Euphemia Thomas-Tsagli

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.

Hypoxia-inducible factor 1α/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis

Hypoxia‐inducible factor (HIF)‐1α is a transcription factor that promotes ischaemia‐driven angiogenesis. The aim of this study was to determine the relation of HIF‐1α to vascular endothelial growth factor (VEGF; an important angiogenic molecule in brain tumours), p53 expression, angiogenesis, proliferative potential and clinical outcome in a large series of diffuse astrocytomas. Expression of HIF‐1α, VEGF, Ki‐67 (a proliferation‐associated marker) and p53 was determined immunohistochemically in 83 adult patients with supratentorial diffuse astrocytomas. Microvessels, highlighted by means of anti‐CD34 immunohistochemistry, were enumerated with computer‐assisted image analysis. Although HIF‐1α and VEGF were expressed in the majority of cases, their levels increased significantly with increasing grade and proliferative potential. HIF‐1α positively correlated with microvessel counts and VEGF with total vascular area and the presence of rounder vessel sections. There was a positive correlation of VEGF with p53 expression in astrocytomas and anaplastic astrocytomas. In univariate analysis, both VEGF and HIF‐1α were associated with shortened survival in the entire cohort, but lost significance when grades II/III and grade IV were analysed separately. Multivariate analysis revealed that the combination of HIF‐1α with grade was a significant prognostic indicator. HIF‐1α expression may be used to refine the prognostic information provided by grade in patients with diffuse astrocytomas. Its adverse prognostic effect is most likely mediated by hypoxia, the driving force for HIF‐1α accumulation.

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.

WAF1/p21 protein expression is an independent prognostic indicator in superficial and invasive bladder cancer.

The inhibitor of cyclin-dependent kinases WAF1 gene product p21 is able to arrest mammalian cell cycle by mediating p53 and other factors. The prognostic value and interrelationships between p21 expression and various parameters in bladder cancer have not been fully elucidated. We retrospectively investigated the immunohistochemical expression of p21 protein in consecutive paraffin sections from 131 transitional cell carcinomas (TCCs) and related it to p53 protein expression, clinicopathologic parameters, proliferative fraction, and survival. Positivity was displayed in 45% of cases, among which one fourth was accompanied by p53 accumulation. p21 expression was statistically related to advanced T category. No association was shown between p21 and p53 or proliferation rate. Low grade invasive TCCs tended to be more often p21 positive than high grade invasive TCCs. Most superficial tumors displayed neither p21 nor p53 expression, whereas the combined phenotypes p53/p21+ and p53+/p21− predominated among invasive tumors. P21 labeling index emerged by multivariate analysis as the single independent indicator of shortened overall (P = 0.0294) and disease-free (P = 0.0414) survival in superficial TCCs. Conversely, in invasive tumors, loss of p21 expression was a predictor of shortened disease-free survival (P = 0.0234) and was associated with poor outcome when accompanied by p53 accumulation (P = 0.0033). In conclusion, our results indicate that p21 activation occurs early in tumorigenesis, appears associated with invasiveness, and is capable of cell cycle control in TCCs mostly through p53-dependent pathways. Finally, p21 expression, alone or in combination with p53 and irrespective of other clinicopathologic parameters, plays distinct roles in determining clinical outcome in superficial and invasive tumors, suggesting that urothelial bladder cancer represents two different diseases.

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.