Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

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.

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.

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

INTRODUCTION: Discordant 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 PRESENTATION: We 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. CONCLUSION: Our case emphasizes the usefulness of investigating simultaneous specimens from different anatomic sites from the same patient and the relevant diagnostic role of splenectomy.

Prethymic cytoplasmic CD3 negative acute lymphoblastic leukemia or acute undifferentiated leukemia: a case report.

Acute undiffentiated leukemia (AUL) is an acute leukemia with no more than one membrane marker of any given lineage. Blasts often express HLA-DR, CD34, and/or CD38 and may be positive for terminal deoxynucleotidyl transferase (TdT). The expression of CD34, HLA-DR, and CD38 has been shown in pro-T-ALL, although in this case, blasts should also express CD7 and cyCD3. However, some cases of T-ALL without CD3 in the cytoplasm and all TCR chain genes in germ line configuration are reported, features that fit well with a very early hematopoietic cell. We report a case of acute leukemia CD34+/-HLADR+CD7+CD38+cyCD3- in which a diagnosis of AUL was considered. However the blasts were also positive for CD99 and TCR delta gene rearrangement which was found on molecular studies. Therefore a differential diagnosis between AUL and an early cyCD3 negative T-ALL was debated.

Multiparameter immunophenotyping by flow cytometry in multiple myeloma: The diagnostic utility of defining ranges of normal antigenic expression in comparison to histology.

BACKGROUND: Numerous studies have reported on the immunophenotype of plasma cells (PCs) in monoclonal gammopathy of undetermined significance (MGUS) and in plasma cell myeloma (PCM), but very few have examined the immunophenotype of normal PCs. In this study, an objective definition of normal range of expression for each antigen was found on normal control PCs. Using these new ranges of normal expression (new method) is different from using a static 20% of PCs cut-off for all antigens as described in the literature (traditional method). These newly calculated normal ranges for each antigen were applied to our data, and compared to histologic and immunohistochemical findings. METHODS: Bone marrow samples from 46 patients with PC neoplasms and 15 normal controls were studied. A minimum of 100 PC were analyzed for each patient and control sample. An 8-color staining method was applied to study the immunophenotype of PCs, using a BD FACSCanto II. RESULTS: By the new ranges of normality calculated in this study it was determined that different antigens have different level of expression on polyclonal PCs. CD19 correlated with histology by both the traditional and new methods, but had superior correlation by the new method. CONCLUSIONS: This report is the first 8-color immunophenotypic study of PCM in which a "range of normal expression" for each antigen is defined. This is a critical step to help distinguish between a normal and neoplastic PC immunophenotype and discern which antigens are of diagnostic importance.

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.

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.

Meningeal relapse in a case of B acute lymphoblastic leukemia: the role of CD56 expression.

BACKGROUND: Blasts from B acute lymphoblastic leukemia (B-ALL) may express CD56 in about 10% of cases. The presence of this marker at diagnosis is associated with an increased risk of meningeal relapse. A case is described of B-ALL which was CD56 negative at diagnosis, and expressed this marker when isolated meningeal relapse was diagnosed. CASE REPORT: A 53-year-old female patient presented with neurological symptoms during maintenance therapy for B-ALL. Peripheral blood, bone marrow, and cerebrospinal fluid (CSF) were subjected to both morphological and flow cytometric analyses. The latter was carried out by a wide routine panel of MoAbs which was the same as the one at diagnosis and included CD56. Isolated meningeal relapse was diagnosed since blast cell infiltration was detected in the CSF, but not in the peripheral blood and bone marrow samples. Blast cells showed an immunological phenotype similar to that at diagnosis (cyCD79a+, CD79b+, CD19+, CD22+, CD34+, CD10+, CD20+), but characterized by the acquisition of CD56 on the surfaces of more than 90% of cells. CONCLUSIONS: This case shows that CD56 can be expressed at relapse of B-ALL and that its presence likely enables leukemic cell binding to the central nervous system (CNS). This phenomenon may be responsible for the isolated CNS relapse diagnosed in this patient.

Carcinoma and multiple lymphomas in one patient: establishing the diagnoses and analyzing risk factors.

Multiple malignancies may occur in the same patient, and a few reports describe cases with multiple hematologic and non-hematologic neoplasms. We report the case of a patient who showed the sequential occurrence of four different lymphoid neoplasms together with a squamous cell carcinoma of the lung. A 62-year-old man with adenopathy was admitted to the hospital, and lymph node biopsy was positive for low-grade follicular lymphoma. He achieved a partial remission with chemotherapy. Two years later, a PET-CT scan showed a left hilar mass in the lung; biopsy showed a squamous cell carcinoma. Simultaneously, he was diagnosed with diffuse large B cell lymphoma in a neck lymph node; after chemo- and radiotherapy, he achieved a complete response. A restaging PET-CT scan 2 years later revealed a retroperitoneal nodule, and biopsy again showed a low-grade follicular lymphoma, while a biopsy of a cutaneous scalp lesion showed a CD30-positive peripheral T cell lymphoma. After some months, a liver biopsy and a right cervical lymph node biopsy showed a CD30-positive peripheral T cell lymphoma consistent with anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. Flow cytometry and cytogenetic and molecular genetic analysis performed at diagnosis and during the patient's follow-up confirmed the presence of two clonally distinct B cell lymphomas, while the two T cell neoplasms were confirmed to be clonally related. We discuss the relationship between multiple neoplasms occurring in the same patient and the various possible risk factors involved in their development.

CD45 expression in low-grade B-cell non-Hodgkin's lymphomas.

CD45 is a glycoprotein expressed in all lymphohemopoietic cells. Its expression increases during B-lymphocyte ontogeny. Few data are available about CD45 expression in the various types of low-grade B-cell non-Hodgkin's lymphomas (NHL). Low levels of CD45 have been reported in pathologic lymphocytes from typical chronic lymphocytic leukemia (CLL) and higher levels of this antigen have been observed in some cases of atypical CLL and in some cases of other types of NHL. One hundred and seven bone marrow samples of NHL with bone marrow infiltration were investigated: 45 typical CLL, 15 atypical CLL, 9 mantle cell lymphomas (MCL), 1 MCL with CD23 expression, 18 marginal zone lymphomas (MZL), 6 lymphoplasmacytic lymphomas (LPL), 6 follicular lymphomas (FL), and 7 hairy cell leukemias (HCL). CD45 expression was evaluated by flow cytometry: pathologic lymphocytes were identified on the basis of specific immunophenotypic profile, CD19/K or CD19/lambda co-expression. Results were expressed as median fluorescence intensity (MFI) along a 1024 linear scale. CD45 expression was measured also on autologous T-lymphocytes and a "CD45 index" was calculated as the ratio MFI of pathologic B-lymphocytes/MFI of T-lymphocytes, to normalize the results obtained. We found four CD45 expression patterns: very low in typical CLL; relatively low in MCL; intermediate intensity in MZL, LPL, and FL; very high expression in HCL. Among the atypical cases, very high CD45 expression was found in one case of CD23-negative CLL, in CD23-positive MCL, and CLL with atypical morphology. The results indicate different levels of maturation in low-grade NHL and may help to characterize such neoplasias.