Isolation and characterization of a CD34+ sub-clone in B-cell lymphoma.

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy in the US. Many types remain incurable despite response to initial therapy and achievement of complete remission (CR). Advanced laboratory techniques like multicolor flow cytometry (FCM) and polymerase chain reaction (PCR) have demonstrated persistence of rare malignant cell population post therapy. However, the functional and biological characteristics of this population have not been elucidated. Established B-lymphoma cell lines (B-NHL) and patient-derived samples (PDS) were analyzed using 8-color FCM. CD34+ sub-population was enriched using in vitro exposure to 2-chlorodeoxyadenosine (2-CdA) and by CD34 magnetic beads. Genetic analysis of cell fractions was done by karyotyping and array comparative genomic hybridization (aCGH). Sensitivity to chemotherapy was assayed by short-term in vitro exposure to chemotherapy. Clonogenicity was determined by soft agar colony formation assay, and proliferation was determined using DNA staining with propidium iodide and FCM. FCM demonstrated the presence of a minute sub-clone of monotypic B-cells that express CD34 in B-NHL cell lines (3 of 3) and in PDS (8 of 8). This sub-population enriched up to 50 fold in vitro by exposure to 2-CdA and up to 80% purity by CD34 magnetic bead column isolation. Except for CD34 expression, this population expressed identical phenotype and genotype to parent cells, but was more proliferative, Hoechst 33342-positive, clonogenic, and resistant to chemotherapy compared with the CD34- population. The isolated CD34+ monotypic B-cells may contribute to resistance of certain NHL to treatment and should be targeted by potential new drugs for NHL.

High Ki67 proliferation index but not cell-of-origin subtypes is associated with shorter overall survival in diffuse large B-cell lymphoma.

BACKGROUND: CD10, BCL6, and MUM1 are commonly used immunohistochemical stains for classifying diffuse large B-cell lymphoma (DLBCL), which is useful in predicting outcome. Conflicting reports of the prognostic value of other markers such as BCL2, CD23, and Ki67 proliferation index have been reported. Our objective was to correlate these immunostains and Hans classification with response to therapy and overall survival. MATERIALS AND METHODS: A retrospective study of patients diagnosed with DLBCL from 2008-2014 at a tertiary-care cancer hospital. The slides with the IHC stains were reviewed by two independent pathologists. The clinical outcomes--assessed independently--were response to therapy and overall survival. The treatment response evaluation was based on the new Lugano classification. Statistical analyses were conducted using the Fisher's exact test and Kaplan-Meier survival curves. Significance was set at P < 0.05. RESULTS: Forty-one patients were included in the study with a known Hans classification, available clinical data, and at least 5-year follow-up. CD10 immunostain was reported in all patients, whereas CD23 was the least reported in only four patients. No significant association was observed between CD10, BCL6, MUM1, BCL2, and both Response to therapy and overall survival. Owing to few cases reported CD23 immunostain, further analysis of association is not reported. High Ki67 proliferative index of >80% was statistically significantly associated with shorter overall survival and not statistically significant associated with no response to therapy. Hans classification subtypes were not predictive in regard to therapy response. CONCLUSION: High Ki67 expression (>80%) was associated with shorter overall survival in DLBCL. Hans classification subtypes were not predictive.

Comparative assessment of conventional chromosomal analysis and fluorescence in situ hybridization in the evaluation of suspected myelodysplastic syndromes: a single institution experience.

BACKGROUND: Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic neoplasms, roughly half of which harbor cytogenetic abnormalities with diagnostic, prognostic, and therapeutic significance. Fluorescence in situ hybridization (FISH) for the most commonly seen abnormalities (5/5q, -7/7q, +8, and -20/20q-) is routinely performed alongside conventional cytogenetics (CC) in the evaluation of suspected MDS despite conflicting reports of its relative contribution compared to CC alone. OBJECTIVES: To assess the additional diagnostic and prognostic value of performing concurrent FISH versus CC alone in cases of suspected MDS. MATERIALS AND METHODS: A total of 127 bone marrow samples submitted to our cytogenetic laboratory with a presumptive diagnosis of MDS were evaluated by concurrent CC and an MDS FISH panel. RESULTS: CC was used as the gold standard method with 100% sensitivity in detecting suspected MDS-associated cytogenetic abnormalities. FISH alone had a sensitivity of 76%, whereas CC alone achieved a sensitivity of 97%. The addition of FISH did not change the diagnosis nor change the Revised International Prognostic Scoring System score in any patient. Moreover, in 12 cases identified as positive by both CC and FISH, CC identified multiple chromosomal aberrations of clinical significance not interrogated by the FISH probe panel. CONCLUSION: CC alone is sufficiently sensitive in detecting suspected MDS-associated cytogenetic abnormalities that influence clinical decision-making. Routine FISH testing does not provide a significant increase in test sensitivity when an adequate karyotype is obtained. Therefore, FISH testing is best reserved for suspected MDS cases lacking sufficient metaphases.

Molecular Diagnosis of Hematopoietic Neoplasms: 2018 Update.

Diagnosis of hematologic malignancies have matured to encompass molecular as well as phenotypic characteristics. Cytogenetic abnormalities are considered common events in this regard. These abnormalities generally consist of structural chromosomal abnormalities or gene mutations, which often are integral to the pathogenesis and subsequent evolution of an individual malignancy. Improvements made in identifying and interpreting these molecular alterations have resulted in advances in the diagnosis, prognosis, monitoring, and therapy for cancer. As a consequence of the increasingly important role of molecular testing in hematologic malignancy management, this article presents an update on the importance and use of molecular tests, detailing the advantages and disadvantages of each test when applicable.

Simultaneous hepatosplenic T-cell lymphoma and myelofibrosis.

Hepatosplenic T-cell lymphoma (HSTL) is a rare T-cell neoplasm of the lymphoid system. This type of lymphoma is characterized by sinusoidal infiltration of spleen, liver, bone marrow and lymph nodes by neoplastic lymphocytes. Here, we discuss a patient who had a left axillary lymph node biopsy with characteristic histological and immunohistochemical features of HSTL. In addition, infiltrating neoplastic T-cells and simultaneous characteristic features of myelofibrosis (MF) were also present in the bone marrow biopsy specimen. In contrast to secondary MF, primary MF is a progressive disease and may significantly affect the prognosis of coexisting HSTL. There are few reports in the literature talking about mild bone marrow fibrosis in association with T cell lymphoma, however marked increase in bone marrow fibrosis and HSTL never being reported. This case is shedding light on HSTL and marked increase in bone marrow fibrosis.

Molecular diagnosis of hematopoietic neoplasms.

Cytogenetic abnormalities are considered to be common events in hematologic malignancies. These abnormalities generally consist of structural chromosomal abnormalities or gene mutations, which often are integral to the pathogenesis and subsequent evolution of an individual malignancy. Improvements made in identifying and interpreting these molecular alterations have resulted in advances in the diagnosis, prognosis, monitoring, and therapy for cancer. As a consequence of the increasingly important role of molecular testing in hematologic malignancy management, this article presents an update on the importance and use of molecular tests, detailing the advantages and disadvantages of each test when applicable.

Human immunodeficiency virus type 1 infection alters enzymatic and ultrastructural features of peripheral blood monocytes.

INTRODUCTION: Human immunodeficiency virus-1 (HIV-1) infected monocytes are now believed to serve as a reservoir for HIV-1 infection, and to play a role in viral rebound phenomena in certain groups of patients who failed or stopped highly active antiretroviral therapy (HAART). Data characterizing the morphological changes of peripheral blood monocytes in HIV-1-infected individuals are limited. MATERIALS AND METHODS: In this study, we collected monocytes from 21 asymptomatic HIV-1-infected individuals with CD4 count more than 500 cells/mm(3) and healthy individuals. The monocytes ultrastructural morphologic changes and α-naphthyl butyrate esterase (ANBE) activity were compared between the two groups. RESULTS: In monocytes from patients infected with HIV-1, activity of α-naphthyl butyrate esterase (ANBE) was markedly increased compared with normal monocytes. In both light microscopic and ultrastructural studies, the cytoplasm of monocytes from HIV-1-infected patients contained a haphazard appearing network of thin fibrils. Cell surface expression of the activation marker HLA-DR molecule was upregulated. There were no discernible differences between the cell surface expression of CD4, CD14, and CD16 molecules comparing normal monocytes to those from HIV-1-infected patients. α-naphthyl butyrate esterase (ANBE) was markedly increased compared with normal monocytes. In both light microscopic and ultrastructural studies, the cytoplasm of monocytes from HIV-1-infected patients contained a haphazard appearing network of thin fibrils. Cell surface expression of the activation marker HLA-DR molecule was upregulated. There were no discernible differences between the cell surface expression of CD4, CD14, and CD16 molecules comparing normal monocytes to those from HIV-1-infected patients. CONCLUSIONS: Possibly, changes in the activity of ANBE along with a disrupted appearing cytoplasmic fibril network contribute to monocyte dysfunction in HIV-1-infected patients.

P38 mitogen activated protein kinase expression and regulation by interleukin-4 in human B cell non-Hodgkin lymphomas.

UNLABELLED: The prevalence and regulation of p38 mitogen activated protein kinase (MAPK) expression in human lymphomas have not been extensively studied. In order to elucidate the role of p38 MAPK in lymphomagenesis, we examined the expression of native and phosphorylated p38 (p-p38) MAPK in cell lines derived from human hematopoietic neoplasms including B cell lymphoma-derived cell lines using Western blot analysis. The p-p38 MAPK protein was also analyzed in 30 B cell non-Hodgkin lymphoma (NHL) tissue biopsies by immunohistochemistry. Our results show that the expression of p38 MAPK was up-regulated in most of the cell lines as compared with peripheral blood lymphocytes, while the expression of p-p38 MAPK was more variable. A subset of B cell NHL biopsies showed increased expression of p-p38 MAPK relative to reactive germinal center cells. Interleukin-4 (IL-4) induced a dose-dependent increase in the expression of p-p38 MAPK (1.6- to 2.8-fold) in cell lines derived from activated B cell-like diffuse large B cell lymphoma (DLBCL) but not those from germinal center-like DLBCL. No change was seen in native p38 MAPK. The in vitro kinase activity of p38 MAPK, however, was induced (1.6- to 3.2-fold) in all five cell lines by IL-4. Quantitative fluorescent RT-PCR demonstrated that all four isoforms of p38 MAPK gene were expressed in the lymphoma cell lines, with p38gamma and p38beta isoforms being predominant. IL-4 stimulation increased the expression of beta, gamma, and delta isoforms but not alpha isoform in two cell lines. In conclusion, there is constitutive expression and activation of p38 MAPK in a large number of B-lymphoma-derived cell lines and primary lymphoma tissues, supportive of its role in lymphomagenesis. The differential IL-4 regulation of p38 MAPK expression in cell lines derived from DLBCL may relate to the cellular origin of these neoplasms. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12308-009-0049-5) contains supplementary material, which is available to authorized users.

Primary melanoma of small intestine masquerading as gastrointestinal stromal tumor: a case report and literature review.

Malignant melanoma of the gastrointestinal tract is a rare entity among intestinal neoplasms. Primary intestinal melanoma is difficult to differentiate from metastatic melanoma, especially given that the primary cutaneous lesion has the potential to regress and disappear. In addition, melanoma by itself is a great mimicker of other neoplastic conditions and may create a major diagnostic challenge when presenting at an intra-abdominal location. Here we report a case of small intestinal melanoma in a 74-year-old female who presented with symptoms of intestinal bleeding and a preoperative clinical and radiological diagnosis of gastrointestinal stromal tumor. The initial frozen section diagnosis also favored gastrointestinal stromal tumor, however furthermore histological and immunohistochemical stain evaluation confirmed the diagnosis of intestinal melanoma.

HIV infection of mononuclear cells is calcium-dependent.

Strategies that prevent initial HIV infection of cells are greatly needed. In this study, we determined the requirement of divalent cations for HIV infection of and attachment to peripheral blood mononuclear cells (PBMC), which contain several types of HIV-infectable cells-CD4(+) T cells, monocytes and dendritic cells. EDTA, added only during PBMC exposure to HIV, reduced infection by an average of 92%. The reduction of infection by EDTA was accompanied by a reduction in HIV binding to PBMC; R5, X4 and dual-tropic HIV binding to PBMC were inhibited by >85%. EGTA similarly reduced HIV binding to PBMC, while addition of Ca(2+) or Mn(2+), but not Mg(2+), fully restored binding. Virus attachment was inhibited in a dose-dependent manner by trypsin treatment of PBMC, indicating protein involvement in HIV binding. In contrast, mannan or soluble ICAM-1 did not inhibit HIV binding to PBMC. These data indicate that a Ca(2+)-dependent cell-surface protein(s) is responsible for the majority of HIV attachment to and infection of PBMC. Further studies of this are likely to reveal novel strategies to prevent infection of PBMC.

Activation by inflammatory stimuli increases neutrophil binding of human immunodeficiency virus type 1 and subsequent infection of lymphocytes.

Resting neutrophils bind human immunodeficiency virus type 1 (HIV-1) and efficiently transfer infection to lymphocytes. The present study shows that a brief activation by inflammatory stimuli increases the neutrophil binding levels of both R5 and X4 isolates of HIV-1 at least twofold. The binding occurs independently of CD4, gp120, and incubation temperature and is observed with HIV-1 propagated either in lymphocytes or in HEK293 cells. Significantly, HIV-1 bound to the activated neutrophils accelerates the infection of activated lymphocytes compared to free HIV-1 or to HIV-1 bound to resting neutrophils. It is proposed that these events may contribute to the increased risk of HIV-1 transmission at sites of mucosal infection.