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.

Topoisomerase II alpha expression in breast ductal invasive carcinomas and correlation with clinicopathological variables.

Topoisomerase II alpha (topo IIa) is an enzyme that in normal cells is expressed predominantly in the S/G2/M-phase of the cell cycle. In malignant cells, in vitro studies have indicated that the expression of topo II alpha is both higher and less dependent on the proliferation state in the cell. To study the expression of topo IIa and the relationship between that expression-and other variables in cases of breast ductal invasive carcinomas, 50 fine-needle aspiration biopsies were performed from the same number of female patients, diagnosed cytologically and confirmed histologically after surgery. The same cases were studied immunocytochemically using monoclonal antibodies to topo IIa and Her2/neu (CB11) by the alkaline phosphatase method (APAAP). Topo IIa was found in 32 cases (64%) of the carcinomas studied. An overexpression between topo IIa and Her2/neu was found (p < 0.005). A relationship between topo IIa expression, histological grade and lymph node status (LNs) was also found (p < 0.005). Increased topo IIa expression seems to be related to an aggressive form of breast cancer featuring Her2 amplification and lymph node metastasis.

Lens epithelial apoptosis and cell proliferation in human age-related cortical cataract.

PURPOSE: To probe the presence of apoptosis in the epithelium of human lenses with age-related cortical cataract as well as to assess cell proliferation, a predicted consequence of apoptotic cell death, in this specific cell population. METHODS: DNA fragmentation was assessed using terminal digoxigenin-labeled dUTP nick end labeling (TUNEL) in capsulotomy specimens obtained from patients who underwent either extracapsular cataract extraction for the removal of adult-onset cortical cataract (n=27) or clear lens extraction for the correction of high myopia (n=25). Cell proliferation was assayed in 23 epithelia of cataractous lenses, and 20 epithelia of non-cataractous lenses with the proliferation marker MIB1, a monoclonal antibody against the nuclear antigen Ki-67 that is detected throughout the cell cycle but is absent in the resting (G0) cell. RESULTS: TUNEL staining was observed in 25 (92.6%) specimens of cataractous lenses, whereas cells undergoing apoptosis were identified in 2 (8%) of the epithelia from non-cataractous lenses. Only two MIB1-positive samples were detected, one of which was a capsule obtained during intracapsular cataract extraction. CONCLUSIONS: The epithelium of human lenses with cortical cataract undergoes low rate apoptotic death. This limited epithelial apoptosis is unlikely to result in any significant cell density decrease since epithelial gaps are likely to be replaced by cell proliferation at the germinative zone of the anterior lens capsule. Nevertheless, the accumulation of small-scale epithelial losses during lifetime may induce alterations in lens fiber formation and homeostasis and result in loss of lens transparency.

Vascular density and the response of breast carcinomas to mastectomy and adjuvant chemotherapy.

Using morphometric analysis of histological preparations, in a retrospective study vascular indices, expressing the extent of vascularisation, were determined for a number of mammary carcinomas. The indices were found to be related to the survival of the patients treated with modified radical mastectomy in combination with pre- and postoperative chemotherapy, the cases with low indices having the shorter survival. The impaired access of the cytotoxic agents to cells in the deficiently vascularised tumours was considered as an explanation. It was concluded that determination of vascular density in tumours may have a prognostic value in regard to the treatment response, and may be helpful in choosing the appropriate treatment.

Variation of vascular density within and between tumors of the uterine cervix and its predictive value for radiotherapy.

Vascular density in different regions of a number of uterine cervix carcinomas was determined by morphometric analysis of stained histologic sections. Variance analysis indicated a larger inter- than intra-tumoral inhomogeneity of the vasculature, suggesting a certain individual vascularization pattern in these tumors. As indicated by a retrospective study of archival biopsies, this pattern has a predictive value in regard to the efficacy of radiotherapy.

Correlation of histoenzymological studies with the response to chemotherapy and survival in breast cancer patients.

The enzyme activity of cancerous tissue and the adjacent normal epithelium of 40 patients with breast cancer was determined. This enzymatic activity was correlated with the responsiveness of those tumors to the chemotherapy. It was found that the presence of cytochrome oxidase and alkaline phosphatase and the absence of leucine aminopeptidase, beta-glucuronidase and dehydrogenases in cancerous tissues was related to good response. On the contrary, the absence of alkaline phosphatase and cytochrome oxidase and the presence of leucine aminopeptidase, beta-glucuronidase and dehydrogenases in the cancerous tissues was related to poor response and therefore to poor survival.

Vascularization and curability of stage III and IV nasopharyngeal tumors.

In a retrospective study the capillary density was determined in biopsies from 25 cases of nasopharyngeal tumor, stages III and IV. Determinations were made on stained histological sections by counting the capillaries within a standard field size and at standard magnification. A statistically significant correlation was found to exist between capillary density and eventually with chemotherapy. No correlation between survival and age or sex of the patients, or stage of tumor, was noted. It is concluded that capillary density in nasopharyngeal tumors has a predictive value.

Changing pattern of cytokeratin 7 and 20 expression from normal epithelium to intestinal metaplasia of the gastric mucosa and gastroesophageal junction.

It is currently unclear whether intestinal metaplasia at the esophagogastric junction and in the distal esophagus represent a continuum of the same underlying disease process, i.e., gastroesophageal reflux, or constitute different entities with a different pathogenesis. Biopsies below the Z line might show specialized epithelium in some patients and the question is whether this is another form of short segment Barrett's esophagus or whether it is related to a generalized atrophic process of the stomach. Data from recent studies regarding the expression of cytokeratin CK7 and CK20 in intestinal metaplasia (IM) found at the gastroesophageal junction are conflicting. Prompted by these data we undertook the present study: a) to evaluate the expression of CK7 and CK20 in IM of the gastric cardia and to compare the findings with those in patients with Barrett's esophagus and IM of the gastric corpus and antrum mucosa; and b) to evaluate the immunophenotype of non-intestinalized cardiac mucosa and to compare it with that of normal gastric epithelium. We studied the expression of CK7 and CK20 on biopsy specimens from patients with long-segment Barrett's esophagus (n=17) and surgical resection and biopsy specimens of gastric cardia (n=15), corpus (n=14) and antrum (n=22) from patients with histological evidence of IM. Eighty-four biopsy specimens from 42 patients (antrum n=15, corpus n=20, cardia n=7) without evidence of IM were studied as a control group. We observed an immunophenotype characterised by diffuse moderate to strong CK7 staining on the surface and crypt epithelium combined with strong CK20 staining on the surface and superficial part of the crypts in 94.1% (16/17) of the cases with long-segment Barrett's esophagus, but in none of the 36 cases with IM in distal stomach (antrum and corpus). IM in the gastric cardia expressed the immunophenotype seen in IM of the gastric mucosa in 93.3% (14/15) of the cases. On the other hand, normal cardiac epithelium expressed patchy strong CK7 staining on the surface epithelium and on both, superficial and deep parts of the pits combined with patchy strong CK20 staining on the surface epithelium and superficial pits, a feature permitting distinction of the normal cardiac epithelium from those of the normal gastric antrum and corpus epithelium. We conclude that the expression of cytokeratins 7 and 20 can be used to distinguish the origin of IM of the gastroesophageal junction. The CK7/20 immunophenotype of IM in the gastric cardia closely resembles that of the IM in the gastric antrum and corpus and is different from IM in long-segment Barrett's esophagus. In contrast, the CK7/20 immunophenotype of the cardiac epithelium is different from that of the gastric antrum and corpus mucosa, suggesting that cardiac epithelium might not be a native normal gastric epithelium but one that is acquired as a consequence of longstanding inflammation. Changing pattern of CK7 and CK20 expression from normal to intestinalized epithelium suggests that IM arising from cardiac epithelium might have distinctive features.

Metastasis of malignant melanoma to a transitional cell carcinoma of the urinary bladder.

A case of malignant melanoma metastatic to a transitional cell carcinoma of the urinary bladder in a 28-year-old female is reported. This is the sixth reported case involving malignant melanoma metastasis in another neoplasm. The pertinent literature is reviewed.

Cytological diagnosis of malignant mixed müllerian tumor of the uterus in ascitic fluid.

The malignant mixed Mullerian tumour (MMMT) is a rare and aggressive neoplasm of the uterus, seen in postmenopausal women. In this case, an uncommon neoplasm was diagnosed cytologically in the ascitic fluid of a woman 58 years old and was confirmed histologically after hysterectomy and bilateral adnexectomy.

C-erbB-2: expression in patients with breast carcinoma in comparison to patients with benign breast diseases.

C-erbB-2 (Her-2 or c-neu) expression was studied by immunohistochemistry on FNA specimens of 20 breast ductal carcinomas, 20 fibroadenomas and 20 atypical fibrocystic lesions of the breast. Twelve cases of breast carcinomas, six fibroadenomas and five atypical fibrocystic lesions were found to display c-erbB-2 staining. A significant difference was found among c-erbB-2 index of breast carcinomas (mean 70,25), fibroadenomas (mean 43,83) and atypical fibrocystic disease (mean 37,4). We also found variations in c-erbB-2 expression, among individual cases of breast carcinomas, concerning the number and the intensity of carcinoma cells. It would be interesting to correlate these variations in c-erbB-2 expression with the prognosis of breast carcinomas.

Immunohistochemical expression of p53, bcl-2, mdm2 and waf1/p21 proteins in colorectal adenocarcinomas.

The present study was undertaken to examine the distribution of p53, p21, mdm-2 and bcl-2 protein expression in human colorectal adenocarcinomas in order to obtain combined information about the immunophenotypes characterising these tumours. Formalin-fixed, paraffin-embedded tissue sections from 52 cases of colorectal adenocarcinomas were stained using immunohistochemical methods for the detection of p53, p21/waf1, mdm2 and bcl-2 proteins. P53, p21/waf1, mdm2 and bcl-2 proteins were expressed in 35/52, 45/52, 9/52 and 27/52 cases, respectively. All nine mdm2+ cases expressed p53 and p21 proteins as well. The three patterns observed in p53/p21 expression were: p53+/p21+, p53+/p21- and p53-/p21+ in 28, 7, and 17 cases, respectively. Consequently, p53+/mdm2-/p21+, p53+/mdm-/p21- and p53-/mdm2-/p21+ immunophenotypes were expressed in 19, 7, and 17 cases respectively. Four patterns of p53/bcl2 expression were identified: p53+/bcl2+, 20 cases; p53+/bcl2-, 15 cases; p53-/bcl2+, 7 cases; p53-/bcl2-, 10 cases. It was noteworthy that 9 of the 10 p53-/bcl2-tumours had negative lymph node status. The present results suggest that both p53 dependent and p53-independent induction of p21 expression may be involved in the molecular mechanisms controlling these tumours. High expression of the p53 protein in colorectal carcinomas could be due not only to p53 gene mutations but also to binding to mdm2 protein which leads to p53 protein stabilisation. In addition, tumours with p53-/bcl2- immunophenotype are frequently associated to negative lymph node status and seem to be less aggressive.

p53 protein expression in breast carcinomas. Comparative study with the wild type p53 induced proteins mdm2 and p21/waf1.

The aim was to investigate the pattern of expression of p53 protein and two wild-type (wt) p53-induced proteins (mdm2 and p21/waf1), as an indirect way of assessing p53 gene status in breast carcinomas. Formalin-fixed paraffin embedded tissue from 102 cases of breast carcinomas comprising mostly ductal carcinomas (88 cases) was stained by immunohistochemistry for p53, mdm2 and p21/waf1 proteins. We found p53, mdm2 and waf1/p21 protein expression in 33/102, 20/102 and 38/102 breast carcinomas, respectively. Parallel p53/mdm2 protein expression was found in 9 cases. Five were also p21/waf1 positive. Discordant p53+/ mdm2-protein expression was found in 24 cases. Nine were p21/waf1 positive and the remaining fifteen were p21/waf1 negative. The patterns mdm2+/p53-/p21- and p21+/p53-(+)/mdm2- were found in 6 and 20 cases, respectively. Parallel p53/mdm2/p21 protein expression may represent breast carcinomas with wt p53 gene 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+/mdm2-/p21- protein expression may represent breast carcinomas with p53 gene mutations unable to activate expression of mdm2 and p21 proteins. Breast carcinomas with p53+/mdm2/p21+ protein expression may have either wt p53 with deregulated mdm2 gene expression or mutated p53 gene with p53-independent p21 expression. Cases with only mdm2 expression may represent tumours with mdm2 gene amplification or overexpression and cases with only p21 expression may reflect p53-independent regulation of p21 protein.

Expression of p53, p21, mdm2, Rb, bax and Ki67 proteins in lymphomas of the mucosa-associated lymphoid (MALT) tissue.

We have investigated by immunohistochemistry 38 cases of B-cell MALT-NHL comprising 23 high grade (HG) and 15 low grade-(LG) tumours for the expression of p53, mdm2, p21, Rb, Ki67, bcl2 and Bax proteins. P53, mdm2 and p21 proteins were found in at least 5% of the tumour cells in 13/23, 2/23 and 11/23 HG tumours, respectively. These proteins were detected in very rare tumour cells in LG tumours. The following patterns were recorded in HG tumours: p53+/p21+/mdm2+ (2 cases), p53+/p21+/mdm2- (7 cases), p53+/p21-/mdm2- (4 cases), p53-/p21-/mdm2- (18 cases) and p53-/p21+/mdm2-(2 cases). Proliferative Ki67 index and Rb protein expression were higher in HG than in LG MALT-NHL. Bcl2 protein was expressed in all LG MALT-NHL, whereas only 2/23 HG MALT-NHL were bcl2 positive in most tumour cells. Bax protein was expressed in all MALT-NHL with HG tumours being positive in higher proportion of tumour cells than LG tumours. These findings show that significant expression of p53, mdm2, p21,Ki67 and Rb proteins occurs more frequently in aggressive histotypes of MALT-NHL. The parallel Rb/Ki67 expression suggests that Rb protein expression in MALT-NHL is normally regulated in relation to the proliferative growth fraction of the tumours. The pattern p53+/p21+/mdm2 +/- may represent MALT-NHL with wild type (wt) p53 gene since mdm2 and p21 proteins are inducible by wt p53 gene. The pattern p53+/mdm2-/p21-may represent MALT-NHL with p53 gene mutations unable to activate expression of mdm2 and p21 proteins. MALT-NHL with the p53-/mdm2-/p21 + pattern may be consistent with p53-independent p21 expression. Bax protein expression in all MALT-NHL suggests a role for this protein in the pathogenesis of these tumours.

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.

P53 expression in patients with malignant and benign breast diseases.

Immunohistochemical expression of p53 protein was studied in FNA specimens of 20 breast ductal carcinomas, 20 fibroadenomas and 20 atypical ductal hyperplasia of the breast. Nine cases of breast carcinomas (45%), five fibroadenomas (25%) and four atypical ductal hyperplasia (20%) were found to be p53-immuno-positive. A statistically significant difference was found among p53 staining index of breast carcinomas (mean 72.55%), fibroadenomas (mean 41.2%) and atypical ductal hyperplasia (mean 34%). Variations in p53 expression among individual breast carcinomas was found, and these variations may correlate with prognosis.

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.

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.

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.

Morphologic differences between latent membrane protein-1 (LMP-1)-positive and negative tumour cells in Epstein-Barr virus (EBV)-related childhood Hodgkin's disease. A morphometric study.

The values of five cellular morphometric parameters (longest and shortest cytoplasmic axis, cellular circumference, area and roundness coefficient) were compared between 20 Latent Membrane Protein 1 (LMP-1)-positive and an equal number of LMP-1-negative Reed-Sternberg and Hodgkin (HRS) cells for each of 13 cases of Hodgkin's disease (HD) occurring in children (aged 3-15 years); the presence of Epstein-Barr virus (EBV) encoded EBER mRNAs had previously been detected in all cases using RNA in situ hybridisation (RISH), while the presence of LMP-1 was immunohistochemically detected using the alkaline phosphatase-antialkaline phosphatase (APAAP) method. The longest and shortest axis, circumference and area were larger in LMP-1-positive than in LMP-1 negative HRS cells, while the roundness coefficient of LMP-positive HRS cells was smaller than that of LMP-1 negative cells. All differences were statistically highly significant when univariate (paired comparisons) t-test were used. Multivariate analysis (Hotelling's T2 test) showed all differences (except the roundness coefficient) to be significant both at the 5% and 1% level of significance. These results provide a numerical basis for the alteration brought by the expression of LMP-1 in the cellular skeleton of tumour (HRS) cells in EBV-related childhood HD cases.