ALBERT

Description: Background: Chronic lymphocytic leukemia (CLL) is characterized by phenotypic and functional defects of immune cells, which often emerge into increased susceptibility to infections and autoimmunity, and also contribute to immune evasion of cancer cells. The BTK inhibitor ibrutinib exerts its anti-tumor activity via the targeting of key pathways in CLL cells. In addition, ibrutinib has also shown immune modulatory properties suggesting the ability to partially restore immune functions in CLL. Currently, available data are mainly limited to the activity exerted by ibrutinib on conventional T cells, whereas little is known on the effects induced on other non-neoplastic immune cell populations. Aim: The aim of this study was to perform a comprehensive and longitudinal analysis of the immune changes occurring in multiple lymphoid populations in a broad cohort of CLL patients treated with ibrutinib. Methods: We included 22 CLL patients with progressive disease (P-CLL) and eligible to ibrutinib therapy. Peripheral blood samples were collected from patients at baseline and after 1, 6 and 12 months of treatment with ibrutinib. For comparison, we also analyzed 7 healthy donors (HD) and 10 treatment-naïve CLL patients with stable disease not requiring treatment (S-CLL). The percentages and the absolute numbers of CLL cells, T cells, γδ (Vδ1 and Vγ9Vδ2) T cells, T regulatory cells (Tregs), natural killer (NK) and NK-T cells, as well as the expression of activation markers and immune checkpoint molecules were assessed by flow cytometry. The cytotoxic function of Vγ9Vδ2 T cells was evaluated using the CD107 assay. Statistical analyses were carried out by paired t-test. Results: Median age of enrolled patients was 70 years (range 42-80). The median lymphocyte count at study entry was 35.7 x 109/L (range 1.8-178) and the median number of previous treatment regimens was 2 (range 0-5). After 12 months of ibrutinib, 20 out of 22 (91%) patients achieved at least a partial response. The mean absolute number of CLL cells started to decrease by month 6 and became significantly lower than the baseline value by month 12. We also observed a parallel reduction of the total count of CD4+ T cells, CD8+ T cells and Tregs which reached statistical significance for the CD4+ T-cell compartment at the 12-month timepoint. Overall, ibrutinib treatment had no impact on the absolute numbers of Vδ1 and Vγ9Vδ2 T cells, NK and NK-T cells over time. In our cohort, we observed no change in the differentiation subset distribution of conventional CD4+ and CD8+ T cells, Tregs, Vδ1 and Vγ9Vδ2 T cells after 6 and 12 months of ibrutinib treatment. At baseline, we observed in the P-CLL cohort a significantly higher surface expression of the early activation marker CD69, both in the leukemic cell compartment and on T cells, NK and NK-T cells compared to S-CLL and HD. CD69 expression significantly decreased on CLL cells, T cells and NK-T cells already after 1 month of ibrutinib treatment, and on NK cells after 6 months (Figure 1A-D). The expression of the costimulatory molecule NKG2D was not modulated by ibrutinib treatment in any immune cell compartment. Among checkpoint molecules, the expression of CD96 was significantly reduced after 12 months of ibrutinib treatment on T lymphocytes and on NK, NK-T, and Vγ9Vδ2 T cells, whereas TIGIT, PD-1, TIM-3 and BTLA were not modulated (Figure 1E-H). In addition, when we restricted the analysis to patients showing a response in terms of lymphocyte count (i.e. 〉50% reduction in 6 months) (11 out of 22 patients) we observed a recovery of CD16 surface expression on NK cells (Figure 1I, blue graph), a reduced expression of the co-inhibitory molecule TIGIT on Tregs (Figure 1J, blue graph) and a normalization in the mean values of CD4+ and CD8+ T cells, all becoming significant after 12 months of treatment. From a functional standpoint, we observed, after 12 months of treatment, an improvement in the cytotoxic function of Vγ9Vδ2 T cells in response to IL-2 and zoledronic acid, which was not associated to a modulation of their proliferative ability. Conclusions: Our data suggest that in CLL patients the anti-tumor activity of ibrutinib is paralleled by a dampening of immune exhaustion features, which is more evident in patients showing a more profound decrease in leukemic cell counts, and by a recovery of Vγ9Vδ2 T cell cytotoxic functions. These ibrutinib-induced effects might be exploited in the context of cellular immunotherapeutic strategies. Disclosures Mauro: Abbvie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Jannsen: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Scarfo:AstraZeneca: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Gaidano:Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesys: Consultancy, Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Foà:Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Boccadoro:Amgen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Metastatic disease of the bone is a rare complication of chronic lymphocytic leukemia (CLL), it may be result from richter's transformation or metastatic from non lymphoid malignancies. CLL is the most common form of adult leukemia, with the median age of 70 years at diagnosis [Siegel et al. 2013]. The diagnosis is established by blood counts, blood smears, and immunophenotyping of circulating B-lymphocytes.The result is the increased number of lymphocytes in the peripheral blood, leukocytosis with absolute lymphocytosis, the increase of the lymphnodes, the increase in size of the spleen. The diagnosis of chronic lymphocytic leukemia B requires the presence of Clonal B cells in the peripheral blood at or above 5,000 / ul for at least 3 months. Typing immunophenotypical pathological lymphocytes are positive for surface antigens CD5, CD19, CD23, weakly positive for CD20 and CD22, generally negative FMC7 and CD79b; also expressing surface immunoglobulins. The Rai and Binet staging systems, which are established by physical examination and blood counts, have been recognized as standards for deciding whether to begin treatment. Patients with active or symptomatic disease or with advanced Binet or Rai stages require therapy. For fit patients, chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab represents the current standard therapy. For unfit patients, treatment with an anti-CD20 antibody (obinutuzumab, rituximab, ofatumumab) plus a milder chemotherapy (Chlorambucil) may be applied. At relapse, if the treatment-free interval exceeds two to three years, the initial treatment may be repeated, if the disease relapses earlier, drugs such as bendamustine (plus rituximab), alemtuzumab, lenalidomide, ofatumumab, ibrutinib, or idelalisib, must be choosen. Patients with a del(17p) or TP53 mutation can be treated with ibrutinib or a combination of idelalisib and rituximab. in relapsing patients with TP53 mutations or del(17p) or patients that are refractory to repeated chemoimmunotherapies, an allogeneic SCT may be considered [Hallek M 2015]. In this article we show a case of a 66-year-old man with CLL and a bone localization. In 2011 diagnosis of CLL, Rai Stage 0, Binet Stage A. Principal characteristics at diagnosis: HB 13.2 g /dl, White Blood Cells 15.800 / mm3, lymphocytes 61%, neutrophils 32%, monocytes 4%, platelets 141.000/mm3; normal hepatic end renal function; flowcytometric immunophenotyping of the peripheral blood revealed B-cell CLL; prognostic factors: CD38 negative, ZAP70 positive, rearrangement of the immunoglobulins mutated; FISH: negative; CT chest / abdomen / pelvis: presence of multiple aorto-pulmonary and axillary adenopathies (max diameter of 2 centimeters); bone marrow biopsy: infiltration of CLL equal to 60% of global cellularity. The patient was only observed until January 2015, when he was hospitalized due to acute anemia, requiring supportive therapy, and right foot pain . So it was decided to re-evaluate the whole disease in order to decide whether to start chemotherapy. The disease was staged again with instrumental and laboratory tests: presence of renal insufficiency, egd and colonoscopy negative, Coombs' test negative, bone marrow biopsy confirmed the diagnosis of chronic lymphocytic with bone marrow infiltration of 90%, abdomen ultrasound showed only moderate splenomegaly. On February, persistence of right foot pain and appearance of swelling, assessed by the orthopedic as a suspected algic and dystrophic syndrome. So he suggested to perform scintigraphy which revealed: pronounced inflammatory osteometabolic reaction of the right tibia/fibula/ankle third distal which could be referred, in the first evaluation, to algic and dystrophic syndrome. However, a local biopsy was performed: localization of chronic lymphocytic leukemia. On March 2015 a total body TC showed 2 nodular calcifications in the right lung lobe, multiple right paratracheal, barety space, aortopulmonary and axillary adenopathies. Prostate size increased. In order to study carefully the liver and prostate lesions, an ultrasound abdomen was performed that documented only enlarged spleen, normal size liver, free of focal disease, increased prostate due to symmetric bilobate hypertrophy . After the second cycle of chemotherapy, prolonged thrombocytopenia, so he continues only with a radiotherapy program. Disclosures No relevant conflicts of interest to declare.

Description: The aim of our perspective observational study was identify a fast molecular biomarker of tumorigenic-proliferative haematological disorders at on set using a non-invasive method. At this end, to better discriminate between myeloproliferative or lymphoproliferative hematological disease, from May to July 2014, we collected peripheral blood mononuclear cells (PBMCs) from patients at the first medical examination and without drug therapy. The patients were divided into groups on the basis of the diagnosis. Group 2 (n=8) included patients suffered from mixed disorders such as myeloproliferative neoplasms (MPD) associated to monoclonal gammopathy of undetermined significance (MGUS). Group 3 (n=8) included patients with only MGUS, group 4 included 9 patients with only primary myelofibrosis (M). Healthy donors (Group 1, n=16) were considered as normal subject or calibrator in molecular analysis. Their PBMCs were used to perform relativegene expression profile of a transcriptome involved in apoptotic control, stem-cell differentiation, immune network, inflammation and leukemogenesis, in order to detect whether these may serve to label the patient groups. A multigene expression assay (47 genes) was carried out with the TaqM,an® Low Density Array Fluidic card. In Group 3 (MGUS), 6 genes were differentially expressed (BMI-1, FLT3, FZD1, FZD5, ICAM1, IMP3). Also, we compared variance, main value for each gene in each group and T-test that showed a significant differential expression pattern (p

Description: Introduction:Biosimilars of filgrastim have been available since 2008 and are now in widespread use also for stem cells (SCs) mobilization. There are no previously published data on the use of biosimilar G-CSF for peripheral blood (PB) SCs collection only in patients with Multiple Myeloma (MM) as they always had been presented aggregated with other different hematological malignancies. Here we report the use of a biosimilar filgrastim (Zarzio®) compared with a matched historical control group in PBSCs in patients with de-novo MM treated with reference product (Neupogen®) and scheduled to receive a double autologous transplantation. Material and Methods: A total of 26 consecutive patients received biosimilar filgrastim after cyclophosphamide (CTX) 4g/m2 chemotherapy for PBSCs mobilization between January and July 2014, this group was compared with a matched historical control cohort (n=26) who had been treated with originator filgrastim between 2012 and 2013. In both groups G-CSF was administered 5 μg/kg/day until almost a minimal count of 4 x106 CD34+ cells/kg was collect. Monitoring of peripheral blood CD34+ cell concentrations began as soon as WBC recovery reached 1x109/L and leukapheresis was initiated when the CD34+cell concentration reached 10/mL. Results: Characteristics (age, gender, body weight, type of induction CHT, radiation therapy) were similar in both groups. The median peak CD34+ cells value was 187.8 ± 159.5 x ml in the biosimilar group compared with 223.7 ± 206.5 x ml in the originator group (P=n.s.). The median number of leukapheresis necessary to harvest a minimal count of 4 and 8 x10E6 CD34+/kg was similar in both biosimilar and originator groups: 1.4 ± 0.6 vs 1.3 ± 0.6 (P=n.s.) and 1.8 ± 0.8 vs 1.4 ± 0.7 (P=n.s), respectively. One and 3 patients failed to mobilize 4 and 8 x 10 E /kg CD34+ cells in the biosimilar group compared with 3 and 8 patients in the originator group (P=n.s), respectively). Short-term hematological reconstitution was evaluated for all transplanted patients. Number of CD34+ cells reinfused at the first transplant were 4.8 ± 0.75x10E6/Kg in the biosimilar group compared with 5.1 ± 0.75x10E6/Kg in the originator group (P=n.s.). Median time to neutrophil engraftment (〉500/μl) was similar in both the biosimilar and originator groups (10.7 ± 0.8 vs 10.3 ± 1.1 days; P=n.s), as was platelet recovery (13.6 ± 1.8 vs13.4 ± 1.2 days; P=n.s. Conclusions:Our study shows that biosimilar filgrastim administered following chemotherapy is equivalent to originator filgrastim for autologous stem cell mobilization and PBSCs collection when used with the same schedules and doses in a MM real life setting. Long-term follow-up is required to confirm the safety of biosimilar and originator G-CSF. Disclosures No relevant conflicts of interest to declare.

Description: Background: High-dose melphalan (HDM) is the standard therapy for autologous stem cell transplantation (ASCT) in multiple myeloma (MM), although the optimal conditioning regimen remains yet to be identified. Bendamustine (BENDA) has a proved activity in hematological malignancies including both first line and relapsed MM. Methods: We conducted a phase II trial, adding BENDA to HDM before second ASCT, in a tandem ASCT strategy, in 32 patients with "de-novo" MM. All patients received a bortezomib-based induction therapy. High-dose cyclophosphamide (CY) and G-CSF were used to mobilize stem cells. Four to 6 weeks after the administration of CY, patients received HDM (200 mg/mq), followed by ASCT. Three to 6 months after the first transplantation, patients received a second ASCT with BENDA (200 mg/m2) to HDM (140 mg/m2) as conditioning regimen (BM). Results: The median age was 56 years (range 40 to 66). Overall, there was no transplant related mortality. The incidence of neutropenic fever and mucositis (grade 1-2) was 46.9% and 81.2%, respectively. No mucositis grade 3-4 was observed. Median number of days to neutrophil and platelet engraftment were 11 and 12, respectively. After the second transplantation, the complete response (CR) improved to 62.5%. Overall response rate was 90.6%. After a median follow-up of 18,2 months, 4 patients had progressed and 1 died. Median progression free survival (PFS) was not reached and actuarial 2-year PFS and OS was 78% and 90%, respectively. Conclusion: Bendamustine plus melphalan is feasible as conditioning regimen for second ASCT in MM and should be explored for efficacy in a phase III study. Longer follow-up is needed to evaluate conversion rate and survival. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Lenalidomide (Len) and low-dose Dexamethasone (dex) (Rd) in continuous is a new standard of care for elderly newly-diagnosed multiple myeloma (NDMM) patients (pts), as established by FIRST trial (Facon et al, Blood 2018). Methods and results This is a retrospective, multicentric study conducted in Italy with the aim of evaluating efficacy and tolerability of Rd in a real-life population. Thirty-seven centers were involved and data of 429 pts are available. Pts were considered eligible for the study when completing at least 2 cycles of Rd regimen. Table 1 summarizes the characteristics of pts at time of MM diagnosis. Median age was 78 years (range 57-92), 36.6% had an ECOG PS≥2, creatinine clearance (ClCr) was ULN (P=0.01) and ClCr2 still impact on OS (p

Description: Background Elderly multiple myeloma (MM) patients are an heterogeneous population. Aging is associated with an increased frequency of co-morbidities, frailty and disability, with negative impact on treatment tolerance and outcome. A simple and reliable scoring system, based on geriatric assessment, has been developed to predict survival and used also to predict the risk of severe toxicities or treatment discontinuation in elderly newly diagnosed MM patients treated with lenalidomide-, bortezomib- or carfilzomib-based induction regimens. Methods Patients with newly diagnosed MM, ineligible for high-dose therapy and autologous stem cell transplantation due to age (≥65 years) or coexisting co-morbidities, enrolled in 3 prospective multicenter trials, were included in the analysis. Up-front dose reductions were performed according to patients age (full doses for patients ≤75 years and reduced for patients 〉75 years). Details on treatment regimens and results of these studies have previously been reported (Gay F et al EHA 2013, Larocca A et al EHA 2013, Bringhen S et al EHA 2013). At diagnosis, a geriatric assessment had been performed, to assess co-morbidities, cognitive and physical conditions. Results 869 patients were included in the analysis: 659 enrolled in the lenalidomide-based, 152 in the bortezomib-based and 58 in the carfilzomib-based trial. Median age was 74 years, and 44% of patients were older than 75 years. Median follow-up was 18 months. In univariable analysis, the risk of death was higher in patients aged 75-80 (Hazard Ratio, HR 1.37, p=0.11), and in patients older than 80 years (HR 2.75, p

Description: Background: In multiple myeloma (MM), different clinical parameters and molecular prognostic factors can predict disease course and response to therapy. The classification of myeloma patients includes laboratory parameters associated with higher tumor activity, resistance to therapy and proliferative competence. Tumor circulating plasma cells (TCPC) in MM patients showed a strong correlation with a more aggressive disease. Aim: For the first time, we quantified the amounts of TCPC with single platform flow cytometric method and evaluated their relationship with patients' baseline characteristics and response to therapy before maintenance. Methods: Whole peripheral blood samples from 413 newly diagnosed MM patients ≤65 years enrolled in the UNITO-MM-01/FORTE trial were collected. Patients were randomized [1:1:1; stratification: International Staging System (ISS) and age] to ARM A: carfilzomib-cyclophosphamide-dexamethasone (KCyd) followed by melphalan 200 mg/m2 and autologous stem-cell transplantation (MEL200-ASCT) and consolidation with 4 KCyd; ARM B: carfilzomib-lenalidomide-dexamethasone (KRd) followed by MEL200-ASCT and 4 KRd; ARM C: 12 KRd cycles. Enrollment was completed in March 2017; data cut-off was November 30, 2018. For the single platform tube, the antibody combination CD38PC7/CD138PC5.5/ CD45KO/CD56PE/CD19PB was mixed with 100µL of EDTA peripheral blood, dispensed with reverse pipetting, and incubated for 15 min, added with 500µL of lysing solution and, after 15 min, 100µL of flow count fluorospheres were dispensed with reverse pipetting and cells acquired with Navios flow cytometer. Intracytoplasmic tube was set up to confirm the clonality of CPC. Results: Circulating plasma cells (CPC) were quantified in 413 samples, with median values of 0.03% (range: 0-51%) and 2.37/mm3 (range: 0-6272/mm3). White blood cells were 5710/mm3 (range: 1752-26102/mm3); total events acquired 1285000 (range: 40000-2000000); median CPC events were 58 (range: 0-441000); cellular events acquired were 190000 (range: 4428-1300000). In 390 out of 413 samples (94.4%), CPC were detected; 272 samples (66%) showed TCPC with a median of 1.24/mm3 (range 0.06- 6272/mm3). Patients were sorted according to different baseline characteristics and the medians of absolute TCPC were compared. The most statistically significant differences (p

Description: Background: Bendamustine has a proved activity in hematological malignancies including both first line and relapsed multiple myeloma (MM). Moreover Bendamustine is generally well tolerated, with the majority of adverse events mainly limited to myelosuppression. Recently Mark et al published a phase I trial adding escalating doses of Bendamustine to the current standard conditioning of Melphalan 200 mg/m2 (HDM) in patients with MM at their first transplant. No transplant related mortality (TRM) was observed and the regimen was well tolerated. Recently was stated that double autologous stem cell transplantation (ASCT) improve the outcome of patients achieving a poor response after first ASCT. Although HDM is the standard for conditioning in MM, in some cases the CR rate is lower than expected. In this trial we evaluate feasibility and efficacy of the association of Bendamustine and Melphalan (BM) as conditioning regimen to second ASCT in patients with MM. Methods: This study was approved by local ethic committee.Between January and June 2014, 12 patients with MM underwent second ASCT following BM as conditioning regimen. The median age was of 56 years (range 40-66), 8 male and 4 female with a diagnosis of MM, stage IIIA (n=7), IIIB (n=4) and IIA (n=1). All patients received a bortezomib-containing regimen as first-line induction therapy and received Melphalan 200 mg/m2 as conditioning regimen before the first ASCT. All patients urderwent second ASCT following Bendamustine (100 mg/m2 days -4 and -3) and Melphalan (140 mg/m2 day -2) as conditioning regimen. G-CSF were given at day 5 after transplant. Results: A median number of 4.9x106/kg of CD34+ cells (range: 4-6.2) was infused. All patients engrafted, with median time to reach neutrophil〉500/µl and platelet 〉20.000/µl of 12 days (range, 11 to 15) and 14 (range, 11 to 19), respectively. Overall, the BM regimen was well tolerated. Almost all patients experienced mucositis, nausea and diarrhea but all events were of grade 1 or 2 (grade 3 diarrhea occurred in only 1 patient). Four patients (33%) had fever (grade 1 or 2) that was clinically identified in 2 cases (pneumonia and cystitis). The median time of duration of fever was 4 days (range, 2-6). No TRM occurred after at least day + 90 after transplant. Overall, no added toxicity were observed between the first and second ASCT. At day + 90 after first and second transplant response rate was respectively: 1 CR, 10 VGPR, 1 PR and 4 CR, 5 VGPR, 1 PR, 2 too early. Three patients in VGPR after first transplant converted to CR after second ASCT. Conclusion: Bendamustine plus Melphalan is feasible as conditioning regimen for second ASCT in patients with MM. The regimen was well tolerated and the toxicity profile of ASCT with conditioning regimens BM or Melphalan is comparable. Longer follow-up is needed to evaluate conversion rate and survival. References: Mark TM et al. A phase 1 study of bendamustine and melphalan conditioning for autologous stem cell transplantation in multiple myeloma. Biol Blood Marrow Transplant. 2013 May;19(5):831-7. Disclosures No relevant conflicts of interest to declare.

Description: Background: Chronic lymphocytic leukemia (CLL) is characterized by phenotypic and functional defects of immune cells, which often emerge into increased susceptibility to infections and autoimmunity, and also contribute to immune evasion of cancer cells. Ibrutinib is a selective inhibitor of BTK that shows activity via its direct effects on crucial survival pathways in CLL cells. In addition to its anti-neoplastic effects, ibrutinib has also shown to have immunomodulatory properties. Currently available data are mainly limited to the activity exerted by ibrutinib on conventional T cells, whereas little is known on the effects induced on other non-neoplastic immune cell populations. Aim: The aim of this study was to evaluate the in vivo immunomodulatory effects of ibrutinib treatment on different immune compartments in patients with CLL Methods: We included 11 CLL patients with progressive disease (PD CLL) and eligible to ibrutinib therapy. PB samples were collected from patients at baseline and after 1, 6 and 12 months of treatment with ibrutinib. For comparison, we also analyzed 5 healthy donors (HD) and 6 treatment-naïve CLL patients with stable disease (SD CLL), who were not fulfilling criteria for treatment start. We assessed the percentages and the absolute numbers of CLL cells, T cells, γδ (Vδ1 and Vγ9Vδ2) T cells, T regulatory cells (Tregs), natural killer (NK) and NK-T cells by flow cytometry using population-specific markers. The expression of activation markers and immune checkpoint receptors (i.e. CD69, PD-1, CD96, TIGIT, NKG2D) was evaluated by flow cytometry as well. Results: Median age of patients was 69 years (range 44-75). The median lymphocyte count at study entry was 49 x 109/L (range 1,8-110) and the median number of previous treatment regimens was 2 (range 0-5). After 12 months of ibrutinib, 10 out of 11 (91%) patients achieved at least a partial response. The mean absolute number of CLL cells started to decrease by month 6 and became significantly lower than baseline value at 12 months of ibrutinib therapy. At the baseline, leukemic cells from PD CLL had significantly higher surface expression of the early activation marker CD69 and of the immune checkpoint molecule PD-1 compared to SD CLL and HD. After 6 months of treatment, the percentage of CLL cells expressing CD69 and PD-1 was normalized (reached values not significantly different from HD) (Fig 1A,B). We observed a gradual reduction of the total count of CD4+ and CD8+ T cells during ibrutinib treatment, becoming significant at 12 months. At the same timepoint, the expression of CD69, which was higher on T cells from PD CLL prior to therapy compared to SD CLL and HD, was normalized. Concurrently, a significant reduction in the surface levels of the inhibitory immune checkpoint molecule CD96 was observed (Fig 1C,D). Ibrutinib treatment had no impact on the absolute numbers of NK and NK-T cells. Compared to SD CLL and HD, NK cells from PD CLL showed a higher expression of CD69 before treatment start. In addition, they were characterized by increased levels of the immune checkpoints CD96 and TIGIT, and by reduced expression of the Fc receptor CD16, that is involved in the ADCC process, and the activating receptor NKG2D. After 6 months of treatment, the expression of CD69, CD16 and NKG2D on NK cells were restored (Fig 1E-G), whereas TIGIT and CD96 were not yet significantly modulated. Concerning the Tregs, a trend toward a reduction in the absolute number was detected after 12 months of ibrutinib treatment, compared to the baseline. The percentage of Tregs expressing the co-inhibitory molecule TIGIT was higher in PD CLL at the time of treatment start and was normalized by 12 months of ibrutinib therapy (Fig 1H). Lastly, we assessed ibrutinib effects on γδ T cells. The absolute numbers of both Vδ1 and Vγ9Vδ2 T cells remained unchanged during patients' treatment. Similar to conventional T lymphocytes, Vγ9Vδ2 T cells showed a decrease in the expression of CD96 after 12 months of ibrutinib administration (Fig 1I). Conclusions: our data suggest that the anti-tumor activity of ibrutinib is paralleled by a valuable immunomodulatory effect, leading to a partial recovery of phenotypic alterations that are hallmarks of immune exhaustion. Further studies to investigate the ability of ibrutinib to restore the functionality of the described immune cell compartments and to explore clinical correlations are currently ongoing on an enlarged cohort of treated patients. Figure 1. Figure 1. Disclosures Mauro: abbvie: Other: board member; janssen: Other: board member. Gaidano:Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Foà:ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; NOVARTIS: Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; INCYTE: Other: ADVISORY BOARD; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD. Boccadoro:AbbVie: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Mundipharma: Research Funding; Novartis: Honoraria, Research Funding. Coscia:Janssen, Karyopharm: Research Funding; Abbvie, Gilead, Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees.