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.

The 5-year survival rate of non-small cell lung carcinoma (NSCLC) has only marginally improved during the past two decades, despite advances in surgery and chemoradiotherapy, Major efforts are currently directed toward biological characterization of these tumors to define biomarkers able to add further prognostic information, thus improving new therapeutic protocols, We analyzed the predictive relevance of the microvessel count (MC), bcl-2 and p53 proteins, proliferative activity, and usual postsurgical parameters on recurrence and overall survival in a series of 70 patients with NSCLC. The expression of biological parameters (p53, bcl-2, proliferative activity, and MC) was detected using immunohistochemistry on paraffin-embedded and frozen sections from the tumors treated with surgical resection alone until relapse, In the univariate analysis, the histotype, tumor status, node status, p53, bcl-2, and MC have been shown to significantly affect progression and death, In the multiple logistic regression analysis, the MC (P < 0.000001), tumor status (P < 0.005), and node status (P < 0.0002) influenced the overall survival while prediction of relapse was strongly revealed by tumor status (P < 0.005), nodal metastatic involvement (P < 0.000001), and the assessment of the vascular count (P < 0.0004), These data have allowed the creation of a multivariate model which may add more information on risk of recurrence and death in patients with NSCLC and can form the basis for future randomized clinical trials.

Epidermal growth factor receptor (EGFr) levels were evaluated in paraffin-embedded tumour specimens of nonsmall 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.

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.

The growth of newly formed vessels, or neoangiogenesis, represents an important step in both physiological and pathological situations: in particular, tumour growth and metastasis require angiogenesis. Microvessel count (MC), which represents a measure of tumour angiogenesis, has been associated with metastatic spread in cutaneous, mammary, prostatic, head and neck, and early-stage lung cancer. In this study, the role of tumour angiogenesis as a prognostic indicator was examined in 253 primary non-small cell lung cancer (NSCLC) patients. Microvessels were counted by highlighting endothelial cells with anti-Factor VIII monoclonal antibody (MAb) in methacarn-fixed tumour samples. In univariat analysis, MC (P<0.000001), sex (P=0.0036), histotype (P<0.014), tumour status (P<0.007), and vessel invasion (P<0.019) were significantly related to hilar and/or mediastinal nodal involvement. However, in the stepwise logistic regression analysis, MC (P<0.00003) retained the most important influence on nodal metastasis. The overall survival analysis calculated by the Kaplan-Meier method revealed that tumours with high MC (>25 vessels/field) were significantly associated with increased death risk (log-rank test P=0.00067; Cox's test P=0.00046; Gehan's Wilcoxon test P=0.00108). In 94 patients, the development of metastatic disease during follow-up was significantly related to MC. Indeed, patients who developed metastasis during follow-up showed a higher MC, either as a dichotomous (P=0.01) or as a continuous (P=0.003) variable, than patients who had developed no metastasis at the time of the analysis. Moreover, in the stepwise logistic regression analysis, MC retained the most important influence on distant metastases. Microvessel count, as a method for the quantitation of tumour angiogenesis, has an important prognostic role in non-small cell lung carcinomas.

Cathepsin D expression has been assessed by immunohistochemistry in 108 cases of Non-Small Cell Lung Cancer (NSCLC) and its expression has been related to conventional prognostic factors such as tumor size, tumor grade, histotype and nodal involvement. The cancer cells in 41.6% of the tumors showed a granular, cytoplasmic cathepsin D (C-D) positivity. Moreover, some benign macrofage-like stromal cells expressed similar positivity. The C-D immunoreactivity was significantly associated with non-squamous histotype, small size, less advanced and more differentiated cancers. Additionally, in non-squamous tumors we observed a higher significant expression in early-staged (T1-2, NO) than in more advanced neoplasias. No significant associations were found between C-D and Ki-67, PCNA immunoreactivity, DNA ploidy or flow cytometric S-phase data. These findings suggest that NSCLCs also express C-D but, as previously reported for breast cancers, the immunohistochemical C-D expression has a good prognostic significance unlike the results obtained by cytosolic evaluation.

p53 is a tumor suppressor gene, located in the short arm of chromosome 17, which encodes for a nuclear protein involved in the control of cellular growth. Mutations in p53 gene are the most common genetic alterations in a several human cancers, including Non Small Cell Lung Cancer (NSCLC). However, up to now, the role of p53 in the tumour's behaviour and its progression has not been completely clear. We performed immunohistochemical staining for mutated p53 using two monoclonal antibodies, PAb1801 and PAb240, in fresh tumour specimens from 103 consecutive patients who underwent surgery for resectable NSCLC. PAb1801 detects both the normal and mutant form of p53, while PAb240 is specific only for the mutant form and recognizes a denaturation-resistant epitope located between aminoacids 156-335. Both antibodies showed a mainly nuclear staining in neoplastic cells but not in surrounding uninvolved lung tissues. 68 out of 100 (68%) and 37 out of 103 (35.9%) of the cases were positive with PAb1801 and with PAb240, respectively. Tumours from patients with hilar-mediastinal lymph node involvement showed a higher p53 expression, detected by PAb1801, than those without nodal metastases (p = 0.04). Moreover, tumours expressing more than 60% of positive cells with both antibodies showed a significant increase of nodal involvement (p = 0.1; p = 0.03). Furthermore, p53 expression was significantly related to post-surgical stage (p Tumor Stage) (p = 0.04). In addition, we did not find any correlation between p53 expression and proliferating activity evaluated by PCNA, Ki-67 and DNA flow cytometric cell cycle. In conclusion, the evaluation of p53 oncogene expression may identify individuals whose resectable NSCLCs have a more aggressive tumour behaviour.

We examined 50 non-small cell lung cancers and the matching serum from the same patients for the levels of p53 antigen. Histological sections of the tumor specimens were stained with anti-p53 specific antibodies, and the sera were tested by ELISA. We observed a correlation between the expression of the p53 protein in the tumors and the presence of circulating p53 antigen, using either a wild type-(P=0.024) or a mutant specific (P=0.007) ELISA test. The p53 serum levels were also significantly associated (P=0.019) with the extent of tumor necrosis, suggesting that circulating p53 may be derived by shedding from dead tumor cells. The levels of mutant p53 detected in the serum were significantly higher in patients with lymph node involvement (P=0.012) and with late-staged disease. (P=0.05). No association was found with tumor size and extent. Testing for serum levels of mutant p53 could provide additional prognostic information and could be used as as novel molecular diagnostic tool in the management of non-small cell lung cancer.

Mutations in the p53 tumour suppressor gene, with consequent accumulation of the p53 protein, are frequently observed in non-small cell lung cancer (NSCLC). Little is known, however, about the timing of their appearance or their maintenance through cancer progression and metastatic spread. We have examined the normal epithelium and a panel of bronchial lesions, including dysplastic, neoplastic, and metastatic lesions, for p53 immunoreactivity and for expression of proliferating cell nuclear antigen (PCNA). No p53 immunoreactivity was found in normal and hyperplastic epithelium, nor in squamous metaplastic lesions. Twenty out of 30 invasive tumours and 13 out of 17 in situ carcinomas adjacent to an invasive tumour showed p53 immunoreactivity. There was a strict correlation between the level of p53 expression in the non-invasive and the invasive components of the tumours. Five out of eight pairs of primary tumours and matching metastases expressed p53, at identical levels in both compartments. These data indicate that p53 overexpression can occur in the earliest recognized phase of NSCLC and that the alteration is maintained during progression from in situ to invasive carcinoma and metastatic spread. PCNA expression increased from early to advanced phases of NSCLC. High PCNA immunoreactivity was observed in tumours expressing high p53 levels. A significant association was observed for PCNA expression between preinvasive and invasive lesions.

Objective:To determine the neoplastic nature of Kaposi's sarcoma (KS). A highly vascularized lesion, KS is frequently associated with AIDS, indicating HIV products may be involved. Design and methods:We determined the angiogenic properties of KS cell-secreted products and the HIV-1-tat gene product in vivo. Cell-free secreted products (KS–CM) from cultured epidemic and sporadic KS spindle cells or recombinant (r) HIV-1 tat protein were injected into mice with a matrix support (Matrigel). Results:KS–CM produced lesions carrying all the phenotypic hallmarks of KS, as observed by light and electron microscopy: spindle-shaped cells, haemorrhages and an inflammatory infiltrate, as well as Factor VIII-positive endothelial cells lining new blood vessels. Electron microscopy indicated an initial granulocyte invasion, with spindle-cell migration and neocapillary formation in the centre of the matrix. These lesions required the cofactor heparin; KS–CM or heparin alone were poorly angiogenic. A less intense angiogenesis, with lower cellularity and few granulocytes, was observed in basic fibroblast growth factor (bFGF)/heparin lesions, indicating that factors other than bFGF are present in the KS spindle-cell products. When the collagenase inhibitor tissue inhibitor of metalloproteinases (TIMP)-2 was added to the sponges, KS–CM-induced angiogenesis was reduced by approximately 65% and bFGF-induced angiogenesis inhibited completely. Recombinant HIV-1 tat protein, a growth factor for KS cells, induced vascularization that was also enhanced by heparin, implying that HIV-1 tat could contribute to the aetiology of HIV-associated KS. Conclusions:KS-like lesions were obtained by injecting cell-free secreted products, suggesting that KS is a 'self-propagating' proliferative lesion caused by a cytokine imbalance and not a true neoplasm. Heparin-binding factors appear to be involved, and HIV-1 tat angiogenic properties implicate this molecule in AIDS-associated KS. Inhibition of KS–CM-induced KS-like lesions by TIMP-2 suggests that metalloproteinase inhibitors could be potential therapeutic agents for KS.

The histological examinations performed in our Institute in the years 1985-1991 and concerning thyroid diseases pertain to 2930 patients. These were considered clinically benign in 2463 cases and malignant in 467. After histological control, the actually benign cases resulted to be 2335, whereas carcinomas were 588 (495 papillary, 61 follicular, 17 anaplastic, 15 medullary). Six lymphomas and 1 fibrosarcoma have been also observed. A comparison between clinical and morphological diagnosis for thyroids thought to be clinically benign has shown a diagnostic overlapping of 93.8%, with absolute agreement for Basedow's diseases, whereas thyroiditis is the disease most often not diagnosed preoperatively. Incidental carcinomas were 132, with an attendance related directly to the number of samples randomly taken from the surgical specimen. In 538 cases of carcinoma it has been possible to evaluate the intraglandular spread of the tumor: bilateral multifocal cases were 18.4%, with an incidence of latero-cervical metastases (41.4%) nearly twice as that observed in monolateral cases (24.2%). Post-surgical examination of the whole gland in 381 patients treated with a total thyroidectomy for carcinoma has discovered 20 false positive cases: 11 were operated on only according to clinical and instrumental examinations, 2 on the basis of just the cytological results, and 7 on the basis of cytologically positive evidence though refuted by intraoperative frozen sections. In the 381 cases taken into account, intraoperative frozen sections have been done 256 times: the results have been confirmed by the histological post-surgical examination of the whole gland in 94.5% of the cases. No false positive cases did occur. False negative cases were 7, due to inadequate sampling (in the operating room or in the pathology lab). Delayed diagnoses were 4, whereas truly false negative cases have been in all 3 (1.2%). The greater part of our data has been disaggregated in two different periods (1985-1988 and 1989-1991) to point out any change with time in the parameters under observation. The most relevant reflections relating to the time were the following: (1) per cent increase of bilateral multifocal carcinomas (13.9 vs. 21.8%), (2) per cent decrease of latero-cervical metastases in monolateral carcinomas (31.8 vs 17.7%), (3) frank decrease in the median value of incidental carcinomas' diameters (2.2 cm in the first vs. 0.6 cm in the last year), (4) frank decrease of false positive cases among patients operated on without prior cytological or histological verification (6/21 vs. 1/27), (5) remarkable improvement of the rate of correlation between intraoperative frozen section diagnosis and post-surgical histological examination (92.1 vs. 97.4%).(ABSTRACT TRUNCATED AT 400 WORDS)

The prognostic significance of traditional and newer tumor cell-related biologic parameters, like deoxyribonucleic acid ploidy (flow cytometry), proliferative activity (expression of proliferating cell nuclear antigen by immunohistochemistry), mitotic count, and intratumoral or peritumoral (or both) blood or lymphatic vessel invasion by tumor cells was investigated in 95 consecutive patients who had T1 NO MO non-small-cell lung cancer and who had operation alone between 1975 and 1985. The median follow-up for the entire group is now 8.3 years, and overall 5-, 10-, and 15-year-survivals were 75 %, 69 %, and 61 %, respectively. Twenty-two patients died of either local (n = 3) or systemic (n = 19) recurrent non-small-cell lung cancer, 5 of non-cancer-related causes, 2 of new primary lung cancer, and 1 of an extrathoracic cancer. By multivariate analysis, blood vessel invasion by tumor cells (p = 0.0001) and mitotic count (p = 0.016) were independent predictors of survival; by contrast, the disease-free survival was influenced only by blood vessel invasion (p = 0.0004). The relative risk of death of recurrent non-small-cell lung cancer for low-risk patients (n = 79) was 13.3 (95 % confidence interval, 6.1 to 28.7) times lower than that of high-risk patients (n = 16) (p < 0.0001). The relative risk of manifesting recurrent disease as distant metastasis for high-risk patients was 25.64 (95 % confidence intervals, 8.4 to 77.6) times higher than that of their low-risk counterparts (p < 0.0001). These results provide a rationale for effective systemic adjuvant treatment in completely resected T1 N0 M0 non-small-cell lung cancer tailored to the individual patients' risk of development of recurrent non-small-cell lung cancer.

The morphological analysis of 223 cases of chronic calculous cholecystitis has drawn attention to the following outstanding facts: (a) elementary lesions are numerous (above all ulceration, mucosal erosion, antral and intestinal metaplasia) and put together a complex sight; (b) mild, moderate and severe dysplasias have a surprising high incidence (67%), even in the age-group under 40; (c) dysplasia is more common in the gallbladders bearing parallel hyperplastic and metaplastic mucosal changes, if compared with gallbladders where these changes are lacking; (d) thickness of gallbladder's wall and incidence of dysplasia are in direct relation; (e) echographic preoperative assessment of wall's thickness has predictive value for presence of dysplasia; (f) CEA and CA 19-9 have a progressively increasing expression in the various degrees of dysplasia.

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) hy 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.