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Malnutrition is frequent in ovarian cancer (OC) patients and may compromise post-operative outcomes. The aim of this study is to evaluate the impact of pre-operative immunonutrition on the surgical outcome of OC patients, and on their nutritional, inflammatory and peripheral blood immune status.A prospective study was performed between September 2016 and April 2020. Immune-enhancing enteral nutrition was administered to 42 patients before surgery according to their nutritional status assessed by the Malnutritional Universal Screening Tool. Biochemical and hematological monitoring was performed before and after immunonutrition. Post-operative outcomes were assessed and compared with those of a similar group of patients treated without nutritional support.Of the 42 immune-nourished patients, 23 (54.8%) had a low, 11 (26.2%) an intermediate and 8 (19%) a high risk of malnutrition. After the immunonutritional intake, significant variations of prealbumin, creatinine and white blood cells were detected. All T cell populations had an increasing trend, in particular CD3Pre-operative immunonutrition improves the surgical outcome of OC patients. After immunonutrition, an increase of CD3

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large ( 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Multiple myeloma (MM) is a hematological disease characterized by the proliferation and accumulation of malignant plasmacells (PCs) in the bone marrow (BM). Despite widespread use of high-dose chemotherapy in combination with autologous stem cell transplantation (ASCT) and the introduction of novel agents (immunomodulatory drugs, IMiDs, and proteasome inhibitors, PIs), the prognosis of MM patients is still poor. CD38 is a multifunctional cell-surface glycoprotein with receptor and ectoenzymatic activities. The very high and homogeneous expression of CD38 on myeloma PCs makes it an attractive target for novel therapeutic strategies. Several anti-CD38 monoclonal antibodies have been, or are being, developed for the treatment of MM, including daratumumab and isatuximab. Here we provide an in-depth look atCD38 biology, the role of CD38 in MM progression and its complex interactions with the BM microenvironment, the importance of anti-CD38 monoclonal antibodies, and the main mechanisms of antibody resistance. We then review a number of multiparametric flow cytometry techniques exploiting CD38 antigen expression on PCs to diagnose and monitor the response to treatment in MM patients.

The Coronavirus Disease 2019 (COVID-19) pandemic is a global threat to healthcare systems, requiring hospitalization in sub intensive and intensive care for respiratory syndrome in 25-30% of patients and accounting for a lethality up to 15%.In this retrospective study the clinical characteristics of 215 COVID-19 patients were correlated with the peripheral blood immune status.Different groups of COVID-19 patients may be identified on the basis of clinical behavior and a strong correlation between groups and age and comorbidities as well as with the immune profile is demonstrated. A lower age correlates with a lower severity of the disease differentiating between patients who may be quarantined at home and those requiring hospitalization. An older age (>82 years) together with a higher number of comorbidities is associated to a very severe prognosis. The absolute number of CD3+, CD4+, CD8+ T lymphocytes was progressively decreasing according to the severity of the disease and a CD3+ and CD8+ threshold indicating very severe cases is suggested.

Allogeneic hematopoietic cell transplantation (allo-HCT) is an adoptive immunotherapy strategy whose effectiveness relies on graft-versus-tumor (GVT) effect. We explored the feasibility of enhancing GVT after allo-HCT by peptide vaccination. Two myeloma patients were transplanted with a fludarabine-total body irradiation conditioning regimen and vaccinated with an HLA-A*0201-restricted modified survivin nonapeptide, plus montanide as adjuvant. At time of first vaccination, one patient had just attained serological remission despite documented relapse after transplant, while the other patient was in stable disease. Both patients had an immune response to vaccination: the frequency of survivin-specific CD8+ T cells increased between second and sixth vaccination and accounted for 0.5–0.8% of CD8+ cells; CD8+ cells were functional in ELISPOT assay. The first patient persists in complete remission with a follow-up of >5 years, while the second patient did not have a clinical response and vaccination was halted. We analyzed the T-cell receptor (TCR) repertoire of the first patient by spectratyping and found that vaccination did not affect the diversity of TCR profile, indicating that survivin clonotypes were probably spread in multiple TCR families. We generated a limited number (n = 4) of survivin-specific T cell clones: three were reactive only against the modified peptide, whereas one clone recognized also the naive peptide. Peptide vaccination is safe and applicable after allo-HCT and elicits an efficient antigen-specific T cell response without causing graft-versus-host disease.

Cytokine-induced killer (CIK) cells consist of a heterogeneous population of polyclonal T lymphocytes displaying NK phenotype and HLA-unrestricted cytotoxic activity against a broad range of tumors. We sought to determine whether transduction of CIK cells with T cell receptor (TCR) genes specific for tumor-associated antigens could generate effector cells endowed with a double mechanism of tumor recognition. HLA-A2-restricted TCR-transduced (TD) CIK directed against the melanoma antigens Mart1 and NY-ESO1 were generated by lentiviral transduction and successfully expanded over a 3-4-week period. TD-CIK cells were both CD3(+)/CD56(-) and CD3(+)/CD56(+) (31±8% and 59±9%, respectively), indicating that both major histocompatibility complex (MHC)-restricted T cells and MHC-unrestricted CIK could be targeted by lentiviral transduction. At the end of the culture, the majority of both unmodified and TD-CIK displayed an effector memory phenotype, without considerable expression of replicative senescence and exhaustion markers. Functionally, TD-CIK specifically recognized tumor cells expressing the relevant antigen as well as maintained their MHC-unrestricted tumor activity. The cytotoxic activity of TD-CIK against HLA-A2(+) melanoma cell lines was significantly higher than the untransduced counterparts at a low effector:target ratio (cytotoxic activity of TD-CIK was from 1.9- to 4.3-fold higher than untransduced counterparts). TD-CIK were highly proficient in releasing high amount of IFN-γ upon antigen-specific stimulation and were able to recognize primary melanoma targets. In conclusion, we showed that (1) the reproducibility and simplicity of CIK transduction and expansion might solve the problem of obtaining adequate numbers of potent antitumor effector cells for adoptive immunotherapy; (2) the presence of both terminal effectors as well as of less differentiated progenitors might confer them long survival in vivo; and (3) the addition of an MHC-restricted antigen recognition allows not only targeting tumor surface antigens but also a wider range of cytoplasmic or nuclear antigens, involved in tumor proliferation and survival. TD-CIK cells with a double mechanism of tumor recognition are an attractive and alternative tool for the development of efficient cell therapeutic strategies.

Background Paroxysmal nocturnal hemoglobinuria (PNH) diagnostic guidelines recommend single-tube five- to six-color or two-tube four-color assays. PNH clones are detectable in only a fraction of patients at risk, and screening for new PNH cases can be complex and expensive. In this multicenter study, we have validated a simplified, one-tube two-color FLAER-based assay suitable for PNH screening. Methods Six laboratories received samples containing spiked PNH leukocyte clones to be analyzed in parallel with a common six-color cocktail (FLAER/CD24/CD45/CD64/CD15/CD14) and a simplified two-color mixture (FLAER/CD15), a shared calibration procedure, and a common analysis protocol. Replicate precision and sensitivity tests were performed on PNH patients, from undiluted to 1:10 000. Specificity tests were performed on normal donors to identify the possible sources of artifacts. Results The performance comparison between six-color and two-color assays showed an excellent agreement for granulocyte PNH clones. Dilution experiments showed an accurate detectability down to 0.01% sensitivity level for granulocyte PNH clones and to 1% for monocytes. Specificity experiments disclosed that basophils and platelets can contaminate the monocyte gate and generate false PNH events. Conclusions A simplified two-color (FLAER/CD15) PNH screening test has been validated in a highly standardized multicenter study and proved feasible and effective in ongoing regional programs. Precision, sensitivity, and specificity of the simplified test for granulocytes were comparable to the more complex and expensive six-color assay and applicable for screening also in peripheral laboratories. The diagnostic confirmation of PNH should be always performed by a reference center using the established technique on all cell lineages.

Introduzione: il latte d'asina non viene abitualmente consumato dalla popolazione e non sono mai stati riportati casi di allergia a questo alimento. In questo case report descriviamo come una giovane adulta si sia sensibilizzata al latte d'asina, mantenendo la tolleranza per il latte vaccino. Caso clinico: una biotecnologa di 37 anni impiegata in un laboratorio di manipolazione di alimenti ha iniziato a manifestare sintomi oculorinitici e talora asmatiformi durante la manipolazione di latte di asinaliquido/in polvere, circa 8 mesi dopo aver iniziato a lavorare a contatto con questo alimento. In un'occasione, in seguito ad ingestione di latte di asina UHT, ha lamentato comparsa immediata di vellichio faringeo e successivamente tosse e sibilo toracico. Da allora non ha più ingerito né maneggiato latte d'asina, mentre ha continuato ad assumere e tollerare il latte vaccino e i suoi derivati. Indagini e risultati: gli skin prick test per latte vaccino, e per lattoalbumina, lattoglobulina, caseina vaccine sono risultati negativi. I test prick-by-prick per latte vaccino e latte d'asina (2 controlli sani), sono risultati positivi per latte d'asina. Le IgE specifiche per latte vaccino, alfa-lattoalbumina vaccina, beta-lattoglobulina vaccina, caseina vaccina, latte di capra e latte di pecora sono risultate negative. E' stato effettuato l'IgE immunoblot utilizzando l'estratto di latte vaccino e il latte d'asina crudo, che ha mostrato la presenza di bande reattive al latte d'asina, ma non al latte vaccino. il BAT (basophil activation test)effettuato con il latte d'asina é risultato positivo e negativo quando effettuato conil latte vaccino. Conclusioni: oltre a costituire il primo caso documentato di allergia alimentare al latte d'asina, questo caso clinico conferma la possibilità della sensibilizzazione per via inalatoria /contatto nello sviluppo di allergia alimentare.

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer.

OBJECTIVES Morphology and cytogenetics are currently used to define prognosis in myelodysplastic syndromes (MDS). However, these parameters have some limits. Flow cytometry has been recently included in the diagnostic panel for MDS, and its prognostic significance is under evaluation. METHODS Marrow aspirates from 424 MDS patients were analyzed by flow cytometry to evaluate the impact of bone marrow cell immunophenotype on overall survival (OS) and leukemia-free survival (LFS). The immature compartment of myeloblasts was analyzed by the quantitative expression of CD34 (

The role of the proinflammatory interleukin (IL)-18 in cancer progression remains controversial; we thus examined the hypothesis that impaired antitumor immune response in pancreatic carcinoma patients is related to elevated levels of its natural inhibitor IL-18 binding protein (BP) and/or to alteration in the IL-18 receptor complex expression and function. IL-18 and IL-18 binding protein isoform a (BPa) was assessed in pancreatic carcinoma patients at various disease stages, and after surgery/chemotherapy; free bioactive IL-18 concentrations were calculated. IL-18 receptor complex expression in lymphocyte subsets was analyzed and signaling function was assessed versus healthy donors. Carcinoma cells exhibited below normal IL-18BPa expression and above normal IL-18 expression. Circulating IL-18BPa and IL-18 were above controls. Unexpectedly, free unbound IL-18 serum levels were correlated with disease severity and poor survival. IL-18BPa levels were unchanged by surgery but free IL-18 levels were elevated. Gemcitabine with 5-fluorouracil or oxaliplatin, but not alone, increased IL-18 and free IL-18 levels statistically significantly, without affecting IL-18BPa. Spontaneous/induced IL-18 receptor alpha and receptor beta expression in peripheral blood lymphocyte subsets from patients with advanced disease were near-normal, although CD4+ and CD8+ cells were fewer in percentage, and fully functional in inducing interferon-gamma. IL-18 is proposed as novel adjuvant cancer therapy, but free IL-18 levels are increased in the blood of pancreatic carcinoma patients, despite elevated IL-18BP levels, and are associated with poor survival; this highlights recent experimental insights into the prometastatic and proangiogenic effects of IL-18, and suggests that careful preclinical studies are needed to determine the proper application of IL-18 in cancer therapy.

We used a proteomic approach for identifying molecules involved in the pathogenesis of chronic lymphocytic leukemia (CLL). We investigated 14 patients who were completely concordant for IgV(H) mutational status (unmutated vs. mutated), CD38 expression (positive vs. negative), and clinical behavior (progressive vs. stable); these patients were characterized as having either poor or good prognoses. The 2 patient subsets differed in the expression of hematopoietic lineage cell-specific protein 1 (HS1). In patients with poor prognoses, most HS1 protein was constitutively phosphorylated, whereas only a fraction was phosphorylated in patients with good prognoses. This difference was investigated in a larger cohort of 26 unselected patients. The survival curve of all 40 patients analyzed revealed that patients with predominately phosphorylated HS1 experience a significantly shorter median survival time. As HS1 is a protein pivotal in the signal cascade triggered by B cell receptor (BCR) stimulation, we studied its pattern of expression following BCR engagement. Normal mature B cells stimulated by anti-IgM shifted the non- or less-phosphorylated form of HS1 toward the more phosphorylated form. Naive B cells showed both HS1 forms while memory B cells expressed mainly the phosphorylated fraction. These data indicate a central role for antigen stimulation in CLL and suggest a new therapeutic target for patients with aggressive disease.

The HER2/c-ErbB-2 proto-oncogene is overexpressed in 25–30% of human breast cancers. We previously reported the c-ErbB-2 transcript in mononuclear cells (MNC) from bone marrow (BM), peripheral blood (PB), and mobilized PB (MPB). Here, we describe extensively the expression pattern of c-ErbB-2 mRNA and protein in normal adult hematopoietic tissue and cord blood (CB)-derived cells. Quantitative reverse transcriptase-polymerase chain reaction shows that the c-ErbB-2 transcript is expressed in hematopoietic cells at low levels if compared with normal epithelial and breast cancer cells. The c-ErbB-2 protein was detected predominantly in MNC from PB and CB by Western blot analysis. Flow cytometry revealed that CD15+, CD14+, and glycophorin A+ subpopulations express c-ErbB-2 protein, whereas lymphocytes are c-ErbB-2-negative. The c-ErbB-2 expression is higher in CB MNC. More than 90% of BM- and MPB-derived CD34+ progenitors are c-ErbB-2-negative; by contrast, 5–40% of CB-derived CD34+ progenitors express c-ErbB-2. We found that c-ErbB-2 protein is up-regulated during cell-cycle recruitment of progenitor cells. Similarly, it increases in mature, hematopoietic proliferating cells. This study reports the first evidence that the c-ErbB-2 receptor is correlated to the proliferating state of hematopoietic cells. Studies in progress aim to clarify the role of c-ErbB-2 in regulation of this process in hematopoietic tissues.

Chronic lymphocytic leukemia (CLL) has a variable clinical course. CD38 expression and IgV(H) gene mutational status are independent predictors of prognosis, but their relationships and the CD38 cutoff level are unknown. Using cytofluorography, we analyzed CD38 in 148 patients, in 108 of whom we were able to evaluate IgV(H) Mutations, make correlations with disease history, and assess cumulative survival. Three different patient groups were identified by the CD38 expression pattern: a group homogeneously CD38(-), a group homogeneously CD38(+), and a group characterized by a bimodal profile, because of the concomitant presence of variable proportions of 2 distinct populations, one CD38(+) and one CD38(-). In CD38 bimodal expression patients the CD38(+) subset was significantly more represented in the bone marrow than in the peripheral blood. For IgV(H) mutations, 11.4% of CD38-, 84.6% of CD38(+), and 68.0% of CD38 bimodal expression patients had no mutation. CD38 expression, 19V(H) mutational status, and traditional prognostic factors were concordant. The progression rate was 12.9% for CD38-, 75.0% for CD38(+), and 63.3% for CD38 bimodal expression patients. Only 25.8% of the CD38(-) patients but 63.3% of the bimodal and 75.0% of CD38(+) patients were treated The presence of a CD38(+) population, albeit small, correlated with the development of auioimmune manifestations. T I he CD38(-) group has hot yet reached the median survival, which is 183 months in the CD38(+) group and 156 months in the CD38 bimodal expression group, regardless of the size of the CD38(+) population. The presence of a distinct CD38(+) population within the leukemic clone, rather than a numerical cutoff definition, correlates with IgV(H) gene mutational status and, irrespective of its size, identifies CLL patients who will have progressive disease. (C) 2003 by The American Society of Hematology.

We developed a panel of lentiviral vectors that displayed tetracycline-regulated transgene expression over two orders of magnitude in bulk, non-selected populations of transduced cells in vitro and in vivo. The robust expression and homogeneous response indicated that most transduced vector genomes were transcription competent and responsive to regulation, providing the lentiviral vector with a novel competitive advantage for gene transfer. After ex vivo transduction and transplantation of cord blood CD34+ cells into NOD/SCID mice, reporter gene expression could be switched "on" and "off" in human hematopoietic cells in vivo for prolonged times, proving integration of the regulated expression system into long-term repopulating cells. By vector injection into established tumor grafts, we achieved efficient delivery and quantitative regulation of transgene expression in vivo. By these approaches, gene function studies can now be performed in in vivo models of human hematopoiesis and cancer. In the future, regulated lentiviral vectors will improve the safety and efficacy of gene therapy.

Prolactin (PRL) shares structural and functional features with haemopoietic factors and cytokine peptides. Dendritic cells (DC) are involved in both initiating the primary and boosting the secondary host immune response and can be differentiated in vitro from precursors under the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus other factors. Because PRL has been shown to functionally interact with GM-CSF, we have addressed its role on GM-CSF-driven differentiation of DC. Monocytic DC precursors from peripheral blood mononuclear cells (PBMC) were enriched either by adhesion to a plastic surface or CD14-positive selection and cultured for 7 days in serum-free medium containing GM-CSF, interleukin (IL)-4 and PRL, alone or in combination. Cells with large, veiled cytoplasm, expressing major histocompatibility complex (MHC) class II and the costimulatory molecules CD80, CD86 and CD40 and lacking the monocyte marker CD14, were considered as having the phenotype of cytokine-generated DC. Functional maturation was assessed by proliferation and interferon-gamma (IFN-gamma) release of allogeneic T lymphocytes. Physiological (10-20 ng/ml) concentrations of PRL interacted synergistically with GM-CSF and the effect was similar to that induced by IL-4 on GM-CSF-driven DC maturation. When used alone, the physiological concentrations of PRL were inhibitory, whereas higher concentrations (80 ng/ml) were stimulatory. The synergistic effect of PRL may in part be caused by its ability to counteract the down-modulation of the GM-CSF receptor observed in serum-free conditions. These data provide further evidence of the significance of PRL in the process of T lymphocyte activation.

Prolactin (PRL) interacts with lymphocyte-signaling molecules and cytokines. Previous work has shown independent and synergistic effects of PRL on the generation of IL-2-driven anti-tumor lymphokine activated killer (LAK) activity by peripheral blood mononuclear cells (PBMC). The potential importance of PRL as a biological immunomodifier, however, is challenged by its ability to influence normal lymphocyte mitogenesis and hence lymphoid tumor growth. Since non-Hodgkin's lymphoma (NHL) cell lines were efficiently killed by LAK generated with native (n) or recombinant (r) human PRL combined with low, per se ineffective doses of IL-2, we have addressed here the question of whether PRL acts as a growth factor for LAK targets. NHL cells were analyzed for: 1. expression of the PRL receptor (PRL-R); 2. responsiveness to nPRL or rPRL; 3. constitutive expression and release of PRL; 4. existence of a PRL autocrine loop. PRL-R, defined by multiple antibodies, was detected in 3 of 12 NHL cell lines. However, nPRL or rPRL, in a wide range of concentrations (0.75-50 ng/ml), were not mitogenic for growth-arrested, PRL-R positive NHL cell lines. PRL mRNA was detected by RT-PCR in 10 of the 12 cell lines examined with a higher frequency among AIDS-related NHL cell lines. PRL protein in the immunoprecipitate of (35)S-methionine-labeled cell lysates and supernatants paralleled mRNA expression, and Western blotting analysis showed the presence of the pituitary/lymphocyte non-glycosylated (23.5 kDa) and glycosylated (25 kDa) isoforms. Experiments with blocking antibodies showed the independence from endogenous PRL for NHL cell growth.

DNA flow cytometry and the monoclonal antibody D07 were applied in formalin-fixed, paraffin-embedded specimens from 34 primary male breast carcinomas to verify whether DNA ploidy and p53 expression were associated with survival and proliferative activity. They were compared with tumor clinicopathologic features, sex steroid hormone receptors and cell proliferative activity, assessed by the counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against the proliferating cell nuclear antigen and the monoclonal antibody MIB-1. A significant correlation was found between survival and tumor ploidy (median survival, 77 months for diploid but only 38 months for aneuploid cases; P = .03) and p53 expression (median survival, 95 months for cases with p53 scores ⩽ 14.06% versus 33 for cases with p53 scores > 14.06%; P = .0004; median survival, 99 months for p53 negative vs 39 for positive cases; P = .007). Tumor histological grade ( P = .006), AgNOR counts ( P = .0001), PC10 scores ( P = .002), and MIB-1 scores ( P = .001) were also associated with prognosis. In the multivariate analysis, only p53 scores ( P = .001) or p53 immunopositivity ( P = .003) and AgNOR counts ( P = .022) retained an independent prognostic significance. Aneuploid tumors had higher AgNOR counts ( P = .002), PC10 ( P = .007), MIB-1 ( P = .006), and p53 scores ( P = .01) than diploid cases. A linear relationship was observed between p53 scores and AgNOR counts ( r = .41; P = .014), PC10 ( r = .46; P = .005), and MIB-1 scores ( r = .44; P = .011). These results indicate that DNA ploidy and p53 expression are associated with survival and cell proliferative activity in male breast carcinoma. Quantitative parameters, such as DNA ploidy, p53 scores, AgNOR counts, PC10, and MIB-1 scores substantially improve the prognostic significance of the traditional parameters in male breast carcinoma.

Our previous studies have shown that prolactin (PRL), a pituitary and lymphocyte hormone and a ligand of the cytokine/hemopoietin receptors (R) superfamily, acts synergistically with interleukin (IL)-2 on the development of lymphokine activated killer (LAK) cells and enhances the effects of GM-CSF and IL-3 on myeloid progenitors' proliferation and differentiation. More recently, we have demonstrated that GM-CSF and IL-3 increase the sensitivity of acute myeloid leukemic (AML) cells to LAK activity. Together, these findings have prompted us to study the role of PRL on the target arm of the LAK response. We show here that CD33+ blasts from AML patients express membrane PRL-R and that the PRL/PRL-R interaction is followed by increased susceptibility to natural killer (NK) (p < 0.02) and LAK (p < 0.001) cells. As predicted from the dimerization model of PRL-R and in agreement with previous reports, the response of AML blasts to PRL was bell-shaped with a trend peak at 25 ng/ml. Although enhanced lysis occurred at the target recognition level, it was not accompanied by changes in the MHC class I, cellular adhesion molecules, or myeloid differentiation antigens. Cell cycle recruitment and lysis increased concurrently in three cases studied, suggesting a modulatory action of PRL on the expression of putative cycle-related NK/LAK-target structures. Together, these data strengthen the role of PRL in the LAK response.

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

AIMS--To verify the correlation between MIB-1, Ki67, and proliferating cell nuclear antigen (PCNA-PC10) scores and S-phase fraction in intermediate grade non-Hodgkin's lymphomas (Working Formulation F); and their reliability in differently processed tissues. METHODS--Forty one non-Hodgkin's lymphomas were classified as (F) intermediate grade malignant lymphomas according to the Working Formulation; mitotic counts and percentage of large cells were assessed for each case. Sections from formalin fixed, paraffin wax embedded tissues were stained with anti MIB-1 monoclonal antibody, after microwave oven processing, and anti-PCNA (PC10) monoclonal antibody using an avidin-biotin immunoperoxidase (ABC) method. One thousand cells from 10 representative fields were scored. Frozen sections from surgical specimens were stained with Ki67 monoclonal antibody using the ABC method; the fraction of Ki67 positive cells was calculated scoring 1000 cells. Flow cytometry analysis (FCM) was performed on cell suspensions from fresh tissues. Correlations between data were estimated using linear regression. RESULTS--A linear correlation was found between MIB-1 and Ki67 scores (r = 0.92; p < 0.00001); between MIB-1 and PCNA scores (r = 0.79; p < 0.00001); and between MIB-1 score and S-phase fraction (r = 0.51; p = 0.0006). A linear correlation was also found between Ki67 and PCNA scores (r = 0.85; p < 0.00001); between Ki67 score and S-phase fraction (r = 0.6; p = 0.0002); and between PCNA score and S-phase fraction (r = 0.74; p < 0.00001). A correlation was found between mitotic counts and MIB-1 (r = 0.56; p = 0.0001), PCNA (r = 0.51; p = 0.0007), or Ki67 scores (r = 0.47; p = 0.002); between the percentage of large cells and MIB-1 (r = 0.49; p = 0.0009), PCNA (r = 0.6; p = 0.00003), and Ki67 scores (r = 0.53; p = 0.0003) and S-phase fraction (r = 0.55; p = 0.0002). CONCLUSION--MIB-1, Ki67, and PCNA (PC10) scores and S-phase fraction are highly correlated and equally well represent the proliferative activity of intermediate grade non-Hodgkin's lymphomas in differently processed material. MIB-1 and PCNA stains can be applied even on small biopsy specimens. MIB-1 produces homogenous staining without background; it also strongly stains mitotic figures. It can be performed on routinely processed tissues, permitting the simultaneous evaluation of the morphology and tumour cell kinetics. The wide standard deviations of the proliferative indices found for intermediate grade NHL suggest that this category probably includes various degrees of malignancy.

Background aims Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34 + -derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. Methods The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34 + cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34 + cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. Results Gene expression analysis of in vitro -expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14 + subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo , we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. Conclusions Our data suggest that combining basal and expanded BM-derived CD34 + cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.

Morphology and cytogenetics are currently used to define prognosis in myelodysplastic syndromes (MDS). However, these parameters have some limits. Flow cytometry has been recently included in the diagnostic panel for MDS, and its prognostic significance is under evaluation.Marrow aspirates from 424 MDS patients were analyzed by flow cytometry to evaluate the impact of bone marrow cell immunophenotype on overall survival (OS) and leukemia-free survival (LFS). The immature compartment of myeloblasts was analyzed by the quantitative expression of CD34 (3% vs. ≥3%), CD117, and CD11b(-) /CD66b(-) (5% vs. ≥5%); myeloid maturation was analyzed by the expression of CD11b(+) /CD66b(++) (15% vs. ≥15%) and CD11b(+) /CD66b(+) (25% vs. ≥25%).In univariate analysis, the expression of immaturity markers (CD34(+) , CD117(+) , and CD11b(-) /CD66b(-) ) was associated with shorter LFS and OS (P 0.0001); higher expression of differentiation markers (CD11b(+) /CD66b(++) and CD11b(+) /CD66b(+) ) was associated with longer LFS (P 0.0001 and P = 0.0002, respectively) and OS (P 0.0001). In multivariate analysis, expression of CD34(+) (P = 0.007), CD117(+) (P = 0.013), and CD11b(+) /CD66b(++) (P = 0.023) retained independent prognostic value for OS, while only the expression of CD34(+) was a prognostic factor for LFS (P = 0.0003). Two different risk groups were defined according to the presence of 0-1 or ≥2 of these factors with significant different LFS and OS (P 0.0001). This score showed prognostic value in predicting survival even in subanalysis according to IPSS and WHO subgroups.Flow cytometric analysis in MDS may provide meaningful prognostic information. Blast percentage expressed as CD117(+) or CD34(+) cells and the quantitative assessment of myeloid maturation showed prognostic value for survival.

CD38, a nonlineage-restricted surface glycoprotein, is an ecto-enzyme (ADP ribosyl cyclase/cADPR hydrolase/EC 3.2.2.6) that regulates cytoplasmic Ca2+ and cell-cell interactions. The molecule also delivers trans-membrane signals, despite a structural ineptitude to the scope. To reconcile these issues in a unitarian model, we compared the effects of CD38 signaling in circulating and residential T lymphocytes, the latter represented by those colonizing the intestinal lamina propria. Results are as follows: 1) LP T cells express an enzymatically active form of CD38, characterized by a modified ratio between cyclase and hydrolase functions; 2) LP T cells do not mobilize Ca2+ upon CD38 ligation, as seen in PB T cells (this condition is due to a lack in activation of PLC- g, constantly observed in PB T lymphocytes); 3) The early steps of CD38 signaling involve activation of lck, syk, and LAT; 4) Late events include synthesis and release of IL-2, IL-4, IL-5, IL-10, IFN-g and GM-CSF; 5) The uniqueness of the CD38 pathway in LP T cells is not caused by impaired interactions with the CD31 ligand. The differences observed concern the signaling machinery that CD38 exploits for its own use and not the interplay with its ligand.

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.

Interferon-gamma (IFN-gamma) is produced by activated T lymphocytes and plays a regulatory role in immune responses. The nature and location of cells that express the IFN-gamma receptor (R) and respond to this lymphokine are not well documented. The distribution of human IFN-gamma-R (HuIFN-gamma-R) was, therefore, investigated in situ by immunohistochemistry, using affinity-purified rabbit polyclonal antibodies directed against the extracellular domain of the receptor. In lymphoid organs, IFN-gamma-R expression is restricted to the B cell areas of lymph nodes, adult and fetal spleen, tonsils, appendix, and mucosa-associated lymphoid tissue of the small bowel. Macrophages and other reticular cells in lymphoid tissues and other organs are strongly positive for IFN-gamma-R, whereas its expression was consistently negative in the cortical and medullary thymocytes. Two-color flow cytofluorometric analysis of blood, lymph node, tonsil, spleen and thymus cells confirms that most B lymphocytes are IFN-gamma-R positive, whereas T lymphocytes are negative. However, after in vitro activation, peripheral blood T cells become IFN-gamma-R+. In non-lymphoid organs, IFN-gamma-R is expressed on endothelial cells of the medium- and small-size vessels. In epithelial tissues, high expression of IFN-gamma-R is detected on trophoblastic epithelium, glandular cells of stomach, ileum and colon, lung alveolar cells, salivary duct cells, renal tubular cells, and endometrial mucosa cells. Hepatocytes are weakly positive, while squamous epithelial cells are negative. The distribution of the HuIFN-gamma-R is discussed in view of the known functions of IFN-gamma.

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

The observation that the architecture of the cardiovascular and nervous systems is drawn by common guidance cues and the closeness between neural progenitors and endothelial cells in the vascular niche strongly suggests the existence of links between endothelial and neural cell fates. We identified an embryonic stem cell-derived discrete, nonclonal cell population expressing the two vascular endothelial growth factor receptors neuropilin-1 (Nrp1) and Flk1 that differentiates in vitro toward endothelial or neural phenotypes depending on microenvironmental cues. When microinjected in the chick embryo, Nrp1(+) cells integrate within the host, developing vessels and brain, and acquire endothelial and neural markers, respectively. These results show that precursors of endothelial cells and precursors of neural cells arise from the same pool of differentiating embryonic stem cells and share the expression of Nrp1 and Flk1. These data reinforce the parallelism between vascular and nervous system at the level of cell fate and commitment and open new perspective in regenerative medicine of neurovascular diseases.

Donor-derived cytokine-induced killer (CIK) can be infused as adoptive immunotherapy after hematopoietic cell transplant (HCT). Promising results were recently reported in HLA-identical HCT, where mild grafts versus host (GVH) events were observed. To extend this strategy across major HLA barriers (e.g. HLA-haploidentical HCT), further studies on CIK cells' alloreactivity are needed. We hypothesized that alloreactivity and anti-tumor activity of CIK cells segregate within two different cell subsets and could consequently be separated according to CD56 and CD3 expression. We tested CIK cells expanded from seven patients who underwent HCT as treatment of metastatic colorectal cancer. We found that CIK cells maintained their alloreactivity across major HLA barriers when tested as bulk population; after CD56-positive selection, anti-tumor activity was restricted to the CD3+/CD56+ cell fraction and alloreactivity versus HLA-mismatched PBMC was restricted to the CD3+/CD56- cell fraction. Bulk CIK cells from engrafted patients did not exhibit alloreactivity in response to host- or donor-derived PBMC, confirming their low potential for GVH across minor HLA barriers. Moreover, we tested if CIK cells expanded from engrafted patients after HCT were as effective as donor-derived ones and could be considered as an alternative option. The expansion rate and tumor cell killing was comparable to that observed in sibling donors. In conclusion, depletion of CD3+/CD56- cells might reduce the risk of GVH without affecting the tumor-killing capacity and could help extending CIK infusions across major HLA barriers. Engrafted patients after HCT could also be considered as an effective alternative option to donor-derived CIK cells.

Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.

The phenotype of the lymphoid cell component of 35 thymomas was investigated by analyzing cryostat sections and lymphocyte suspensions. The morphology in each case was determined by examining multiple tissue samples from different parts of the tumor. Structural heterogeneity was shown in 14 thymomas, and a homogeneous morphology of cortical or medullary or mixed types in the others. To assess whether this heterogeneity was correlated with differences in the lymphoid phenotype, we analyzed both lymphocyte suspensions and frozen sections from the same samples. Phenotypical differences in the suspensions of each thymoma in the heterogeneous group were noted and similar differences were also observed in the cryostat sections. Phenotypical abnormalities were found in some thymomas. They consisted of the simultaneous expression of cortical and medullary markers, which was most marked in the heterogenous mixed-type thymomas invading the lung. Furthermore, the global phenotype was tested on a pool of lymphocyte suspensions in all thymomas. This procedure distinguished cortical, medullary and intermediate cortico-medullary immunophenotype models which closely correlated with the tumor histology. It was concluded that, due to the frequent structural and immunological heterogeneity of thymomas, correct assessment of their lymphoid component requires a two-step analysis. This comprises: 1) individual suspensions from samples taken from different areas of the same thymoma, and 2) a pool of these suspensions. The first step will reveal the different immunological characteristics. In the second, the lymphocyte phenotype, which may vary widely throughout the tumor, will be represented in its totality. These findings may be of great help in predicting clinical patterns, especially possible malignant evolution.

Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome–positive patients and 6 BCR/ABL–positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in ∼80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL–positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8+ T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8+ T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells. [Cancer Res 2007;67(11):5300–7]

Multicolour flow cytometric analysis enabled the identification of monoclonal B-cell lymphocytosis (MBL), frequently resembling chronic lymphocytic leukaemia, at a rather high frequency in peripheral blood (PB) samples from an elderly population. PB T lymphocytes from 103 otherwise healthy subjects > 65 years of age and 51 younger donors (< 65 years) were analysed. Besides CD4(+) and CD8(+) single positive (SP) cells, CD4(+)CD8(+) double positive (DP) mature T lymphocytes were present in both series and could be further distinguished into CD4(high)CD8(low) and CD4(low)CD8(high) subsets. An age-dependent increase of both DP T-cell subsets was observed, while SP T cells remained stable throughout life. Flow cytometry and polymerase chain reaction analysis of the TRBV expression profiles showed the presence of a TRBV restriction within CD4(+)CD8(+) DP cells in more than half (53/103; 55.3%) of the individuals > 65 years of age, regardless the actual number of DP T cells observed. Clonal expansions were more prominent within the CD4(high)CD8(low) subset, accounting for most circulating DP clones (47/103; 45.6%). A few cases showed more than one (up to three) monoclonal expansion. Clonal CD4(low)CD8(high) DP T-lymphocyte expansions were detected in only 10/103 samples (9.7%) and showed a close phenotypic similarity to the rare T-cell large granular lymphocyte leukaemias. The similarities between DP clones and MBL in the elderly may help to better understand the mechanisms of immunosenescence and their relationships with the development of lymphoproliferative disorders.

The eradication of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) predicts for improved outcome. However, the wide variety of MRD techniques makes it difficult to interpret and compare different clinical trials. Our aim was to develop a standardized flow cytometric CLL-MRD assay and compare it to real-time quantitative allele-specific oligonucleotide (RQ-ASO) Immunoglobulin heavy chain gene (IgH) polymerase chain reaction (PCR). Analysis of 728 paired blood and marrow samples demonstrated high concordance (87%) for patients off-therapy. Blood analysis was equally or more sensitive than marrow in 92% of samples but marrow analysis was necessary to detect MRD within 3 months of alemtuzumab therapy. Assessment of 50 CLL-specific antibody combinations identified three (CD5/CD19 with CD20/CD38, CD81/CD22 and CD79b/CD43) with low inter-laboratory variation and false-detection rates. Experienced operators demonstrated an accuracy of 95.7% ( specificity 98.8%, sensitivity 91.1%) in 141 samples with 0.01 - 0.1% CLL. There was close correlation and 95% concordance with RQ-ASO IgH-PCR for detection of CLL above 0.01%. The proposed flow cytometry approach is applicable to all sample types and therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD-negativity in real time. These techniques may be used as a tool for assessing response and comparing the efficacy of different therapeutic approaches.

A pilot study was conducted to evaluate safety and activity of nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) in colorectal carcinoma (CRC) and to determine whether a T-cell response to a tumor-associated antigen (TAA) was induced. Fifteen patients with metastatic CRC underwent HCT from human leukocyte antigen (HLA)-matched siblings after a nonmyeloablative conditioning regimen. All patients engrafted with a median donor T-cell chimerism of 72% at day +56. Eight patients experienced grades II to IV acute graft-versus-host disease (GVHD). Despite progressive disease before HCT, partial remission and disease stabilization longer than 90 days were observed in 1 and 3 patients, respectively. Induction of TAA-specific T cells was evaluated with a carcinoembryonic antigen (CEA)-specific HLA-A(*)0201 pentamer in 6 patients with CRC. CEA-specific CD8(+) T cells were detected in 3 of 3 patients concomitant with GHVD onset, but not in 3 of 3 patients without GVHD. They were also not detected in 9 of 9 control patients with GVHD who received transplants for diagnoses other than CRC. Antitumor activity of CEA-specific T cells was also validated in vitro. In one patient, the induction of CEA-specific T cells was associated with a decrease of serum CEA levels and a partial response. Thus, graft-versus-host reactions associated with allogeneic HCT can trigger the generation of T cells specific for CEA, and this may be associated with a clinical response.

ZAP-70 tyrosine kinase is involved in signalling pathways following T-cell receptor stimulation and was originally described only in T cells and natural killer cells. ZAP-70 expression has been reported in normal mouse B lineage cells and in human malignant B lymphocytes, mainly in chronic lymphocytic leukemia (CLL) where it correlates with clinical outcome. We analyzed several B-cell lines and ex vivo malignant B cells, ranging from acute lymphoblastic leukemia to multiple myeloma and reflecting different stages of B-cell differentiation, and they showed ZAP-70 expression regardless their maturation stage. We then analyzed by Western blot and flow cytometry different human normal B-lymphocyte subpopulations: naive, germinal center and memory B cells from tonsils, CD19(+) CD5(+) cells from cord blood and CD19(+) lymphocytes from peripheral blood. All expressed ZAP-70 protein, though at different levels depending on their differentiation, activation and tissue localization. In addition, ZAP-70 expression levels could be modulated following stimulation via the B-cell receptor. These findings implicate a potential role of ZAP-70 in the signalling pathway of B lymphocytes at different maturational stages, indicate that ZAP-70 expression is not a CLL-specific feature among B-cell malignancies and suggest that the absence of ZAP-70 rather than its presence should be considered abnormal for malignant B lymphocytes.

Background. The role of normal lymphocytes in the development of malignancies is investigated with increasing interest, with particular focus on lymphoproliferative disorders. A few recent reports, including a large study from our group, have shown that the lymphocyte/monocyte ratio (LMR) is often altered in diffuse large B cell lymphoma (DLB-CL) and a reduced LMR at diagnosis is associated with a poor response to therapy and a marked reduction in the overall survival expectancy. Reduction of LMR is primarily due to decreased levels of circulating lymphocytes, although an increase of circulating monocytes may also concur in reducing the LMR value. The present study was undertaken to further characterize the lymphocyte subpopulations in DLB-CL and in the other germinal-center derived Follicular Lymphoma (FL). In particular, the aim was to investigate possible abnormalities of either B or T-lymphocytes that might be present in patients with DLB-CL or FL at disease onset. Patients and Methods. Peripheral blood (PB) samples were obtained at diagnosis from 23 DLB-CL and 15 FL without leukemic involvement; among them 11/23 DLB-CL and 12/15 FL did not display disease involvement also in their bone marrow (BM). In addition, 25 PB and 20 BM were obtained as controls from age matched healthy donors or from subjects undergoing diagnostic procedures without any evidence of hematological or solid malignancy. Multicolor flow cytometry (MFC) was used, to evaluate the PB distribution and the absolute value of the main cell categories, i.e.: CD3+ T cell, CD3-CD56+ NK cells, and CD19+CD20+ B cells; among these latter, the following subsets were investigated: transitional (CD38+high/CD24+high/IgD+/CD27-), naive (CD38+/CD24+/IgD+/CD27-), memory IgD+ (CD38-/+CD24+/IgD+/ CD27+), and memory IgD- (CD38-/CD24+high/IgD-/CD27+). Similarly, MFC was used to identify B-lymphocyte subsets in BM samples (B cell precursors as CD19+CD20-CD10+ and mature B cells as CD19+CD20+CD10-). Results. The absolute number of circulating B cells was significantly reduced in both FL (median 86 cell/ul) and DLB-CL (median 72 cell/ul) compared to normal age-matched controls (median 233 cells/ul), as detailed in the Figure 1. The low absolute number of circulating B cells did not correlate with a low LMR or with bone marrow involvement. Despite the generalized reduction in absolute number, the proportion of transitional and naïve B cell subsets was similar or slightly increased in B-NHL, while the percentage of memory B cell was deeply reduced in both FL and DLB-CL compared to normal controls. The amount of BM CD19+ B cells, evaluated as percentage on the whole lymphocyte population, was similar in both FL and DLBCL (15% and 15.9% respectively) and not significantly different from normal controls (18.9%). Interestingly, the percentage of B cell precursors (CD19+CD20-CD10+) within the B cell compartment was similar in FL and normal BM (50% vs 43.1%, p=ns), while it was marginally reduced in DLB-CL (23.7% vs 43.1%, p=ns). T and NK cells were not significantly different in DLB-CL and FL and in normal controls. Conclusions. Our data suggest that the homeostasis of normal B-cell compartment is impaired in FL and DLBCL, mainly at the peripheral level. The circulating memory B cells seem to be the B-cell subset affected in FL and DLB-LC. The mechanism underlying the reduction of circulating B lymphocyte number remains unknown. It might be hypothesized that FL and DLB-CL affect the microenvironment in which normal B cell differentiation occurs, resulting in the hampered production or increased elimination of differentiating B-cells. This hypothesis seems supported by the observation that the B cell population is not significantly altered in BM. Further study should be addressed to verify the impact of B-cell reduction on the long-term outcome of DLB-CL e FL and whether increasing circulating B-cells may improve the response to therapy. In addition the results here reported should be considered in relation with the prolonged B-cell impairment associated with the widely employed chemo-immunotherapy treatments for B-cell lymphoma. Figure 1. Absolute count values of total PB B lymphocyte in normal control (dottet bar), DLBCL (vertical line bar) and FL (diagonal line bar). Figure 1. Absolute count values of total PB B lymphocyte in normal control (dottet bar), DLBCL (vertical line bar) and FL (diagonal line bar). Disclosures No relevant conflicts of interest to declare.

We studied the biological features and the immunophenotype of a cell culture established from the lesion of soft tissues of a woman affected by Gorham-Stout syndrome. We found that these cells belonged to a monocytic lineage with some characteristics of immature osteoclasts and were able to release large amounts of osteoclastogenic and angiogenic molecules that may contribute to disease progression. Introduction: Gorham-Stout syndrome is a rare disease characterized by osteolysis and proliferation of vascular or lymphatic vessels, with a severe outcome. Its etiology and the identification of the cell types involved are completely unknown. Materials and Methods: A cell culture from a lesion of soft tissues was established, and its behavior in vitro and in immunodeficient mice was studied. We analyzed (1) the cell phenotype by flow cytometry; (2) the adhesive and migratory properties on different substrates; (3) the ability to differentiate into mature osteoclasts; (4) the production of osteclastogenic and angiogenic molecules; (5) the in vivo angiogenic activity of the cells subcutaneously implanted in mouse in a Matrigel plug; and (6) the ability to recapitulate the disease when transplanted in nude mice. Results and Conclusions: The established culture consisted of a morphologically homogeneous cell population belonging to a monocytic lineage having some features of an osteoclast-like cell type. Cells had an invasive phenotype, were angiogenic, and produced osteoclastogenic (IL-6, TGF-β1, IL-1β) and angiogenic (vascular endothelial growth factor-A {VEGF-A}, CXCL-8) molecules when challenged with inflammatory cytokines. Immunodeficient mice injected with these cells did not show any bone lesions or vascular alteration, but had high amounts of circulating human IL-6 and VEGF-A. Cells isolated from a cutaneous lymphangiomatosis did not show any of these findings. These data suggest that cells of monocyte-macrophage lineage play an essential role in the pathogenesis of Gorham-Stout disease, whose progression is propelled by cytokine circuits that accelerate angiogenesis and osteoclastogenesis.

The responsiveness and diversity of peripheral B-cell repertoire decreases with age, possibly because of B-cell clonal expansions, as suggested by the incidence of serum monoclonal immunoglobulins and of monoclonal chronic lymphocytic leukemia (CLL)-like B lymphocytes in clinically silent adults. We phenotyped peripheral blood cells from 500 healthy subjects older than 65 years with no history or suspicion of malignancies and no evidence of lymphocytosis. In 19 cases (3.8%) a kappa/lambda ratio of more than 3:1 or less than 1:3 was found: 9 were CD5(+), CD19(+), CD23(+), CD20(low), CD79b(low), slg(low) (classic CLL-like phenotype); 3 were CD5(+), CD19(+), CD23(+), CD20(high), CD79b(low), slg(low) (atypical CLL-like), and 7 were CD5(-), CD19(+), CD20(high), CD23(-), CD79b(bright), FMC7(+), slg(bright) (non-CLL-like). In 2 subjects, 2 phenotypically distinct unrelated clones were concomitantly evident. No cases were CD10(+). Polymerase chain reaction (PCR) analysis demonstrated a monoclonal rearrangement Of Ig(H) genes in 15 of 19 cases. No bcl-1 or bcl-2 rearrangements were detected. Using a gating strategy based on CD20/CD5/CD79 expression, 13 additional CLL-like B-cell clones were identified (cumulative frequency of classic CLL-like: 5.5%). Thus, phenotypically heterogeneous monoclonal B-lymphocyte expansions are common among healthy elderly individuals and are not limited to classic CLL-like clones but may have the phenotypic features of different chronic lymphoproliferative disorders, involving also CD5(-) B cells. (C) 2004 by The American Society of Hematology.

Gene transfer into T lymphocytes is currently being tested for the treatment of lymphohematologic disorders. We previously showed that suicide gene transfer into donor lymphocytes infused to treat leukemic relapse after allogeneic hematopoietic stem cell transplantation allowed control of graft-versus-host disease. However, the T-cell receptor (TCR) activation and sustained proliferation required for retroviral vector transduction may impair the half-life and immune competence of transduced cells and reduce graft-versus-leukemia activity. Thus, we tested lentiviral vectors (LVs) and stimulation with cytokines involved in antigen-independent T-cell homeostasis, such as interleukin 7 (IL-7), IL-2, and IL-15. Late-generation LVs transduced efficiently nonproliferating T cells that had progressed from G0 to the G1 phase of the cell cycle on cytokine treatment. Importantly, IL-2 and IL-7, but not IL-15, stimulation preserved physiologic CD4/CD8 and naive-memory ratios in transduced cells with only minor induction of some activation markers. Functional analysis of immune response to cytomegalovirus (CMV) showed that, although CMV-specific T cells were preserved by all conditions of transduction, proliferation and specific killing of autologous cells presenting a CMV epitope were higher for IL-2 and IL-7 than for IL-15. Thus, LV transduction of IL-2 or IL-7 prestimulated cells overcomes the limitations of retroviral vectors and may significantly improve the efficacy of T-cell–based gene therapy.

Growth and survival of chronic B-cell tumors are favored by the malignant cell's capacity to respond to selected microenvironmental stimuli provided by nontumoral bystander cells. To investigate which mechanisms operate in these crosstalks and whether they are malignancy-related or reproduce the mechanisms used by normal B cells we have studied the expression and functional role of semaphorin CD100 (now called Sema4D) in chronic lymphocytic leukemia (CLL) cells and normal CD5+ B cells. We demonstrate here that (1) leukemic and normal CD5+ B lymphocytes uniformly express CD100; (2) the CD100 high-affinity receptor Plexin-B1 is expressed by bone marrow stromal cells, follicular dendritic cells, and activated T lymphocytes, and is thus available to CD100+ lymphocytes in different specific microenvironments; and (3) upon interaction between CD100 and Plexin-B1 both CLL and normal CD5+ B cells increase their proliferative activity and extend their life span. These findings establish that Plexin-B1 is an easily accessible receptor for CD100 within the immune system. The encounter of CD100+ leukemic cells with Plexin-B1 may promote the proliferation and survival of malignant cells. The crosstalk operated by the CD100/Plexin-B1 interaction is not malignancy related but reproduces a mechanism used by normal CD5+ B cells.

A remarkable immune property of the hormone/cytokine prolactin (PRL) is its ability to modulate the cytotoxic activity of natural killer (NK) cells. This effect is observed with purified CD56+ cell populations stimulated by PRL or with peripheral blood mononuclear cells (PBMC) stimulated by PRL plus suboptimal concentrations of IL-2. The effect of PRL on NK cells can be referred to: i) increased proliferation of the γ/δ TCR positive NK subpopulation (CD16+CD3+), ii) increased release of IFN-γ, which acts as a potent autocrine maturation factor for lymphokine activated killers (LAK) and iii) activation of IRF-1, which regulates transcription of perforin, one of the effectors molecules of cell-mediated cytotoxicity. Modulation of MHC-restricted cytotoxic T lymphocytes (CTL) by PRL has recently been described. Acquisition of antigen presenting capabilities by monocytes is increased by physiological PRL in conjunction with GM-CSF, which results in increased IFN-γ response of T lymphocytes to alloantigen. IFN-γ release and cytotoxicity of CTL-sensitized in vitro against the tumor associated carcinoembrionic antigen (CEA) are also potentiated by the same concentrations of PRL. As a general trend, both NK cell mediated and MHC-restricted cytotoxicities are inhibited by high concentrations of PRL, so is Th1 cytokines production. CEA positive tumors produce PRL, which does not seem to be necessary for autocrine growth stimulation. It is tempting to speculate that by producing excess PRL the tumor hampers the local host tumor defense, thus creating a microenvironment favourable to its undisturbed growth.

The natural history of B-chronic lymphocytic leukemia (CLL) is not entirely explained by intrinsic defects of the neoplastic cell, but is also favored by microenvironmental signals. As CLL cells retain the capacity to respond to CD40 ligand (CD40L) and as CD4(+) T cells are always present in involved tissues, we asked whether malignant CLL cells might produce T cell-attracting chemokines. We studied the chemokine expression of CD19(+)/CD5(+) malignant B cells from peripheral blood (PB), lymph nodes (LN) or bone marrow (BM) of 32 patients and found a major difference. LN- and BM-, but not PB-derived cells, expressed a readily detectable reverse transcription-PCR band for CCL22 and one for CCL17 of variable intensity. CD40 ligation of PB cells induced the mRNA expression of both CCL22 and CCL17. CCL22 was also released in the culture supernatants. These supernatants induced the migration of activated CD4(+), CD40L(+) T cells expressing the CCL22 receptor, CCR4. T cell migration was abrogated by anti-CCL22 antibodies. Immunohistochemistry and cytofluorography studies revealed that a proportion of CD4(+) T cells in CLL LN and BM expressed CD40L. Our data demonstrate that malignant CLL cells chemo-attract CD4(+) T cells that in turn induce a strong chemokine production by the leukemic clone, suggesting a vicious circle, leading to the progressive accumulation of the neoplastic cells.