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The mutual interactions between the host immune system and cancer cells may either promote or control oncogenesis. Cancer immunotherapies remain viable approaches to sustain patient survival. However, positive clinical response of phase I/II clinical trials remains still to low. The Double negative T cells (DNTs) have emerged as new subset of T cells that contributes specifically to anti- tumor immunity since involved in immune regulation and tolerance acting as regulatory T cells (Treg) and/or cytotoxic T cells. DNTs express either αβ or γδ T-cell receptors (TCR) or lack CD4, CD8 and CD56. Functional studies showed that DNTs might have a direct in vitro anti-tumor activity against lymphoma and melanoma cells. However no data are available on their prognostic significance, modulation during the therapy response and their ability to kill tumor cells in cooperation with other immune cells such as DCs to explain their role in human anti-lymphoma immunity. Knowledge of the DNTs role in tumor surveillance and as prognostic factor for clinical outcome has a strong clinical valence and might allow us to design new approaches of therapeutic strategy. The aim of this study was to evaluate the DNTs during therapy response in lymph nodes (LN), bone marrow (BM) and peripheral blood (PB) of both lymphoma (Ly), Renal Cell Carcinoma (RCC) and Metastatic Melanoma (MM) patients (pts) in order to assess their role on clinical outcome, progression and tumor surveillance. PB and BM samples of 90 Ly pts, with NHL and cHL were collected at diagnosis and during the follow-up (1-6-12 months after chemo- or immuno-chemotherapy therapy). PB samples of 16 healthy donors were collected as control. Twenty fresh LN tissues from pts clinically suggestive of Ly were quickly processed. To evaluate the interaction of DNTs with tumor burden either 22 samples from RCC pts and 30 samples from MM pts were collected. The ontogeny, functional attitude and TCR clonality of DNT subsets (TCRαβ+ and TCRγδ+) were evaluated by following antibodies: CD3, CD4, CD8, CD56, CD45, TCRαβ, CD45Ra, CD45Ro, CCR7, CD27, CD28, CD30, CD69, GITR, CD95, CD178, CD152, IFN-γ, TNF-α, perforin and granzyme B. For functional studies, DNTs were purified from PBMCs through a negative selection and then cultured for 2 weeks. Data were acquired by 8-colour flow cytometer and analysed using Kaluza software. Other immune cells such as DCs, MDSCs and Treg was also detected to evaluate the correlation with DNTs. 7 cases of LNs received a finally diagnosis of reactive follicular hyperplasia (RFH) so they were considered as controls. All pts provided their informed consent in accordance with the Declaration of Helsinki. We observed a significant decrease (p =0.006) of circulating αβ-DNTs in untreated Ly pts (20.5 cells/ul ± 4.8) as compared with healthy controls (31.3 cells/ul ± 3.4) (fig.1-2) and their number seemed to be modulated during the follow-up. Their frequency in 1-month post-cht or disease relapsed pts was significantly decreased (p=0.006) as compared with the diagnosis as well as when compared indolent with aggressive histotypes (p=0.0001). In HL pts the frequency of αβ-DNT was significantly increased as compared with other histotypes (p=0.005) (Fig.3) Furthermore, the αβ-DNTs in LNs of Ly pts were significantly reduced as compared to to RFH-LNs (p=0.006) (fig.5) and are related to the aggressiveness of the disease. When evaluated either in MM and RRC pts the αβ-DNTs were significantly decreased (p=0.001) as compared with healthy controls and more interestingly when compared with Ly pts (p=0.001) given the greater immunological impairment of RCC/MM tumor burden (fig.6). Finally, ex vivo expanded DNTs acquired an immunomodulatory cytokine profile, characterized by the secretion of IFN-gamma and granzyme B, which are known as central components of anti-tumor immune responses supporting the hypothesis of αβ-DNT potential use in immunotherapeutic strategies (fig.4) Our preliminary data, showed an inverse correlation between the frequency of circulating αβ-DNTs and the tumor condition as well as that they could play an important role in both the development and the progression of lymphomas. This is the first study that compares the frequency of αβ-DNTs in three different pathologies correlating to tumor burden. In addition, it is likely that ex-vivo expanded DNTs exert an anti-tumor activity thus suggesting their possible use as a new strategy for adoptive immune-therapy. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

BACKGROUND. Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm characterized by an aberrant protein (BCR–ABL) which is a constitutively active tyrosine kinase. According to the latest ELN recommendations for the management of CML, molecular response (MR) is best assessed according to the International Scale (IS) as the ratio of BCR-ABL1 transcripts to ABL1 transcripts, or other internationally recognized control transcripts. It is expressed and reported as BCR-ABL1% on a log scale where 10%, 1%, 0.1%, 0.01%, 0.0032%, and 0.001% correspond to a decrease of respectively 1 (MR1), 2 (MR2), 3 (MR3), 4 (MR4), 4.5 (MR4.5) logs below the standard baseline that was used in the IRIS study. Recent advances in the proteomic field have allowed us to better understand the biology of several cancer types and/or discover new candidate biomarkers, but very few data are available in CML. AIMS. The purpose of this study was to evaluate a possible correlation between depth of MR and proteomic profile in sera samples obtained from the peripheral blood and bone marrow of CML patients. PATIENTS AND METHODS Samples were consecutively and prospectively obtained from 20 CML patients observed between January and June 2014 at the Hematology Unit of the National Cancer Research Centre “Istituto Tumori Giovanni Paolo II” in Bari, Italy. Each individual involved in the study signed an informed consent form authorizing the Institute to utilize their biological tissues for research purposes. All patients at diagnosis displayed the classic t(9;22) Ph chromosome according to standard cytogenetics. The BCR/ABL transcript at RT-PCR was b3a2 in 13 patients and b2a2 in 7 patients. Peripheral blood and bone marrow samples were centrifuged within 30 minutes of sample taking. Serum specimens were immediately collected and frozen at −80°C. Twenty sera from peripheral blood were sampled from 5 patients in MR1 response, four in MR2, eight in MR3, two in MR4 and 1 patient at diagnosis; for eleven patients serum from bone marrow was also available; in particular 2 were sampled from patients in MR1, 3 in MR2, 4 in MR3, 1 in MR4 and 1 at diagnosis. Patients were grouped in two cohorts: the first comprised those with lower molecular response to MR3 (group A: 10 patients) and the second greater than or equal to MR3 (group B: 10 patients). The association of proteomic profile with molecular response was performed using the SELDI ToF Mass Spectrometry platform. Each specimen was spotted on an IMAC30 metal affinity protein-chip, prepared according to the manufacturer's instructions, and analyzed in duplicate. RESULTS Fourteen differentially expressed peaks were highlighted when comparing peripheral sera from group A and group B, but none was statistically significant. When comparing 11 available serum samples from the bone marrow of groups A (6) and B (5), four peaks (m/z 10629, m/z 3889, m/z 7772, m/z 7987) were reported as differentially expressed in a statistically significant way (p CONCLUSIONS These preliminary data suggest that an over-expression of m/z 11092 in serum obtained from peripheral blood and bone marrow could be associated with a deeper molecular response; further investigations are needed on a larger number of patients in order to confirm or refute our results and, to definitively characterize the peak at m/z 11092. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is a clonal disease characterized by an increased number of malignant plasma cells (M-PCs) that accumulate in bone marrow (BM). High dose of chemiotherapy followed by autologous peripheral blood stem cell transplantation produces a high rate of complete remissions; however the duration of such responses does not usually exceed three years due to the persistence of residual tumor cells, known as minimal residual disease (MRD), responsible for tumor relapse. The aetiology of malignancy remains unknown, although the BM microenvironment plays a critical role in the genesis by stimulating the proliferation of M-PCs and inhibiting the apoptosis. The aim of this study is to analyse and compare the phenotype of M-PCs with that of normal PCs as well as to correlate it with clinical outcome of MM patients in order to identify significant phenotypic differences able to 1) contribute to the differential diagnosis of monoclonal gammopathies by clearly identifying and characterizing M-PCs; 2) identify new prognostic markers; 3) evaluate MRD and monitor treatment efficacy; 4) identify new potential specific markers of M-PCs suggesting antibody-based targeted therapies. A total of 45 patients (pts) were enrolled in the study and their BM samples collected at the diagnosis: 15 patients with active MM, 10 with non-active MM, 10 with MGUS and 10 controls. The BM samples of MM patients were prospectively collected following each course of therapy, and in particular at time +100 after HDT/ASCT, as well as at the time of relapse or progression. At each consultation a multiparametric flow cytometric immunophenotype evaluation was performed using the following monoclonal antibodies: CD56, CD19, CD138 (CD38), CD45 and immunoglobulin κ and λ light chains to identify PCs . The CD117, CD40 and CD27 antibodies were used to distinguish distinct populations of PCs. A direct 8-color method was applied to study the immunophenotype of PCs, utilizing a Navios Cytometer (Beckaman Coulter), and analysing with Kaluza software. Immunophenotype data were compared with them of immunofixation (IMF) and polymerase chain reaction (PCR) approaches. The study was approved by the local Ethics Committee and all patients provided their informed consent in accordance with the Declaration of Helsinki. Significant immunophenotype changes were found in the marrow infiltration of M-PCs, and, mainly, in the aberrant expression of CD27 and CD40/CD117. Almost all patients (93,3%) with active MM showed the characteristic immunophenotype of M-PCs with aberrant expression of CD56 and concomitantly lack of CD19 and CD27. When the expression of CD27 was evaluated in M-PCs following the therapy and compared with both IMF and PCR data seemed to be modulated by disease relapse/progression or disease treatment (fig.1) resulting a significant increased (P=0.006) in pts with complete response (CR) (IMF and PCR negative) as compared with them relapsed and lost with myeloma progression. An inverse correlation was observed by evaluating the expression of CD40 and CD117 that seemed be modulated by disease treatment. Following the therapy the simultaneous expression of CD40/CD117 was observed in almost pts (86%) with negative IMF and positive PCR. Conversely this characteristic immunophenotype fingerprint was absent in all pts with CR. However the expression of CD117 in CD38+CD138+ cells was greater in MGUS or non-active MM pts than in pts in CR (p In the present study, we assumed that the contribution of different antigens assessed simultaneously would give more information to diagnosis of MM pts and to better stratify for risk of relapse/progression in order to initiate the most appropriate treatment, and to monitor treatment efficacy. CD19 and CD27 together resulted in optimal accordance with IMF and PCR approaches. Conversely CD40/CD117 showed a good accordance with disease response to the treatment. Our next goal will be to enrol more patients in order to generate a model of forecasting that evaluates simultaneous all three antigens assigning each one a specific weight in the contribution of diagnosis/prognosis. This will be useful to identify a immunophenotype fingerprint on which to design antibody-based targeted therapies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Many aspects of lymphoma pathophysiology indicate mutual interactions between the host immune system and lymphoma cells. These interactions may either promote or control lymphomagenesis. An unconventional subset of CD4-CD8- double-negative T cells (DNTs) has been recently described to contribute specifically to anti-tumor immunity. Indeed, DNTs are involved in immune regulation and tolerance as well as in host defense and inflammation, acting as regulatory T cells and/or cytotoxic T cells. DNTs are T lymphocytes which express either αβ or γδ T-cell receptors (TCR) and lack CD4, CD8 and CD56. In healthy human donors and murine models, they constitute about 1-5% of the lymphocytes in peripheral blood and in lymphoid organs. No data are available on the role of DNT cells in human anti-lymphoma immunity. Information from murine models suggests that expanded DNT cells would not impair host immunity against lymphoma and would perhaps stimulate it. DNT cells have also demonstrated to have a direct in vitro anti-tumor activity against lymphoma. Few data are available on the prognostic significance of DNTs in lymphomas, on their interaction with other immune cells, and on their functional attitude. Aims The aim of this study was to assess the frequency and the functional attitude of circulating DNTs in Lymphoma patients and healthy donors as controls, in order to assess the role of DNTs on clinical outcome and progression. Methods To test this population as prognostic factor on clinical outcome and progression of lymphoma disease, peripheral blood (PB) and bone marrow (BM) samples of 46 Lymphoma patients (pts), with non-Hodgkin's Lymphomas and classical Hodgkin Lymphoma were selected and prospectively collected at diagnosis and after one month till the end of chemo- or immuno-chemotherapy therapy. Blood samples were collected also at the time of relapse or progression. As control PB samples of 16 healthy donors were collected. Circulating DNT subsets (TCRαβ+ and TCRγδ+) were characterized for their ontogeny, tolerogenic or cytotoxic attitude and TCR clonality by staining with the following conjugated monoclonal antibodies (MoAbs) for surface and intracellular markers: CD3, CD4, CD8, CD56, CD45, TCRαβ, CD45Ra, CD45Ro, CCR7, CD27, CD28, CD30, CD69, GITR, CD95, CD178, CD152, IFN-γ, TNF-α, granzyme B, and perforin. Isotype-matched MoAbs were used as staining controls. For functional studies, DNTs were purified from PBMCs of patients through a negative selection by using specific MACS microbeads and then cultured for 2 weeks in complete medium supplemented with anti-CD3 (OKT3), rhIL-2 and rhIL-4. Data were acquired using an 8-colour flow cytometer and analyzed using Kaluza software. Data were compared among the groups using the Mann-Whitney non-parametric test or Kruskal–Wallis one-way analysis of variance. The study was approved by the local Ethics Committee and all patients provided their informed consent in accordance with the Declaration of Helsinki. Results The percentage (mean + SE) of DNTs in BM (2.367 ± 0.5891) of Lymphoma pts was lower than in PB samples (3.421 ± 0.981). Moreover we observed a significant decrease (p = 0.006) of circulating αβ-DNTs in pts with untreated lymphoma (23.7 ± 3.7) as compared with healthy controls (31.3 ± 3.4), and their number seemed to be modulated by disease relapse/progression or disease treatment. (fig.1). In Hodgkin's Lymphoma circulating αβ-DNTs were significantly increased as compared with other histotypes (p = 0.0001) (fig.2). Circulating αβ-DNTs were significantly decreased (p=0.006) in serial samples collected after treatment or at the time of disease relapse. Interestingly, after ex vivo expansion, DNTs acquired an immunomodulatory cytokine profile, characterized by the secretion of IFN-γ and granzyme B which are known as central components of anti-tumor immune responses (fig.3). Conclusions To date, no data have been reported on DNT phenotypic and functional characterization in Lymphoma patients. Our study has demonstrated for the first time that αβ-DNTs could play an important role in both the development and the progression of lymphomas. In addition, based on our preliminary results, it is likely that ex-vivo expanded DNTs exert an anti-tumor activity thus suggesting their possible use as a new strategy for adoptive immune-therapy. Disclosures: No relevant conflicts of interest to declare.