Elisa Cannizzo

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.

The Role of CD19 and CD27 in the Diagnosis of Multiple Myeloma by Flow Cytometry: A New Statistical Model

We have developed a new statistical diagnostic model that examines the correlation between immunophenotype and clonality as detected by flow cytometry (FC) and histology, defining the diagnostic role of FC in multiple myeloma (MM). The 192 bone marrow samples from patients and control subjects were studied for routine diagnostic analysis of MM; a minimum of 100 plasma cells (PCs) were analyzed for each patient sample. A direct 7- or 8-color method was applied to study the immunophenotype of PCs, utilizing a FACSCanto II (BD Biosciences, San Jose, CA). Samples were labeled with fluorochrome-conjugated monoclonal antibodies (AmCyan, Pac Blue, fluorescein isothiocyanate, phycoerythrin [PE], PECy7, peridinin-chlorophyll protein, allophycocyanin [APC], and APC-Cy7) to the following antigens: CD138, CD81, CD200, CD221, CD45, CD38, CD28, CD19, CD27, CD117, CD38, CD33, CD20, CD56, CD10, and immunoglobulin κ and λ light chains. Among all antigens tested, CD19 and CD27, when applied to our model, resulted in optimal concordance with histology. This model defines the effective diagnostic role FC could have in MM and in the detection of minimal residual disease.

Abnormal phenotype of bone marrow plasma cells in patients with chronic myeloid leukemia undergoing therapy with Imatinib

Imatinib induces several effects on the immune system, including hypogammaglobulinemia and has been associated with multiple myeloma in some patients. We studied the phenotype of plasma cells from patients with chronic myeloid leukemia (CML) undergoing therapy with Imatinib mesylate (Glivec). Bone marrow samples from 30 CML patients were evaluated and plasma cells were identified by multiparametric flow cytometry. In 21 patients an abnormal plasma cell phenotype, characterized by the absence of CD19, was registered, with 12 patients expressing also the CD56 molecule. A significant correlation between abnormal plasma cell phenotype and reduced gamma-globulin levels was found. Immunofixation was always negative. Therapy with Imatinib for CML seems to induce a plasma cell phenotype with the same characteristics as monoclonal gammapathies. These findings deserve further studies and suggest to monitor plasma protein electrophoresis and gamma-globulin levels in all patients treated with Imatinib.

CD19/CD8 Coexpression in B-Chronic Lymphocytic Leukemia

and persistence of IgH rearrangement. The unusual CD19/CD8 association was found during a phase of reexpansion of the disease, with splenomegaly and significant bone marrow infiltration. Because of psychological problems, only therapy with rituximab (375 mg/m 2 weekly, 4 courses) was given. A good response was achieved, with clearance of bone marrow infiltration, persistence of IgH rearrangement and, interestingly, disappearance of the CD19/C8-positive clone. In both cases the phenotypic aberration was studied by both CD19-FITC/ CD8-PE and CD8-FITC/CD19-PE combinations, using MoAb purchased from Becton-Dickinson. Most of the CD19/CD8-positive B-CLL cases reported so far were characterized by nonprogressive disease, but in some patients chemotherapy was necessary to control disease progression and a good response to treatment was not always obtained [1, 5] . Our experience shows that CD19/CD8 coexpression in B-CLL may be found not only at diagnosis, but also during followup (in our case after a previous treatment) and that the CD19/CD8-positive clone can be sensitive not only to fludarabine, as suggested by Islam et al. [6] , but also to nonaggressive therapy such as rituximab. We agree with Parisi-Duchene et al. [1] about the necessity to study larger series of B-CLL, using a complete panel including MoAb directed against T cell markers. In We read with great interest the paper by Parisi-Duchêne et al. [1] , dealing with 5 cases of B-chronic lymphocytic leukemia (B-CLL) with aberrant expression of CD8 on B cells. This interesting finding has been reported in few cases [1–6] and may be found in about 2% of B-CLL. However, its frequency may be underestimated, because the expression of T cell markers such as CD3, CD4 and CD8 is not systematically analyzed in many laboratories. In our laboratory a routine monoclonal antibody (MoAb) panel including CD3, CD4 and CD8 is always used both at diagnosis and during the follow-up of all types of B cell non-Hodgkin lymphomas. After a revision of 115 B-CLL cases observed in the period 2004–2006, only 2 (1.7%) were characterized by CD19/CD8 coexpression. The first patient was a 75-year-old female with mild lymphocytosis, Rai stage 0, Matutes score 5 and nonprogressive disease. CD19/CD8 coexpression was found at diagnosis and confirmed during followup (4 analyses over 2 years). The second patient was a 48-year-old male without CD19/CD8 coexpression at diagnosis. The patient presented with Rai stage II and Matutes score 5, and underwent chemotherapy because of progressive disease. After treatment with the association of fludarabine, cyclophosphamide and rituximab the patient achieved a good partial remission, with less than 10% of CD19/CD5/CD23-positive lymphocytes Accepted: October 11, 2006 Published online: December 12, 2006

Discordant lymphoma consisting of splenic mantle cell lymphoma and marginal zone lymphoma involving the bone marrow and peripheral blood: a case report

IntroductionDiscordant lymphomas are rare entities characterized by the simultaneous presence of two distinct types of lymphomas in different anatomic sites. We describe a very rare case of simultaneous occurrence of splenic mantle cell lymphoma and marginal zone lymphoma involving the bone marrow and peripheral blood.Case presentationWe report the case of a 60-year-old asymptomatic Caucasian woman in whom discordant lymphomas were discovered when a slight lymphocytosis and a conspicuous splenomegaly were observed. The different morphological, immunophenotypical and immunohistochemical features found in the different pathologic samples obtained from peripheral blood, bone marrow and spleen sections made it possible to differentiate two types of non-Hodgkin B-cell lymphomas: a mantle cell lymphoma infiltrating the spleen and a marginal zone lymphoma involving both the bone marrow and peripheral blood. Since a similar IgH gene rearrangement was found both in the bone marrow and in the spleen, the hypothesis of a common origin, followed by a different clonal selection of the neoplastic lymphocytes may be taken into consideration.ConclusionOur case emphasizes the usefulness of investigating simultaneous specimens from different anatomic sites from the same patient and the relevant diagnostic role of splenectomy.

Il referto citofluorimetrico

RiassuntoIn Medicina di Laboratorio il momento del referto, oltre a rappresentare un evento essenziale nella validazione clinica di un risultato, assume anche il preciso significato di “giudizio interpretativo” formulato dallo specialista di laboratorio. Deve quindi essere ritenuto l’atto conclusivo di un processo tecnico, che ha prodotto risultati numerici derivanti da analisi (biochimiche, ematologiche, immunologiche ecc.) eseguite su materiali biologici. In un sistema di gestione della qualità il referto costituisce l’atto scritto conclusivo e ufficiale di un iter diagnostico all’interno di un percorso clinico rivolto al beneficio di un paziente.Le applicazioni diagnostiche citofluorimetriche sono numerose e riguardano un ampio spettro di patologie e di substrati da analizzare: emopatie maligne, malattie linfoproliferative, studio della malattia minima residua, sottopopolazioni linfocitarie sono solo alcuni importanti campi di utilizzo delle metodiche citofluorimetriche; molteplici sono anche i substrati analizzabili, potendosi effettuare studi su numerosi comparti cellulari e su diversi costituenti subcellulari.Il GdS Citofluorimetria intende proporre alcune raccomandazioni per la stesura del referto citometrico, che spesso risente troppo della personalizzazione dell’operatore ed è una fase nevralgica nel percorso diagnostico-terapeutico del paziente.SummaryIn Laboratory Medicine, the “report” time is a fundamental event in the clinical evaluation of a result, and also signifies “interpretative judgement” formulated by a laboratory specialist. The report itself must therefore be considered the conclusive act of a technical process that has produced numerical results deriving from the analysis of biological materials (biochemical, hematologic, immunological, etc.). In a quality management system, the report is the conclusive and official written act in a diagnostic workup within the context of a clinical pathway taken in order to benefit the patient.Diagnostic cytofluorimetric applications are heterogeneous, covering a wide range of diseases and substrates for analysis: malignant hemopathy, lymphoproliferative disorders, minimal residual disease, and lymphocytic subpopulations are among the important fields of cytofluorimetric methods; the substrates are also heterogenous, allowing cytometric studies on many cellular compartments, and on different subcellular constituents.The aim of cytofluorimetric GdS is to make recommendations for drawing up the cytometric report, which is of extreme importance in the diagnostic and therapeutic course of the patient, but is often compromised by the individual approach of the operator.

CD8 expression in B-cell non-Hodgkin's lymphomas.

Dear Editor, T cell antigen expression in B-cell non-Hodgkin’s lymphomas (NHLs) is an interesting biological feature. CD5 is normally expressed by B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL), but may also be expressed in few cases of other NHLs, such as follicular lymphomas (FL) [1] and diffuse large B-cell lymphoma (DLBCL) [2]. The presence of other T antigens seems to be less common. Inaba et al. [3] reported 101 cases of NHLs, classified using the working formulation, with nodal and extranodal localizations, and found that CD2 was coexpressed in three cases and CD7 in seven patients. In that study, only two bone marrow specimens were analyzed and CD8 expression was never detected. The presence of CD8 seems to be a peculiar feature of some CLL cases and approximately 40 cases have been reported [4–6]. Very little information is available about the frequency of CD8 on B cells from the other NHLs, although Hoffman et al. [7] reported two cases of CD8-positive MCL. The evaluation of T cell-associated markers is not used in consensus protocols for the flow cytometric immunophenotyping of NHLs [8] and is not systematically analyzed. In our laboratories, a routine MoAb panel, including CD3, CD4, CD8, and CD16/56, is always used (both at diagnosis and during follow-up), and lymphocytes are gated using the CD45/SSC gating strategy. Thus, the percentage of T cell antigen-binding lymphocytes is always evaluated on the whole lymphocyte population and an abnormal antigen expression can easily be detected. Because of the lack of information about the frequency of aberrant CD8 expression in NHLs, we revised the clinical files of cases characterized by bone marrow infiltration, classified according to the R.E.A.L. system. In the period 2004–2006, 384 bone marrow samples with lymphomatous infiltration were obtained from 137 patients with B-CLL and 223 patients with other NHLs (FL, n=27; marginal zone lymphoma, MZL, n=70; MCL, n=40; hairy cell leukemia, HCL, n=32; lymphoplasmacytic lymphoma, n=41; DLBCL, n=13). Diagnosis was made by flow cytometry, morphology (bone marrow biopsy and immunohistochemistry), and PCR assays including IgH rearrangement, bcl-1/JH and bcl-2/JH translocations. Flow cytometry immunophenotyping was carried out by a three-color method with the following MoAb panel (FITC, PE, PerCP): CD3/CD16– 56/CD45; CD4/CD8/CD45; CD3/CD4/CD45; CD3/CD8/ CD45; CD5/CD19/CD45; CD10/CD20/CD45; FMC7/ CD22/CD45; CD19/CD23/CD45; CD19/CD38/CD45; CD103/CD25/CD45; CD25/CD11c/CD45; K/CD19/CD45; λ/CD19/CD45. When necessary, the CD19/CD8/CD45, CD8/CD19/CD45, and CD5/CD8/CD19 MoAb combinations were used, with different MoAb clones and different Ann Hematol (2008) 87:577–579 DOI 10.1007/s00277-008-0457-0