Immunohistochemical staining for terminal deoxynucleotidyl transferase (TDT). An enhanced method in routinely processed formalin-fixed tissue sections

This report describes an improved technique for sensitive and specific localization of terminal deoxynucleotidyl transferase (TdT) in routinely processed paraffin-embedded, formalin-fixed tissue sections using DNAse pretreatment and the avidin-biotin complex (ABC) technique. This method is useful in identifying lymphoblastic lymphomas (14/15 cases positive), with all other B- and T-cell lymphomas tested negative for the reaction. Used in conjunction with monoclonal antibodies immunoreactive for T- and B-cells in paraffin sections this technique should prove helpful in immunophenotyping malignant lymphomas where fresh tissue is unavailable for study.     

An immunocytochemical study of T-cell lymphomas using monoclonal and polyclonal antibodies effective in routinely fixed wax embedded tissues

Formalin fixed and paraffin wax embedded tissue from 24 cases of T-cell lymphoma diagnosed using immunocytochemistry on cryostat sections was examined using a panel of eight monoclonal and three polyclonal antisera. The monoclonal antibodies UCHL1 and MT1 proved to be comparable and reliable markers of neoplastic cells in T-cell lymphomas. The B-cell specific marker, MB1, strongly stained all cells in two cases of pleomorphic large cell T-cell lymphoma, large cells in two cases of pleomorphic mixed medium and large cell lymphoma, and isolated clusters of blast cells in four cases of T-zone and angioimmunoblastic lymphadenopathy-like T-cell lymphoma. The cells stained by MB1 expressed T suppressor/cytotoxic surface markers on frozen section. Epithelial membrane antigen, as detected by a polyclonal anti-EMA and the monoclonal antibody HMFG2, was expressed in 36% of tumours especially those of monomorphic large cell and pleomorphic large cell phenotype. Single granules or finely dispersed cytoplasmic granularity was seen in four tumours using the anti-granulocyte reagent Leu M1. Tumour cells in one case stained in a pattern identical to Reed-Sternberg cells in Hodgkin's disease. Granular alpha-1-antitrypsin staining was found in 10 cases of pleomorphic large cell and monomorphic large cell lymphoma. No staining was observed using anti-lysozyme or the monoclonal macrophage specific marker Mac411. Monomorphic and pleomorphic large cell lymphomas tended to show a common immunophenotype with the majority of cells co-expressing alpha-1-antitrypsin HLA-DR and epithelial membrane antigen. Scattered large transformed blast cells in cases of angioimmunoblastic lymphadenopathy-like T-cell lymphomas and T-zone lymphomas shared a similar immunophenotype with the large cell lymphomas. Using a panel of monoclonal antibodies effective in paraffin embedded tissue, diagnostically useful staining profiles which correlate with the morphological phenotype can be established in T-cell lymphomas.     

Heterogeneity of CD3 antigen expression in T-cell lymphoma

CD 3 antigen expression was studied in a series of 98 T-cell lymphomas, using polyclonal antibodies which recognize this molecule in routinely processed, paraffin-embedded, tissue. We identified 40 cases in which CD3 was present on only a proportion of the neoplastic cells. This phenomenon of heterogeneous CD3 expression was commonest in pleomorphic T-cell lymphomas (22/42 cases) and in CD30 (Ki-1)-positive lymphomas (5/11 cases), and was less frequently observed in mycosis fungoides (4/18 cases) and not seen in T-cell lymphoblastic lymphoma (0/9 cases). CD3 expression was often related to cell morphology, with CD3 antigen being present on the smaller neoplastic cells but absent from the larger ones. The diagnostic significance of these observations is that, on occasion, it may be possible to diagnose a lymphoma as being of T-cell origin in paraffin sections by demonstrating a minor subpopulation of CD3-positive neoplastic cells.     

Emmprin, a cell surface inducer of matrix metalloproteinases (MMPs), is expressed in T-cell lymphomas

Degradation of the extracellular matrix by matrix metalloproteinases (MMPs) is a crucial step in tumour invasion and metastasis. In human carcinomas, tumour cell-fibroblast interactions (TFIs) have been demonstrated to play a role in the up-regulation of MMP levels in tumours, and emmprin is a surface molecule on tumour cells that stimulates nearby fibroblasts to produce MMP-1, 2, and 3. T-cell lymphomas frequently show extranodal organ involvement and skin invasion, but a role for TFIs in their invasion has not been examined in detail. This study investigated TFIs in T-cell lymphomas with special reference to emmprin expression and MMP production. Immunohistochemically, only germinal centre cells and some histiocytes expressed emmprin in non-neoplastic lymph nodes (ten cases), while all T-cell lymphomas [14 cases of adult T-cell leukaemia/lymphoma (ATLL), six cases of lymphoblastic lymphoma, seven cases of anaplastic large cell lymphoma, and nine cases of angio-immunoblastic T-cell lymphoma] expressed emmprin strongly and diffusely. FACS analysis of peripheral blood from normal individuals revealed that small fractions of B-cells, T-cells, and monocytes expressed emmprin, whereas emmprin-expressing T-cells were much increased in number, and expressed this protein to a higher level, in ATLL patients. In vitro co-cultures of emmprin-positive HTLV-1-transformed lymphocytes (MT-2) and emmprin-negative human fibroblasts enhanced the production of pro-MMP-2 (gelatinase A) and active MMP-2, compared with cultures of either cell type alone. This stimulation was inhibited by an activity-blocking peptide against emmprin. Moreover, in histopathological sections from patients with ATL skin involvement, MMP-2 was demonstrated in fibroblasts around infiltrating ATL cells, but not in fibroblasts in non-diseased areas. In conclusion, emmprin is overexpressed by T-lymphoma cells, when compared with normal counterparts, and facilitates MMP-2 production via interactions with fibroblasts, which could play a role in stromal invasion by lymphoma cells.     

Diagnosis of T-cell lymphoma using beta F1, anti-T-cell receptor beta chain antibody

The reactivity of a new monoclonal antibody to the T-cell beta chain antigen receptor (beta F1) with routinely processed paraffin sections from patients with T-cell lymphoma is described. Staining of tumour cells was seen in 36/47 cases of T-cell lymphoma. No staining was seen in any cases of B-cell lymphoma (0/21 cases), nine of which had previously been shown to react with other T-cell antibodies (MT1/UCHL1). We conclude that beta F1 is a specific marker for demonstrating a T-cell histogenesis of lymphoma and with advantages over other currently available antibodies reactive with paraffin sections.     

Specific phenotyping of T-cell proliferations in formalin-fixed paraffin-embedded tissues. Use of antibodies to the T-cell receptor beta F1.

Antibody beta F1 to a common framework determinant of the beta subunit of the T-cell receptor (TCR) was used as a specific phenotypic marker for T-cell differentiation in malignant lymphomas. Sensitivity of immunoperoxidase staining in paraffin sections was enhanced by pronase pretreatment, overnight incubation of primary antibody in Tween 20, and use of streptavidin horseradish peroxidase complexes to amplify the reaction. All 43 cases of B-cell lymphoma were negative for TCR. Reed Sternberg (RS) cells in 3 of 20 cases of Hodgkin's disease exhibited cell membrane staining for TCR (all nodular sclerosis type), further evidence that some RS cells may be T-cell derived. Twenty-nine of 44 cases of T-cell lymphoma expressed TCR (66%). These included 11 of 12 cases of peripheral T-cell lymphoma (PTCL) of small and mixed cell type, 8 of 9 cases of lymphoepithelioid cell (Lennert's) lymphoma, and 2 of 4 cases of T-cell lymphoblastic lymphoma. Loss of immunoreactivity for TCR occurred in lymphomas of large or activated T-cell type, including 7 of 9 cases of T-cell immunoblastic lymphoma and 3 of 4 cases of large cell PTCL. Antibody beta F1 is a specific and relatively sensitive marker of T-cell phenotype in formalin-fixed paraffin sections of malignant lymphomas.     

Expression of DNA-PKcs and Ku86, but not Ku70, differs between lymphoid malignancies

We have investigated the role of DNA-dependent protein kinase (DNA-PK) and related it to proliferation and maturation of different lymphoid malignancies. DNA-PK and Ki-67 protein content was investigated in tumour samples of lymphoid malignancies, obtained from patients with low- and high-grade lymphomas, acute lymphoblastic leukaemia and multiple myeloma. All patients were untreated before sampling. Normal bone marrow, reactive tonsillar tissue and ordinary lymph node tissue were used as controls. We show here that lymphoid malignancies display differences in DNA-PK protein expression. Low-grade lymphoma, appearing as chronic lymphocytic leukaemia (CLL) displayed a significantly lower frequency of cells staining positive for DNA-PKcs and Ku86, but surprisingly not for Ku70, compared with acute lymphoblastic leukaemia (ALL) cells. When material from individual CLL patients was investigated, cells from lymph nodes showed a higher frequency of positive cells with respect to all DNA-PK subunits, compared with CLL cells infiltrating the bone marrow. High-grade lymphoma lymph node samples showed an increased frequency of cells staining positive for DNA-PKcs, Ku86 and Ki-67 compared with lymph node samples from low-grade lymphoma patients. Again, no difference in the Ku70 levels between the two lymphoma entities was noted. In multiple myeloma, the frequency of cells with positive staining for DNA-PKcs was similar to that detected in ALL and high-grade lymphoma. We conclude that with the exception of multiple myeloma, expression of DNA-PK coincides with the degree of maturation of lymphoid malignancies. In low- and high-grade lymphoma, DNA-PK is associated with the proliferation rate.     

Leu-M1 antigen expression in acute leukaemias

Leu-M1 is a differentiation antigen present in human myelomonocytic cells. Seventy-seven acute leukaemias were retrospectively stained with anti-Leu-M1 using the immunoperoxidase technique on Bouin-fixed paraffin-embedded sections. The subjects were 44 acute lymphoblastic leukaemias (ALL) and 33 acute myeloid leukaemias (AML) previously characterized by cytochemical and immunologic (cell suspension) methods. Leu-M1 was positive in all the AML and in half of the ALL cases. These results suggest that Leu-M1 does not allow differentiation between AML and ALL. For the ALL cases Leu-M1 was positive in 15/28 B-cell types, 4/12 T-cell type and 3/4 'null'-cell type cases. Thus, this antibody is of no assistance in defining types B, T, or 'null' in ALL. Leu-M1 was also studied on paraffin sections of 34 high grade malignant lymphomas. The antibody was negative in all 13 B-cell lymphomas (lymphoblastic: 6; immunoblastic: 7) and in all 4 'null' cell lymphomas. It was positive in 4/9 peripheral T-cell type, the other T-cell lymphomas (lymphoblastic: 5; immunoblastic: 3) remaining negative. Thus, Leu-M1 may be positive in T-cell lymphomas but it is negative in B-cell lymphomas and is always negative in B or T lymphoblastic types. It seems that lymphoblasts are Leu-M1 negative in non-Hodgkin's lymphoma and may be Leu-M1 positive in leukaemias.     

Terminal deoxynucleotidyl transferase in non-Hodgkin's lymphoma

To investigate the specificity of terminal deoxynucleotidyl transferase (TdT) as a marker for lymphoblastic lymphoma in the non-Hodgkin's lymphomas, the authors assayed malignant cells from 142 patients with non-Hodgkin's malignant lymphoma, as well as cells from normal lymphoid tissue controls. Neoplastic cells from all 25 patients with lymphoblastic lymphoma were TdT positive. Of the remaining 117 patients with malignant lymphoma of other histologic subtypes, only one patient, with diffuse, large cell immunoblastic lymphoma, had TdT-positive cells. This patient's cells had positive results only by the enzyme assay and were TdT-negative by both immunofluorescence and immunoperoxidase assays. Of 56 patients whose cells were studied by both enzyme assay and an immunoassay method for TdT detection, a discordant result was obtained only on cells from the previously mentioned patient with large cell immunoblastic lymphoma. To determine the optimal immunoassay method, the authors studied malignant cells from 24 patients with non-Hodgkin's lymphoma for the presence of TdT by three different methods: immunofluorescence, peroxidase-antiperoxidase, and avidin-biotin-complex immunoperoxidase. Fifteen of the 24 patients had lymphoblastic lymphoma. The avidin-biotin-complex method produced superior staining, with no false-negative results. False-negative results were seen with both the immunofluorescence and peroxidase-antiperoxidase methods. Other advantages of the avidin-biotin-complex method included a marked decrease in background and non-specific staining and the ability to use higher dilutions of antisera.     

Clinicopathological analysis of 143 primary malignant lymphomas in the small and large intestines based on the new WHO classification

AIM: To study the clinicopathological and immunohistochemical features of 143 cases of primary small and large intestinal non-Hodgkin's lymphoma (NHL) in Japanese patients who presented between 1981 and 2000. METHODS AND RESULTS: The new World Health Organization (WHO) classification was used to classify NHL. The patients included 109 males and 34 females, with an average age of 54.1 years. Tumour sites were as follows: ileocaecal (n = 51, 35.7%), ileum (n = 29, 20.3%), rectum (n = 13, 9.1%), and duodenum (n = 11, 7.7%). Macroscopically, 124 cases (86.7%) were classified as tumorous type, 12 (8.4%) as diffuse infiltration type (erosion, superficial ulceration), five (3.5%) as polyposis type, and only two cases (1.4%) as ulceration type. Immunohistochemically, 122 lesions (85.3%) were of B-cell phenotype and 21 lesions (14.7%) were of T-cell phenotype. According to the WHO classification, of the B-cell lymphomas, 84 cases (68.9%) were large cell, 16 (13.1%) were Burkitt, 10 (8.2%) were marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT), and seven (5.7%) were mantle cell tumours. Among the T-cell lymphomas, 15 (71.4%) were of unspecified type, two (9.5%) were natural killer type, two were anaplastic large-cell lymphomas, one was lymphoblastic, and one was an adult T-cell leukaemia lymphoma. The survival rate for T-cell lymphomas was poorer than for B-cell lymphomas. Among the B-cell lymphomas, mantle cell lymphoma tended to have a poorer prognosis, whereas MALT lymphomas had a better prognosis than other B-cell tumour types. CONCLUSIONS: Our retrospective study of patients with primary malignant lymphomas in the small and large intestines has illustrated the clinical features and outcomes of patients with this disease.     

Primary malignant lymphomas of the central nervous system: a histological and immunohistological study of 12 cases

Paraffin sections of surgical and autopsy material from 12 cases of primary non-Hodgkin's lymphomas of the central nervous system were examined for histopathological diagnosis and for the demonstration of cytoplasmic immunoglobulins. According to the Kiel classification, there were five cases of lymphoplasmacytoid polymorphous lymphoma, five of immunoblastic lymphoma, one of lymphoblastic lymphoma of convoluted cell type. There was also one of the recently described multilobated lymphoma. An immunohistological study of light and heavy chains by peroxidase-antiperoxidase (PAP) technique and avidin-biotin complex (ABC)technique was performed. Intracellular immunoglobulins were demonstrated in seven cases: four cases were classified as immunoblastic lymphomas and three cases as lymphoplasmacytoid lymphomas. Negative immunoglobulin staining was observed in five cases: two lymphoplasmacytoid lymphomas, one immunoblastic, one lymphoblastic of convoluted cell type and one multilobated. A 'monoclonal' pattern of immunoglobulin staining was detected in six cases. One case, classified as immunoblastic lymphoma, showed 'bitypic' staining for kappa and lambda chains. It was concluded that primary CNS non-Hodgkin's lymphomas of the present series showed morphological and immunohistological features similar to those of malignant lymphomas arising in extraneural sites. In particular, the presence in our series of a multilobated lymphoma, as a primary CNS tumour, is emphasized.     

Immunoperoxidase detection of CD10 in Precursor T-lymphoblastic lymphoma/leukemia: a clinicopathologic study of 24 cases

CONTEXT: CD10 was originally reported in non-T-cell lymphoblastic lymphomas/leukemias. It has since been identified, however, in a minority of cases of T-lympho-blastic lymphoma/leukemia and other hematopoietic and nonhematopoietic entities. The usual method for the detection of CD10 previously required fresh tissue. A new antibody for CD10 (56C6) in paraffin embedded tissue sections, however, has recently become available. OBJECTIVE: To study the expression of CD10 in paraffin sections of T-lymphoblastic lymphoma/leukemia using monoclonal antibody 56C6. DESIGN: Twenty-four cases of T-lymphoblastic lymphoma/leukemia in various anatomic sites were studied. Immunohistochemical analysis with CD10 and a panel of other hematolymphoid antibodies was performed in all 24 cases. Gene rearrangement studies for the T-cell receptor by the polymerase chain reaction were performed in 18 of 24 cases. RESULTS: All cases were positive with at least 2 T-cell markers. In 15 (63%) of 24 cases CD10 was positive. T-cell receptor gene rearrangement was detected in 10 of 18 cases. CONCLUSIONS: Immunodetection of CD10 in T-lympho-blastic lymphoma/leukemia using monoclonal antibody 56C6 is common. This finding is useful in the evaluation of T-cell neoplasms.     

High-grade non-Hodgkin''s lymphoma of B-cell type. I. Histopathology

One hundred and twenty-eight cases of high-grade malignant B-cell lymphoma were studied with a plastic re-embedding technique and classified according to the Kiel classification. The cytological details could be better recognized than in the original paraffin sections, thus permitting a more precise definition of the various lymphoma types. The entities centroblastic and immunoblastic lymphoma are more precisely defined and supplemented by the addition of several new variants. In contrast to the present Kiel classification we separate Burkitt's from lymphoblastic lymphoma. In all cases investigated, the B-cell nature of the tumour cells was proven by immunohistochemistry using monoclonal antibodies. The four entities of high-grade malignant B-cell lymphoma described in this paper are: (1) centroblastic lymphoma with four morphological variants (monomorphic, polymorphic, multilobated and centrocytoid); (2) immunoblastic lymphoma with three morphological variants (with or without plasmacytic differentiation, with many lymphocytes); (3) Burkitt's lymphoma and the closely related Burkitt's lymphoma-like lymphoma with plasmablastic differentiation; and (4) lymphoblastic lymphoma. Only the centroblastic lymphomas (in 17%) showed occasional follicular growth pattern, which further confirms the view that they are derived from germinal centre cells.     

Immunocytochemical study of 12E7 in small round-cell tumours of childhood: an assessment of its sensitivity and specificity

12E7 is a monoclonal antibody to the MIC2 gene product and can be applied to formalin-fixed, paraffin-embedded tissue. The diagnostic utility of 12E7 as a marker of Ewing's sarcoma and peripheral neuroectodermal tumour was assessed. Immunocytochemical studies were performed on 120 small round-cell tumours from children and adolescents. Immunoreactivity for 12E7 was seen in 13 of 15 Ewing's sarcomas, 14 of 15 peripheral neuroectodermal tumours, four of 14 embryonal rhabdomyosarcomas, seven of 11 T-lymphoblastic lymphomas and one T-cell acute lymphoblastic leukaemia. Immunoreactivity was located on the cell-membrane of Ewing's sarcomas, peripheral neuroectodermal tumours and lymphoid tumours while rhabdomyosarcomas showed weak, cytoplasmic staining in differentiated rhabdomyoblasts. Studies on alveolar rhabdomyosarcomas ( n = 10), acute myeloid leukaemias (3), B-lymphoblastic lymphomas (8), blastema-rich nephroblastomas (9), neuroblastomas (20) and retinoblastomas (10) as well as single examples of B-cell acute lymphoblastic leukaemia, Ki-1 anaplastic lymphoma of indeterminate phenotype and intra-abdominal desmoplastic tumour with divergent differentiation were negative. 12E7 is a sensitive marker for the Ewing's sarcoma/peripheral neuroectodermal group of tumours and is useful in distinguishing them from neuroblastoma and blastema-rich nephroblastoma. However, immunopositivity for 12E7 should be interpreted in conjunction with the results of neural and lymphoid markers.     

CD30 (Ki-1) expression in adult T-cell leukaemia/lymphoma is associated with distinctive immunohistological and clinical characteristics

Twenty-one patients with CD30 (Ki-1) positive lymphoma were studied from a group of 91 patients with adult T-cell leukaemia/lymphoma. The patients were grouped into three types: diffuse CD30 positive anaplastic large cell lymphoma in 11 patients (group 1); pleomorphic type lymphoma with diffuse CD30 expression in five patients (group 2); and pleomorphic type lymphoma with positive CD30 expression in large cells but negative in medium-sized and small cells in five patients (group 3). The patients with diffuse CD30 positive lymphomas (groups 1, 2) frequently presented with extranodal tumours (68.8%) and lymph node enlargement greater than 2 cm in diameter (50%), and rarely with leukaemic changes, bone marrow involvement and hypercalcaemia (one case of each). Patients in group 3 rarely had extranodal tumours, but had frequent leukaemic changes. Expression of intercellular adhesion molecule (ICAM-1; CD54) by the lymphoma cells in 13 patients (81.3%) with diffuse CD30 positive lymphomas, was significantly higher than that in 33 patients (9.1%) with CD30 negative adult T-cell leukaemia/lymphomas. No positive reaction for epithelial membrane antigen (EMA) was found in the lymphoma cells of CD30 positive cases. The overall survival in patients with diffuse CD30 positive lymphomas was better than that of CD30 negative adult T-cell leukaemia/lymphoma patients, but showed no significant difference. These findings suggest that diffuse CD30 positive adult T-cell leukaemia/lymphoma has unusual clinical and immunohistological findings. It is also speculated that local tumour formation and leukaemic changes in such diffuse CD30 positive cases are influenced by CD54 (ICAM-1) expression by the lymphoma cells.     

Immunohistochemical staining of non-Hodgkin's lymphoma with monoclonal antibodies specific for the leucocyte common antigen

Forty cases of non-Hodgkin's lymphoma (NHL) have been stained with monoclonal antibodies (F8-11-13, F-10-89-4 and Dako LC) to the Leucocyte Common Antigen (LC) in both cryostat and paraffin sections with an immunoperoxidase technique. In cryostat sections all B-cell lymphomas (25/25) reacted with F10-89-4 and Dako LC, whilst the majority (23/25) stained with F8-11-13; of the T-cell lymphomas studied, all reacted with F10-89-4 (6/60 and Dako LC (4/4), however 2/6 did not react with F8-11-13. Similar variability in reaction was observed in malignant histiocytosis where 1/3 did not react with F8-11-13 whilst all three reacted with F10-89-4 and Dako LC. In paraffin sections (using the two MCabs F8-11-13 and Dako LC) three of the 25 B-cell lymphomas failed to stain with either F8-11-13 or Dako LC (one lymphocytic lymphoma and two lymphomas showing plasmacytic differentiation). The remainder of the B-cell lymphomas reacted with both antibodies. Six out of 12 T-cell lymphomas did not stain with either F8-11-13 or Dako LC, using our standard immunoperoxidase procedure. Staining with Dako LC was however detected in all cases of T-cell lymphoma when incubation with primary antibody was extended from thirty minutes (standard) to overnight. This study confirmed that LC can be detected in all NHL in cryostat sections, and that in the majority of B-cell NHL the higher molecular weight component of LC was demonstrable using F8-11-13. Difficulty in detecting LC determinants after tissue processing for paraffin sections in a number of cases of NHL, especially those of T-cell type or showing plasmacytic differentiation, suggests that lack of reaction with these antibodies does not always preclude the diagnosis of lymphoma.     

Monoclonal antibody HML-1 reactivity with adult T-cell leukaemia/lymphoma and other lymphomas

Human T-cell leukaemia/lymphoma virus type 1 (HTLV-1), a causative virus of adult T-cell leukaemia/lymphoma (ATLL), is known to be transmitted by breast-feeding. Using a monoclonal antibody HML-1 which labels human intestinal intra-epithelial T lymphocytes, we have immunohistochemically examined ATLL tissues in order to evaluate the possibility that HTLV-1 infected intestinal T cells are the origin of ATLL cells. Previously this antibody was reported to react with intestinal T-cell malignant lymphomas but not with peripheral tumours, or any B-cell lymphomas. We investigated 181 patients with malignant lymphomas and found that 19 out of 113 ATLLs were positive for HML-1. T-cell malignant lymphomas excluding ATLL also reacted with HML-1 (7/24), but all the B-cell lymphomas 0/33) and non-neoplastic lymph node and skin lesions (0/10) were negative for HML-1. In patients with ATLL and other T-cell malignant lymphomas, the positivity level of HML-1 was relatively higher in stomach (3/7) and tonsil (2/6) than that in lymph nodes (15/100) and skin (8/47). We observed one HML-1 positive ATLL patient with tumour formation in the skin and lymphadenopathy and marked infiltration of the large intestine but minimal involvement of other organs. Although HML-1 was frequently expressed in gastric infiltration of ATLL, the level of positivity was too low in lymph nodes to support the hypothesis that HTLV-1 infected intestinal T cells are the origin of ATLL cells. Some of the HML-1 positive ATLL cases co-expressed CD30. Furthermore, three of six cases of Ki-1 lymphoma (large anaplastic cell lymphoma) were positive for HML-1. We conclude that expression of HML-1 in ATLL reflects an activated state of the lymphoma cells, but not the intestinal origin of ATLL cells.     

CD69 expression correlates with expression of other markers of Th1 T cell differentiation in peripheral T cell lymphomas

CD69, a marker of early T cell activation, is associated with Th1 T cell differentiation. Previously we found that peripheral T cell lymphomas could be subdivided based on the expression of markers of Th1 versus Th2 differentiation, including CXCR3, CD134/OX40, CCR4, and CD30. Here we report immunohistochemical staining for CD69 in frozen and paraffin sections of peripheral T cell lymphomas that exhibit immunoreactivity for markers of Th1 or Th2 differentiation. CD69 expression correlated with immunoreactivity for other Th1 differentiation markers in 18 of 19 frozen specimens of peripheral T cell lymphomas (P = 0.0005). In 10 of these cases in which paraffin-embedded tissue was available for study, CD69 immunohistochemical staining of paraffin sections correlated with frozen section expression. CD69 immunostaining was performed on paraffin sections from 53 additional cases of peripheral T cell lymphoma and correlated with immunoreactivity for other Th1 differentiation markers (P < 0.0001) and was associated with specific subtypes of peripheral T cell lymphoma, including angioimmunoblastic lymphoma, Lennert's lymphoma, and mycosis fungoides/Sezary syndrome, previously noted to express Th1 differentiation-associated markers. Anaplastic large cell lymphoma, both systemic and cutaneous, which typically exhibits immunoreactivity for markers of Th2 expression, was negative for CD69 immunostaining in 22 of 24 cases. CD69 immunostaining results support previous findings that a subset of T cell lymphomas exhibits immunophenotypic features of either Th1 or Th2 T cell differentiation. In addition, CD69 is a useful immunohistochemical marker for specific T cell lymphomas in frozen and paraffin-embedded tissue.     

Detection of Epstein–Barr virus small RNAs in routine paraffin sections using non-isotopic RNA/RNA in situ hybridization

The Epstein–Barr virus (EBV) is associated with an increasing range of reactive and neoplastic lesions. There is a need for a sensitive and specific method for detecting latent EBV in routine histological sections. We report the use of a highly sensitive paraffin section RNA/RNA in situ hybridization (ISH) technique using digoxigenin-labelled antisense riboprobes for demonstrating EBV encoded-small RNAs (EBERs), EBV gene products that are transcribed in abundance during latent EBV infection. We applied EBER-ISH to 846 paraffin embedded specimens, including cases of reactive lymphoid hyperplasia ( n = 28), infectious mononucleosis (16), Burkitt's lymphoma (44), immunodeficiency-associated lymphomas in transplant recipients (9) and AIDS patients (128), Hodgkin's disease (130), CD30 antigen positive lymphomas (106), peripheral T-cell lymphomas (104), sporadic B-cell non-Hodgkin's lymphomas (162), undifferentiated nasopharyngeal carcinoma (86), salivary gland lymphoepithelioma (11), and oral hairy leukoplakia (5). Strong, reproducible EBER staining was seen in EBV latently infected cells in archival surgical biopsy and autopsy specimens. EBER-ISH is specific, has a sensitivity comparable to that of the polymerase chain reaction, and is now the method of choice for the in situ detection of latent EBV infection.     

T-cell lymphoma in Hashimoto's thyroiditis

An elderly Caucasian woman with a 2-year-history of hypothyroidism, treated with thyroxine, presented with a rapidly growing mass in the thyroid. The morphological and immunological features of this thyroid tumour were those of a peripheral T-cell lymphoma with an immunophenotype commonly associated with HTLV-1 positive-adult T-cell leukaemia/lymphoma, although serology for HTLV1 antibody was negative. Monoclonal gene rearrangements were demonstrated with T-cell receptor beta- and gamma-specific primers. There are several interesting features in this case (i): although primary B-cell lymphomas (MALT-associated lymphomas) of thyroid are a well-recognized sequel to thyroiditis, primary T-cell lymphomas are rare, even in areas of the world where adult T-cell lymphomas predominate; (ii) the tumour showed the typical immunophenotype of an HTLV-1 positive T-cell lymphoma but the patient is English, has not visited endemic areas, and is serologically negative for HTLV–1; (iii) the residual thyroid gland showed a florid lymphocytic thyroiditis with Hu¨rthle cell change, typical of Hashimoto's thyroiditis; (iv) unlike other reports of thyroid T-cell lymphoma, which have presented with stage III-IV disease, this tumour presented in the favourable clinical stage of IE.