E- to N-cadherin switch in melanoma is associated with decreased expression of phosphatase and tensin homolog and cancer progression.

BACKGROUND: Cadherin switch in melanoma, with loss of E-cadherin and upregulation of N-cadherin, is believed to underlie melanoma cell detachment from the epidermis and promotion of dermal and vascular melanoma invasion. The tumour suppressor phosphatase and tensin homolog (PTEN) has been suggested as a potential regulator of this cadherin switch. OBJECTIVES: To study the biological and clinical implications of cadherin switch and PTEN expression in melanoma progression. METHODS: We constructed tissue microarrays from primary tumour samples from 394 formalin-fixed paraffin-embedded melanomas diagnosed between 2001 and 2006. Median follow-up was 10 years. Tissue microarray sections were stained by immunohistochemistry for E-cadherin, N-cadherin and PTEN, and expression was analysed semiquantitatively. RESULTS: Breslow thickness correlated strongly with reduced/absent PTEN expression (P < 0·0001), low E-cadherin expression (P < 0·0001), high N-cadherin expression (P < 0·0001) and the combination of low E-cadherin and high N-cadherin expression (cadherin switch profile; P = 0·001). There was a significant association between reduced/absent PTEN and the presence of the cadherin switch profile (P = 0·03). In univariate analyses, low E-cadherin expression significantly predicted an adverse overall relapse-free (P = 0·04), melanoma-specific (P = 0·03) and distant-metastasis-free (P = 0·01) survival; reduced/absent PTEN predicted an adverse overall relapse-free survival (P = 0·006), and the cadherin switch profile predicted adverse melanoma-specific (P = 0·005) and distant-metastasis-free (P = 0·01) survival. In multivariate analysis, the cadherin switch profile was an independent prognostic marker of melanoma-specific (P = 0·04) and distant-metastasis-free survival (P = 0·02). CONCLUSIONS: Cadherin switch and reduced/absent PTEN expression are associated in melanoma, and both factors may play important roles in the progression of melanoma.

HIV-associated lymphoma: histopathology and association with Epstein-Barr virus genome related to clinical, immunological and prognostic features.

All 51 cases of HIV-related malignant lymphoma in Denmark diagnosed from 1983 to 1989 were reviewed. There were 12 Burkitt-type lymphomas, 30 immunoblast-rich lymphomas and 9 other lymphomas. Patients with immunoblast-rich lymphomas had significantly lower CD4 cell counts (median 60 vs. 188 x 10(6)/l, P less than 0.05), and more often a history of previous AIDS-defining illnesses (50% vs. 0%, P less than 0.005), compared with patients with Burkitt-type lymphomas. Epstein-Barr virus (EBV) DNA was demonstrated in 14 of 19 immunoblast-rich tumours, and in 2 of 7 Burkitt-type lymphomas (P = 0.10). Compared with EBV DNA-negative tumours EBV DNA-positive tumours were associated with lower CD4 cell counts (median 39 vs. 188 x 10(6)/l, P = 0.01). It is concluded that two main types of HIV-related malignant lymphoma exist. One is associated with severe immunosuppression, is often of immunoblast-rich morphology, and may be linked to EBV, whereas the other may occur in the absence of immunosuppression, is often of Burkitt-type morphology, and is probably not linked to EBV. In addition to these two main types, other non-Hodgkin lymphomas and Hodgkin's disease do occur.

Preventing acute rejection, Epstein-Barr virus infection, and posttransplant lymphoproliferative disorders after kidney transplantation: use of aciclovir and mycophenolate mofetil in a steroid-free immunosuppressive protocol.

BACKGROUND: A widely held view is that any increase in the potency of an immunosuppressive agent will lead to an increase in infection and malignancy, such as life-threatening Epstein-Barr virus (EBV) induced posttransplant lymphoproliferative disorders (PTLD). We tested this paradigm by studying the effect of adding mofetil to a steroid-free protocol under cover of high-dose aciclovir prophylaxis on the number of acute rejections, EBV infections and PTLDs after kidney transplantation. METHODS: EBV serology was performed in 267 consecutive renal transplantations (1990-1997). All were treated with cyclosporine with an initial 10-day antilymphocyte globulin course, supplemented from September 1995 with MMF. In 208 consecutive transplantations after June 1992 aciclovir 3200 mg/day was given for 3 months posttransplantation. RESULTS: After an observation period of up to 7 years we found that: (1) primary or reactivated EBV infection (PREBV) was correlated to acute rejection (treated with OKT3; P<0.00005) and to the incidence of PTLD (P=0.03; P=0.01, if Hodgkin's disease is included); (2) aciclovir protected against PREBV (P<0.00005) and (3) adding mofetil to the immunosuppressive protocol reduced PREBV further (P=0.0001), (4) in 78 transplantations treated with cyclosporine/antilymphocyte globulin/mofetil we observed only 10 acute rejections (P=0.0001), 10 PREBVs (P<0.00005), and no PTLDs compared with the cyclosporine/antilymphocyte globulin group (P=0.04). CONCLUSIONS: Supplemental immunosuppression with mofetil protects against acute rejection. In combination with aciclovir, there is also a reduction in the number of PREBVs, apparently as a result of both direct viral prophylaxis and better rejection control, and in the incidence of EBV-induced PTLD.

Undifferentiated carcinoma of the salivary gland in Greenlandic Eskimos: demonstration of Epstein-Barr virus DNA by in situ nucleic acid hybridization.

Paraffin sections of 11 undifferentiated salivary gland carcinomas of lymphoepithelioma type (malignant lymphoepithelial lesion) arising in Greenlandic Eskimos (Inuit) were examined for the presence of Epstein-Barr virus (EBV) using in situ nucleic acid hybridization with a 35S-labeled EBV-specific probe. Epstein-Barr virus genomes were detected in each case in malignant epithelial cells, but were not found in lymphoid stroma or in residual benign salivary epithelium. Eight undifferentiated salivary gland carcinomas from non-Eskimo patients (including two with lymphoepithelioma-like features) were negative for EBV-DNA. Our results confirm the existence of a consistent and specific association between EBV and tumor cells of undifferentiated salivary gland carcinoma of lymphoepithelioma type arising in Greenlandic Eskimos.

B cell associated monoclonal antibody L26 may occasionally label T cell lymphomas.

Monoclonal antibody L26 has been shown to be a very sensitive marker for B lymphocytes in formalin-fixed, paraffin-embedded tissue. Most studies have found that the antibody is also highly specific for B cells, although a few examples of L26-positive T cell lymphoma (TCL) have been reported. We have studied L26 reactivity in 50 TCLs (all previously extensively immunophenotyped on frozen sections) and found positive labelling in 4 cases (3 pleomorphic, medium and large cell type with surface membrane staining; 1 T-anaplastic large cell type with cytoplasmic staining). The finding that L26 may give surface labelling in occasional TCLs (particularly of the pleomorphic, medium and large cell type) indistinguishable from that seen in B cell lymphomas emphasises the importance of always using diagnostic MAbs in combination if the risk of misinterpretation of lymphoma cell lineage is to be minimised.

The expression of placental alkaline phosphatase (PLAP) and PLAP-like enzymes in normal and neoplastic human tissues. An immunohistological survey using monoclonal antibodies.

The immunohistological expression of placental alkaline phosphatase (PLAP) and PLAP-like enzyme was studied in frozen sections from a wide variety (n = 254) of normal and malignant tissues using monoclonal antibodies reactive with PLAP (H317) and PLAP/PLAP-like enzyme (H17E2; H315). PLAP/PLAP-like reactivity was seen in normal thymus, and foetal and neonatal testis, and in 21 out of 22 malignant germ cell tumours (GCTs), but was also found in normal endocervix, normal Fallopian tube and in 28 out of 167 non-GCTs (particularly in ovarian and proximal gastrointestinal tract tumours). Positivity for true PLAP (as demonstrated with H317) was seen in term placenta, in endocervix, and in Fallopian tube (but not in other normal tissues) and was commonly found in ovarian and proximal gastrointestinal tract tumours. Reactivity with H317 was unusual in malignant GCTs (2 out of 22 cases). These findings confirm that PLAP/PLAP-like positivity is a highly sensitive immunohistological marker for malignant GCTs, but one which by itself is of only moderate specificity. Furthermore, expression of true PLAP is rare in GCTs and favours instead an origin from the ovary or proximal gastrointestinal tract. The results also indicate that the predominant heat-stable alkaline phosphatase species in normal foetal and neonatal testis, and in thymus has a similar immunohistological profile to that found in malignant GCTs, and is a PLAP-like enzyme ("germ cell alkaline phosphatase") distinct from true PLAP. The occurrence of this marker in GCTs would appear to reflect increased eutopic production of an enzyme present in trace amount in corresponding normal tissues rather than a genuine example of ectopic expression.

Acute tubulointerstitial nephritis: phenotype of infiltrating cells and prognostic impact of tubulitis.

The prognostic impact of tubulitis and the phenotype of the infiltrating cells in the tubules were studied in ten percutaneous renal biopsies from six patients with acute tubulointerstitial nephritis (ATIN). The inflammatory cell subsets in the tubules and interstitium (CD3+, CD4+, CD8+, CD20+, CD45RO+, CD56+, CD57+, CD68+ and TIA-1+ cells), the expression of vimentin and the proliferation-associated antigen Ki-67 by cortical tubular cells, and the grade of tubulitis, interstitial infiltration and fibrosis were analysed. Cytotoxic injury to tubular cells in the vicinity of tubular-wall-localized lymphocytes was studied ultrastructurally. ATIN was drug-induced in three patients, related to Legionella infection in two and idiopathic in one patient. Four patients recovered, one with reduced renal function. Two patients developed end-stage renal disease. CD8+ and CD4+ lymphocytes, and a smaller number of macrophages, infiltrated the tubules. The predominant lymphocyte subset in the tubules was the same as in the interstitium. Cytotoxic injury to tubular cells was not seen electron microscopically. The tubular cells exhibited increased proliferative activity and expressed vimentin, indicating non-specific tubular damage. The cell subset, the severity of tubulitis, and the tubular expression of vimentin were not related to outcome. The main prognostic factor was the severity of the interstitial fibrosis. Tubulitis in ATIN may be a harmless non-immune reaction, mediated by tubular expression of cytokines, together with adhesion and other molecules.

Detection of Epstein-Barr virus genomes in AIDS related lymphomas: sensitivity and specificity of in situ hybridisation compared with Southern blotting.

Eighteen cases of AIDS related, non-Hodgkin's lymphomas were examined for the presence of Epstein-Barr virus (EBV) genomes using in situ hybridisation with a 35S-labelled probe. The results were compared with those obtained independently by Southern blot analysis with a 32P-labelled probe of frozen tissue from the same tumours. Technically satisfactory results were obtained with both methods in 15 lymphomas. EBV DNA was detected in seven of 15 (47%) cases by in situ hybridisation and in eight of 15 (53%) cases by Southern blotting (including all the cases positive by in situ hybridisation). The results of EBV DNA detection by the two techniques were identical in 14 of 15 (93%) cases. In situ hybridisation gave no false positive results. This study shows that the sensitivity and specificity of in situ hybridisation for the detection of EBV genomes in AIDS related lymphomas approaches that of Southern blotting, even when using routinely processed archival, paraffin wax embedded material.

Non-invasive investigation of liver disease in haemophilic patients.

Liver biopsy specimens previously taken from 16 haemophilic patients with chronic non-A, non-B hepatitis were reviewed. The degree of fibrosis correlated with serum procollagen III peptide (sPIIIP) concentrations, measured both at the time of biopsy and 4.25 years later. Two patients with extremely high sPIIIP concentrations had collateral veins on computed tomography, suggesting portal hypertension. Twenty eight of 47 patients (60%) had splenomegaly on computed tomography, and of 28 patients in whom intravenous contrast medium was used, seven (25%) had collateral oesophageal veins. Serum procollagen III peptide estimations and computed tomography, both non-invasive investigations, indicated that hepatic fibrosis and portal hypertension had developed in a proportion of haemophilic patients with non-A, non-B hepatitis. Infection with the human immunodeficiency virus (HIV) may modify the course of this presumably cytopathic virus infection of the liver.

Influence of Epstein-Barr virus encoded latent membrane protein 1 on the expression of CD23 antigen, ICAM-1 and LFA-3 in Hodgkin and Reed-Sternberg cells. A morphometric analysis.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP 1) is expressed in Hodgkin and Reed-Sternberg (HRS) cells in about one half of Hodgkin's disease (HD) cases. In vitro, LMP 1 induces B-cell expression of CD23 antigen, ICAM-1 and LFA-3. To evaluate the influence of LMP 1 on the expression of these molecules in HRS cells in vivo, we performed a quantitative frozen section immunohistological study comparing the numerical density (cells per unit area) of HRS cells expressing the CD23 antigen, ICAM-1 and LFA-3 in 14 LMP 1-positive and 13 LMP 1-negative HD cases. CD23 antigen was demonstrated in HRS cells in five LMP 1-positive and three LMP 1-negative cases (not significant). The relative density of HRS cells tended to be lower in the LMP 1-positive than in the LMP 1-negative cases, but this did not reach significance (0.2 > 2p > 0.1). All recognizable HRS cells expressed ICAM-1 and LFA-3 irrespective of LMP 1 status. We conclude that expression of CD23 antigen and LMP 1 are not coordinated in HD. Although LMP 1 may have some influence on CD23 antigen expression, it is unlikely that the latter is of importance in the putative EBV induced growth transformation of HRS cells in vivo.

High-throughput immunophenotyping of 43 ferret lymphomas using tissue microarray technology.

To validate the use of the tissue microarray (TMA) method for immunophenotyping of ferret lymphomas, a TMA was constructed containing duplicate 1-mm cores sampled from 112 paraffin-embedded lymphoma tissue specimens obtained from 43 ferret lymphoma cases. Immunohistochemical (IHC) expression of CD3, CD79alpha, and Ki-67 (MIB-1) was determined by TMA and whole mount (WM) staining of each individual case for result comparison. There was a high correlation between CD79alpha and CD3 results comparing ferret TMA and WM sections (kappa statistic 0.71-0.73 for single-core TMA and 0.79-0.95 for duplicate-core TMA) and between continuous data from Ki-67 staining of ferret TMA sections and WM sections (concordance correlation coefficients 0.77 for single cores and 0.87 for duplicate cores). Subsequently, a panel of commercially available antibodies was applied to the TMA for the analysis of expression in ferret lymphomas. The results of this study confirmed previously published results suggesting specific cross-reactivity of the applied IHC markers (CD3, CD79alpha, Ki67) with ferret lymphoma tissue. Other IHC markers (CD45Ro, bcl2, bcl10, MUM1, CD30, vimentin) were also expressed in subsets of the included ferret lymphomas. Further studies are necessary to determine the usefulness of these markers for diagnostic and prognostic evaluation of ferret lymphomas. In conclusion, the TMA technology was useful for rapid and accurate analysis of protein expression in large archival cohorts of ferret lymphoma cases.

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.

A survey of Epstein-Barr virus gene expression in sporadic non-Hodgkin's lymphomas. Detection of Epstein-Barr virus in a subset of peripheral T-cell lymphomas.

This study analyzes the association of Epstein-Barr virus (EBV) with non-Hodgkin's lymphoma (NHL) arising in patients without pre-existing overt immunodeficiency. The authors examined 201 lymphomas (105 high-grade B-cell, 82 peripheral T-cell, 7 high-grade non-B-cell, non-T-cell, and 7 hairy-cell leukemia) for EBV gene expression by immunohistologic procedures using monoclonal antibodies to EBV latent, immediate early, and replicative infection antigens. Transformation-associated EBV latent membrane protein 1 (LMP 1) was detected in 13 (6%) NHL, comprising 4 (4%) high-grade B-cell, 8 (10%) peripheral T-cell, and 1 non-B-cell, non-T-cell lymphomas. Anaplastic large-cell lymphoma of T-cell type was consistently LMP 1-negative. EBV nuclear antigen 2 was demonstrated in only three (1%) cases. Induction of replication as defined by expression of the immediate early BamHI Z leftward reading frame 1 (BZLF1) protein was detected in five cases, but early (EA) and late (VCA and MA) lytic cycle antigens were only found in two cases and in one case, respectively. The presence of EBV was confirmed by in situ DNA hybridization in 9 of 11 EBV antigen-positive lymphomas. This study shows the surprisingly frequent presence of EBV in peripheral T-cell NHL in European patients without pre-existing overt immunodeficiency. Interestingly, most sporadic B-cell NHL are not associated with the virus. Furthermore, the usefulness of selected monoclonal antibodies for the routine immunohistological diagnosis of EBV infection was confirmed.

Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma.

Ki-1 (CD30) antigen expression has been considered to be restricted to hematopoietic tissues including the recently described anaplastic large cell lymphoma and Reed-Sternberg (RS) cells in Hodgkin's disease. Its presence on some activated lymphocytes in non-neoplastic lymphoid tissues has been used as evidence that such cells might represent the physiologic counterpart of RS cells. In this study expression of CD30 antigen in 117 nonhematopoietic tumors was investigated using monoclonal antibody Ber-H2. The antigen was regularly expressed in frozen section (strongly) and paraffin section (less strongly) by embryonal carcinomas (8 of 10 studied) and the embryonal elements of mixed germ cell tumors (4 of 4), but not in other types of germ cell tumors (0 of 11) or nonhematopoietic tumors (0 of 92). Normal adult, neonatal, and fetal testes were negative for CD30 antigen, as were other fetal tissues and placenta. Ki-1 antibody gives similar results in frozen section. These findings have implications for theories suggesting an origin of RS cells from activated lymphocytes. They are also important for determining the diagnostic significance of CD30 positivity in a tumor of unknown origin, and suggest possible new uses for CD30 antibodies in routine diagnostic immunohistology.

The association between Epstein-Barr virus and Chinese Hodgkin's disease.

Epstein-Barr virus (EBV) can be detected in Hodgkin and Reed-Sternberg (HRS) cells in about one-half of cases of Hodgkin's disease (HD) in Western countries. To determine whether EBV is also associated with HD in a developing country such as China, we studied paraffin sections from 28 Chinese cases of HD for expression of latent membrane protein-I (LMP-I) and EBV-encoded small RNA (EBER-I), using immuno-histology and RNA/RNA in situ hybridization respectively. The cases were selected from a large series of Chinese lymphomas following histological and immunophenotypical revision. EBV gene expression was found in HRS cells in 17/28 cases, and was related to histological sub-type, being present in 10/11 of mixed cellularity, 6/14 nodular sclerosis, 0/1 lymphocytic predominance, 0/1 lymphocytic depletion, and 1/1 unclassified HD. The 2 methods for detecting EBV gene expression gave similar results, except in one case of nodular sclerosis, in which HRS cells were negative for EBER-I, but weakly positive for LMP-I. In 5/12 cases with EBER-negative HRS cells, rare small or medium-sized lymphocytes expressed EBER-I but not LMP-I. These results suggest that (i) Chinese HD is frequently associated with EBV; (ii) the proportional frequency and sub-type distribution of EBV-positive HD are similar in China and in the West; (iii) both LMP-I immunohistology and EBER in situ hybridization reliably detect EBV in HRS cells in routine biopsies, but the former is simpler and less resource-consuming to perform.

Activation of Epstein-Barr virus replication in Hodgkin and Reed-Sternberg cells.

Recent evidence has shown that Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) in a substantial number of cases and that in these cases EBV DNA is localized exclusively to Hodgkin and Reed-Sternberg (RS) cells. The virus genome is not silent in RS cells because two EBV latent gene products, latent membrane protein (LMP) and EB early region (EBER) transcripts, have recently been reported to be expressed in RS cells. However, little information is available about the possible activation of EBV replicative genes in HD. This prompted us to investigate HD biopsies from 96 patients for expression of replicative gene products. Cryostat sections were immunostained with monoclonal antibodies to protein BZLF1, which controls the switch between EBV latency and replication, and also to LMP. LMP was demonstrated in RS cells in 47 cases (49%). Three of the LMP-positive cases (6%), but none of the LMP-negative cases, expressed the BZLF1 protein. BZLF1 positively was confined to rare RS cells. These three cases showed no detectable early, virus capsid, or membrane antigens. Our findings show that activation of EBV immediate early genes occurs only infrequently in RS cells, indicating that control of viral latency is not severely impaired in HD patients.