HSP-70, C-myc and HLA-DR expression in patients with cutaneous malignant melanoma metastatic in lymph nodes.

HSP-70, C-myc and HLA-DR were examined in patients with cutaneous malignant melanoma metastatic to lymph nodes. Lymph-nodal fine-needle aspiration biopsies (FNABs) were analyzed and the results were correlated to other variables, such as the gender of the patients, Clark level and Breslow thickness of the primary tumor. Thirty cases of metastatic melanoma in lymph nodes from 30 patients with cutaneous malignant melanoma were studied. All patients (100%) had microscopic regional nodal metastasis and a recurrence of the lesion during the first two years. The HSP-70, C-myc and HLA-DR expressions were investigated immunocytologically, using the APAAP (alkaline phosphatase) method on the FNAB samples. The immunocytochemical expressions of HSP-70 protein, C-myc oncogene, and HLA-DR antigen were found in 18 cases (60%), in 14 cases (43.3%) and in 12 cases (40%), respectively. Clark levels were significantly associated with HSP-70 protein (< 0.01), C-myc oncogene expression (< 0.05) and HLA-DR antigen (< 0.01) expression. The HLA-DR antigen was also found to be related (< 0.05) to higher Breslow thickness (> 1.5 mm). The clinical course of malignant cutaneous melanoma is related to the expression of these indices, which seem to play a significant role in the metastasis and prognosis of this aggressive tumor. The immunocytochemical expression of HSP-70 in the malignant melanoma tumor could be of particular value in the identification of patients with poor prognosis.

Immunohistochemical detection of p53, mdm2, waf1/p21, and Ki67 proteins in bone marrow biopsies in myelodysplastic syndroms, acute myelogenous leukaemias and chronic myeloproliferative disorders.

The aim oof this study was to investigate the immunohistochemical expression of p53, mdm2, and waf1/p21 proteins in myelodysplastic syndromes (MDS), acute myelogenous leukaemias (AML), and chronic myeloproliferative disorders (CMPD). Paraffin-sections of bone marrow biopsies from 30 cases of MDS (6 cases of RAEB and RAEB-T) 22 AML (4 cases occurring in the setting of MDS), 16 chronic myeloproliferative disorders (CMPD), and 10 cases without alterations were investigated by immunohistochemistry for p53, waf1/p21, mdm2 and Ki67 proteins. P53 was detected in immature myeloid cells in 6/30 MDS (20%) and in 6/22 AML (27%) while it was not expressed in CMPD. Of the 6 p53 positive AML, 3 occurred as evolution of MDS and 3 were de novo acute leukaemias. Waf1/p21 was detected in 5/22 (23%) AML in immature myeloid cells. Waf1/p21 was also expressed in 18/30 (60%) MDS and 10/16 (63%) CMPD in variable proportion (5-25%) of the mature myeloid cells and megakaryocytes. Waf1/p21 was not detected in immature myeloid cells in MDS and CMPD. Mdm2 protein was expressed in 3/30 (10%) MDS in the immature myeloid cells and in 1/22 AML in blastic cells. The combined immunophenotypes of immature myeloid cells of MDS were: p53+/mdm2+/waf1-: 3, p53+/mdm2-/waf1-: 3, while the immunohistochemical patterns of AML were: p53+/mdm2-/waf1-: 4, p53+/mdm2+/waf1+: 1, p53+/mdm2-/waf1+: 1, p53-/mdm2-/waf1+: 3. Ki67/MIB1 staining was found in at least 30% of immature myeloid cells in MDS and AML and in at least 20% of these cells in CMPD. In conclusion, our results indicate that p53 protein is overexpressed in the myeloid lineage in a proportion of AML and MDS, while is not detected in CMPD and normal bone marrow, p53 expression was much more frequent in AML occurring as an evolution of MDS than in de novo AML. The combined immunophenotypes of p53 positive AML and MDS suggest that p53 overexpression may be due to mutation, in some AML and MDS cases with the p53+/mdm2-/waf1- phenotype. However, it would be also possible that p53 protein accumulation is not related to p53 mutation but to inhibition of p53/mdm2 binding due to mdm2 defects and/or other events related to cell stress signals. On the other hand, waf1/p21 protein overexpression without p53 expression in some AML could be p53-independent and may represent an attempt to control the high proliferation rate which was evidenced by Ki67/MIB1 immunostaining. However, the possibility of p21 to arrest cell-cycle, in these cases of AML, seems to be overridden, suggesting that cell-cycle deregulation may be involved in a proportion of AML.

Cytotoxic protein expression in non-Hodgkin's lymphomas and Hodgkin's disease.

Recent studies have shown that some peripheral T-cell lymphomas (PTCL) could be derived from lymphocytes with cytotoxic potential. Therefore, we have investigated by immunohistochemistry 34 cases of PTCL including 2 cases of hepatosplenic gamma delta PTCL and 5 cases of sinonasal NK-cell lymphomas as well as 7 cases of T-lymphoblastic lymphomas (T-LBL) for the expression of the cytotoxic proteins TIA-1 and granzyme B. In addition, 50 cases of Hodgkin's disease (HD) were investigated in order to see if these cytotoxic proteins are expressed by Hodgkin and Reed-Sternberg (HRS) cells. Expression of the TIA-1 is characteristic of cytotoxic cells regardless of their activation status whereas expression of granzymes is highly increased in activated cytotoxic cells. All the five cases of sinonasal NK-cell lymphomas expressed TIA-1 and granzyme B in most tumour cells. The two gamma delta PTCL cases expressed TIA-1 protein in most tumour cells but not granzyme B. Of the 32 other PTCL, 9 cases showed cytotoxic protein expression in tumour cells. These cases comprised 2 pleomorphic medium large cell (PML) (1 nodal and 1 intestinal) and 7 CD30 positive anaplastic large cell lymphomas (ALCL) (5 nodal and 2 cutaneous). Cytotoxic protein expression in our series appeared to be related to the location since 10/18 (55%) extranodal PTCL and NK-NHL and only 6/21 (28%) nodal PTCL expressed TIA-1, and related to histology since, in nodal PTCL, this pattern was observed in most anaplastic (5/6 cases) and in a few pleomorphic (1/9 cases) lymphomas, but not in AILD-type NHL (0/6 cases). The 7 cases of T-LBL did not express cytotoxic proteins in tumour cells. EBV was detected by EBER RNA in situ hybridization (RISH) in tumour cells in all 5 sinonasal NK-NHL and in scattered atypical cells in all 6 cases of AILD. Two of the 50 cases of HD weakly expressed TIA-1 and granzyme B in a proportion of HRS cells. EBV was detected by RISH in 19/50 cases of HD but no correlation was found between EBV status and expression of cytotoxic proteins in HRS cells. However, the finding that granzyme B positive cells were found very rarely in close vicinity of HRS cells suggests that the function of activated cytotoxic cells is locally inhibited by the HRS cells and/or the reactive cells in the vicinity of HRS cells. Taken together our data suggest that: a) sinonasal NK-cell NHL represent tumours of activated cytotoxic NK-cells, b) the hepatosplenic gamma delta PTCL represent tumours of nonactivated cytotoxic gamma delta T-cells, c) a small proportion of other PTCL, mostly anaplastic large cell lymphomas represent tumours of cytotoxic T-cells and d) only very few cases of HD expressing cytotoxic proteins in a proportion of tumour cells, could be derived from activated cytotoxic cells.

P21/waf1 protein expression in nasopharyngeal carcinoma. Comparative study with PCNA, p53 and MDM-2 protein expression.

We investigated the immunohistochemical expression of p21/waf1 protein in 59 cases of nasopharyngeal carcinomas (NPC) and compared p21 expression with PCNA, p53 and mdm2 protein expression. We found p21, PCNA, p53 and mdm2 in 59/59, 59/59, 18/59 and 12/59 nasopharyngeal carcinomas, respectively. We observed a tendency to a relationship between high expression of PCNA (> 25% positivity in tumour cells) and low expression of p21 protein. Parallel p53/p21 protein expression was found in 18 cases. Twelve were also mdm2 positive. This pattern may represent NPC with wild type (wt) p53 since mdm2 and p21 proteins are inducible by wt p53 gene. In these cases p53 protein expression may be due to stabilisation to mdm2 protein. This could be important in the pathogenesis of these cases since mdm2 may deregulate the p53-dependent growth suppressive pathway. Discordant p53-/p21+ protein expression was found in 41 cases. All were also mdm2 negative. This pattern suggests immunohistochemically undetectable wt p53 gene which is able to induce p21 protein expression.

MIB1 (Ki-67) expression in non-Hodgkin's lymphomas.

The MIB1 monoclonal antibody which is used as a marker of cell proliferation was studied by immunohistochemistry on formalin-fixed and paraffin embedded biopsy specimens of lymph nodes in 40 high- and 46 lowgrade cases of non-Hodgkin's lymphomas (NHL) classified according to the Kiel classification. All cases were found to display nuclear MIB1 staining. A statistically significant difference (P < 0.005) was found between high- and low grade NHLs and this indicates that the high- grade NHL display a higher proliferation rate than low grade. In addition, remarkable variations in MIB1 expression were found among individual cases of the same histological group. These data may suggest that MIB1 index can help in the individual approach of the proliferation rate of each tumour and this may be an important parameter in association with clinical and other laboratory parameters for predicting the biological behaviour of non-Hodgkin's lymphomas.

Expression of p53 and mdm-2 proteins in Hodgkin's Disease. Absence of correlation with the presence of Epstein-Barr virus.

The expression of p53 and mdm-2 proteins was analysed in parrafin sections from 39 cases of Hodgkin's disease (HD) and compared to the presence of Epstein-Barr Virus (EBV). P53 protein was found in Hodgkin and Reed-Sternberg (HRS) cells in 12/39 cases. Mdm-2 protein was found in HRS cells in 10/39 cases. EBV-encoded EBER1-2 mRNAs and LMP-1 protein expression were found in HRS cells in 16/39 cases. In view of the LMP-1 oncogenic potential in vitro, these findings suggest that EBV may be involved in the pathogenesis of a proportion of HD cases. The coexpression of mdm-2 and p53 proteins was found in HRS cells in 10 cases, whereas in 27 cases neither was identified and in 2 cases there was no coexpression of mdm-2/p53. The simultaneous p53/mdm-2 protein expression, in view of previous findings which showed that most cases of HD display no p53 gene mutations, suggests that mdm-2 protein expression may be one of the factors responsible for the stabilisation of p53 protein in these cases. This could be important, in the pathogenesis of these cases of HD, since mdm-2 may deregulate the p53 dependent growth suppressive pathway. Mdm-2-/ p53+ protein expression may reflect the stabilisation of p53 protein by proteins other than mdm-2, mutations in the p53 gene making it unable to activate mdm-2, or the deregulation of the mdm-2 gene. No relationship was found between the presence of EBV and the expression of p53 and/or mdm-2 proteins.

Major histocompatibility complex (MHC)-I and MHC-II expression in Hodgkin's disease in relation to the presence of Epstein-Barr Virus (EBV).

We have investigated the immunohistochemical expression of beta2-microglobulin and HLA-DR proteins in Hodgkin's disease (HD) in relation to the expression of the EBV-encoded EBER1-2 mRNAs and the LMP-1 protein. beta2-microglobulin is expressed in association with MHC-I molecules on most nucleated cells and HLA-DR belongs to the MHC-II molecules which are expressed mostly on antigen-presenting cells. Formalin-fixed paraffin embedded tissue from 39 cases of lymphonodal HD were stained by immunohistochemistry for beta2-microglobulin, HLA-DR and LMP-1 proteins and by RNA in situ hybridization for EBER1-2 mRNAs. beta2-microglobulin positive staining was found in Reed-Sternberg and Hodgkin cells (HRS cells) in 18/39 cases of HD. HLA-DR positive staining was found in HRS cells in all cases of HD. EBER1-2 transcripts and LMP-1 protein were detected in HRS cells in 16/39 cases of HD. No correlation as found between the presence of EBER 1-2 transcripts or the LMP-1 protein and the detection of beta2-microglobulin and HLA-DR proteins in HD. Thus, EBV does not seem to use downregulation of MHC-I to avoid the T-cell cytotoxic immune response in HD. In addition, EBV does not seem to be the only factor responsible for the HLA-DR expression in HRS cells of HD, although it could participate in the induction of the expression of HLA-DR molecule in the EBV positive cases of HD.

MDM-2 protein expression in nasopharyngeal carcinomas. Comparative study with p53 protein expression.

Aims-To investigate the immunohistochemical expression of MDM-2 protein in comparison with that of p53 protein in nasopharyngeal carcinomas.Methods-Formalin fixed, paraffin wax embedded tissue from 59 cases of nasopharyngeal carcinoma was stained by immunohistochemistry for MDM-2 and p53 proteins.Results-The tumours were divided histologically into seven cases of keratinising nasopharyngeal carcinoma (KNPC), 14 cases of non-keratinising nasopharyngeal carcinoma (NKNPC), and 38 cases of undifferentiated nasopharyngeal carcinoma (UNPC). MDM-2 nuclear expression was observed in 0/7 KNPC, 1/14 NKNPC, and 11/38 UNPC. p53 nuclear expression was observed in 1/7 KNPC, 2/14 NKNPC, and 15/38 UNPC. Parallel MDM-2 and p53 expression was found in 12 cases (11 UNPC and one NKNPC). Discordant MDM-2-/p53 + expression was found in six cases (four UNPC, one NKNPC, and one KNPC), and absence of expression of both proteins in the remaining 41 cases.Conclusions-Expression of MDM-2 and p53 proteins may be associated with the level of tumour cell differentiation in nasopharyngeal carcinoma. Simultaneous expression of MDM-2/p53 in a proportion of UNPC suggests that MDM-2 protein may be responsible for stabilisation of p53 protein in these cases, in view of the previous demonstration of the p53 gene in germ line configuration. This could be important in the pathogenesis of these cases, since MDM-2 may deregulate the p53 dependent growth suppressive pathway. Discordant MDM-2-/p53 + expression in a few cases of nasopharyngeal carcinoma may reflect stabilisation of p53 protein by other proteins, or p53 mutations unable to activate MDM-2.

Beta 2-microglobulin and HLA-DR expression in relation to the presence of Epstein-Barr virus in nasopharyngeal carcinomas.

We have investigated the immunohistochemical expression of beta 2-microglobulin and HLA-DR proteins in nasopharyngeal carcinoma (NPC) in relation to the expression of the Epstein-Barr virus (EBV)-encoded EBER 1-2 mRNAs and LMP-1 protein. beta 2-Microglobulin is expressed in association with MHC-I molecules on most nucleated cells and HLA-DR belongs to the MHC-II molecules which are expressed mostly on antigen-presenting cells. Formalin-fixed paraffin-embedded tissue from 37 NPC cases was stained with immunohistochemistry for beta 2-microglobulin, HLA-DR and LMP-1 proteins and by RNA in situ hybridization for EBER 1-2 mRNAs. beta 2-Microglobulin-positive staining was found in neoplastic cells in all cases. In 19/37 NPC cases the positive staining was found in most neoplastic cells. In the remaining 18 NPC cases more than 25% of the neoplastic cells showed significantly reduced or negative staining in comparison to the normal epithelium and infiltrating small lymphocytes. HLA-DR-positive staining was found in 27/37 NPC cases. EBER 1-2 transcripts were deteced in neoplastic cells in 13/37 NPC cases. LMP-1 expression in tumour cells was found in 4/13 EBER1-2-positive cases. No correlation was found between the presence of EBER1-2 transcripts or LMP-1 protein and the beta 2-microglobulin-reduced expression in NPC. Thus, EBV does not seem to use downregulation of MHC-I to avoid the T cell cytotoxic immune response in NPC. HLA-DR expression was observed in all 13 EBV-positive cases of NPC, suggesting that EBV may participate in the induction of HLA-DR expression in a proportion of NPC.

Frequent detection of Epstein-Barr virus (EBV), EBER transcripts and latent membrane protein-1 (LMP-1) in tumor cells in Hodgkin's disease arising in childhood.

Paraffin sections from 22 cases of Hodgkin's disease (HD) and 30 cases of non-Hodgkin's lymphomas (NHL) occurring in childhood (3-15 years old) were examined for the presence of Epstein-Barr Virus (EBV) encoded EBER mRNAS and Latent Membrane Protein-1 (LMP-1) using RNA in situ hybridization (RISH) and immunohistochemistry, respectively. In 12/22 (54%) cases of HD the EBER transcripts were detected in most Reed-Sternberg and Hodgkin (HRS) cells as well as in some scattered smaller lymphoid cells. In all these cases the LMP-1 protein was detected exclusively in HRS cells. Three additional cases of HD were found to be EBER RISH positive only in a few scattered small lymphoid cells, the LMP-1 staining being negative in these cases. The EBER and LMP-1 positivity in HRS cells were present in 0/1 of lymphocyte predominant, 4/10 (40%) of nodular sclerosis and 8/11 (72%) of mixed cellularity of HD. No EBER RISH signal was found in tumor cells of the 30 cases of NHL. In four of them only a few scattered small lymphoid cells were EBER RISH positive. LMP-1 reactivity was not detected in any NHL. These results provide evidence for an association between EBV and a sizeable proportion of childhood Hodgkin's disease and show that this association is more frequent in mixed cellularity subtype. Furthermore, the detection of the LMP-1 protein in HRS cells in view of the LMP-1 transforming potential, suggests that EBV may be involved in the pathogenesis of a substantial proportion of cases of HD occurring in childhood.