Immunophenotyping of Nigerian cases of non-Hodgkin''s lymphomas on paraffin sections

One hundred cases of routinely fixed and processed non-Hodgkin's lymphoma from Nigeria were immunostained with a small panel of monoclonal antibodies against B-, T- and macrophage antigens. The aims of the study were to assess the suitability of stored material from a country like Nigeria for immunohistochemical examination and the ability of the antibody panel to evaluate the distribution of B- and T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. There were no tumours of macrophage lineage. Four cases gave satisfactory staining of reactive lymphoid cells but no reactivity with malignant cells and thus were not phenotyped. The remaining nine cases gave no staining of neoplastic or reactive cells, suggesting that they were unsuitable for immunohistochemical study, presumably because of inappropriate fixation and handling. We concluded that a panel of three monoclonal antibodies is suitable for routine immunostaining of conventionally fixed and processed blocks in Third World countries and will give diagnostically useful information in approximately 95% of cases.     

Immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue sections. A comparison with genotypic analysis.

Several monoclonal antibodies (MoAbs) are now available for immunophenotyping non-Hodgkin's lymphomas (NHLs) in paraffin-embedded tissue sections. To determine the reliability of these reagents in predicting the genotype, 44 cases of NHL were studied with the alkaline phosphatase-anti-alkaline phosphatase technique with the use of the following MoAbs: leukocyte common antigen (CD45), Mac 387, L26, 4KB5, MB1, MB2, LN2, UCHL1, MT1, and MT2. The lineage of the neoplastic cells was determined in all cases by gene rearrangement studies for immunoglobulin heavy chain and for the T-cell receptor beta-chain. Genotypic results showed B-cell lineage in 33 cases (75%), T-cell lineage in 6 cases (14%), and mixed or undetermined lineage in 5 cases (11%). A concordance of lineage assignment by paraffin section immunophenotyping with gene rearrangement studies was observed in 37 of 39 (95%) lymphomas with an unequivocally defined genotype. MoAb L26 was the most sensitive in detecting B-cell genotype; MoAbs MT1 and UCHL1 were the most sensitive and specific, respectively, in detecting T-cell genotype. The authors conclude that lineage assignment of NHLs in paraffin sections is reflective of the corresponding genotype when an appropriate panel of MoAbs is used.     

Immunophenotyping of acute leukemias using paraffin-embedded tissue sections

In a study of 55 patients with either acute lymphoid leukemia (ALL; 25 cases) or acute myeloid leukemia (AML; 30 cases), paraffin-embedded bone marrow particle sections were examined with a panel of monoclonal and polyclonal antibodies reactive toward lymphoid and myeloid-associated antigens, using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. All cases were previously classified according to the French-American-British (FAB) Co-operative Group, and cases of ALL were immunophenotyped by flow cytometry. Results indicated that myeloid-associated antibodies (Mac 387, KP 1 [CD68], antielastase, antilactoferrin, and antilysozyme) did not react with any case of ALL, M1-AML, or M6-AML, whereas at least one of these antibodies reacted with 20 of 21 (95%) cases of M2, M3, M4, and M5-AML. Anti-glycophorin C marked cases of M6-AML, whereas anti-CD3 labeled T-cell ALL. None of the antibodies tested specifically identified cases of B-cell ALL. The authors conclude that use of a selected panel of antibodies on paraffin-embedded bone marrow particle sections may be of value in the diagnosis and immunophenotypic classification of many cases of acute leukemias.     

Differentiating Ewing's sarcoma from other round blue cell tumors using a RT-PCR translocation panel on formalin-fixed paraffin-embedded tissues.

Ewing's sarcoma is a common malignancy of bone and soft tissue that occurs most often in children and young adults. Differentiating Ewing's sarcoma from other round blue cell tumors can be a diagnostic challenge because of their similarity in histology and clinical presentation. Thus, ancillary molecular tests for detecting disease-defining translocations are important for confirming the diagnosis. We analyzed 65 round blue cell tumors, including 53 Ewing's sarcoma samples from 50 unique cases. Samples were processed for RNA from archived formalin-fixed paraffin-embedded tissue blocks. Real-time RT-PCR assays specific for Ewing's sarcoma (EWS-FLI1, EWS-ERG, EWS-ETV1, EWS-ETV4, and EWS-FEV), synovial sarcoma (SYT-SSX1 and SYT-SSX2), and rhabdomyosarcoma (PAX3-FKHR and PAX7-FKHR) were tested across the samples. The translocation panel had a sensitivity of 81% (43 out of 53 samples) for diagnosing Ewing's sarcoma when using the histological criteria as the 'gold' standard. None of the Ewing's specific translocations were found in the non-Ewing's samples (100% specificity). Of the 43 samples with translocations detected, 26 (60%) had an EWS-FLI1 type 1 translocation, 13 (30%) had an EWS-FLI1 type 2 translocation, 3 (7%) had an EWS-ERG translocation, 1 had an EWS-ETV1 translocation, and 1 sample had both an EWS-FLI1 type 1 and type 2 translocation. Our real-time RT-PCR assay for detecting sarcoma translocations has high sensitivity and specificity for Ewing's sarcoma and has clinical utility in differentiating small round blue cell tumors in the clinical lab.     

Immunologic surface markers in non-Hodgkin''s lymphomas.

Tissues from 21 patients with non-Hodgkin's lymphomas were examined for immunologic cell surface markers. Patterns of distribution of complement receptor (CR) B lymphocytes and Fc receptor (FcR)-bearing histiocytes in tumor tissue were evaluated and compared to routine histologic preparations of the tumors and to normal tissue. The lymphomatous infiltrates from all 6 cases of nodular, poorly differentiated lymphocytic lymphoma (NPDLL) consisted of dense populations of CR B lymphocytes. Involved tissue from 7 of 8 patients with diffuse, poorly differentiated lymphocytic lymphoma (DPDLL) was predominately comprised of CR B lymphocytes. Discrete nodules of CR B cells were present in a lymph node replaced by DPDLL. FcR were identified on the cells from 1 of 3 cases of histiocytic lymphoma. None of the 4 cases of undifferentiated lymphoma possessed demonstrable surface markers in tissue section; however, the cell suspension from 1 case contained a high percentage of CR B cells. Both CR and T cell markers were present on the cells of DPDLL of childhood.     

Prognostic importance of DNA flow cytometry in non-Hodgkin''s lymphomas.

Eighty one cases of non-Hodgkin's lymphoma were examined by DNA flow cytometry, using fixed embedded histological tissue. The frequency of detection of DNA aneuploidy and the values for S phase fractions depended on the histological subtype and grade of lymphoma. Twenty two of the patients with low grade centroblastic/centrocytic non-Hodgkin's lymphoma had repeat biopsies. Eleven of these patients remained histologically and cytometrically stable, but the remaining eleven transformed into high grade non-Hodgkin's lymphoma. The mean value for the S phase fraction in the initial biopsy specimens from patients which transformed was higher than that for patients whose lymphomas remained stable (p less than 0.001). It is proposed that estimates of S phase fraction prospectively identify patients with low grade non-Hodgkin's lymphoma at risk from transformation.     

Nature of non-B, non-T lymphomas: an immunohistological study on frozen tissues using monoclonal antibodies

In a previous study employing conventional immunological marker analysis we found that 17% of high grade malignant lymphomas were devoid of cytoplasmic and membrane immunoglobulin and also sheep erythrocyte receptors. Cryostat sections from 24 of these cases (four of low grade and 20 of high grade malignancy) were stained with a panel of 30 monoclonal antibodies and six polyclonal antisera using a sensitive immunoperoxidase method. All tumours expressed the leucocyte common antigen (detected by monoclonal antibody 2D1) and all lacked epithelial cytokeratin (monoclonal antibody LE61), confirming their haematopoietic origin. All but one of the lymphomas expressed antigens characteristic of either B cells (17 cases) or T cells (six cases), while one case (morphologically a centroblastic lymphoma) had an unusual dual phenotype in which strong staining for T6 (marker of immature T cells) was associated with expression of the pan B lymphocyte antigens detectable with To15, anti-B1, anti-Leu12. This case was therefore classified as a B cell lymphoma showing aberrant expression of the T6 antigen. The pan B cell antibodies (To15, anti-B1, anti-Leu12) all appeared highly specific and sensitive, but the simultaneous use of all three monoclonal antibodies was necessary to detect the B cell nature in each of the 18 lymphomas. A wider panel of monoclonal antibodies was required to detect T lymphomas since these often disclosed atypical and restricted phenotypes. To15 and UCHT1 were the most reliable antibodies for the detection of B and T cell neoplasms, respectively. We conclude that most, if not all, "non-B, non-T" lymphomas are of either B or T lymphocyte origin and that monoclonal antibodies provide indispensable tools in their classification and diagnosis.     

Laboratory strategies for efficient handling of paraffin-embedded tissues for molecular detection of clonality in non-hodgkin lymphomas.

We herein present a technical strategy to optimize DNA isolation from paraffin-embedded tissue (PET). This includes the choice of adequate buffers for proteinase K digestion and multiplex PCR amplifications for assessing the appropriateness of DNA extracts for subsequent PCR assays for detecting clonality. We found that the association of proteinase K digestion in nonionic buffer and subsequent extract dilutions accounted for 79% of successful amplifications. A final efficiency of 88% was achieved by additional organic extractions and/or re-extractions. Comparisons were carried out with control DNA extracts from fresh samples to assess the efficiency of each clonality assay. Immunoglobulin CDRIII rearranged region amplification was more efficient for pregerminal center B-cell lymphomas in contrast to CDRII rearrangement detection, which was more effective for germinal and postgerminal lymphomas. T-cell clonality detection by TCRgamma PCR was less efficient in PET samples than in fresh tissues showing that DNA integrity is more critical for TCR than for IGH amplification. Two inconclusive cases without phenotypic markers and two other atypical lymphoproliferations masked by reactive T cells were diagnosed as plasmablastic lymphomas and as monoclonal B-proliferations, respectively, due to IGH rearrangements.     

Use of monoclonal antibodies for analyzing the distribution of the intermediate filament protein vimentin in human non-Hodgkin''s lymphomas.

A series of human non-Hodgkin's lymphomas was examined for immunoreactivity with monoclonal antibodies to the intermediate filament protein vimentin with the use of an avidin-biotin immunoperoxidase method. The lymphoid cell nature of each tumor was established with the use of a panel of monoclonal antibodies to lymphoid cell differentiation antigens. There were 28 B-cell and 2 T-cell lymphomas in the series; of the 30 tumors, 11 (37%) were immunoreactive for vimentin. There was no correlation between vimentin immunoreactivity and the histopathologic type of lymphoma. In some tumors, there was nonspecific stromal immunoreactivity for vimentin, but the neoplastic lymphocytes were not immunoreactive. The selective expression of vimentin in non-Hodgkin's lymphomas may be due to masking of the appropriate epitopes or to selective expression of the vimentin gene in certain tumors. On the basis of these results, monoclonal antibodies to vimentin appear to be of limited usefulness in establishing the diagnosis of non-Hodgkin's lymphoma.     

Pathological variables determining the prognosis of non-Hodgkin''s lymphomas

The aim of the study was to assess the role of pathological grade, cell proliferation, ploidy, immunophenotype and site in determining the prognosis of non-Hodgkin's lymphomas. Of particular interest was the relative value of grades derived from the Kiel classification as opposed to the National Cancer Institute (NCI) working formulation. The study consisted of 181 cases, treated in a relatively uniform way over an 18-month period spanning 1986. Using life table analysis, both NCI working formulation grade and Kiel grade correlated strongly with survival. However, the differences between grades were entirely due to an excess of early deaths in the high-grade and intermediate-grade categories. In patients surviving greater than 0.1 years (37 days), phenotype, site, ploidy and cell proliferation had no effect on survival. There was no evidence that intermediate-grade tumours, when subdivided into Kiel low- and high-grade types, differed in survival from tumours graded as low- or high-grade by both methods. However, NCI working, formulation high-grade tumours, especially those with a high proliferation rate, formed a group with a very high likelihood of death within 0.1 years.     

Epitope mapping of HBsAg using a panel of human anti-HBs antibodies.

Mapping of B cell epitopes on HBsAg was performed using a panel of human anti-HBs antibodies. Synthetic peptides representing different regions of HBsAg failed to inhibit the binding of two antibodies which recognized non-conformational HBsAg determinants in dot-blot ELISA and HBsAg polypeptide bands in immunoblot analysis. Cross-inhibition studies using five of the antibodies conjugated to horseradish peroxidase suggested that at least three different epitopes are recognised by the panel of antibodies, two of which are within the 'a' group determinant.     

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.     

Morphologic and immunophenotypic characterization of primary brain lymphomas using paraffin-embedded tissue

Primary lymphomas of the brain constitute about 1% of all primary intracranial neoplasms, but recent studies suggest an increasing incidence. Most cases are associated with an immunosuppressed state. We reviewed 29 cases of primary brain lymphoma from the Yorkshire Health Authority Region between 1970 and 1988 and found a striking increase in incidence over this period. No overt evidence of immunosuppression was found in any case. All were non-Hodgkin's in type and were classified morphologically using Kiel criteria and immunophenotypically using a panel of antibodies. Cryo-preserved tissue was available in five cases for parallel immunophenotyping. The majority of tumours were high-grade lymphomas together with three of lymphoplasmacytoid type. Thirteen tumours showed a striking pleomorphic morphology with plasmacytoid features. A reactive, predominantly perivascular monomorphic T-cell population was seen in all tumours. Most tumours were of B-cell lineage. No cases of Hodgkin's disease, T-cell or histiocytic lymphoma were present. Light chain restriction was present in only 46% of cases. The results of tumour immunophenotyping on cryostat sections were comparable with those from paraffin blocks. Our study emphasizes the value of a panel of antibodies reactive in paraffin-embedded tissue, allowing simultaneous evaluation of morphology and immunophenotype, and suitable for small biopsies received from stereotactic procedures.     

Cytokeratin expression and vimentin content in large cell anaplastic lymphomas and other non-Hodgkin''s lymphomas.

The immunophenotypes of 74 malignant lymphomas (9 Hodgkin's disease, 19 low-grade B-cell, 20 high-grade B-cell, 8 T-cell, and 18 large cell anaplastic lymphomas [LCAL]) have been characterized with antibodies against leucocyte differentiation antigens, keratin, and vimentin. All the non-LCAL were CD45 positive and keratin negative. The LCALs had a more varied immunophenotype, with CD45 present only in 11 of 18 cases and keratin present in 5 of 18 of these rare lymphomas. The lymphoid origin of these latter cases was proven by gene rearrangement studies. All LCALs were CD30+, and, where tested, vimentin positive. Of four different vimentin monoclonal antibodies tested, V9 and MVI stained the highest number of lymphomas. Positive staining of tumor cells was seen in 61 of 71 cases. Vimentin-negative cases included Burkitt's as well as some follicular lymphomas.     

Analysis of large-cell lymphomas using monoclonal and heterologous antibodies.

Fifteen cases of large-cell lymphoma, diagnosed as centroblastic (5), B-immunoblastic (5) or true histiocytic (5). lymphoma and one case of malignant histiocytosis were studied with monoclonal antibodies. Each diagnosis was based on morphological as well as marker studies. A panel of monoclonal and heterologous antibodies against T lymphocyte differentiation antigens (Leul, Leu2a, Leu3a, OKT4, OKT8, TA1), B lymphocyte subsets (BA1, BA2, HLA-DR, alpha C3b receptor antiserum, surface immunoglobulins), the common acute lymphoblastic leukaemia antigen (CALLA), monocytes/macrophages (OKM1, anti-human monocyte 1, TA1, Mac1, HLA-DR, anti-C3b receptor), myeloid cells (VIM-D5, elastase, OKM1) and the cells of the Langerhans cell/interdigitating reticulum cell series (OKT6, NA1/34). The results show a specific staining pattern for true histiocytic lymphoma (histiocytic sarcoma). Centroblastic and B-immunoblastic lymphomas showed gradual differences with mostly strong staining for HLA-DR and weak with anti C3b receptor for B-immunoblastic lymphomas in contrast to centroblastic lymphomas. Staining with BA1 and BA2 indicated immunological heterogeneity in these lymphomas. The number of admixed cells was usually low with few B cells and a shift in the ratio helper/inducer to suppressor/cytotoxic T cells in favour of the suppressor/cytotoxic subset.     

Immunophenotyping large B-cell lymphomas. Flow cytometric pitfalls and pathologic correlation

Large cell lymphomas often challenge the diagnostic flow cytometrist. The purposes of this study were to improve our protocols for diagnosing large cell lymphomas and to correlate flow cytometric (FC) data with demographic and histologic features. We identified 63 cases of large B-cell lymphoma between January 1, 1995, and July 30, 1999, and reviewed the diagnostic slides and FC light scatter and staining patterns. The 51 lymphomas with adequate material for systemic review fell into 2 light scatter patterns: "clear cut," with large abnormal cells (high forward scatter relative to normal lymphocytes), 17 cases (33%); and "complex," 34 cases (67%). Clear-cut cases were more mitotically active (average of 42 vs 25 per 10 high-power fields), with higher cellularity. Apoptosis, geographic necrosis, and sclerosis were present histologically in many cases, regardless of FC findings. We conclude that morphologic features of large cell lymphomas do not predict which cases will be difficult to diagnose by FC. Gating strategies can be critical to improve the diagnostic yield.     

Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies

There is considerable confusion in the literature about the size of the myotonic dystrophy protein kinase (DMPK) and its localization within tissues. We have used a new panel of monoclonal antibodies (mAbs) to begin to resolve these issues, which are important for understanding the possible role of DMPK in myotonic dystrophy. Antisera raised against the catalytic and coil domains of DMPK recognized a major 55 kDa protein and a minor 72-80 kDa doublet on western blots of human skeletal muscle. Ten mAbs, five against the catalytic domain and five against the coil region, recognized only the 72-80 kDa doublet. The 72 kDa protein was present in all tissues tested, whereas the 80 kDa component was variably expressed, mainly in skeletal and cardiac muscles. The 72 kDa protein was absent in a DMPK knockout mouse and was greatly increased in a transgenic mouse overexpressing human DMPK, confirming its identity as authentic DMPK. Two mAbs against the catalytic domain recognized only the more abundant 55 kDa protein, which was found only in skeletal muscle. Nine out of 10 mAbs located DMPK to intercalated discs in human heart, an affected tissue in myotonic dystrophy. However, co-localization of DMPK with acetylcholine receptors at neuromuscular junctions was not observed with any of the mAbs. Subcellular fractionation and sedimentation analysis suggest that a major proportion of the DMPK in skeletal muscle and brain is cytosolic.