PDGFRß promotes oncogenic progression via STAT3/STAT5 hyperactivation in anaplastic large cell lymphoma.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T cell lymphoma commonly driven by NPM-ALK. AP-1 transcription factors, cJUN and JUNb, act as downstream effectors of NPM-ALK and transcriptionally regulate PDGFRß. Blocking PDGFRß kinase activity with imatinib effectively reduces tumor burden and prolongs survival, although the downstream molecular mechanisms remain elusive. METHODS AND RESULTS: In a transgenic mouse model that mimics PDGFRß-driven human ALCL in vivo, we identify PDGFRß as a driver of aggressive tumor growth. Mechanistically, PDGFRß induces the pro-survival factor Bcl-xL and the growth-enhancing cytokine IL-10 via STAT5 activation. CRISPR/Cas9 deletion of both STAT5 gene products, STAT5A and STAT5B, results in the significant impairment of cell viability compared to deletion of STAT5A, STAT5B or STAT3 alone. Moreover, combined blockade of STAT3/5 activity with a selective SH2 domain inhibitor, AC-4-130, effectively obstructs tumor development in vivo. CONCLUSIONS: We therefore propose PDGFRß as a novel biomarker and introduce PDGFRß-STAT3/5 signaling as an important axis in aggressive ALCL. Furthermore, we suggest that inhibition of PDGFRß or STAT3/5 improve existing therapies for both previously untreated and relapsed/refractory ALK+ ALCL patients.

[Genetic evolution of in situ follicular neoplasia to t(14;18)-positive aggressive B-cell lymphoma]. / Die genetische Evolution der in situ follikulären Neoplasie zum t(14;18)-positiven aggressiven B­Zell-Lymphom.

BACKGROUND: In situ follicular neoplasia (ISFN) is a t(14;18)(q32;q21)+ precursor lesion of follicular lymphoma (FL), which in turn can transform into diffuse large B­cell lymphoma (DLBCL). For DLBCL that arise de novo, no precursor lesion is known. Given the high frequency of the t(14;18) translocation in de novo DLBCL as well, we investigated whether they can also arise from ISFN without FL as an intermediate step. OBJECTIVES: To investigate the clonal evolution of ISFN to DLBCL - transformed from FL and de novo. MATERIALS AND METHODS: Identification of ISFN lesions in patients with DLBCL was performed by BCL2 staining of reactive lymphoid tissues. ISFN and DLBCL were subsequently analyzed by fluorescence in situ hybridization, clonality analyses, sequencing of the t(14;18) breakpoint, and targeted next-generation sequencing. RESULTS: 10 cases with paired ISFN and DLBCL samples were identified, 6 of which were de novo DLBCL and 4 transformed from FL. 3 DLBCL carried MYC-rearrangements in addition to the t(14;18) and were classified as high-grade B­cell lymphoma (HGBL). The clonal relationship of ISFN and DLBCL/HGBL was confirmed for all cases. CREBBP, KMT2D, EZH2, TNFRSF14, and BCL2 were the genes most frequently mutated, with the distribution of private and shared mutations pointing to 2 different scenarios of clonal evolution. In most cases, DLBCL/HGBL, ISFN, and, if also present, FL had evolved divergently from a common progenitor, whereas linear evolution was less frequent. CONCLUSION: We show for the first time that t(14;18)+ DLBCL/HGBL can arise directly from ISFN without FL as an intermediate step and that during this progression, divergent evolution is common.

[Clonal evolution of early lymphoproliferations]. / Klonale Evolution früher Lymphoproliferationen.

The identification and molecular characterisation of premalignant precursor lesions of lymphomas, such as monoclonal gammopathy of unknown significance (MGUS) and the so-called in situ lymphoproliferations, has made significant progress in the recent years. The in situ follicular neoplasia (ISFN), the best-characterised entity, is by definition not identifiable by morphology and represents a t(14;18)+ precursor lesion of follicular lymphoma with characteristic immunophenotype, low potential for progression, and already identifiable secondary genetic alterations. The use of high-throughput genetic techniques on microdissected tissues has generated novel insights into clonal evolution and biological progression of early lesions and documented that an isolated genetic analysis is insufficient to understand the complexity of proliferations.

An unusual presentation of a common infection.

A 40-year-old Ghanaian woman presented with fever and exanthema. She had anemia, leukopenia, increased erythrocyte sedimentation rate (ESR), creatinin kinase, lactate dehydrogenase (LDH), and liver enzymes. She was diagnosed with schistosomiasis and was cured with praziquantel. During the following years, she developed polymyositis, chronic nephritis, and life-threatening perimyocarditis. High numbers of Epstein-Barr virus (EBV)-encoded RNA copies were demonstrated in CD8+ T-lymphocytes from endomyocardial biopsies. There was no evidence of any underlying immunosuppression or an EBV-related malignancy. Chronic active EBV infection was diagnosed, a clinical picture not described in an adult African previously. Interestingly, among all therapy attempts, only rituximab was effective at stabilizing the disease.

[B-cell neoplasms with plasmacellular and plasmablastic differentiation]. / B-Zell-Neoplasien mit plasmazellulärer und plasmablastischer Differenzierung.

Plasma cell malignancies are tumors of terminally differentiated B-cells in which the neoplastic plasma cells are the dominant and proliferating tumor cell component. Plasma cell myeloma (PCM) is one of the most common hematological neoplasms and typically does not cause diagnostic problems. A morphologically and immunophenotypically detectable plasmacellular orplasmablastic differentiation is, however, commonly observed in a wide range of mature B-cell lymphomas. A confident separation of the distinct entities requires the integration of clinical and morphological findings as well as an adequate phenotyping of both the plasma cell and the B-cell component if present. Detection of lymphotropic viruses, specific translocations and novel molecular markers, such as the MYD88 L265P mutation occurring in the vast majority of lymphoplasmacytic lymphomas complement our diagnostic repertoire. In this review we describe the most commonly observed diagnostic problems in separating small B-cell lymphomas from PCM and high-grade B-cell non-Hodgkin lymphoma (B-NHL) with plasmablastic differentiation from extramedullary spread of aggressive PCM and provide helpful criteria for routine diagnostics.

[In situ lymphoma and other early stage malignant non-Hodgkin lymphomas]. / "In-situ-Lymphome" und andere Frühstadien maligner Non-Hodgkin-Lymphome.

The increasing use of immunohistochemical and molecular investigations of lymphatic tissues results in more frequent detection of early lymphoid proliferations. These show some but not all features of malignant lymphomas without fulfilling the diagnostic criteria for the diagnosis of lymphoid malignancy. In addition to well-known premalignant B-cell proliferations, such as monoclonal gammopathy of unknown significance (MGUS) and monoclonal B-cell lymphocytosis (MBL), so-called in situ lymphomas have recently been described with minimal infiltrates of clonal B-cells in morphologically reactive lymphoid tissues which show the phenotypic and genetic features of specific B-cell lymphoma subtypes and often show a characteristic topographical distribution. This article addresses a group of clonal lymphoproliferations with usually localized disease and excellent clinical prognosis, such as pediatric follicular lymphoma and nodal marginal zone lymphoma. Another group of early lesions not addressed in this review are virally induced lymphoproliferations which represent a grey zone between purely reactive lesions and malignant lymphomas and may pose significant diagnostic as well as clinical problems. In this review diagnostic criteria for early or in situ lesions and their distinction from partial infiltration by malignant lymphoma are described.

Peach extract induces systemic and local immune responses in an experimental food allergy model.

Peach allergy is among the most frequent food allergies in the Mediterranean area, often eliciting severe anaphylactic reactions in patients. Due to the risk of severe symptoms, studies in humans are limited, leading to a lack of therapeutic options. This study aimed to develop a peach allergy mouse model as a tool to better understand the pathomechanism and to allow preclinical investigations on the development of optimized strategies for immunotherapy. CBA/J mice were sensitized intraperitoneally with peach extract or PBS, using alum as adjuvant. Afterwards, extract was administered intragastrically to involve the intestinal tract. Allergen provocation was performed via intraperitoneal injection of extract, measuring drop of body temperature as main read out of anaphylaxis. The model induced allergy-related symptoms in mice, including decrease of body temperature. Antibody levels in serum and intestinal homogenates revealed a Th2 response with increased levels of mMCPT-1, peach- and Pru p 3-specific IgE, IgG1 and IgG2a as well as increased levels of IL-4 and IL-13. FACS analysis of small intestine lamina propria revealed increased amounts of T cells, neutrophils and DCs in peach allergic mice. These data suggest the successful establishment of a peach allergy mouse model, inducing systemic as well as local gastrointestinal reactions.

[Bone marrow biopsy: processing and use of molecular techniques]. / Knochenmarkbiopsie: Aufarbeitung und Einsatzmöglichkeiten molekularpathologischer Methoden.

The rapid technological development in diagnostic pathology, especially of immunohistochemical and molecular techniques, also has a significant impact on diagnostic procedures for the evaluation of bone marrow trephine biopsies. The necessity for optimal morphology, combined with preservation of tissue antigens and nucleic acids on one hand and the wish for short turnaround times on the other hand require careful planning of the workflow for fixation, decalcification and embedding of trephines. Although any kind of bone marrow processing has its advantages and disadvantages, formalin fixation followed by EDTA decalcification can be considered a good compromise, which does not restrict the use of molecular techniques. Although the majority of molecular studies in haematological neoplasms are routinely performed on bone marrow aspirates or peripheral blood cells, there are certain indications, in which molecular studies such as clonality determination or detection of specific mutations need to be performed on the trephine biopsy. Especially, the determination of B- or T-cell clonality for the diagnosis of lymphoid malignancies requires stringent quality controls and knowledge of technical pitfalls. In this review, we discuss technical aspects of bone marrow biopsy processing and the application of diagnostic molecular techniques.

An impending crisis in the provision of histopathology expertise for mouse functional genomics.

The generation of new mouse models of human disease is accelerating rapidly, due to the completion of whole-genome sequencing efforts and technological advances in the manipulation of the mouse genome. We sought to investigate manpower issues in the provision of histopathology expertise for mouse functional genomics and compared this to the perceived demand from principal investigators (PIs). Through the European Commission (EC)-funded PRIME pathology training initiative, two questionnaires were devised to collect information from pathologists and EC-funded PIs on the current provision of mouse histopathology expertise in Europe and the demands for this service. We find that pathological analysis is being performed almost exclusively by professionally qualified pathologists, generally employed in clinical diagnostic posts, where the work is undertaken as collaboration outside of their contractual commitments but without previous training in veterinary or comparative pathology. The results indicate that there is a lack of both trainees and provision of specialist training in this field. Unsurprisingly, the availability of diagnostic expertise and advice falls far short of the number of genetically engineered mice (GEM) being generated for analysis. We analyse these results with reference to previous studies and discuss solutions for the future recruitment, training and funding for pathologists in mouse functional genomics in Europe.

Progress in updating the European Radiobiology Archives.

PURPOSE: The European Radiobiology Archives (ERA), together with corresponding Japanese and American databases, hold data from nearly all experimental animal radiation biology studies carried out between 1960 and 1998, involving more than 300,000 animals. The Federal Office for Radiation Protection, together with the University of Cambridge have undertaken to transfer the existing ERA archive to a web-based database to maximize its usefulness to the scientific community and bring data coding and structure of this legacy database into congruence with currently accepted semantic standards for anatomy and pathology. METHODS: The accuracy of the primary data input was assessed and improved. The original rodent pathology nomenclature was recoded to replace the local 'DIS-ROD' (Disease Rodent) formalism with Mouse Pathology (MPATH) and Mouse Anatomy (MA) ontology terms. A pathology panel sampled histopathological slide material and compared the original diagnoses with currently accepted diagnostic criteria. RESULTS: The overall non-systematic error rate varied among the studies between 0.26% and 4.41%, the mean error being 1.71%. The errors found have been corrected and the studies thus controlled have been annotated. The majority of the original pathology terms have been successfully translated into a combination of MPATH and MA ontology terms. CONCLUSIONS: ERA has the potential of becoming a world-wide radiobiological research tool for numerous applications, such as the re-analysis of existing data with new approaches in the light of new hypotheses and techniques, and using the database as an information resource for planning future animal studies. When the database is opened for new data it may be possible to offer long-term storage of data from recent and future animal studies.

Flavopiridol, a novel cyclin-dependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis.

Flavopiridol (HMR 1275) has been identified recently as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Flavopiridol inhibits most cyclin-dependent kinases (cdks) and displays unique anticancer properties. Here, we investigated whether this compound was effective against head and neck squamous cell carcinomas (HNSCC). Exposure of HNSCC cells to flavopiridol diminished cdc2 and cdk2 activity and potently inhibited cell proliferation (IC50 43-83 nM), which was concomitant with the appearance of cells with a sub-G1 DNA content. Moreover, DNA fragmentation and TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) reaction confirmed that flavopiridol induces apoptosis in all cell lines, even on certain HNSCC cells that are insensitive to apoptosis to DNA-damaging agents (gamma-irradiation and bleomycin). A tumorigenic HNSCC cell line was used to assess the effect of flavopiridol in vivo. Treatment (5 mg/kg per day, intraperitoneally) for 5 d led to the appearance of apoptotic cells in the tumor xenografts and caused a 60-70% reduction in tumor size, which was sustained over a period of 10 wk. Flavopiridol treatment also resulted in a remarkable reduction of cyclin D1 expression in HNSCC cells and tumor xenografts. Our data indicate that flavopiridol exerts antitumor activity in HNSCC, and thus it can be considered a suitable candidate drug for testing in the treatment of refractory carcinomas of the head and neck.

Gene dosage reductions of Trf1 and/or Tin2 induce telomere DNA damage and lymphoma formation in aging mice.

This corrects the article DOI: 10.1038/leu.2015.173.

The target cell of transformation is distinct from the leukemia stem cell in murine CALM/AF10 leukemia models.

The CALM/AF10 fusion gene is found in various hematological malignancies including acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant AML model as B220+ lymphoid cells with B-cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis, we inserted the CALM/AF10 fusion gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre-induced panhematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B-lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed coexpression of the B-cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of two to three additional mutations per leukemia, including activating mutations in known oncogenes such as FLT3 and PTPN11. Our results show that the COL for CALM/AF10 leukemia is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10+ leukemia.

Prethymic adult lymphoblastic lymphoma. A clinicopathologic and immunohistochemical analysis.

The clinical, histologic, and immunohistologic features of three cases of pre-T-cell (CD7+/CD2-) lymphoblastic lymphoma in adults are reported. The patients were adults over age 50 years who had a relatively indolent nodal disease, partial involvement of lymph nodes, and primitive immunophenotype. The phenotype of the three cases was TdT+, HLA-DR+, CD34+, CD71+, CD38+, and CD7+, most resembling the normal prothymocyte, and in contrast to normal thymocytes, which generally coexpress CD1+, CD4+, and CD8+. The prethymic T-cell character was further supported by germline T-cell receptor beta and gamma chain genes. In contrast to most reported cases with this early immunophenotype, these patients had nodal disease but not peripheral blood involvement. Two of the three cases were associated with Langerhans' cell histiocytosis (histiocytosis X), a previously unreported association. Because of the Langerhans' cell histiocytosis and the relatively indolent clinical presentation, the differential diagnosis in all cases included both a benign process and a lower-grade lymphoma. Recognition of this unusual form of adult lymphoblastic lymphoma is essential for correct diagnosis and treatment.

Epstein-Barr virus-positive primary gastrointestinal Hodgkin's disease: association with inflammatory bowel disease and immunosuppression.

Inflammatory bowel disease (IBD) is associated with an increased risk of lymphoma, which is usually extraintestinal but sometimes may involve the diseased bowel itself. Most lymphomas described in this setting are of non-Hodgkin's type, but rare cases of Hodgkin's disease (HD) have been reported. We describe the clinicopathologic and molecular features of four patients with primary gastrointestinal HD. Three patients had preexistent Crohn's disease (CD), for which two of them had received immunosuppressive therapy. The fourth patient had a longstanding history of diverticulitis and myasthenia gravis and was receiving immunosuppressive therapy for the latter. Multifocal involvement of the bowel by HD was noted in all four cases. Disease was staged as IVA in one patient, IIIB in one patient, and IE in one patient, and the fourth patient died in the postoperative period before further workup. Two patients received chemotherapy, one of whom was dead at 9 months, whereas the other has no evidence of disease at 25 months' follow-up. The patient with IE disease did not receive any therapy because only a few microscopic foci of disease were present and is also without any evidence of disease at 17 months. The Reed-Sternberg (RS) cells in all four cases expressed CD30, CD15, EBER-1, and LMP-1; two of four were focally CD20-positive. VJ-polymerase chain reaction for immunoglobulin heavy chain (IgH) rearrangement showed a polyclonal pattern in all four cases. In two cases, laser capture microdissection was used to isolate individual RS and Hodgkin's cells, which contained rearranged immunoglobulin genes, confirming a B-cell genotype. Whereas one case showed a dominant clonal band present in all isolates, cells from the patient with stage IE disease clearly showed a polyclonal population of RS cells. Our findings indicate that HD arising in the setting of IBD or chronic inflammation is the result of an Epstein-Barr virus-driven lymphoproliferation, analogous to that found in other immunodeficient states. Disordered immunoregulation inherent to CD and immunosuppressive therapy for the latter may contribute to its development. The finding of polyclonal RS cells in a patient with early stage disease and apparent cure by surgical resection versus monoclonal RS cells in the patient with disseminated disease suggests that HD in the setting of immunodeficiency also may show molecular progression, in a manner similar to that occurring in conventional B-cell lymphoproliferative disorders arising in the same setting.

Peripheral T-cell lymphoma with Reed-Sternberg-like cells of B-cell phenotype and genotype associated with Epstein-Barr virus infection.

We report three cases of nodal peripheral T-cell lymphoma (PTCL) with Reed-Sternberg-like (RS-like) cells of B-cell pheno- and/or genotype. Histologic analysis in all cases revealed diffuse nodal effacement by atypical lymphoid cells of variable size. Two of the three cases had features of angioimmunoblastic T-cell lymphoma (AILT). Large mononuclear and binucleated cells with prominent eosinophilic nucleoli and abundant cytoplasm resembling classic RS cells and mononuclear variants were scattered throughout all biopsies. The lymphoma cells in the three cases were of T-cell lineage (CD3+, CD43+, and CD45RO+). The RS-like cells from all cases were CD30 and CD15 positive. In contrast to the neoplastic T cells, the RS-like cells lacked all T-cell markers and in two cases were positive for CD20. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) and EBER 1 (2/2) were detected in the RS-like cells in all cases. The neoplastic T cells were negative for EBV. Polymerase chain reaction (PCR) analysis demonstrated clonal rearrangements of the T-cell receptor gamma chain gene in the three cases. PCR analysis of microdissected RS-like cells for immunoglobulin heavy chain gene rearrangements in cases 1 and 3 showed an oligoclonal pattern. The presence of RS-like cells in PTCL represents a diagnostic pitfall, because in one case this observation led to a misdiagnosis of Hodgkin's disease (HD). The oligoclonal expansion of EBV-infected cells may be related to underlying immunodeficiency associated with T-cell lymphomas and AILT in particular. This phenomenon may provide the basis for some cases of Hodgkin's disease after T-cell lymphomas and suggests that they are clonally unrelated neoplasms. The expression of LMP1 appears to be crucial for the immunophenotype and probably for the morphology of the RS and RS-like cells appearing in diverse lymphoid malignancies, including HD, chronic lymphocytic leukemia, and PTCL.

Cell kinetic study of thymic epithelial tumors using PCNA (PC10) and Ki-67 (MIB-1) antibodies.

We performed an immunohistochemical cell kinetic study with monoclonal antibodies to proliferating cell nuclear antigen (PCNA)-PC10-and Ki-67-MIB-1-on 62 thymic epithelial tumors, to evaluate whether there is correlation between the proliferation indices of the neoplastic epithelial cells and histological subtype, stage, and risk of relapse. The 62 cases of thymic epithelial tumors were classified as medullary thymoma (4 cases), composite (mixed) thymoma (17 cases), organoid thymoma (predominantly cortical) (11 cases), cortical thymoma (10 cases), well-differentiated thymic carcinoma (18 cases), and poorly differentiated thymic carcinoma (2 cases). Labeling indices were expressed as percentage of epithelial cells with positive nuclear immunostaining by random counting of 1,000 epithelial tumor cells, using an oil immersion 100 x objective. PCNA labeling indices were consistently higher than those of Ki-67, and they correlated with each other. Well-differentiated thymic carcinoma showed higher labeling indices (3.11% +/- 3.53%) by Ki-67 antibody compared with the medullary type (0.60% +/- 0.07%) (P < .05) but there were no statistically significant differences between the other histological subtypes. Stage IV cases showed higher PCNA labeling indices (PCNA: 11.07% +/- 7.35%, Ki-67: 6.86% +/- 5.87%) than cases of the other stages (P < .05), but there were no statistically significant differences in either labeling index between the other stages. The number of patients who relapsed was too small to permit meaningful correlation between labeling indices and relapse. Our results indicate that the differences in biological behavior of the different histological subtypes of thymic epithelial tumors may be in part explained by differences in tumor growth fraction. Analysis of a larger group of patients will be required to determine whether proliferation fraction as determined by this method can be used to predict outcome in individual cases.

Thymoma--morphologic subclassification correlates with invasiveness and immunohistologic features: a study of 122 cases.

We applied the classification system of Marino and Müller-Hermelink (Virchows Arch A Pathol Anat Histopathol 407:119-149, 1985) and Kirchner and Müller-Hermelink (Prog Surg Pathol 10: 167-189, 1989) to 122 thymic epithelial tumors to evaluate the utility of this classification and to determine its correlation with invasiveness, follicular lymphoid hyperplasia, myasthenia gravis, and immunophenotype. The majority of cases could be classified according to this scheme: eight cases (7%) were medullary thymoma, 34 cases (28%) were mixed thymoma, 20 cases (16%) were predominantly cortical (organoid) thymoma, 22 cases (18%) were cortical thymoma, 32 cases (26%) were well-differentiated carcinoma (WDC), two cases (2%) were high-grade carcinoma, and four cases (3%) were unclassifiable. We report a detailed histologic analysis of these subtypes, with criteria for diagnosis. We propose that the term "organoid thymoma" be replaced by "predominantly cortical thymoma." We also observed a previously unrecognized subtype of WDC with spindle-shaped epithelial cells, which must be distinguished from medullary thymoma. There was a strong correlation between histologic subtype and invasion (P < .0001). All medullary and mixed thymomas were either completely encapsulated (64%) or had only invasion through the capsule (36%). Organoid and cortical thymomas showed an intermediate degree of invasiveness, with 36% invasive into mediastinal fat and 14% into adjacent structures. All the cases of WDC were invasive either through the capsule (19%) or into adjacent structures (81%); five cases (16%) also showed distant spread in the form of pleural seeding (four cases) or lymph node metastasis (one case). Microscopic evaluation disclosed invasion in a substantial number of cases (28.5%) thought by the surgeon to be encapsulated. The presence of follicular lymphoid hyperplasia, either within the tumor, in the residual normal thymus, or in both, was a significant predictor of myasthenia gravis (P < .0015); there was an increased association of myasthenia gravis and thymomas with cortical type epithelium (organoid thymoma, cortical thymoma, and WDC). There was no correlation between epithelial cell morphology and antigen expression; however, the immunophenotype of the associated lymphocytes corresponded closely to the morphologic classification of the thymomas. The Müller-Hermelink morphologic classification can be applied to most cases of thymoma and strongly predicts invasive behavior. The correlation of lymphocyte immunophenotype with the morphologic classification of the epithelial cells suggests that it is biologically accurate.

Human immunodeficiency virus-associated Hodgkin's disease contains latent, not replicative, Epstein-Barr virus.

Severe immunodeficiency is associated with reactivation of latent Epstein-Barr virus (EBV) that is manifested by virus replication. It is unknown whether EBV replication also occurs in the Hodgkin's disease (HD) tissue of patients infected with the human immunodeficiency virus (HIV). Therefore, we studied paraffin-embedded lymph nodes from 13 cases of HIV-associated HD to determine the latent or replicative state of EBV infection. All patients were seropositive HIV-infected men; additional clinical information was available for 12 patients. The risk factor(s) for HIV infection were homosexuality (n = 7), intravenous drug abuse (n = 2), homosexuality and intravenous drug abuse (n = 1), sexual promiscuity (n = 1), or hemophilia (n = 1). Advanced clinical stage and B symptoms were common at the time of initial diagnosis of HD. The histological subtype of Hodgkin's disease was universally mixed cellularity, except for a single case classified as nodular sclerosis. Seven cases exhibited foci of relative lymphoid depletion. Five cases contained foci of necrosis. Reed-Sternberg (RS) cells and RS cell variants were positive for CD30/BerH2 and negative for CD45/LCA, CD45RO/UCHL1, and CD20/L26 in all cases. Tumor cells were positive for CD15/LeuM1 in seven cases. In all 13 cases, RS cells and RS cell variants were infected by latent EBV as shown by in situ hybridization to EBV-encoded ribonucleic acid (EBER1). In 12 of 13 cases neoplastic cells coexpressed EBV latent membrane protein 1 (LMP1). EBV replication was examined by two different methods: immunohistochemistry to identify EBV-encoded BZLF1 protein and in situ hybridization to detect EBV BHLF1 transcripts. No positivity in RS or RS cell variants was detected with either assay of EBV replication (95% confidence interval [CI] = 0% to 23%). The findings confirm that EBV is detected more frequently in HIV-associated HD when compared with immunocompetent patients with HD. The findings also suggest that EBV is tightly latent within RS and RS cell variants of HIV-associated HD. It appears that factors other than host immune status are important in maintaining EBV latency in HIV-associated HD.

High prevalence of a 30-base pair deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 gene and of strain type B EBV in Mexican classical Hodgkin's disease and reactive lymphoid tissue.

Depending on geographic location and patient age Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV), mostly type A EBV, in 20% to 100%. The highest prevalence occurs in children of developing countries. Molecular analysis of the oncogene coding for the latent membrane protein 1 (LMP-1) revealed a 30-base pair (bp) deletion in up to 46% of EBV-positive HD. We investigated the presence of EBV in a series of Mexican classical HD (n = 57) and reactive lymphoid tissues (n = 20) from a private and a public hospital with special emphasis on the prevalence of the 30-bp deletion and the type of EBV. EBV infection was analyzed at the cellular level by Epstein-Barr encoded early RNA transcripts (EBER) in situ hybridization (ISH) and by LMP-1 protein immunohistochemistry (IHC). Molecular analysis of the LMP-1 gene configuration was performed by polymerase chain reaction (PCR) with primers spanning the site of the deletion and subsequent Southern and/or dot blot hybridization using wild-type and deletion-specific probes. The prevalence of type A and type B EBV was investigated by PCR-analysis for divergence in the coding region of Epstein-Barr nuclear antigen (EBNA)-2. EBV was detected in Hodgkin- and Reed-Sternberg cells (H-RS) by LMP-1 IHC and/or EBER ISH in 35/57 (61%) Mexican HD including 18/32 (56%) with nodular sclerosis, 15/20 (75%) with mixed cellularity and 2/4 (50%) with lymphocyte depletion. In addition, LMP-1 gene sequences were detected by PCR in 9 cases of HD without LMP/EBER expression by H-RS cells and in 17/20 (85%) reactive lymph nodes, supposedly originating from rare latently infected B cells. Surprisingly, the 30-bp LMP-1 deletion was found in 28/35 (80%) EBV-positive HD. This deletion, however, was also found in all 9 (100%) HD with H-RS cells negative for EBV and in 10/17 (59%) reactive lymph nodes. Thus, the overall LMP-1 del prevalence in reactive tissue is 73% (19/26). Typing of EBV was successful in 26 cases of EBV-positive HD, 10 of these were infected by type B EBV (38%). Of the reactive lymphoid tissue, 9 (47%) were infected by type A, and 10 (53%) by type B; All 20 cases (100%) associated with type B, whether neoplastic or reactive, displayed the LMP-1 del variant compared with 18/25 (72%) infected by type A EBV. To our knowledge, this is the highest incidence for both the LMP-1 deletion variant and the infection by type B EBV in HD reported so far worldwide. Our data suggest that EBV infection contributes to the pathogenesis of the majority of Hodgkin's disease cases in Mexico. The specific tumorigenic role of the LMP-1 deletion variant, however, is doubtful with regard to its high frequency in nonneoplastic lesions. Moreover, type B infection frequently occurs in Mexican HD and reactive lymphoid tissue and is consistently associated with the deletion variant pointing to a pathogenetic role of this combined genotype.