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Proclamazione del vincitore della borsa di studio 2019
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Borsa di Studio “Damiano per l’Ematologia” 2019

Proclamazione del Vincitore

L’autrice del progetto, primo classificato, ha rinunciato alla borsa di studio, per cui è risultato vincitore il progetto “Evaluation of the immunomodulatory effects of new targeted drugs in patients with chronic lymphocytic leukemia”. L’autrice è la Dr.ssa Valentina Griggio, che svolgerà il suo lavoro presso la Divisione Universitaria di Ematologia dell’Università degli Studi di Torino, diretta dal Prof. Mario Boccadoro, sotto la supervisione della Dr.ssa Candida Vitale.

Testo del progetto

a) Title

Evaluation of the immunomodulatory effects of new targeted drugs in patients with chronic lymphocytic leukemia

b) Introduction

Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in Western Countries, and it is characterized by complex underlying immune dysregulations, which often emerge into clinically manifest immune defects and/or autoimmune phenomena, and also favor disease progression. A number of studies reported abnormalities in the phenotype of CD4+ and CD8+ T cells, with the accumulation of terminally differentiated effector memory T cells, and a relative decrease of naïve precursors [1]. Recent findings have identified the PD-1/PD-L1 axis as an important component that contributes to dysfunctional interactions between CLL cells and host T lymphocytes, leading to a pronounced Th2 skewing of the T-cell responses [2]. Tregs influence CLL progression, especially in patients with advanced disease who have increased numbers of Tregs in the peripheral blood [3]. With regard to innate immune responses, dysfunctional Vγ9Vδ2 T cells and their altered subset distribution in CLL patients have already been reported [4]. New targeted drugs characterized by unprecedented anti-tumor efficacy have been recently approved or are in late stage of development for the treatment of CLL. Interestingly, beside the direct anti-neoplastic effect, these molecules may exert complex activities on the host immune system. Ibrutinib is a small selective BTKi that blocks signal transduction from B-cell receptor (BCR), impairing CLL cells growth and inducing apoptosis. Preclinical animal studies have demonstrated that ibrutinib has highly promising activity in experimental models of autoimmune diseases, such as rheumatoid arthritis and lupus, by targeting not only B lymphocytes but also monocytes and macrophages, which are important BTK-expressing effector cells [5, 6]. Moreover, data show that ibrutinib, in addition to blocking BTK, is an irreversible inhibitor of interleukin-2–inducible kinase (ITK), driving a Th1-skewed profile in CD4+ T-cell populations isolated from CLL patients [7]. Recent studies demonstrate that ibrutinib treatment markedly increased CD4+ and CD8+ T cell numbers, also reducing the ratio of Treg to CD4+ T cells [8]. Idelalisib is an inhibitor of PI3kδ isoform that impairs cellular growth and proliferation. Noteworthy, the molecular targets of idelalisib are not restricted to the B-cell compartment. In fact, the targeted inhibition of PI3Kδ decreases the production of various inflammatory and anti-apoptotic cytokines by T cells, and counteracts the infiltrative capacity of macrophages both at the tumor site and within the target tissues of autoimmune reactions [9-13]. Venetoclax is a selective BH3 mimetic antagonist of Bcl-2 with a specific anti-tumor activity against CLL cells. However, the control on apoptosis pathway exerted by the Bcl-2 family proteins is not restricted to tumor cells. Interestingly, Bcl-2–associated dysregulation of lymphocyte apoptosis can contribute to the pathogenesis of autoimmune diseases, and ABT737, a potent inhibitor of Bcl-2, Bcl-xL, and Bcl-w significantly reduces disease severity in tissue-specific and systemic animal models of autoimmunity [14]. The intrinsic apoptosis pathway is differently regulated among conventional T cells, NK cells and Tregs, and the survival of distinct T cell subpopulations depends on different Bcl-2 proteins. ABT-737 induces in murine models a tolerogenic milieu, which can be exploited to alleviate graft-versus-host disease, to prevent allograft rejection, and to induce immunological tolerance in combination with bone marrow transplantation [15]. Along with the advances in the development of new targeted drugs, in the recent years also the field of immunotherapy gained a renewed attention, leading to the approval of chimeric antigen receptor (CAR)-T cell based therapies [16, 17] and immune checkpoints inhibitors. CAR-T cells are currently under investigation for both hematological and solid tumors, but new approaches to improve their efficacy and persistency, and reduce their side effects are needed.

c) Aims of the project

The present project is articulated in 2 Aims:

Aims 1.

To evaluate the immunomodulatory effects exerted in vitro by idelalisib, ibrutinib and venetoclax on the following parameters: a) percentage, absolute number and subset distribution of conventional T cells; b) percentage and absolute number of Tregs and unconventional T cells; b) expression of immune checkpoint

molecules (i.e. PD-1, CTLA-4, LAG3, TIGIT, CD96, TIM-3) on T and NK cells; c) proliferative and cytotoxic properties of conventional and unconventional T cells.

Task 2.

To evaluate the immunomodulatory effects exerted by targeted drugs currently used in the clinical practice (i.e. idelalisib, ibrutinib and venetoclax) on different T-cell populations during patients’ treatment. The following parameters will be assessed: a) percentage, absolute number and subset distribution of conventional T cells; b) percentage and absolute number of Tregs and unconventional T cells; b) expression of immune checkpoint molecules (i.e. PD-1, CTLA-4, LAG3, TIGIT, CD96, TIM-3) on T and NK cells; c) proliferative and cytotoxic properties of conventional and unconventional T cells. Immune parameters and their treatment-induced modifications will be correlated with CLL prognostic factors and outcome variables.

This project aims at better understanding the pleiotropic immunomodulatory effects of new targeted drugs in chronic lymphocytic leukemia (CLL), both in vitro, and during treatment of patients. The effects induced by targeted drugs, especially on conventional and unconventional T cell-compartments will be correlated with outcome data and side effect profile. From the translational standpoint, the demonstration of a positive effect of targeted drugs on the host immune system may provide the rationale for their inclusion in combination strategies, aimed at improving the efficacy of immunotherapeutic approaches - such as CAR T cell-based therapies - in CLL, and in other hematologic and solid tumors.

d) Methods

Peripheral blood samples will be collected from patients with CLL after their informed consent, in accordance with the Declaration of Helsinki and approval by the local Institutional Review Board. The diagnosis of CLL will be defined according to the International Workshop on CLL-National Cancer Institute (IWCLL/NCI) guidelines. Samples will be obtained from CLL patients with an available clinical record.

Samples from treatment-naïve patients will be used for in vitro experiments (Task 1), whereas samples from patients treated with targeted drugs will be collected at baseline and at different timepoints during treatment (e.g. 1 month and 3 months) and used for ex vivo evaluations (Task 2).

Cytofluorimetric analyses and functional assays will be performed on peripheral blood mononuclear cells (PBMC). PBMC will be isolated by density gradient centrifugation on Ficoll-Hypaque. Cells will be analyzed without further manipulation, or will be exposed in vitro to ibrutinib, idelalisib and venetoclax and analyzed at defined timepoints (e.g. 24, 48, and 72 hours). Cytofluorimetric analysis will be performed on PBMC to study the expression of the following molecules: CD3, CD4, CD8, CD25, CD27, CD45RA, CD16, CD56, TCRγδ, Vδ1, Vγ9Vδ2, PD-1, CTLA4, LAG3, TIGIT, CD96, TIM-3.

PBMC will be cultured in vitro with polyclonal stimuli (e.g. OKT3 and anti-CD28 antibody for T cells, K562 cell line for NK cells, α-galactosylceramide for NK-T cells, and zoledronic acid for Vγ9Vδ2 T cells) and the expansion of various cell subset will be evaluated through carboxyfluorescein succinimidyl ester (CFSE) proliferation assay. Different assessment will be performed to evaluate the functional properties of immune cells, such as CD69 expression evaluation, CD107 degranulation assay, and detection of intracellular production of IFNγ, TNFα and IL-2.

Relevant biological and clinical data of patients will be collected. Appropriate statistical tests will be applied to evaluate the modulation in consecutive measurements of variables for the same patient and to compare biological parameters between different groups of patients. Survival will be estimated by the Kaplan-Meier method and the difference between groups will be assessed by log-rank test. An adequate number of experiments will be performed to grant the achievement of meaningful results. The statistical significance will be defined as a p value <0.05.

e) References

1. Nunes, C., et al., Expansion of a CD8(+)PD-1(+) replicative senescence phenotype in early stage CLL patients is associated with inverted CD4:CD8 ratios and disease progression. Clin Cancer Res, 2012. 18(3): p. 678-87.

2. Brusa, D., et al., The PD-1/PD-L1 axis contributes to T-cell dysfunction in chronic lymphocytic leukemia. Haematologica, 2013. 98(6): p. 953-63.

3. D'Arena, G., et al., Regulatory T-cell number is increased in chronic lymphocytic leukemia patients and correlates with progressive disease. Leuk Res, 2011. 35(3): p. 363-8.

4. Coscia, M., et al., Dysfunctional Vgamma9Vdelta2 T cells are negative prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells. Blood, 2012. 120(16): p. 3271-9.

5. Cheng, S., et al., BTK inhibition targets in vivo CLL proliferation through its effects on B-cell receptor signaling activity. Leukemia, 2014. 28(3): p. 649-57.

6. Hutcheson, J., et al., Modulating proximal cell signaling by targeting Btk ameliorates humoral autoimmunity and end-organ disease in murine lupus. Arthritis Res Ther, 2012. 14(6): p. R243.

7. Dubovsky, J.A., et al., Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes. Blood, 2013. 122(15): p. 2539-49.

8. Long, M., et al., Ibrutinib treatment improves T cell number and function in CLL patients. J Clin Invest, 2017. 127(8): p. 3052-3064.

9. Reif, K., et al., Cutting Edge: Differential Roles for Phosphoinositide 3-Kinases, p110g and p110d, in Lymphocyte Chemotaxis and Homing. J Immunol, 2004. 173(4): p. 2236-2240.

10. Okkenhaug, K., et al., The p110delta isoform of phosphoinositide 3-kinase controls clonal expansion and differentiation of Th cells. J Immunol, 2006. 177(8): p. 5122-8.

11. Ali, K., et al., Inactivation of PI(3)K p110delta breaks regulatory T-cell-mediated immune tolerance to cancer. Nature, 2014. 510(7505): p. 407-11.

12. Mouchemore, K.A., et al., Specific inhibition of PI3K p110delta inhibits CSF-1-induced macrophage spreading and invasive capacity. Febs j, 2013. 280(21): p. 5228-36.

13. Suarez-Fueyo, A., et al., Inhibition of PI3Kdelta reduces kidney infiltration by macrophages and ameliorates systemic lupus in the mouse. J Immunol, 2014. 193(2): p. 544-54.

14. Bardwell, P.D., et al., The Bcl-2 family antagonist ABT-737 significantly inhibits multiple animal models of autoimmunity. J Immunol, 2009. 182(12): p. 7482-9.

15. Gabriel, S.S., et al., Distinctive Expression of Bcl-2 Factors in Regulatory T Cells Determines a Pharmacological Target to Induce Immunological Tolerance. Front Immunol, 2016. 7: p. 73.

16. Geyer, M.B., et al., Concurrent therapy of chronic lymphocytic leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia utilizing CD19-targeted CAR T-cells. Leuk Lymphoma, 2017: p. 1-5.

17. Chu, F., J. Cao, and S.S. Neelalpu, Versatile CAR T-cells for cancer immunotherapy. Contemp Oncol (Pozn), 2018. 22(1a): p. 73-80.

Period: 12 months

Giudizio del Comitato Scientifico:



Rilevanza dell’argomento


Originalità del progetto






Riproducibilità e applicabilità dei risultati al trattamento dei pazienti


Punteggio addizionale


Breve commento del Comitato Scientifico (testo originale):

The project aims to answer very relevant questions that may have an impact on the management of patients with CLL.

It should be noted that several data are already available on the immunologic effects of ibrutinib (e.g., Lee-Verges Int J Cancer 2018, De Weerdt Blood 2018) and idelalisib (e.g. Dong J Clin Invest 2018, Martinelli Haematologica 2018, Lim JCI Insight 2018), maybe less for venetoclax. In addition, combinations of CAR-T cell therapy and checkpoint blockade at least with ibrutinib are being tested and preliminary results have been presented.

CV della candidata

Il CV della Dr.ssa Griggio è così riassumibile: presso l’Università di Torino conseguimento della laurea magistrale in Biotecnologie Mediche e del Dottorato di Ricerca in Medicina Molecolare, Laurea triennale in Biotecnologie Sanitarie presso l’Università di Padova. Esperienza di 7 anni di studio e lavoro presso la Divisione Universitaria di Ematologia dell’Università degli Studi di Torino, inerente gli aspetti farmacologici e terapeutici della Leucemia >Linfatica Cronica. La Dr.ssa Griggio è titolare di 6 pubblicazioni peer-reviewed su riviste indexate con hI > 10.

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