ALBERT

Description: The recent advances in cytogenetic and molecular diagnostic techniques has provided a better understanding of biology of CLL and a better prediction of disease progression and refractoriness but they are not easily accessible to all Institutions. CLL cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B cell receptor (BCR) signaling. An increased number of studies describe the emerging role of neutrophils and monocytes as mediators of the inflammation process and antitumor immunity modulation that support tumorigenesis and tumor progression. The neutrophil to lymphocyte ratio (NLR), has been recently published as new marker of systemic inflammation associated with outcome in different cancer types. In our study we retrospectively evaluated the prognostic significance of peripheral blood neutrophils, monocytes and non-neoplastic lymphocytes in a unicentric, unselected, CLL cohort of 400 CLL patients (160 were treated). The study was approved by the institutional board review;informed consent was obtained from patients. Using the blood count and the Flow cytometric analysis reports, peripheral blood absolute neutrophil count (ANC), absolute monocyte count (AMC) and absolute T-lymphocyte count (ALC-CD3+) were evaluated and the neutrophil to T-lymphocyte ratio (NLR), the monocyte to T-lymphocyte ratio (MLR) and the neutrophil to monocyte ratio (NMR) were calculated. Clinical and biological data from all patients were also retrieved. Serial count and ratio were evaluated at diagnosis, during follow up and at relapse to make a better comparison with the major clinical and prognostic markers commonly used. The median ratio for NLR 2.67 and patients with pre-treatment NLR 〉 3 had a shorter time from diagnosis to treatment initiation. CLL patients showed an increase in the absolute number of monocytes compared to normal controls (788±65 cells/μL vs 469±51 cells/μL, p=0.005) and MLR appeared higher in advanced stage (stage Binet C) and bulky disease (p=0.06). High level of both NLR and MLR (cut off 〉3) correlated with the presence of a complex karyotype detected by FISH (p=0.03, p=0.016). NLR ratio was associated with the absence of serum prognostic markers, such as the expression of CD38, ZAP-70 and CD49d. This result, apparently conflicting, could strengthen the NLR as an independent prognostic factor. NMR was higher in asymptomatic patients (absence vs. presence of B symptoms, p=0.02) and this data resulted independent from disease stage. NMR median value was significantly higher in untreated patients than in patients who received treatment (p=0.01), strengthening the hypothesis that this ratio is associated with a more indolent form of disease. In this contest, the subset of patients with CD49d positive disease showed the lowest NMR value. This data seems of relevance, being CD49d a recently discovered and validated prognostic marker. ANC/ALC and AMC/ALC ratio significantly increased at relapse compare to baseline (NLR average onset 2.3±0.4 vs 3.4±0.6 at relapse). The median NMR value showed conversely the opposite trend: NMR is reduced in first relapse (NMR average onset 7.2±0.4 vs. 5.4±0.5 at relapse). These combined ratios reflect both the inflammatory status, the immune response and the microenviromental network that contribute to aggressive tumor biology and disease progression. They are simple, cheap, easily measured and reproducible and can be integrated into daily clinical practice as new prognostic markers for CLL. Disclosures Chiarenza: Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Description: Introduction Our previous work showed that in Multiple Myeloma (MM) the immune function is impaired, including immunosuppressive properties of granulocytes due to their increased amount of arginase-1 and reduced phagocytic activity (Parrinello, manuscript in preparation). It is currently unknown if granulocyte dysfunction occurs in progression from MGUS to MM. Aim Providing a gene expression profile of mature granulocytes isolated from peripheral blood at the steady-statein MGUS and MM. Methods Using oligonucleotide microarrays we first evaluated the gene expression profile of granulocytes at the steady state in 5 MM, 3 MGUS and 3 healthy subjects matched for sex and age. Then, we validated the first up-regulated gene PROK-2, obtained from preliminary findings in granulocytes from peripheral blood in 85 consecutive newly diagnosed MGUS (N=45), MM (N=40) and 15 healthy subjects, in RT-PCR (validation set). Results We found 708 genes differentially expressed (467 up- and 241 down regulated) in MGUS versus healthy granulocytes at the steady state. The set of annotated, differentially expressed genes could befunctionally organized by “gene ontology” (http://www.geneontology.org/) into the following major categories: i) receptors and signal transduction (including up-regulation of CD14, Toll-like receptor 5 (TLR-5), IL-7 Receptor (CD127), IL-11 receptor, TGF-beta receptor 2, hematopoietic cells kinase (HCK), IFNAR1); ii) negative regulation of adaptive immune response (including up regulation of CD127, STAT6, IFNAR1, OSCAR, PROK-2 and down regulation of p50, p65,NFKBIA, IL8, ELK-1, HIF-1 alpha, CEBP-beta, CEBP-zeta). In MM samples we confirmed a statistically significant up-regulation of PROK-2 (a key molecule of VEGF-independent angiogenesis), CD14 (mediator hypersensitive innate immune response to lipopolysaccharide) and HCK (the hematopoietic cell kinase, involved in neutrophil migration and degranulation). In the validation set, PROK-2 expression was two times higher in MGUS than healthy subjects (p=.02) and up to ten times higher in MM (p=.001). In MM patients, increased levels of PROK-2 were positively associated with advanced bone disease and unfavourable cytogenetics. Conclusion Granulocytic impairment is present in MGUS and worsened in MM patients due to increased expression of genes that negatively regulate adaptive immune response. PROK-2 is a key molecule involved in the granulocyte dysfunction and could be involved in the progression from MGUS to MM. Disclosures Musto: Celgene: Honoraria; Janssen: Honoraria.

Description: Abstract 1239 Poster Board I-261 Background Purine analogues, in particular fludarabine, are considered the gold standard of treatment of CLL. However, fludarabine therapy is sometimes complicated by autoimmune haemolytic anemia (AHA). The mechanism of this side effect is not clear but it is conceivable that a fludarabine-induced suppression of some regulatory systems, including T-reg, is responsible for this phenomenon. In addition, we have observed that patients affected by autoimmune diseases such as AHA or PTI have a reduced number of T lymphocytes bearing the CD200 antigen that is considered a tolerogenic molecule. In this perspective, we evaluated the variation of T-reg and CD200+ T lymphocytes induced by incubating in vitro peripheral blood mononuclear cells (PBMC) of CLL patients and normal subjects with purine analogues and other drugs active against CLL. Method PBMC obtained from patients with chronic lymphocytic leukaemia (CLL) (n=9) and from normal adult (n=6) were isolated by density gradient and cultured in RPMI supplemented with 10% FBS and 1% of penicillin streptomicyn. Cells were then incubated for 24 hours with drugs at two concentrations: bendamustine (1 and 50 μg/ml) campath (1 and 5 μg/ml) prednisone ( 1 and 10 nM), fludarabine (0,25 and 10 μg/ml) pentostatin (3 and 60 μg/ml). The cytotoxicity was evalutated after 24 hours by trypan Blue and flow cytometry. T-reg cells were identified as CD4+/CD25+/FoxP3+ T cells and expressed as a percentage of the CD4+T-cell population. Results Although all of these drugs induced lymphocytes cytotoxicity, fludarabine, prednisone, and campath reduced also the percentage of T-reg and CD200+ T –lymphocytes, while bendamustin and especially pentostatin induced the same cytotoxicity but spared T-reg populations and CD200+ T-lymphocytes. Table I indicates results obtained with the highest concentration of drugs. In conclusion, pentostatin and bendamustine seems to be active drugs against CLL and their usage shouldn't be complicated by autoimmune phenomena. Disclosures No relevant conflicts of interest to declare.

Description: Background Extensive sequencing data highlighted the recurrence of mutations resulting in constitutive MAP kinase signaling in chronic lymphocytic leukemia (CLL), and proposed these mutations as late drivers of CLL progression (X.S. Puente, Nature, 2015; D.A. Landau, Nature, 2015). Although some preliminary studies suggested KRAS, NRAS and BRAF mutations as possibly associated with trisomy 12 (TRI12) and/or unmutated (UM) IGHV genes (C.D. Herling, Blood, 2016; K. Takahashi, Blood, 2018), dedicated analysis of these aspects is still missing. Aim To correlate the occurrence of KRAS, NRAS and BRAF mutations with specific clinico-biological features in CLL. Methods The study included 534 primary CLL purposely enriched with 300 TRI12 cases: 190 cases with TRI12 as sole chromosomal aberration (TRI12only) and 110 cases harboring TRI12 plus other chromosomal aberrations (TRI12plus). The cohort was also enriched in cases with NOTCH1 aberrations (n=214) to evaluate their possible role in RAS-RAF mutations incidence. Amplicon-based targeted next-generation sequencing assay was performed using Miseq Illumina on DNA from purified CLL samples. Analyzed amplicons mapped in the hotspot regions of KRAS, NRAS (exons 2, 3 and 4) and BRAF (exons 11 and 15) genes. Variant allele frequency (VAF) was obtained by Miseq report. CLL cases were characterized for IGHV mutational status, main cytogenetic abnormalities, NOTCH1 and TP53 aberrations. Time to first treatment (TTFT) data were correlated with molecular findings. Results We found 90 missense point mutations (Fig.1) in 64 CLL cases, with prevalence of KRAS (44 mutations in 38 patients), followed by BRAF (31 mutations in 24 patients) and NRAS (15 mutations in 13 patients). Nearly all mutations have been previously associated with gain-of-function phenotype and increased RAS/ERK downstream signaling. The co-occurrence of 2 mutated genes (mainly KRAS and BRAF) were observed in 11 patients. Mutations were mainly subclonal (median VAF 6.15%, range 1.3%-61.6%) with only one third of mutations (27/90) above 15% VAF. A high association between the presence of KRAS/NRAS/BRAF mutations and UM IGHV and the presence of TRI12 was observed. Overall, 87.3% of KRAS/NRAS/BRAF mutated cases had a UM IGHV (p

Description: Abstract 2720 Introduction: Bendamustine is a promising agent that has been showed high efficacy in combination with Rituximab in indolent lymphomas; however experiences on its use in combination with other cytotoxic agents are scant, particularly in FL. A previous FIL trial showed that a brief R-FND induction chemoimmunotherapy with only 4 courses of R-chemotherapy followed by 4 rituximab doses as consolidation can achieve high CR and PFS rates in FL patients, supporting the feasibility of this regimen in the elderly (Vitolo et al, ASH 2011). These promising results prompted FIL to investigate safety and efficacy of a similar combined brief regimen substituting Bendamustine for Fludarabine aimed at reducing toxicity and maintaining efficacy. Patients and methods: From September 2009 to November 2011, 76 elderly patients (age 65–80) with advanced stage FL at diagnosis were enrolled. Inclusion criteria were: untreated grade I, II and IIIa FL; advanced disease requiring treatment (stage III/IV) or stage II including at least one of the following conditions: bulky disease (〉7 cm), active disease with rapid lymph node progression, lactate dehydrogenase or β2 microglobulin above normal, systemic symptoms and extranodal involvement. A comprehensive geriatric assessment (ADL, I-ADL, CIRS) was also performed at diagnosis and only “FIT” patients were enrolled into the study. Treatment plan was: 4 monthly courses of R-BM (375 mg/m2 Rituximab day 1, 90 mg/m2 Bendamustine days 1–2, 8 mg/m2 Mitoxantrone day 1), followed by consolidation with 4 weekly Rituximab infusions. Polymerase chain reaction (PCR) for BCL2/IgH rearrangement was performed on bone marrow samples at diagnosis, after R-BM and after consolidation. Results: At the time of analysis, 69/76 patients are evaluable for clinical characteristics, response to treatment and toxicity. Median age was 71 years (range 65–80); 26 males, 43 females; WHO grading was as follow: I 16%, II 55% and IIIa 29%; 19% had advanced stage II disease, 27% stage III and 54% stage IV; 49% had BM involvement, 19% B symptoms and 7% leukemic dissemination; 58% patients had no comorbidity, 20% one and 22% two or more comorbidities. According to FLIPI patients were: 12% at low, 30% at intermediate and 58% at high risk. PCR analysis was carried out in 64 patients at diagnosis: 58% were Bcl-2 positive. Sixty three patients completed the planned treatment and the whole program was completed according to the planned time in 60/63 patients. Six patients did not complete the treatment: 1 for progressive disease, 4 for adverse events (2 haematological toxicity with prolonged neutropenia; 1 CMV colitis and 1 for infection and concomitant worsening of pre-existing oral pemfigo) and 1 patient for worsening of performance status. Overall response after R-BM + Rituximab was observed in 64 (96%) patients with 52 (75%) patients in complete remission and 12 (18%) in partial remission (PR). Response to 4 cycles of R-BM was as follow: 27 (39%) in CR, 37 (54%) in PR and 5 (7%) patients in stable (SD) or progressive disease. Of the 40 patients in PR or SD after 4 R-BM, 26 (65%) converted to CR following further Rituximab consolidation. At the time of this analysis, of the 37 patients Bcl-2 rearranged at diagnosis, 19 (51%) were evaluable for molecular response during and after treatment. PCR negativity was achieved in 16/19 (84%) patients after R-BM and in 18/19 (95%) patients at the end of treatment. A total of 521 courses of R-BM and Rituximab were given. The most frequent severe toxicity (CTC grade 3–4) was neutropenia reported in 18% of the courses; nevertheless only four serious infections were recorded (all due to bacterial agents). Pulmonary and cardiac toxicities were 〈 1%. Two deaths were recorded: one due to lymphoma after second line treatment and one due to hepatic carcinoma occurred 3 months after completion of therapy. Conclusions: A brief course of chemo-immunotherapy R-BM followed by Rituximab consolidation is safe and effective with a high CR rate in elderly patients with untreated advanced stage FL. Planned future analysis of the entire study will give further information on late toxicity and outcome. Disclosures: Off Label Use: In Europe use of Bendamustine in first-line treatment of follicular lymphoma is off-label. Drug was provided free by Mundipharma. Vitolo:Roche: Membership on an entity's Board of Directors or advisory committees.

Description: Background. TP53 mutations (TP53mut) along with 17p13 deletion (del17p) are strong predictors of poor survival and refractoriness to chemo-immunotherapies (CIT) in chronic lymphocytic leukemia (CLL), and their analyses should be always performed before treatment. Studies based upon ultra-deep-NGS have shown that TP53mut can be present at very low level in CLL cell populations, although their detrimental clinical impact in this setting is still matter of debate (Rossi, Blood 2014), and ERIC recomendations discourage to report TP53 mutations if subclonal Malcikova, Leukemia 2018). Aim. To investigated the presence and clinical relevance of clonal/subclonal TP53 aberrations in a large CLL cohort. Methods. The study includes 1,058 out of 1,613 CLL patients (509 treated with standard CIT) diagnosed between 1991 and 2018, and consecutively referred to a single institution for del17p analyses by FISH (167-kb 17p13 orange probe, MetaSystems), and TP53mut by ultra-deep NGS (MiSeq Illumina; median coverage 〉2,000X with an amplicon-based strategy covering exons 2-11 using 40ng DNA/test) in CD19-purified (〉85% pure) CLL samples, collected before treatment (as per ERIC recommendations). For TP53mut analyses, FASTQ files were aligned to the Hg19 reference with Burrows-Wheeler Aligner-MEM algorithm, and allele variants called by FreeBayes (Garrison & Marth, arXiv 2102) with non-stringent parameters. To calculate random/systematic errors we generated a specific database with all the variant allele frequencies (VAF) observed in a subset of TP53 wild type (wt) subjects (n=362). TP53mut were accepted if: i) validated by Fisher exact test after Bonferroni correction (p 12.5%) and 67 subclonal (all clones 0.4% for the clinical impact of TP53mut (Fig.D), and the c-index of combined clonal/subclonal TP53mut (0.645) was significantly higher than the c-index of clonal TP53mut alone (0.602; P10% of nuclei had significantly shorter OS than cases with del17p in 0.5% as the best cutoff. In keeping with this cutoff, TP53 mutated patients experienced a significantly shorter OS than wt patients. Again cases with clonal and subclonal mutation experienced the same OS (Fig.I). Importantly, the cutoff found in the training cohort was able to reproduce the very same results also in the validation cohort both in term of mutation per se and in terms of clonal and subclonal TP53 mutations (Fig.J). Conclusion. i) By applying ERIC recommendations and a rigorous pipeline of analysis, TP53mut impacted on OS also with VAF 10% of nuclei. These cutoffs may be employed for the clinical management of CLL patients. Figure Disclosures Di Raimondo: Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board.

Description: Imatinib mesylate (IM), a tyrosine kinase inhibitor, currently used in chronic myeloid leukaemia (CML) may also affect the growth of other cellular systems besides CML cells. It has been reported that IM may affect bone tissue remodeling mainly by an inhibitory activity on osteoclastogenesis. We therefore evaluated possible effects of IM on osteoblatic differentiation of Mesenchymal Stem Cells derived from bone marrow (BMSCs). BMSCs are multi-potent non-haematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes and nervous cells. Culture of normal human BMSCs were treated with osteogenic medium (OM) with or without IM 1 μM. By RT-PCR, we assessed mRNA expression of osteogenic markers such as RUNX2, osteocalcin (OCN) and osteopontin (OPN) at 7, 14, and 21 days of culture. BMSCs treated with OM and IM 1 μM showed increased levels of osteogenic markers mRNA as compared to BMSCs cultured with OM only (RUNX2 ctrl=0,37±0,02 vs OM+IM=0,79±0,06; OCN ctrl=0,36±0,04 vs OM+IM= 0,97±0,03; OPN ctrl=0,94±0,01 vs OM+IM= 1,55±0,13). We also evaluated the OCN levels, and the OPG (osteoprotegerin)/RANKL (receptor activator of nuclear factor-kappa B ligand) ratio (OPG/RANKL ratio) in the surnatant of the culture at day 21 by ELISA assays. OPG/RANKL ratio (OM alone = 55,4±0,4 vs OM+IM = 65,3±0,7; P

Description: In 2001, the GIMEMA Group started a phase II study to evaluate Imatinib both in adult (study A, 18–60 years) and elderly (Study B, 〉61 years) patients with Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL). In study A, adult patients with Ph+ ALL received the standard GIMEMA induction (VCR, DNR, PDN, L-ASP) and consolidation (MTZ and high-dose Ara-C) treatments, without imatinib. All patients in hematological complete remission (HCR) after the consolidation, either with or withouth a donor, received imatinib p.o. at the dosage of 800 mg/day for 6 months. After completing the 6-months therapy, in the absence of safety concerns, if the investigator considered the patient eligible for treatment, the therapy with imatinib was continued. A total of 22 patients have been enrolled [7 in molecular complete remission (MCR) and 15 still BCR/ABL+]: 18 are still in HCR after a median follow up of 20 months (2 – 26). In particular, all the 7 patients PCR- when started imatinib are still in HCR (6 PCR- and 1 becoming PCR+), after a median follow-up of 19 months (7.6 – 24) and eleven of the 15 PCR+ patients are in CHR after a median follow-up of 19 months (1.2 – 26). Of the remaining 4 PCR+ patients, 1 died in CR after an alloSCT and 3 patients relapsed at 4, 9 and 11 months, respectively. The probability of disease-free survival at 2 years is 79% (C.I. 95%: 58,4 – 100,3). Figure Figure In study B, planned for elderly patients usually not eligible for intensive treatments, the induction therapy consisted of imatinib 800 mg/day p.o. associated to prednisone 40 mg/sqm/day for 30 days, withouth any other chemotherapy. Up to date only 12 patients have been enrolled. Median age was 67.5 yrs (61–78), 9 were females. Eleven (92%) obtained a CR with the prednisone-Imatinib association induction treatment, 1 patient discontinued the treatment for toxicity. The post-remissional treatment mainly consisted of imatinib alone: 8 patients are still in CR, after a median follow up of 7 months (4 – 15), 2 relapsed at 4 and 4 months and 1 died in CR due to a 2nd neoplasia. In conclusion, these studies, planned 3 years ago to verify the feasibility and toxicity of imatinib in adult and elderly Ph+ ALL patients, clearly suggest a strong activity of Imatinib also in this disease, not only giving the possibility of inducing a CR as monotherapy, but even of maintaining long-lasting hematological and molecular CR withouth allogeneic stem cell transplantation. Further studies are mandatory to finally identify the best way to integrate imatinib in the entire treatment strategy of this disease.

Description: Abstract 3386 It has been demonstrated that long term treatment of CML patients with Imatinib (IM) is associated with altered bone and mineral metabolism. The mechanisms that are responsible for this effect are not fully understood but an inhibition of the PDGF-Rβ (Platelet Derived Growth Factor- Receptor beta) axis has been documented. In order to evaluate if stimulation of osteoblastogenesis is a common feature of other tyrosine kinase inhibitors (TKIs) approved for the treatment of patients with CML, we evaluated the osteoblatic differentiation of Mesenchymal Stem Cells derived from adult bone marrow donors (BM-MSCs) after in vitro treatment with Dasatinib (DA), Nilotinib (NI) or Bosutinib (BO). BM-MSCs were induced to differentiate in osteoblastic cells by treatment with osteogenic medium (OM) with or without DA, NI or BO. BM-MSCs was induced to differentiate in osteoblastic cells by treatmet with osteogenic medium (0.2 mM ascorbic acid, 0.1 μ m dexamethasone and 10 mM β-glycerophosphate, OM) with or without DA 2nM, BO 5nM or NI 100nM. After 21 days of treatment, in the cultures treated with DA e NI for 21 days we have observed a significant increase of extracellular mineralization and of osteogenic markers such as bone morphogenetic protein (BMP2) (p