Clonal X-chromosome inactivation suggests that splenic cord capillary hemangioma is a true neoplasm and not a subtype of splenic hamartoma.

Splenic hamartoma is a rare tumor-like lesion composed of structurally disorganized red pulp elements. It has been hypothesized that two other splenic lesions, cord capillary hemangioma and myoid angioendothelioma, may fall within the spectrum of splenic hamartoma, simply representing morphological variants. In this study, we compared the vascular and stromal composition of cord capillary hemangioma and myoid angioendothelioma with those of classical hamartoma. In addition, we assessed the clonal vs polyclonal nature of the lesions in nine female cases by performing clonality analysis for X-chromosome inactivation at the human androgen receptor locus (HUMARA) on laser-assisted microdissected samples. In 15 of 17 cases, increased reticulin and/or collagen content was observed. The classical hamartoma cases showed a vasculature predominantly composed of CD8+ CD31+ CD34- splenic sinuses, whereas cases of cord capillary hemangioma and myoid angioendothelioma contained many CD8- CD31+ CD34+ cord capillaries, but very little CD8+ vasculature. All cases lacked expression of D2-40 and Epstein Barr virus-encoded RNA. All cases showed a proliferation index of ≤5% by Ki-67. Cases of classical hamartoma lacked significant perisinusoidal expression of collagen IV and low-affinity nerve growth factor receptor. Both markers were variably expressed in the other lesions. Increased CD163-positive histiocytes were found in four cases (three cord capillary hemangiomas and one myoid angioendothelioma). HUMARA analysis was informative in all nine tested cases, of which three cases showed a non-random X-chromosome inactivation pattern, indicating clonality. All three clonal cases were cord capillary hemangiomas. Our study has shown that in spite of considerable morphologic heterogeneity and overlapping features, classical hamartoma and cord capillary hemangioma and myoid angioendothelioma are different in terms of their vascular and stromal composition. Clonality analysis supports a true neoplastic origin for the cord capillary hemangioma. A larger study using additional immunohistochemical and molecular studies is necessary to further evaluate the biological significance of the current findings.

Immunophenotypic analysis of the Kaposi sarcoma herpesvirus (KSHV; HHV-8)-infected B cells in HIV+ multicentric Castleman disease (MCD).

AIMS: Kaposi sarcoma herpesvirus (KSHV) is aetiologically related to Kaposi sarcoma, classical and extracavitary primary effusion lymphoma (PEL; EC-PEL) and multicentric Castleman disease (MCD), entities preferentially occurring in HIV-infected individuals. Characterization of HIV-associated PELs/EC-PELs suggests that the KSHV-infected malignant cells originate from a pre-terminal stage of B-cell differentiation. However, only limited phenotypic studies have been performed on HIV+ MCD, including for PR domain containing 1 with zinc finger domain/B lymphocyte-induced maturation protein 1 (PRDM1/BLIMP1), a key regulator of terminal B-cell differentiation. The aim was to characterize KSHV-infected cells in 17 cases of HIV+ MCD. METHODS AND RESULTS: Double immunohistochemistry and immunohistochemistry-in situ hybridization were used to characterize the KSHV-infected cells in MCD; the results were compared with the phenotypic profiles of 39 PELs/EC-PELs and seven PEL cell lines. Whereas the immunophenotype of KSHV-infected cells in MCD and malignant KSHV+ PEL cells was similar (PAX5, Bcl-6-; PRDM1/BLIMP1, IRF4/MUM1+; Ki67+), the MCD KSHV-infected cells differed, as they expressed OCT2, cytoplasmic lambda immunoglobulin; variably expressed CD27; lacked CD138; and were Epstein-Barr virus negative. CONCLUSIONS: Although both PEL and MCD originate from KSHV-infected pre-terminally differentiated B cells, these findings, with previously reported genetic studies, indicate HIV+ MCD may arise from extrafollicular B cells, whereas PELs may originate from cells that have traversed the germinal centre.

Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration.

Emerging data suggest that VEGF receptors are expressed by endothelial cells as well as hematopoietic stem cells. Therefore, we hypothesized that functional VEGF receptors may also be expressed in malignant counterparts of hematopoietic stem cells such as leukemias. We demonstrate that certain leukemias not only produce VEGF but also express functional VEGFR-2 in vivo and in vitro, resulting in the generation of an autocrine loop that may support leukemic cell survival and proliferation. Approximately 50% of freshly isolated leukemias expressed mRNA and protein for VEGFR-2. VEGF(165) induced phosphorylation of VEGFR-2 and increased proliferation of leukemic cells, demonstrating these receptors were functional. VEGF(165) also induced the expression of MMP-9 by leukemic cells and promoted their migration through reconstituted basement membrane. The neutralizing mAb IMC-1C11, specific to human VEGFR-2, inhibited leukemic cell survival in vitro and blocked VEGF(165)-mediated proliferation of leukemic cells and VEGF-induced leukemic cell migration. Xenotransplantation of primary leukemias and leukemic cell lines into immunocompromised nonobese diabetic mice resulted in significant elevation of human, but not murine, VEGF in plasma and death of inoculated mice within 3 weeks. Injection of IMC-1C11 inhibited proliferation of xenotransplanted human leukemias and significantly increased the survival of inoculated mice. Interruption of signaling by VEGFRs, particularly VEGFR-2, may provide a novel strategy for inhibiting leukemic cell proliferation.

Competitor mRNA fragments for quantitation of cytokine specific transcripts in cell lysates.

Synthetic RNAs (sRNAs) specific for four human cytokines were constructed and used as an exogenous internal standard in a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The sequences of the sRNA and the target mRNA were identical except for a duplication or deletion of approximately 100 nucleotides. The size difference between these two templates permitted easy electrophoretic separation of their PCR products. The sRNA has polyadenylated sequences at the 3' end and can be added directly either to a cell lysate before RNA purification or to a reverse transcription reaction. One pair of primers is used to amplify the internal standard and the target simultaneously, and the ratio of the two PCR products remains constant throughout the amplification. This technique can be applied to quantitate specific mRNA in as few as 10 cells when the exogenous control is added directly to cell lysates. This method is sensitive, accurate and adaptable for quantitation of other transcripts.

ICSH guidelines for the standardization of bone marrow immunohistochemistry.

Bone marrow (BM) tissue biopsy evaluation, including trephine biopsy and clot section, is an integral part of BM investigation and is often followed by ancillary studies, in particular immunohistochemistry (IHC). IHC provides in situ coupling of morphological assessment and immunophenotype. The number of different IHC tests that can be applied to BM trephine biopsies and the number of indications for IHC testing is increasing concurrently with the development of flow cytometry and molecular diagnostic methods. An international Working Party for the Standardization of Bone Marrow IHC was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines for the standardization of BM IHC based on currently available published evidence and modern understanding of quality assurance principles as applied to IHC in general. The guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus and represent further development of the previously published ICSH guidelines for the standardization of BM specimens handling and reports.

Cytokeratin-positive large-cell lymphomas of B-cell lineage. A study of five phenotypically unusual cases verified by polymerase chain reaction.

Five cases of clinically aggressive, keratin-positive malignant lymphomas of B-cell type with unusual immunophenotypes were studied. All cases were extranodal: two from the stomach, one from soft tissue, one from the skin, and one from the spleen. These tumors were undifferentiated large-cell neoplasms that showed reactivity for low-molecular-weight keratin 8, but they were negative for keratin 19; three cases were also positive for epithelial membrane antigen. The immunohistochemical diagnosis was complicated by the fact that two of these cases lacked reactivity for leukocyte common antigen and three were CD20 negative. These findings simulated the immunophenotype of a carcinoma and led to an initial misdiagnosis of carcinoma. Although only two cases showed immunohistochemical evidence of B-cell lineage (CD20+), all five cases were documented as B-cell lymphomas on the basis of the clonal immunoglobulin heavychain gene rearrangement, as demonstrated by polymerase chain reaction (PCR) in all the cases and by Southern blot hybridization in three cases; all cases were negative for T-cell markers, and three cases showed germline configuration for T-cell receptor beta-chain. One case was strongly CD30 positive and represented large-cell anaplastic lymphoma of B-cell type. Our results show that some B-cell lymphomas can have unusual and confusing immunophenotypes, including keratin positivity and leukocyte antigen negativity. Use of PCR-based molecular genetic demonstration of clonal immunoglobulin heavychain gene rearrangement is helpful in establishing the correct diagnosis in such cases.

Double immunolabeling with cytokeratin and smooth-muscle actin in confirming early invasive carcinoma of breast.

Histopathological identification of invasive breast carcinoma in its earliest phases is fraught with pitfalls. Preinvasive malignant lesions complicated by radial scar, sclerosing adenosis, and lobular cancerization, among other lesions, may simulate invasive carcinoma. Fibrosis, inflammatory reaction, and other stromal changes around in situ carcinoma may mask microinvasive foci on routine stains. Conventional immunohistochemistry to demonstrate basement membrane or myoepithelial cell layer may not, by itself, be unequivocally diagnostic of invasion. We performed a novel double immunoenzyme labeling technique using an avidin-biotin complex peroxidase-diaminobenzidine system for smooth-muscle actin followed by an alkaline phosphatase anti-alkaline phosphatase-new fuchsin system for cytokeratin antigen on formalin-fixed, paraffin-embedded histology sections to evaluate 32 such problematic cases. The initial histologic impression with hematoxylin and eosin staining alone was as follows-first group: microinvasive carcinoma-10; second group: carcinoma in situ--"stromal invasion cannot be ruled out"--15; third group: frankly infiltrating carcinoma of various grades and morphologic types-6. The last group served as positive control for invasion. One fibroadenoma with fine-needle-aspiration-induced artifact simulating stromal invasion was also included. The double immunoenzyme labeling technique imparted a dark brown color to the myoepithelial cells and a vivid red color to the epithelial cells, making individual or loosely cohesive groups of malignant epithelial cells infiltrating the stroma easily detectable, whereas their in situ counterparts were contained within dark brown myoepithelial boundaries. The TNM 1997 definition of pT1mic, i.e., extension of malignant cells in the stroma with no focus measuring >0.1 cm, was followed to classify microinvasion. In the first group, microinvasion was confirmed in six cases but was not demonstrable in four. In the second group, definite invasion was identified in five cases, ruled out in nine, and in one case the suspicion of early invasion could not be entirely ruled out even after double immunoenzyme labeling. Thus, it was possible to render a definite opinion regarding presence or absence of invasion in 24 of 25 (96%) cases diagnosed as or suspected to be microinvasive. The precise and simultaneous elucidation of topography between malignant cells and myoepithelial cells on a single permanent section makes this technique a useful diagnostic tool in the evaluation of those cases of breast carcinoma that exhibit equivocal invasion.

Primary B cell lymphoma of the thyroid and its relationship to Hashimoto's thyroiditis.

Twenty-one cases were selected from 236 thyroidectomies with a diagnosis of Hashimoto's disease for detailed clinicopathologic study on the basis of "early" changes in three cases and an unusually heavy lymphoplasmacytic infiltrate in 18 cases. These cases were studied in conjunction with ten cases of high-grade non-Hodgkin's lymphoma of the thyroid. Immunoglobulin light chain restriction was demonstrated in five cases of Hashimoto's thyroiditis and the diagnosis was accordingly changed to low-grade lymphoma. All ten high-grade lymphoma cases were of B phenotype and light chain restriction could be demonstrated in eight of them. The study revealed close homology between the lymphoplasmacytic infiltrate in Hashimoto's thyroiditis and normal mucosa-associated lymphoid tissue (MALT). Like lymphomas of mucosal sites, thyroid lymphoma appears to be derived from the parafollicular ("centrocyte-like") B cells. The high-grade thyroid lymphomas appear to be derived from low-grade tumors. There were close histologic, immunohistologic, and clinical similarities between low- and high-grade non-Hodgkin's lymphomas of the thyroid and those appearing in mucosal sites. This study confirms the close association between Hashimoto's thyroiditis and B cell lymphoma of the thyroid gland and suggests that this tumor belongs to the group of non-Hodgkin's lymphomas derived from MALT.

Primary B-cell lymphoma of salivary glands and its relationship to myoepithelial sialadenitis.

A detailed morphologic and immunohistochemical study has been carried out on salivary glands excised from 20 cases in which the initial histologic diagnosis was either myoepithelial sialadenitis (MESA) or salivary gland lymphoma (SGL). The results have shown that these cases, except one that had been diagnosed as MESA, showed a spectrum of changes ranging from focal lymphoid infiltrates, designated as early MESA, through established MESA with dense, extensive lymphoid infiltration, to lymphoma. The distribution of the lymphoid infiltrate in early MESA was related to ducts and mimicked Peyers patches. In established MESA, this infiltrate became confluent with the formation of prominent epimyoepithelial islands. The evolution of lymphoma was characterized by an expanded population of centrocyte-like (CCL) cells that showed light chain restriction. Like other lymphomas of mucosa-associated lymphoid tissue, to which they bear a striking resemblance, salivary gland lymphomas may remain localized for prolonged periods with a tendency to local recurrence rather than to distant spread. These properties may be explained by the histogenesis of these tumors from CCL cells that appear to be of similar lineage of splenic marginal zone cells.

[Intermediate lymphocytic lymphoma]. / Chloniaki o posrednim stopniu zróznicowania (Intermediate Lymphocytic Lymphoma, ILL).

The characteristic features are presented of intermediate lymphocytic lymphomas which have suggested the pathomorphological and clinical peculiarity of this disease entity. They occur in two forms: diffuse and nodular which are two phases in the development of the disease. The phenotypic and karyotypic characteristics of these lymphomas resemble those of chronic lymphocytic leukaemia, but their more malignant course is similar to that of centrocytic lymphomas.

Call for a European programme in external quality assurance for bone marrow immunohistochemistry; report of a European Bone Marrow Working Group pilot study.

BACKGROUND AND AIMS: In diagnostic immunohistochemistry (IHC), daily quality control/quality assurance measures (QC/QA) and participation in external quality assurance programmes (EQA) are important in ensuring good laboratory practice and patient care. Bone marrow trephine biopsies (BMTB) have been generally excluded from EQA programmes for diagnostic IHC due to a lack of standards for tissue processing. The European Bone Marrow Working Group (EBMWG) has set up an EBMWG IHC Committee with the task of exploring the plausibility of an EQA programme for BMTB IHC in Europe. METHODS: 28 laboratories participated in a web-based anonymous survey; 19 laboratories submitted a total of 109 slides stained for CD34, CD117, CD20, CD3, Ki-67 and a megakaryocyte marker of choice. RESULTS: Eight different fixatives and nine different decalcification methods were used. While 93% of participants believed that they produced excellent results in BMTB IHC, only 4/19 (21%) laboratories did not have any poor results. CD117 and Ki-67, with 53% and 50% poor results, respectively, were the most problematic immunostains, while CD20 was the least problematic, with only 11% poor results. CONCLUSIONS: The EBMWG IHC Committee calls for a reduction in the tissue processing methods for BMTB and establishment of an EQA programme for BMTB IHC to help diagnostic IHC laboratories calibrate their tests according to expert recommendations. This is especially necessary in the light of recent introduction of predictive IHC tests in BMTB.

Pseudolymphomas of the stomach: a lesion characterized by progressively transformed germinal centres.

Pseudolymphomas of the stomach are frequently associated with chronic peptic ulcer. Out of the 11 cases studied, there were characteristic pathohistological signs of chronic ulceration in eight and suspicion of chronic ulcer in three. Thus it seems that pseudolymphomas frequently represent an exaggerated lymphocytic reaction to chronic ulcer. The pseudolymphomatous reaction is essentially a follicular hyperplasia with the gradual development of progressively transformed germinal centres. These large nodular structures are usually found in the submucosa and, at first inspection, the transformed follicle can easily be mistaken for a possibly malignant diffuse lymphomatous lesion. However, once the existence of the condition is realized, its recognition by the pathologist should be easy. A heavy plasma cell infiltration of polytypic immunoglobulin type associated with progressively transformed germinal centres might be a consequence of long-standing antigenic stimulation.

[Etiopathogenesis of Hodgkin's disease in the light of current studies]. / Etiopatogeneza ziarnicy zlosliwej w swietle wspólczesnych badan.

Several problems concerning the etiology and pathogenesis of malignant granulomatosis have been presented with particular attention paid to immune disturbances in the course of this disease. Some modern theories of the origin of Reed-Sternberg neoplastic cells have been also discussed.

Primary pulmonary lymphoma: a re-appraisal of its histogenesis and its relationship to pseudolymphoma and lymphoid interstitial pneumonia.

The clinical, morphological and immunohistochemical features of 15 cases of pulmonary lymphoproliferative disease are described. The diagnosis of primary pulmonary lymphoma was based in 13 cases on the demonstration of light chain restriction and in two cases on morphological characteristics. Many patients had a prolonged clinical course without significant clinical or radiographic deterioration, a feature associated with malignant lymphomas of mucosa-associated lymphoid tissue in other sites. Lymphoepithelial lesions were characteristic and malignant cells had the features of centrocyte-like cells, similar to those described in gastric and salivary gland lymphomas. Germinal centres were present in three cases: some were partially overgrown by centrocyte-like cells but residual polyclonal follicle centre cells and dendritic reticulum cells were still detectable. It is suggested that primary pulmonary lymphoma arises from centrocyte-like cells normally present in bronchus-associated lymphoid tissue. In addition to the malignant population, reactive follicles and polytypic plasma cells are frequently present and may prejudice interpretation of immunohistochemical features. In the light of these findings, cases previously diagnosed as pseudolymphoma or lymphoid interstitial pneumonia require careful assessment and the majority are, in reality, examples of primary pulmonary lymphomas.

Analysis of gamma delta+ T cells in peripheral blood of children with perinatal human immunodeficiency virus (HIV) infection.

The present study examined CD8 antigen expression and variable (V) gene segment usage by T cell receptor (TCR)-gamma delta+ lymphocytes in peripheral blood of symptomatic children with perinatal HIV infection. The relative number of gamma delta+, CD8+ T cells in most of the infected children was higher than that in uninfected children from HIV+ or HIV- mothers and correlated with the immunodeficiency status of the patients. Infected infants and children over 1 year old also showed an increased proportion of V delta 1-J delta 1+ T lymphocytes. CD8 expression on those cells was higher in infected than in uninfected infants and children. Sequence analysis of the delta gene rearrangement of the predominant V delta 1 family in peripheral blood of three HIV+ donors revealed extensive junctional diversity. These results suggest that the V delta skewing in the majority of HIV+ children reflects peripheral expansion of V delta 1-J delta 1+ T lymphocytes early in life, which might be involved in the mechanisms of HIV-induced immunodeficiency.

BCL-6 protein is expressed in precursor T-cell lymphoblastic lymphoma and in prenatal and postnatal thymus.

The organization and expression of the BCL-6 gene in normal and neoplastic thymic T cells has not been fully determined. We examined 8 precursor T-cell lymphoblastic lymphomas (T-LBLs) and found significant BCL-6 expression in 4 cases. Three of the BCL-6(+) cases expressed a common thymocyte phenotype (CD4(+), CD8(+)), and one expressed a precursor thymocyte phenotype (CD4(-), CD8(-)). In 6 cases evaluated, including those expressing BCL-6, molecular analyses demonstrated a germline configuration of the BCL-6 gene and a wild-type BCL-6 gene first exon-intron boundary region. We also evaluated 12 normal prenatal and postnatal thymuses for BCL-6 protein. BCL-6 was expressed by most cortical thymocytes and by scattered medullary thymocytes. BCL-6(+) cortical and medullary thymocytes also expressed CD2, CD3, CD4, CD5, CD7, or CD8. We further analyzed the pattern of BCL-2 and BCL-X(L) expression and their coexpression with BCL-6 in normal thymus and T-LBL and compared it to that of follicle centers of reactive lymph nodes and follicular lymphoma. BCL-6(+) cortical thymocytes coexpressed BCL-X(L) but not BCL-2. All 4 BCL-6(+) T-LBLs and 4 BCL-6(-) T-LBLs coexpressed BCL-2 and BCL-X(L). Conceivably, T-LBLs may arise through clonal expansion of cortical thymocytes normally expressing the BCL-6 protein. The pattern of BCL-6, BCL-2, and BCL-X(L) expression in cortical thymocytes is highly reminiscent of germinal centers, and the abnormal coexpression of BCL-2, BCL-X(L), and BCL-6 in T-LBL is analogous to coexpression in follicle center cell lymphomas, suggesting that coexpression of these anti-apoptotic genes may contribute to the pathogenesis of T-LBL.

Gene transfer studies of T cell receptor-gamma delta recognition. Specificity for staphylococcal enterotoxin A is conveyed by V gamma 9 alone.

gamma delta T cells bearing the V gamma 9 gene segment have been shown to recognize staphylococcal enterotoxin A (SEA) and a range of other Ags including mycobacterial Ags. We have established an experimental system to analyze the recognition properties of human TCR-gamma delta on a molecular level by transferring the receptor from its original T cell into a Jurkat T cell host that does not express an endogenous TCR. Three groups of transfectants that express the same delta-chain, V delta 1, but different gamma-chains (V gamma 9-J2-C gamma 2, V gamma 3-J2-C gamma 2, and V gamma 9-JP-C gamma 1) together with the endogenous CD3 were obtained. The transfectant T cells each expressing different gamma delta receptors all produced IL-2 after stimulation with plastic bound anti-CD3 Ab, but only those expressing V gamma 9 responded to stimulation with SEA in the presence of an autologous lymphoblastoid B cell line. In addition, transfectants that expressed V delta 2 combined with V gamma 9 could also respond to SEA. These results indicate that the V gamma 9 portion of the receptor, independent of the J region and C region or the delta-chain, is responsible for recognizing SEA.

Diminished HIV-specific CTL activity is associated with lower type 1 and enhanced type 2 responses to HIV-specific peptides during perinatal HIV infection.

The early development of symptoms and the rapid progression of disease in some vertically infected infants are thought to reflect in part the immaturity of their immune systems. We examined the relationship between HIV-specific CTL activity and the profile of cytokine production induced by mAb to CD3 and HIV envelope (env) peptides P18 and T1 in PBMC derived from 0.6- to 3.6-yr-old children with perinatal HIV infection. Cellular immunity against HIV was demonstrated only during early stages of disease, whereas the responses were either undetectable or at background levels in HIV-infected children with rapidly progressing disease and in uninfected children of HIV+ and HIV- mothers. Levels of IL-2 mRNA in anti-CD3 mAb- and env peptide-induced PBMC varied and were increased in the infected children with high frequencies of HIV-specific CTL precursors. Analysis of IFN-gamma and IL-4 production by CD4+ T cell clones obtained from cultures stimulated with anti-CD3 mAb or the env peptides showed an increased proportion of Th2 and Th0 clones in HIV-infected children with lower HIV-specific CTL activity, whereas children with high CTL activity had increased numbers of Th1 clones. The results of these studies suggest that decreases in CTL activity to the virus might be associated with the induction of a type 2 cytokine response. These findings underline the role of cytokines in the generation of HIV-specific CTL responses and may be important for the development of immunomodulatory and vaccine strategies to interrupt vertical transmission of HIV.

Transformation of primary human endothelial cells by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is invariably present in Kaposi's sarcoma lesions. KSHV contains several viral oncogenes and serological evidence suggests that KSHV infection is necessary for the development of Kaposi's sarcoma, but cellular transformation by this virus has not so far been demonstrated. KSHV is found in the microvascular endothelial cells in Kaposi's sarcoma lesions and in the spindle 'tumour' cells, which are also thought to be of endothelial origin. Here we investigate the biological consequences of infecting human primary endothelial cells with purified KSHV particles. We find that infection causes long-term proliferation and survival of these cells, which are associated with the acquisition of telomerase activity and anchorage-independent growth. KSHV was present in only a subset of cells, and paracrine mechanisms were found to be responsible for the survival of uninfected cells. Their survival may have been mediated by upregulation of a receptor for vascular endothelial growth factor. Our results indicate that transformation of endothelial cells by KSHV, as well as paracrine mechanisms that are induced by this virus, may be critical in the pathogenesis of Kaposi's sarcoma.