ALBERT

Description: Key Points Lyn’s overexpression mediates resistance to apoptosis by promoting phosphorylation and dimerization of procaspase 8 in B-CLL cells.

Description: Key PointsIn T-LGLL, autologous LGL-depleted PBMCs release high levels of IL-6 contributing to the constitutive STAT3 activation in leukemic LGL. Leukemic LGLs show SOCS3 down-modulation, which is responsible for lack of the negative feedback mechanism controlling STAT3 activation.

Description: T-cell depleted hematopoietic stem cell transplantation from haploidentical donors (haplo-HSCT) has been reported to benefit from the graft-versus-leukemia effect mediated by natural killer (NK) cells when donor displays NK alloreactivity versus the recipient. NK alloreactivity is mediated by NK receptors, namely Killer Ig-like receptors (KIR) which are specific for allotypic determinants that are shared by different HLA-class I alleles (referred to as KIR ligands). It is known that KIR2DL1 recognizes HLA-C alleles characterized by Lys at position 80 (C2 group), KIR2DL2/3 recognize HLA-C alleles characterized by Asn at position 80 (C1 group), KIR3DL1 recognizes HLA-B alleles sharing the Bw4 supertypic specificity (Bw4 group) and KIR3DL2 recognizes HLA-A3 and –A11 alleles. KIR2D/3DL are inhibitory receptors that, upon engagement with the cognate ligand, inhibit lysis. Activating KIRs, highly homologous in the extracellular domain to the inhibitory counterparts, are KIR2DS1, KIR2DS2 and KIR3DS1, but only KIR2DS1 has been shown to specifically recognize C2 group of alleles expressed on B-EBV cells. We analyzed 21 children with leukemia receiving haplo-HSCT from a relative after a myeloablative conditioning regimen; in all pairs, the expression of a given KIR ligand (HLA class I allele) of the donor was missing in the patient (i.e. KIR ligand-mismatched haplo-HSCT). T-cell depletion was performed through positive selection of CD34+ cells; no pharmacological immune suppression was employed after HSCT. KIR genotype of all donors was evaluated to detect the presence of the various inhibitory and activating KIR genes. Phenotypic analyses were performed on NK cells derived from the donor and the patient at different time points after HSCT. Thanks to the availability of new mAbs able to discriminate between the inhibitory and the activating forms of a certain KIR, we could identify the alloreactive NK cell subset at the population level. These alloreactive NK cells express the KIR specific for the KIR ligand-mismatch (permissive inhibitory KIR) and the activating KIR (if present), while they do not express all inhibitory KIR specific for the patient HLA alleles and NKG2A. Thus, in most instances, we could precisely identify the size of the alloreactive NK cell subset in the donor and in the reconstituted repertoire of the recipient. Functional assays were performed to assess alloreactivity, using appropriate B-EBV cell lines and, if available, patient’s leukemia blasts. In some cases, also NK cell clones were extensively studied, for phenotype and receptor involvement in killing activity. We found that, in most transplanted patients, variable proportions of donor-derived alloreactive NK cells displaying anti-leukemia activity were generated and maintained even at late time-points after transplantation. Donor-derived KIR2DL1+ NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3+ NK cells displayed poor alloreactivity against leukemia cells carrying HLA alleles belonging to the C2 specificity. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3+ (or by NKG2A+) NK cells that co-expressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role for the KIR2DS2 activating receptor in leukemia cell lysis could not be established. Taken together, these findings provide new information on NK alloreactivity in haplo-HSCT that may greatly impact on the selection of the optimal donor.

Description: Natural killer (NK) cells from patients with familial hemophagocytic lymphohistiocytosis because of PRF1 (FHL2, n = 5) or MUNC13-4 (FHL3, n = 8) mutations were cultured in IL-2 prior to their use in various functional assays. Here, we report on the surface CD107a expression as a novel rapid tool for identification of patients with Munc13-4 defect. On target interaction and degranulation, FHL3 NK cells displayed low levels of surface CD107a staining, in contrast to healthy control subjects or perforin-deficient NK cells. B-EBV cell lines and dendritic cell targets reveal the FHL3 NK-cell defect, whereas highly susceptible tumor targets were partially lysed by FHL3 NK cells expressing only trace amounts of Munc13-4 protein. Perforin-deficient NK cells were completely devoid of any ability to lyse target cells. Cytokine production induced by mAb-crosslinking of triggering receptors was comparable in patients and healthy control subjects. However, when cytokine production was induced by coculture with 721.221 B-EBV cells, FHL NK cells resulted in high producers, whereas control cells were almost ineffective. This could reflect survival versus elimination of B-EBV cells (ie, the source of NK-cell stimulation) in patients versus healthy control subjects, thus mimicking the pathophysiologic scenario of FHL.

Description: T large granular lymphocytes leukemia (T-LGLL) and NK-type chronic lymphoproliferative disorder (CLPD-NK) are rare diseases characterized by the abnormal expansion of large granular lymphocytes (LGLs) with cytotoxic activity, belonging to T and NK lineage, respectively. Currently, the etiology of these diseases is still largely unknown. Several data support the hypothesis that the inciting event is represented by the persistence of antigenic stimulation, maintained by the abnormal release of cytokines (mainly IL-6 and IL-15), establishing an inflammation status not achieving resolution. Recently, we showed that IL-6 and soluble IL-6Rα were highly expressed and released by patients’ LGL-depleted peripheral blood mononuclear cells (PBMC), accounting for a trans-signaling process. IL-6 trans-signaling is critically involved in inflammatory disease and promotes the transition from acute to chronic inflammation. Additionally, LGL proliferation is maintained for an impairment of the apoptotic machinery due to the activation of many survival signaling pathways, including JAK/STAT and RAS/MEK/ERK pathways. In some patients (both T-LGLL and CLPD-NK) STAT3 hot-spot mutations, inducing STAT3 activation, have been demonstrated. With this as a background, we investigated the IL-15 contribution to sustain IL-6 trans-signaling and in turn inflammation. We analyzed the relationships between STAT3 mutations, IL-6 and IL-15 in disease progression to assess the hypothesis that these findings characterize different stages of LGL disease. Thirty T-LGLL and 15 CLPD-NK patients were included in this study. Patients were subdivided according to the percentage of LGLs in PBMCs (LGL range: 35-90%). By ELISA in patients’ plasma, we showed that IL-6 concentrations were significantly higher in patients characterized by a disease with less than 60% circulating LGLs (35.7 ± 11.4 pg/ml with respect to patients with LGLs 〉60%: 9.1 ± 2.7 pg/ml; p

Description: Multiple myeloma is an incurable disease characterized by proliferation of clonal malignant plasma cells (PC). Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification but the clinical and prognostic value of immunophenotyping in MM remains questionable. We have analyzed the prognostic impact of a relatively new marker as CD69. CD69 is a type II membrane protein. T cells express CD69 rapidly upon stimulation of the T-cell receptor (TCR), which is why CD69 has been mostly regarded as an activation marker. The precise role of CD69 in immunity has not been determined because its ligand is unknown, but an emerging role of CD69 in Multiple Myeloma (MM) has been postulated. Previous laboratoristic data, using tumor lines derived from murine model with genotypic and immunophenotypic features of resistance to bortezomib, showed that as the neoplastic plasma cells (PC) develop bortezomib resistance, they have a germinal center B cell like immunophenotype, including decreased to absent expression of CD69. Interestingly the activation antigen CD69 associates with and inhibits the function of Sphingosine 1-phosphate (S1P). S1P is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. Other data showed that MM cells express the S1P receptors, S1P1, S1P2 and S1P3. Furthermore, S1P protects MM cells against dexametason-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time and concentration-dependent manner in human MM cell lines. In a previous abstract, we described for the first time in a clinical report, the CD69 expression on pathological PCs of MM patients. Our preliminary data also suggested an intriguing role of CD69 in patients treated with chemotherapy in different stages of disease. In this study, we report a larger setting of 97 patients where we confirmed the expression of CD 69 in 48 of them (49%) (see table I). Immunophenotyping was carried out by a 6-color method, using a FacsCanto II cytometer and the FacsDiva software. PCs were identified as CD138+/CD38+ events after an initial gate which included events with low SSC in the CD45/SSC cytogram. The MoAb panel also included CD19, CD20, CD117, CD56, cytoplasmic light chains K and Lambda. PerCP-Cy5.5-conjugated CD69 was evaluated on phenotypically abnormal plasma cells (i.e. CD19-, CD45- or dim), which were resulted to be clonally restricted. Results were considered positive when the percentage of positive cells was 〉 20%. After an induction regimen of treatment with four cycles of VDT (bortezomib, dexametasone, thalidomide), 69 patients were evaluable. 40/69 (65%) of patients obtained at least of a very good partial response or better (Responding pts). In this subgroup of patients 30/45 (66.6%) showed the expression of CD 69. On the contrary only in a little part of partial or less responding patients (NR pts) 9/24 (37.5%) CD69 was detected (see table II) (p=0.02 using a chi squared test and p=0.019 using a Fisher's exact test). Data on cytogenetic abnormalities, including del(13q), t(4;14) and del(17p), detected by fluorescence in situ hybridization, were available in 〉90% of patients. Clinical data were available in all patients and CD69 maintained its association with different response, independently of other prognostic variables. In conclusion CD69 is often expressed in PCM cases, and the expression of this marker is useful to reveal poor prognostic categories and delineate a risk stratification. This molecule could represent an emerging clinical factor to identify different outcomes in patients affected by MM and treated with the modern drugs. Table I Pts Characteristics Total CD69+ 97 48/97 Sex Male 51(52%) Female 46(48%) Clinical status MM non evaluableMM after VTD 28/9769/97 in VGPR/CR 45/69 in PR/SD/PD 24/69 Table II Pts treated with VTD Responding pts NRpts 45 24 CD69+ 39/69 30/45 9/24 CD69- 30/69 15/45 15/24 Disclosures No relevant conflicts of interest to declare.

Description: It is thought that the production of a variety of cytokines by the malignant Reed-Sternberg cells and the surrounding tissue contributes to the abnormal immune response which is a clinical and pathological feature in Hodgkin’s lymphoma. Single nucleotide polymorphism in the 5′-promoter region of cytokine genes are key factors for cytokine production and may modify the biology of the disease. Recently, differences in the prognosis according to the Interleukin-10 (IL-10) genotype have been shown in patients with diffuse large B cell lymphomas (Lech-Maranda et al, Blood2004; 103:3529). We assessed the distribution of frequencies of polymorphic allele variants in the genes of IL-10 (A-1082G and C-592A), TNF-alpha (C-863A and G-308A) and IL-6 (G-174C) in 218 patients with Hodgkin’s lymphoma and analysed for associations with patient charcteristics and prognosis. The polymorphism were analyzed using a multiplex amplification and mismatched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique as described by Tseng et al (Tissue Antigens2002; 59:280). DNA was extracted either from peripheral blood or paraffin-embedded lymph node biopsies from 218 patients with Hodgkin’s lymphoma (median age 32 years, range 13–71 years; 104 females and 114 males). 206 patients were treated with standard chemotherapy regimens: 123 patients received ABVD, 33 pts a modified Stanford V regimen (substituting 6 mg/m2 metchloramine with 650 mg/m2 cyclophosphamide), 29 pts MOPP (±ABVD), 21 pts BEACOPP. The distribution of allele frequencies in Hodgkin’s lymphoma at position -592 of the IL-10 gene was as follows: 43 % were homozygous for the CC genotype, 41 % were heterozygous and 16 % were homozygous for the AA genotype. The IL10 -592AA genotype was associated with a decreased progression-free survival (p=0.0057). The probability of progression-free survival at median time of observation of 4 years for patients homozygous for the IL-10 -592 AA genotype was 33 % (95 % C.I, 14–54 %), while for heterozygous patients and for patients homozygous for the −592 C allele it was 74% (95% C.I., 61–83). When the analysis was restricted to 123 patients treated with ABVD chemotherapy, essentially the same differences in progression-free survival were observed. No associations between genotype distributions of TNF alpha at position −308 and −863, IL-6 at position −174, and IL-10 at position −1082 and clinical variables and prognosis were found. In univariate analysis of established prognostic factors, stage proved to be of prognostic value in our patient group (limited disease in stage I–IIA vs advanced disease in stage IIB–IV, p=0.012) The Cox multivariate analysis showed that IL-10 -592AA genotype and stage were independent prognostic factors (p=0.01 and 0.018, respectively). Our study indicates that the cytokine genotype can predict clinical outcome in patients with Hodgkin’s lymphoma and points to the importance of the genetic background of the host.

Description: INTRODUCTION Cortactin is an actin-binding protein involved in several cell functions, i.e. the assembly and the organization of cytoskeleton. Its overexpression was observed in several human cancers and experimental data support the role of cortactin in metastatic capability through the regulation of cell motility and the release of matrix metalloproteinase-9 (MMP-9). The activity of this protein is regulated by its phosphorylation in Tyr residues by Src kinase family. We previously demonstrated that in leukemic cells from B-CLL patients the Src kinase Lyn is overexpressed, activated and involved in the resistance to apoptosis. Recently, we found that cortactin is overexpressed in patients with B-CLL. Here we investigated the involvement of cortactin in the release of MMP-9 and, therefore, in the progression of B-CLL. METHODS Blood samples were collected from 10 controls and 34 B-CLL patients. Informed consent was obtained according to the Declaration of Helsinki. Untouched peripheral blood B cells were purified using the RosetteSep for human B cells isolation kit. The samples that were used had at least 95% of normal CD19+ or neoplastic CD5+/CD19+ cells, as assessed by flow-cytometry (FC). The purified B cells (2×106 cells/ml) were cultured in RPMI medium with or without CXCL12 (100ng/ml) and Ibrutinib (5μM) for the evaluation of MMP-9 production using ELISA. MMP-9 release by neoplastic B cells was also investigated after cortactin silencing in 4 patients which expressed high level of cortactin. The protein was silenced by SMARTpool siRNA collection (Dharmacon, Thermo Scientific), according to the manufacturer's instructions. Immunohistochemical (IHC) staining was performed on formalin-fixed, paraffin-embedded tissue sections using a fully automated platform. RESULTS By IHC and FC we confirmed the increased expression of cortactin in patients with respect to controls, moreover we defined three groups of patients according to different expression levels of Cortactin. We found that the release of MMP-9 by neoplastic B cells correlated to the expression of cortactin after 5 and 24-hrs culture. To investigate whether cortactin was involved in MMP-9 secretion in B-CLL, a cortactin-targeted siRNA silencing system was used to knockdown this protein in 4 patients with high cortactin expression. We found that following cortactin knockdown, leukemic cells showed a defect in MMP-9 secretion, as assessed by ELISA test. This protease also showed a decreased gelatinolytic activity in culture medium, supporting the hypothesis that cortactin is involved in the regulation of MMP-9 secretion in B-CLL malignant cells. We also demonstrated that the incubation of leukemic cells with PP2 or Btk decreased Tyr phosphorylation level of cortactin and shut down the release of MMP-9 in culture medium, also following CXCL12 triggering. Interestingly, we found that cortactin levels were significantly reduced following 3 months of Ibrutinib treatment and remained lower till the last clinical control, at 6 months of treatment. Consistent with these data we demonstrated i) a significant reduction in MMP-9 levels after in vivotreatment with Ibrutinib ii) a strong correlation between Cortactin expression and MMP-9 levels in CLL patients before and following Ibrutinib therapy. Finally, we also observed a redistribution of lymphocytes from the tissues to the periphery and a consequent reduction of lymphadenopathy in 4 cases where the levels of cortactin were higher than those observed in the patient who did not show enlarged lymph nodes at the time of diagnosis. CONCLUSIONS The overexpression of cortactin in neoplastic B cells and the correlation between cortactin levels, activity and MMP-9 release suggest a role of this protein in metastatic invasion and in the B-CLL aggressiveness. In addition, cortactin might represent a biomarker for diagnosis and prognosis and a target for new therapeutic strategies. Disclosures No relevant conflicts of interest to declare.