ALBERT

Description: BACKGROUND: The multiple myeloma (MM) represents a process in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. It is known that tumor progression are determined by a favorable tumor microenvironment (TME) and in this scenario fibroblasts represent the principal cellular component in the TME. A particular subpopulation of fibroblasts, cancer associated fibroblasts (CAFs), has recently raised the interest of many researchers due to their active participation in tumor growth and invasion and their association with higher malignancy grade, and poor prognosis. Recent findings indicate that the urokinase plasminogen activator (u-PA), and the urokinase receptor (u-PAR) are critical in cell invasion and degradation processes. Degradation and remodeling of the surrounding tissues are crucial in the early steps of tumor progression by facilitating expansion of the tumor mass, tumor cell proliferation, migration, and invasion. uPAR is expressed by multiple tumor associated cell types found in tumors. Targeting uPAR expressed on tumor-associated cells may be as important as targeting uPAR expressed on tumor cells and may lead to enhanced antitumor activity especially in those tumor types expressing uPAR on both types of cells. METHODS: Cell purification and cultures: BM mononuclear cells (BMMCs) were isolated by Ficoll-Hypaque gradient from heparinized bone marrow (BM) aspirates from 10 patients with relapse/refractory MM, 7 patients with asymptomatic MM, 7 with remission MM, 10 with MGUS. Fibroblasts were purified from BM stromal cells BMSCs through anti-fibroblasts-microbeads, and culture in DMEM medium with 10% FBS. Cell phenotype analysis: CAFs were analyzed on heparinized bone marrow aspirates and they were identified by FSP1 and α-smooth muscle actin (α-SMA) expression on gated CD45-population. Expression of alpha-SMA in BM purified fibroblasts was also demonstrated by immunofluorescence staining. Immunofluorescence: CAFs were cultured in DMEM medium, fixed in paraformaldehyde, and permeabilized according to routine methods. The primary antibodies were anti–uPAR, and anti–α-SMA. Fibroblast nuclei were stained with DAPI. Quantitative PCR analysis: Complementary DNA was prepared from 1 ug total RNA using a GoScript reverse transcription system. The relative quantity of uPA, uPAR, MMP-2, α-SMA and vimentin messenger RNA were measured using the Applied Biosystems 7500 Fast Real-Time PCR System and determined by the comparative Ct method using 18S ribosomal RNA as the normalization gene. The study was approved by the local Ethics Committee and all patients provided their informed consent in accordance with the Declaration of Helsinki. RESULTS: Cell phenotype analysis:Flow cytometry analysis showed that CAFs were increased in patients with relapse-MM compared to patients with asymptomatic, remission MM and MGUS suggesting that CAFs expansion is involved in MM progression. CAFs activation: The increased frequency of alpha-SMA in CAFs of relapsed MM patients was demonstrated by the immunofluorescence analysis. Overall, these results suggest that MM activation is associated with the overexpression of uPAR. (Fig.1) CAFs activation was also demonstrated by Real Time PCR and the figure 2 shows the overexpression of activation molecules as well as proinvasive systems in CAF of relapsed MM in comparison of MGUS and asymptomatic MM. CONCLUSIONS: In MM developement and progression the BM niche appears to play an important role in differentiation, migration, proliferation, and drug resistance of the malignant PCs. The main goal of this proposal was to globally approach the expression of CAFs’ activation and proinvasive systems in the initiation and progression of MM. Our results highlight an important mile stone on the phisiopatology of MM progression demonstrating that the CAF-activated phenotype is associated with an over-expression of the most important pro-invasive systems, and in particular the relapsed-MM CAFs seem to overexpress the fibrinolytic pattern of invasive tumor-like cells. On the basis of these results we aim to develop a biological model which can become a potential therapeutic target. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: The combination schedule of rituximab, fludarabine and cyclophosphamide is considered the standard therapy for fit and young untreated chronic lymphocytic leukemia (CLL) patients. However, although this therapy improves progression free survival (PFS) and overall survival (OS), it has been associated with increased toxicity: 76% of the patients experienced at least one grade 3 or 4 event. Recently, encouraging clinical results in terms of safety and efficacy have been obtained using bendamustine in combination with rituximab (R-B) in untreated CLL patients. PURPOSE: We performed a multicentre retrospective study to assess safety and efficacy of R-B in a large group of untreated CLL patients. METHODS: One hundred and thirty six untreated CLL patients were recruited from 16 Italian Institutions and included in the present analysis. The median age was 69 years (range 43–85), with 49.3% of patients older than 70 years; 53.7% of cases were male. All patients had active disease as defined by the NCI-WG, and 27.2% were at Binet stage C. FISH data, available in 86/136 cases, identified a del(17p) in 5.1% of the patients and del(11q) in 5.9%. Sixty-four patients (47.1%) had a creatinine clearance ≤70 mL/min. Fifty-six % of cases showed a WHO performance status (PS) of I-II and 61% a CIRS comorbidity index 〉0. RESULTS: Among the 136 patients, 90 cases (66.2%) received B at the dosage of 90 mg/m2 on day 1 and 2 every 28 days, the remaining 46 cases received B at the dosage of 70 or 80 mg/m2. R was administered at the dosage of 375 mg/m2 on day 1 of all cycles in 59.6% of cases, while 40.4% received 375 mg/m2 on day 1 of the first cycle and 500 mg/m2 on day 1 of the other cycles. A total of 725 cycles were administered with a median number of 6 cycles per patient. Eighteen patients (13.2%) required early discontinuation of therapy before the sixth cycle for serious infections (n=7), persistent hematological toxicity (n=5), grade 4 dermatological toxicity (n=3), withdrawal of consent (n=2), hypersomnia and depression (n=1). Grade 3 or 4 adverse events for neutropenia, thrombocytopenia, and anemia were documented in 25.8%, 25.8%, and 16.2% of patients, respectively. Grade 3 or 4 severe infections occurred in 6.6% of patients. The overall response rate (ORR) was 93.4%, 48 patients (35.3%) achieved a complete response (CR), 79 (58.1%) a partial response (PR), 8 (5.9%) a stable disease (SD) and in 1 patient (0.7%) no response assessment was performed due to death prior to terminating therapy. In the high-risk group with del17p, 5/7 (71.4%) cases achieved a PR and 2 a SD. Age 〉70 years (P=0.026) and del17p (P=0.007) were the only two parameters significantly associated with a lower response rate, while del11q and unmutated IGHV, as well as creatinine clearance 0 did not seem to impact on achievement of response. The only parameter predictive of the probability of achieving a CR was an age

Description: 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.

Description: 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

Description: 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.

Description: Background The combination of immunomodulatory drugs (IMiDs(r)) and alkylating agents have significantly improved outcomes of patients with relapsed or refractory multiple myeloma (RRMM). Bendamustine, an agent sharing properties of alkylators and purine analogous, and lenalidomide, an immunomodulator drug, are highly effective for the treatment of lymphoprolipherative disorders. Some clinical trials have shown good responses to the combination of bendamustine with steroids and novel agents (thalidomide, lenalidomide and bortezomib) for the treatment of RRMM (Knop S 2005, Lentzsch S 2012, Pönisch W 2013), but further research is needed to confirm the best combination, the optimal schedule of administration, and to better define the safety profile. Method A multicenter phase I/II trial tested the combination of escalating doses of bendamustine and lenalidomide, and a fixed dose of dexamethasone (BdL) in repeating 4-weeks cycles as treatment for RRMM. The drug dosages were chosen taking into account the haematological toxicity of both, and that patients were heavily pretreated. The phase I trial was conducted using a 3+3 cohort design beginning at a dose level 0 of intravenous bendamustine 40 mg/m²/day, days 1 and 2, oral lenalidomide 10 mg/day, days 1-21 and oral dexamethasone 40 mg/day, days 1, 8, 15, and 22 every 4 weeks. Patients were treated for 4 cycles + 2 cycles if the response was stable disease (SD) or better. In the phase II study patients were treated at the maximum tolerated dose (MTD) to evaluate the antimyeloma activity of BdL. Endpoints of the study included safety profile, overall response rate (ORR), progression-free survival (PFS), duration of remission (DR) and overall survival (OS). Results A total of 41 patients (17 in phase I, and 24 in phase II) were enrolled between October 2011 and July 2013. Four patients withdrew their informed consent: 3 before starting treatment and 1 after the fifth cycle. Twenty-eight out of 37 are currently evaluable. Median age of these patients was 68 (43-87 years), with a male/female ratio of 12/16. Sixteen patients had IgG MM, 10 had IgA, and 2 patients had light chain MM. Median number of previous treatments was 3; 9 patients were previously treated with thalidomide and 6 with lenalidomide; 19 with bortezomib and 11 underwent autologous stem cell transplant. The MTD established by the phase I study was dose level 0. To date, with a median follow up of 10 months, ORR is 50% (3 CR, 2 VGPR, 9 PR) and 1 SD. The median OS has not been reached yet. One-year OS is 78% (95% confidence interval [CI], 57%-89%). Median PFS is 10 months (95% CI, 5-12 months) with one-year PFS of 40% (95% CI, 21%-59%). Median duration of remission is 9.6 months with one-year DR of 44% (95% CI, 12%-73%; Figure 1). Toxicity data was available for 32 pts. Grade 3/4 adverse events (AEs) observed in ≥ 10% of patients included neutropenia, thrombocytopenia and anemia. The major non-hematological toxicity was renal dysfunction (6%). A total of 8 pts died during the study: 5 due to toxicity and 3 for progression of disease. Conclusions This study confirms that the combination of bendamustine with lenalidomide and low dose dexamethasone is effective in RRMM patients, even when administered after regimens containing novel agents. The dose of bendamustine in our study is lower (40 mg vs. 75 mg) that of previously published data (Knop S 2005, Lentzsch S 2012, Pönisch W 2013). However, we would like to emphasize that in our both phase I and phase II study we observed a consistent toxicity even utilizing 40 mg of bendamustine and 10 mg of lenalidomide. The toxicity was mainly hematological. Thus far, age, number of previous treatments, or the previous use of alkylating agents did not correlate with observed toxicity. In conclusion with a 50% ORR and 3 CR this is a very promising therapeutic option in RRMM requiring careful management of the patients. Disclosures: No relevant conflicts of interest to declare.