Diagnostic application of two-color flow cytometry in 161 cases of hairy cell leukemia.

Recent immunophenotypic studies of hairy cell leukemia (HCL) have suggested specific patterns of immunoreactivity that may aid in diagnosis. We studied peripheral blood (PB) from 161 cases of HCL using two-color direct immunofluorescence flow cytometry and an extended panel of antibody combinations. Circulating hairy cells were identified by immunophenotypic features in 92% of the cases and could be detected even when representing < or = 1% of circulating lymphocytes. The 133 cases with > or = 2% detectable hairy cells were analyzed in detail. HCL showed a uniform and unique B-cell phenotype, with each of the following features identified in 99% to 100% of cases: (1) positive staining for B-ly7, coexpressed with CD19; (2) very intense, uniform expression of CD11c, with CD19; (3) moderately intense staining for CD25, with CD19; (4) very intense staining for CD22; (5) moderate to very intense staining for CD20; and (6) moderately intense monoclonal surface Ig. Phenotypic variability existed in expression of CD10 (26%) and CD5 (4%). Based on these features, HCL was easily distinguished from 50 cases of chronic lymphocytic leukemia (CLL). Although CLL exhibited frequent expression of CD11c (74%) and CD25 (68%), the intensity of staining was significantly less than HCL. Furthermore, CLL was uniformly positive for CD5 and showed weak staining for CD20, CD22, and surface Ig. B-ly7 proved to be the most specific marker, reacting with 100% of HCL cases, but absent in all cases of CLL. We conclude that two-color flow cytometry with specific antibody combinations is an efficacious method for characterization and sensitive detection of hairy cells in PB. Application of the phenotypic criteria described should help to increase accuracy in diagnosis of HCL.

Malignant lymphoma in Hawaii-Japanese: a retrospective morphologic survey.

A retrospective morphologic survey (1973-1983) of 146 cases of malignant lymphoma among the Hawaii-Japanese (migrant Japanese and their offspring) was conducted to determine whether differences in the incidence and cytologic types of malignant lymphoma exist when compared to those of native Japanese (lifetime residents of Japan). The age-adjusted incidence rates for malignant lymphoma among the Hawaii-Japanese were similar to rates for U.S. whites. However, higher rates for follicular centre cell (FCC) lymphoma with a follicular pattern were observed in the Hawaii-Japanese population when compared with rates for native Japanese. On the basis of the cytologic types of the Lukes-Collins classification, non-Hodgkin's lymphomas among the Hawaii-Japanese resembled those of Western countries, rather than those of Japan. B-cell lymphomas predominated (72 per cent), while T-cell types comprised 23 per cent of cases. Follicular centre cell types were encountered most often (59 per cent), and the small cleaved FCC subtype was the most common (30 per cent). The high degree of follicularity (29 per cent) was at variance with the consistently low rates reported in Japan. This may be explained, in part, by higher rates of nodal lymphomas among the Hawaii-Japanese. Of the T-cell lymphomas, diffuse large cell types (T-cell immunoblastic sarcoma, T-IBS), often with cytologic pleomorphism, were relatively frequently encountered (16 per cent), and comprised 15 per cent of non-Hodgkin's lymphomas; this observation necessitates special clinical and epidemiologic consideration in view of the large Japanese migration to Hawaii from HTLV-I endemic regions of southern Japan. No registered cases of non-Hodgkin's lymphoma or of Hodgkin's disease were documented in Hawaii-Japanese subjects under the age of 15 years. The age-adjusted incidence rates for Hodgkin's disease among the Hawaii-Japanese were similar with those of native Japanese. Nodular sclerosis was the most frequent histologic subtype. The difficulty in distinguishing between Hodgkin's disease and non-Hodgkin's lymphoma, particularly when immunologic cell surface markers are not available, is addressed. Low rates for chronic lymphocytic leukemia among the Hawaii-Japanese were confirmed. Not one well-documented case was identified in the 11-year period surveyed.

Lymphocyte immunophenotyping of B-cell lymphomas: a flow cytometric analysis of neoplastic and nonneoplastic cells in 271 cases.

We have reviewed our experience with 271 B-cell lymphomas to determine the effectiveness of flow cytometry in the characterization of these malignancies. Flow cytometric immunophenotyping of the total lymphocyte and/or large lymphocyte populations confirmed the morphologic diagnosis of B-cell lymphoma in 92% of cases, which included 79% monoclonal and 13% surface immunoglobulin (SIg)-negative lymphomas. Light chain monoclonality was most frequent in low grade and follicular center cell (FCC) lymphomas, while SIg-negative cases were most common in high grade and non-FCC types. Low grade lymphomas of all histologic types had high median percentages of neoplastic cells and low T-cell percentages. Conversely, high grade lymphomas exhibited lower and less uniform median percentages of B-cells, with higher numbers of T-cells and lower CD4/CD8 ratios than low grade lymphomas. Several differences in B- and T-cells were observed between specific high grade histologic types. FCC lymphomas with a diffuse pattern had lower percentages of CD4+ cells and lower CD4/CD8 ratios than cases with a follicular pattern. Thus, immunophenotypic differences were observed between histologic types or groups with known differences in clinical course and prognosis. We conclude that flow cytometry provides reliable information on neoplastic and nonneoplastic cells in lymph nodes involved by B-cell lymphomas.

Enzyme immunoassay for detection of monoclonal immunoglobulin in lymph nodes. A comparison with histologic and DNA gene rearrangement studies.

This article describes a 1-hour enzyme immunoassay (ELISA) for identifying lymph nodes in which a single immunoglobulin light chain isotype is abnormally predominant. Eighteen lymph nodes representing a variety of reactive hyperplasias and B-cell neoplasms were analyzed with the ELISA and submitted for B- and T-cell gene rearrangement studies at the CT beta, JH and J kappa (JK) loci. In all 18 specimens, there was agreement in the results of the ELISA, the histologic diagnoses, and the results of gene rearrangement studies. In three of the B-cell lymphomas identified by histologic, ELISA, and DNA studies, surface marker phenotyping by flow cytometry did not indicate the presence of a monoclonal cell population. It is concluded that the ELISA correlates well with DNA gene rearrangement studies and is a rapid, simple, and accurate method for identifying lymph nodes containing monoclonal immunoglobulin. Moreover, it is proposed that the ELISA has certain advantages over other methods for determining light chain clonality in clinical specimens, and it is therefore useful to distinguish between benign lymphoid hyperplasia and monoclonal B-cell neoplasms.

Small noncleaved follicular center cell lymphoma: Burkitt's and non-Burkitt's variants in the US. II. Pathologic and immunologic features.

The morphologic criteria for the two variants of small noncleaved follicular center cell (SNC FCC) lymphoma in the Lukes-Collins classification, Burkitt's (BL) and non-Burkitt's variants (NBL), were evaluated and related to the results of multiparameter laboratory and clinical studies. Forty-two patients were studied: 25 cases were classified as BL according to World Health Organization (WHO) criteria. Seventeen cases were classified as NBL on the basis of greater variability in nuclear size and shape, more prominent nucleoli, and greater variation in the amount of cytoplasm. Neoplastic follicles were present in three cases of BL and two of NBL, indicating an FCC origin for this lymphoma. Electron microscopic examination confirmed the light microscopic features. Immunoglobulin (Ig) monoclonality, as demonstrated by immunofluorescence (surface Ig) and/or immunoperoxidase staining for cytoplasmic immunoglobulin (CIg), was demonstrated in 21 of 24 (87.5%) of BL and 13 of 16 (71%) of NBL. Clinically, BL presented more frequently in extranodal sites and with gastrointestinal involvement than NBL. Bone marrow involvement was more common in NBL patients. Both groups had advanced stage disease at diagnosis. The median survival was 10.5 months in BL and 7.7 months in NBL. The results of this study indicate that BL and NBL are biologically related variants of SNC FCC lymphoma but have different presentations, which may be clinically significant.

Extensive deposition of eosinophil peroxidase in Hodgkin's and non-Hodgkin's lymphomas.

To determine whether occult eosinophil degranulation occurs in lymphomas, the authors performed immunohistologic and cytochemical studies on cryostat sections from 25 consecutive lymph node biopsies. A glucose-oxidase immunohistochemical technique was employed with a murine monoclonal antibody specific for human eosinophil peroxidase (EPO). In 7 cases of Hodgkin's disease, 3 cases of T-immunoblastic sarcoma, and 1 case of small cleaved follicular center cell lymphoma with sclerosis, there was extensive and striking extracellular deposition of EPO in a granular and fibrillar pattern within the connective tissue. Similar degranulation of eosinophils was not observed in any of the benign lymph node specimens or other B-cell lymphomas. It is concluded that eosinophils extensively degranulate and release EPO in some types of lymphoma.

Immunohistologic identification of phenotypic antigens associated with Hodgkin and Reed-Sternberg cells. A paraffin section study.

A combination of two monoclonal antibodies, designated LN-1 and LN-2, were used in an attempt to identify the corresponding antigens in Hodgkin and Reed-Sternberg cells. The LN-1 antibody has been shown in previous studies in our laboratories to identify follicular center cells, whereas LN-2 marks certain B-cell subpopulations as well as interfollicular histiocytes. Utilizing the peroxidase-antiperoxidase (PAP) technique, paraffin-embedded sections were examined representing 39 cases of various histologic subgroups of Hodgkin's lymphoma following immunostaining with LN-1, LN-2, and the antibody to S-100. Of these 39 cases, the LN-2 antibody was found to consistently mark the majority of Hodgkin and Reed-Sternberg cells. LN-1 was found to identify Hodgkin and Reed-Sternberg cells in a smaller number of cases. In no instance were Hodgkin or Reed-Sternberg cells found to mark for the S-100 protein. The use of LN-1 and LN-2 antibodies facilitated the identification of Hodgkin and Reed-Sternberg cells and produced additional information regarding the phenotypic nature of these cells.

Lymph-node infarction and malignant lymphoma: a multicentre survey of European, English and American cases.

A multicentre study of 51 cases of lymph-node infarction seen in the 30-year period 1956 to 1985 was conducted in order to assess both the short- and long-term prognostic implications of the condition. In 14 cases malignant lymphoma was found synchronously with the infarct. Of the remaining 37 patients with apparently 'benign' lymph-node infarction only six showed manifestations of malignant lymphoma in the follow-up time studied (mean = 48 months; range 1-156 months). These subsequent malignant lymphomas all occurred within 2 years of the lymph-node infarction. A postal enquiry and collation of other cases in the medical literature indicates that a minority (26 of 81) have developed malignant lymphoma, and that these lymphomas, too, have all appeared within 2 years. Thorough examination of both the infarcted lymph nodes and others resected at the same time is mandatory in order to exclude concomitant or underlying malignant lymphoma. Two years after lymph-node infarction the risk of malignant lymphoma is negligible.

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.

Large noncleaved follicular center cell lymphoma. Clinical features in 53 patients.

To clarify the clinical characteristics of large noncleaved lymphoma (LNC-FCC; intermediate grade, large cell, noncleaved, Working formulation), 53 patients were studied. Thirty-one were male and 22 female. Median age was 54 years. Initial symptoms included lymphadenopathy (40%), pain (34%), and B symptoms (21%). Stage I disease was present in 6, Stage II in 9, Stage III in 14, and Stage IV in 24 (72% Stage III or IV). Gastrointestinal (GI) tract involvement was present in 13. Central nervous system (CNS) disease was present at diagnosis in two patients, occurred during therapy in two, and was the sole site of relapse in two. Bone marrow involvement was found in 7 of 50 patients (14%). Complete remission was attained in 60% of all patients. Twenty-nine Stage III and IV patients received intensive multiagent chemotherapy; complete remission (CR) was attained in 69%. In contrast, zero of nine patients with Stage III or IV disease who did not receive an anthracycline-containing regimen, attained CR. Median survival for the entire group was 25 months. It was concluded that, in our patients with LNC-FCC, GI involvement was prominent (25%) and CNS disease was not uncommon (11%). Long-term disease-free survival may be achieved in patients with more advanced disease after the administration of anthracycline-containing combination chemotherapy.

Damage to secondary lymphoid follicles in AIDS-related persistent generalized lymphadenopathy, as revealed by the behaviour of dendritic reticulum cells possessing immunoreactive acid cysteine-proteinase inhibitor.

It has been shown previously that dendritic reticulum cells (DRC) in human secondary lymphoid follicles possess an immunoreactive acid cysteine-proteinase inhibitor (ACPI). In the present study, lymph nodes from 12 patients with AIDS-related persistent generalized lymphadenopathy (PGL) were investigated in order to detect whether or not any alterations occur in ACPI-immunoreactive DRC in this disorder. In the majority of PGL cases, profound alterations were found, the main characteristics of which were erosion, partial or total disruption of lymphoid follicles. However, similar though much less marked alterations were also found in some control cases. It is concluded that this type of follicular damage is a common and characteristic feature in PGL. It is not specific to PGL, however, but represents rather a special type of reaction in lymphatic tissue. The advantage of ACPI immunohistology for demonstrating the DRC pattern is that it can be performed on routinely fixed and paraffin-embedded tissues.

Retrovirus and malignant lymphoma in homosexual men.

We report malignant lymphoma in 27 homosexual men, of whom 22 had high-grade lymphomas (B-cell immunoblastic sarcoma or small non-cleaved lymphoma) and five had low-grade disease. Antibody to human T-cell lymphotropic virus type III (HTLV-III) was present in 13 (87%) of 15 with high-grade lymphoma and in two (40%) of five with low-grade disease. In contrast, only one (9%) of 11 "control" heterosexual patients with high-grade lymphoma had antibody to HTLV-III, while such antibody was found in none of 40 asymptomatic heterosexual controls and in 17 (55%) of 31 asymptomatic homosexual men. Of the homosexual lymphoma patients, 85% presented with disease in extranodal sites, including the central nervous system and rectum, and 81% had reversed T-helper/suppressor ratios. Median survival, despite treatment, is eight months. The acquired immunodeficiency syndrome-related lymphomas in homosexual men are extranodal, high-grade, B-lymphoid tumors, associated with exposure to HTLV-III and unusual clinical characteristics.

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.

Dendritic reticulum cells in AIDS-related lymphadenopathy.

One of two cases of acquired immune deficiency syndrome-related persistent generalized lymphadenopathy revealed a profoundly altered pattern of dendritic reticulum cells as demonstrated by immunoreactive acid cysteine proteinase inhibitor. The alterations could be related to totally or partially destructed lymphoid secondary follicles.

Monoclonal antibodies reactive with B-lymphocytes and histiocytes in paraffin sections.

Representative sections of normal lymph nodes, cases of reactive lymphadenopathy, and Hodgkin's disease were examined using two monoclonal antibodies reactive in paraffin sections using an immunoperoxidase technique. Antibody LN-1 recognizes B-lymphocytes in follicles; antibody LN-2 recognizes a broader spectrum of B-cells, and also shows positivity with some monocytes/histiocytes. The pattern of immunostaining with these antibodies is described with particular reference to Hodgkin The pattern of immunostaining with these antibodies is described with particular reference to Hodgkin and Reed-Sternberg cells. Some of the implications are discussed.

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.

Heterogeneity of response and survival in diffuse histiocytic lymphoma after BACOP therapy (bleomycin, doxorubicin, cyclophosphamide, vincristine, prednisone).

Diffuse 'histiocytic' lymphoma (DHL) is heterogeneous pathologically, consisting of four subtypes within Lukes-Collins; large-cleaved (LC), large non-cleaved (LNC), immunoblastic sarcoma of B cells (B-IBS), and immunoblastic sarcoma of T-cells (T-IBS). This heterogeneity is also recognized in the Cooperative Working Formulation on non-Hodgkin's lymphoma. Prior studies have suggested clinical heterogeneity of DHL as well, although conclusions were hampered by small numbers, and lack of therapeutic uniformity. We treated 57 patients with advanced DHL, using BACOP: 22 LNC, 16 T-IBS, 13 B-IBS, six LC. Complete remission rate for LNC was 64 per cent (14/22); B-IBS was 54 per cent (7/13); LC was 33 per cent (2/6); T-IBS was 25 per cent (4/16). (p = 0.10). Median survival for LNC was 27.8 months, B-IBS was 25.9, LC was 14, T-IBS was 12.0. The survival was significantly shorter for T-IBS patients when compared to the others (p = 0.01). By multi-variate analysis, histologic subtype (p = 0.02), age (p = 0.03), and stage (p = 0.06) were significant and independent prognostic variables in predicting survival. We conclude that LNC may respond the most favourably to BACOP, whereas alternative regimens appear necessary for patients with T-IBS.

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.

B- and T-lymphocytes in Hodgkin's disease. An immunohistochemical study utilizing heterologous and monoclonal antibodies.

Immunohistologic techniques utilizing monoclonal antibodies have made possible the identification of leukocytes by their phenotypic characteristics rather than by morphologic features alone. A panel of antibodies for T-lymphocyte, B-lymphocyte, and monocyte/histiocyte markers has been applied to frozen sections of nodular sclerosing and mixed cellularity Hodgkin's disease to identify more precisely the various cell types present in tissues involved in Hodgkin's disease. The majority of lymphocytes expressed detectable T-cell phenotypic markers, with a predominance of the "helper" phenotype (Leu 3/OKT4) in most cases. Lymphocytes reacting with anti-B-cell antibodies were also demonstrated; their distribution is described here and has not previously been reported. The anti-B- and anti-T-cell antibodies used in this study failed to give positive reactivity with Reed-Sternberg cells. However, one of the anti-monocyte antibodies (Leu-M1) reacted with diagnostic Reed-Sternberg cells in some cases. The patterns of staining observed varied widely within the two histologic types of Hodgkin's disease, leading to a conclusion that this disease may be more heterogeneous than is currently suspected.