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We evaluated the correlation between immunohistochemical BCL2, BCL2 E17, MCL1 (Bcl2-related protein family) and BAX (effector proapoptotic protein) expression and intensity of staining in plasma cells (PC) in MM patients (pts) who were eligible for autologous stem cell transplantation (ASCT) with DD4, CD8, and FOXP3 positive Tregs lymphocytes in bone marrow (BM) microenvironment, morphologic features of PC, clinical parameters, and outcomes. Immunohistochemical staining of BCL2, BCL2 E17, MCL1, BAX, CD4, CD8, FOXP3 and Ki67 was performed in BM biopsy of 36 newly diagnosed MM patients who were eligible for ASCT between 2012-2017 at University Hospital Merkur. Pearson's and Spearmans's coefficients of correlation, t-tests, one-way independent sample ANOVAs, mixed ANOVAs, moderation analyses, chi-square tests of independence and Cox's proportional hazards were used to analyze the data. Median age of pts was 57 years (95% CI 52-60), 15 pts were female with equal distribution in rISS I-III (12 pts). Twelve (33%) pts had high risk cytogenetics: del 13q34 (8 pts; 22%), del 17p (5 pts, 13,8%), t(4; 14) (4 pts; 11.1%); t(16;14) (4 pts; 11.1%); 4 pts (11.1%) had t(11;14). The median percentage of PC (PC ptc) in BM was 60 (95% CI 45 - 80); 25 pts (69.4%) had diffuse type of infiltration (TI) in BM; and 23 pts (63.8%) had high grade of PC differentiation (grade 2/3). The median percentage of Ki67 in PC was 3% of PC (95% CI 2 - 4, range 1 - 80). The median percentage of BCL2 positive PC in BM was 50 (95% CI 25 - 60) with high intensity of staining in 23 pts (63.8 %); BCL2 E17 positive PC in BM was 60 (95% CI 60 - 70) with high intensity of staining in 33 pts (91.6%); MCL1 positive PC in BM was 40 (95% CI 20 - 60) with high intensity of staining in 21 pts (58.3%); and BAX positive PC in BM was 17.5 (95% CI 6 - 40) with high intensity of staining in 18 pts (50%). Median CD4 positive T-cells was 8.5 (95% CI 4 - 18); CD8 T-cells 24 (95% CI 18 to 37); and FOXP3 Tregs 1 (95% CI 1 - 2, range 1 - 31). Nineteen pts (52.7 %) underwent tandem ASCT, 34 pts (94.4%) received VCD induction; 2 pts received MP induction; and 1 pts received VD induction. Overall response rate (≥partial response) to induction therapy was 94.4%; ≥ very good partial response rate was 80.5%. Overall response rate (≥partial response) to ASCT was 86.1%; complete response rate was 50%. Median overall survival time was 35.40 months (95% CI 26.61-165.39). Expression of BCL2, BCL2 E17, MCL1 and BAX proteins was significant and positively, moderately to strongly associated (p < 0.05), except for BCL2E17 and BAX association (r (36) = 0.319, p = 0.058). BCL2 and MCL-1 expression was more correlated when PC were well differentiated, grade 1 (p = .016). Higher PC pct was correlated with higher intensity of BLC2 staining (t (34) = 2.51, p = 0.017). Patients with Ki67 >10% had a higher PC pct in BM (t (34) = 2.04, p = 0.049). Higher grade of PC in BM correlated with higher PC pct in BM (t (34) = 2.63, p = 0.012). A higher MCL1 expression was found in MM with high-risk cytogenetic changes (t (34) = 2.09, p = 0.045). Cytoplasmic granular MCL1 staining had greater MLC 1 staining intensity than the diffuse TI (χ 2 (1) = 6.60, p = 0.017). Low BCL2, BCL2E17, MCL1 and BAX expression was found in MM stage rISS1 compared to rISS3 (p < 0.05), and low BCL2 and MCL-1 expression in MM stage rISS1 compared to ISS2 (p < 0.05). Patients with non-diffuse TI had worse response to ASCT compared to their response to induction therapy than patients with diffuse TI (χ 2 (2) = 6.39, p = 0.041). Although we observed high number of CD4 and CD8 T-cell in BM microenvironment and aberrant CD4:CD8 ratio in favor to CD8 positive T-cell, it had no impact on other variables in study. We found significant correlation between BCL2E17 expression and response to ASCT (r s = -0.417, p = 0.011), with higher protein expression being associated with worse outcomes. BCL2 contributed to worse survivability (χ 2 (1) = 7.18, p = 0.007), while BCL2 E17, MCL1 and BAX expression, as well as the number of CD4 and CD8 T-cells, did not predict survival (p > 0.05). Results showed aberrant expression of the BCL2 family members that may plays a role in application strategies for MM therapy. Bcl-2 inhibits Bax but we found aberrant BAX expression in MM that may be result of pre-condition or hormesis like responses in stressful micro-environments that serve to increase apoptotic resistance. Further investigation of the role Bcl2- proteins family in the biology of MM and its impact on clinical and therapeutic outcomes is warranted. Disclosures Jaksic: Roche, Oktal-Pharma/Celtrion, Sandoz: Consultancy, Honoraria.

There is a high number of clinical and biological parameters with impact on prognosis in B-CLL and number of successful prognostic models were developed (clinical stages, CLL IPI, MDACC score, etc.). However, rapidly changing therapeutic landscape with more successful targeted terapines with different modes of action, render most of these models developed in era of chemo and chemoimmunotherapy less useful. Recently simple prognostic score (BALL score) based on 4 parameters (LDH>UNL, B2microglobulin>5mcg/l, Hemoglobin High cost of novel drugs in less affluent countries led to a higher criterion for reimbursement. Until recently in Croatia ibrutinib was reimbursed only for early relapse (less than 24 months from last therapy) or refractory disease with additional criteria for high risk disease including Rai stages III and IV or high tumor burden demonstrated by TTM score >15. TTM score (www.b-cll.org) is old, simple and continuous parameter useful for prognosis and response assessment (Jaksic B et al BJH 1980) and because it cover tumor mass in all major lymphoid compartments and allow tumor distribution assessment (Jaksic O et al, Haematologica 2001) it can be very useful for response assessment to novel agents were redistribution of lymphocytes can be significant (Jaksic O BJH 2014). In other to evaluate usefulness of novel BALL score and old TTM score in real life setting where the novel drug is available only to patients with higher risk defined by respective scores, we have evaluated series of 42 RR CLL patients treated with ibrutinib at our institution since March 2015. There were 15 females and 27 males, median age 70 years (range 53 to 82), 9 patients had 17p deletion, median TTM was 14 (range 1.2-28), 20 patients had Rai stage III/IV. Median follow up was 24 months, and maximal 64 months. Since all patients were in early relapse or refractory, we have actually used simplified BALL score based on only 3 parameters. There was no significant relationship between BALL and TTM in our patient population. In our patient cohort low risk BALL score had only 3 patients, 28 patients had intermediate risk and 11 high risk and it showed only marginal discriminative power (p=0.054) While TTM>15 did not show discriminative power (p=0.14). When we added TTM>18 as an additional parameter (point) to BALL score, this modified score (0-3 low/intermediate - 23 patients and 3-4 high risk - 19 patients) showed significant discriminative power (p=0.002) (Figure 1). Observed results indicate that: 1) performance of prognostic models may be significantly dependent on discriminative power of its components and if these components are criteria for initiation of therapy with novel agent(i.e. only high risk group defined by these parameters) it significantly lower its prognostic power, and vice versa 2) such criteria for therapy selection/initiation until these criteria are met may actually mean that we treat patients with novel agents when these are less effective. We have shown that addition of TTM significantly improves BALL score. Refinement of prognostic scores with parameters that may have predictive power for novel drugs, as well as adaptation criteria for reimbursement in future studies (including pharmacoeconomics) may further improve quality of care of B-CLL patients. Disclosures Jaksic: Roche: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Pejsa:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pliva: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alvogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oktal Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Chronic lymphocytic leukemia (CLL) occurs in older individuals with a median age at diagnosis of 72 years. In recent years, there has been considerable progress in the frontline therapy of elderly/physically unfit patients with CLL. The German CLL11 trial showed that addition of obinutuzumab to chlorambucil (G-Clb) prolongs progression free survival (PFS) and overall survival (OS) compared to chlorambucil alone or in combination with rituximab. More recently, obinutuzumab together with ibrutinib or venetoclax were shown to be superior to G-Clb with regard to PFS, but there was no advantage in terms of OS. In this retrospective, multinational and multicenter co-operative study the European Research Initiative on CLL (ERIC) and the Israeli CLL Study Group (ICLLSG) evaluated the efficacy of frontline treatment with G-Clb in patients with CLL, in a "real-world" setting. Our analysis excluded CLL patients with documented del(17p) or TP53 mutations since they are no longer treated with chemotherapy. Results: A total of 437 treatment-naïve patients with CLL from 51 medical centers located in 13 countries were included. The median age of this patient population was 75.9 years; 59.7% were men, median CIRS total score was 8 and estimated creatinine clearance 61.1 mL/min. Seventy four patients had Binet stage A (17.2%), 167 (38.8%) stage B and 190 (44.1%) stage C. Results of FISH and IGHV mutational status were available for 332 and 115 patients, respectively. High-risk cytogenetics, del(11q) was documented in 18.7% patients and IGHV-unmutated gene in 64.4%. The vast majority of patients were treated with G-Clb (N=408) and the rest with obinutuzumab monotherapy (G-monotherapy, N=29). The clinical overall response was 86.5%, including clinical complete and partial responses in 41.6% and 45.8% of cases, respectively. The median observation time was 14.1 months (m) and the median PFS of the entire cohort was 27.6m (95% CI, 24.2-31.0). The PFS for G-Clb was significantly better than G-monotherapy (P=0.001; HR=0.38, 95% CI: 0.22-0.67), being the 2-year PFS estimates 61.8% and 52.8%, respectively. The median PFS was significantly shorter for patients with del(11q) (19.2m) compared to those with normal FISH (not reached, P5cm, G-monotherapy, reduced cumulative dose of obinutuzumab and status less than CR, were independently associated with shorter PFS. Seventy patients (16%) received a second-line treatment. The median OS for the entire cohort has not been reached yet and 2-year OS estimate is 88%. In conclusion, in a "real-world" setting, frontline treatment with G-Clb achieves PFS comparable to that reported in clinical trials. Inferior outcomes were observed in patients with high-risk disease [del(11q) and/or IGHV-unmutated] and those treated with G-monotherapy. Thus, even today in the era of novel drugs, G-Clb can be considered a legitimate frontline treatment in unfit CLL patients with low-risk disease [non-del(11q) and IGHV-mutated]. Disclosures Herishanu: Roche: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Simkovic:Roche: Honoraria; University Hospital Hradec Kralove: Employment; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy, Honoraria. Mauro:Gilead: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Jannsen: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Research Funding. Scarfo:AstraZeneca: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Tedeschi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen spa: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; SUNESIS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BeiGene: Honoraria. Gimeno Vázquez:JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Assouline:F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Levato:Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria. Rigolin:Gilead: Speakers Bureau; Gilead: Research Funding; AbbVie: Speakers Bureau. Loscertales:Janssen: Honoraria; Roche: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria; Gilead: Honoraria. Ghia:Dynamo: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy; Gilead: Consultancy, Honoraria, Research Funding.

The chronic lymphocytic leukemia (CLL) niche is a closed environment where leukemic cells derive growth and survival signals through their interaction with macrophages and T lymphocytes. Here, we show that the CLL lymph node niche is characterized by overexpression and activation of HIF-1α, which increases adenosine generation and signaling, affecting tumor and host cellular responses. Hypoxia in CLL lymphocytes modifies central metabolic pathways, protects against drug-driven apoptosis, and induces interleukin 10 (IL-10) production. In myeloid cells, it forces monocyte differentiation to macrophages expressing IRF4, IDO, CD163, and CD206, hallmarks of the M2 phenotype, which promotes tumor progression. It also induces IL-6 production and enhances nurturing properties. Low oxygen levels decrease T-cell proliferation, promote glycolysis, and cause the appearance of a population of PD-1+ and IL-10-secreting T cells. Blockade of the A2A adenosine receptor counteracts these effects on all cell populations, making leukemic cells more susceptible to pharmacological agents while restoring immune competence and T-cell proliferation. Together, these results indicate that adenosine signaling through the A2A receptor mediates part of the effects of hypoxia. They also suggest that therapeutic strategies to inhibit the adenosinergic axis may be useful adjuncts to chemotherapy or tyrosine kinase inhibitors in the treatment of CLL patients.

Our interest in chronic lymphocytic leukemia (CLL) derives primarily from the exploitation of human diseases as strategic models for defining the in vivo biological roles of CD38. Using this model, we showed that CD38 triggers robust proliferation/survival signals modulated through the interactions with the CD31 ligand expressed by nurselike cells and by the stromal/endothelial components. By analyzing a cohort of 56 patients with clinically and molecularly characterized CLL, we show that (1) patients with CD38+/ZAP-70+ are characterized by enhanced migration toward Stromal derived factor-1α (SDF-1α)/CXCL12; (2) CD38 ligation leads to tyrosine phosphorylation of ZAP-70, showing that these markers are functionally linked; (3) ZAP-70 represents a limiting factor for the CD38 pathway in the CLL context, as shown by studying CD38-mediated signal transduction in 26 molecularly characterized patients; and (4) the CLL subgroup of patients defined on the basis of migratory potential is marked by a specific genetic signature, with a significant number of differentially expressed genes being involved in cell-cell interactions and movement. Altogether, the results of this work provide biological evidence for why the combined analysis of CD38 and ZAP-70 expression as determined in several clinical trials results in more dependable identification of patients with CLL who have aggressive disease.

We read with great interest the paper by Stirewalt et al[1][1] analyzing the prognostic significance of different Fms-like tyrosine kinase (FLT3) gene internal tandem duplication (ITD) size in acute myeloid leukemia (AML). There is a considerable variability in ITD size, and Stirewalt et al[1][1]

Chronic lymphocytic leukemia (CLL), is characterized by the expansion of mature B lymphocytes present in blood, bone marrow and lymphoid organs. Clinical behavior is highly heterogeneous, thus requiring timely identification of high-risk patients. NOTCH1 encodes a trans-membrane receptor acting as a ligand-activated transcription factor. NOTCH signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the NOTCH intra-cellular domain (NICD). Signaling is terminated by phosphorylation of the PEST domain of NOTCH1, triggering its ubiquitination and proteasomal degradation. Whole exome sequencing approaches have revealed NOTCH1 mutations in 5-10% newly diagnosed CLL cases, with their prevalence increasing to 15-20% in progressive or relapsed patients. The most frequent mutation is 7544-7545delCT frameshift deletion in exon 34, resulting in disruption of the C-terminal PEST domain. Truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated signaling. The present study was undertaken with the aim to compare NOTCH1 expression and functional role in CLL patients harboring wild type (WT) or mutated (M) NOTCH1 gene. NOCTH1 mRNA and surface protein were expressed at comparable high levels in peripheral blood (PB) CLL cells obtained from NOTCH1 M and WT patients, consistent with a more general requirement of NOTCH1 signaling in this leukemia. However, at a variance of NOTCH1 WT cases, NOCTH1 M patients displayed remarkable accumulation of both the intermediate molecular species of the activated NOCTH1 receptor, as well as of the active NICD. Consistently, by gene expression profiling NOCTH1 M patients displayed significantly higher levels of HES1 and DTX1, the main NOTCH1 target genes. Overall, these data suggest a more active signaling pathway in NOTCH1 M CLL than in NOTCH1 WT cases. Expression of NOTCH1 and of its target gene (DTX1) varied across disease compartments, being higher in CLL cells obtained from the lymph nodes (LN), as compared to paired samples derived from the PB or the bone marrow (BM). By immunohistochemical analyses of primary LN tissue samples, NOTCH1 M CLL showed an intense nuclear staining as opposed to the more cytoplasmic distribution observed in NOTCH1 WT samples. These data suggest a more active NOTCH1 signaling in CLL residing in the LN microenvironment and confirm the functional effect of NOTCH1 mutations in vivo. When PB CLL cells were cultured in vitro in the absence of any supporting layer or stimulation, they showed a rapid down regulation of the NOTCH pathway, with complete loss of NICD after 24 hours paralleled by a sharp decrease in HES1 and DTX1 transcription. Consistently, levels of presenilin-1 (PSEN1), the catalytic subunit of the g-secretase complex, were also down-regulated offering a partial mechanistic explanation for the NICD loss. NOTCH1 mRNA levels remained unchanged, with accumulation of the receptor at the plasma membrane. These effects were independent of NOTCH1 mutation status and suggested the dependence of NOTCH1 signaling activation upon in vivo microenvironmental interaction, even in NOTCH1 M CLL. Within primary LN biopsies from CLL patients, the NOTCH1 ligand, was highly expressed on CD68+ elements of myeloid origin. This observation prompted the in vitro recreation of a lymphoid niche by co-culturing Jagged1+ nurse-like cells (NLC) with autologous CLL cells. Under these conditions, NOTCH1 activity in CLL cells was sustained over time, as shown by Q-PCR analyses of DTX1 and PSEN1. Moreover, NLCs protect NOTCH1 M CLL cells from fludarabine-induced apoptosis. This microenvironment-induced chemoresistance was prevented by pre-treatment of NOTCH1 M CLL cells with specific g-secretase inhibitors, to block NOTCH1 activation. Taken together, these results show that the 7544-7545delCT mutation in the PEST domain of NOTCH1 has a stabilizing effect on NOTCH1 signaling pathway. They also show that micro-environmental interactions are critical in activating NOTCH1 pathway both in the M and WT patients. Lastly, these results show that NOTCH1 signals micht create local conditions that favour drug resistance, thus making NOTCH1 a potential molecular target in CLL. Disclosures: No relevant conflicts of interest to declare.

B-CLL patients have variable tumor mass distribution within major cell compartments, and as a consequence different clinical presentation. Adhesion molecules (AM) expression phenotype of integrins (CD11a, CD11b, CD11c, CD18, CD29, CD49c, CD49d and CD61), ICAMs (CD54, CD102 and CD50), CD38 and CD31 was determined in samples taken from peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) by two/three color flow cytometry in 33 B-CLL patients. Differences for particular AM expression between compartments were quantified to express respective intensity and direction (gradients). We found following significant differences: stronger expression for CD11a and CD102 in PB than in BM, while CD54 was stronger in BM than in PB; CD11b and CD102 were stronger in PB than in LN, opposite to CD54 and CD38; CD102 was stronger in BM than in LN, while CD18, CD11a, CD11c and CD38 were stronger in LN then in BM. Observed gradients were compared with clinical and laboratory parameters: PB lymphocytosis, LN size, Spleen size, Total Tumor Mass score (TTM), TTM distribution (TD), BM failure (BMF), Rai and Binet stages. Peripheral blood lymphocytosis positively correlated with LN to PB gradient for CD11a, CD11b, CD18 and CD31 (p