Combined polymerase chain reaction approach for clonality detection in lymphoid neoplasms.

The present study analyzes the efficiency of a combination of four immunoglobulin heavy chain (IgH) gene polymerase chain reaction (PCR) primer systems and a multiplex T-cell receptor gamma chain (TRG) gene PCR for detection of clonality in 409 samples (234 paraffin sections, 175 bone marrow aspirates) of different lymphomas. Using the four IgH PCR systems together, clonality was detected in all samples of B-cell chronic lymphocytic leukemias, hairy cell leukemias, common acute lymphoblastic leukemias, and Burkitt-like B-cell lymphomas. Clonality was detected in all bone marrow aspirates with lymphoplasmacytoid immunocytoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and unclassifiable low-grade B-cell lymphomas. The combined IgH gene PCR approach allowed clonality detection in 78.2% of myelomas, 75% of Burkitt lymphomas, 74.4% of diffuse large B-cell lymphomas, 68.7% of follicular center lymphomas, 50% of posttransplant lymphomas, 28.6% of anaplastic large cell lymphomas, 29% of T-cell lymphomas, and 18.8% of Hodgkin diseases. The combination of the four IgH gene primer systems with the multiplex TRG gene PCR allowed detection of clonality in 84.2% of B-cell neoplasms, 92.1% of T-cell non-Hodgkin lymphomas, and 18.8% of Hodgkin diseases, which was much more efficient than single PCR protocols.     

Pax5 expression in non-Hodgkin's lymphomas and acute leukemias

The Pax5 gene encodes the B-cell-specific activator protein which is a key regulator in development and differentiation of B-cell. We studied the expression of Pax5 in hematologic malignancies to evaluate the diagnostic utility as a B cell marker. Materials included 70 B cell lymphomas, 26 T cell lymphomas, 53 acute leukemias, and 6 multiple myelomas (MMs). Representative areas from the paraffin embedded tissues were selected for tissue microarray, and the expressions of Pax5 was immunohistochemically evaluated. Pax5 was strongly expressed in most of the B cell lymphomas; 44 of 47 diffuse large B cell lymphomas (93.6%), 15 of 16 marginal zone B cell lymphomas (93.8%), all 3 mantle cell lymphomas, 2 follicular lymphomas, and 2 Burkitt's lymphomas (100%). However, Pax5 was expressed in only one of 26 T cell lymphomas. Among leukemias, it was expressed in 10 of the 14 B acute lymphocytic leukemias (ALLs) (72.4%), but also in 3 of the 6 T ALLs (50%), 13 of the 26 acute myelogenous leukemias (AMLs) (50%) and in all 3 ALL arising in chronic myelogenous leukemias and 4 mixed B ALL and AML. In MMs, Pax5 was negative in all cases. We concluded that Pax5 is very useful B cell marker in classification of lymphomas, but not of acute leukemias.     

Low-grade lymphomas. Expression of developmentally regulated B-cell antigens

A series of low-grade B-cell lymphomas was analyzed for a battery of immunologic determinants by flow cytometry and immunohistochemistry. Histologically distinctive subclasses of these lymphomas, well-differentiated lymphocytic (WDL), intermediately differentiated lymphocytic (IDL), and follicular center cell (FCC) lymphoma, were found to be readily distinguishable by their expression of immunologic determinants that are known to be developmentally regulated in normal B cells. Although all cases expressed monoclonal surface immunoglobulin (sIg), HLA-DR, and the surface membrane proteins recognized by antibodies B1 (p32) and BA1, staining with other monoclonal antibodies revealed unique immunologic phenotypes for each subclass: WDL p65 (Leu 1)+, p24 (BA2)-; IDL p65+, p24+; FCC p65-, p24-. Additionally, the fluorescence intensities (number of determinants per cell) obtained for sIg, BA-1, and B1, but not HLA-DR, were significantly different among the three lymphoma subclasses. The relative fluorescence intensities of each of these three markers followed the same pattern: FCC greater than IDL greater than WDL. Taken together, these distinguishing features suggest that low-grade B-cell lymphomas represent arrested, and possibly sequential, stages of B-cell differentiation.     

Immunological and cytogenetic studies in two cases of B-cell acute lymphoblastic leukemia (Burkitt's type)

Immunological and cytogenetic studies were performed on two patients who presented with L-3 acute lymphocytic leukemia (Burkitt-type). Surface marker studies showed that both had B-cell leukemias. The blast cells in Case 1 expressed monoclonal IgM kappa surface immunoglobulin and in Case 2, IgG kappa. In the first case, cytogenetic analysis of bone marrow revealed the presence of a rare variant translocation involving the short arm of chromosome 2 and the long arm of chromosome 8 in all the metaphases examined. This is the second report of such a translocation in Burkitt's leukemia. The 8;14 translocation reported in classical Burkitt's lymphoma and other B-cell lymphomas was present in all the bone marrow metaphases in the second case.     

Significance of lymphoglandular bodies in bone marrow aspiration smears

The presence of lymphoglandular bodies (LGB) or S?derstr?m bodies is often stated to be a feature of lymphoid processes. In our experience, LGB are typically identified in B-cell processes but not in T-cell lymphomas or myeloid leukemias. We reviewed 136 bone marrow aspirate smears. The number of LGB per five high-power fields was counted, and median counts for B-cell processes, non-B-cell processes, myeloid leukemias, and T-cell malignancies were obtained and compared by the Wilcoxon rank sum test. Bone marrow aspirate smears involved with B-cell malignancies contained a median of 30 (range, 1-250) LGB per five high-power fields. Compared to myeloid leukemias (median, 11; range, 1-253) and T-cell malignancies (median, 7; range, 0-41), the differences were statistically significant (P < 0.001 and P = 0.01, respectively). While lymphoglandular bodies can be seen in a variety of malignant hematopoietic and nonhematopoietic disorders, they are found in significantly greater numbers in B-cell malignancies.     

Intermediate lymphocytic lymphoma: an immunohistologic study with comparison to other lymphocytic lymphomas

In an immunohistologic analysis of 13 cases of intermediate lymphocytic lymphoma (ILL), the immunophenotype of ILL was compared to the immunophenotypes of other B-lymphocytic lymphomas and the normal lymphoid follicle to determine the normal cell in the scheme of B-cell differentiation that corresponds to ILL. The characteristic immunophenotype of ILL was surface IgM +/- D+, cytoplasmic immunoglobulin -, B1+, BA1+, B2-, BA2-, B4+, Leu 14+, HLA-DR+, Leu 1+, and common acute lymphoblastic leukemia associated (CALLA) antigen -. The immunophenotype of ILL was similar to that of lymphocytes in normal primary follicles and the mantle zones of secondary follicles. The "immature" phenotype of ILL was identical to that of small lymphocytic lymphoma, which strongly supports their close lineage relationship. In contrast, the "mature" phenotypes of the follicular center cell and lymphoplasmacytoid lymphomas suggest that they correspond to normal cells at later stages of differentiation. Our findings indicate that B-lymphocytic lymphomas recapitulate the normal stages of B-cell differentiation. The cell of ILL appears to be an immature B cell that homes to, and resides in, primary follicles and the mantle zones of secondary follicles. The cytologic, architectural, immunologic, and clinical features of ILL indicate that it should be included as a separate category in the International Working Formulation.     

B-cell neoplasms of the lymphocytic, lymphoplasmacytoid, and plasma cell types: immunohistologic analysis and clinical correlation

The relation between chronic lymphocytic leukemia (CLL, lymphocytic lymphoma (SL), plasmacytoid lymphocytic lymphoma (LP), plasmacytoma (PL), and multiple myeloma (MM) was investigated with cryostat sections stained with antibodies to immunoglobulin heavy and light chains and the B-cell differentiation antigens B1, B2, Ia, T1, and CALLA. Neoplasms were subclassified according to plasmacytoid features, leukemia (CLL) site of involvement (nodal or extranodal), serum monoclonal immunoglobulin, or clinical evidence of MM. The results defined two groups of lymphocytic lymphomas without plasmacytoid features (16 cases). Ten of these lymphomas were associated with CLL. Nine involved lymph nodes, all expressed IgM, five expressed IgD, nine were B2-positive, eight were T1-positive, and all were B1- and Ia-positive. Six of the lymphomas were not associated with CLL. Five of these tumors were extranodal, all were T1- B1+ B2- Ia+, five expressed IgM without IgD, and one contained IgG. These differences in clinical and immunologic phenotypes suggest that CLL and SL without CLL may be related to different stages of B-cell differentiation. T1 appeared to be a marker for CLL, since all T1-positive neoplasms were leukemic. Lymphomas with plasmacytoid features (ten cases) were more often extranodal, and none was leukemic. The immunologic phenotypes were heterogeneous: all of these lymphomas were T1-negative, most were IgM+ IgD-, three were B2-positive, and all were Ia-positive. The plasma cells in five lymphomas with marked plasmacytoid features were B1-negative; they were Ia-positive in four and Ia-negative in one. These data suggest that LP is a heterogeneous group, reflecting B cells at diverse stages of differentiation. Ten plasmacytomas, nine of which were associated with MM, differed from LP in showing heavy chain class switching; all were T1- B1- B2-, and all but one were Ia-negative. These results are consistent with the existence of two pathways or stages of B-cell differentiation: one that generates IgM-producing plasma cells, as seen in the primary immune response or in response to pokeweed mitogen, and one that generates IgG- or IgA-positive plasma cells, as seen in the late primary or secondary immune response. Plasmacytoid lymphocytic lymphoma reflects the first, while PL/MM reflects the second pathway. B1 appears to be lost before Ia in terminal plasma cell differentiation.     

Characterization of Non-Hodgkin''s Lymphomas Using Multiple Cell Markers: Immunologic, Morphologic, and Cytochemical Studies of 72 Cases

Tissues from 72 cases (87 specimens) of various non-Hodgkin's lymphomas were analyzed for cell markers using multiple techniques. Cell suspensions were evaluated for E, EAC, and IgGEA rosette forming cells; Fc receptor cells; and surface immunoglobulin bearing cells. Cryostat section studies topographically defined EAC binding cells. Cytochemical determinations and immunoperoxidase methods for detection of intracellular immunoglobulin and lysozyme complemented other techniques in evaluating infiltrates containing large neoplastic cells. B-cell malignancies comprised 58 cases (80%) of this series and included well and moderately well differentiated lymphocytic lymphomas (10/10); nodular (23/23) and diffuse (10/18) poorly differentiated lymphocytic lymphomas; and lymphomas of mixed lymphocytic-“histiocytic” (3/3), “undifferentiated” (3/3), and “histiocytic” (9/13) types. Nodular lymphomas were characterized as B-cell neoplasms but also revealed a prominent population of T lymphocytes (39 ± 12%). Alkaline phosphatase activity, a cytochemical marker for lymphoid cells of follicular cuffs, was most consistently observed in B-cell lymphomas of moderately well differentiated lymphocytic type (4/6 cases). In some diffuse lymphomas, cryostat section studies (EAC rosettes) suggested a pre-existing nodular proliferation. One unusual B-cell lymphoma of large cell type exhibited IgGEA rosette formation and a strong receptor for the Fc portion of IgG. Ten lymphomas (14%) were of T-cell type and were represented by cases of diffuse poorly differentiated lymphocytic lymphoma (5/18, including 3 lymphoblastic lymphomas), Sézary syndrome (1), mycosis fungoides (1), and a cytologically distinctive large cell (“histiocytic”) lymphoma (3/13). Acid phosphatase activity was a consistent marker for the T-cell malignancies, some of which also revealed α-naphthyl butyrate esterase activity. No true histiocytic lymphomas were detected. Three cases of diffuse poorly differentiated lymphocytic lymphoma and one “histiocytic” lymphoma were null.     

Cytokeratin expression in hematological neoplasms: a tissue microarray study on 866 lymphoma and leukemia cases

Aberrant expression of cytokeratins (CK) is known to occasionally occur in malignant lymphomas. The monoclonal mouse-anti-human CK cocktail CK22 recognizes keratin polypeptides with a wide range of molecular weights and can be applied in diagnostic panels for tumors of unknown origin. Using tissue microarray technology, we tested 1059 lymphoma and acute leukemia cases, covering the most common disease entities, for aberrant CK expression, using CK22. In total, 866 of the arrayed cases were evaluable (80%), and 13 positive cases (1.5%) were found: 1 out of 230 Hodgkin lymphomas (0.4%), 1 plasma cell myeloma, 2 out of 326 diffuse large B-cell lymphomas (0.6%), 5 out of 18 mantle cell lymphomas (26%), 3 out of 70 small cell lymphomas/chronic lymphocytic leukemias (4%) and 1 out of 27 peripheral T-cell lymphomas, not otherwise specified (4%). Immunostaining was finely granular in most cases, and the total amount of positively staining cells exceeded 10% only in the cases of Hodgkin lymphoma and plasmocytoma. All CK22-positive cases, except for one mantle cell lymphoma, expressed the specific simple epithelial CK8 but not the basal/stratified epithelial CK5/6. Aberrant CK expression can be encountered in a small subset of otherwise characteristic B- and T-cell lymphomas, but not in acute leukemias, which should be considered in difficult differential diagnostic settings.     

Position effect in translocation (2;8) in acute lymphocytic leukemia with kappa light chain immunoglobulin expression

We report a correlation between t(2;8) translocation in acute lymphocytic leukemia and kappa light chain immunoglobulin production. Since the kappa chain genes are on chromosome #2, this, as well as data on Burkitt lymphoma, points to the possibility of position effect on the level of gene action. Chromosome #2 in the translocation together with chromosome #8 is concerned with malignancy, while the normal homologous chromosome #2 transcribes kappa chains. This model applies to B cell leukemias and lymphomas with changes in chromosome #2 which will predictably express kappa chains. The model also applies to B-cell malignancies with changes in chromosome #22 which will predictably express lambda chains.     

Molecular and virologic characteristics of lymphoid malignancies in children with AIDS

PURPOSE: To characterize AIDS-associated lymphoid malignancies in children. PATIENTS AND METHODS: We studied lymphomas and B-cell leukemias from 25 children with AIDS for immunoglobulin heavy chain gene clonality, c-myc oncogene abnormalities, and presence of HIV and Epstein-Barr virus. RESULTS: Monoclonal immunoglobulin gene rearrangements were identified in 22 of 23 cases tested, the single exception being one of mucosa-associated lymphoid tissue. Immunoglobulin gene/c-myc translocations were found in 3 of 4 cases of B (surface immunoglobulin-positive)-acute lymphoblastic leukemia, 8 of 11 small noncleaved cell lymphomas, and 1 of 5 large cell lymphomas. Mutations of c-myc were found in 2 of 13 small noncleaved cell lymphomas, 1 of 2 Epstein-Barr virus-positive mucosa-associated lymphoid tissue neoplasms, and 1 of 4 Epstein-Barr virus-negative B-acute lymphoblastic leukemia. Six small noncleaved cell lymphomas, both mucosa-associated lymphoid tissue neoplasms and one of large cell lymphoma had high levels of Epstein-Barr virus in tumor tissue. Hodgkin's disease tissue and B-acute lymphoblastic leukemia tumors were negative for EBV. Proviral HIV-1 was not detected in any tumor. CONCLUSIONS: AIDS-associated lymphoid malignancies in children appear to have a different distribution of histologic subtypes than adult HIV-infected individuals, fewer large cell lymphomas occur in children. The small noncleaved cell lymphomas exhibit a lower frequency as well as different locations of c-myc mutations than AIDS-associated small noncleaved cell lymphomas in adults.     

Hairy cell leukemia. Diagnosis of bone marrow involvement in paraffin-embedded sections with monoclonal antibody DBA.44.

The new monoclonal antibody DBA.44 recognizes an unknown fixation-resistant B-cell differentiation antigen expressed by mantle zone lymphocytes, reactive immunoblasts, monocytoid B cells, and a small proportion of high- and low-grade lymphomas. Among node-based lymphomas, the strongest membrane staining was observed in centroblastic, immunoblastic, and monocytoid B-cell lymphomas. In studying bone marrow biopsy specimens from 166 patients with hairy cell leukemia, strong positive staining of surface membrane 'hairy' features of leukemic cells was observed in routinely fixed and decalcified bone marrow biopsy specimens of nearly all cases. The antibody distinguished hairy cell leukemia from the more common B-cell chronic lymphocytic leukemia and bone marrow infiltrates of typical lymph node-based lymphomas by immunomorphologic criteria. DBA.44 was valuable to (1) confirm the diagnosis of hairy cell leukemia, (2) estimate the bone marrow density of hairy cell leukemia before and after treatment, and (3) make the diagnosis of hairy cell leukemia in ambiguous cases, which are all properties that indicate its usefulness in the practice of diagnostic hematopathology.     

Lack of surface immunoglobulin light chain expression by flow cytometric immunophenotyping can help diagnose peripheral B-cell lymphoma

We determined the prevalence and significance of finding B cells without surface immunoglobulin (SIg) light chain expression. The flow cytometry database at Johns Hopkins Medical Institutions was searched for cases in which immunoglobulin light chain staining was performed to rule out a B-cell malignant neoplasm between January 1994 and February 2000. We excluded plasma cell dyscrasias, precursor B-cell acute lymphoblastic leukemia/lymphomas, and hematogones. Cases with more than 25% of B cells lacking SIg light chain expression were retrieved. Polymerase chain reaction assays for immunoglobulin heavy chain gene rearrangements were performed in SIg-negative cases with available tissue blocks. We identified 36 cases; all represented lymphoma. Their diagnoses included diffuse large B-cell lymphoma (20), HIV-related lymphoma (5), follicular lymphoma (5), Burkitt lymphoma (2), monomorphic posttransplant lymphoproliferative disorder (1), chronic lymphocytic leukemia/small lymphocytic lymphoma (1), marginal zone B-cell lymphoma (1), and low grade B-cell lymphoma (1). Of the 17 SIg-negative cases with amplifiable DNAs, 12 (71%) showed a clonal immunoglobulin heavy chain gene rearrangement. SIg-negative B-cell lymphomas are rare. Complete absence of SIg light chain expression in a mature B cell proliferation can be used as a surrogate marker to help diagnose peripheral B-cell lymphoma.     

Immunoglobulin expression in B-cell lymphoma. Immunohistochemical study of 345 cases.

Immunoglobulin expression was studied in a series of 345 cases of B-cell lymphoma by immunohistochemical studies and correlated with the histopathologic classification with the use of the Working Formulation. Immunoglobulin expression was present in 254 cases (74%) of B-cell lymphomas (IgM kappa, 122; IgM lambda, 82; light chain only, 40; mu heavy chain only, 10). Immunoglobulin expression occurred with the greatest frequency in lymphomas of small lymphocytic, small cleaved cell, and small noncleaved cell histologic types (93%, 100%, 100%, respectively) and occurred with the least frequency in lymphomas of large cell (cleaved and noncleaved) and immunoblastic histologic types (59% each). Other lymphomas demonstrated intermediate frequencies of immunoglobulin expression. An excess of cases expressing lambda light chain was noted overall (kappa-lambda ratio of 1.3; expected, 2.0) and was particularly evident for intermediate lymphocytic and follicular mixed histologic types (kappa-lambda ratios of 0.8 and 0.9, respectively). Immunoglobulin expression in B-cell lymphomas varies as a function of cellular differentiation as reflected in the histologic type and grade of the Working Formulation. An excess of cases expressing lambda light chain in specific histologic categories suggests the possibility that lymphocytes bearing the lambda light chain rearrangement may be more susceptible to certain types of lymphomatous transformation.     

Primary mediastinal non-Hodgkin's lymphomas: a morphologic and immunologic study of 19 cases

Nineteen, primary, non-lymphoblastic, non-Hodgkin's lymphomas were investigated by conventional morphologic studies as well as immunologic studies using the application of a battery of monoclonal antibodies to frozen tissue sections. Seventeen of the lymphomas were diffuse large cell; one was large cell immunoblastic and one was a follicular and diffuse lymphoma of intermediate differentiation. Thirty-seven percent of the lymphomas showed prominent sclerosis, sometimes associated with the superior vena cava syndrome. Six of the cases showed evidence of immunoglobulin production with light chain restriction. Twelve additional cases were shown to be of B-cell lineage by B1/T015 expression but did not show evidence of immunoglobulin production. One case was a T-cell lymphoma of helper phenotype. Ia expression was found in 14 of 18 cases studied.     

Diagnostic usefulness and prognostic impact of CD200 expression in lymphoid malignancies and plasma cell myeloma

The membrane glycoprotein MRC OX-2 (CD200) is expressed in several lymphoid malignancies. However, the diagnostic usefulness and potential prognostic importance of CD200 expression have not been rigorously examined. We show that CD200 is uniformly expressed in chronic lymphocytic leukemia (CLL) and absent in mantle cell lymphoma (MCL). It is important to note that expression of CD200 is retained even in CLLs with immunophenotypic aberrancies, making CD200 a particularly useful marker for discrimination between these cases and MCL. CD200 is expressed in nearly all precursor B-lymphoblastic leukemias, with aberrant overexpression or underexpression compared with normal B-cell progenitors in 55% of cases. More than 70% of plasma cell myelomas (PCMs) expressed CD200, and loss of CD200 expression in PCM may be associated with more clinically aggressive disease. CD200 is expressed in several hematolymphoid neoplasms. Analysis of its expression has several diagnostic and potentially prognostic applications in the flow cytometric evaluation of lymphoid malignancies.     

Expression of transmembrane adaptor protein PAG/Cbp in diffuse large B-cell lymphoma: immunohistochemical study of 73 cases

PAG/Cbp is a transmembrane adaptor protein involved in proximal immune signaling. It is expressed in reactive germinal centers (GC) of secondary lymphatic follicles and related malignant lymphomas. We studied PAG/Cbp expression in GC-like and non-GC-like diffuse large B-cell lymphoma (DLBCL) subtypes. Seventy-three cases of DLBCL identified among 155 malignant lymphomas were classified as GC-like DLBCL (CD10+ or CD10-, bcl-6+, and MUM1-) and non-GC-like DLBCL (CD10-, MUM1+ or CD10-, bcl-6+, MUM1+). PAG/Cbp was detected by monoclonal antibody MEM-255 following routine immunohistochemical procedures. Thirty-five of 40 GC-like DLBCLs (88%) and 20 of 33 non-GC-like DLBCL cases (61%) expressed PAG/Cbp. Four of 12 bcl-6-negative non-GC-like DLBCL cases (33%) were PAG/Cbp positive, and only 4 of 20 bcl-6-positive non-GC-like DLBCL cases (25%) were PAG/CBP negative. All 37 FL and all 5 Burkitt's lymphomas (BL) expressed PAG/Cbp, whereas all 6 mantle cell lymphomas (MCL) and 4 of 5 chronic lymphocytic leukemias (CLL/SLL) were PAG/Cbp negative. PAG/Cbp is a reliable GC marker. Its expression correlates with GC-like DLBC phenotype in a significant majority of cases. It is typically absent in MCL and SLL/CLL.     

Monoclonal antibody phenotyping of B-cell non-Hodgkin's lymphomas. The Southeastern Cancer Study Group experience

This report describes the experience of the Southeastern Cancer Study Group (SECSG) with the frozen-section immunoperoxidase phenotyping of 162 cases of B-lineage non-Hodgkin's lymphomas. The authors used a panel of 13 different markers with varying degrees of specificity for B lymphocytes and B-cell neoplasms. All lymphomas were classified according to the International Working Formulation. Several antibodies, including anti-immunoglobulin, B1, Leu 12, and Leu 14 were B-cell-specific markers that were generally pan-reactive. Several other monoclonal antibodies, however, were selectively reactive with subpopulations of B-cell lymphomas. Three "selective-B" antigens (BA1, p24, CALLA) were found on about half of the B-cell lymphomas tested, while another three (HB31, transferrin receptor, C3d receptor) were found on about two-thirds of the lymphomas tested. Leu 1 reacted with 18% of the B-cell lymphomas, particularly the small lymphocytic lymphomas. When the reactivity of the monoclonal antibodies was compared with the histologic classification, two important points became apparent. First, with the large panel of antibodies, there was tremendous phenotypic diversity even among histologically similar tumors. Second, however, not all possible combinations of antibody phenotypes were encountered. That is, clusters of antigenic phenotypes were seen, and these phenotypes correlated to some degree with the histologic diagnosis of the tumor. Small lymphocytic and follicular lymphomas tended to be phenotypically distinct, although there was some overlap. Intermediate- and high-grade lymphomas were phenotypically more diverse. The more common phenotypes of lymphomas encountered could not be reconciled with any simple linear scheme of neoplastic B-cell differentiation.     

Immunohistologic cellular phenotypes of lymphoproliferative disorders. Comprehensive evaluation of 564 cases including 257 non-Hodgkin's lymphomas classified by the International Working Formulation

The plethora of classifications for non-Hodgkin's lymphomas (NHLs) and controversy regarding the merits of the individual classification schemes has led to the articulation of an International Working Formulation for NHL classification by a working group sponsored by the National Cancer Institute. This classification is based on both architectural and cytologic features and has been shown to have clinical relevance, but it is not an immunologic approach. With the use of frozen sections and both polyclonal and monoclonal antibodies, a comprehensive immunohistologic study was made of 564 biopsy specimens 1) for determination of the utility of the principle of monoclonality in differentiating benign from malignant lymphoproliferative disorders, 2) for definition of the immunohistochemical phenotypes of histologically benign and malignant cellular proliferations, and 3) for evaluation of the immunologic phenotype of 257 non-Hodgkin's lymphomas classified by the International Working Formulation. Two hundred seven "reactive benign" lymphoproliferations demonstrated polyclonal immunostaining. Monoclonal kappa light chain immunostaining was demonstrated in 3 of 4 cases classified as atypical hyperplasia, two of which had coexistent NHL or subsequently developed overt NHL. Frozen tissue sections were found to be essential for demonstration of immunoglobulin and glycoprotein membrane antigens. The results of immunohistochemical studies were readily integrated with the International Formulation. Although diffuse mixed and small lymphocytic lymphomas were immunologically heterogeneous (both T- and B-cell), follicular lymphomas were invariably of B-cell type, and immunoblastic lymphomas originating from homogeneous T- and B-cell populations were identified.