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The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphological appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. CD49d, the chain of the 41 integrin heterodimer, is one of the main interactors between CLL cells and accessory cells in the microenvironmental sites and one of the main predictors of overall survival. In particular, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues eventually delivering prosurvival signals and protecting CLL cells from drug-induced damages. Treatment strategies targeting the 41 integrin could represent an interesting option in CLL. In this context, the recombinant anti-CD49d antibody natalizumab demonstrated the potential to overcome stromal cell-induced resistance of B cell lymphoma cells against cytotoxic drugs and rituximab in vitro. Moreover, a specific interest for the CD49d molecule raises from the clinical activity of the recently proposed inhibitors of kinases downstream the BCR that has been recently related with the inside-out activation of the α4β1 integrin. In the review, we addressed in detail the role of CD49d in CLL cells, including clinical impact, relationship with specific cytogenetic features, and CD49d-dependent interactions in lymph node and bone marrow microenvironment responsible for growth- and survival- supporting signals, eventually influencing CLL prognosis and therapeutic options.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphologic appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. A combination of genetic lesions is primarily responsible for the first step(s) of neoplastic transformation, along with microenvironmental signals, which concurrently operate by enhancing proliferation and/or inhibiting apoptosis. In this context, CD49d is known to play a pivotal role in mediating both cell–cell and cell–matrix interactions in CLL-involved tissues, eventually delivering pro-survival signals and protecting CLL cells from drug-induced damages. In the present review, we address, in detail, CD49d activities in the CLL microenvironment, CD49d functional and physical interactions with other microenvironmental receptors (including CD38 and B-cell receptor), and the relationship of CD49d expression with specific cytogenetic features in CLL.

CD49d is a negative prognosticator in chronic lymphocytic leukemia (CLL), expressed by ∼40% of CLL cases and associated with aggressive, accelerated clinical courses. In this study, analyzing CD49d expression in a wide CLL cohort (n = 1200) belonging to different cytogenetic groups, we report that trisomy 12 CLL almost universally expressed CD49d and were characterized by the highest CD49d expression levels among all CD49d+ CLL. Through bisulfite genomic sequencing, we demonstrated that, although CD49d+/trisomy 12 CLL almost completely lacked methylation of the CD49d gene, CD49d–/no trisomy 12 CLL were overall methylated, the methylation levels correlating inversely to CD49d expression (P = .0001). Consistently, CD49d expression was recovered in CD49d– hypermethylated CLL cells upon in vitro treatment with the hypomethylating agent 5-aza-2′-deoxycytidine. This may help explain the clinicobiological features of trisomy 12 CLL, including the high rates of cell proliferation and disease progression, lymph node involvement, and predisposition to Richter syndrome transformation.

Genetic lesions and B-cell receptor (BCR) signaling are both oncogenic drivers in chronic lymphocytic leukemia (CLL). However, scant data are available on preferential associations between specific genetic alterations and stereotyped BCR subsets. By analyzing 1419 cases, 2 CLL subsets (2 and 8) harboring stereotyped BCR are enriched in specific molecular alterations influencing disease course. SF3B1 mutations are the genetic hallmark of IGHV3-21-CLL belonging to subset 2 (52%) but are evenly represented in nonstereotyped IGHV3-21-CLL. Trisomy 12 (87%) and NOTCH1 mutations (62%) characterize IGHV4-39-CLL belonging to subset 8 but occur with the expected frequency in IGHV4-39-CLL with heterogeneous BCR. Clinically, co-occurrence of SF3B1 mutations and subset 2 BCR configuration prompts disease progression in IGHV3-21-CLL, whereas cooperation between NOTCH1 mutations, +12, and subset 8 BCR configuration invariably primes CLL transformation into Richter syndrome. These findings provide a proof of concept that specific stereotyped BCR may promote or select molecular lesions influencing outcome.

The identification of new genetic lesions in chronic lymphocytic leukemia (CLL) prompts a comprehensive and dynamic prognostic algorithm including gene mutations and chromosomal abnormalities and their changes during clonal evolution. By integrating mutational and cytogenetic analysis in 1274 CLL samples and using both a training-validation and a time-dependent design, 4 CLL subgroups were hierarchically classified: (1) high-risk, harboring TP53 and/or BIRC3 abnormalities (10-year survival: 29%); (2) intermediate-risk, harboring NOTCH1 and/or SF3B1 mutations and/or del11q22-q23 (10-year survival: 37%); (3) low-risk, harboring +12 or a normal genetics (10-year survival: 57%); and (4) very low-risk, harboring del13q14 only, whose 10-year survival (69.3%) did not significantly differ from a matched general population. This integrated mutational and cytogenetic model independently predicted survival, improved CLL prognostication accuracy compared with FISH karyotype (P < .0001), and was externally validated in an independent CLL cohort. Clonal evolution from lower to higher risk implicated the emergence of NOTCH1, SF3B1, and BIRC3 abnormalities in addition to TP53 and 11q22-q23 lesions. By taking into account clonal evolution through time-dependent analysis, the genetic model maintained its prognostic relevance at any time from diagnosis. These findings may have relevant implications for the design of clinical trials aimed at assessing the use of mutational profiling to inform therapeutic decisions.

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. Although initial genetic events are considered primarily responsible for the first step(s) of neoplastic transformation, the development and progression of the CLL clone are thought to be affected by various micro-environmental signals that regulate proliferation and survival of malignant B cells. In the present review, we focus on specific interactions of CLL cells with the microenvironmental component, as they occur through the usage by CLL cells of specific molecular structures whose expression has been associated with prognosis, including: i) interactions of CLL cells via the surface BCR and dependent on specific molecular features of the BCR itself and/or the presence of the BCR-associated molecule ZAP- 70; ii) non-BCR-dependent proliferative and/or pro-survival interactions of CLL cells by CD49d and CD38. An overview of the putative drugs that could be employed to target specific molecules involved in CLL cells/tumor microenvironment interactions is also proposed in the closing chapter of the review.

CD49d/α4-integrin is variably expressed in chronic lymphocytic leukemia (CLL). We evaluated its relevance as independent prognosticator for overall survival and time to treatment (TTT) in a series of 303 (232 for TTT) CLLs, in comparison with other biologic or clinical prognosticators (CD38, ZAP-70, immunoglobulin variable heavy chain (IGHV) gene status, cytogenetic abnormalities, soluble CD23, β2-microglobulin, Rai staging). Flow cytometric detection of CD49d was stable and reproducible, and the chosen cut-off (30% CLL cells) easily discriminated CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (P < .001) and ZAP-70 (P < .001), or with IGHV mutations (P < .001), was independent prognosticator for overall survival along with IGHV mutational status (CD49d hazard ratio, HRCD49d = 3.52, P = .02; HRIGHV = 6.53, P < .001) or, if this parameter was omitted, with ZAP-70 (HRCD49d = 3.72, P = .002; HRZAP-70 = 3.32, P = .009). CD49d was also a prognosticator for TTT (HR = 1.74, P = .007) and refined the impact of all the other factors. Notably, a CD49dhigh phenotype, although not changing the outcome of good prognosis (ZAP-70low, mutated IGHV) CLL, was necessary to correctly prognosticate the shorter TTT of ZAP-70high (HR = 3.12; P = .023) or unmutated IGHV (HR = 2.95; P = .002) cases. These findings support the introduction of CD49d detection in routine prognostic assessment of CLL patients, and suggest both pathogenetic and therapeutic implications for CD49d expression in CLL.

IGHV3-21–using chronic lymphocytic leukemia (CLL) is a distinct entity with restricted immunoglobulin gene features and poor prognosis and is more frequently encountered in Northern than Southern Europe. To further investigate this subset and its geographic distribution in the context of a country (Italy) with both continental and Mediterranean areas, 37 IGHV3-21 CLLs were collected out of 1076 cases enrolled by different institutions from Northern or Central Southern Italy. Of the 37 cases, 18 were identified as homologous (hom)HCDR3–IGHV3-21 CLLs and were found almost exclusively (16 of 18) in Northern Italy; in contrast, 19 nonhomHCDR3–IGHV3-21 cases were evenly distributed throughout Italy. Clinically, poor survivals were documented for IGHV3-21 CLLs as well as for subgroups of mutated and homHCDR3–IGHV3-21 CLLs. Negative prognosticators CD38, ZAP-70, CD49d, and CD79b were expressed at higher levels in homHCDR3 than nonhomHCDR3–IGHV3-21 cases. Differential gene expression profiling (GEP) of 13 IGHV3-21 versus 52 non–IGHV3-21 CLLs identified, among 122 best-correlated genes, TGFB2 and VIPR1 as down- and up-regulated in IGHV3-21 CLL cases, respectively. Moreover, GEP of 7 homHCDR3 versus 6 nonhomHCDR3–IGHV3-21 CLLs yielded 20 differentially expressed genes, with WNT-16 being that expressed at the highest levels in homHCDR3–IGHV3-21 CLLs. Altogether, IGHV3-21 CLLs, including those with homHCDR3, had a peculiar global phenotype in part explaining their worse clinical outcome.

Mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes, along with CD38 expression, is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL). Configuration of IgVH genes displaying 2% mismatch has been shown to correlate with longer survivals. In a series of 64 B-CLLs, we failed to confirm the prognostic value of the IgVH gene mutational status by using the suggested cutoff. However, the IgVH mutational status maintained its prognostic value only when evidence of antigen-driven selection could be documented. This was accomplished by applying statistical methods aimed at evaluating a significant skewing of replacement mutations from framework to complementary determining regions, as it occurs during germinal center differentiation of B cells. These data caution against wide application of the 2% somatic mutation cutoff as a prognostic determinant without demonstration of antigen-driven selection.

Zaja: Amgen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria; Abbvie: Honoraria.

Background

Zaja: Novartis: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria.

Background

Chiarenza: Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

D'Arena: Janssen-Cilag: Honoraria. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Shanafelt:Genentech: Research Funding; Janssen: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

No relevant conflicts of interest to declare.

Rossi: Gilead: Honoraria, Research Funding; Janssen: Honoraria; AbbVie: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Massaia:Janssen: Other: advisory board; Roche: Other: advisory board, research support; Gilead: Other: advisory board. Boccadoro:Mundipharma: Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Coscia:Gilead: Honoraria; Janssen: Honoraria; ROCHE: Honoraria, Other: Advisory board; Mundipharma: Honoraria; Karyopharm: Research Funding.

Lo Coco: Teva: Consultancy, Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; Novartis: Consultancy; Baxalta: Consultancy; Pfizer: Consultancy.

Background.In chronic lymphocytic leukemia (CLL), NOTCH1mutations associate with clinical resistance to anti-CD20 immunotherapy in FCR combination (Stilgenbauer et al., Blood, 2014, Dal Bo et al., AHO, 2014), that can be ascribed to a NOTCH1mutation-driven repression of CD20 levels by HDACs (Pozzo et al., Leukemia, 2016). Recently, novel recurrent mutations have been identified in the 3'untranslated region of NOTCH1(3'UTR NOTCH1mutations), determining a novel splicing event within the last NOTCH1exon (Puente et al., Nature, 2015), leading to an impaired degradation of NOTCH1 protein.

Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau.

Trisomy 12 is the third most common cytogenetic abnormality in CLL with several distinguishing features including abormal morphology, high prevalence of NOTCH1mutations (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 Mimplicate constitutive activation of NOTCH1signaling 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 Mand bax/bcl-2; 2) to correlate NOTCH1 Mand bax/bcl-2 with biological and clinical prognosticators; 3) to determine progression-free survival (PFS) and overall survival (OS) upon NOTCH1 Mand bax/bcl-2; 4) to evaluate NOTCH1 Mand 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 Mwas investigated with ARMS PCR for c.75447545delCT and by Sanger sequencing of NOTCH1exon 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 Mwere significantly associated with lymphocyte doubling time <12 months (p=0.0077 and p=0.0006), with beta-2microglobulin >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 Mor bax/bcl-2 <1.5 and IGHVunmutated 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 Mvs NOTCH1wild 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 Mand bax/bcl-2 showed synergistic prognostic properties, since NOTCH1 Mand 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 Mare 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 IGHVstatus (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.

Background.NOTCH1mutations have been associated with an aggressive clinical course in chronic lymphocytic leukemia (CLL), and may predict anti-CD20 immunotherapy failure. Although about 80% of NOTCH1mutations are 2-bp deletions (delCT) causing a sequence frameshift within the PEST domain, other mutations may occur involving either the PEST domain, i.e. frameshift mutations other than delCT (FS), missense (MS) and nonsense (NS) mutations, or, more recently described, the 3'UTR NOTCH1 sequence (UTR).

Marchetti: GILEAD: Consultancy, Research Funding; JANSSEN: Other: tavel, accomodation, expenses; SANOFI: Membership on an entity's Board of Directors or advisory committees; GILEAD: Other: teaching, Research Funding; NOVARTIS: Research Funding. Gaidano:MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding. Boccadoro:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees. Massaia:Gilead: Research Funding; Janssen: Honoraria; Roche: Honoraria. Coscia:Roche: Honoraria, Other: Advisory board; Mundipharma: Honoraria.

No relevant conflicts of interest to declare.

Gaidano: Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding.

Luminari: Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees.

Background. Risk assessment in chronic lymphocytic leukemia (CLL) is determined by the presence/absence of several negative prognostic factors, including the unmutated (UM) IGHVmutational status, TP53 deregulation by mutation and/or deletion of the 17p (17p-) chromosome, and high CD49d expression. Nevertheless, clinical heterogeneity can be observed also in the context of low-risk CLL, identified according to the absence of the aforementioned negative prognosticators. Thus, identifying novel markers that may predict an indolent clinical course in the context of low-risk CLL cases can be of key clinical relevance. The CD150/SLAMF1, CD305/LAIR1, and CD307b/FCRL2 molecules have been independently reported as molecules associated with a mutated IGHV status, and with longer time to first treatment in CLL. However, the prognostic relevance of the combined CD150/CD305/CD307b expression in predicting overall survival (OS) in the context of low-risk CLL remains to be explored.

Differences in tumor proliferation and apoptosis levels explain the heterogeneous clinical course of CLL reflecting both genetic differences and the activity of external signals mainly through the B-cell receptor pathway (Herishanu et al, Blood, 2011; Rossi et al, Blood, 2013). Therefore high dynamic proliferation rate and impaired apoptosis represent crucial mechanisms both in the chemoresistance and in the progressive disease (Messmer et al, J Clin Invest 2005; Cervantes-Gomez et al, Clin Cancer Res, 2015). The today availability in clinical use both of ibrutinib, a potent Bruton tyrosine kinase (BTK) inhibitor, which blocks cell proliferation and trafficking (Cheng S et al, Leukemia, 2013; Burger et al, Blood, 2014) and venetoclax (ABT-199), a novel potent oral anti-bcl-2 peptidomimetic (Seymour et al, J Clin Oncol, 2014) as well as their possible synergistic combination (Portell et al, Blood, 2014), prompted us to analyze the real impact of the proliferative rate (Ki67 percentages) and the apoptosis (bax/bcl-2) on CLL prognosis. The primary aims of our research were: 1) to correlate Ki67 and bax/bcl-2 with clinical and biological prognostic factors; 2) to determine progression free survival (PFS) and overall survival (OS) upon Ki67 and bax/bcl-2; 3) to evaluate Ki67 and bax/bcl-2 as independent prognosticators. Therefore we investigated 606 patients, median age 66 years (range 33-89), 340 males and 266 females. With regard to modified Rai stages at diagnosis, 220 patients had a low stage, 369 an intermediate stage and 17 a high stage. Ki67 was evaluated by flow cytometry in terms of percentages of CD19+CD5+ CLL cells and the threshold of positivity was set at >5% (range 0.10-22.7). Also bax/bcl-2 was calculated by flow cytometry, dividing mean fluorescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value >1.5 (range 0.27-6.10). One hundred-ten patients were Ki67+ (18.2%) and 321 were bax/bcl-2+ (53%). Both higher Ki67 and lower bax/bcl-2 were significantly associated with intermediate/high Rai stage, beta-2microglobulin (B2M)>2.2 mg/dl and soluble CD23>70 U/ml (P<0.0001). Moreover, higher Ki67 and lower bax/bcl-2 were greatly represented within the high risk (del11q or del17p) cytogenetics (P<0.0001). Noteworthy, strong correlations were found between higher Ki67 or lower bax/bcl-2 and IGHVunmutated status (P<0.0001) or NOTCH1(P<0.0001 and P=0.00003) or TP53mutations (P<0.0001 and P=0.00001). With regard to clinical outcome, significant shorter PFS and OS were observed in patients with higher Ki67 (2% vs 44% and 43% vs 83% at 12 years; P<0.0001) and lower bax/bcl-2 (13% vs 57% and 58% vs 91% at 12 years; P<0.0001). Noteworthy, bax/bcl-2 and Ki67 showed synergistic prognostic properties, since bax/bcl-2>1.5 plus Ki67<5% identified a CLL subset at best prognosis with regard to OS (94% vs 39% at 12 years; P<0.00001, Figure), so suggesting that apoptosis and proliferation may be key pathways for combined targeted therapies. The two discordant subsets (bax/bcl-2<1.5/Ki67<5% and bax/bcl-2>1.5/Ki67>5%) showed an intermediate outcome (67% and 63% at 12 years, respectively, Figure). In multivariate analysis of OS (593 patients), bax/bcl-2 ratio (P=0.004) and Ki67 (P=0.02) together with age (P=0.0002), B2M (P=0.005), cytogenetics (P=0.003), IGHVstatus (P=0.0005) and TP53(P=0.0006) were confirmed as independent prognostic factors. Therefore, both the proliferative marker Ki67 and the apoptotic index bax/bcl-2, performed by flow cytometry, are powerful prognosticators showing synergistic clinical effects. Experiments of apoptosis, proliferation and trafficking testing ABT-199 and ibrutinib on CLL cells in vitroare in progress at our Institutions (Gattei et al, unpublished data). In conclusion, the modern treatment strategies in CLL should aim to block proliferation and trigger apoptosis concurrently (i.e. ibrutinib and venetoclax) in order to achieve longer overall survival.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Background:stabilizing mutations of NOTCH1have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV(IGHV-UM), immuno-chemorefractory or advanced disease phase CLL, and have been associated with particularly unfavourable prognosis (Rossi et al, Blood, 2012; Del Poeta et al, Br J Haematol, 2013; Stingelbauer et al, Blood, 2014). In CLL, all NOTCH1mutations disrupt the C-terminal PEST domain (about 80% of which are a 7544-7545delCT frameshift deletion) and cause an accumulation of an active NOTCH1 isoform.

Background.NOTCH1mutations are found in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases and in Richter Syndrome transformations (Rossi et al. Blood, 2013). In CLL, NOTCH1mutations have been recently associated with clinical resistance to anti-CD20 immunotherapy (Stilgenbauer et al. Blood, 2014, Dal Bo et al. AHO, 2014). In lymphoproliferative disorders, susceptibility to rituximab is determined by CD20 levels (Golay et al. Blood, 2001), which are in turn epigenetically modulated via HDAC (Shimizu et al., Leukemia, 2010).

activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012).to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL.the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation.in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels.NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 3880

Expression of the T-cells constitutive ZAP-70 protein by CLL cells has been the focus of many studies in the last few years, due to its ability to stratify indolent and more aggressive disease subsets. Although the strength of ZAP-70 as independent negative prognosticator was demonstrated by several studies, concerns about its use derive from a lack of multicentric standardization of flow-cytometric protocols. Analyses in clinical settings are usually performed according to two methods, respectively evaluating the percentage of CLL cells expressing ZAP-70 compared to isotypic control (ISO-method), or to autologous T-cells (T-method). Of note, while the two methods yield concordant results in most patients, a fraction of cases may be discordant as for evaluation of ZAP-70 positivity. Moreover, either method suffers of an operator dependent variability, mainly related to subjectivity in cursor placement to determine the percentage of ZAP-70+ cells. The aim of this study was to compare the ISO- and T-methods with the expression of ZAP-70 evaluated as Mean-Fluorescence-Intensity (MFI) Ratio between gated T and CLL cells (T/B-Ratio-method), and to assess the prognostic significance of the three approaches.Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed with BD-DiVa software on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set). Notably, in the two cohorts, ZAP-70 assessment was accomplished using two different antibody combinations and instrumentations for data acquisition and analysis.ZAP-70 expression was reviewed in the test set by applying the ISO- and T-methods. Utilizing respectively 11% for ISO-method and 20% of ZAP-70+ cells for T-method, both selected as optimal cut-offs with prognostic relevance by ROC-analysis and maximally selected log-rank statistics, 66 (ISO-method) and 60 (T-method) ZAP-70+ cases were defined. By applying the T/B-Ratio-method, a value of 3.0 was identified as the optimal prognostic cut-point. According to this value, 73 ZAP-70+ cases (i.e. with T/B-Ratio<3.0) were identified in the test set. Univariate analyses resulted in a better separation of ZAP-70+ vs ZAP-70− CLL patients utilizing the T/B-Ratio-method (p=5.6×10−6), compared to T- (p=1.27×10−5) or ISO- (p=0.009) methods. In multivariate analyses with RAI stage, β2microglobulin, IGHV, FISH, CD38 and CD49d, ZAP-70 was selected as independent risk factor, irrespective of the readout employed for evaluation of ZAP-70 expression; however, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio-method was applied (significative hazard ratio=2.72 vs 2.19 with the T-method, or 2.11 with ISO-method). To confirm these findings, we analyzed the 341 cases of the validation set with T-method (cut-off=20%) and T/B-Ratio-method (cut-off=3.0). Analyses yielded 180 (T-method), and 127 (T/B-Ratio-method) ZAP-70+ cases. Univariate analyses also on this cohort resulted in a better separation with T/B-Ratio-method (p=7.77×10−16) than with T-method (p=1.23×10−12). Of note, ROC-analysis and maximally selected log-rank statistics confirmed also in this patient series, the 20% and 3.0 values as optimal cut-offs capable to separate CLL patients into two classes with different treatment probabilities.We suggest to evaluate ZAP-70 expression in routine settings using the T/B-Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B-Ratio value as optimal cut-off to discriminate ZAP-70+ (T/B-Ratio less than 3.0) from ZAP-70− (T/B-Ratio more/equal than 3.0) casesNo relevant conflicts of interest to declare.

In the last years, the B cell receptor (BCR) has become a key molecule in chronic lymphocytic leukemia (CLL), given the correlation between mutational status of immunoglobulin heavy chain variable (IGHV) genes and disease prognosis. Recently, a fraction of CLL has been shown to preferentially express specific IGHV genes, often in a non-random combination with homologous heavy chain complementarity-determining region-3 (HCDR3) and peculiar light chains. Some of these stereotyped BCR mark CLL subsets with peculiar clinical behavior regardless of IGHV mutations. These data suggest a role for BCR in defining the clinical and biological features of CLL, also beyond the mutational status of IGHV genes.A HCDR3-driven clustering of 1,426 IG sequences (1,398 patients) was performed using ClustalX(1.83). Time to treatment (TTT) intervals, Rai staging, IGHV mutational status, CD38, ZAP-70, and karyotype abnormalities evaluated by FISH were available for 617 patients. Gene expression profiling (GEP) and quantitative real-time PCR experiments (QRT-PCR) were performed on purified CLL cells.IGHV3-23 was totally absent in 71 identified stereotyped clusters despite being the second most frequently used IGHV gene, such distribution was significantly skewed (p<0.0001), compared with the distribution of IGHV genes belonging to stereotyped BCR clusters observed in our series. Although 109/134 IGHV3-23 were mutated (M), alignment of IGHV sequences revealed a high degree of conservation in the context of the 13 AA positions involved in superantigen binding by IGHV3 subgroup genes, suggesting that the majority of M IGHV3-23 cases maintained the capacity to mediate superantigen recognition and binding. Median TTT (73 months) of 43 M IGHV3-23 CLL was significantly shorter than median TTT (253 months, p=0.0153) of 333 M CLL, as well as of 326 M CLL in which 7 cases belonging to the bad prognosis IGHV3-21/IGLV3-21 cluster were excluded (253 months, p=0.0082). Multivariate Cox proportional hazard analyses selected IGHV3-23 usage (p=0.029), Rai stage (p<0.0001) and FISH group (p<0.0001) as independent markers of disease progression for 376 M CLL, and for the cohort in which 7 M CLL from the IGHV3-21/IGLV3-21 cluster were excluded. Comparing 5 M IGHV3-23 and 22 M non-IGHV3-23 CLL for their differential GEP, 212 genes were selected, 108 up-regulated and 104 down-regulated in M IGHV3-23 CLL. Using the “Gene-Ontology Tree Machine” platform, a set of growth/tumor suppressor genes (PDCD4, TIA1, RASSF5), all down-regulated in M IGHV3-23 CLL, was constantly found in several gene-ontology categories related to apoptosis. QRT-PCR confirmed a significant down-regulation of these genes in 15 M IGHV3-23 compared to 35 M non-IGHV3-23 CLL. Given the notion that PDCD4 and TIA1 are among the genes under control of miR-15a and miR-16-1 a “Gene Set Enrichment Analysis” carried out on the 212 differentially expressed genes, confirmed that M IGHV3-23 samples were significantly deprived in genes whose expression is under control of miR-15a and miR-16-1. Accordingly, QRT-PCR experiments performed on 15 M IGHV3-23 and 35 M non-IGHV3-23 CLL revealed significant higher levels of both miR-15a (p=0.0007) and miR-16-1 (p=0.0031) in M IGHV3-23 cases. No difference was found in the distribution of patients with 13q14 deletion between M IGHV3-23 CLL and M non-IGHV3-23 CLL (p=0.19). Considering the cases used for microRNA expression experiments (data available in 47/50 cases), 8/15 M IGHV3-23 CLL bore the 13q14 deletion in more than 20% of nuclei, against 19/32 cases in the group of M non-IGHV3-23 CLL (p=0.94).Expression of IGHV3-23 marks a subset of M CLL with a worse prognosis; such a peculiar clinical behavior may be related to superantigen stimulation combined with down-regulation of specific growth/tumor suppressor genes and up-regulation of miR-15a and miR-16-1.No relevant conflicts of interest to declare.

CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells.CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays.IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells.CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study.No relevant conflicts of interest to declare.

In chronic lymphocytic leukemia (CLL), CD49d, often in association with CD38, has been shown to mark a disease subset with poor prognosis. Functionally, both molecules act as counter-receptors for surface structures (i.e. VCAM-1/CD106 and CD31) usually expressed by the endothelial/stromal component of tumor micro-environment. We have recently identified a micro-environmental circuitry which involves CD38 triggering, and eventually determines an enrichment of the VCAM-1/CD106-expressing endothelial component detected in the context of CLL infiltrates found in bone marrow biopsies. Data was also provided that CD49d/VCAM-1 interactions are active in delivering pro-survival signals to CD49d-expressing CLL cells (Zucchetto et al, Cancer Res, 69, 4001, 2009). In this study, we investigated the amount of circulating progenitors with endothelial phenotype in CLL samples with different CD49d and CD38 expression levels.Peripheral blood (PB) samples from 91 CLL cases purposely selected with WBC>25,000/μl (B cells absolute lymphocyte count >10,000/μl) were evaluated by multiparametric flow cytometry for the absolute count of circulating CD34+ cells (ISHAGE protocol in single platform). Whenever possible (i.e. if a cluster of at least 100 CD34+ cells was detectable), a further characterization was performed (4-6 colours flow cytometry) for circulating endothelial cells (CEC), identified as a CD34+CD45low cell population co-expressing one of the following endothelial markers: CD309/VEGFR-2, CD144/VE-cadherin, CD106/VCAM-1 and CD146/Muc-18. CD49d and CD38 expression by CLL cells was considered positive if exceeding the standard cut-off value of 30% of positive cells.PB absolute CD34+ cell counts were 7.5±7.5/μl in CD49d+ CLL (32 cases), vs. 3.3±2.7/μl in CD49d− CLL (59 cases; p=2.6×10−4), or 9.4±8.7/μl in CD49d+ CLL (30 cases) vs. 4.6±2.9/μl in CD49d− CLL (18 cases; p=0.004) when only cases phenotyped for CEC were considered. Furthermore, when samples were stratified also for CD38 expression, values of circulating CD34+ cells increased to 10.6±10.1/μl in CD38+CD49d+ CLL (11 cases) vs. 3.1±2.4/μl in CD38−CD49d− (51cases; p=1×10−5). Regarding the absolute quantification of CEC, a CD49d+ phenotype again marked the CLL subset with the highest CEC count, as identified by the expression of either the CD309/VEGFR-2 (CEC counts 1.7±2.3/μl in CD49d+ CLL vs. 0.5±0.5/μl CD49d− CLL; p=0.009) or the CD144/VE-cadherin (CEC counts 0.8±1/μl in CD49d+ CLL vs. 0.3±0.5/μl in CD49d− CLL; p=0.057) endothelial markers on CD34+CD45low cells. Notably, CEC from CD49d+ CLL expressed CD106/VCAM-1 in virtually all cells (1.6±2.4/μl), while the other marker of endothelial activation CD146/Muc-18 was detected in a fraction of CEC only (0.4±0.9/μl).CD49d and CD38 expression by CLL cells identify a disease subset with significantly higher number of both circulating CD34+ cells and CEC. This phenomenon could be explained considering several aspects: i) the sharing of common phenotypic markers between CLL cells and CD34+ progenitors, including CD38 and CD49d, which could be responsible for a displacement of CD34+ progenitors in the context of micro-environmental niches; ii) the known capacity of CLL cells, especially with a unmutated IGHV gene status and/or a CD38+CD49d+ phenotype to produce pro-angiogenic factors including Ang-2; iii) the rare PB cells expressing CD34 and CEC markers may represent CLL cell precursors with tumor-initiating cell features. Studies are currently ongoing to dissect among these hypothesesNo relevant conflicts of interest to declare.

p53 plays a key role in determining the clinical features of B cell chronic lymphocytic leukemia (CLL). Disruption of p53 by point mutations, deletion at 17p13, or both, occurs in a fraction of cases at diagnosis and predicts poor survival and chemorefractoriness. In cells with functional p53, p53 activity is inhibited through interaction with MDM2. In fact, p53 can be activated upon exposure of cells to inhibitors of p53/MDM2 interaction, like Nutlins. Exposure of CLL cells to Nutlin-3 is effective in raising the levels of p53 protein with subsequent induction of cell cycle arrest and/or apoptosis, independently of the most relevant prognostic markers. The aim of the present study was to analyze the gene expression profile (GEP) induced by Nutlin-3 exposure in primary CLL cells from p53wt and p53del/mut cases.purified cells from 24 PB CLL samples, all characterized for IGHV mutational status, CD38 and ZAP-70 and p53 mutations (16 p53wt CLL, 8 p53del/mut CLL of which 6 with del17p13 and p53 mutations, 1 with del17p13 alone, and 1 with p53 mutations alone), were exposed to 10 mM Nutlin-3 for 24 hours. GEP was performed using a dual labelling strategy; the differential expression of the below reported genes were validated by quantitative real-time PCR.i) signature of Nutlin-3 exposure in p53wt CLL: 144 differentially expressed genes (143 up-regulated, 1 down-regulated) were correlated with response to Nutlin-3. Among the over-expressed genes, several genes were related to apoptosis (e.g. BAX, BBC3, E124, IKIP, FAS, LRDD, FLJ11259, TRIAP1, GADD45, TP53INP1, ISG20L1, ZMAT3, TNFRS10C, TNFRSF10B/TRAIL-R2), while other genes (e.g. MDM2, CDKN1A, PCNA) were up-regulated by Nutlin-3 as a part of a negative feed-back mechanism. Of note, this signature was not shared by 3/16 p53wt cases (identified as “non-responder” p53wt CLL) and 7/8 p53del/mut cases (identified as “non-responder” p53del/mut CLL); consistently, cells from these cases were also significantly resistant to the in-vitro cytotoxic effects of Nutlin-3; ii) signature of Nutlin-3 “non-responder” p53wt CLL: by comparing the constitutive GEP of 13 “responder” versus 3 “non-responder” p53wt CLL, we obtained 278 differentially expressed genes, 149 up-regulated and 129 down-regulated in “non-responder” p53wt CLL. Among up-regulated genes, we focused on MDM4/MDMX, a gene whose product was known to have an inhibitor activity of p53-dependent transcription and to form Nutlin-3 resistant complexes with p53. Among down-regulated genes, validations were made for BIRC4BP, whose product is known to act as an antagonist of the anti-apoptotic protein XIAP; iii) signature of Nutlin-3 “non-responder” p53del/mut CLL: by comparing the constitutive GEP of 13 “responder” versus 7 “non-responder” p53del/mut cases, we obtained 72 differentially expressed genes, 26 up-regulated and 46 down-regulated (31/46 located at the 17p segment) in “non-responder” p53del/mut CLL. Validations were made for several genes whose products display pro-apoptotic activities (e.g. PSMB6, RPL26 and ZBTB4, located at 17p segment, and GNAZ located at chromosome 22) among down-regulated genes, and ARHGDIA, whose gene product displays anti-apoptotic activities and mediates cellular resistance to chemotherapeutic agents, among up-regulated genes. Notably, CLL cells (n=43) displayed constitutively higher levels of MDM4/MDMX (p<0.0001) and ARHGDIA (p=0.0002) transcripts than purified normal B cells (n=15), irrespectively to the major biologic prognosticators.specific gene-sets and GEP were documented to be associated with response or resistance to Nutlin-3 exposure in p53wt or p53del/mut CLL. These findings may help to identify novel molecular targets for CLL therapy.No relevant conflicts of interest to declare.

The simultaneous over-expression of CD38 and CD49d (integrin α4) was recently shown to be part of the molecular signature characterizing a chronic lymphocytic leukemia (CLL) subgroup with bad prognosis. The high correlation between CD38 and CD49d expression levels in CLL, and the known propensity of both CD38 and integrins to form supramolecular complexes, prompted us to investigate whether specific CD49d and CD38 interactions occur on the CLL membranes, and whether the resulted molecular complexes have a pathogenetic role in part explaining the aggressiveness of CD38/CD49d expressing CLL cells.Confocal microscopy and biochemical approaches were used to study the membrane organization of CD49d and CD38 in primary CLL cells and other B cell lines. Detection of apoptosis was performed by flow cytometry.Co-capping experiments in CLL cells and in the cell line models Raji and RPMI-8226 demonstrated a membrane relationship between CD38 and CD49d. Anti-CD49d monoclonal antibodies (mAbs) induced capping in about 75% of CLL cells, with a 80% redistribution of CD38 in the context of the capping area. The same capping area stained for CD29 (integrin β1) indicating that CD49d is part of the α4β1 (CD49d/CD29) complex. It also contained CD81, confirming the known association between this tetraspanin and α4 integrins. Similar results were obtained with the cell lines Raji and RPMI-8226, both constitutively expressing CD38 and CD49d at high levels. The lateral association between CD38 and CD49d was next confirmed at the biochemical level by co-immunoprecipitation experiments with anti-CD49d mAbs and subsequent staining of immunoprecipitates by anti-CD38 mAbs. To further check whether the integrity of lipid rafts is necessary for the physical association between CD38 and CD49d, CLL cells were treated with methyl-β-cyclodestrin (MβCD) to deprive cell membranes of cholesterol, and cell lysates with octyl-D-glucopyranoside (ODG) to solubilize raft-associated proteins. Treatment with ODG revealed that a discrete amount of CD38/CD49d complexes could also be constitutively found outside raft domains. Consistently, co-capping experiments showed that CD49d-CD38 association was unaffected by lipid rafts disruption by MβCD. CD38-CD49d association was also maintained after polarization of CD49d to its natural ligand, as witnessed by a striking co-localization of CD49d and CD38 in cell uropods formed by CLL and Raji cells left to adhere and spread onto CS-1 fibronectin (FN) fragments, which specifically bind CD49d integrins. Finally, we tested whether the simultaneous engagement of CD38 and CD49d could enhance the protection against spontaneous apoptosis of cultured CLL. Purified CLL cells from six patients were cultured in the presence of anti-CD38 mAb or CS-1 FN fragment, either alone or in combination, in order to engage CD38 and CD49d alone or simultaneously. Analysis of cell apoptosis after a 72-hour culture, showed 74%±1.7 viable cells in the absence of any stimuli, and a substantial improvement of cell viability (94%±0.5) after exposure to both anti-CD38 mAb and CS-1 fragment, compared to a slight protection from apoptosis obtained by CD38 (80±1.1) or CD49d (79±1.1) independent engagements (p<0.01). Conclusions: CD49d and CD38 are physically associated both inside and outside the membrane cholesterol-rich raft domains. Such a physical association suggests a cooperative role for CD38 and CD49d which may explain at least in part the bad clinical outcome of the CLL subset coexpressing these molecules.No relevant conflicts of interest to declare.

Abstract 2350

CD38, a negative prognostic marker for patients with CLL, has been demonstrated to be a key molecule in the interactions occurring in the context of tumor microenvironment, mediating both survival and migratory signals for CLL cells. By taking advantage of gene expression profiling studies (GEP) comparing 11 CD38pos (CD38>30%) and 15 CD38neg (CD38<10%) CLLs, we identified as over-expressed in CD38pos CLL cells: i) genes for the two C-C chemokines CCL3 and CCL4 (median-log difference, MLD-CCL3= 3.5; MLD-CCL4=4.4); real-time quantitative PCR (RTQ-PCR) of selected cases confirmed GEP results; ii) the gene for CD49d (MLD=4.4); a high correlation between CD38 and CD49d protein expression, also characterizing the CLL series of the present study, has been reported previously. In vitro experiments, performed on purified tumor cells from additional 11 CD38pos CLL cases cultured for 14 (t14) and 24 (t24) hours in the presence of either the agonist anti-CD38 monoclonal antibody (mAb) IB4 or the non-agonistic anti-CD38 mAb IB6 as control, demonstrated upregulation of CCL3/CCL4 transcripts at t14 (CCL3: mean fold increase=18, p=0.041; CCL4: mean fold increase=13.8, p=0.005), as assessed by RTQ-PCR, and an increased release of CCL3/CCL4 proteins at t24 (CCL3: mean =0.9 ng/mL, mean fold increase=14, p=0.003; CCL4: mean =1.7 ng/mL, mean fold increase=49, p=0.01), as assessed by ELISA. Consistently, immunohistochemistry (IHC) analysis performed in bone marrow biopsies (BMB) from 20 CLL patients (10 CD38pos and 10 CD38neg cases) showed detectable levels of CCL3 in 8 cases, all but one belonging to the CD38pos group (p=0.02). Expression of the CCL3/CCL4 specific receptors CCR1 and CCR5 was examined by flow cytometry in peripheral blood cell subpopulations from 30 CLL (12 CD38pos and 18 CD38neg). Irrespectively of CD38 expression by CLL cells, monocytes showed the highest expression levels for CCR1 and, although at a lesser extent, CCR5. Consistently, CCL3 was able to attract CLL-derived monocytes by in-vitro chemotaxis experiments, and a higher number of infiltrating CD68pos macrophages were found in BMB of CD38pos compared to CD38neg CLLs (p=0.016). In parallel experiments, conditioned media (CM) from CCL3-stimulated macrophages were collected; these CM were able to induce expression of the CD49d-ligand VCAM in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (ADMEC). As shown by ELISA, TNFalpha was among the cytokines contained in macrophage-CM. This citokine was likely responsible for VCAM up-regulation by HUVEC and ADMEC, as suggested by TNFalpha neutralization experiments leading to a suppression of VCAM-1 induction in endothelial cell models. Again, IHC analysis of CLL BMB showed a meshwork of VCAM-1-positive cells more prominent in the context of lymphoid infiltrates of CD38pos, as compared to CD38neg cases (p=0.002). To verify whether CD49d engagement through VCAM-1 could enhance the protection against spontaneous apoptosis of CLL cells in vitro, we cultured purified CD38pos/CD49dpos CLL cells from 5 cases onto VCAM-1-transfected L cells or mock-transfected L cells. Results demonstrated a substantial improvement in cell viability after CD49d engagement: as high as 70%±25 cells were viable after 10 days of culture on L-VCAM cells compared to 50%±25 in control conditions (p=0.009). Altogether, these results identify molecules involved in a functional cross-talk between CD38/CD49d-expressing CLL and cells of the tumor microenvironment. This interplay may eventually affect survival and recirculation of tumor cells via the CD49d/VCAM pair.

CLL subsets with stereotyped HCDR3 and IGH/IGK-L gene combinations have been reported among cases with both mutated (M) and unmutated (UM) IGHV gene status. Here we evaluated the characteristics of stereotyped combinations in 1428 IG sequences (848 M and 580 UM) from 1399 CLL patients ordered according to a HCDR3-driven clustering, performed aligning HCDR3 amino acid sequences (codons 107–117 IMGT) by the multiple sequence alignment software ClustalX (1.83). Clusters with stereotyped HCDR3 were selected for having a mean alignment score>60/100. We identified 73 different clusters with stereotyped HCDR3, 32/73 with >3 cases (range 3–32) for a total of 247 cases (17.4%), 81 with M and 166 with UM IGHV genes; 17/32 clusters (200 cases) were previous described, while 15 (47 cases) were novel. Seventeen/32 clusters (156 cases) were composed by UM cases (UM/M = 153/3), 14/32 clusters (59 cases) were of M cases (UM/M = 3/53), and a single cluster (32 IGHV3-21 cases) was a mixed one (UM/M=10/22). In our series, 64% of cases (158/247) utilized four specific IGHV genes, i.e. 1–69 (60/169 cases, all UM, split into 6 clusters), 3–21 (32/55 cases, all in a single cluster), 4–34 (19/129 cases, 17 M and 2 UM, split into 2 clusters), 4–39 (17/55 cases, 14 M and 3 UM, split into 3 clusters) or mixed IGHV genes of the IGHV1 family/clan (IGHV1-2/1-3/1-18/1-46/7-4), all in a single cluster (30/153 cases, all UM). Clinical data (time-to-treatment, TTT) were available for 637/1399 patients. UM cases (251) had significantly shorter TTT (p<0.00001), as compared to M (386) cases. Similarly, TTT were significantly shorter (p<0.0005) in 113 CLL patients belonging to clusters (Hom) as compared to 492 CLL patients not belonging to clusters (Het), although these differences were no longer observed when TTT of Hom vs. Het cases were compared in the context of M (Hom/Het = 34/336) and UM (Hom/Het = 79/156) CLL. Patients belonging to the IGHV3-21 cluster had TTT similar to 241 UM cases and significantly shorter (p<0.0001) than 370 M CLL. Shorter TTT were also documented in 15 cases belonging to the IGHV3-21 cluster when compared to 13 IGHV3-21-expressing Het cases either considered altogether or split into M and UM cases (p=0.03). Finally, patients belonging to the IGHV1-2/1-3/1-18/1-46/7-4 mixed cluster had shorter TTT in comparison either with all the other M and UM cases, or with the cases expressing the same IGHV genes but with Het HCDR3 gene, even if this latter group was split in M and UM cases (p = 0.002). The identification of stereotyped HCDR3 CLL suggests a role of specific antigen(s) in defining the biological and clinical features of this disease.