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B cell receptor (BCR) signaling is recognised as a central pathway in the pathogenesis of chronic lymphocytic leukaemia (CLL), the most common leukemia in the western world. In CLL, the BCR pathway is activated by antigens in the tissue microenvironment to promote the maintenance and expansion of leukemic cells. The impressive inhibition of clonal expansion of CLL cells and the disease control achieved with tyrosine kinase inhibitors that block BCR signaling provided further evidence of the role of BCR in CLL. Indoleamine 2,3-dioxygenase 1 (IDO1) is the inducible and rate-limiting enzyme involved in the catabolism of tryptophan to kynurenine. It was discovered as a modulator of the innate immune response during infection. Subsequent discoveries have implicated IDO1 as a player in acquired immune tolerance. IDO1 activity has been identified in several types of cancer cells as well as in their tumor-surrounding microenvironment. We have previously investigated and characterized the impact of IDO1 expression in CLL. Our results show that CLL microenvironmental stimuli positively modulate IDO1 expression in leukemic cells and that increased IDO1 activity leads to sustained survival of malignant lymphocytes and impaired drug sensitivity through the autocrine/paracrine activation of the aryl hydrocarbon receptor by kynurenine (Atene CG et al. 2022). To assess the functional and clinical significance of IDO1 enzymatic activity in CLL, we used several inhibitors that are currently in late-stage clinical trials. Using BMS-986205 (BMS), an irreversible inhibitor of IDO1 with the best cell-based potency, in primary patient cells, we observed not only a reduction in kynurenine production due to enzymatic inhibition of IDO1, but also lower levels of IDO1 RNA and protein following BCR stimulation with α-IgM. As we have previously shown that BCR activation can induce IDO1 expression, we treated CLL cells with increasing doses of BMS for 3 hours and then added α-IgM to the culture. BMS is able to counteract the pro-survival effect of BCR triggering, leading to significant apoptosis of CLL cells, while the viability of CLL cells in the absence of external stimuli was not significantly modulated. Focusing on the complex signaling cascade activated by BCR engagement, we observed that BMS impaired the phosphorylation of upstream kinases such as SYK and BTK, but also CD79a, CD19 and BLNK, in addition to downstream effectors such as AKT. We therefore investigated which factors that negatively regulate the BCR pathway are modulated by BMS treatment. By analyzing both mRNA and protein levels, we measured a significant induction of the phosphatases PTPN6, PTPN22 and SHIP1. Since BMS also showed a downregulation of total SYK expression, we are analyzing the expression of the proto-oncogene CBL, an E3 ubiquitin ligase known to be involved in the negative regulation of SYK. Further studies should be undertaken to determine whether, in addition to its role as an IDO1 inhibitor, BMS may also have the ability to indirectly modulate and disrupt BCR signaling. Efforts are underway to identify the proteome-wide direct target of BMS in CLL cells. In conclusion, our limited and preliminary data suggest that BMS could be considered as a potential therapeutic strategy to target CLL cells with a double hit: by blocking the pro-survival effect of BCR signaling, one of the driving forces for CLL cell survival and proliferation, and by inhibiting IDO1/kynurenine action to restore sensitivity to key leukemia treatments.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin [IL] 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin [IL] 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL.

The clinical relevance of angiopoietin-2 (Ang2) in chronic lymphocytic leukemia (CLL) was previously suggested by the association between high Ang2, and shorter progression-free survival reported in small series of patients. Here, we evaluated Ang2 glycoprotein levels in plasma samples collected from a multicentric cohort of CLL patients (n = 316) using an enzyme-linked immunosorbent assay method, and we investigated its prognostic role in relation to time to first treatment (TTFT) and overall survival. Based on a cutoff equal to 2459 pg/mL, we divided our cohort in 2 subsets (high and low Ang2) composing 100 (31.6%) and 216 (68.4%) patients, respectively. High Ang2 was predictive of reduced TTFT (P < .001) and overall survival (P = .002). Multivariate analysis confirmed that high Ang2 was an independent prognosticator for TTFT (hazard ratio = 1.739; 95% confidence interval, 1.059-2.857; P = .029). Significant associations were found between high Ang2 and advanced Binet stages (P < .001), high β2-microglobulin (P < .001), unmutated variable region of immunoglobulin heavy chain gene status (P < .001), high CD38 and ζ-chain-associated protein kinase 70 expression (P < .001 and P = .003), and intermediate/high cytogenetic risk (P = .005). Moreover, Ang2 added prognostic power to other conventional prognosticators and helped to refine prognosis among CLL subsets with both high and low vascular endothelial growth factor plasma levels. Ang2 plasma level may be a useful independent prognosticator for CLL.

The clinical relevance of angiopoietin-2 (Ang2) in chronic lymphocytic leukemia (CLL) was previously suggested by the association between high Ang2, and shorter progression-free survival reported in small series of patients. Here, we evaluated Ang2 glycoprotein levels in plasma samples collected from a multicentric cohort of CLL patients (n = 316) using an enzyme-linked immunosorbent assay method, and we investigated its prognostic role in relation to time to first treatment (TTFT) and overall survival. Based on a cutoff equal to 2459 pg/mL, we divided our cohort in 2 subsets (high and low Ang2) composing 100 (31.6%) and 216 (68.4%) patients, respectively. High Ang2 was predictive of reduced TTFT (P< .001) and overall survival (P= .002). Multivariate analysis confirmed that high Ang2 was an independent prognosticator for TTFT (hazard ratio = 1.739; 95% confidence interval, 1.059-2.857; P= .029). Significant associations were found between high Ang2 and advanced Binet stages (P< .001), high β2-microglobulin (P< .001), unmutated variable region of immunoglobulin heavy chain gene status (P< .001), high CD38 and ζ-chain-associated protein kinase 70 expression (P< .001 and P= .003), and intermediate/high cytogenetic risk (P= .005). Moreover, Ang2 added prognostic power to other conventional prognosticators and helped to refine prognosis among CLL subsets with both high and low vascular endothelial growth factor plasma levels. Ang2 plasma level may be a useful independent prognosticator for 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.

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, TGFB2and VIPR1as 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-16being 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.

Background. Approximately 70% of newly diagnosed chronic lymphocytic leukemia (CLL) patients present in early Binet or Rai stage, may never require treatment, and may have a life expectancy similar to that of the general population. Two independent and recent studies have identified the clinical and immunogenetic variables associated with shorter time to first treatment (TTFT) in Binet A and Rai 0 CLL (Condoluci et al., Blood2020; Cohen et al., Haematologica2020). However, the clinical impact of gene mutations in predicting TTFT is not completely understood.

Introduction

Abruzzese: Incyte:Consultancy, Membership on an entity's Board of Directors or advisory committees;Pfizer:Consultancy, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Membership on an entity's Board of Directors or advisory committees;Bms:Honoraria.

We recently reported that Swedish VH3-21-using chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite VH mutation status. To investigate this further, we analyzed the VH and VL gene rearrangements in 90 VH3-21+ patients from Sweden, Germany, Italy, United States, Finland, and Australia and correlated these data with survival and other prognostic markers. Sixty-three percent exhibited mutated VH genes and 37% unmutated VH genes. Fifty (56%) patients displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif DANGMDV. Also, a highly biased Vλ2-14 use was evident in 72% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although VH3-21+ CLLs have a remarkably predominant λ expression, analyses of kappa deleting element indicated a conserved light-chain rearrangement order. The overall survival was poor in the VH3-21+ cohort (median survival, 88 months), with no significant difference in relation to mutation status or CDR3 homology. High ZAP-70 and CD38 expression was found in both mutated and unmutated VH3-21+ cases as well as a slight increase of 11q-aberrations. In summary, highly restricted B-cell receptors and worse outcome characterize VH3-21+ CLLs independent of geographic origin and mutation status.

We recently reported that Swedish VH3-21-using chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite VHmutation status. To investigate this further, we analyzed the VHand VLgene rearrangements in 90 VH3-21+patients from Sweden, Germany, Italy, United States, Finland, and Australia and correlated these data with survival and other prognostic markers. Sixty-three percent exhibited mutated VHgenes and 37% unmutated VHgenes. Fifty (56%) patients displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif DANGMDV. Also, a highly biased Vλ2-14 use was evident in 72% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although VH3-21+CLLs have a remarkably predominant λ expression, analyses of kappa deleting element indicated a conserved light-chain rearrangement order. The overall survival was poor in the VH3-21+cohort (median survival, 88 months), with no significant difference in relation to mutation status or CDR3 homology. High ZAP-70 and CD38 expression was found in both mutated and unmutated VH3-21+cases as well as a slight increase of 11q-aberrations. In summary, highly restricted B-cell receptors and worse outcome characterize VH3-21+CLLs independent of geographic origin and mutation status.

Structural analysis of naturally processed peptides bound to the HLA class I and class II molecules of chronic myeloid leukemia (CML) blast cells was performed to characterize the antigen processing and autoantigen repertoire in this hematopoietic malignancy. Self-peptides derived from the carboxy-terminal end of the breakpoint cluster region (bcr) protein, as well as several differentiation stage- and tissue-specific self-antigens characteristic of early stages of myeloid differentiation, such as c-fes, c-pim, granulocyte-macrophage colony-stimulating factor receptor a chain, proteinase 3, and cathepsin G, were identified. A common characteristic of several of the high copy-number self-peptides identified in this study is the participation of their parent proteins in signal transduction or myeloid effector function. Because bcr-abl junctional peptides bind to a limited number of major histocompatibility complex (MHC) class I alleles, an effective peptide-based immunotherapy strategy for CML requires identification of further tumor-associated or tissue-specific peptide antigens binding to common MHC alleles such as HLA-A2. The differentiation stage- and tissue-specific MHC-bound peptides found in this study, as well as the naturally processed proteins from which they are derived, may represent autoantigens towards which T-cell responses may potentially be developed for immunotherapy of hematopoietic malignancies such as CML.

Human myeloid progenitor cells temporarily express HLA class II molecules during the differentiation pathway to granulocytes and macrophages. The significance of major histocompatibility complex (MHC) class II molecules at this stage of development is unknown. As a first stop of inquiry into their function, we have characterized the profile of major self-peptides bound to the HLA-DR molecules expressed by KG-1 cells, a line that shares many of the phenotypic characteristics of colony-forming unit-granulocyte-macrophage progenitors. Searches of protein data bases showed that all matching peptides bound to the HLA- DR molecules of KG-1 cells corresponded to intracellular, rather than exogenous or transmembrane, precursor proteins. Because the absence of a conventional self-peptide repertoire could be related to altered trafficking of class II molecules, the biosynthesis of HLA-DR and the invariant chain proteins was determined. The MHC class II associated invariant chain protein is synthesized normally in KG-1 cells, but processed fragments of invariant chain, class II-associated invariant chain peptides (CLIPs), occupy the antigen-binding groove of KG-1 class II molecules at a much lower frequency compared with that of mature antigen-presenting cells. Low CLIP occupancy of HLA-DR is a characteristic shared by KG-1 cells, normal CD34+ progenitor cells, and HLA-DR+ breast carcinoma cells. The unusual profile of MHC class II bound peptides and the low level of CLIP bound to HLA-DR suggest that the antigen-processing pathway of KG-1 is different from that characterized in professional antigen-presenting cells and that exogenous antigen-processing may be a developmentally acquired characteristic in the myeloid lineage.

We investigated the pathogenesis of isolated erythrocytosis of 14 yr duration in a 28-yr-old man. The increase in red cell mass was attributed to increased erythropoietin production. An extensive search for recognized causes of secondary erythrocytosis was unrevealing. Family members were found to be hematologically normal. After reduction of the circulating red cell mass by 20%, erythropoietin activity nearly quadrupled, thus suggesting a normal erythropoietin response to phlebotomy. When bone marrow cells of the patient were cultured in plasma clots in the absence of added erythropoietin, endogenous erythroid colony formation was observed, a pattern previously believed to be specific for polycythemia vera bone marrow cells. Our observations suggest that the erythrocytosis in this individual is best explained by an abnormal “servoregulatory” mechanism of erythropoietin production. In addition, this is the first instance in which the rule that endogenous erythroid colony formation is correlated with the diagnosis of polycythemia vera has not held.

We investigated the pathogenesis of isolated erythrocytosis of 14 yr duration in a 28-yr-old man. The increase in red cell mass was attributed to increased erythropoietin production. An extensive search for recognized causes of secondary erythrocytosis was unrevealing. Family members were found to be hematologically normal. After reduction of the circulating red cell mass by 20 %. erythropoietin activity nearly quadrupled, thus suggesting a normal erythropoietin response to phlebotomy. When bone marrow cells of the patient were cultured in plasma clots in the absence of added erythropoietin, endogenous erythroid colony formation was observed, a pattern previously believed to be specific for polycythemia vera bone marrow cells. Our observations suggest that the erythrocytosis in this individual is best explained by an abnormal “servoregulatory” mechanism of erythropoietin production. In addition, this is the first instance in which the rule that endogenous erythroid colony formation is correlated with the diagnosis of polycythemia vera has not held.

The bleeding time is currently the only clinically available comprehensive test to explore primary hemostasis. It is currently performed mostly as a screening procedure before surgery, to detect otherwise unknown defects in platelet-vessel wall interactions, but its use in this specific setting has been seriously questioned by recent reanalyses of previously published literature. We studied the relationship of the bleeding time from a standardized cutaneous incision with other parameters of bleeding derived from the analysis of the bleeding time curve and prospectively investigated possible correlations of these alternative parameters, as well as of the bleeding time, with a number of indices of actual bleeding during or after coronary bypass surgery. Four parameters (bleeding time, total bleeding, peak bleeding rate, and time to peak bleeding) were derived from the analysis of bleeding time curves measuring blood losses from a standardized cutaneous incision at 30-second intervals in 118 subjects. Parameters from the bleeding time curve were subsequently obtained in duplicate as a preoperative assessment in 40 patients with a negative bleeding history and no recent intake of non-steroidal anti- inflammatory drugs who were undergoing elective pure coronary bypass surgery performed by the same operator. These parameters were correlated in simple linear regression analysis with estimates of surgical bleeding (chest tube drainage, transfusion requirements, percentage of hematocrit, percentage of platelet level decrease, and times to hematocrit and platelet level nadir) and then, in multiple regression analysis, with indices of operation complexity (number of bypasses, total duration of the operation, and duration of the extracorporeal circulation). Bleeding time was significantly correlated, among parameters derived from the bleeding time curve, with total bleeding and peak bleeding rate, but not with time to peak bleeding. Bleeding time, total bleeding, and peak bleeding rate were similarly affected by acute interventions with intravenous aspirin (500 mg) and sublingual nitroglycerin (0.3 mg). None of these parameters, which were obtained in duplicate in each patient preoperatively, was significantly related to actual indices of bleeding at surgery. Thus, in patients with a negative history of bleeding and no recent intake of non-steroidal anti-inflammatory drugs, higher values for bleeding time and bleeding time-related parameters are not associated with higher indices of perioperative and postoperative bleeding at coronary bypass surgery. Therefore, we do not recommend the use of the test in this setting to predict perioperative or postoperative bleeding.

The bleeding time is currently the only clinically available comprehensive test to explore primary hemostasis. It is currently performed mostly as a screening procedure before surgery, to detect otherwise unknown defects in plateletvessel wall interactions, but its use in this specific setting has been seriously questioned by recent reanalyses of previously published literature. We studied the relationship of the bleeding time from a standardized cutaneous incision with other parameters of bleeding derived from the analysis of the bleeding time curve and prospectively investigated possible correlations of these alternative parameters, as well as of the bleeding time, with a number of indices of actual bleeding during or after coronary bypass surgery. Four parameters (bleeding time, total bleeding, peak bleeding rate, and time to peak bleeding) were derived from the analysis of bleeding time curves measuring blood losses from a standardized cutaneous incision at 30-second intervals in 118 subjects. Parameters from the bleeding time curve were subsequently obtained in duplicate as a preoperative assessment in 40 patients with a negative bleeding history and no recent intake of non-steroidal anti-inflammatory drugs who were undergoing elective pure coronary bypass surgery performed by the same operator. These parameters were correlated in simple linear regression analysis with estimates of surgical bleeding (chest tube drainage, transfusion requirements, percentage of hematocrit, percentage of platelet level decrease, and times to hematocrit and platelet level nadir) and then, in multiple regression analysis, with indices of operation complexity (number of bypasses, total duration of the operation, and duration of the extracorporeal circulation). Bleeding time was significantly correlated, among parameters derived from the bleeding time curve, with total bleeding and peak bleeding rate, but not with time to peak bleeding. Bleeding time, total bleeding, and peak bleeding rate were similarly affected by acute interventions with intravenous aspirin (500 mg) and sublingual nitroglycerin (0.3 mg). None of these parameters, which were obtained in duplicate in each patient preoperatively, was significantly related to actual indices of bleeding at surgery. Thus, in patients with a negative history of bleeding and no recent intake of non-steroidal anti-inflammatory drugs, higher values for bleeding time and bleeding time-related parameters are not associated with higher indices of perioperative and postoperative bleeding at coronary bypass surgery. Therefore, we do not recommend the use of the test in this setting to predict perioperative or postoperative bleeding.

We compared the immunophenotypic and karyotypic features of 25 cases of minimally differentiated acute myeloid leukemia (AML-M0) with those of 247 cases comprising all AML French-American-British (FAB) classification. Myeloperoxidase (MPO) was detectable with a specific monoclonal antibody in all cases of AML-M0, whereas CD13 and CD33 were both negative in 4 of the 25 cases. Thus, anti-MPO reliably detects minimal myeloid differentiation in AML-M0. CD34 and terminal deoxynucleotidyl transferase (TdT) were more frequently expressed in AML-M0 (96% and 68% of the cases, respectively) than in the other FAB subsets (P < . 001 for both). By contrast, GP-170 and CD7 were less frequently expressed in AML-M0 than in FAB classes such as M1, M4, and M5 (P = .02 and .003, respectively). A total of 80% of AML-M0 cases carried lymphoid markers (including TdT), and 48% showed a coordinate positivity for two or more of them. CD2, CD5, CD10, and CD19 were expressed in a similar fashion among the different FAB groups, whereas CD4 expression was significantly more frequent in AML-M0, AML-M4, and AML-M5 (P = .014). AML-M0 was characterized by a more frequent occurrence of complex karyotypes. In addition, approximately 20% of cases had TdT positivity, complex karyotypes, and anomalies of chromosome 5 and/or 7, a pattern not observed in the other FAB subsets. Finally, 80% of anomalies of chromosome 5 and/or 7 in AML-M0 were comprised within complex karyotypes, whereas only 13% of the remaining FAB cases carried this feature. In summary, AML-M0 frequently expresses immunophenotypic and karyotypic aspects that are likely to identify a “stem cell” pattern.

Human myeloid progenitor cells temporarily express HLA class II molecules during the differentiation pathway to granulocytes and macrophages. The significance of major histocompatibility complex (MHC) class II molecules at this stage of development is unknown. As a first step of inquiry into their function, we have characterized the profile of major self-peptides bound to the HLA-DR molecules expressed by KG-1 cells, a line that shares many of the phenotypic characteristics of colony-forming unit-granulocyte-macrophage progenitors. Searches of protein data bases showed that all matching peptides bound to the HLA-DR molecules of KG-1 cells corresponded to intracellular, rather than exogenous or transmembrane, precursor proteins. Because the absence of a conventional self-peptide repertoire could be related to altered trafficking of class II molecules, the biosynthesis of HLA-DR and the invariant chain proteins was determined. The MHC class II associated invariant chain protein is synthesized normally in KG-1 cells, but processed fragments of invariant chain, class II-associated invariant chain peptides (CLIPs), occupy the antigen-binding groove of KG-1 class II molecules at a much lower frequency compared with that of mature antigen-presenting cells. Low CLIP occupancy of HLA-DR is a characteristic shared by KG-1 cells, normal CD34+progenitor cells, and HLA-DR+breast carcinoma cells. The unusual profile of MHC class II bound peptides and the low level of CLIP bound to HLA-DR suggest that the antigen-processing pathway of KG-1 is different from that characterized in professional antigen-presenting cells and that exogenous antigen-processing may be a developmentally acquired characteristic in the myeloid lineage.

Structural analysis of naturally processed peptides bound to the HLA class I and class II molecules of chronic myeloid leukemia (CML) blast cells was performed to characterize the antigen processing and autoantigen repertoire in this hematopoietic malignancy. Self-peptides derived from the carboxy-terminal end of the breakpoint cluster region (bcr) protein, as well as several differentiation stage- and tissue-specific self-antigens characteristic of early stages of myeloid differentiation, such as c-fes, c-pim, granulocyte-macrophage colony-stimulating factor receptor α chain, proteinase 3, and cathepsin G, were identified. A common characteristic of several of the high copy-number self-peptides identified in this study is the participation of their parent proteins in signal transduction or myeloid effector function. Because bcr-abl junctional peptides bind to a limited number of major histocompatibility complex (MHC) class I alleles, an effective peptide-based immunotherapy strategy for CML requires identification of further tumor-associated or tissue-specific peptide antigens binding to common MHC alleles such as HLA-A2. The differentiation stage- and tissue-specific MHC-bound peptides found in this study, as well as the naturally processed proteins from which they are derived, may represent autoantigens towards which T-cell responses may potentially be developed for immunotherapy of hematopoietic malignancies such as CML.

Introduction: Chronic lymphocytic leukemia (CLL) is a dynamic disease in which monoclonal B cells proliferate within the pseudo-follicular centers in lymphoid organs and then they accumulate due to an intrinsic defect of apoptosis. Leukemic cells are considered as “addicted to the host” since extrinsic signals from the microenvironment strongly influence the establishment of a progressive immunosuppression for malignant cell growth and survival.

Luppi: Gilead Sci: Consultancy, Speakers Bureau; MSD: Consultancy; Sanofi: Consultancy; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy; Novartis: Consultancy, Speakers Bureau. Marasca:Gilead Sci: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Shire: Consultancy, Honoraria; Janssen: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria.

BACKGROUND:

Luppi: Gilead Sci., MSD, Pfizer, Novartis, Abbvie, Sanofi, Daiichi Sankyo, Jazz Pharmaceuticals: Honoraria. Marasca:Janssen and Gilead Sci, Abbvie, Roche and Shire: Honoraria, Research Funding.

BACKGROUND:

Background.It has been shown that serum ferritin and heart iron content are good indicators of bone mineral density (BMD) in patients with beta thalassemia major (TM). In the present study we explored for the first time the relationship between pancreatic iron and BMD and bone markers in TM.

Background.A common link between bone and heart health has been detected in the general population. In the present study we evaluated the relationship between bone mineral density (BMD) and cardiac iron, cardiovascular complications, and cardiovascular risk factors in patients with thalassemia major (TM).

Chronic lymphocytic leukemia (CLL) accounts for about 30% of all adult leukemias and is the most common hematologic malignancy in the Western countries. Interferon regulatory factor 4 (IRF4, also known as MUM1) is a transcriptional regulator of immune system development and function. Different IRF4 concentrations underlie the generation of alternative cell fate in mature B cells localized in secondary lymphoid organs, i.e. IRF4 “kinetic control” model. IRF4 at lower levels promotes germinal center formation and class switch recombination, whereas at higher levels IRF4 inhibits Bcl-6 and induces Blimp-1 to facilitate plasma cell development. Recent studies suggest a possible role of IRF4 in CLL pathogenesis. In New Zealand Black (NZB) IRF4+/- mouse model, CLL development is dramatically accelerated and IRF4+/- CLL cells showed hyper-responsiveness to B-cell receptor (BCR) activation.

Pepe: Chiesi Farmaceutici and ApoPharma Inc.: Other: Alessia Pepe is the PI of the MIOT project, that receives no profit support from Chiesi Farmaceutici S.p.A. and ApoPharma Inc.. De Franceschi:F. Hoffmann-La Roche Ltd, Basel, Switzerland: Research Funding.

Introduction.We aimed to prospectively assess if the male gender was associated with an higher risk of progressive cardiac iron accumulation, development of biventricular dysfunction and myocardial fibrosis assessed by CMR, and development of cardiac complications including heart failure (HF), arrhythmias and pulmonary hypertension (PH).

Burger: Boehringer Ingelheim Pharma: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Noxxon: Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Research Funding; Gilead: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Marasca:Janssen: Honoraria, Other: support for travel to congresses.

No relevant conflicts of interest to declare.

Introduction:In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages with a M2-like phenotype, associated to prominent immunosuppressive functions, and play a crucial role in CLL survival. Ibrutinib is a potent inhibitor of BTK and ITK kinases and is able to counteract the microenvironmental-derived pro-survival effects in CLL cells. One of the main effects of ibrutinib treatment is attenuated retention and homing of CLL cells to tissue compartments, which ultimately leads to mobilization of CLL cells from microenvironmental niches into the peripheral blood.

No relevant conflicts of interest to declare.

Maffei: Celgene: support for travel to congresses Other. Marasca:Celgene: Honoraria, Research Funding.

Cancer-associated microenvironment provides to malignant cells the right cocktail of signals to increase survival, reprogram their metabolism and escape the control of the immune system. Nicotinamide phosphoribosyltransferase (NAMPT) is the only human enzyme that metabolizes nicotinamide, the main NAD precursor, in a rate-limiting step in NAD biosynthesis. Beside this canonical activity, NAMPT can be secreted in the extracellular milieu (eNAMPT), where it exerts cytokine/adipokine-like actions in different tumor models, as well as in acute and chronic inflammatory-metabolic diseases. Here we investigated the functional role of eNAMPT in the microenvironment of chronic lymphocytic leukemia (CLL), an indolent lymphoproliferative disorder, strongly dependent on a growth supportive environment.

Lenalidomide is an immunomodulatory agent clinically active in CLL patients. The specific mechanism of action is still undefined, but includes the modulation of microenvironment. In CLL patients, nurse-like cells (NLCs) differentiate from CD14+ mononuclear cells and nurture/protect CLL cells from apoptosis. NLCs resemble M2 macrophages with potent immunosuppressive functions.

Off Label Use: Lenalidomide, a thalidomide analogue, is an immunomodulatory drug (IMiD) with antitumoural activity reported in various malignant disorders including multiple myeloma and myelodysplastic syndrome (MDS). At preclinical level, lenalidomide has shown to decrease the production of several prosurvival cytokines. This drug is also reported to modulate an effector cell immune response through the activation of T and natural killer (NK) cells, inducing apoptosis directly on tumour cells. Currently available data indicate that Lenalidomide is active also in heavily pre-treated CLL patients with unfavourable prognostic factors. However, in order to optimize the therapeutic index of Lenalidomide treatment in CLL patients, it will be necessary to identify features of tumour cells that differ between responder and non responder patients. Hence, we reported results from a multicenter, phase II study designed in order to identify potential predictive factors correlating with response to Lenalidomide treatment in relapsed/refractory CLL. Marasca:CELGENE CORPORATION: Honoraria, Research Funding.

Abstract 1782

Abstract 1782

Maffei: CELGENE CORPORATION: Research Funding. Off Label Use: Lenalidomide, a thalidomide analogue, is an immunomodulatory drug (IMiD) with antitumoural activity reported in various malignant disorders including multiple myeloma and myelodysplastic syndrome. At preclinical level, lenalidomide has shown to decrease the production of several prosurvival cytokines. This drug is also reported to modulate an effector cell immune response through the activation of T and natural killer cells, inducing apoptosis directly on tumour cells. Currently available data indicate that lenalidomide is active also in heavily pre-treated CLL patients. However, in order to reduce toxicity and to optimize the therapeutic index of lenalidomide treatment in CLL patients, it is necessary to identify features of tumour cells that differ between responder and non responder patients. Hence, we propose a multicenter, phase II study designed in order to identify potential predictive factors correlating with response and toxicity to Lenalidomide treatment in relapsed/refractory CLL patients. Martinelli:CELGENE CORPORATION: Research Funding. Debbia:CELGENE CORPORATION: Research Funding. Rigolin:CELGENE CORPORATION: Research Funding. Rizzotto:CELGENE CORPORATION: Research Funding. Castelli:CELGENE CORPORATION: Research Funding. Bonacorsi:CELGENE CORPORATION: Research Funding. Bulgarelli:CELGENE CORPORATION: Research Funding. Fiorcari:CELGENE CORPORATION: Research Funding. Zucchini:CELGENE CORPORATION: Research Funding. Santachiara:CELGENE CORPORATION: Research Funding. Forconi:CELGENE CORPORATION: Research Funding. Rossi:CELGENE CORPORATION: Research Funding. Laurenti:CELGENE CORPORATION: Research Funding. Palumbo:CELGENE CORPORATION: Research Funding. Vallisa:CELGENE CORPORATION: Research Funding. Cuneo:CELGENE CORPORATION: Research Funding. Gaidano:CELGENE CORPORATION: Research Funding. Luppi:CELGENE CORPORATION: Research Funding. Marasca:CELGENE CORPORATION: Research Funding.

IGHV1-69 gene identifies the most frequent IGHV gene in chronic lymphocytic leukemia (CLL) and identifies the paradigm of unmutated CLL (U-CLL), being used in roughly 1/3 U-CLL. It is often rearranged to form subsets of stereotyped HCDR3 patterns, likely selected and transformed from the natural naïve B-cell repertoire (Blood. 2010; 115:71-7). Being unmutated, IGHV1-69 CLL are hypothetically expected to have competent tumor B-cell receptors (BCR) and to progress more rapidly. However, it has not been investigated if progression occurs similarly in all the subsets.we aimed to investigate the prognostic significance of mutational status and of stereotypic B-cell receptors in IGHV1-69+ CLL.Nucleotide sequences of the tumor IGHV1-69/D/J rearrangement, clinical and molecular prognostic parameters at diagnosis and clinical status at follow-up of 294 IGHV1-69+ CLL patients were obtained from 22 hematological Institutions in Italy. CLL B-cell derived IGHV1-69 rearrangements were scanned for HCDR3 stereotypic patterns and assigned to subsets according to the criteria by Murray et al (Blood. 2008; 111: 1524–1533). Enpoint of outcome was time to progression requiring first treatment according to NCI criteria (TTFT) in Rai stage 0 CLL.Of 294 IGHV1-69+ CLL, 264 (89,8%) were unmutated, 168 (57,1%) were assigned to subsets, of which subsets 7 (n=23, 7,8%), 6 (17, 5,8%), 3 (13, 4,4%), 5 (10, 3,4%) and 9 (10, 3,4%) were the most frequent. CD38, ZAP70, normal or sole del13, +12, del11 and del17p scored positive in 109/264 (58,7%), 139/245 (56,7%), 128/248 (50,5%), 51/248 (20,6%), 43/248 (17,3%) and 34/248 (13,7%). CLLs were reclassified as 18 (6,1%) clinical MBL, 101 (34,4%) Rai 0, 155 (52,7%) Rai I-II and 20 (6,8%) Rai III-IV CLL. Subset 6 was also UM in 16/17 (94,1%) cases. Prevalence of CD38 (p<.001), ZAP70 (p=.016), normal or sole del13 (p<.001), +12 (p=.026), del11 (p=.011), and clinical high risk CLL (p=.025) were lower in IGHV1-69 M-CLL than in IGHV1-69 U-CLL. TTFT was significantly shorter in stage 0 IGHV1-69 U-CLL than in IGHV1-69 M-CLL (49 vs 144 months, p<.001, while it was not different between CLL assigned or not to subsets (65 vs 55 months, p=.346). However, specific analysis of individual subsets revealed differential outcomes (p=.005). Among all, it emerged that subset 6 had a TTFT equivalent to IGHV1-69 M-CLL (p=.29) and significantly longer than stage 0 IGHV1-69 U-CLL (median not reached vs 48 months, p=.017).our analysis documents and confirms that unmutated status of IGHV, and not stereotypy, is a relevant prognosticator of outcome (TTFT) in CLL. In the IGHV1-69 CLL it exclude a role of IGHV gene use for CLL progression. However, the good prognosis of Rai 0 U-CLL assigned to subset 6 suggests a differential clinical benign course of this particular subset, irrelevant of unmutated status. One possibility is that the IGHV1-69/D3-16/J3 rearrangements of subset 6 produce a tumor-specific BCR with stereotypic HCDR3 patterns that are anergized by antigen while circulating in the peripheral blood in early stage (Rai 0) CLL.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 3611

Abstract 1371

No relevant conflicts of interest to declare.