S. Vignati

Bcl-2 protein: a prognostic factor inversely correlated to p53 in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) prognosis is strictly related to well-established clinicopathological parameters which have unfortunately become insufficient in the prognostic evaluation of this type of cancer. As p53 and bcl-2 gene deregulations are frequently involved in several types of epithelial malignancies, we investigated the Bcl-2 and p53 protein expression in 91 and 101 cases of NSCLC respectively. The expression was then compared with established indicators of prognosis and biological behaviour of the tumours. No relationship was observed between Bcl-2 and either clinicopathological or biological parameters such as histology, grading, tumour status, nodal metastasis and proliferative activity evaluated by scoring proliferating cell nuclear antigen expression and Ki-67 immunoreactivity. However, the mean Bcl-2 expression was significantly lower in patients who developed metastasis during follow-up or died of metastatic disease (P = 0.006 and P = 0.01 respectively). Moreover, survival probability was higher in patients who expressed the Bcl-2 protein (P = 0.0002). In contrast with this, p53 protein accumulation was observed in tumours with metastatic nodal involvement (P = 0.02) or in patients who developed metastasis during follow-up (P = 0.01), although no correlation was found between p53 expression and overall survival. An inverse relationship was also found between Bcl-2 and the anti-oncogene protein product p53 (P = 0.01). Thus, a high proportion of NSCLCs express p53 and Bcl-2 proteins and their expression may have prognostic importance.

Neoangiogenesis and p53 protein in lung cancer: their prognostic role and their relation with vascular endothelial growth factor (VEGF) expression.

Following up-regulation of an angiogenesis inhibitor by the wild-type p53 protein proven recently, we have analysed on the one hand the prognostic impact of microvessel count (MC) and p53 protein overexpression in non-small-cell lung carcinoma (NSCLC) progression and, on the other hand, the inter-relation between the microvascular pattern and the p53 protein expression. Moreover, we assessed the expression of vascular endothelial growth factor (VEGF), one of the pivotal mediators of tumour angiogenesis, in order to investigate its relation to p53 protein expression and MC. Tumours from 73 patients resected for NSCLC between March 1991 and April 1992 (median follow-up 47 months, range 32-51 months) were analysed using an immunohistochemical method. In univariate analysis, MC and p53 accumulation were shown to affect metastatic nodal involvement, recurrence and death significantly. Multiple logistic regression analysis showed an important prognostic influence of MC and nodal status on overall (P = 0.0009; P = 0.01) and disease-free survival (P = 0.0001; P = 0.03). Interestingly, a strong statistical association was observed between p53 nuclear accumulation and MC (P = 0.0003). The same inter-relationship was found in non-squamous histotype (P = 0.002). When we analysed the concomitant influence of MC and p53 expression on overall survival, we were able to confirm a real predominant role of MC in comparison with p53. With regard to VEGF expression, p53-negative and lowly vascularized tumours showed a mean VEGF expression significantly lower than p53-positive and highly vascularized cancers (P = 0.02). These results underline the prognostic impact of MC and p53 protein accumulation in NSCLC and their reciprocal inter-relationship, supporting the hypothesis of a wild-type p53 regulation on the angiogenetic process through a VEGF up-regulation.

Epidermal Growth Factor Receptor (EGFr) Expression in Non-small Cell Lung Carcinomas Correlates with Metastatic Involvement of Hilar and Mediastinal Lymph Nodes in the Squamous Subtype

Epidermal growth factor receptor (EGFr) levels were evaluated in paraffin-embedded tumour specimens of non-small cell lung cancer (NSCLC) from 176 patients who underwent surgical resection. The EGFr expression was evaluated by immunocytochemical assay using a monoclonal antibody which recognises the external domain of the receptor. EGFr immunoreactivity was significantly higher in squamous than in non-squamous cell carcinomas (P = 0.0009). Hilar and/or mediastinal nodal involvement was found in 29 of 105 (27.4%) squamous cancers, and in this group of patients, the mean of EGFr positive cells was significantly higher than that of patients without nodal involvement (P = 0.01). No significant correlations were found between the expression of EGFr and other clinicopathological or biological parameters such as T-status, grading, proliferative activity. EGFR is suggested to represent a useful indicator of nodal metastasis in NSCLC.

The expression of proliferating cell nuclear antigen in paraffin sections of peripheral, node-negative non-small cell lung cancer.

Cell proliferation of 40 peripheral, node‐negative non‐small cell lung cancers (NSCLC) treated with surgery alone was investigated by immunohistochemical analysis with the monoclonal antibody (MoAb) PC10, which recognizes a proliferating cell nuclear antigen (PCNA) in formalin‐fixed and paraffin‐embedded material. Results were correlated with DNA ploidy and S‐phase fraction (SPF) analyzed by DNA flow cytometric study. Mitotic count (MC) was analyzed by light microscopic study and histopathologic features. PCNA immunoreactivity was seen in all samples and confined to the nuclei of cancer, but not to the surrounding, tumor‐negative cells; its frequency ranged from 0–70% (median, 15%), and tumors expressed either a low (0–25%, n = 25) or intermediate (26–75%, n = 15) proliferative activity. There was no relationship between PCNA immunoreactivity and tumor stage or among size, histologic type, and mitotic count (MC). Tumors with intratumoral blood vessel invasion (BVI) showed a significantly higher (P < 0.005) PCNA immunoreactivity than BVI‐negative tumors. PCNA scores were significantly higher (P < 0.005) in DNA aneuploid (n = 22) than in DNA diploid (n = 18) tumors and correlated significantly with the SPF of DNA aneuploid tumors (r = 0.825, P < 0.0001), but not with diploid tumors (r = 0.002, P = < 0.9). Intermediate proliferating tumors had a significantly higher (P < 0.01) MC than their counterparts. In univariate analysis, significant predictors of survival were tumor classification (T1 versus T2), tumor size (less than or equal to 2.6 cm versus more than 2.6 cm), BVI (BVI‐negative versus BVI‐positive), MC (less than or equal to 8 versus more than 8), and PCNA immunoreactivity (low versus intermediate). DNA ploidy and SPF did not influence survival significantly. Only PCNA immunoreactivity retained its independent level of significance (P = 0.02) by multivariate analysis. It was concluded that PCNA immunostaining is a simple and clinically useful method for estimating cell proliferation in formalin‐fixed, paraffin‐embedded tissue of resected peripheral, node‐negative NSCLC.

Bcl2 and p53 regulate vascular endothelial growth factor (VEGF)-mediated angiogenesis in non-small cell lung carcinoma.

The aim of this study was to investigate the expression of p53 and bcl2 proteins in a series of 107 non-small cell lung cancers (NSCLC), and to relate such protein expression to neovascularisation and the expression of vascular endothelial growth factor (VEGF). Moreover, we analysed the prognostic impact of these biological parameters on overall survival, both in univariate and multivariate analyses. An inverse association was found between bcl2 expression and microvessel count (MVC; P = 0.0004) and bcl2 and VEGF (P = 0.007). In contrast, a significant association was found between p53 expression and MVC (P = 0.03) and p53 and VEGF expression (P = 0.04). In univariate analysis, nodal status (P < 0.000001), MVC (P < 0.000001), bcl2 (P = 0.002), p53 (P = 0.03) and VEGF expression (P < 0.000001) significantly affected overall survival, but in multivariate analysis only MVC and VEGF expression retained their prognostic influence. Our results suggest that bcl2 and p53 possibly control the development of tumour angiogenesis in NSCLC, with putative mediation by VEGF. Moreover, the important influence of angiogenesis in the progression of NSCLC is further highlighted.

bcl-2, p53 and proliferating cell nuclear antigen expression is related to the degree of differentiation in thyroid carcinomas

Thyroid carcinomas are heterogeneous in terms of histology, clinical presentation, treatment response and prognosis. Since bcl-2 and p53 gene alterations are frequently involved in both lymphoid and epithelial malignancies, we analysed the expression of bcl-2, p53 and proliferating cell nuclear antigen (PCNA) in a group of 134 patients with thyroid neoplasms. The same markers were evaluated in fetal and adult normal thyroids as well as in 40 benign lesions. The study was carried out by immunocytochemistry on archival material using antibodies against bcl-2 and p53 protein on tissue sections of 40 adenomas (As), 20 medullary carcinomas (MCs), 70 well-differentiated carcinomas (WDCs), 20 poorly differentiated carcinomas (PDCs) and 24 undifferentiated carcinomas (UCs). bcl-2 immunoreactivity was detected in 36 out of 40 (90%) As, 20 out of 20 (100%) MCs, 60 out of 70 (85.7%) WDCs, 20 out of 20 (100%) PDCs, and 8 out of 24 (33.3%) of UCs. p53 expression was present in 11.4% of WDCs, 5% of PDCs, 5% of MCs and 62.5% of UCs. By contrast, no p53 immunoreactivity was detected in 40 adenomas and in all the normal thyroid tissues studied. We observed a positive correlation between the expression of p53 and PCNA (r = 0.42; P = 0.035) in a group of UCs, but not in WDCs, PDCs and MCs. Neither p53 nor bcl-2 expression were correlated with clinicopathological parameters, such as age, sex, pTNM and survival. Our results suggest that in tumours of the follicular epithelium p53 and bcl-2 protein abnormalities are associated with more advanced carcinomas and especially with undifferentiated carcinomas, while they are only rarely altered in tumours of the parafollicular C cells.

Prognostic significance of tumoral angiogenesis in completely resected late stage lung carcinoma (Stage IIIA‐N2): Impact of adjuvant therapies in a subset of patients at high risk of recurrence

Angiogenesis plays a critical role in human tumor growth and metastasis. Microvessel count (MC), as a measure of tumor angiogenesis, has been significantly correlated with metastatic disease in cutaneous, mammary, prostatic, head and neck, and early stage lung carcinoma.

Neoangiogenesis: A putative marker of malignancy in non-small-cell lung cancer (NSCLC) development

Several studies have documented a relevant prognostic role of microvessel count (MC) in non‐small‐cell lung carcinomas (NSCLC). However, no evidence has been reported about the involvement of neo‐angiogenesis in the development of bronchial cancers. The aim of this study was to analyze microvessel density both in normal and in pathological features of the bronchial tree detected concomitantly with carcinomas. In a group of 34 patients resected for NSCLC, 48 bronchial lesions (hyperplasia, squamous metaplasia, moderate dysplasia and in situ carcinoma) were identified. In addition, 20 samples of normal bronchial epithelium from the same patients were analyzed. A monoclonal antibody was used in order to identify microvessels in the most intense areas of neovascularization from the bronchial specimens. MC was also analyzed in invasive components. An increased number of microvessels was observed from normal to dysplastic epithelium, including in situ carcinoma. Mean MC was significantly lower in normal, hyperplastic and squamous metaplastic epithelium than in dysplastic epithelium and in situ carcinoma. In particular, no differences were observed between normal and hyperplastic/metaplastic components, whereas a statistically significant difference appeared between the latter and dysplastic lesions. Moderate dysplasia and in situ carcinoma showed a number of microvessels in the lamina propria of their mucosa which were not significantly different from the invasive component, whereas hyperplastic/metaplastic lesions presented a much lower number of microvessels than invasive cancer. From these data it appears that normal bronchial epithelium and lesions associated with cancers of the bronchial tree show neovascularization in their stromal component. Hyperplasia and squamous metaplasia, unlike dysplasia and in situ carcinoma, show a low microvessel count, and they cannot represent precursor or incipient changes in the bronchial epithelium before the fully developed in situ stage has also been reached.

Bio-morphological events in the development of the human female mammary gland from fetal age to puberty

Abstract Bio-morphological understanding of the developing human mammary glands may clarify some aspects of breast pathology, including cancer. In particular, some epidemiological data suggests that during fetal growth an altered intrauterine hormonal status, especially a change in estrogen status, could predispose to carcinogenesis. In an attempt to achieve new information on early breast growth, a series of developing human breasts have been analyzed, namely: 4 fetal breasts (28–32 weeks of gestational age), 7 infant breasts (7 h to 2 years) and 1 puberal breast (12 years). In addition to the morphological features, we studied the immunohistochemical expression of some markers involved in morphogenesis, such as MIB-1 for cell proliferation, bcl-2 for apoptosis control, CD34 for vasculogenesis, estrogen (ER) and progesterone (PR) receptors for hormonal profile, and smooth-muscle actin for myoepithelial differentiation. The results were as follows: (a) lobules, absent between 28 weeks and 2 days, were well evident at 2 years of age and at puberty; (b) myoepithelial cells appeared from 28 weeks onward and persisted later with no modification in quantity and distribution; (c) epithelial cell proliferation was constantly low; (d) in all breasts inner epithelial cells showed diffuse bcl-2 positivity, while basal myoepithelial-like cells were generally negative; (e) all breasts were well vascularized with two different patterns: periductal vascularization (PDV) and interductal vascularization (IDV), IDV being always present, whereas PDV was found only in infant breasts; (f) ER and PR were almost absent in fetal and infant breasts, while their expression was high in the epithelial cells of the puberal breast; (g) stromal cells had no hormonal receptors and were heterogeneous for proliferation and bcl-2 expression. Interestingly, two fetal breasts showed high proliferation and high ER expression, respectively, in their epithelial compartment. This could be the expression of an altered hormonal environment in utero, representing a basis for possible subsequent cancer initiation.

Tumor angiogenesis and biologic markers in resected stage I NSCLC

OBJECTIVE Microvessel count (MC), as a measure of tumor angiogenesis, has been shown to be significantly correlated with metastatic disease in cutaneous, mammary, prostatic, head and neck cancer. We have previously assessed the role of intensity of angiogenesis as predictor of metastasis in surgically resected T1N0M0 NSCLC. We needed to confirm its value, in a prospective larger study on Stage I NSCLC, before its utilization as a prognostic tool for further clinical investigations. METHODS In the present report we prospectively investigated 227 patients (206 males, 21 females; median age 65 years) with Stage I NSCLC treated only by radical surgery between March 1991 and December 1994 with utmost care for some biological characteristics (proliferative activity, the blood vessel invasion, angiogenesis and the p53 protein expression). RESULTS The operative procedures consisted of 62 pneumonectomies, 148 lobectomies and 17 segmentectomies or wedge resections. With a median follow-up of 36 months (range 15-60), eighty patients have already experienced a local (n = 22) or systemic (n = 58) relapse. Univariate analysis revealed that T factor (T1 versus T2)(P = 0.008) and angiogenesis count (< or = versus > median, 17) (P = 0.0006) were significant predictors of survival. The same variables were also significant predictors of long Disease Free Survival (P = 0.006 and P = 0.004, respectively). On multivariate analysis, however, only the microvessel count retained its level of prognostic significance as regards both overall (P < 0.01) and disease-free survival (P < 0.01). CONCLUSIONS The present study corroborates the role of angiogenesis in the metastatic spread of NSCLC and emphasizes its value in the identification of patients in whom surgery should be supplemented by systemic treatment.