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1.
Front Oncol ; 11: 685786, 2021.
Article in English | MEDLINE | ID: mdl-34150651

ABSTRACT

Cerebrospinal fluid (CSF) flow cytometry has a crucial role in the diagnosis of leptomeningeal disease in onco-hematology. This report describes the flow cytometry characterization of 138 CSF samples from patients affected by non-Hodgkin lymphoma, negative for disease infiltration. The aim was to focus on the CSF non-neoplastic population, to compare the cellular composition of the CSF with paired peripheral blood samples and to document the feasibility of flow cytometry in hypocellular samples. Despite the extremely low cell count (1 cell/µl, range 1.0-35) the study was successfully conducted in 95% of the samples. T lymphocytes were the most abundant subset in CSF (77%; range 20-100%) with a predominance of CD4-positive over CD8-positive T cells (CD4/CD8 ratio = 2) together with a minority of monocytes (15%; range 0-70%). No B cells were identified in 90% of samples. Of relevance, a normal, non-clonal B-cell population was documented in 5/7 (71%) patients with primary central nervous system lymphoma at diagnosis (p<0.0001), suggesting a possible involvement of blood-brain barrier cell permeability in the pathogenesis of cerebral B-cell lymphomas. The highly significant differences between CSF and paired peripheral blood lymphoid phenotype (p<0.0001) confirms the existence of an active mechanism of lymphoid migration through the meninges.

3.
Breast Cancer Res ; 19(1): 46, 2017 04 11.
Article in English | MEDLINE | ID: mdl-28399903

ABSTRACT

BACKGROUND: Cancer is a mosaic of tumor cell subpopulations, where only a minority is responsible for disease recurrence and cancer invasiveness. We focused on one of the most aggressive circulating tumor cells (CTCs) which, from the primitive tumor, spreads to the central nervous system (CNS), evaluating the expression of prognostic and putative cancer stem cell markers in breast cancer (BC) leptomeningeal metastasis (LM). METHODS: Flow cytometry immunophenotypic analysis of cerebrospinal fluid (CSF) samples (4.5 ml) was performed in 13 consecutive cases of BCLM. Syndecan-1 (CD138), MUC-1 (CD227) CD45, CD34, and the putative cancer stem cell markers CD15, CD24, CD44, and CD133 surface expression were evaluated on CSF floating tumor cells. The tumor-associated leukocyte population was also characterized. RESULTS: Despite a low absolute cell number (8 cell/µl, range 1-86), the flow cytometry characterization was successfully conducted in all the samples. Syndecan-1 and MUC-1 overexpression was documented on BC cells in all the samples analyzed; CD44, CD24, CD15, and CD133 in 77%, 75%, 70%, and 45% of cases, respectively. A strong syndecan-1 and MUC-1 expression was also documented by immunohistochemistry on primary breast cancer tissues, performed in four patients. The CSF tumor population was flanked by T lymphocytes, with a different immunophenotype between the CSF and peripheral blood samples (P ≤ 0.02). CONCLUSIONS: Flow cytometry can be successfully employed for solid tumor LM characterization even in CSF samples with low cell count. This in vivo study documents that CSF floating BC cells overexpress prognostic and putative cancer stem cell biomarkers related to tumor invasiveness, potentially representing a molecular target for circulating tumor cell detection and LM treatment monitoring, as well as a primary target for innovative treatment strategies. The T lymphocyte infiltration, documented in all CSF samples, suggests a possible involvement of the CNS lymphatic system in both lymphoid and cancer cell migration into and out of the meninges, supporting the extension of a new form of cellular immunotherapy to LM. Due to the small number of cases, validation on large cohorts of patients are warranted to confirm these findings and to evaluate the impact and value of these results for diagnosis and management of LM.


Subject(s)
Biomarkers, Tumor , Breast Neoplasms/pathology , Meningeal Neoplasms/metabolism , Meningeal Neoplasms/secondary , Mucin-1/metabolism , Neoplastic Stem Cells/metabolism , Syndecan-1/metabolism , Adult , Aged , Cell Count , Cerebrospinal Fluid/cytology , Female , Flow Cytometry , Humans , Immunohistochemistry , Immunophenotyping , Leukocytes/metabolism , Leukocytes/pathology , Meningeal Neoplasms/diagnosis , Middle Aged , Mucin-1/cerebrospinal fluid , Mucin-1/genetics , Neoplasm Staging , Prognosis , Syndecan-1/cerebrospinal fluid , Syndecan-1/genetics
4.
J Exp Clin Cancer Res ; 35(1): 128, 2016 08 27.
Article in English | MEDLINE | ID: mdl-27567676

ABSTRACT

BACKGROUND: Brain stereotactic biopsy (SB) followed by conventional histopathology and immunohistochemistry (IHC) is the gold standard approach for primary central nervous system lymphoma (PCNSL) diagnosis. Flow cytometry (FCM) characterization of fine-needle aspiration cytology and core needle biopsies are increasingly utilized to diagnose lymphomas however, no biological data have been published on FCM characterization of fresh single cell suspension from PCNSL SB. The aim of this study was to establish the feasibility and utility of FCM for the diagnosis and characterization of brain lymphomas from a tissue samples obtained by a single SB disaggregation. METHODS: Twenty-nine patients with a magnetic resonance suggestive for PCNSL entered the study. A median of 6 SB were performed for each patient. A cell suspension generated from manual tissue disaggregation of a single, unfixed, brain SB, was characterized by FCM. The FCM versus standard approach was prospectively compared. RESULTS: FCM and IHC showed an high degree of agreement (89 %) in brain lymphoma identification. By FCM, 16 out of 18 PCNSL were identified within 2 h from biopsy. All were of B cell type, with a heterogeneous CD20 mean fluorescence intensity (MFI), CD10 positive in 3 cases (19 %) with surface Ig light chain restriction documented in 11 cases (69 %). No false positive lymphomas cases were observed. Up to 38 % of the brain leukocyte population consisted of CD8 reactive T cells, in contrast with the CD4 positive lymphocytes of the peripheral blood samples (P < 0.001). By histopathology, 18 B-PCNSL, only one CD10 positive (5 %), 1 primitive neuroectodermal tumor (PNET) and 10 gliomas were diagnosed. A median of 6 days was required for IHC diagnosis. CONCLUSION: Complementary to histopathology FCM can contribute to a better characterization of PCNSL, although necrosis and previous steroid treatment can represent a pitfall of this approach. A single brain SB is a valid source for accurate FCM characterization of both lymphoma and reactive lymphocyte population, routinely applicable for antigen intensity quantification and consistently documenting an active mechanism of reactive CD8 T-lymphocytes migration in brain lymphomas. Moreover, FCM confirmed to be more sensitive than IHC for the identification of selected markers.


Subject(s)
Brain Neoplasms/diagnosis , Flow Cytometry/methods , Glioma/diagnosis , Lymphoma/diagnosis , Neuroectodermal Tumors/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Biopsy, Fine-Needle , Biopsy, Large-Core Needle , Female , Humans , Male , Middle Aged , Prospective Studies , Sensitivity and Specificity , Young Adult
5.
Leuk Res ; 30(7): 785-94, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16527350

ABSTRACT

Leukemic bcr-abl positive dendritic cells (DCs) are likely to be present in vivo in chronic myelogenous leukemia (CML) patients, but no data are available on their functional qualities. We analyzed the circulating BDCA-1+ myeloid DC compartment in 15 chronic phase CML patients. Phenotypic features of CML DCs were comparable with that of normal DCs, except for the CD80 and CD40 antigens, significantly under-represented in CML patients. Nonetheless, no differences were found between normal samples and leukemic DCs in the allostimulatory ability, as well as in the production of cytokines and polarization of T cell responses. CML DCs were analyzed by fluorescence in situ hybridization (FISH) and found positive for the bcr-abl translocation. However, when bcr-abl+ DCs were tested for their ability to stimulate an autologous T-cell response in vitro, we could not detect a specific recognition. We conclude that an apparently normal circulating DC compartment carrying the Ph+ chromosome can be identified in CML patients; however, these cells appear unable to trigger a protective anti-leukemic immune response in autologous T cells.


Subject(s)
Antigens, CD1/immunology , Dendritic Cells/immunology , Fusion Proteins, bcr-abl/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Translocation, Genetic/genetics , Antigens, CD1/drug effects , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytokines/immunology , Dendritic Cells/cytology , Dendritic Cells/drug effects , Humans , Immunophenotyping , In Situ Hybridization, Fluorescence/methods , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Lipopolysaccharides/pharmacology , Phenotype , Sensitivity and Specificity
6.
Br J Haematol ; 125(6): 720-8, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15180861

ABSTRACT

Dendritic cells (DCs) are the most potent antigen-presenting cells and are therefore an attractive option as antigen carriers in vaccination protocols. Chronic lymphocytic leukaemia (CLL) represents a potential good target for these approaches. The present study was designed to investigate the feasibility of generating in vitro fully functional DCs from peripheral blood (PB) monocytes of CLL patients at different phases of the disease. Although functional DCs could be obtained from CLL samples, in patients with active disease the expression of some co-stimulatory molecules appeared to be reduced. In contrast, DCs from CLL patients in remission showed no difference from those of normal controls. Moreover, patients with active disease produced DCs with reduced allostimulatory ability when compared with normal ones, whereas the functional capacities appeared to be restored in CLL DCs from remission patients. To more precisely assess the possible inhibitory effect of CLL cells on DC development, the influence of autologous leukaemic CD19(+) cells on the generation of monocyte-derived CLL DCs in vitro was investigated. The addition of CLL neoplastic cells markedly affected monocyte-derived DC maturation. In conclusion, monocytes from CLL patients with active disease give rise to DCs, which show phenotypic and functional defects that are not observed in remission CLL patients. These results need to be taken into account in the design of DC-based immunotherapeutic approaches in CLL.


Subject(s)
Antigens, CD19/immunology , Dendritic Cells/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Adult , Aged , Alemtuzumab , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized , Antibodies, Monoclonal, Murine-Derived , Antibodies, Neoplasm/therapeutic use , Antineoplastic Agents/therapeutic use , Biomarkers/analysis , Cancer Vaccines , Case-Control Studies , Clone Cells , Coculture Techniques , Female , Flow Cytometry , Humans , Immunotherapy, Adoptive , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Male , Middle Aged , Rituximab
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