Sabrina Aliberti

The usefulness of flow cytometric CD10 detection in the differential diagnosis of peripheral T-cell lymphomas.

We studied the histologic and multiparameter flow cytometry (MFC) features of 12 cases of angioimmunoblastic T-cell lymphoma (AITL), 13 of mature T-cell lymphoma, and 25 control cases of reactive lymphoid hyperplasia to evaluate the role of CD10 in the differential diagnosis of peripheral T-cell lymphomas (PTCLs). A characteristic immunophenotypic profile (CD2+/CD4+) with recurrent phenotypic aberrancies (eg, CD3 and CD7 loss) was identified in most AITL cases; MFC documented CD10 coexpression on T cells in 10 (83%). Mature T-cell lymphoma showed a more heterogeneous altered immunophenotypic pattern, and 2 cases of PTCL, unspecified, had clear evidence of aberrant CD10 expression on T cells. A small physiologic CD3+/CD4+/CD10+ T-cell population was detected by MFC in all control cases tested (range, 0.28%-4.71%), suggesting that a normal subset of peripheral CD10+ T cells exists. CD10 was a highly sensitive but incompletely specific phenotypic marker for diagnosing AITL; the differential diagnosis of PTCL, unspecified, must be related with traditional histologic features. A small number of CD10+ T cells in reactive lymph nodes suggests that this subpopulation may be the normal counterpart of neoplastic T cells in AITL. The biologic role of CD10+ T cells should be studied further.

Immune Reconstitution and Early Infectious Complications Following Nonmyeloablative Hematopoietic Stem Cell Transplantation

Abstract Non-myeloablative stem cell transplantation (NMT) has been increasingly used in compromised patients who would otherwise have been unable to undergo allotransplant. There is little understanding of the kinetics of immune reconstitution and its influence on infective complications following NMT. The aim of present study was to evaluate lymphocyte subset reconstitution over the first 12 months post-transplant in 15 adult patients receiving NMT with comparison to that of 30 patients grafted with a conventional hemopoietic stem cell transplantation (HSCT). NMT recipients were conditioned with fludarabine-based conditioning regimens. Peripheral blood stem cell (PBSC) was the source of stem cells in 13 NMT recipients and in 24 conventional HSCT recipients. Absolute numbers of helper (CD4+) T cells, naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+) T cells as well as suppressor (CD8+) T cells, CD19+ B cells and NK cells were comparable in the two groups at all time points after transplantation. A median value of 200 CD4+ T cells/μl was achieved at 2 months post-transplant by the NMT and HSCT recipients. The CD4:CD8 ratio remained severely depressed throughout the study period. Almost all CD4+ lymphocytes expressed CD45RO antigen in the both groups of patients B lymphocytes showed low counts throughout the entire study period in both groups. Bacteremia and CMV antigenemia occurred respectively in 13 and 36% of the patients in the NMT group and in 15 and 39% of the patients in the HSCT group. Our preliminary data indicate that patients receiving a NMT have a lymphocyte reconstitution similar to that observed in patients who received a conventional HSCT. The incidence of bacteremia and CMV infection were not significantly different between the groups. Nevertheless, due to the small sample size, these results should be considered suggestive rather than definitive.

Usefulness of Multiparametric Flow Cytometry in Detecting Composite Lymphoma Study of 17 Cases in a 12-Year Period

Composite lymphoma (CL) is a rare occurrence of 2 or more morphologically and immunophenotypically distinct lymphoma clones in a single anatomic site. A retrospective analysis of 1,722 solid tissue samples clinically suggestive of lymphoma was carried out in our institute during a 12-year period to evaluate the efficacy of flow cytometry (FC) in identifying CL. We report 17 CL cases. A strong correlation between morphologic findings and FC was observed in 13 cases (76%). In the 4 cases diagnosed as non-Hodgkin lymphoma plus Hodgkin lymphoma, although FC did not detect Reed-Sternberg cells, it accurately identified the neoplastic B- or T-cell component. In 3 cases, FC indicated the need to evaluate an additional neoplastic component that was not morphologically evident. Our data demonstrate that FC immunophenotyping of tissues may enhance the performance of the diagnostic morphologic evaluation of CL. To the best of our knowledge, this is the first report in the literature of a wide series of CL studied also by FC.

Tissue flow cytometry immunophenotyping in the diagnosis and classification of non‐Hodgkin's lymphomas: A retrospective evaluation of 1,792 cases

A retrospective analysis of 1,792 solid tissues suggestive of lymphoma, submitted over a 12‐year period, was carried out and flow cytometry (FC) results were compared with histologic findings. The final histologic diagnosis of cases documented in this report is as follows: 1,270 non‐Hodgkins lymphomas (NHL); 17 composite lymphomas; four NHL plus carcinomas; five post‐transplant lymphoproliferative disorders; 105 Hodgkins lymphomas (HL); eight acute leukemias; 42 tissue cancers; and 341 non‐neoplastic diseases. A strong correlation between morphology and FC data was observed among hematological malignancies (1,268/1,304, 97.2%) with the exception of HL. Among B‐NHL, FC detection of clonally restricted B‐cell allowed the identification of lymphomas that were not histologically clear and the differential diagnosis between follicular lymphoma and reactive hyperplasia. A high correlation level (r = 0.83; P < 0.0001) was obtained in comparing proliferation results obtained by FC and immunohistochemistry. Among T‐NHL, FC detection of an aberrant phenotype direct histologic diagnosis in cases having less than 20% of neoplastic cells. In nine cases, FC suggested the need to evaluate a neoplastic population, not morphologically evident. Results show that FC routinely performed on tissue samples suspected of lymphomas is a fundamental adjunct to morphology in the diagnosis of NHL and may enhance the performance of the histologic evaluation so as to achieve the final diagnosis. To the best of our knowledge, this is the first report in the literature of a wide series of tissues also studied by FC.

A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

Aberrant expression of CD8 in B-cell non-Hodgkin lymphoma: a multicenter study of 951 bone marrow samples with lymphomatous infiltration.

T-cell antigen expression can be observed in B-cell non-Hodgkin lymphoma (B-NHL). Although CD5 is expressed in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, the presence of other T-cell antigens is less common. This article reports a retrospective multicenter analysis in which flow cytometry was used to evaluate aberrant CD8 expression on the pathologic B cells of 951 bone marrow samples from patients with various types of B-NHL. In a total of 18 patients, CD8 was coexpressed: 10 had B-CLL; 1, small lymphocytic lymphoma (SLL); 1, marginal zone lymphoma; 1, lymphoplasmacytic lymphoma; 2, diffuse large B-cell lymphoma; and 3, follicular lymphoma. There was a 1.89% overall frequency of CD8 coexpression in which B-CLL/SLL had a higher frequency (3.03%) than did the other B-cell neoplasms (1.18%). Most cases were characterized by a favorable outcome.

Ten Antibodies, Six Colors, Twelve Parameters: A Multiparameter Flow Cytometric Approach to Evaluate Leptomeningeal Disease in B-cell Non-Hodgkin's Lymphomas

Flow cytometry (FC) is considered a sensitive and specific technique for the detection of occult lymphoma cells in cerebrospinal fluid (CSF).

Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature

Stacchini A, Barreca A, Demurtas A, Aliberti S, di Celle P F & Novero D 
(2012) Histopathology 60, 452–459 
Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature

Utilility of flow cytometry as ancillary study to improve the cytologic diagnosis of thyroid lymphomas.

To evaluate the efficacy of the use of flow cytometry (FC) immunophenotyping together with fine‐needle aspiration cytology (FNAC) in the diagnosis of thyroid lymphoma.

Immunophenotyping of paucicellular samples.

Immunophenotyping of paucicellular samples may represent a diagnostic challenge in the flow cytometry (FC) laboratory routine, as the scarcity of cells limits the number of tests that can be performed. Specimens such as fine needle aspirates (FNA), human body fluids (BF), cerebrospinal fluid (CSF), or ocular fluid (OF) sent for FC investigations in the case of suspicion of lymphoma, or for lymphoma monitoring, may contain very low numbers of cells. In these cases, it is mandatory to obtain the largest amount possible of useful information from a single tube. The basic protocol described in this unit provides a method that combines the use of multiple monoclonal antibodies (MAbs) with a Boolean gating strategy to identify and quantify the main lymphocyte populations, as well as to detect lymphomatous B cells or any aberrant T cell expression, if present, in paucicellular samples. Curr. Protoc. Cytom. 68:9.46.1‐9.46.14.