ALBERT

Description: Introduction: Immune reconstitution after stem cell transplantation (SCT) plays a crucial role in host defense against microbial agents. The thymus atrophy occurring with ageing represents a limit for de novo T-cell reconstitution, although the reactivation of thymic function after BMT is documented. The proper strategy to improve the thymic output is currently matter of debate. Pre-clinical and clinical evidences suggest that Zinc oral supplementation may contribute to thymic reactivation and to improve the T-mediated cellular defense against pathogens. We tested the role of Zinc oral supplementation in immune reconstitution, using as model the autologous SCT in multiple myeloma. Methods: From January 2014 to May 2016, we prospectively enrolled 18 patients (12 male, 6 female; average age: 58 years, range 43-72) undergoing single MEL 100 or 200 auto-SCT after one or two lines of therapy (VTD, lyposomal anthracycline and lenalidomide) and stem cell collection (mobilizing therapy: cyclophosphamide 3 g/sqm and G-CSF). The trial, prospective and randomized, has been approved by local ethics committee (EUDRACT: 2014-004499-47). All patients undersigned the informed consent. The clinical trial was carried out in accordance with Helsinki declaration. Randomization was effected at day 0 (day of PBSC infusion). Results: nine patients were treated from day +5 to day +100 after transplant with 600 mg/day of Zinc sulfate (uncoated tablets), whereas nine patients received only standard antimicrobial prophylaxis. Peripheral blood samples were collected in both groups at day +30 (t2) and day +100 (t3). Eight-colour flow cytometry was performed for CD3, CD4, CD8, CD45RA, CD45R0, CD27, CD28, CD25, CD127, with specific gates to identify specific lymphocyte populations (T naïve, T central memory, T effector memory, T terminal memory). The lymphocyte's population's count was statistically analysed intra- and inter-group with Wilcoxon test. Droplet-digital PCR for TRECs was performed on lymphocytes isolated by peripheral blood. A qPCR for viral load of Torquetenovirus (TTV), a harmless virus whose viral load is related to immunodepression, was performed. Zinc serum level was measured. Results: The only significant difference in the clinical features between the 2 groups was the mean age (58 years in the control group, 63 years in the sample). The recovery of naïve CD4 cells was visible in both groups from day +30 to day +100, but a significant increase was detected only in the Zinc group. Similarly, TRECs showed a significant increase in the treated group. CD8+ T cells showed a notable decrease until day 100 in the control group, specifically the CD8 naïve, memory, and effector memory populations. TTV load in the control group increased in inverse proportion to the decrease of CD8 population. In the Zinc group there were no differences between the TTV loads at the two time points. As a result, the viral load of TTV was higher in the control group than in the Zinc group at day +100. Zinc serum levels were normal in all patients. Conclusions: data show that Zinc plays a role in a faster recovery of TRECs and CD4 naïve T cells after autologous transplantation. Furthermore, Zinc seems to prevent the expected CD8 decrease of the control group, thus probably explaining the TTV reactivation. Zinc-deficiency was not observed in any of the patient; however, a Zinc supplementation may contribute to a better T-cell reconstitution. To the best of our knowledge, this is the first study describing the role of Zinc after stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (PC). Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification but the clinical and prognostic value of immunophenotyping in MM remains questionable. We have analyzed the prognostic impact of a relatively new marker as CD69. CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous laboratoristic data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. In a previous abstract, we described for the first time in a clinical report, the CD69 expression on pathological PCs of MM patients. Our preliminary data also suggested an intriguing role of CD69 in patients treated with chemotherapy in different stages of disease. In this study, we report a larger setting of 97 patients where we confirmed the expression of CD 69 in 48 of them (49%) (see table I). Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was 〉 20%. After an induction regimen of treatment with four cycles of VDT (bortezomib, dexametasone, thalidomide), 69 patients were evaluable. 40/69 (65%) of patients obtained at least of a very good partial response or better (Responding pts). In this subgroup of patients 30/45 (66.6%) showed the expression of CD 69. On the contrary only in a little part of partial or less responding patients (NR pts) 9/24 (37.5%) CD69 was detected (see table II) (p=0.02 using a chi squared test and p=0.019 using a Fisher's exact test). Data on cytogenetic abnormalities, including del(13q), t(4;14) and del(17p), detected by fluorescence in situ hybridization, were available in 〉90% of patients. Clinical data were available in all patients and CD69 maintained its association with different response, independently of other prognostic variables. In conclusion CD69 is often expressed in PCM cases, and the expression of this marker is useful to reveal poor prognostic categories and delineate a risk stratification. This molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs. Table I Pts Characteristics Total CD69+ 97 48/97 Sex Male 51(52%) Female 46(48%) Clinical status MM non evaluableMM after VTD 28/9769/97 in VGPR/CR 45/69 in PR/SD/PD 24/69 Table II Pts treated with VTD Responding pts NRpts 45 24 CD69+ 39/69 30/45 9/24 CD69- 30/69 15/45 15/24 Disclosures No relevant conflicts of interest to declare.

Description: Background: Multiple myeloma is still today an incurable disease. The many therapeutic techniques and new therapies proposed in recent years have extended survival but did not allow for healing. Further study allowed to demonstrate that a maintenance could be useful to control the progression of disease. However, there is no clear indication for which maintenance has to be used after a first line of induction therapy. The technique of allograft, used in patients at highest risk, demonstrates that the immune response to the residual disease plays a key role in the success of this technique. Among the major players in response to myeloma, in allogeneic stem cell transplantation, gamma delta lymphocytes play a significant action: complete response after allogeneic few months later (also the molecular level) happen in parallel with the presence in the bone marrow of a significant proportion of lymphocytes with gamma delta oligoclonal expression of TCR rearrangements. Zoledronic acid induces proliferation of these cells by the production of several cytokines, in particular interleukin-2 (IL-2). Furthermore, T lymphocytes Vdelta2 are proved to be crucial antineoplastic mediators and, after expansion in vitro, capable of controlling tumor growth in animal models. These data confirm the hypothesis that gammadelta lymphocytes have a role in controlling the growth of myeloma plasma cells and can be active on the residual disease after autologous stem cell transplant. We planned to evaluate the role of the association of Zoledronate and IL-2 in vivo as post ASCT maintenance therapy in patients with newly diagnosed multiple myeloma (NDMM). Methods: This is a single arm phase II multicenter ongoing study of the combination of IL-2 with zoledronic acid as maintenance therapy for NDMM patients post ASCT. The primary objective was to establish safety and efficacy of IL-2 as maintenance therapy. The secondary objective was to evaluate the immunological expansion of gamma delta lymphocytes. Eligible patients had undergone ASCT, with melphalan as a preparative regimen. At July 2016, forty two patients in very good partial remission (VGPR) have been enrolled in the study (total planned enrollment: 43 pts) and started maintenance therapy 90-180 days post ASCT. Maintenance schedule included IL2 and zoledronic acid. IL2 was administered at a fixed dose of 2 x 106UI from day 1 to day 7 for the first cycle and with the maximum tolerated dose (up to a max of 8 x 106UI) from day 1 to day 7 for subsequent cycles (dose escalation of 25% in each cycle in the absence of toxicity). Zoledronic acid was infused 4 mg iv on day 2. This dosing regimen is repeated every 28 days until disease progression. Adverse events were graded by NCI-CTCAE v4. Response was assessed by the modified International Uniform Response Criteria. Results and toxicity: 42 patients (pts) have been enrolled with a median age of 59 (range 42-72); 50% were male and 50% female. All the 42 pts have received a median of 11 cycles (range 1-23). Of the 42 pts 21 remain on therapy (data at July 2016), 21 pts are off study: 9 due to progressive disease (PD) and 12 due to consent withdrawal. Among the 9 pts with PD, the median PFS post ASCT was 12 months (2-18 months). Of the 42 pts, 33 (79%) not progressed after a median of 13 months (range 1-33) and the median PFS has not been reached. 7/42 patients (17%) reached complete remission. Peripheral and bone marrow analysis of gamma delta lymphocytes expansion to evaluate the level of immune response is still under examination. Grade 1/2 hematologic adverse events (AEs) included: grade 1 (G1) anemia (3 pts), G1 neutropenia (3). Grade 1/2 drug-related non-hematologic AEs included: G1 fever (25) G2 fever (8); G2 constitutional symptoms (joint pains) (20); G2 constipation (4); G1/2 nausea (10); G1 fatigue (15), G1/2 cutaneous rash (2). Conclusions: Long term administration of combination of IL-2/zoledronate as maintenance therapy post ASCT is feasible. The incidence of non hematologic adverse events (in particular fever) were manageable with no dose escalation of IL-2 over 5 x 106UI. This immunological approach, without any chemotherapeutic drug, seems to be able to control the disease and to obtain the complete remission in a subgroup of myeloma patients. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION Multiple myeloma (MM) is considered an incurable disease. Despite the introduction of novel agents allowed deeper response, high-dose chemotherapy and autologous stem cell transplantation (ASCT) remain the standard of care for patients (pts) in good clinical conditions. The most used strategies to mobilize stem cells from bone marrow (BM) into peripheral blood are high-dose cyclophosphamide (HD-CTX) plus G-CSF and G-CSF plus plerixafor (G-CSF+P). The goal of this retrospective study is to investigate whether the two different mobilization strategies have an impact on the clearance of monoclonal PCs in the apheresis products and on pts' outcome. PATIENTS AND METHODS We analyzed 62 pts (median age 61, range 41-75, 37 males and 25 women) diagnosed with MM and treated with ASCT between Mar 2014 and Mar 2018 at our Hematology Division (Pisa, Italy). All pts received induction therapy with at least 4 cycles of bortezomib, thalidomide and dexamethasone (VTD). 9/62 pts obtained a less than partial response (PR) and received lenalidomide-based regimens. After induction, 8 (12,9%) pts achieved complete remission (CR), 26 (41,9%) were in PR, 28 (45,2%) obtained a very good partial response (VGPR). 43/62 fit pts received HD-CTX (2-3 g/sqm) on day 1 followed by G-CSF (30 MU/day) started on day 4 until day 7, increased to 60 MU/day from day 8 until the end of apheresis. In 19/62 pts, after 4 days of G-CSF (60 MU/day) administration and not sufficient mobilization, we added plerixafor (0,24 mg/kgbw) for up to 4 consecutive days. In 43/62 pts we collected apheresis samples (10μl) analyzed through flow citometry to enumerate clonal residual PCs. The panel used to asses clonality included: CD138 Per-Cp, CD38 APC, CD19 PE-Cy7, CD45 APC-Cy7, cytoplasmic immunoglobulin K chain and L chain. RESULTS At the end of the peripheral blood stem cell (PBSC) collection, pts treated with HD-CTX presented a higher CD34+ absolute count (p=0.0489) and achieved the threshold of 5x106 CD34+ cells/kgbw in a significantly (p=0.006) higher percentage. We found a nearly significant (p=0.0517) lower count of CD34+ PBSCs in pts who received lenalidomide-based regimens before the mobilization. Performing flow citometry on apheresis samples, we observed that the number of the harvested clonal PCs showed a significant correlation (p=0.0115) with the occurrence of post-ASCT relapse. ROC curve analysis investigating the predictive effect of the number of pathological PCs on disease relapse showed an area under the curve of 0,6978 (95% CI 0.5392-0.8564; p=0.0267). Neither BM residual PCs detectable on BM biopsies performed before apheresis (r=-0.1323; p=0.609) nor the type of mobilization scheme (p=0.707) had an impact on the proportion of clonal PCs in the graft. Additionally, we did not observe any statistically significant difference in progression free- (PFS) (p=0.8276) and overall survival (OS) (p=0.2475) between the HD-CTX and G-CSF+P groups. DISCUSSION PBSC mobilization has a succession rate 〉 85%. Despite the use of HD-CTX to increase PBSC yields and decrease tumor burden, there is not clear evidence of a superior mobilization strategy. Additionally, HD-CTX has a not negligible toxicity and approximately 10% of the pts require hospitalization. Conversely, G-CSF+P is a safe and effective approach also in poor mobilizers. In our study, we observed a significative difference in the apheresis yields (p=0.0489) and in the percentage of pts who achieved the threshold of 5x106 CD34+ cells/kgbw (p=0.006) in favor of HD-CTX. Additionally, the detection of harvested residual clonal PCs could be a promising strategy to recognise pts more likely to relapse after ASCT. Nonetheless, we failed to demonstrate a superior effect of HD-CTX in the clearance of harvested clonal PCs, in agreement with the absence of a different pts' outcome amongst the two mobilization strategies. In conclusion, the choice between the two regimens is challenging and requires careful consideration of multiple factors. Overall, young fit pts, especially in the high-risk setting, should be treated with all appropriate modalities including chemiomobilization followed by double-ASCT. Conversely, in pts candidate to a single-ASCT it is reasonable to use G-CSF+P, since HD-CTX does not improve PFS and OS and add toxicity. The absence of an in-vivo purging effect on apheresis products of chemiomobilization further strengthens a chemotherapy-free mobilization. Disclosures Galimberti: Roche: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau.

Description: Abstract 3482 Three different types of PNH have been described so far, based upon the presence of hemolysis and bone marrow failure (BMF): a) florid PNH (hemolysis+/BMF-), b) PNH in the context of a BMF syndrome (hemolysis+/BMF+), c) subclinical PNH (hemolysis-/BMF+). In any case, the presence of a PNH clone is a prerequisite and Flow Cytometry (FCM) plays a crucial role in detecting and monitoring it with high sensitivity and specificity. In 2010, an Italian archive of FCM-detected PNH clones (http://int.clonepnh.com/) was created on a multi-laboratory basis. Eighty-one laboratories participated in the project. The aim was twofold: a) to provide a large dictionary of Italian PNH clones; b) based upon stringent rules regarding the compilation of records, to obtain an auto-educational effect on participating laboratories. Here, we describe the cellular composition and the clonal evolution of 295 type III (complete defect of GPI-linked proteins) PNH clones identified during the study. Forty-nine of these clones (16.6%) were accompanied by a PNH-II clone (partial defect of GPI-linked proteins). Hemoglobinuria was the most frequent (27%) reason for testing (RFT), followed by aplastic anemia (AA, 13%), MDS (13%), hemolytic anemia (12%), unexplained cytopenia (UC, 10%), BMF (5%), atypical venous thrombosis (5%), other (15%). Fluorescent Aerolysin (FLAER) was used since 2007, with an increasing % of utilization, from 4% to 60% of cases. CD24 utilization also progressively increased. CD59 was the most used antigen for RBC typing. The most used gating strategies were based upon physical parameters for RBC, CD45 and/or CD33 vs side scatter for granulocytes and monocytes. The 295 clones were categorized into 3 classes according to their size, determined as the percent of PNH cells in peripheral granulocytes: 0–10% (112 clones, or 38%, defined as “small”), 10.1%–70% (69 clones, or 23%, defined as “intermediate”), 70.1%–100% (115 clones, or 39%, defined as “large”). This distribution was significantly different from that expected on the basis of a random distribution within the three classes (i.e. 10%, 60% and 30%): chi square was 51 with a p value

Description: Bone marrow biopsy, (BMB) is essential to detect bone marrow (BM) infiltration in B-cell non-Hodgkin lymphomas (NHLs). Flow cytometry (FC) and PCR for clonal IgH rearrangement are considered as ancillary methods, but there is increasing evidence for their clinical usefulness. Observations dealing with combined use of the three methods still are lacking. Thus we carried out a retrospective study about the usefulness of an integrated approach to detect BM infiltration in NHLs. 193 patients suffering from NHLs (79 at presentation, 114 after chemotherapy alone or with: Rituximab, Campath-1, autologous BM transplantation), who had undergone simultaneous execution of BMB, and FC, and PCR from the myeloaspirate on the same iliac crest, were evaluated. BMB was carried out according to standard methods (infiltration pattern and immunohistochemistry). FC was performed using three-color staining, including CD45, to identify: κ/λ ratio, specific phenotype for CLL, MCL and HCL. PCR included identification of IgH rearrangement (CDR3 and VH families), BCL-1/JH translocation for MCL and BCL-2/JH translocation for follicular lymphoma. BMB, FC and PCR agreed in 142 cases and showed infiltration in 74 and lack of infiltration in 68. Cases at presentation were characterized by higher percentages of concordance than cases during the post-chemotherapy (84,8% vs 65.6%). Discrepant results were obtained in 51 cases (26.4%), 13 at presentation and 38 after treatment. In 17 specimens (8.8% of all cases, 33.3% of discordant cases), BM infiltration was detected only by PCR. In 12 of these samples (3 untreated and 14 treated) small B-cell percentages (0.50 ± 0.72, mean ± SD; range 0.02–3.00%) were present at FC. The remaining 5 cases (2.6%) were characterized by a lack of surface Ig expression and absence of specific phenotype: BMB was negative but IgH was clonal. 2 other cases with lack of surface Ig expression (for 7 cases in total, 3.6%), BMB-/PCR- were identified. Conversely, in 10 samples (5.2% of all cases, 19.6% of discordant specimens) PCR failed to detect BM infiltration, which was demonstrated by both FC and BMB. These specimens were characterized by high B-cell percentages (7 ± 8.25, mean ± SD; range 0.3–26.0%) and were obtained from 5 untreated and 5 treated patients. The remaining discordant cases were: 7 treated cases with BMB+/PCR+ and FC-; 6 cases (3 treated and 3 untreated) with FC+ and BMB-/PCR-; 3 treated cases with BMB- and FC+/PCR+; 3 cases (1 untreated and 2 treated) with BMB+ and FC-/PCR-. In the 3 treated cases with lack of amplification by PCR, the following results were observed: FC+/BMB+ in 1 case; FC-/BMB- in the remaining 2. Our data show that no single method is able to identify all cases of BM involvement in NHLs. BMB is actually considered the gold standard, however the combination of the three assays can increase the yields of detection of minimal residual disease. In fact, considering the three assays together as gold standard, BMB alone has a sensibility of 82.1%, a specificity of 96,3%, with 1.6% of false positive but 10.4% of false negative. The PCR can increase the sensibility and FC can than be considered as a valid confirmation assay, able to solve the cases when BMB and PCR show discrepancy. To conclude, the three assays are necessary to evaluate BM infiltration in NHLs, because BMB alone underestimate the BM involvement, especially following treatment.

Description: CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. CD69 has not been yet studied in human multiple myeloma, though it has been shown that human chronic lymphocytic lymphoma cells, when induced toward a plasma cell phenotype with tetradecanoyl phorbol acetate (TPA) have increased CD69 expression. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. Therefore, we analyzed the CD69 expression on pathological PCs, from bone marrow samples of 43 patients, by flow cytometry with two aims: to evaluate the real expression of CD69 on pathological PCs and to determine the clinico-pathological significance of this molecule. Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was 〉 20%. 22 of 43 pts (see table I, group A) were MM resistant/refractory to at least two different chemotherapy regimens (including bortezomib in all patients). 21 patients (table I, group B) were smouldering multiple myeloma (SMM) or MM in at least very good partial response (VGPR) after first line treatment. CD69 was detected on bone marrow PCs in 19 of the 43 patients evaluated (44%). Of the 19 patients with CD69+ (see table II) only 6 (27%) were in the group of refractory/resistant MM, while the majority of these advanced patients, 16/22 (73%), had an absent expression of CD69. On the contrary in the group of SMM/VGPR/CR MM 13 patients (62%) were CD69+ (p=0.04, using a Chi squared test with Yates correction). At the best of our Knowledge this is the first clinical report that confirms CD69 expression on pathological PCs of MM patients. Our preliminary data also suggest an intriguing role of CD69, this molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs. Table IPts CharacteristicsGroup AGroup B2221SexMale8(36%)11(52%)Female14(64%)10 (48%)Clinical statusSMMMM inVGPR/CR9 (43%)12 (57%)Relapsed/refractory22(100%)Number of Previous Therapy (range)3,5 (2-6)1 (0-1)Previous Bor regimenSMM0MM inVGPR/CR12(100%)Relapsed/refractory22(100%)Previous Lena regimenSMM0MM inVGPR/CR0Relapsed/refractory17(77%) Table II Pts Results Group A Group B 22 21 CD69+ 19/43 (44%) 6 (27%) 13 (62%) CD69-24/43 (56%) 16 (73%) 8 (38%) Disclosures No relevant conflicts of interest to declare.