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Biomedical subjects

Mark Raffeld

Publications and source records attributed to Mark Raffeld.

At least 37 records · Page 2Linked to original sources
PubMed2005

Mediastinal gray zone lymphoma: the missing link between classic Hodgkin's lymphoma and mediastinal large B-cell lymphoma.

In recent years, overlap in biologic and morphologic features has been identified between classic Hodgkin lymphoma (cHL) and B-cell non-Hodgkin lymphoma. Nevertheless, the therapeutic approaches for these diseases remain different. We undertook a study of "mediastinal gray zone lymphomas" (MGZL), with features transitional between cHL nodular sclerosis (NS) and primary mediastinal large B-cell lymphoma (MLBCL) to better understand the morphologic and immunophenotypic spectrum of such cases. Twenty-one MGZL cases were identified over a 20-year period. We also studied 6 cases of composite or synchronous lymphoma with two distinct components at the same time (cHL-NS and MLBCL) and 9 sequential cases with MLBCL and cHL-NS at different times. All patients had a large mediastinal mass. Immunohistochemical studies focused on markers known to discriminate between cHL and MLBCL, including B-cell transcription factors. VJ-PCR was performed in 8 cases to look at clonality of the immunoglobulin heavy chain gene (IgH). Of the gray zone cases, 11 had morphology reminiscent of cHL-NS, but with unusual features, including a large number of mononuclear variants, diminished inflammatory background, absence of classic Hodgkin phenotype, and strong CD20 expression (11 of 11). Ten cases had morphology of MLBCL, but with admixed Hodgkin/Reed-Sternberg and lacunar cells, absent (3 of 10) or weak (7 of 10) CD20 expression, and positivity for CD15 in 7 cases. B-cell transcription factor expression in the gray zone cases more closely resembled MLBCL than cHL with expression of Pax5, Oct2, and BOB.1 in all but 1 case studied (14 of 15). MAL staining was found in 7 of 10 MGZL, and in at least one component of 6 of 7 evaluable composite or sequential MLBCL/cHL cases. Two cases of sequential lymphoma showed rearrangements of the IgH gene of identical size: one in which MLBCL was the first diagnosis and one in which MLBCL was diagnosed at relapse, indicating clonal identity for the two components of cHL and MLBCL. There is accumulating evidence that MLBCL and cHL are related entities. Further support for a relationship between MLBCL and cHL-NS is provided by composite and metachronous lymphomas in the same patient, as well as the existence of MGZL with transitional morphology and phenotype.

Adolescent↗
PubMed2005

Squirrel monkeys support replication of BK virus more efficiently than simian virus 40: an animal model for human BK virus infection.

We performed experiments to test the suitability of squirrel monkeys (Saimiri sciureus) as an experimental model for BK virus (BKV) and simian virus 40 (SV40) infection. Four squirrel monkeys received intravenous inoculation with BKV Gardner strain, and six squirrel monkeys received intravenous inoculation with SV40 777 strain. Eight of 10 monkeys received immunosuppression therapy, namely, cyclophosphamide subcutaneously either before or both before and after viral inoculation. The presence of viral infection was assessed by quantitative real-time PCR amplification of viral DNA from blood, urine, and 10 tissues. We found that squirrel monkeys were susceptible to infection with BKV, with high viral copy number detected in blood and viral genome detected in all tissues examined. BKV genome was detected in urine from only one monkey, while three monkeys manifested focal interstitial nephritis. BKV T antigen was expressed in renal peritubular capillary endothelial cells. By contrast, SV40 was detected at very low copy numbers in only a few tissues and was not detected in blood. We conclude that the squirrel monkey is a suitable animal for studies of experimental BKV infection and may facilitate studies of viral entry, pathogenesis, and therapy.

Animals↗
PubMed2005

Mesothelin is overexpressed in pancreaticobiliary adenocarcinomas but not in normal pancreas and chronic pancreatitis.

Mesothelin, a cell surface glycoprotein present on normal mesothelial cells, has been reported to be expressed in pancreatic adenocarcinomas. We conducted this study to fully characterize mesothelin expression in surgically resected, formalin-fixed, paraffin-embedded tissue specimens of 18 pancreatic adenocarcinomas, 9 adenocarcinomas of the ampulla of Vater, 12 adenocarcinomas of the common bile duct, and 17 cases of chronic pancreatitis. Mesothelin immunostaining was performed using the antimesothelin monoclonal antibody 5B2. All 18 cases (100%) of pancreatic adenocarcinomas showed mesothelin expression, as did 8 (89%) of 9 cases of ampullar adenocarcinoma and all 12 cases (100%) of common bile duct adenocarcinoma. In all cases of pancreaticobiliary adenocarcinoma, the adjacent normal pancreas did not stain for mesothelin. Of 17 specimens of chronic pancreatitis, 16 were negative for mesothelin expression, and 1 case showed weak mesothelin staining of fewer than 5% of normal pancreatic ducts. Our results demonstrated mesothelin expression in the majority of pancreaticobiliary adenocarcinomas and no expression in normal pancreatic tissues and in chronic pancreatitis.

Adenocarcinoma↗
PubMed2004

Tissue inhibitor of metalloproteinase 1 (TIMP-1) promotes plasmablastic differentiation of a Burkitt lymphoma cell line: implications in the pathogenesis of plasmacytic/plasmablastic tumors.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a stromal factor with multiple functions. Overexpression of TIMP-1 correlates with aggressive clinical behavior of a spectrum of tumors. Here, for the first time, we address the role of TIMP-1 in the pathogenesis of B-cell lymphomas. An Epstein-Barr virus (EBV)-negative Burkitt lymphoma cell line with ectopic TIMP-1 expression (TIMP-1JD38) was used to identify genes induced/repressed by TIMP-1. Differentially expressed genes were analyzed by cDNA microarray, and they were validated by immunohistochemistry, flow cytometry, and Western blotting. Analysis revealed changes of genes coding for B-cell growth/differentiation, transcription, and cell cycle regulators. TIMP-1 repressed expression of germinal center (GC) markers CD10, Bcl-6, PAX-5 and up-regulated plasma cell-associated antigens CD138, MUM-1/IRF-4, XBP-1, and CD44, suggesting a plasma cell differentiation. This is accompanied by activation of signal transducer and activator of transcription 3 (STAT-3) and switch to cyclin D2 expression. However, TIMP-1JD38 cells expressed an inactive form of XBP-1, lacking antibody production/secretion. This incomplete plasmacytic differentiation occurs without altering cell proliferation, and despite c-Myc deregulation, indicating an arrested plasmacytic/plasmablastic stage of differentiation. Further validation in human lymphoma cell lines and in primary B-cell tumors demonstrated a predominant TIMP-1 expression in tumors with plasmacytic/plasmablastic phenotypes, including multiple myelomas. These findings strongly support TIMP-1 as an important factor in the pathogenesis of plasmacytic/plasmablastic tumors.

B-Lymphocytes↗
PubMed2004

Elevated serum-soluble interleukin-2 receptor levels in patients with anaplastic large cell lymphoma.

Levels of serum soluble interleukin 2 receptor (sIL-2R) provide a reliable marker of disease activity in patients with hairy cell leukemia and adult T-cell leukemia/lymphoma. The malignant cells in patients with anaplastic large cell lymphoma (ALCL) express CD30 and are usually positive for expression of CD25. We measured serum sIL-2R and soluble CD30 (sCD30) levels in patients with ALCL treated with EPOCH (etoposide, prednisone, Oncovin, Cytoxan, hydroxydaunorubicin) infusional chemotherapy. Serum sCD30 levels were elevated and decreased in response to therapy as previously reported. Serum sIL-2R levels were elevated in 7 of 9 patients with ALCL and decreased in response to treatment. Baseline serum sIL-2R levels varied but correlated well with serum sCD30 levels (r = 0.97). Patients positive for the anaplastic lymphoma kinase (ALK) gene showed elevated sIL-2R levels, whereas those negative for ALK had normal serum sIL-2R levels and their tumors lacked CD25 expression. Serum sIL-2R levels were elevated in both patients with recurrent disease.

Adult↗
PubMed2004

Prognostic impact of ANX7-GTPase in metastatic and HER2-negative breast cancer patients.

PURPOSE: ANX7-GTPase located on chromosome 10q21 is significantly altered and associated with hormone-refractory metastatic prostate cancers. Therefore, we investigated whether levels of ANX7 correlate with breast cancer progression and survival EXPERIMENTAL DESIGN: A diagnostic tumor tissue microarray containing 525 human breast tissue specimens at different stages of the disease was assayed for ANX7 using immunocytochemical methods with ANX7 monoclonal antibody. A separate prognostic tumor tissue microarray containing 553 human breast tissue specimens annotated with clinicopathological parameters was assayed for ANX7, HER2, estrogen receptor, progesterone receptor, and p53 protein. RESULTS: We report here for the first time that the expression of ANX7-GTPase is significantly enhanced and associated with the presence of metastatic disease (P < 0.0001) in the 525 human breast tissue specimens analyzed. Furthermore, using a separate 553 case retrospective prognostic tumor tissue microarray, we found that increased ANX7 expression is also significantly associated with poor overall patient survival (P < 0.014). This is particularly true when restricted to patients in whom the BRE clinical grade is 2 (P < 0.001) or for whom there is a lack of HER2 expression (P < 0.002). Finally, Cox regression analysis shows that as the expression of ANX7 rises, the probability of survival decreases by more than 10-fold for those patients with HER2-negative tumors. These latter patients represented 66% of the population affected with breast cancer in this study. CONCLUSIONS: High levels of ANX7 in tumor correlate strongly with poor survival of HER2-negative patients and the most aggressive forms of breast cancer. This is the first study to demonstrate that ANX7 antibody has the potential for development into an in vivo diagnostic and therapeutic tool. This simple and reliable immunohistochemical assay may therefore become an important biomarker for metastatic breast cancer diagnosis and management of HER2-negative breast tumor patients.

Adult↗
PubMed2004

Demonstration that mast cells, T cells, and B cells bearing the activating kit mutation D816V occur in clusters within the marrow of patients with mastocytosis.

Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering.

Adult↗
PubMed2004

Similarities of prosurvival signals in Bcl-2-positive and Bcl-2-negative follicular lymphomas identified by reverse phase protein microarray.

Overexpression of Bcl-2 protein has been known to play a role in the pathogenesis of follicular lymphoma (FL). However, 10-15% of FLs are negative for Bcl-2 by immunohistochemistry, raising the possibility that another gene product(s) may provide prosurvival signal(s). We used reverse phase protein microarray to analyze lysates of follicle center cells isolated by laser capture microdissection from: Bcl-2+ FL, Bcl-2- FL and reactive follicular hyperplasia (FH) (nine cases each group). TUNEL assay confirmed similar and reduced levels of apoptosis in Bcl-2+ FL and Bcl-2- FL, indicating the likelihood of Bcl-2-independent inhibition of apoptosis. Arrays were quantitatively analyzed with antibodies to proteins involved in the apoptotic pathway. As expected, Bcl-2 levels were up to eight-fold higher in Bcl-2+ FL than in FH and Bcl-2- FL. However, there was no difference in levels of Mcl-1 and survivin among these three groups. Bcl-X(L) showed a trend for increased expression in Bcl-2- FL as compared with Bcl-2+ FL, although the differences did not reach statistical significance (P>0.1). The increase in Bcl-X(L) may provide an alternative antiapoptotic signal in FL negative for Bcl-2 protein. Interestingly, Bax expression was higher in FL (Bcl-2+ or -) than in FH (P=0.001). Notably, phospho-Akt (Ser-473) was increased in FL (Bcl-2+ or -) (P<0.03) with increased phospho-Bad (Ser-136), as compared with levels in FH. The activation of the Akt/Bad pathway provides further evidence of prosurvival signals in FL, independent of Bcl-2 alone. These data suggest that nodal FL represents a single disease with a final common biochemical pathway.

Apoptosis↗
PubMed2004

Human herpesvirus-6-associated acute lymphadenitis in immunocompetent adults.

In contrast to other causes of herpetic lymphadenitis, the histological features associated with human herpesvirus-6 (HHV-6) infection have remained elusive since its discovery in 1986. We describe the histologic and phenotypic changes associated with acute HHV-6 lymphadenitis in two immunocompetent adults who presented with fever, fatigue, generalized lymphadenopathy, and elevated liver enzymes. Serologic tests for human immunodeficiency virus, acute Epstein-Barr virus, and cytomegalovirus infection were negative. Lymph node biopsies were consistent with viral lymphadenitis. Intranuclear and cytoplasmic inclusions were identified in CD4-positive T lymphocytes in expanded paracortical areas. Immunohistochemical staining with monoclonal antibody to the HHV-6 gp60/110 kDa envelope glycoprotein showed that the inclusions were positive for viral antigen. Electron microscopy demonstrated numerous viral particles in the cytoplasm and nucleus, characteristic of Herpesviridae family. Clustering of viral particles was observed, which has previously been reported only in infected tissue culture cells. PCR followed by sequencing of DNA extracted from the lymph nodes identified the virus as HHV-6, type B. This is the first report that documents distinctive histologic features of HHV-6 lymphadenitis and demonstrates that the cells harboring the virus in vivo are CD4-positive T lymphocytes.

Adult↗
PubMed2004

Utilization of polymerase chain reaction on archival cytologic material: a comparison with fresh material with special emphasis on cerebrospinal fluids.

Use of the polymerase chain reaction (PCR) for the detection of B- and T-cell clonality, Epstein-Barr virus (EBV) and Human Herpes Virus 8 (HHV 8) infection is gaining increasing importance as a diagnostic modality. These tests are usually performed on fresh specimens. There are instances when fresh material is not available and there is a clinical utility for the performance of PCR on archival material via slide scrape lysates (SSL). However, the suitability of archival material may be questioned. Records were searched for all archival cytology cases submitted for SSL molecular diagnostics tests since 1998. Results for each case were analyzed for PCR amplification status and individual test results. A randomly chosen control group of equivalent cytologic samples submitted fresh was evaluated for comparison of amplification status. In all, 241 PCR runs were performed on SSL of archival material from 112 cytologic samples (89 cerebrospinal fluids (CSFs), 13 fine-needle aspirates (FNAs), 10 effusions). Out of these samples, 95 (85%) had amplifiable DNA, as assessed by a positive reaction for glyceraldehyde phosphate dehydrogenase (GAPDH). For the control group, 320 PCR runs were performed on 112 fresh cytologic samples (89 CSFs, 13 FNAs, 10 effusions). In total, 102 samples (91%) had amplifiable DNA. There was no statistical difference in the amplification yield between the two groups (P = 0.2177). A morphologic review of 16 of the 17 SSL archival cytologic cases that did not show amplification revealed 11/16 to be of sparse cellularity. Molecular diagnostic tests are performed routinely on fresh cytologic samples with excellent results. At times critical decisions on patient care may need to be made when fresh tissue is not available for molecular diagnostic tests. SSL of archival cytologic material can be used with excellent results for molecular diagnostic tests when fresh material is not available or when the cytologic diagnosis needs further clarification.

B-Lymphocytes↗
PubMed2004

Clonal T-cell populations and increased risk for cytotoxic T-cell lymphomas in B-CLL patients: clinicopathologic observations and molecular analysis.

Chronic lymphocytic leukemia (CLL) is associated with increased risk of malignancy, but the occurrence of other lymphomas, in particular T-cell lymphomas, is rare. We identified 7 cases of peripheral T-cell malignancy associated with B-cell-derived CLL from the files of two institutions over a 20-year period. The presence of both B and T lymphoproliferative disorders was confirmed in all cases by immunophenotype and in 6 cases by gene rearrangements. Six patients developed peripheral T-cell lymphoma (PTCL), unspecified, during the course of CLL (10-168 months). In all 5 evaluable cases, the cells had a cytotoxic T-cell phenotype; the sixth case was CD56+, but TIA-1 and Granzyme B could not be studied. A seventh patient with CLL developed mycosis fungoides, and an aggressive NK cell leukemia. To investigate possible risk factors for the development of PTCL, we screened 100 unselected peripheral blood samples from newly diagnosed CLL patients by PCR for the presence of clonal T cell populations. We found evidence of clonal T-cell expansion in 8 patients and increased lymphocytes with large granular lymphocyte morphology in 7 of 8 cases. The immunophenotype was assessed by multicolor flow cytometry and in 4 cases the T-cell expansion was composed of either CD3+/CD8+ or CD3+/CD4-/CD8- cells. The cytotoxic nature of the clonal T-cell expansions in the peripheral blood correlates with the cytotoxic nature of the PTCLs, but their role in the subsequent development of T-cell lymphomas is still unclear. PTCL following CLL should be distinguished from typical Richter syndrome, which it can mimic clinically.

Aged↗
PubMed2003

Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling.

Colon and ovarian cancers can be difficult to distinguish in the abdomen, and the distinction is important because it determines which drugs will be used for therapy. To identify molecular markers for that differential diagnosis, we developed a multistep protocol starting with the 60 human cancer cell lines used by the National Cancer Institute to screen for new anticancer agents. The steps included: (a) identification of candidate markers using cDNA microarrays; (b) verification of clone identities by resequencing; (c) corroboration of transcript levels using Affymetrix oligonucleotide chips; (d) quantitation of protein expression by "reverse-phase" protein microarray; and (e) prospective validation of candidate markers on clinical tumor sections in tissue microarrays. The two best candidates identified were villin for colon cancer cells and moesin for ovarian cancer cells. Because moesin stained stromal elements in both types of cancer, it would probably not have been identified as a marker if we had started with mRNA or protein profiling of bulk tumors. Villin appears at least as useful as the currently used colon cancer marker cytokeratin 20, and moesin also appears to have utility. The multistep process introduced here has the potential to produce additional markers for cancer diagnosis, prognosis, and therapy.

Adenocarcinoma↗
PubMed2003

The proapoptotic factor Nix is coexpressed with Bcl-xL during terminal erythroid differentiation.

Transcriptional profiles of cultured primary human erythroid cells were examined to identify those genes involved in the control of erythroid growth during the terminal phase of maturation. Our in silico screening strategy indicated that a hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during erythropoiesis. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than reticulocytes. Polymerase chain reaction (PCR)-based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we propose that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation.

Apoptosis↗
PubMed2003

Highly effective treatment of acquired immunodeficiency syndrome-related lymphoma with dose-adjusted EPOCH: impact of antiretroviral therapy suspension and tumor biology.

The outcome of acquired immunodeficiency syndrome-related lymphomas (ARLs) has improved since the era of highly active antiretroviral therapy, but median survival remains low. We studied dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) with suspension of antiretroviral therapy in 39 newly diagnosed ARLs and examined protein expression profiles associated with drug resistance and histogenesis, patient immunity, and HIV dynamics and mutations. The expression profiles from a subset of ARL cases were also compared with a matched group of similarly treated HIV-negative cases. Complete remission was achieved in 74% of patients, and at 53 months median follow-up, disease-free and overall survival are 92% and 60%, respectively. Following reinstitution of antiretroviral therapy after chemotherapy, the CD4+ cells recovered by 12 months and the viral loads decreased below baseline by 3 months. Compared with HIV-negative cases, the ARL cases had lower bcl-2 and higher CD10 expression, consistent with a germinal center origin and good prognosis, but were more likely to be highly proliferative and to express p53, adverse features with standard chemotherapy. Unlike HIV-negative cases, p53 overexpression was not associated with a poor outcome, suggesting different pathogenesis. High tumor proliferation did not correlate with poor outcome and may partially explain the high activity of dose-adjusted EPOCH. The results suggest that the improved immune function associated with highly active antiretroviral therapy (HAART) may have led to a shift in pathogenesis away from lymphomas of post-germinal center origin, which have a poor prognosis. These results suggest that tumor pathogenesis is responsible for the improved outcome of ARLs in the era of HAART.

Acquired Immunodeficiency Syndrome↗
PubMed2003

ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile.

The presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of chronic lymphocytic leukemia (CLL) cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Ig-unmutated CLL) often have progressive disease, whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. Pilot gene expression profiling studies in patients with CLL identified genes that were differentially expressed between the Ig-unmutated and Ig-mutated CLL subtypes. Here, we have profiled an expanded cohort of 107 patients and show that ZAP-70 is the gene that best distinguishes the CLL subtypes. Ig-unmutated CLL expressed ZAP-70 5.54-fold more highly than Ig-mutated CLL (P < 10(-21)). ZAP-70 expression correctly predicted IgVH mutation status in 93% of patients. ZAP-70 expression and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis. In 7 patients, ZAP-70 expression and IgVH mutation status were discordant: 4 Ig-mutated CLLs had high ZAP-70 expression and 3 Ig-unmutated CLLs had low ZAP-70 expression. Among these ZAP-70 "outliers," those with Ig-mutated CLL had clinical features that are uncharacteristic of this CLL subtype: 2 required early treatment and 2 used a mutated VH3-21 gene, an IgVH gene that has been associated with progressive disease. We developed reverse transcriptase-polymerase chain reaction and immunohistochemical assays for ZAP-70 expression that can be applied clinically and would yield important prognostic information for patients with CLL.

ADP-ribosyl Cyclase↗