Targeting siglecs--a novel pharmacological strategy for immuno- and glycotherapy

The immune system must be tightly held in check to avoid bystander tissue damage as well as autoreactivity caused by overwhelming immune reactions. A novel family of immunoregulatory, carbohydrate-binding receptors, the Siglecs (sialic acid binding immunoglobulin-like lectins), has received particular attention in light of their capacity to mediate cell death, anti-proliferative effects and to regulate a variety of cellular activities. Siglec receptors are mainly expressed on leukocytes in a cell type-specific and differentiation-dependent manner. Siglecs might potentially be exploited as targets of novel immune- and glycotherapeutics for cell-directed therapies in autoimmune and allergic diseases, as well as in haematologic malignancies. Here we present novel insights on structural and functional characteristics, expression patterns and evolutionary aspects of Siglecs and their ligands. Pharmacological strategies using Siglec agonistic cross-linking therapeutics, such as monoclonal or engineered antibodies, intravenous immunoglobulin (IVIG), or glycomimetics are discussed. Modulation of immune responses by targeting Siglecs using agonistic or antagonistic therapeutics may have important clinical implications and may pave the way for novel pharmacological avenues for the treatment of autoimmune and allergic diseases or for tumor immunotherapy.

'leaky Gut' In Hematological Malignancies.

36

Suppression of lymphoma and epithelial malignancies effected by interferon gamma

The immunosurveillance of transformed cells by the immune system remains one of the most controversial and poorly understood areas of immunity. Gene-targeted mice have greatly aided our understanding of the key effector molecules in tumor immunity. Herein, we describe spontaneous tumor development in gene-targeted mice lacking interferon (IFN)-gamma and/or perform (pfp), or the immunoregulatory cytokines, interleukin (IL)-12, IL-18, and tumor necrosis factor (TNF). Both IFN-gamma and pfp were critical for suppression of lymphomagenesis, however the level of protection afforded by IFN-gamma was strain specific. Lymphomas arising in IFN-gamma deficient mice were very nonimmunogenic compared with those derived from pfp-deficient mice, suggesting a comparatively weaker immunoselection pressure by IFN-gamma. Single loss of IL-12, IL-18, or TNF was not sufficient for spontaneous tumor development. A significant incidence of late onset adenocarcinoma observed in both IFN-gamma- and pfp-deficient mice indicated that some epithelial tissues were also subject to immunosurveillance.

Outpatient treatment with intravenous antimicrobial therapy and oral levofloxacin in patients with febrile neutropenia and hematological malignancies

The purpose of this study was to evaluate outcomes such as success of the initial therapy, failure of outpatient treatment, and death in outpatient treatment during intravenous antimicrobial therapy in patients with febrile neutropenia (FN) and hematological malignancies. In addition, clinical and laboratory data and the Multinational Association for Supportive Care of Cancer index (MASCC) were compared with failure of outpatient treatment and death. In a retrospective study, we evaluated FN following chemotherapy events that were treated initially with cefepime, with or without teicoplanin and replaced by levofloxacin after 48 h of defervescence in patients with good general conditions and ANC > 500/mm(3). Of the 178 FN episodes occurred in 126 patients, we observed success of the initial therapy in 63.5% of the events, failure of outpatient treatment in 20.8%, and death in 6.2%. The success rate of oral levofloxacin after defervescence was 99% (95 out of 96). Using multivariate analysis, significant risks of failure of outpatient treatment were found to be smoking (odds ratio (OR) 3.14, confidence interval (CI) 1.14-8.66; p = 0.027) and serum creatinine levels > 1.2 mg/dL (OR 7.97, CI 2.19-28.95; p = 0.002). With regard to death, the risk found was oxygen saturation by pulse oximetry < 95% (OR 5.8, IC 1.50-22.56; p = 0.011). Using the MASCC index, 165 events were classified as low risk and 13 as high risk. Failure of outpatient treatment was reported in seven (53.8%) high-risk and 30 (18.2%) low-risk episodes (p = 0.006). In addition, death occurred in seven (4.2%) low-risk and four (30.8%) high-risk events (p = 0.004). Ours results show that MASCC index was able to identify patients with high risk. In addition, non-smoking, serum creatinine levels a parts per thousand currency sign1.2 mg/dL, and oxygen saturation by pulse oximetry a parts per thousand yen95% were protection factors.

Clinico-pathological discrepancies in the diagnoses of solid malignancies

Autopsy is a valuable tool in evaluating diagnostic accuracy. Solid malignancies may have a protracted presentation, and diagnosis frequently requires imaging and deep-sited biopsies; clinical and postmortem diagnosis discrepancies may occur in a high rate in these diseases. Here, we analyzed the occurrence of clinico-pathological discrepancies in the diagnoses of solid malignancies in a Brazilian academic hospital. We reviewed charts and autopsy reports of the patients that died from 2001 to 2003 with at least one solid neoplasm. Patients were classified in concordant and discordant cases regarding cancer diagnosis. Discordant cases were categorized in undiagnosed cases (no suspicion of cancer) and in misdiagnosed cases (clinical suspicion of cancer but incompletely diagnosed). Among the 264 patients with a single non-incidental solid neoplasm, the clinico-pathological discrepancy rate was 37.1%. Liver (22.5%), lung (19.4%), and pancreatic cancer (15.3%) were the most frequent malignancies in the discordant group. Misdiagnosis category comprised 68% of the discordant cases, i.e., there was no correct knowledge about the tumor primary site and/or the histological type during life. Our data show that a high rate of discrepancies occurs in solid malignancies. Autopsies may provide the basis for a better understanding of diagnostic deficiencies in different circumstances. (C) 2008 Elsevier GmbH. All rights reserved.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.

Microdialysis and response during regional chemotherapy by isolated limb infusion of melphalan for limb malignancies

This study sought to use a microdialysis technique to relate clinical and biochemical responses to the time course of melphalan concentrations in the subcutaneous interstitial space and in tumour tissue (melanoma, malignant fibrous histiocytoma, Merkel cell tumour and osteosarcoma) in patients undergoing regional chemotherapy by Isolated Limb Infusion (ILI). 19 patients undergoing ILI for treatment of various limb malignancies were monitored for intra-operative melphalan concentrations in plasma and, using microdialysis, in subcutaneous and tumour tissues. Peak and mean concentrations of melphalan were significantly higher in plasma than in subcutaneous or tumour microdialysate. There was no significant difference between drug peak and mean concentrations in interstitial and tumour tissue, indicating that there was no preferential uptake of melphalan into the tumours. The time course of melphalan in the microdialysate could be described by a pharmacokinetic model which assumed melphalan distributed from the plasma into the interstitial space. The model also accounted for the vascular dispersion of melphalan in the limb. Tumour response in the whole group to treatment was partial response: 53.8% (n = 7); complete response: 33.3% (n = 5); no responses 6.7% (n = 1). There was a significant association between tumour response and melphalan concentrations measured over time in subcutaneous microdialysate (P < 0.01). No significant relationship existed between the severity of toxic reactions in the limb or peak plasma creatine phosphokinase levels and peak melphalan microdialysate or plasma concentrations. It is concluded that microdialysis is a technique well suited for measuring concentrations of cytotoxic drug during ILI. The possibility of predicting actual concentrations of cytotoxic drug in the limb during ILI using our model opens an opportunity for improved drug dose calculation. The combination of predicting tissue concentrations and monitoring in microdialysate of subcutaneous tissue could help optimise ILI with regard to post-operative limb morbidity and tumour response. (C) 2001 Cancer Research Campaign http:,//www.bjcancer.com.

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore, our results confirm the potential usefulness of PCR serial screening and the clinical applicability in everyday routine. PCR screening offers a noninvasive repeatable aid to the diagnosis of IFI.

Prospective study of strongyloidosis in patients with hematologic malignancies

Immunocompromised individuals infected with Strongyloides stercoralis may develop severe hyperinfection or disseminated disease with high mortality. Patients with hematological malignancies are at risk because of immunodepression produced either by the disease or its treatment. A prospective study was undertaken at the Hospital de Clínicas de Porto Alegre, from July 1994 to July 1995. Seventy-two (HIV negative), had 3 stool samples collected at different days and had not received recent anti-helmintic therapy. Larvae, isolated in a modified Baermann method, were found in 6 patients, with a resultant prevalence of 8.3%. No complicated strongyloidosis was documented. The positive result for S. stercoralis larvae was significantly associated (p < 0.001) with eosinophilia. Knowledge of prevalence figures and incidence of severe disease is important to adequate guidelines for empirical treatment besides the rigorous search for strongyloidosis in patients with hematological malignancies.

Malignancies in HIV/AIDS patients attending an outpatient clinic in Vitória, State of Espírito Santo, Brazil

INTRODUCTION: The present study investigated cancer prevalence and associated factors among HIV-infected individuals attending an AIDS outpatient clinic in Vitória, State of Espírito Santo, Brazil. METHODS: A sectional study was conducted among HIV infected adults attending an AIDS outpatient clinic in Vitória, State of Espírito Santo, Brazil. Demographic, epidemiological and clinical data were abstracted from medical records, including cancer diagnoses; nadir and current CD4 cell count, HIV viral load, time on antiretroviral treatment (ART), type of ART and smoking status. RESULTS: A total of 730 (91.3%) patients were included in the study. Median age was 44.0 [interquartile range (IQR): 35-50.3] years; median time since HIV diagnosis was 5.5 years (IQR: 2-10); 60% were male; and 59% were white. Thirty (4.1%) cases of cancer were identified of which 16 (53%) were AIDS defining cancers and 14 (47%) were non-AIDS defining malignancies. Patients diagnosed with cancer presented higher chance of being tobacco users [OR 2.2 (95% CI: 1.04-6.24)]; having nadir CD4 ≤200 cells/mm³ [OR 3.0 (95% CI: 1.19-7.81)] and higher lethality [OR 13,3 (95% CI: 4,57-38,72)]. CONCLUSIONS: These results corroborate the importance of screening for and prevention of non-AIDS defining cancers focus in HIV-infected population, as these cancers presented with similar frequency as AIDS defining cancers.

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Polysomy 8 defines a clinico-cytogenetic entity representing a subset of myeloid hematologic malignancies associated with a poor prognosis: report on a cohort of 12 patients and review of 105 published cases.

Tetrasomy, pentasomy, and hexasomy 8 (polysomy 8) are relatively rare compared to trisomy 8. Here we report on a series of 12 patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or myeloproliferative disorder (MPD) associated with polysomy 8 as detected by conventional cytogenetics and fluorescence in situ hybridization (FISH). In an attempt to better characterize the clinical and hematological profile of this cytogenetic entity, our data were combined with those of 105 published patients. Tetrasomy 8 was the most common presentation of polysomy 8. In 60.7% of patients, polysomy 8 occurred as part of complex changes (16.2% with 11q23 rearrangements). No cryptic MLL rearrangements were found in cases in which polysomy 8 was the only karyotypic change. Our study demonstrates the existence of a polysomy 8 syndrome, which represents a subtype of AML, MDS, and MPD characterized by a high incidence of secondary diseases, myelomonocytic or monocytic involvement in AML and poor overall survival (6 months). Age significantly reduced median survival, but associated cytogenetic abnormalities did not modify it. Cytogenetic results further demonstrate an in vitro preferential growth of the cells with a high level of aneuploidy suggesting a selective advantage for polysomy 8 cells.

β-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3).

BACKGROUND: Invasive fungal infections (IFIs) are life-threatening complications in patients with hemato-oncological malignancies, and early diagnosis is crucial for outcome. The compound 1,3-β-D-glucan (BG), a cell wall component of most fungal species, can be detected in blood during IFI. Four commercial BG antigenemia assays are available (Fungitell, Fungitec-G, Wako, and Maruha). This meta-analysis from the Third European Conference on Infections in Leukemia (ECIL-3) assessed the performance of BG assays for the diagnosis of IFI in hemato-oncological patients. METHODS: Studies reporting the performance of BG antigenemia assays for the diagnosis of IFI (European Organization for Research and Treatment of Cancer and Mycoses Study Group criteria) in hemato-oncological patients were identified. The analysis was focused on high-quality cohort studies with exclusion of case-control studies. Meta-analysis was performed by conventional meta-analytical pooling and bivariate analysis. RESULTS: Six cohort studies were included (1771 adult patients with 414 IFIs of which 215 were proven or probable). Similar performance was observed among the different BG assays. For the cutoff recommended by the manufacturer, the diagnostic performance of the BG assay in proven or probable IFI was better with 2 consecutive positive test results (diagnostic odds ratio for 2 consecutive vs one single positive results, 111.8 [95% confidence interval {CI}, 38.6-324.1] vs 16.3 [95% CI, 6.5-40.8], respectively; heterogeneity index for 2 consecutive vs one single positive results, 0% vs 72.6%, respectively). For 2 consecutive tests, sensitivity and specificity were 49.6% (95% CI, 34.0%-65.3%) and 98.9% (95% CI, 97.4%-99.5%), respectively. Estimated positive and negative predictive values for an IFI prevalence of 10% were 83.5% and 94.6%, respectively. CONCLUSIONS: Different BG assays have similar accuracy for the diagnosis of IFI in hemato-oncological patients. Two consecutive positive antigenemia assays have very high specificity, positive predictive value, and negative predictive value. Because sensitivity is low, the test needs to be combined with clinical, radiological, and microbiological findings.