Panayota Christodoulou

The role of p53, MDM2 and c-erb B-2 oncoproteins, epidermal growth factor receptor and proliferation markers in the prognosis of urinary bladder cancer.

The immunohistological expression of p53 and MDM2 oncoproteins was examined in paraffin embedded tissue from 106 patients with transitional cell carcinoma of the urinary bladder and was related to various clinicopathological features, the expression of proliferation associated markers (proliferating cell nuclear antigen - PCNA - and Ki-67), c-erb B-2 oncoprotein and epidermal growth factor receptor (EGFR), as well as to survival. MDM2 immunoreactivity was seen in 38% of our cases, and in 14% was accompanied by p53 positive immunohistochemistry. The rate of p53 positivity was associated with grade, stage and papillary status, whereas MDM2 immunopositivity increased with grade and stage (Ta VS T1), and MDM2 labeling index (LI) with stage. MDM2 expression was related to p53 expression and less strongly to proliferation rate (Ki-67 LI). The simultaneous p53 and MDM2 expression was more frequently observed in higher grade and stage tumours. C-erb B-2, EGFR and proliferation marker expression increased with grade, stage and non-papillary configuration. In univariate analysis high grade, solid growth pattern, advanced T-category, cystectomy, EGFR and Ki-67 expression were linked to shorter overall survival but only Ki-67 LI, along with T-category and type of therapy, had independent prognostic value. C-erb B-2 expression and stage were the two independent predictors of disease-free survival and Ki-67 LI and EGFR LI the independent predictors of post-relapse survival. For patients with superficial tumors PCNA LI emerged as the single independent determinator of survival. p53 and MDM2 expression did not appear to have any significant impact on survival, although the simultaneous expression of p53 and MDM2 turned out to be a highly significant parameter of shortened overall survival in univariate analysis.

Prognostic implications of microvessel morphometry in diffuse astrocytic neoplasms

Astrocytic brain tumours, particularly malignant astrocytomas, are recognized to be highly vascular neoplasms with potent angiogenic activity. Recent research has shown that quantification of microvessel density (MVD), as a measure of the degree of angiogenesis, constitutes a strong prognostic indicator in patients with astrocytomas. However, the significance of other morphometric aspects of microvessel network has not been tested so far. In this report, histological sections from 70 astrocytomas (grades II to IV), immunostained for CD34, were evaluated by image analysis for the quantification of MVD, total vascular area (TVA), and microvascular branching, as well as several morphometric parameters related to vessel size or shape. Minor axis length increased with grade (P = 0.045) but MVD and TVA presented a peak in grade III (P = 0.033 and P < 0.001, respectively). Size and shape related parameters affected survival in univariate analysis of grade IV and grades II/III, respectively. In multivariate analysis, only branching counts, along with age and grade, were the independent predictors of survival. Although MVD, TVA and branching counts were adversely related to disease‐free survival in grades II and III (univariate analysis), only TVA remained statistically significant in multivariate analysis. It is concluded that TVA and branching counts are prognostically more informative than MVD for patients with diffuse astrocytic tumours.

Differential Expression of bcl-2 Family Proteins in Bladder Carcinomas: Relationship with Apoptotic Rate and Survival

OBJECTIVE To elucidate the role of various bcl-2 family molecules in the regulation of apoptosis and the progression of urothelial cancer, in relation to standard prognosticators. METHODS Paraffin-embedded archival tissue from 103 N0M0 consecutive patients with invasive bladder cancer (28 T1, 57 T2, 13 T3 and 5 T4) was immunostained for bcl-2, bax, bcl-XL, bcl-Xs, p53, Ki-67 and with an anti-single stranded DNA monoclonal antibody recognizing the apoptotic cells. Survival analysis was restricted to T2-T4 tumours. Patients were followed-up until death (n = 27) or for a mean (+/- S.D.) follow-up of 37.6 (+/- 17.4) months. Within this period, 39 patients relapsed after a mean (+/- S.D.) period of 13.6 (+/- 12.3) months. RESULTS Most tumours were immunoreactive for bax (73.1%) and bcl-XL (80.9%) whereas bcl-2 and bcl-XS expression was comparatively less common (44.4 and 28.9%, respectively). The bcl-XL and bcl-XS positivity was related to high grade (P = 0.007) and advanced stage (P = 0.010), respectively. On the contrary, bax and bcl-2 positivity was unrelated to stage or grade. Apoptotic rate was independently influenced only by p53, bcl-2 and proliferation rate. In multivariate analysis of T2-T4 urothelial carcinomas (UC)s, only bax along with T-category and age were the significant predictors of disease-free survival. Increased apoptosis and T-category were also independently related to the overall survival in T2-T4 UCs. CONCLUSIONS The expression of bcl-2 family members appears to be differentially regulated in association with UC evolution. Most importantly, bax immunostaining offers additional information to that provided by traditional prognosticators, with regard to disease-free survival of T2-T4 UCs.

Prognostic implications of aberrations in p16/pRb pathway in urothelial bladder carcinomas: a multivariate analysis including p53 expression and proliferation markers.

Objective: To assess the prognostic value of the expression of two negative regulators of the cell cycle, namely CDKN2/INK4a gene product (p16) and retinoblastoma gene product (pRb), in urinary bladder cancer in relation to clinicopathological parameters, proliferative fraction and p53 protein accumulation. Methods: Paraffin sections from 139 patients with urothelial carcinomas were stained immunohistochemically with antibodies to p16 (F12), pRb (PMG3–245), p53 (DO1), PCNA (PC10) and Ki–67 (MIB–1). Results: Diminished p16 and pRb expression occurred in 29 and 74% of cases, respectively, being associated with advanced stage but not with histological grade, papillary status or proliferation rate. In most cases (53%) with some fault in the p16/pRb pathway, only one gene was affected. A double–negative p16/pRb phenotype was comparatively uncommon (25%) and was usually seen in T3–T4 tumours. In survival analysis (either univariate or multivariate) aberrant p16 expression was an adverse prognostic parameter only in T3–T4 tumours. In contrast, the abnormal p16/pRb and p53/p16 phenotypes were linked to a diminished overall and disease–free survival (univariate analysis); p53/p16 abnormal expression was also found to be an independent predictor of reduced survival in muscle–invasive tumours, while proliferation markers were the only parameters with independent significance in superficial (Ta–T1) tumours. Conclusion: Our results suggest that lack of p16 immunoexpression, when combined with p53 accumulation, plays an important role in determining the clinical outcome in muscle–invasive urothelial carcinomas.

Expression of retinoblastoma gene product and p21 (WAF1/Cip 1) protein in gliomas: correlations with proliferation markers, p53 expression and survival.

Abstract Using immunohistochemistry we evaluated the expression of two negative regulators of the cell cycle, the retinoblastoma gene product (pRb) and the WAF1/Cip1 gene product (p21), in consecutive paraffin sections from 54 gliomas (49 astrocytomas and 5 oligodendrogliomas) and related it to clinicopathological parameters, proliferative fraction, p53 expression and survival. Survival analysis was restricted to the group of diffuse astrocytomas (48 patients). pRb expression did not correlate with histological type, grade or p53 expression, while a moderately strong correlation existed between pRb expression and the percentages of proliferating cell nuclear antigen (PCNA) and MIB-1-positive cells. In 30% of cases we observed diminished pRb expression (i.e., a low pRb/Ki-67 ratio), irrespective of grade or histological type. p21 protein was elevated in 50% of cases, especially within the higher grades. The percentage of p21-positive cells was not related to histological type or grade but correlated loosely with PCNA and pRb expression. A p53-negative/p21-negative phenotype was characteristic of oligodendrogliomas and low-grade astrocytomas, whereas the p53-positive/p21-positive, p53-positive/p21-negative and p53-negative/p21-positive phenotypes were almost equally distributed among high-grade tumors. In survival analysis (either univariate or multivariate) diminished pRb expression was not a statistically significant prognostic indicator. In contrast, p21 expression emerged as an important indicator of shortened disease-free survival, in both univariate and multivariate analyses. Moreover, the double-positive p53/p21 phenotype tended to be associated with a shorter overall survival. Our results suggest that Rb gene deregulation does not significantly affect prognosis but p21 expression may play an important role in disease-free survival of astrocytoma patients.

Detection of apoptotic cells in archival tissue from diffuse astrocytomas using a monoclonal antibody to single-stranded DNA

Precise quantitation of apoptotic cells in gliomas is necessary to determine the role of apoptosis in tumour growth, prognosis, and treatment. This study investigated the incidence of baseline apoptosis in relation to proliferation status, p53 expression, standard clinicopathological parameters, and outcome, in a series of 61 patients with diffuse cerebral astrocytomas. Apoptotic fractions were quantified immunohistochemically by means of a novel monoclonal antibody recognizing exposed single‐stranded (ss) regions in the DNA of apoptotic cells during heating. Proliferative activity was expressed as the percentage of Ki‐67‐positive cells. Tissues consisted of primary formalin‐fixed, paraffin‐embedded astrocytoma specimens. The apoptotic index (AI) increased with grade, proliferative activity, and p53 expression. Increased AI tended to be accompanied by a shortened overall and disease‐free survival in univariate analysis in glioblastoma multiforme and astrocytoma/anaplastic astrocytoma, respectively. Multivariate analysis demonstrated that increased AI was an independent predictor of adverse significance in overall and disease‐free survival. These results implicate apoptotic rate in astrocytoma aggressiveness and show that the assessment of apoptotic potential by means of anti‐ssDNA monoclonal antibody provides valuable prognostic information independently of standard parameters or tumour proliferation status. Copyright

Comparative assessment of proliferating cell nuclear antigen immunostaining and of nucleolar organizer region staining in transitional cell carcinomas of the urinary bladder. Correlation with other conventional prognostic pathologic parameters.

The expression of two cell proliferation indices, the proliferating cell nuclear antigen (PCNA), using the monoclonal antibody PC-10 in the immunoperoxidase method, and the nucleolar organizer regions (NORs), using the colloid silver nitrate staining technique, was assessed in formalin-fixed paraffin-embedded material of 50 transitional cell urinary bladder carcinomas. A relationship was found between the histologic grade and each of the two indices used. A significant difference was observed, particularly between carcinomas of grade II and III (p < 0.001). A relationship was also demonstrated between each of PC-10 and AgNOR scores and the clinical stage, but it was attributed mostly to the close correlation of the latter with the histologic grade of these tumors. The linear correlation coefficient between PC-10 and AgNOR scores was 0.757 (p < 0.001). Our results suggest that PC-10 and AgNOR scores may be important prognostic indices in urinary bladder cancer.

Proliferating cell nuclear antigen and nucleolar organizer regions in CNS tumors: correlation with histological type and tumor grade. A comparative study of 82 cases on paraffin sections.

We investigated the expression of proliferating cell nuclear antigen (PCNA) and the number of nucleolar organizer regions (NORs) in 82 cases of CNS tumors. PCNA is a nuclear protein maximally elevated in the S phase of the cell cycle and recognized immunohistochemically in paraffin sections by the monoclonal antibody PC-10. On the other hand, NORs are loops of DNA that carry the rRNA genes and can be demonstrated in paraffin sections using an argyrophilic method (AgNORs). The present study shows a significant correlation of PCNA index and of AgNOR number with the histological grade (PCNA: I versus II, p < 0.01; II versus III, p < 0.01; and III versus IV, p < 0.05; AgNORs: I versus II, p < 0.001; II versus III, p < 0.05; and III versus IV, p < 0.001). Higher values of PCNA index (0.01 < p < 0.05) were found in recurrent tumors. Metastatic carcinomas were characterized by high PCNA indices and AgNOR numbers, similar to grade IV tumors, whereas in CNS lymphomas the malignancy grade was reflected in PCNA indices and AgNOR numbers. A wide range of PCNA and AgNOR values has been observed within each histological type and grade, probably reflecting variations in the biological behavior, but little overlap in PCNA values was present between grades II and III. The latter finding might be of importance in distinguishing between low- and high-grade CNS tumors. The linear regression coefficient between PCNA index and AgNOR number was excellent (0.91). We suggest that PCNA and AgNORs may be successfully applied in routine material to assess the growth potential of CNS tumors. Their prognostic value, however, must be validated with clinical studies.

Retinoblastoma gene product and P21 (WAF1, CIP1) protein expression in non Hodgkin's lymphomas: a multivariate survival analysis.

We evaluated immunohistochemically the expression of two negative regulators of the cell cycle, namely retinoblastoma gene product (pRb) and WAF1/Cip1 gene product (p21), in paraffin sections from 93 patients with non-Hodgkins lymphomas (NHL) and related it to clinicopathological parameters, proliferative fraction, p53 expression and survival. Patients were followed until death (n=33) or for an average of 52 months (60–160). Rb labelling index (LI) increased with malignancy grade and proliferative activity but was unrelated to other clinicopathological parameters. In 33% of cases, especially those of the aggressive groups, we observed diminished pRb expression (i.e. low pRb/Ki-67 ratio). p21 expression on the other hand correlated only with histological grade, Rb LI and p53 LI. In multivariate analysis, Rb LI was a negative predictor of disease-free survival but was linked to a higher probability of complete response. However, diminished pRb expression as well as p21 expression were not statistically significant prognostic indicators. Our results suggest that pRb as a cell cycle related molecule may play an important role in determining prognosis and therapeutic response in NHL patients.

Mitosin and DNA topoisomerase IIalpha: two novel proliferation markers in the prognostication of diffuse astrocytoma patient survival.

The expression of two novel proliferation-associated markers, mitosin and topoisomerase II&agr; (Topo II&agr;), was evaluated immunohistochemically in consecutive paraffin sections from 60 diffuse astrocytomas (grades 2 to 4) in relation to clinicopathologic parameters, proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB-1) expression and survival. The percentage of mitosin and Topo II&agr;–positive cells (LI) increased with grade and Ki-67 LI, but could not discriminate between grade 3 on the one hand and grades 2 or 4 on the other hand. In 51% of cases, Ki-67 LI exceeded Topo II&agr; LI, especially within grade 4. Topo II&agr; and mitosin expression was adversely related to overall and disease-free survival in the entire cohort and in grades 2/3. However, only Topo II&agr; LI affected disease-free survival in grade 4 tumors. Multivariate analysis selected only mitosin LI along with the age of the patient, as the independent parameters predicting overall survival, whereas Topo II&agr; emerged as the single independent predictor of disease-free survival. It is concluded that the proliferative potential of astrocytomas, as measured by mitosin and Topo II&agr; immunostaining, conveys useful prognostic information, in addition to that obtained by standard clinicopathologic parameters.