Abstract: Trisomy 12 is the third most common cytogenetic abnormality in CLL with several distinguishing features including abormal morphology, high prevalence of NOTCH1 mutations (NOTCH1 M) and increased expression of the alpha-integrins CD11a and CD49d (Balatti, 2012; Zucchetto, 2013). Trisomy 12 marks a disease subset characterized by high rates of cell proliferation, disease progression and Richter syndrome transformation (Rossi, 2013). Noteworthy, NOTCH1 M implicate constitutive activation of NOTCH1 signaling which triggers apoptosis resistance and increased survival of CLL cells (Rosati, 2009). Moreover, bax/bcl-2 ratio (bax/bcl-2), marker of apoptosis, predicts chemoresistance and progressive disease in CLL (Del Principe, 2016). The today availability in clinical use of venetoclax (ABT-199), a potent oral anti-bcl-2 peptidomimetic (Seymour, 2014), prompted us to analyze the real impact of the apoptosis (bax/bcl-2) on trisomy 12 CLL prognosis. The primary aims of our research were: 1) to demonstrate a significant association between NOTCH1 M and bax/bcl-2; 2) to correlate NOTCH1 M and bax/bcl-2 with biological and clinical prognosticators; 3) to determine progression-free survival (PFS) and overall survival (OS) upon NOTCH1 M and bax/bcl-2; 4) to evaluate NOTCH1 M and bax/bcl-2 as independent prognostic factors. Therefore we investigated 653 CLL patients, median age 66 years, 363 males and 290 females. Using the 5% cutoff for the FISH cytogenetic abnormalities, 140 (21.4%) cases were classified as trisomy 12 CLL. Bax/bcl-2 was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value 〉 1.5 (range 0.32-5.30). The presence of NOTCH1 M was investigated with ARMS PCR for c.75447545delCT and by Sanger sequencing of NOTCH1 exon 34. CD49d was 〉 20% in almost all trisomy 12 pts (116/140; 83%). Forty-five patients were NOTCH1 M (32.1%) and 45 were bax/bcl-2 positive (32.1%). There was a very strong correlation between bax/bcl-2 〈 1.5 and NOTCH1 M (42/45; p 〈 0.0001), corroborating the close dependence of the lacking apoptosis on NOTCH1 M. Both lower bax/bcl-2 and NOTCH1 M were significantly associated with lymphocyte doubling time 〈 12 months (p=0.0077 and p=0.0006), with beta-2 microglobulin 〉 2.2 mg/dl (p=0.0001 and p=0.0007) and LDH 〉 300 U/L (p=0.004 and p=0.00001), thus confirming their significant association both with a high proliferative rate and a high tumor burden. Strong correlations were found between NOTCH1 M or bax/bcl-2 〈 1.5 and IGHV unmutated status (p 〈 0.0001 and p=0.0005) or ZAP-70 〉 30% (p 〈 0.0001 and p=0.00002). With regard to clinical outcome, significant shorter PFS and OS were observed in patients with NOTCH1 M vs NOTCH1 wild type [WT] (0% vs 27% at 10 years, p=0.001 and 26% vs 78% at 14 years, p=0.005) or lower bax/bcl-2 (9% vs 38% at 10 years, p=0.0007 and 45% vs 89% at 14 years, p=0.002). Interestingly, NOTCH1 M and bax/bcl-2 showed synergistic prognostic properties, since NOTCH1 M and bax/bcl-2 〈 1.5 identified a trisomy 12 CLL subset at worst prognosis with regard to PFS (0% vs 40% at 10 years, p=0.00004, Figure) and OS (20% vs 100% at 14 years, p=0.0001, Figure). Therefore, bax/bcl-2 and NOTCH1 M are powerful prognosticators, showing synergistic clinical effects. In multivariate analysis of OS, bax/bcl-2 (p=0.004) together with age (p=0.0006), CD49d (p=0.01) and IGHV status (p=0.03) was confirmed as an independent prognostic factor. In conclusion, the modern strategies to trigger apoptosis and block proliferation, such as venetoclax, may be very effective in this CLL subset, also taking into account that trisomy 12 CD49d+ CLL has abbreviated lymphocytosis with retention of CLL cells within tissues (Thompson, 2014) and consequent poor reduction of lymphadenopathy during treatment with ibrutinib. Figure 1 Figure 1. Disclosures Lo Coco: Teva: Consultancy, Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; Novartis: Consultancy; Baxalta: Consultancy; Pfizer: Consultancy.

Abstract: Introduction: few studies have explored the usefulness of a prognostic index specifically devised for patients with localized DLBCL. The IIL has performed a retrospective analysis of a large group of patients with limited stage DLBCL and developed a new prognostic model. Results: 1,252 patients with localized (Ann Arbor stage I-II) aggressive B-cell lymphoma (IWF: G or H, WHO:DLBCL) diagnosed from 1988 to 2002 and without CNS involvement, treated by 4 cooperative groups and 2 single institutions, are the subject of this analysis. Patient’s median age was 57 years (range, 17–91) and M/F ratio was 1.26. Clinical stage was I in 239 (19%), IE in 303 (24%), II in 356 (28%) and IIE in 354 (28%) patients, respectively. Supradiaphragmatic disease was present in 56% of patients, 13% had 〉 3 nodal sites, 53% had extranodal involvement, and 7% had 〉 1 extranodal site. Bulky disease (≥10 cm) was present in 26% of patients, ECOG-PS 〉 1 in 12% and B symptoms in 14%. Abnormal biochemical data included: elevated LDH (28%), β2-microglobulin (B2M;19%) and ESR (38%) and reduced albumin ( 〈 3.5 g/dL) in 21% of the cases. Patients were treated with anthracyclin-containing regimens ± IF-RT. After a median follow-up of 62 months for alive patients (range 1–183 months), 3 and 5-year OS rates were 73% and 71%, respectively. By univariate analysis the following 11 variables were found to be predictive of a short survival: age ≥65 yrs (P=0,0001), stage II nodal (P=0,0001), number of nodal sites (P 〈 0.0001), poor ECOG-PS (P 〈 0.0001), B symptoms (P=0.0009), bulky disease (P=0.0001), elevated ESR (P=0.0001), LDH (P 〈 0.0001), Radiotherapy (P 〈 0.001), B2M (P=0.007) and reduced serum albumin ( 〈 0.0001). By Cox multivariate analysis, age ≥65 years (p 〈 0.001), stage II nodal (P 〈 0.001), high LDH (P 〈 0.001) and bulky disease (P 〈 0.01) were indipendent risk factors (RF) for a short survival. The prognostic model was calculated with the sum of scores assigned to each variable; a score of 2 was assigned to advanced age, high LDH, and Bulky; for Stage a score of 1 was considered for stage Ie-IIe and 2 for stage II nodal. Three groups of patients with a different probability of survival (P 〈 0.000001) were identified. Patients at low (Score 0–1; 387 pts), intermediate (Score 2–3; 484 pts) or high risk (Score 4–8; 381 pts) had a 5 years OS of 87%, 77%, 51% respectively. The predictive performance of the model was internally validated through a non parametric Bootstrap method and through residues’ analysis. Conclusions: This prognostic model, developed and validated on a very large series of patients with localized DLBCL, will allow us to select appropriate therapeutic approaches on the basis of different risk categories.

Abstract: Abstract 2870 Chronic lymphocytic leukemia (CLL) is a very heterogeneous disease ranging from rapid disease progression leading to death to a nearly normal life expectancy and therefore it is mandatory to find valid prognostic markers. Recent studies showed that activating mutations of NOTCH1 proto-oncogene occur in about 10% CLL at diagnosis and are associated with an unfavorable clinical outcome (Rossi et al, 2012). About 85% of NOTCH1 mutated CLL cases displayed a DCT7544–7545 frameshift deletion (hereafter NOTCH1 mutation), that has been demonstrated to predict NOTCH1 degradation impairment through the truncation of the C-terminal PEST domain. Given the possibility of targeting NOTCH1 with drugs currently under development, the primary endpoints of our research were: 1) to correlate NOTCH1 mutation with other clinical and biological prognostic factors; 2) to determine time to first treatment (TTFT) and overall survival (OS) upon NOTCH1 mutation in univariate analysis; 3) to validate NOTCH1 mutation as an independent prognostic factor. We investigated 463 pts, median age 65 years (range 33–89), 256 males and 207 females. With regard to modified Rai stages at diagnosis, 159 had a low stage, 290 an intermediate stage and 14 a high stage. NOTCH1 mutation was investigated by amplification refractory mutation system (ARMS) PCR at diagnosis or before any chemotherapeutic approach. The ARMS PCR approach was set up in order to identify NOTCH1 mutation when present in at least 10% of the alleles. Using this approach, NOTCH1 mutated pts were 45/463 (9.7%). Considering the association with markers of tumor burden and proliferation, NOTCH1 mutation correlated with intermediate/high Rai stages (37/45; P=0.002), multiple thoracic/abdominal lymphadenopathies and/or splenomegaly (26/45, P=0.003), beta-2 microglobulin 〉 2.2 mg/ml (27/45; P=0.02), lymphocyte doubling time 〈 12 months (19/45; P=0.0006) and soluble CD23 〉 70 U/ml (26/39; P=0.00001). Significant associations were also found with the main biologic prognostic markers in CLL. In this regard, NOTCH1 mutation was associated with an unmutated IGHV status (available for 446 total cases, 30/43; P 〈 0.0001), CD38 〉 30% (26/45, P 〈 0.0001), ZAP-70 〉 20% (33/45; P 〈 0.0001), and CD49d 〉 30% (22/34; P=0.009). Finally, considering associations with specific chromosomal aberrations defined by FISH cytogenetics (available in 417 cases), significant correlations (P=0.003) were found between NOTCH1 mutation and trisomy 12 (14/41; 25%), and del11q (7/41;16% ), whereas only 2/43 NOTCH1 mutated cases presented 17p deletion. With regard to clinical outcome, 30/45 (67%) NOTCH1 mutated pts received chemotherapy vs 193/418 (46%) among NOTCH1 germ line CLL (P=0.01), with 15/45 (33%) vs 48/418 (11%) cases, belonging to the same subgroups, undergoing at least two lines of treatment (P=0.001). Moreover, both significant shorter TTFT and OS were observed in NOTCH1 mutated pts (7% vs 35% at 12 years, P=0.0006 and 34% vs 78% at 14 years, P 〈 0.0001; Figures A, B). To further test the clinical value of NOTCH1 mutation in CLL, we investigated its prognostic impact in bivariate analyses with the main clinical/biological prognosticators. According to these analyses, pts with NOTCH1 mutation showed shorter OS both within the ZAP-70 positive ( 〉 20%, 188 pts) and unmutated IGHV ( 〈 2%, 144 pts) subsets (24% vs 50% at 14 years; P=0.006, and 18% vs 52% at 14 years, P=0.04, respectively; Figures C, D). In multivariate analysis of OS, NOTCH1 mutation (P=0.02) together with age (P=0.001), FISH cytogenetics (P=0.001) and CD38 (P=0.002) was confirmed to be an independent prognostic factor, after correcting for colinearity with IGHV status. Therefore, NOTCH1 mutation, as determined by ARMS PCR, is a novel important prognostic parameter in CLL to be considered in drawing prognostic scores. In addition, NOTCH1 might represent a commendable therapeutic target for specific inhibitors to be employed especially in NOTCH1 mutated CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract: Introduction: Rituximab is a monoclonal antibody commonly used in B-DLCL both in salvage regimens and in combination with first-line chemotherapy. Some studies have reported delays in neutrophil or platelet engraftment after ASCT in patients treated with high dose chemotherapy and Rituximab prior to ASCT. We investigated the effect of adding Rituximab prior to ASCT on peripheral blood stem cell (PBSC) harvest, time to engraftment and short-term toxicity post ASCT. Patients and methods: We analyze stem cell mobilization, engraftment (median time to absolute neutrophil count & gt; 500/mm3 for three consecutive days and platelets & gt; 50.000) and short-term toxicity after ASCT ( & lt; 30 days) in two groups of patients affected by B-DLCL at diagnosis enrolled into two consecutive trials with or without Rituximab in combination with chemotherapy. All patients were & lt; 61 years with B-DLCL at age-adjusted IPI Intermediate-High (IH) or High (H) risk and/or with bone marrow (BM) involvement. Group A: an intensified chemoimmunotherapy regimen R-MegaCEOP (Rituximab 375 mg/m2 day 1, CTX 1200 mg/m2 + EPI 110 mg/m2 + VCR 1.4 mg/m2 day 3 and PDN 40 mg/m2 days 3 to 7) every 14 days with G-CSF support for 4 courses; patients in complete response (CR) or partial response (PR) received two courses of intensified chemotherapy R-MAD (Mitoxantrone 8 mg/m2 + ARA-C 2000 mg/m2/12h + Dexamethasone 4 mg/m2/12h days 1 to 3 and Rituximab 375 mg/m2 day 4 and before PBSC harvest). Group B: MACOP-B 8 weeks + 2 courses of MAD (in patients in CR or PR) without Rituximab. Both groups were given ASCT with BEAM as conditioning regimen. Clinical characteristics at diagnosis were well balanced between the two groups. Results: 68 patients are evaluable: 35 pts in group A (with Rituximab) and 33 pts in group B (without Rituximab). All patients in both groups collected more than 2 x 106 CD34+ cell/kg. Median CD34+ cell/kg in group A was 13 x 106 (range 0-37) compared with 41 x 106 in group B (range 0–253) with no statistically significant difference. Median time to neutrophils engraftment after ASCT was similar in the two groups: 9 days in group A and 10 days in group B. No delay in the platelet recovery was observed in patients treated with chemoimmunotherapy: 15 days in group A vs 16 days in group B. Few severe early toxicities (WHO grade 3–4) were reported with no differences between group A vs B: severe mucositis in 11 pts vs 23 pts, gastrointestinal in 6 pts vs 4 pts. Severe infections occurred in 10 patients: group A 6 patients (2 Gram+ sepsis, 1 Gram- sepsis, 1 FUO, 2 bacterial pneumonia); group B 4 patients (2 FUO, 1 bacterial and 1 viral pneumonia). One patient in group A died of bacterial pneumonia after ASCT. Conclusions: Treatment with Rituximab may be safely included in a pre-ASCT high dose chemotherapy regimen with no delay in stem cell harvest, engraftment and without increased early toxicity after ASCT.

Abstract: Background: The role of bone marrow response in patients with immune thrombocytopenia (ITP) has gained paramount importance since the last 10 years, with the demonstrations that marrow megakaryocytes (MGK) are unable to properly compensate platelets peripheral destruction. TPO receptor agonists (TPOa), namely romiplostim (ROMI) and eltrombopag (EPAG), by stimulating megakaryopoiesis are able to induce a response in 74% to 94% of cases in clinical trials. However, real world use of these drugs has shown frequent changes in individual dose requirement, the possibility of treatment discontinuation, and their effectiveness outside registered indications; moreover, nothing is known about predictors of response. Aim: To evaluate clinical and morphologic predictors of response in a real world cohort of ITP patients treated with TPOa. Methods: ITP patients treated with EPAG and ROMI from September 2009 to May 2018 at seven Italian Centers were evaluated. Clinical and hematologic parameters including treatment response and marrow characteristics were retrospectively collected. Results: Table 1 shows baseline clinical and morphologic characteristics for the 86 cases enrolled, altogether and divided according to the TPOa used: patients were mainly middle-aged females presenting with severe thrombocytopenia; anti-PLT autoantibodies were positive in 32.6% of cases, and 58.1% of cases presented with bleeding, 22% of grade III-IV. All cases had received 1st line treatment with steroids and 43% at list a 2nd line among those listed in Tab1. Pre-TPOa marrow evalutation showed hypocellularity in 30.2% of cases, reticulinic fibrosis in 33.7%, a polyclonal lymphoid infiltrate in 43% (mostly mixed or T-cell), and reduced MGK in 4.7% of patients. Some dysplastic features were present in about 50% of cases, either dysmegakaryopoiesis (46.5%) or dyserythropoiesis (25.6%). Median time from diagnosis to TPOa was 2.4 years (0.1-28.8). Patients were treated for a median of 1.4 years (0.3-10.8), and ORR at 3 months and 9 months were 75.6% (CR 44.2 and PR 31.4%) and 65.1% (CR 36 and PR 29.1%), respectively. Response rates to EPAG and ROMI were comparable. Regarding predictors of response, bone marrow hypocellularity (40 NR vs 21% ORR, p=0.05) and megakaryocytopenia (33 vs 6%, p=0.06) were significantly more frequent among NRs. Other factors associated with poor response were dyserythropoiesis (58 vs 26%, p=0.04) and erythroid hyperplasia (18 vs 8%, p=0.03), and presence of a T cell infiltrate (66 vs 18.9%, p=0.03). Finally, NRs cases showed significantly lower neutrophil counts at baseline (1.9 vs 2.3x103/mmc in ORR, p=0.01), and had been more frequently exposed to cyclosporine or azathioprine (50 vs 18% in ORR, p=0.01). Fifty-five patients are still on treatment, whereas 31 (20 EPAG/11 ROMI) discontinued because of NR or relapse (17), adverse events or intolerance (2); of note, 12 patients with ORR discontinued the drug because of sustained CR, and 7 of them are still in remission. 14/65(21.5%) responding cases (10 EPAG/4 ROMI) lost the response after a median of 6.2 months (1.8-60) and were variably managed (3 splenectomized, 1 switched from ROMI to EPAG, 1 received danazol, 5 were re-treated with EPAG, and the remaining were managed with steroids and supportive treatment). Median RFS was 2.3 years (0.1-10), longer in patients without megakaryocytopenia (9.9+0.5 vs 4.1+0.6, p=0.06), dyserythropoiesis (mean 9.1+0.5 vs 4.9+0.7, p=0.2), and reticular fibrosis (9.6+0.5 vs 5.5+0.6, p=0.08). During EPAG treatment 7 grade adverse events occurred: 2 grade IV (1 stroke with PLT counts of about 30x103/mmc, and 1 NSTEMI 1 month after EPAG discontinuation for sustained CR), 1 grade III pneumonia, and 4 grade I/II transaminase elevation. No events occurred under ROMI. Conclusions: TPOa use in the real world setting confirms their reported efficacy, the option to switch and/or re-treat with either EPAG or ROMI, and the possibility to discontinue the drugs. The presence of hypocellularity and megakaryocytopenia, along with dysplastic features and of a lymphoid T cell infiltrate are associated with a reduced response to TPOa and a shorter RFS. Pre-treatment bone marrow evaluation may give hints to unravel the physiopathologic mechanisms underlying TPOa refractoriness. Disclosures Rossi: MUNDIPHARMA: Honoraria; JANNSEN: Other; AMGEN: Other: ADVISORY BOARD; PFIZER: Other: ADVISORY BOARD; BMS: Honoraria; NOVARTIS: Honoraria; ROCHE: Other: Advisory Board; ABBVIE: Other: ADVISORY BOARD; TEVA: Other: ADVISORY BOARD; CELGENE: Other: ADVISORY BOARD; JAZZ: Other: ADVISORY BOARD; SANOFI: Other: ADVISORY BOARD; GILEAD: Other: ADVISORY BOARD; SANDOZ: Honoraria.

Abstract: Background. Ara-c based chemo-immunotherapy followed by autologous stem cell transplantation (ASCT) is the most effective approach in young mantle cell lymphoma (MCL) patients, though few if any patients are cured. Recent data indicate that subsequent Rituximab maintenance (RM) prolongs PFS and OS (Le Gouill NEJM 2017). Lenalidomide is an oral agent effective in MCL, considered suitable for prolonged maintenance programs, but has never been tested in this setting. The FIL MCL0208 trial (NCT02354313) is a prospective, international randomized, phase III trial, comparing Lenalidomide maintenance (LM) vs observation (OBS) after an intensive Ara-c containing chemo-immunotherapy (R-HDS) program, followed by ASCT in previously untreated MCL patients. Patients and Methods. Adult patients aged 18-65 years, with advanced stage MCL without clinically significant comorbidities were enrolled. Patients received 3 R-CHOP-21, followed by R-HDS i.e. R-high-dose Cyclophosphamide (R-HD-CTX) (4g/m2), 2 cycles of R-high-dose Ara-C (R-HDAC) (2g/m2 q12x3 d). CD34+ cells were collected after the first course of R-HDAC. The conditioning regimen for ASCT was BEAM. After ASCT, responding patients were randomized between LM (15 mg days 1-21 every 28 days) for 24 months or observation. Primary endpoint analysis was scheduled at the occurrence of the 60th PFS event in the randomized population, which occurred on June 20th, 2017 and data were analyzed for the present abstract on March 3rd 2018. Results. Three-hundred three patients were enrolled from May 2008 to August 2015 by 48 Italian and 1 Portuguese Center. Three patients were excluded after central histological review. Median age was 57 years (IQR 51-62), M/F ratio 3.6/1. Ninety-two percent of patients had stage IV, 33% bulky disease ( 〉 5 cm), 33% elevated LDH, and 75% BM infiltration. Ki67 ≥30% was observed in 32%, MIPI was low (L) in 54%, intermediate in 31% and high (H) in 15% of patients. MIPI-c was L in 49%, low-intermediate (LI) in 29%, high-intermediate (HI) in 14%, H in 9%. Nine percent had blastoid variant. Fifty-two (17%) patients interrupted treatment before randomization (8 toxic deaths, 1 death for car accident, 24 progressions and 19 toxicity/refusals). On an ITT basis, the R-HDS + ASCT program induced 78% of CR, 7% of PRs, 10% of PD, 3% of toxic deaths (TRM) and 2% NA. Median follow-up (mFU) from inclusion was 51 months. Three years PFS and OS for the enrolled population were 67% and 84%, respectively. Of 248 patients who received ASCT, 205 were randomized either to LM (n=104) or OBS (n=101) and 43 (17%) were not because of: lack of response (8), refusal/PI decision/delay (8), unresolved infections (3) and inadequate hematopoietic recovery (24). Feasibility and efficacy were assessed on an ITT basis while toxicity was analyzed on subjects receiving at least one Lenalidomide dose. In the LM arm, 53 out of 104 patients did not start or complete the planned maintenance because of death (2), AE (26), PD (7), still ongoing (2), other causes (16). In the OBS arm 32 patients did not complete the observation phase because of death (1), AE (1), PD (20), still ongoing (10), other causes (1). Overall 28% of patients received less than 25% of the planned Lenalidomide dose. Despite suboptimal exposure to study drug, with a mFU from randomization of 35 months, 22 PFS events were recorded in the LM cohort vs 38 in the OBS arm, resulting in a 3y-PFS of 80% (95% CI; 70%-87%) in the LM arm vs. 64% in the OBS arm (95% CI; 53%-73%), stratified HR 0.51; 95% CI 0.30-0.87; p=0.013 (Fig 1A). OS was superimposable in the two arms: 93% vs 86%, stratified HR 0.96, 95% CI 0.44-2.11, p= 0.91 (Fig1B). Two deaths were observed in the LM arm due to pneumonia and thrombotic thrombocytopenic purpura and one in the OBS arm due to pneumonia. Grade 3-4 hematological toxicity was seen in 63% of patients in LM vs 11% in the OBS arm with 59% vs 10% of patients experiencing granulocytopenia. Non-hematological grade 3 toxicity was comparable in the two arms except grade 3-4 infections (11% vs. 4%; Fisher's p=0.10). Second cancers occurred in 7 patients in the LM and 3 in the OBS arm (Fisher's p=0.20). Conclusions. Results from the MCL0208 trial indicate that LM has a clinically meaningful anti-lymphoma activity in MCL. However, the applicability of LM has some limitations in the context of patients undergoing intensified chemoimmunotherapy. Overall these data support the use of a maintenance regimen after ASCT in young MCL patients. Disclosures Ladetto: Roche: Honoraria; Celgene: Honoraria; Acerta: Honoraria; Jannsen: Honoraria; Abbvie: Honoraria; Sandoz: Honoraria. Di Rocco:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Rossi:Novartis: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Janssen: Membership on an entity's Board of Directors or advisory committees, Travel expenses; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Mundipharma: Honoraria; Sandoz: Honoraria; Seattle Genetics: Research Funding; Alexion: Other: Travel expenses. Chiappella:Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Roche: Other: lecture fees; Teva: Other: lecture fees; Nanostring: Other: lecture fees; Amgen: Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees. Rusconi:Celgene: Research Funding. Gomes da Silva:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Institution's payment for consultancy, Travelling support; Celgene: Other: Travelling support; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Roche: Other: Institution's payment for consultancy, Travelling support; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: lecture fees, Research Funding. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Speakers Bureau. Martelli:Sandoz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; F. Hoffman-La Roche: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract: We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin−CD34−) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR-ABL transcript demonstrated that about one third of CD34− cells are leukemic. CML Lin−CD34− cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells, cell cycling, acquisition of clonogenic activity and increased expression of BCR-ABL transcript. Lin−CD34− cells showed hematopoietic cell engraftment rate in immunodeficient mice similar to Lin-CD34+ cells whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell cycle arrest genes, genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin−CD34−cells. Imatinib mesilate did not reduce fusion transcript levels, BCR-ABL kinase activity and clonogenic efficiency of CML Lin− CD34− cells in vitro. Moreover, leukemic CD34− cells survived to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34− leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

Abstract: Regulatory mechanisms governing homing and engraftment of hematopoietic stem cells (HSCs) involve a complex interplay between chemokines, cytokines, growth factors and adhesion molecules in the intricate architecture of bone marrow (BM) microenvironment. HSCs express P2Y and P2X receptors for extracellular nucleotides, which activation by ATP and UTP has been recently demonstrated (Lemoli et al. Blood. 2004) to produce potent stimulatory effects on HSCs. Moreover extracellular nucleotides are emerging as key factors of flogosis phenomena and related chemotactic responses of several cell types, such as dendritic cells, monocytes and endothelial cells. In this study we investigated the biologic activity of extracellular ATP and UTP and their capacity to cooperatively promote SDF-1 (stromal cell-derived factor-1)-stimulated cell chemotaxis. Low concentrations of UTP (10uM) significantly improved, in vitro, HSCs migration. Moreover, UTP inhibits CXCR4 down-regulation of migrating CD34+ cells and increased cell adhesion to fibronectin filaments. Furthermore, in vivo competitive repopulation assays showed that preincubation with UTP significantly improved the homing efficiency of human CD34+ HSCs in nonobese diabetic/severe combined immunodeficient mice. Inhibition assays with Pertussis Toxin from B. Pertussis blocked SDF-1- and UTP-dependent chemotactic responses, suggesting that Gαi proteins may provide a converging signal for CXCR4- and P2Y-activated transduction pathways. In addition, gene expression profiling of UTP-treated CD34+ cells and subsequent in vitro inhibition assays with Toxin B from C. Difficile suggest that RhoGTPase Rac2 and his downstream effectors ROCK1 and ROCK2 are involved in the UTP-promoted, SDF-1-dependent HSCs migration. Taken together, our data suggest that UTP may physiologically modulate HSC migration and homing to the BM, in concert with the chemotactic peptide SDF-1, via the activation of converging signaling transduction pathways between CXCR4 and P2Y receptors, involving Gαi proteins and RhoGTPases.

Abstract: Background. Peripheral T cell Lymphoma (PTCL) represent a major therapeutic challenge. In the previous Verona experience (Todeschini G et al. Cancer2005;104:555–560), the novel intensive regimen HyperCHiDAM Verona897 (high-dose IV MTX 2 g/sqm 400 sqm bolus, 1600 sqm CI day 1 with IV leucovorin rescue; hyperfractionated IV CTX 300 mg/sqm Q12h, dd 2–4; high-dose IV AraC 2g/sqm Q12h, dd 2-4; plus G-CSF) achieved salutary results in refractory/relapsed aggressive lymphomas, in particular in PTCL. Supportive measures included hydratation 3000 ml/sqm/day, antimicrobial prophylaxis comprising oral ciprofloxacin, itraconazole, trimethoprim/sulfamethoxazole. CMV antigenemia (pp65) was monitored 2 times a week. Patients. Following these results, 7 centers belonging to the Italian co-operative group GITIL (Gruppo Italiano Terapie Innovative Linfomi) treated with 2 cycles of HyperCHiDAM Verona897, 33 patients affected by PTCL (17 upfront, 16 refractory/relapsed) followed in the majority of cases by stem cell transplant. Patients: M/F 21/12, median age 49 (19–63) years; histology: PTCL-NOS 15, ALCL ALK-negative 9, EALTC 4, AIL 3, T-NK nasal 1, Angiocentric 1; stage IV 19/33 (57.5%), bone-marrow positive 10/33 (30.3%), extranodal involvement 24/33 (72.7%), high LDH 18/33 (54.5%). Seven patients needing urgent treatment were treated before HyperCHiDAM with 1 cycle of Campath-CHOP (4 patients) or CHOP + L-ASE (3 patients). Results. Upfront patients: after 2 cycles of HyperCHiDAM, CR were 82.3% (14/17), early toxic deaths 0 (1 late toxic death occurred after SCT, due to CMV pneumonia), relapses 3/14. With a median follow-up of 21 months (3–90+), 11/17 (64.7%) patients are disease-free, 10 in first CR, 1 after rescue with stem cell transplant. Three of the CCR patients received HyperCHiDAM alone. Refractory/relapsed patients: CR were 5/16 (31.2%), CCR 4/16 (25%), with a median follow-up of 24 months (5–64+). The progression-free survival was significantly superior in upfront patients (p=0.036). Overall, toxic deaths were 3/33 (9%), 1/17 (5.8%) in upfront and 2/16 (12.5%) in refractory/relapsed patients. One patient had major cerebellar toxicity. Conclusions. The intensive regimen HyperCHiDAM Verona897 is effective in inducing CR in aggressive PTCL, in particular as upfront therapy. The intensiveness of this treatment requires a careful supportive therapy. Following these results, HyperCHiDAM has been included in a national trial for treatment of PTCL, after 2 cycles of Campath-CHOP and before stem cell transplant (allogeneic or autologous depending on the availability of an HLA-matched sibling). The co-operative study is recruiting.

Abstract: Abstract 2692 Cytogenetic aberrations are considered major prognostic indicators for predicting the survival of chronic lymphocytic leukemia (CLL) patients (pts) [Dohner et al, 2000]. Given the difficulties in obtaining abnormal metaphases in CLL, fluorescent in situ hybridization (FISH) with specific probes is generally used to detect the most frequent abnormalities. Interphase FISH (I-FISH) is a rapid and sensitive technique for analysis of chromosome aberrations in CLL, but the limited number of DNA probes available to screen B-CLL results in a high number of cases presenting normal cytogenetics. Moreover, deletion 13q14 on FISH analysis which is the most common cytogenetic abnormality in CLL is a favourable prognostic biomarker when detected as a sole abnormality, but a higher percentage of 13q- nuclei was found to be associated with significantly shorter time to treatment (P 〈 0.001), [van Dike et al, 2010]. On this line, the primary endpoints of our research were: 1) to determine progression free survival (PFS) and overall survival (OS) within the normal FISH cytogenetics subset on the basis both of ZAP-70, CD38, CD69 expression by flow cytometry and IgVH status; 2) to correlate percentages of 13q- nuclei ( 〈 50% or ≥50%) with PFS and OS and finally 3) to confirm I-FISH as an independent prognostic factor. We investigated 400 pts, median age 65 years (range 33–89), 235 males and 165 females. With regard to modified Rai stages, 115 pts had a low stage, 266 an intermediate stage and 19 a high stage. One hundred forty-two pts (35.5%) exhibit a normal karyotype, 133 pts (33.2%) showed an isolated 13q-, 69 pts (17.3%) presented trisomy 12, 30 pts (7.5%) 11q deletion, 20 (5%) 17p deletion and 6 (1.5%) other chromosome abnormalities. Clearly, pts with intermediate/poor cytogenetic abnormalities (trisomy 12, del11q-, del17p-) showed significant shorter PFS and OS (8% vs 33% at 16 years, P 〈 0.0001 and 41% vs 65% at 16 years, P=0.0001) in comparison with normal or del13q- pts. Therefore, in order to better define the apparently homogenous subset of the patients with normal cytogenetics, we analysed ZAP-70, CD38, CD69 and IgVH status within this subgroup. Interestingly, we observed significant longer PFS and OS for ZAP-70 negative pts (60% vs 29% at 10 years, P 〈 0.0001 and 92% vs 23% at 16 years, P=0.0006), for CD38 negative pts (54% vs 22% at 10 years, P=0.0008 and 83% vs 0% at 14 years, P=0.03), for CD69 negative pts (60% vs 23% at 10 years, P=0.0003 and 83% vs 63% at 14 years, P=0.02) and finally also for IgVH mutated cases (64% vs 35% at 10 years, P=0.0002 and 95% vs 45% at 14 years, P=0.04). Moreover, pts with isolated 13q- in 〈 50% of nuclei (60 pts) showed a longer PFS and OS (68% vs 6% at 16 years, P=0.0003 and 92% vs 0% at 18 years, P=0.002; Figure) compared to those with ≥50% of nuclei (72 pts). No significant correlation was found between 13q- percentages and CD38 or ZAP-70 or IgVH status, while it was found with CD69 expression (P=0.01). In multivariate analysis of PFS, FISH cytogenetics was confirmed to be an independent prognostic factor (P=0.002) together with Rai stages (P=0.005), ZAP-70 (P=0.0001), IgVH status (P=0.0002) and CD69 (P=0.007). Therefore, in the clinical practice, ZAP-70, CD38, IgVH status and CD69 are irreplaceable in order to define prognosis and treatment within the large series of CLL pts with normal cytogenetics, probably presenting abnormalities undetectable by FISH. Besides, the percentage of nuclei exhibiting 13q- has to be considered an important predictor of the outcome and the clinical implications of 13q- in CLL seem to appear more complex than originally considerated. In conclusion, a comprehensive scoring prognostic system based on a combination of clinical, genetic, phenotypic parameters and IgVH status improves the separation of prognostic subgroups in CLL already early in the course of the disease. Disclosures: No relevant conflicts of interest to declare.