Open or closed nuclear membrane? A question to help distinguish malignant lymphoma from carcinoma and sarcoma.

OBJECTIVE: To describe a cytomorphologic criterion that may help improve diagnostic safety in morphologic differentiation of non-Hodgkin's lymphoma (NHL) from carcinoma or sarcoma and investigate the significance of this cytomorphologic phenomenon. STUDY DESIGN: Eighty-two smears of NHL, carcinoma and sarcoma smears were examined. Forty-five smears were from patients with carcinoma and 35 from patients with NHL. The remaining 2 smears were from patients with sarcoma. RESULTS: In 40 of 46 smears of carcinoma or sarcoma the nuclear membrane was assessed as "open" by the observer. In 6 smears the membrane was assessed as "closed. " In 30 of 35 smears with histologically confirmed NHL, the membrane was estimated as being closed. In the remaining 5 smears it was assessed as open. The sensitivity of evaluating the parameter as open or closed membrane was 87% and the specificity was 86%. The negative predictive value was 89%, and the positive predictive value was 83%. CONCLUSION: We suggest that the presence of an open or closed nuclear membrane may be helpful in differentiation of malignant lymphoma from carcinoma or sarcoma and may help improve diagnostic safety in daily practice.

Destruction of murine lymphoma cells by allogeneic peritoneal macrophages in vitro: influence of antiserum.

Peritoneal macrophages isolated from intact inbred C57BL/10ScCr mice and from inbred C57BL/10ScCr mice previously immunized with the allogeneic SL2 lymphoma were incubated with SL2 cells with and without antiserum. The mode of interaction of the cells during the first 4 hours was studied ultrastructurally. Normal macrophages interacted with the tumor cells only when antiserum was present. The main feature was a partial enveloping of the tumor cells by thin lamellipodia without any visible cell damage. Immune macrophages without antiserum killed the tumor cells extracellularly by a process of apoptosis and subsequently phagocytized the cell remnants, as described previously. In the presence of antiserum, immune macrophages phagocytized intact tumor cells with very long, slender lamellipodia. Subsequently, the tumor cells underwent intracellular necrosis that seemed to be initiated by the release by the macrophage of complete lysosomes into the phagosome containing the tumor cell. In addition to altering the mode of interaction, antiserum greatly increased the degree of cytotoxicity.

Invasion of lymphosarcoma cells into the perfused mouse liver.

Liver of (C57BL X DBA)F1 mice were perfused in situ with a synthetic hemoglobin-free medium, to which murine lymphosarcoma cells were added. All cells were arrested. They were only found in the sinusoids, predominantly in periportal areas. Many lymphosarcoma cells penetrated the walls of the sinusoids with protrusions that extended into and through the endothelial cells. The protrusions often also invaded hepatocytes, and some cells migrated out of the sinusoids. The percentage of the cells that penetrated endothelium was constant and reproducible (68 +/- 4%) in experiments lasting longer than 90 minutes, but the percentage of these cells that also invaded hepatocytes varied greatly. Parellel experiments in vivo yielded similar results, except that the number of cells that invaded hepatocytes was generally much lower. The advantages of the perfused liver as a model for experimental study of the invasion mechanism were evaluated.

Annulate in four cases of diffuse lymphocytic lymphoma.

Annulate lamellae were observed in 4 cases of diffuse lymphocytic lymphoma. Two lymphomas were well differentiated and 2 were of intermediate differentiation. These lymphomas had additional abnormalities of membranes: specifically, excessive blebbing of nuclear membranes and lattice-like formations of tubular endoplasmic reticulum.

Tumor-associated macrophages as the primary source of lysozyme in the urine of mice bearing GPC-11, a transplantable reticulum cell sarcoma.

Large amounts of lysozyme accumulated in the serum and urine of (NZB X BALB/c)F1 mice with GPC-11, a transplantable reticulum cell sarcoma, type A. We separated GPC-11 cell suspensions on 20% Ludox HS gradients. (HS is one of the nine general grades of Ludox offered by du Pont de Nemours & Co., Wilmington, Del.) We did morphologic, functional, and biochemical experiments to detect oncogenic and enzymatic activity in each fraction. Oncogenic cells did not produce lysozyme. In contrast, macrophages associated with the solid tumor did produce lysozyme. The lysozyme purified from the GPC-11-associated macrophages resembled in size, electrophoretic mobility, and antigenicity the lysozyme purified from the urine of mice with the GPC-11 tumor.

Characterization of a murine ovarian reticulum cell sarcoma of histiocytic origin.

We have studied the M5076 tumor, a transplantable murine reticulum cell sarcoma that arose spontaneously in the ovary of a C57BL/6 mouse. This tumor displays functional and ultrastructural characteristics indicating that it is of macrophage origin. Cells from the M5076 tumor are phagocytic, form rosettes with sheep red blood cells, mediate antibody-dependent cellular cytotoxicity against 51Cr-labeled red blood cells, and display macrophage-like cytotoxicity against syngeneic tumor target cells but do not exhibit any natural killer cell activity. The tumor cells possess lysozyme, nonspecific esterase, and phosphatase activities comparable to that seen in rodent macrophages. Ultrastructural examination revealed phagocytic vacuoles and a lack of tight junctions typical of macrophage morphology. Karyotype analysis showed that M5076 tumor cells are hypodiploid with a high percentage (greater than 80%) of metacentric chromosomes that serve as an excellent marker for identification of these tumor cells.

Primary extranodal soft tissue lymphoma of the extremities.

Since true primary lymphomas of soft tissues are extremely rare and there are few well-documented cases in the literature, we report eight cases to help define the clinical and pathologic features of this type of extranodal lymphoma. Among 7,000 malignant lymphomas seen at Mayo Clinic over a 10-year period, we found eight cases of Stage IAE extranodal malignant lymphoma that presented as a soft tissue mass in the upper or lower extremities. The tumors occurred in the following sites: thigh (n = 4), upper arm (n = 2), ankle (n = 1), and calf (n = 1). Two cases had low-grade histology: small lymphocytic lymphoma and follicular mixed small-cleaved and large-cell lymphoma. An intermediate-grade histology was present in two cases: diffuse mixed small- and large-cell lymphoma and diffuse large-cell (cleaved) lymphoma. Half of the patients had a high-grade lymphoma: diffuse large-cell (n = 3) and large-cell, immunoblastic (n = 1). The results of immunohistochemistry and electron microscopy in selected cases are discussed in the context of the differential diagnosis with other small, round-cell cancers. Four patients died of recurrent lymphoma, one at 7 months, two at 15 months, and one at 4 years. Three patients are alive without evidence of disease at 3, 8, and 13 years. One patient is alive and is receiving chemotherapy for locally recurrent lymphoma 6 months after diagnosis.

A primary immunoblastic T malignant lymphoma of the small bowel, with azurophilic intracytoplasmic granules. A histologic, immunologic, and electron microscopy study.

We report an aggressive primary T-immunoblastic lymphoma of the small intestine without blood involvement or associated celiac disease. Grossly, the tumor was composed of multiple disseminated ulcerated, infiltrating, or protuberant nodular lesions. Immunologic investigation showed that lymphoma cells were of peripheral (post-thymic) T-cell origin and expressed the phenotype associated with cytotoxic-suppressor subset (Leu4/CD3+, Leu9/CD7+, Leu2/CD8+, Leu11/CD16+, Leu 7/NKcells+, FcIgG+, HLA-DR+, anti-Tac/CD25+, Ki-1/CD30-, Leu1/CD5-, Leu5/CD2-, Leu3/CD4-). A particular morphologic feature of this case is the presence of numerous azurophilic granules within the lymphoma cells, identified as lysosomes by cytochemical and ultrastructural studies. In view of recent immunologic evidence that normal cytotoxic/suppressor T-cells selectively reside within the epithelium of the normal bowel and some of them contain azurophilic granules, it could be suggested that our patient's lymphoma represents a malignant counterpart of these lymphocytes. Furthermore, the aggressive character of this T malignant lymphoma (T-ML) could be related to the expression of T-cell activation markers HLA-DR and Tac/CD25 and the proliferation-associated antigen Ki-67 on a high proportion of tumor cells.

Signet ring cell lymphoma. A rare morphologic and functional expression of nodular (follicular) lymphoma.

Nodular lymphomas and diffuse lymphomas of corresponding cellular composition have been shown to arise from follicular center cells. This paper describes a rare functional and morphological expression of malignant lymphomas arising from follicular center cells, namely, immunoglobulin production, an observation for which no detailed description or nanlysis is available in the literature. Furthermore, the unusual signet ring-like appearance of the lymphoma cells, which is due to retention of immunoglobulins within the cytoplasm, may result in an erroneous interpretation of metastatic adenocarcinoma or liposarcoma. Therefore, we are presenting a detailed analysis of light microscopic, histochemical, immunocytochemical, and ultrastructural observations. The lymphomas of all seven patients in our series showed nodular growth patterns; in all but one, diffuse areas were also observed. Five of the lymphomas were classified as poorly differentiated lymphocytic type and two as mixed cell type, according to Rappaport's classification. In four of the seven patients, the majority of the neoplastic cells had a clear vacuolated cytoplasm, and in three of these cases, a few of the neoplastic cells showed immunoperoxidase positivity for monoclonal IgG. This group in particular closely simulated metastatic carcinoma composed of so-called signet ring cells. In the remaining three cases, most of the neoplastic cells contained PAS-positive, Russell body-like monoclonal IgM. Ultrastructurally, the monoclonal IgG appeared as even-sized electron-dense spherules or irregular electron-dense clumps, while the monoclonal IgM appeared as membrane bound, homogeneous, electron-dense material. The implications of these findings and the morphologic features which are helpful in the identification of these lymphomas are discussed.

Spindle cell neoplasms of lymph nodes of probable reticulum cell lineage. True reticulum cell sarcoma?

Primary spindle cell neoplasms involving hematolymphoid organs are extremely rare. We present four cases of spindle cell neoplasms of unusual phenotype arising within lymph nodes. Two of the four cases showed morphologic and immunophenotypic features suggestive of interdigitating reticulum cell lineage; these cases expressed several macrophage antigens and S-100 protein but not CD1. The other two cases showed evidence suggestive of dendritic reticulum cell lineage. Both cases expressed HLA-DR, several macrophage antigens, complement receptors C3b and C3d; one case expressed R4/23; both showed the presence of desmosomes on ultrastructural examination. A germline configuration for the immunoglobulin heavy chain and beta-T--cell receptor genes was detected in all four cases. Of the two patients in the first group, one had local recurrence of tumor; the other died of widespread metastases. Of the two patients in the second group, both are alive and well at 12 and 27 months follow-up, respectively.

Mantle-zone lymphoma. A pattern produced by lymphomas of more than one cell type.

We analyzed the distribution and immunologic phenotype of the neoplastic cells in eight cases of mantle-zone lymphoma. Although in all the cases the follicle centers appeared reactive on routine histologic examination, polyclonal staining for immunoglobulin was found in the follicle centers in only five of the eight cases; in the other three cases the follicle centers were monoclonal. In three cases, the immunologic phenotype was that of centrocytic (diffuse small cleaved cell) lymphoma: IgM+IgD+B1+B2+Ia+Tl+. In one case the phenotype was that of a follicular (centroblastic/centrocytic) lymphoma: IgG+B1+B2+Ia+CALLA+. In the other four cases, the phenotype was IgM+B1+B2+ or B2-Ia+; this phenotype can be seen in diverse B cell lymphomas. The phenotype of normal mantle-zone cells (IgM+IgD+B1+B2+Ia+) was not reproduced by any of the lymphomas. A mantle-zone pattern may be produced by either follicular or diffuse lymphomas of predominantly small cleaved cell type, and does not indicate an origin from the cells of the normal mantle zone.

DNA double strand breaks in fibroblast cell lines, from non-Hodgkin's lymphoma patients, showing increased sensitivity to chronic gamma irradiation.

Cultured skin fibroblast cell lines from two non-Hodgkin's lymphoma patients (NHL) and a normal subject were studied for cell killing, chromosomal aberrations (breaks, translocations, dicentrics and rings) and DNA double strand breaks (dsbs) following chronic gamma irradiation. Compared to the cell line from the normal donor, the NHL patients' fibroblasts showed enhanced radiosensitivity for both cell survival and chromosomal aberrations. While spontaneous breaks were observed in both normal and patients' cells, spontaneous translocations and radiation-induced dicentrics and rings were found only in the latter. Radiation-induced DNA double-strand breaks (dsb) were determined by CHEF electrophoresis. After chronic irradiation with gamma rays the fraction of residual dsb was significantly increased from 1.4% in controls to 1.9% in the NHL cell lines. These data, thus, suggest that the cellular and chromosomal sensitivity to chronic irradiation observed in NHL patients may be due to a deficiency in the repair of a small fraction of DNA double strand breaks.

Proliferative rates of non-Hodgkin's lymphomas as assessed by Ki-67 antibody.

The Ki-67 antibody, a monoclonal antibody that reacts with nuclei in actively proliferating cells, was used in an immunohistochemical study to assess the growth fractions of non-Hodgkin's lymphomas and related disorders. The lowest proliferative indices were found in small lymphocytic lymphoma/chronic lymphocytic leukemia and intermediate lymphocytic/mantle zone lymphoma. An intermediate proliferative index was seen in the follicular lymphomas and diffuse small cleaved cell and diffuse mixed cell lymphomas. A high index was seen in the diffuse large cell lymphoma and lymphoblastic lymphoma. The highest and most consistent proliferative index was seen in small noncleaved cell lymphoma. Cases of reactive follicular hyperplasia had a significantly higher proliferative index than those of follicular lymphoma. We conclude that the Ki-67 antibody has great utility in providing an estimate of the proliferative rate of non-Hodgkin's lymphomas. Prospective studies may show this information to have prognostic value independent of histologic classification.

Nuclear morphologic and morphometric analyses of large noncleaved cell and immunoblastic non-Hodgkin's lymphomas.

Both morphologically and immunologically, non-Hodgkin's lymphoma (NHL) of the large cell type has been shown to be a heterogeneous category. However, the homogeneity of the nuclear parameters, particularly size and condensed chromatin organization, used to classify this subtype of NHL has not been investigated. In fact, objective morphologic techniques have not been systematically applied to verify the segregation of NHL on the basis of nuclear parameters, a concept common to all current classification systems. In this study morphometric image analysis was used to compare the nuclei in 20 specimens from NHLs of the large cell type with those in mantle zone and germinal center lymphocytes from lymph nodes with reactive hyperplasia. Results of the assessment of mean nuclear area in large cell lymphomas revealed that this class is also heterogeneous, with some of the specimens having a nuclear size in the upper range of that for normal small lymphocytes. In addition, in only a few of these specimens was the mean nuclear area within the range of that for fully transformed germinal center lymphocytes. The majority of large cell lymphomas have a nuclear size more characteristic of partially transformed lymphocytes in germinal centers. In addition to indicating inconsistencies in the current diagnostic criteria used in NHL classifications, the results indicate reasons for interobserver variations in clinicopathologic trials; the validity of nuclear size as a prognostic indicator and the biologic basis for classifying NHL as a reflection of normal lymphocyte transformation are also questioned. In terms of patient management, the classifications of NHL currently used require objective reappraisal.

A comparative assessment of proliferating cell nuclear antigen, c-myc p62, and nucleolar organizer region staining in non-Hodgkin's lymphomas: a histochemical and immunohistochemical study of 200 cases.

Proliferating cell nuclear antigen (PCNA) and c-myc p62 oncoprotein are two nuclear proteins expressed in proliferating and transformed cells. They can be recognized immunohistochemically in paraffin sections by the monoclonal antibodies PC-10 and c-myc 1-9E10, respectively. On the other hand, nucleolar organizer regions (NORs) are loops of DNA that carry the r-RNA genes and can be visualized in paraffin sections as black dots (AgNORs) using a silver impregnation method. It has been suggested that the mean number of AgNORs may reflect the cellular kinetics of a tumor. We independently examined 200 cases of non-Hodgkin's lymphomas using the monoclonal antibodies PC-10 and c-myc 1-9E10, as well as the AgNOR method. Our study shows a very significant correlation between PCNA, c-myc expression, and AgNOR count on the one hand and histologic grade on the other (P < .001), although a significant overlap among the three grades exists. PC-10, c-myc 1-9E10, and AgNOR scores are all shown to be linearly related, even though significant discrepancies were observed, and the correlation is stronger between PCNA and AgNORs (PCNA v c-myc p62, r = .551; PCNA v AgNORs, r = .746; c-myc p62 v AgNORs, r = .529; P < .001). A remarkable finding is that the intermediate group of lymphomas is heterogeneous as far as the proliferative rate is concerned: diffuse large cell cleaved/non-cleaved lymphomas (category G of the Working Formulation) are characterized by a significantly higher proliferative index, as evidenced by the elevated PCNA, c-myc p62, and AgNOR scores, in comparison with the other types of intermediate-grade lymphomas (P < .001). However, the proliferative rate is lower than that of the high-grade lymphomas (PCNA, P < .05; c-myc p62, P < .001; AgNORs, P < .005). No significant difference exists between B-cell and T-cell lymphomas except for the higher expression of c-myc p62 in intermediate-grade B-cell lymphomas, obviously due to the higher proliferative rate of diffuse large cell lymphomas. Based on our findings, it appears that the combination of PCNA, c-myc p62, and AgNORs provides an accurate estimate of the proliferative rate of non-Hodgkin's lymphomas in paraffin sections. Clinical studies may show whether this information has prognostic value independent of histologic classification. In addition, our results suggest that category G (diffuse large cell) lymphomas may belong to a malignancy grade higher than the intermediate grade, a suggestion consistent with their more aggressive biologic behavior.

Morphologic studies of lymphocyte nuclei in follicular and diffuse mixed small- and large-cell (lymphocytic-histiocytic) lymphoma.

Twelve examples of mixed small- and large-cell lymphoma (eight follicular, one follicular and diffuse, and three diffuse) were investigated morphometrically using plastic-embedded tissue in order to study nuclear characteristics of lymphocyte populations in this form of non-Hodgkin's lymphoma (NHL) and to test morphologic bases for current NHL classification systems. This study illustrates that there are many inaccuracies, illusions, and misconceptions in the morphologic criteria currently used to classify mixed small- and large-cell lymphoma. A principal finding was that lymphocyte nuclear profiles in mixed-cell lymphomas tend to be smaller in size (P less than .005) and more irregular in shape (P = .0001) than the morphologically similar counterparts in germinal centers of lymph nodes with reactive hyperplasia. Intercase comparison of mixed small- and large-cell lymphomas revealed a considerable range of mean nuclear area values, some of which were within the size range of normal, small lymphocytes. At the magnifications used for morphometric assessment, a high proportion of lymphocyte nuclear profiles had shallow invaginations, but only a limited number of profiles (4% to 14%) had deep (cleaved) indentations. Contrary to current definitions for this subtype of NHL, lymphocytes with "small" nuclei had the same proportion of the nuclear diameter occupied by nuclear invaginations as lymphocytes with "large" nuclei and, in fact, mean nuclear invagination depth was shallower in "small" nuclei than in "large" nuclei. Furthermore, regardless of whether it is nuclear area or shape that is evaluated, lymphocytes in mixed-cell lymphoma do not separate into two populations of small-cleaved and large noncleaved cells. Morphometry reveals that only four of the 12 examples of mixed small- and large-cell lymphoma had a proportion of the lymphocytes in the size range of fully transformed germinal center lymphocytes that exceeded 25%, and none of the cases approached 50% even though the population of lymphocyte nuclei appearing "transformed," and therefore "large," ranged from 28% to 57%. Such results indicate that the large, noncleaved and cleaved component, as seen in histologic sections of mixed small- and large-cell lymphoma, do not have nuclei of uniform size and many, in fact, are not actually large. The morphometric findings indicate reasons for the poor observer reproducibility in classifying this subtype of NHL.

Non-Hodgkin's lymphoma with immunologic phenotype similar to non-T, non-B acute lymphocytic leukemia.

A diagnosis of diffuse poorly differentiated lymphocytic lymphoma was made from a biopsy of a scapular mass on a 24-month-old child. The bone marrow and peripheral blood were not involved in the neoplastic process. Neoplastic cells stained negatively for Sudan black B, myeloperoxidase, periodic acid-Schiff reagent, alpha-naphthyl acetate esterase, and acid phosphatase. In addition, neoplastic cells did not form nonimmune rosettes with sheep erythrocytes or contain surface membrane immunoglobulin. However, neoplastic cells were positive for terminal deoxynucleotidyl transferase and "Ia-like" antigen. We conclude that this non-Hodgkin's lymphoma has a cytochemical and immunologic phenotype similar to that of lymphoblasts from cases of non-T, non-B acute lymphocytic leukemia.

Histiocytic lymphoma (reticulum-cell sarcoma) of the tongue manifesting as macroglossia.

A case of histiocytic lymphoma primarily involving the tongue in a 71-year-old black man is presented. The authors discuss the diagnostic problems in differentiation of such lesions from other small-cell tumors, primary or secondary, that might involve the tongue. Electron microscopic studies were utilized to confirm the diagnosis of histiocytic lymphoma.

Peripheral T cell lymphoma (immunoblastic sarcoma of T cell type): an immuno-ultrastructural study.

Use of monoclonal antibodies directed against T cell antigens for the phenotypic characterization of neoplastic lymphoid cells in peripheral T cell lymphomas is described. Studies of cryostat sections revealed the distribution of T cell subsets in nodal and extranodal infiltrates, and immuno-ultrastructural techniques demonstrated discrete localization of T cell antigens to the cytoplasmic membranes of neoplastic cells. Although histologically similar, the tumors appeared heterogeneous as to their immunologic phenotype, with the majority demonstrating markers for T helper/inducer lymphocytes.

Immunophenotypic analysis of sinonasal non-Hodgkin's lymphomas.

A panel of paraffin effective antibodies recognizing B cells and T cells (LN-2, MB1, L26, MT1, UCHL1, kappa, lambda) was used to characterize the immunophenotypes of 26 sinonasal non-Hodgkin's lymphomas. Seventeen tumors were stage I, five were stage II, one was stage III, and three were stage IV. Nine lymphomas were classified morphologically as large cell, six were large cell immunoblastic, six were small cleaved cell, two were mixed small and large cell, two were small noncleaved cell, and one was lymphoblastic. None were follicular. Twenty-two lymphomas had a B cell immunophenotype, three were T cell neoplasms, and one was immunoreactive only for MT1. This predominance of sinonasal lymphomas with a B cell immunophenotype in patients residing in the United States contrasts with the almost exclusive occurrence of T cell sinonasal lymphomas in Chinese patients living in Hong Kong and Japanese patients residing in regions of Japan that are nonendemic for human T cell leukemia virus-1.