Anomolous staining patterns in immunohistologic studies of malignant lymphoma.

A number of immunoperoxidase studies of malignant lymphomas have reported polytypic light chain staining of neoplastic cells, thus bringing into question the concept that the monoclonality of B cell lymphomas is reflected in their synthesis of monotypic light chain. In this study of a large number of Ig positive lymphomas, staining for a wide variety of antigens has identified clear differences between monotypic Ig synthesizing cells and cells staining polytypically which appear to be taking up Ig from the environment. Attention to the nature of Ig staining and staining for J chain were the two most useful criteria in differentiating Ig synthesis from uptake. The results confirm that malignant B cell lymphomas synthesise monotypic Ig.     

Immunocytochemical methods in the study of lymphoma and related conditions.

The diagnosis and classification of malignant lymphomas is based upon traditional histological criteria evolved over the years by successive generations of pathologists. Immunoperoxidase methods applied to formalin paraffin sections have permitted a direct correlation of these established morphologic criteria with newer immunological concepts of the form and function of the B lymphocyte and its derivatives. Study of material from 500 cases of Hodgkin's disease, non-Hodgkin lymphomas, and myeloma has revealed that many of these conditions may find a common origin in the lymphocyte, and that some malignant cells, previously identified as malignant histiocytes or reticulum cells are rather related to or derived from the transformed lymphocyte or immunoblast. The corresponding tumors are thus more logically designated immunoblastic sarcoma. The study also reveals a claser developmental relationship between multiple myeloma and immunoblastic sarcoma than previously suspected, and suggests that all of the B cell lymphomas occur as part of a continuous spectrum of disease, rather than as separate entities as implied by current histological classifications. The study of immunoglobulin in formalin paraffin sections is illustrative of the great potential of this method in the diagnosis and study of neoplasia in general.     

Cytochemical examination of acid phosphatase and beta-glucuronidase enzymes in low-grade B cell non-Hodgkin's lymphomas

The differential diagnostic significance of acid phosphatase and beta-glucuronidase were studied in 77 cases of low-grade B cell non-Hodgkin's lymphomas. In most cases the results of cytochemical enzyme studies performed on malignant cells of the bone marrow were evaluated. B cell chronic lymphocytic leukaemia, centrocytic and centroblastic/centrocytic lymphomas were characterized by a weak or a negative acid phosphatase and beta-glucuronidase activity. Stronger positivity was observed in immunocytoma and in Waldenstr?m's macroglobulinaemia, while the highest activity was found in multiple myeloma. Hairy cell leukaemia of B cell origin showed intensive tartrate-resistant acid phosphatase activity. The cytochemical examination of these lysosomal enzymes may be useful in the diagnosis of low-grade malignant lymphomas of B cell origin by completing other methods.     

The pathogenesis of diabetes mellitus. Possible usefulness of spontaneous hyperglycemic syndromes in animals.

Systematic studies of the patterns of anatomic distribution, pathways of probable spread, and prognosis of the malignant lymphomas have been greatly aided by the development of new histopathologic classifications and the introduction of more sophisticated and precise diagnostic techniques, such as lymphangiography and laparotomy with splenectomy and retroperitoneal node biopsy. Concomitantly, megavoltage radiotherapy apparatus has made total-lymphoid radiotherapy feasible and practical, and the availability of a widening spectrum of chemotherapeutic agents has ushered in a new era of combination chemotherapy. Collectively, these diagnostic and therapeutic advances have already begun to yield a dramatic improvement in the prognosis of Hodgkin''s disease and the other malignant lymphomas.     

Isolation and dissociation of immune complexes from pleural effusions of lymphoma patients

Summary Immune complexes (IC) isolated from pleural effusions of lymphomas with favorable and unfavorable prognoses were of IgG type. These IC were further dissociated by ion exchange chromatography using 8 M urea. The antibody was found to be a high molecular weight protein (1.5×10⁵ daltons) and reacted with antihuman IgG immunologically while a second peak obtained on ion exchange chromatography may be an antigen moiety with a molecular weight of 3.2×10⁴ daltons as it reacted immunologically with the antibody. Strong cytoplasmic fluorescence was observed with various cell suspensions of lymphomas when reacted with the antibody preparations. The antisera raised against two different antigen fractions prepared from two lymphomas — nHL and LL showed positive fluorescence with both nHL and LL suspensions. The absorption of these rabbit antibodies with individual cell extracts or with antigen preparations also entirely blocked the cytoplasmic staining. The antigen moiety (PK-II) may have a common origin in the disease process. Pleural effusions from patients with unfavorable and favorable prognoses showed identical patterns of separation of IC components.     

Morphometric characterization of nuclei in non-Hodgkin's malignant lymphoma

In addition to the nuclear area and a form factor, four morphometric parameters of nuclear shape (ID, R1, R2 and ND), obtained by the application of the principles of mathematical morphology, were used to characterize the nuclear contours in non-Hodgkin's malignant lymphomas. The values for each parameter were determined in 58 cases of non-Hodgkin's lymphoma categorized according to the Kiel and National Cancer Institute classifications. Small-cell, mixed and large-cell lymphomas could be distinguished on the basis of the mean nuclear area. The shape parameters R1, R2 and ID were efficient discriminators of the large centrocytic (cleaved-cell) lymphomas. Neither size nor shape factors could distinguish between centroblastic and immunoblastic tumors. The good correlation between the morphometric findings and the histopathologic categories suggest that morphometry may provide a quantitative and objective method for grading lymphomas.     

Primary cervical lymphoma: the role of cervical cytology

Two cases of primary malignant lymphoma of the uterine cervix are reported. Both were confirmed by histology as high grade B cell lymphomas. In one case, the diagnosis was made on a second colposcopic biopsy after an initial cervical smear and colposcopic biopsy were negative. In the second case, dyskaryotic cells of uncertain type were identified in a cervical smear taken at colposcopy performed as part of follow up for previous cervical intraepithelial neoplasia (CIN)I. The cytologic features and differential diagnosis of this rare cervical neoplasm are discussed, with emphasis on the role of the Papanicolaou smear in the initial diagnosis of this tumour.     

Flow cytometry as an accurate tool to complement fine needle aspiration cytology in the diagnosis of low grade malignant lymphomas

S. Schmid, M. Tinguely, P. Cione, H. Moch and B. Bode
Flow cytometry as an accurate tool to complement fine needle aspiration cytology in the diagnosis of low grade malignant lymphomas Objective: Diagnosis of low grade non‐Hodgkin B‐cell lymphomas on cytological material may be problematic and in the past frequently required lymph node excision. We analysed our experience of the value of flow cytometry (FC) as an additional tool for the diagnosis of lymphoproliferative processes in the setting of a university cytology division with a busy fine needle cytology service. Methods: Consecutive cytological specimens with FC over a period of 3 years were retrospectively analysed and correlated with histology and follow‐up if available. FC was performed with the following antibodies: CD3, CD4, CD8, CD2, CD7, CD19, CD5, CD10, CD23, lambda and kappa chains. Results: Of 299 probes (273 fine needle aspirations and 26 fluids from 285 patients), 179 cases (60%) were diagnosed as reactive, 91 cases (30%) as malignant or suspicious and 29 cases (10%) as inconclusive. The results of histological examination of the lymph nodes were available in 41 of 91 (45%) malignant or suspicious cases and in 13 of 179 (7%) reactive cytological diagnoses. Cytologically diagnosed malignancy was confirmed in all histologically examined cases. In 12 of 13 reactive cytological cases (92%), a benign process was diagnosed histologically. In 34 of 299 cases (11%) additional molecular investigations of B‐cell clonality or specific translocations were performed. The lymphomas most frequently diagnosed were follicular lymphoma and lymphocytic lymphoma, followed by mantle cell and marginal zone lymphomas. Correlation with histology showed a sensitivity of 98% and a specificity of 100% for cytology in our series. Conclusions: FC is an important additional tool in the cytological diagnosis of lymphoproliferative disorders. The combined approach has a high diagnostic value that allows a reliable subclassification of low grade B‐cell non‐Hodgkin lymphomas.     

Algorithm for a DNA-cytophotometric diagnosis and grading of malignancy

An algorithm for processing data on nuclear DNA content obtained cytophotometrically was developed (1) to obtain an objective discrimination between benign and malignant lesions in conventional cytologic smears secondarily stained according to Feulgen and (2) to obtain an objective degree of tumor malignancy on a continuous scale of malignancy grades. Investigations in 258 malignant tumors (95 malignant lymphomas, 52 uterine cervix carcinomas, 28 prostate carcinomas, 18 breast carcinomas, 45 malignant bone tumors and 19 larynx carcinomas) and in 74 benign lesions in these organs yielded a diagnostic accuracy of no false-positive, no false-negative and 21% suspicious diagnoses. The probability that "suspicious" cases were malignant was 81%. The overall diagnostic accuracy for non-negative cases thus amounted to 100%. Results in 95 patients with different malignant lymphomas and in 16 patients with squamous-cell carcinoma of the larynx demonstrated the prognostic validity of the DNA-grading system.     

Cytodiagnosis of malignant non-Hodgkin's lymphomas in effusions (author's transl)

Body cavity effusions in malignant non-Hodgkin's lymphomas, particularly in the early stages of those neoplasms, are rare in comparison to the far more common effusions in other malignant diseases and in inflammatory processes. Therefore, the cytological differential diagnosis is of great importance. Of 7,000 pleural and 1,700 ascitic effusions, only 42 cases were malignant non-Hodgkin's lymphoma and in 30 lymphoma was suspected. When lymphoma is suspected, particularly low-grade lymphoma, there are great difficulties in making a differential diagnosis. Using the more or less typical cellular and nuclear criteria of the various lymphoma types, an attempt was made to classify the unequivocal lymphomas according to the Kiel classification of malignant non-Hodgkin's lymphomas. In principle these criteria are the same in cytological and histological examinations. In 31 cases the lymphoma subtype could be specified and confirmed in part by subsequent histological examination. Apart from the suspect lymphoma cases (40%), cases of a low grade of malignancy were predominantly observed (28%). Lymphomas with a high grade of malignancy were less frequently encountered (15%), a proportion similar to that of unclassifiable lymphomas (16%). Apparently the cytological and karyological criteria are not yet adequate to classify lymphomas from conventionally prepared cytological specimens with a higher degree of accuracy.     

Lymphoma associated antigen (LAA)--a unique biomarker for lymphomas.

A lymphoma associated antigen (LAA) isolated from pooled lymph nodes of confirmed Hodgkin's and non-Hodgkin's lymphomas has been purified and characterized. Using a xenogenic rabbit anti-serum, enzyme-linked immunosorbent assay (ELISA) and RIA were developed for LAA. LAA was detected in the sera of all confirmed lymphomas, the test being negative for normals, for patients with benign lymphadenitis and various other types of cancers. Except for a very few false positive results, no false negative was observed. LAA was identified in urine, CSF, saliva and gastric juice of a few lymphoma patients, and the test proved to be of diagnostic potential, as for a few patients it had a lead time of a few months over the histological diagnosis. In order to render the LAA test more precise and specific, monoclonal antibodies were generated by both in vitro and in vivo immunization procedures. Seven monoclonals were generated, viz. 7D6, 7D2, 7G2, 7C5, 6G2, 23B7 and 23G11, which exhibited cytoplasmic staining of frozen sections of malignant lymphoid tissues of B cell derived non-Hodgkin's lymphomas. Two of these monoclonal antibodies, 7D6 and 23B7, revealed strong cytoplasmic staining of frozen sections, impression smears and cytospin specimens of B cell non-Hodgkin's lymphomas. The reactivity was very weak or negative for T cell lymphomas. The test was negative for Hodgkin's disease and controls. These results were confirmed by dot blotting, immunoprecipitation and immunofluorescence study. By ELISA with a sensitivity of 15 ng/ml, serum LAA levels for lymphomas were in the range 72-1250 ng/ml. LAA could not be detected in the sera of normals and controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

B cells under influence: transformation of B cells by Epstein-Barr virus

Epstein-Barr virus (EBV) is an extremely successful virus, infecting more than 90% of the human population worldwide. After primary infection, the virus persists for the life of the host, usually as a harmless passenger residing in B cells. However, EBV can transform B cells, which can result in the development of malignant lymphomas. Intriguingly, the three main types of EBV-associated B-cell lymphoma - that is, Burkitt lymphoma, Hodgkin lymphoma and post-transplant lymphomas - seem to derive from germinal-centre B cells or atypical survivors of the germinal-centre reaction in most, if not all, cases, indicating that EBV-infected germinal-centre B cells are at particular risk for malignant transformation.     

Value of demonstration of cytokeratin and leukocyte common antigen in poorly differentiated malignant tumors in fine needle aspirates

Immunoperoxidase staining for cytokeratin and leukocyte common antigen (LCA) was applied to aspirated material from 21 well-differentiated malignant tumors ("control cases," which could be cytologically categorized) and 32 poorly differentiated malignant tumors ("study cases," whose tumor type was not clearly evident from study of the aspirate). Fifteen (48%) of the 32 poorly differentiated malignant tumors were classified as carcinomas (12 cases) or lymphomas (3 cases) by positive staining for cytokeratin or LCA, respectively. Good cytohistopathologic correlation was observed in all 7 control cases and in 11 cases of the study cases for which histologic specimens were available. The addition of staining for desmin helped in categorizing one undifferentiated tumor as a rhabdomyosarcoma. Immunostaining for vimentin was not found to be valuable in the categorization of these tumors.     

Density of intranodal lymphatics and VEGF-C expression in B-cell lymphoma and reactive lymph nodes

Lymphatic vasculature in solid tumors may serve as the pathway for metastatic spread of the cancer to the regional lymph nodes and to distant organs. Controversy still exists whether tumors metastasize through existing lymphatics or through newly formed vessels (lymphangiogenesis). The role of lymphangiogenesis in lymphoma spread and proliferation is not clearly established. VEGF-C is the most potent inducer of lymphangiogenesis. LYVE-1 was shown to be a specific marker for lymphatic vessels in normal and tumor tissue. The aim of the present study was the evaluation of lymph node LYVE-1-positive lymphatic sinus density (LSD) and VEGF-C expression in patients with non-Hodgkin's lymphoma (nHL) and in reactive lymph nodes. Sixty paraffin-embedded lymph nodes from newly diagnosed patients with B-cell nHL were evaluated. Twelve lymph node biopsy specimens from adult patients with reactive lymphonodulitis were used as controls. Sections of lymph nodes were stained immunohistochemically for LYVE-1 and VEGF-C. VEGF-C expression in lymph nodes of nHL patients was low and not significantly different from that in the control (p = 0.6). Moreover, VEGF-C expression did not differ significantly between aggressive and indolent lymphomas (p = 0.53). Similarly we did not find differences in LSD in aggressive nHL and in indolent nHL (p=0.49). The mean LSD in reactive lymph nodes was higher than in nHL (p = 0.03). Only in 2 out of 12 reactive lymph nodes LYVE-1-positive vessels were absent. In all groups we demonstrated a strong positive correlation between VEGF-C and LYVE-1-expression (p = 0.0001). Higher LSD in reactive lymph nodes as compared to those of nHL patients suggests that lymphoma proliferation leads to the destruction of the existing lymphatics rather than to lymphangiogenesis within lymph nodes. NHL are not associated with increased expression of VEGF-C nor increased LYVE-1-positive lymphatic sinuses density within lymph nodes.     

Pleural effusions in non-Hodgkin's lymphoma. A cytomorphologic, cytochemical and immunologic study

Review of the records of 243 cases of cytologically diagnosed non-Hodgkin's lymphomas (NHL) revealed pleural effusions in 21 (8.6%). Cytologic examination of pleural fluid was done in 17 cases, of which 16 were reported as positive. Cytologic examination was supplemented with cytochemical staining (acid phosphatase, alpha naphthyl acetate esterase and periodic-acid-Schiff reactions) and E-rosetting studies in 12 cases. Of the 16 positive cases, 11 were malignant lymphomas consisting of convoluted lymphocytes. Acute lymphatic leukemia of the prothymocytic type (T-ALL) and chronic lymphocytic leukemia of the T-cell type (T-CLL) comprised one case each, and there were three cases of follicular center cell lymphomas, two of the cleaved-cell type and one of the Burkitt-type. Comparison of the cytomorphology of the tumor cells in the pleural effusion with those in fine needle aspiration smears from the solid tumors in 14 cases showed an identical appearance in 13 cases; in one, the Burkitt-type lymphoma, the cells were larger and more pleomorphic in the pleural effusion. This study indicates that the cytologic diagnosis and categorization of NHL of the convoluted-cell type is greatly enhanced by the study of neoplastic lymphocytes in a pleural effusion.     

Immunologic induction of malignant lymphoma: graft-vs-host reaction-induced B cell lymphomas contain integrations of predominantly ecotropic murine leukemia proviruses

The induction of a graft-vs-host reaction in (BALB/c X A)F1 mice by i.v. injection with BALB/c lymphoid cells leads to a lymphoid hyperplasia that may progress to malignant lymphoma. In the present paper, the following aspects of graft-vs-host-reaction lymphomagenesis were studied: 1) the cellular requirements for the induction of lymphomas, 2) their cellular origin, and 3) the role of murine leukemia viruses. The development of graft-vs-host-reaction lymphomas was found to be mediated by donor T cells and to require the presence of histoincompatibility between donor and host. Histologically, the vast majority of these lymphomas were either of follicular center cell or of immunoblastic type, whereas immunoperoxidase studies showed that they were virtually all B cell derived. Most of the lymphomas were of host origin. In the DNA of approximately 80% of the lymphomas, integrated murine leukemia virus proviruses were detected. In the B cell lymphoma DNA, integrated ecotropic proviruses prevailed, but recombinant murine leukemia virus and/or deleted murine leukemia virus genomes were also detected in some tumor DNA.     

EZH2 codon 641 mutations are common in BCL2-rearranged germinal center B cell lymphomas

Mutations at codon 641 of EZH2 are recurrent in germinal center B cell lymphomas, and the most common variants lead to altered EZH2 enzymatic activity and enhanced tri-methylation of histone H3 at lysine 27, a repressive chromatin modification. As an initial step toward screening patients for cancer genotype-directed therapy, we developed a screening assay for EZH2 codon 641 mutations amenable for testing formalin-fixed clinical specimens, based on the sensitive SNaPshot single nucleotide extension technology. We detected EZH2 mutations in 12/55 (22%) follicular lymphomas (FL), 5/35 (14%) diffuse large B cell lymphomas with a germinal center immunophenotype (GCB-DLBCL), and 2/11 (18%) high grade B cell lymphomas with concurrent rearrangements of BCL2 and MYC. No EZH2 mutations were detected in cases of Burkitt lymphoma (0/23). EZH2 mutations were frequently associated with the presence of BCL2 rearrangement (BCL2-R) in both the FL (28% of BCL-R cases versus 0% of BCL2-WT cases, p<0.05) and GCB-DLBCL groups (33% of BCL2-R cases versus 4% of BCL2-WT cases, p<0.04), and across all lymphoma types excluding BL (27% of BCL2-R cases versus 3% of BCL2-WT cases, p<0.003). We confirmed gain-of-function activity for all previously reported EZH2 codon 641 mutation variants. Our findings suggest that EZH2 mutations constitute an additional genetic "hit" in many BCL2-rearranged germinal center B cell lymphomas. Our work may be helpful in the selection of lymphoma patients for future trials of pharmacologic agents targeting EZH2 and EZH2-regulated pathways.     

Classifying antibodies using flow cytometry data: class prediction and class discovery

Classifying monoclonal antibodies, based on the similarity of their binding to the proteins (antigens) on the surface of blood cells, is essential for progress in immunology, hematology and clinical medicine. The collaborative efforts of researchers from many countries have led to the classification of thousands of antibodies into 247 clusters of differentiation (CD). Classification is based on flow cytometry and biochemical data. In preliminary classifications of antibodies based on flow cytometry data, the object requiring classification (an antibody) is described by a set of random samples from unknown densities of fluorescence intensity. An individual sample is collected in the experiment, where a population of cells of a certain type is stained by the identical fluorescently marked replicates of the antibody of interest. Samples are collected for multiple cell types. The classification problems of interest include identifying new CDs (class discovery or unsupervised learning) and assigning new antibodies to the known CD clusters (class prediction or supervised learning). These problems have attracted limited attention from statisticians. We recommend a novel approach to the classification process in which a computer algorithm suggests to the analyst the subset of the "most appropriate" classifications of an antibody in class prediction problems or the "most similar" pairs/ groups of antibodies in class discovery problems. The suggested algorithm speeds up the analysis of a flow cytometry data by a factor 10-20. This allows the analyst to focus on the interpretation of the automatically suggested preliminary classification solutions and on planning the subsequent biochemical experiments.     

Tumor microenvironments,the immune system and cancer survival

The study of cancer immunology has recently been reinvigorated by the application of new research tools and technologies, as well as by refined bioinformatics methods for interpretation of complex datasets. Recent microarray analyses of lymphomas suggest that the prognosis of cancer patients is related to an interplay between cancer cells and their microenvironment, including the immune response.