Dual parameter flow cytometry studies in human lymphomas.

Dual parameter flow cytometry studies using Coulter volume and cell DNA content were carried out in monodisperse cell suspensions of 64 samples of human lymphoma, chronic lymphocytic leukemia, hairy cell leukemia, and benign lymphoid proliferations. Differences in mean Coulter volume among the lymphomas were due both to the intrinsic differences in mean G1 cell Coulter volume and to the presence of increased fractions of larger S and G2 cells, especially among the large B cell lymphomas. However, the relative contribution of large non-G1 cells to the overall population Coulter volume distribution was a relatively minor one; the presence of cells in S did not increase mean Coulter volume by more than 10%, even in samples with high S fractions. There was a good correlation between mean G1 cell Coulter volume and the log of the fraction of cells in S among the B cell lymphomas (r = 0.55). Evidence is presented that within individual samples, large cells proliferate more rapidly than small cells. This was seen in every case, both in the normal samples and in the lymphomas, and in the T cell lymphomas as well as in the B cell lymphomas. Aneuploidy was detected by flow cytometry in 11 cases; in 7 cases the aneuploid cell component could be analyzed separately from the diploid cell component on the basis of cell Coulter volume differences. The aneuploid components of diploid-aneuploid mixtures had higher S fractions than the diploid components in six of seven cases (0.16 +/- 0.04 [SE] vs. 0.08 +/- 0.02). These findings are considered in relation to the histopathological classification of the lymphomas, and in relation to the concept of clonal selection and clonal evolution of tumors.

Surface immunoglobulin on leukemic, leukemoid, and normal granulocytes.

Using a new functional approach for the study of lymphomas and leukemias in which immunologic and cytochemical techniques were employed, we found a consistent surface immunoglobulin pattern of the gamma-, k-, lambda-type on cells from poorly differentiated (acute) and well-differentiated (chronic) granulocytic leukemias. This pattern was also found on nonneoplastic granulocytes from patients with leukemoid reactions as well as on granulocytes from normal individuals. These findings suggested that both leukemia cells and nonneoplastic granulocytes had IgG bound to the cell surface by an Fc receptor. This binding of IgG by granulocytes was not tumor-specific and appeared to correlate both with the degree of differentiation and possibly with the degree of activation of the granulocytes. In addition to raising the basic question of its functional significance, these findings offered an approach for distinction of poorly differentiated granulocytic leukemia from lymphomatous processes.

Morphologic and cytochemical comparison of human lymphoblastoid T-cell and B-cell lines: light and electron microscopy.

Human lymphoblastoid cell lines characterized as T- or B-cells by various markers were compared morphologically and cytochemically by light and electron microscopy. Distinct differences in nuclear morphology, amount of cytoplasm, pyroninophilia, and periodic acid-Schiff (PAS) staining enabled us to discriminate between T- and B-cell lines. T-cells had nuclei with an irregular configuration, stippled heterochromatin, and small or absent nucleoli. The scanty cytoplasm of T-cells contained intensely stained, PAS-positive globules and was less pyroninophilic than the cytoplasm of B-cells. B-cells had more rounded, uniform, vesicular nuclei with prominent nucleoli and peripheral heterochromatin. The cytoplasm of B-cells was abundant and strongly pyroninophilic. Transmission electron microscopy generally confirmed these morphologic differences. These findings supported our contention that consistent cytologic features concordant with immunologic markers make it possible to identify certain lymphomas as being of B- or T-cell origin on purely morphologic grounds.

B-cell lymphoma in two monogamous homosexual men.

Opportunistic infections and malignant neoplasms have been described in homosexual men in association with immunologic abnormalities. We observed the development of malignant B-cell lymphomas in two homosexual men who had had a monogamous relationship for two years. Patient 1 had an aggressive, monoclonal, small, noncleaved, non-Burkitt's lymphoma ("undifferentiated lymphoma"), associated with severe immunocompromise. Patient 2 manifested a monoclonal, small, cleaved, follicular center cell lymphoma, with a follicular pattern, two months later. No common acute infection was detected. Staining for Epstein-Barr nuclear antigen in malignant tissue was negative in the second patient. However, the possibility of a transmissible agent as a causative factor cannot be excluded, and further study of similar patients is warranted.

Human B lymphocytes in giemsa stained preparations.

A mixed antiglobulin procedure has been developed to provide simultaneous study of cell surface immunoglobulin and detailed cytological features in Giemsa stained preparations. The percentage of surface immunoglobulin bearing lymphocytes, monocytes and granulocytes changed dramatically when lymphocytes were incubated at 37 degrees C, or following sheep erythrocyte rosette enrichment procedures.

Primary mediastinal lymphoma in adults.

The incidence of primary mediastinal lymphoma in adults was investigated in 184 patients with non-Hodgkin's lymphoma. This entity was defined as disease within the mediastinum in patients who presented with symptoms due to an enlarging mediastinal mass. Of 184 patients, 17 presented with primary mediastinal lymphoma. All had a diffuse histologic pattern. The most common pathologic type was poorly differentiated lymphocytic lymphoma, diffuse (PDL-D), (11 cases). In nine of these 11 cases the patients had tumors of convoluted lymphocytes. The presentation was rapid in onset, with heart failure, pericarditis, dyspnea and superior vena caval syndrome predominating. Eleven of the 17 were clinical stage I or II, but eight of these had widespread disease on pathologic staging or rapid dissemination soon after diagnosis. In conclusion (1) primary mediastinal lymphoma is always diffuse in histology. (2) The most frequent pathologic type is PDL-D, with convoluted morphology. (3) Compression of vital intra-thoracic structures is common. (4) Although seemingly localized at presentation, this entity usually implies disseminated disease.

Primary central nervous system lymphoma in homosexual men. Clinical, immunologic, and pathologic features.

Primary central nervous system lymphoma constitutes one of the criteria for the acquired immune deficiency syndrome (AIDS), yet a paucity of information is currently available regarding the clinical, immunologic, or pathologic features of these patients. Six homosexual men presenting with primary central nervous system lymphoma were evaluated. Five of these patients presented with altered mental status. All lymphomas were intracranial. B cell immunoblastic sarcoma was found in five. Immune phenotyping studies performed in five patients revealed monoclonal lambda light chain in three, whereas one expressed only IgG heavy chain, and one demonstrated another B cell (LN-1) surface antigen. Hypodense, contrast-enhancing lesions were apparent on computed axial tomographic scanning of the brain, in sharp contrast to isodense or hyperdense lesions reported in primary central nervous system lymphomas without underlying immunodeficiency. Immunologic abnormalities in these patients were similar to those in AIDS presenting as Kaposi's sarcoma or with opportunistic infections. In spite of therapeutic interventions, survival was short, and only one patient is currently alive.

Immunoblastic sarcoma of T- and B-cell types: morphologic description and comparison.

Immunoblastic sarcoma (IBS) is a large cell lymphoma conceptually related to transformed T and B lymphocytes of the extrafollicular compartment of the immune system (immunoblasts). This light microscopic study of a series of 47 immunologically defined cases of IBS was undertaken in an attempt to define more precisely the morphologic features of the T- and B-cell subtypes. A remarkable morphologic spectrum characterized T-IBS (31 cases), which could be divided into two main groups: 1) tumors composed of varying mixtures of small, medium-sized, and large transformed cells; and 2) tumors with more homogeneous populations of medium-sized or large transformed cells. These cells, in all sizes, generally had abundant pale-staining cytoplasm, delicate nuclear membranes, finely dispersed chromatin, and one to several, small or medium-sized, prominent nucleoli. A distinctive background of small, irregular lymphocytes was frequently present. Plasmacytoid differentiation, seen most consistently as amphophilic staining of the cytoplasm, generally characterized B-IBS (16 cases). B-IBS similarly showed a morphologic spectrum that occurred in two main forms: 1) tumors consisting of a spectrum of transformed cells, with the smaller cells often showing the most striking plasmacytoid differentiation; and 2) tumors consisting predominantly of medium-sized to large transformed cells with varying degrees of plasmacytoid differentiation. With this constellation of features, all but two cases of T-IBS and one case of B-IBS were morphologically distinguishable.

The immunologic approach to the pathology of malignant lymphomas.

Presentations of malignant lymphomas in the past did not have the benefit of modern developments in immunology and do not bear any relationship to our modern understanding of immunology. The malignant lymphomas in our immunologic approach are regarded as neoplasms of the immune system and involve principally the T- and B-cell systems and alterations in lymphocyte transformation. The cytologic types of our new classification are an attempt to identify specific functional subtypes of the T- and B-cell systems as defective expressions of their normal counterparts. The results of our multiparameter studies on large case series of non-Hodgkin's lymphomas and those of others have demonstrated that the malignant lymphomas for the most part mark as T- or B-cell types, with the exception of a rare lymphoma of true histiocytic type and the unmarked portion of acute lymphocytic leukemia (ALL) of childhood. They have demonstrated the heterogeneity of the cytologic types of the past that possibly account for the diversity of their clinical manifestations and responses to therapy. The immunologic approach has permitted the identification of homogeneous cytologic types that are emerging as clinical morphologic immunologic entities.

Sensitivity and specificity of immunostaining in the diagnosis of mantle zone lymphoma.

The diagnosis of mantle zone lymphoma is sometimes difficult to make solely on the basis of morphology because the mantle zone pattern may be present in other disorders such as benign mantle zone hyperplasia, follicular center cell (FCC) lymphomas, and Castleman disease. To distinguish mantle zone lymphoma from the other disorders mentioned previously, the authors performed immunoperoxidase studies on B-5-fixed, paraffin-embedded sections or cryostat sections of lymph nodes from nine patients with a diagnosis of mantle zone lymphoma. The results then were compared with the immunostaining pattern seen in FCC lymphomas and various benign lymphoid hyperplasias. A monoclonal proliferation of mantle zone cells, as shown by staining for immunoglobulin light chains, was noted in the mantle zones and interfollicular areas in all six cases from which cryostat sections were available. The cells in the residual follicular centers uniformly had a polyclonal light chain marking pattern. Two novel monoclonal antibodies (LN-1 and LN-2) that identify FCCs and B-cells in B-5-fixed, paraffin-embedded tissues also were used in this study. Of the six cases in which the monoclonal antibodies could be used, the cells in the residual follicles were uniformly LN-1 positive, LN-2 positive, while the mantle zone and interfollicular cells were almost completely LN-1 negative, LN-2 positive. The data suggest that mantle zone lymphomas are a distinctive neoplasm of monoclonal B-cells of non-FCC origin. The authors conclude that immunostaining is a sensitive technic for identifying a malignant neoplasm of B-cells in the mantle zone and interfollicular areas. In addition, the method is relatively specific and useful for distinguishing mantle zone lymphoma from similar-appearing disorders such as benign mantle zone hyperplasias and certain FCC lymphomas.

Reactive lymphoid hyperplasia with single class (monoclonal) surface immunoglobulin.

Lymphoid tissues from 12 patients were diagnosed as reactive lymphoid hyperplasia, but surface immunoglobulin studies revealed monoclonal (single class) immunoglobulin staining patterns. Infectious, autoimmune, and immunodeficient conditions were diagnosed on the basis of histology and clinical features. Such surface immunoglobulin restriction has been used as an indicator of a neoplastic lymphoid proliferation, but the cases of these patients, in whom the histologic diagnosis was benign, emphasize the importance of a multiparameter approach to diagnosis. Although at the time of this report none of the patients still available to follow-up study have developed known lymphoid neoplasms, the possibility that monoclonal SIg patterns are a harbinger of neoplastic disease makes continuing follow-up of such patients important.

Orbital and adnexal lymphomas. A multiparameter approach.

The application of immunologic, cytochemical, and electron-microscopic technics to the study of lymphoid lesions of the orbit and adnexa was found to enhance the accuracy of diagnoses of malignant lymphoma in five challenging referral cases. Although careful specimen collection, ideal fixation, and processing constitute the cornerstone of the morphologic diagnosis of nodal and extranodal lymphomas, the immunologic characterization of these lymphoproliferative disorders is emphasized as an especially useful diagnostic parameter. The occurrence of false-negative diagnoses of pseudolymphoma of the orbital region may be partially explained by a failure to recognize the relatively frequent plasma-cytoid lymphocytic lymphoma, a distinctive entity that may mimic a reactive inflammatory process. All of the cases of malignant lymphoma reported represented stage I or stage II disease, suggesting that primary lymphoma of the orbital region may be a disease with a favorable prognosis.

Immunoglobulin inclusions in non-Hodgkin's lymphomas.

The occurrence and characteristics of intracellular immunoglobulin inclusions in non-Hodgkin's lymphomas are described. Lymphoma cells from three patients with immunoblastic sarcoma contained classic cytoplasmic, periodic acid-Schiff (PAS)-positive Russell bodies. Immunoperoxidase staining revealed monoclonal IgG (k) in two cases and a polyclonal pattern in the third case, where the tumor evolved from a reactive lesion. Unusual cytoplasmic inclusions were observed in the neoplastic cells of two patients with follicular center cell lymphoma. In one case large globular structures lacking a distinct limiting membrane were seen, while the cells of the other patient contained "signet-ring-like" vacuoles filled with microvesicles. Both were PAS-negative and showed monoclonal immunglobulin staining. In two other cases PAS-positive intranuclear inclusions consisting of monoclonal immunoglobulin IgM(k) could be demonstrated. The possible significance of these findings in B-lymphocyte derived neoplasms is discussed.

An immunohistochemical study of hemoglobin A, hemoglobin F, muramidase, and transferrin in erythroid hyperplasia and neoplasia.

The bone marrow biopsy specimens of 35 patients with benign and malignant erythroid hyperplasias were examined for the presence of hemoglobin A, hemoglobin F, muramidase (lysozyme), and transferrin, using an indirect immunoperoxidase method (PAP) on Zenker's-fixed paraffin-embedded bone marrow biopsy specimens and particles. Five cases of each of the following entities were studied: erythroleukemia and erythremic myelosis, acute granulocytic leukemia with maturation (FAB M2), polycythemia rubra vera, myeloproliferative syndrome in childhood, megaloblastic anemia (B12 and folate deficiency), erythroid hyperplasia (regenerating bone marrow and hemolytic anemia), and Ph' chromosome positive chronic granulocytic leukemia. Hemoglobin A was present in both the early and late erythroid precursors in all conditions. Hemoglobin F was the predominant hemoglobin in early erythroblasts of pernicious anemia and in both early and late erythroid elements in erythroleukemia and erythremic myelosis. Small quantities of hemoglobin F were present in a few isolated clusters in other conditions. Staining for hemoglobin F may be useful in identifying immature erythroid precursors and in distinguishing some cases of dysplastic erythroid hyperplasia from neoplasia. Additionally, these findings suggest that the maturational switch in hemoglobin synthesis operates with distinct pathways under different conditions.

Immunohistochemical characterization of reactive and neoplastic mast cells.

Mast cells are connective tissue elements likened to unicellular endocrine organs because of the wide diversity of physiologic and pathologic events associated with the secretion of biologically active compounds. Using an immunoperoxidase method (PAP), we studied tissue from patients with benign and malignant systemic mastocytosis and with a variety of reactive conditions. The following immunoreactive antigens were identified in mast cells: a heparinlike compound or compounds (HLC), prostaglandin, serotonin, and fibronectin. HLC is constantly present, staining mast cells in a granular fashion from most lesions. Serotonin and prostaglandin stain in a diffuse cytoplasmic manner in occasional lesions. Fibronectin is found in a surface location in selected cases. We found no clear association between the immunoreactivity of one compound in mast cells and one clinical symptom, e.g., HCL with bleeding, prostaglandin, or serotonin with systemic vasomotor activity or fibronectin with increased tissue fibrosis. However, patients with localized and systemic disease had symptoms that might have been attributed to more than one compound. Only occasional patients with reactive conditions showed such symptoms. The presence of these compounds, either alone or in combination, did not separate benign from malignant conditions. Other cells within selected tissues also stained with the antibodies tested. Despite the lack of exclusivity, these antibodies are useful in identifying mast cells within tissue sections and may have a role in the study of mast cell constituents.

Mycosis fungoides/Sezary's syndrome progressing to immunoblastic sarcoma. A T-cell lymphoproliferation with both helper and suppressor phenotypes.

Multiparameter studies of an unusual patient exhibiting cutaneous manifestations of both mycosis fungoides and Sezary's syndrome are presented. The neoplastic cells of dermal and nodal infiltrates and peripheral blood expressed both helper and suppressor immunologic phenotypes. Cytofluorographic analysis of cells isolated from lymph node and peripheral blood showed a population of neoplastic cells that were stained with the monoclonal antibodies OKT 3, 4, 8, and 11. Immunoperoxidase staining of frozen sections with monoclonal antibodies Leu 1, 2, and 3 provided a topographical identification of an identically marking population of cells in dermis and lymph node. In light of current models depicting normal T-cell lineage, the authors suggest that the neoplastic population in this patient, expressing both helper and suppressor phenotypes, reflected a phenotypic stage of immunologic maturation (OKT 6-, OKT 10-, OKT 3+, OKT 11+, OKT 4+, OKT 8+) in which the neoplastic cells had not yet segregated into distinctive T-cell subsets. While excess helper activity was suggested by serum hypergammaglobulinemia, in vitro helper and suppressor function was not determined. The range of studies employed illustrates the wide variety of technics required to adequately characterize complex clinico-immunopathologic disorders, as represented by this case, and the wealth of information that can be gleaned.

Biopsy of peripheral lymph nodes.

This retrospective study reviews 653 patients who had peripheral lymph node biopsies for purposes of diagnosis from 1973 to 1977. Overall, 56 per cent of the nodes had benign lesions, 29 per cent had carcinoma, and 15 per cent had lymphoma. Supraclavicular nodes had the highest incidence of metastatic carcinoma (54%), and inguinal nodes had the highest incidence of reactive hyperplasia or lymphadenitis (71%). Detailed distribution according to histological subtypes, age, and sex of patients are presented. Statistically, age is the most useful factor in estimating the probability of whether the lymphadenopathy is due to benign or malignant process. The chance that lymph adenopathy is due to malignant lymphoma is relatively constant among all age groups (11-16). However, the majority of patients younger than 30 years old had Hodgkin's disease, while older patients were more likely to have non-Hodgkin's lymphomas.

Staging laparotomy and splenectomy for Hodgkin's disease.

The major objective of diagnostic laparotomy in Hodgkin's disease is to define the extent of involvement not detectable by nonoperative means. Fifty patients in this institution had operative staging procedures; six for recurrent disease three to 11 years after initial therapy. Twenty-four patients had nodular sclerosis, 23 mixed cellularity, and three had other types. The clinical stages were advanced in 13 patients and decreased in seven patients. Two patients (both had mixed cellularity and systemic symptoms) had positive wedge biopsy of the liver, whereas direct needle biopsy was negative. Nineteen spleens contained Hodgkin's disease but only three could be palpated on physical examination. About half the patients with abnormal lymphangiograms had positive periaortic nodes; lymphangiogram had a false negative rate of 12%. Additional procedures performed included appendectomy, oophoropexy, and resection of Meckel's diverticulum. There was no mortality and only one case had severe postoperative Salmonella septicemia. Our findings are comparable with those reported in the literature.

Immunoblastic sarcoma in patients with systemic lupus erythematosus-like disorders.

Five patients, predominantly older men, had abnormal immune states for prolonged periods of time with manifestations of multisystem involvement, including features of systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome. In the course of their disease, all had malignant lymphoma (immunoblastic sarcoma) develop. The development of lymphoma was associated with generalized lymphadenopathy in four cases.