effect exerted by ibrutinib. Stromal cells further protected IGHV unmutated cells from doxorubicin by upregulating Ras/ERK1–2, RhoA/RhoA kinase, Akt, HIF-1α and P-glycoprotein activities. Mevalonate pathway inhibition with simvastatin abrogated these signaling pathways and reversed the resistance of IGHV unmutated cells to doxorubicin, also counteracting the protective effect exerted by stromal cells. Similar results were obtained via the targeted inhibition of the downstream molecules ERK1–2, RhoA kinase and HIF-1α. Therefore, targeting the mevalonate pathway and its downstream signaling cascades is a promising strategy to circumvent the MDR signature of IGHV unmutated CLL cells.

CD200 included in a 4-marker modified Matutes score provides optimal sensitivity and specificity for the diagnosis of chronic lymphocytic leukaemia

CD200, a transmembrane type Ia glycoprotein belonging to the immunoglobulin superfamily, has been shown to have a differential expression in B‐cell neoplasms. Here, we retrospectively assessed the diagnostic relevance of CD200 on 427 patients with B‐cell chronic neoplasms in leukemic phase (median age, 69 y; range, 35‐97 y). The final diagnosis based on the investigators assessment was chronic lymphocytic leukaemia (CLL) in 75% of cases and non‐CLL in 25% of cases. Sensitivity and specificity for the diagnosis of CLL (vs non‐CLL) were calculated for the following markers: CD200, CD5, CD22, CD23, CD79b, FMC7, and SmIg. CD23 was the only marker without a statistically significant difference between the investigator assessment and the flowcytometric analysis. The other markers were unable—when individually evaluated—to discriminate between CLL and non‐CLL, requiring the integration into a scoring system. The modified score no. 1 (addition of CD200) showed superimposable sensitivity and specificity compared with the Matutes score. The substitution of CD79b (modified score no. 2), surface membrane immunoglobulins (SmIg) (modified score no. 3), and CD79b and FMC7 (modified score no. 4) with CD200 showed that only the modified score no. 4 had both higher sensitivity and higher specificity compared with standard Matutes score. In conclusion, this work defines a simplified score, compared with the classical Matutes score, for the differential diagnosis of chronic B‐cell leukaemia—which only requires 4 markers instead of 5 (CD5, CD23, CD200, and SmIg).

Magic pills: new oral drugs to treat chronic lymphocytic leukemia

ABSTRACT Introduction: A deeper understanding of chronic lymphocytic leukemia (CLL) biology has led to the identification of new promising therapeutic targets. Different classes of molecules are currently under investigation and novel oral drugs have recently been approved or are in a late stage of clinical development. Areas covered: We present biological data illustrating the heterogeneous mechanisms of action of new oral drugs in CLL. Moreover, we provide clinical data from phase I to III studies, and discuss efficacy and side effects profile of these new therapies. Data are derived from peer-reviewed articles indexed in PubMed and from abstracts presented at major international meetings. Expert opinion: Novel oral drugs represent a valuable alternative to chemo-immunotherapy for patients with CLL, especially when high-risk disease features are present and when age or comorbidities preclude the use of standard treatments. Based on data from ongoing clinical trials, the indications of already approved agents will most likely be expanded and new options will soon be available. Moreover, treatment combinations will broaden the therapeutic armamentarium of physicians treating CLL. The availability of multiple choices is of benefit for patients with CLL, but also represents a challenge for the need of choosing the right drug for each patient.

Prognostic relevance of oxidative stress measurement in chronic lymphocytic leukaemia

To evaluate the prognostic significance of oxidative stress (OS) and antioxidant defence status measurement in patients with chronic lymphocytic leukaemia (CLL).