Cancer therapies: basic and clinical perspectives in brain, prostate, and lung tumors: Naples, September, 24-27, 2000.

A continuous flow of theoretical and practical information among basic research, diagnosis, and therapeutic innovation is a crucial process to achieve a timely and effective progress in defeating human cancer. According to this essential concept, the main objective of the Fourth Joint International Cancer Conference "Cancer Therapies: Basic and Clinical Perspectives in Brain, Prostate and Lung Cancer" has been of gathering together basic scientists and clinicians who represent scientific opinion leaders in their field, to present and discuss the most recent scientific achievements in basic and clinical perspectives, advanced diagnostic and therapeutic strategies, and molecular and cellular therapeutic approaches in brain, prostate, and lung cancer.

RB2/p130 gene-enhanced expression down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in vivo.

Angiogenesis is an essential step in the progression of tumor formation and development. The switch to an angiogenetic phenotype can occur as a distinct step before progression to a neoplastic phenotype and is linked to genetic changes such as mutations in key cell cycle regulatory genes. The pathogenesis of the angiogenetic phenotype may involve the inactivation of tumor suppressor genes such as the "guardian of the genome," p53, and the cyclin-dependent kinase inhibitor p16. Retinoblastoma family member RB2/p130 encodes a cell cycle regulatory protein and has been found mutated in different tumor types. Overexpression of RB2/p130 not only suppresses tumor formation in nude mice but also causes regression of established tumor grafts, suggesting that RB2/p130 may modulate the angiogenetic balance. We found that induction of RB2/p130 expression using a tetracycline-regulated gene expression system as well as retroviral and adenoviral-mediated gene delivery inhibited angiogenesis in vivo. This correlated with pRb2/p130-mediated down-regulation of vascular endothelial growth factor protein expression both in vitro and in vivo.

New dimensions in cancer biology and therapy.

The most successful and productive approach to defeat cancer relies on highly integrated and interchanging cooperation between basic research, diagnosis, and therapeutic innovation. Nevertheless, much remains to be done to achieve a consistent and continuous flow of theoretical and practical information among scientists actively involved in these equally relevant fields. The major objective of the International Conferences, "New Dimensions in Cancer Biology and Therapy," has been identified in gathering together basic scientists and clinicians who represent scientific leaders in their field, whose working efforts are focused on specific human malignancies. Thus, in pursuit of this well-defined goal, the third edition of the Conference has focused on human immunodeficiency virus (HIV) and cancer, cutaneous melanoma, and colorectal carcinoma, which are ideal clinical examples for innovative diagnostic and therapeutic intervention, as well as optimal models to unveil new mechanisms of tumor pathogenesis.

Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer.

The retinoblastoma (Rb) family consists of the tumor suppressor pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in 235 specimens of lung cancer show the tightest inverse association between the histological grading in the most aggressive tumor types and pRb2/p130. This led us to study a panel of human lung cancers for mutations in the RB2/p130 gene. Mutations in the Rb-related gene RB2/p130 were detected in 11 of 14 (78.5%) primary lung tumors by single-strand conformation polymorphism and sequence analysis. A Moloney leukemia virus-based retroviral system was set up, and a comparable viral concentration of 1 x 10(7) infectious units/ml was obtained. Retrovirus-mediated delivery of wild-type RB2/p130 to the lung tumor cell line H23 potently inhibited tumorigenesis in vitro and in vivo, as shown by the dramatic growth arrest observed in a colony assay and the suppression of anchorage-independent growth potential and tumor formation in nude mice. The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and a 12-fold reduction in tumor growth after RB2/p130 transduction compared with the Pac-transduced tumors (92% reduction, P = 0.003) and lacZ-transduced tumors (93% reduction, P < 0.001) was found to be statistically significant. These findings provide the missing confirmation that RB2/p130 is a "bona fide" tumor suppressor gene and strengthen the hypothesis that it may be a candidate for cancer gene therapy for lung cancer.

Independent prognostic role of p16 expression in lung cancer.

OBJECTIVES: The cyclin-dependent kinase p16 (also known as Ink4A, Mts1, Cdkn2, and Cdkn4i) has been proposed as a tumor suppressor gene mapped on chromosome segment 9p21. This study evaluated p16 protein expression in 135 lung cancer specimens and investigated potential genetic alterations occurring in this gene. RESULTS: We found altered p16 immunohistochemical expression to be a frequent event in lung cancer and to be independent of either the histologic type or any other clinical-pathologic feature. Western blot analyses performed on about one third of the specimens correlated highly with these results. In addition, we found p16 immunohistochemical expression to be a favorable prognostic factor in lung cancer in that its reduction or loss correlated with a worse outcome for the patients. Polymerase chain reaction amplification and direct sequencing of p16 exons 1 and 2 revealed no mutations, indicating that p16-altered expression in lung cancer is not necessarily linked to mutational events of these genes. CONCLUSIONS: We conclude that p16-altered expression is both an independent and frequent event in lung cancer and may have an important role in tumorigenesis and in malignant progression of a significant proportion of these cancers. However, the actual incidence and relevance of p16 mutations in this neoplasm continues to be debated, and its analysis seems inconclusive. Our results suggest a prognostic role for the immunodetection of this protein on formalin-fixed and paraffin-embedded specimens. They further suggest its routine use in the evaluation of the frequently unpredictable behavior of lung cancer.

Frequent high expression of bax pro-apoptotic protein in non-small cell lung cancer.

Bax, bcl-2 and their homologues regulate a distal step in an evolutionary very well conserved pathway of apoptotic cell death. It plays a crucial role in the balance between proliferation rate and cell viability. Thus in the last years the attention of the scientific community towards these proteins has remarkably increased, in particular in the oncologic field. We developed an immunohistochemical assay allowing us to evaluate the bax expression in formalin fixed and paraffin embedded lung cancer tissues to investigate bax expression in a cohort of 55 patients affected by non-small cell lung cancer. We detected high expression of bax in 72.7% of our patients. When we statistically analyzed our data we did not find any correlation between bax expression and any clinicopathologic parameters (sex, age, TNM status, tumor grade, histological type). In conclusion, our study shows the frequent overexpression of bax, and this highlights the "apoptotic tendency" of cells during the neoplastic proliferation. But, the role of bax in non-small cell lung cancer pathogenesis still remains unclear and further studies of large numbers of patients, including different stage groups, are needed to better define the involvement of this protein in the complex mechanism of lung carcinogenesis.

Bcl-2 protein expression correlates with nodal status in non small cell lung cancer.

We investigated the immunohistochemical expression of bcl-2 in 55 non small cell lung cancer patients in order to understand if the altered expression of this gene is involved in the development of this kind of neoplasm. Our results showed bcl-2 immunopositivity to be a frequent event in non small cell lung cancer (54.5%) with a higher expression in squamous carcinomas compared to adenocarcinomas (p = .04). In addition, we found a positive correlation between bcl-2 expression levels and nodal status (p < .003). We suggest that bcl-2 immunostaining could be considered as marker of loco-regional invasivity.

Prognostic role of cyclin D1 in lung cancer. Relationship to proliferating cell nuclear antigen.

We developed an immunohistochemical assay specific for cyclin D1 and suitable for formalin-fixed and paraffin-embedded sections, to evaluate cyclin D1 expression in a group of 135 surgically resected lung-cancer patients for the purpose of investigating the prognostic role of this protein in lung cancer. In addition, we compared cyclin D1 expression with the expression of proliferating cell nuclear antigen (PCNA), considered to be a reliable index of the proliferation rate. We found cyclin D1 expressed in more than 60% of the neoplastic cells in 26.5% of our specimens. A total of 24.5% of the specimens showed cyclin D1 expression in a percentage of cells ranging from 30 to 60%; 36.7% of the specimens expressed cyclin D1 in less than 30% of the cells; and 12.2% of the specimens expressed cyclin D1 in less than 1% of the evaluated cells. Western blot analyses confirmed the specificity of this assay by correlating statistically in a highly significant fashion with the immunohistochemical results (P = 0.0003). Furthermore, we found a direct relationship between cyclin D1 and PCNA immunodetection (P = 0.0004), which correlated cyclin D1 overexpression with a higher tumor proliferation rate. When we analyzed our data statistically, cyclin D1 expression was found to be a negative prognostic marker (P < 0.00005) whose expression correlates with a shorter patient survival time.

Retinoblastoma-related protein pRb2/p130 and suppression of tumor growth in vivo.

BACKGROUND: The RB/p105 and p107 genes of the retinoblastoma family are tumor suppressor genes whose proteins are inactivated by interaction with T-antigen proteins encoded by polyomaviruses (e.g., simian virus 40 and human JC virus), which have been found to be highly tumorigenic in animals. A variety of indirect evidence suggests that another member of the retinoblastoma gene family, RB2/p130, is also a tumor suppressor gene. To investigate the putative tumor suppressor activity of RB2/p130 more directly, we utilized a tetracycline-regulated gene expression system to control expression of the encoded protein pRb2/p130 in JC virus-induced hamster brain tumor cells and to study the effects of pRb2/p130 on the growth of such tumor cells in nude mice. The ability of pRb2/p130 to interact with JC virus T antigen was also studied. METHODS: Northern blot hybridization analyses were performed on samples of total cellular RNA to measure RB2/p130 and beta-actin messenger RNA levels. Immunoprecipitation and western blot analyses were used to determine T-antigen and pRb2/p130 protein levels and to assess the phosphorylation status of these proteins. Tumor cells were injected subcutaneously into nude mice, and tumor growth, with or without induced expression of pRb2/p130, was monitored. RESULTS: Induction of pRb2/p130 expression brought about a 3.2-fold, or 69% (95% confidence interval = 64%-73%), reduction in final tumor mass in nude mice. We also demonstrated that JC virus T antigen binds hypophosphorylated pRb2/p130 and that stimulation of pRb2/p130 expression overcomes cellular transformation mediated by this antigen. CONCLUSION: Our findings support the hypothesis that RB2/p130 is a tumor suppressor gene.

RB growth control evasion in lung cancer.

We evaluated the expression of pRb by immunohistochemistry in 98 lung cancer specimens already characterized for their p16 and cyclin D1 status. We found the absence of pRb expression to be dependent upon the histological type, being more frequent in SCLCs than in NSCLCs (p < .00005). On the other hand, we failed to find any correlation between the expression of pRb and p16. In addition, we found a positive correlation between the expression of pRb and cyclin D1 (p = .0001). Therefore, we hypothesize that pRb growth control may be overcome by two different mechanisms in lung carcinogenesis: loss of pRb expression or overexpression of cyclin D1.

Simian virus 40 and human cancer.

Deoxyribonucleic acid (DNA) oncoviruses can induce neoplastic transformation by interfering with proliferative proteins. Simian virus 40 (SV40) has been shown to induce brain tumors, osteosarcoma, lymphoid tumors and malignant mesothelioma in hamsters and SV40-like DNA sequences corresponding to the Rb-pocket binding domain of SV40 T-antigen (Tag) have been detected in the same human tumors. Since only a small percentage of people exposed to asbestos fibers develop a malignant mesothelioma, SV40 has been suspected to co-operate with the fibers in the neoplastic transformation or even to itself induce the onset of malignant mesothelioma in patients without expositive history. The mechanism that seems to be involved in the SV40-induced carcinogenesis process is mediated by interaction of Tag, both with p53 and Rb proteins, leading to their functional inactivation that is responsible for the removal of their inhibitory cell cycle effect which determines the increase of the number of cells entering the G1-S phase. Up to now the source of SV40 human infections has not yet been completely identified even though administration from 1957-1965 of SV40 contaminated polio vaccines is highly suspected. Horizontal infection by sexual transmission has been also hypothesized. Due to the important public health implications further investigations are required in order to establish both the source and the carcinogenetic role of simian virus 40 in humans.

Prognostic role of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer.

Despite its potential role as a tumor suppressor, p27 gene, a member of the Cip/Kip family of cyclin-dependent kinase inhibitor genes, has never been found mutated in human tumors. We investigated p27 protein expression in a series of 108 non-small cell lung cancers (57.4% stage 1, 16.7% stage 2, and 25.9% stage 3) to determine whether the lack or altered expression of this protein correlates with neoplastic transformation and/or progression. We performed immunohistochemistry and Western blot analysis of each specimen. We found that tumors expressing low to undetectable levels of p27 contained high p27 degradation activity. When we evaluated the outcome of the patients in relationship to p27 expression, we found p27 to be a prognostic factor correlating with the overall survival times (P = 0.0012). The possibility of a simple assay, such as the immunohistochemical analysis of p27 expression on routinely formalin-fixed, paraffin-embedded specimens, has considerable value for the prognosis of patients who undergo surgical resection. In addition, confirmation of the involvement of the proteasome-mediated proteolysis in p27 degradation should stimulate new strategies of nonsurgical treatments of non-small cell lung cancer.

The retinoblastoma gene family pRb/p105, p107, pRb2/p130 and simian virus-40 large T-antigen in human mesotheliomas.

The oncoprotein of simian virus-40, SV40 large T-antigen (Tag), is reported to target and to inactivate growth suppressive proteins such as the retinoblastoma family and p53 (ref. 4, 5), leading to transformation of human cell lines in vitro, tumor production in rodents, and detection of Tag in several human cancers including mesotheliomas. The retinoblastoma family contains three members, pRb, p107 and pRb2/p130 (ref. 9), that are phosphorylated in a cell cycle-dependent manner, have cell growth suppressive properties and bind to specific members of the E2F family and various cyclins. Even though mesotheliomas are among the most aggressive human cancers, alterations of important cell-cycle "controllers," such as the Rb family genes, have never been reported in these tumors. We found the presence of SV40-like sequences in 86% of 35 archival specimens of mesothelioma. We also demonstrated that SV40 Tag, isolated from frozen biopsies of human mesothelioma, binds each of the retinoblastoma family proteins, pRb, p107 and pRb2/p130, in four of four specimens. We propose that the tumorigenic potential of SV40 Tag in some human mesotheliomas may arise from its ability to interact with and thereby inactivate several tumor and/or growth suppressive proteins.

Role of PCNA in differentiating between malignant mesothelioma and mesothelial hyperplasia: prognostic considerations.

We investigated the PCNA immunoreactivity in 35 specimens of malignant mesothelioma and 20 specimens of mesothelial hyperplasia in order to evaluate the usefulness of this parameter in differentiating between these two mesothelial proliferations, and to determine whether PCNA has any prognostic significance in mesotheliomas. Eleven of the 35 investigated malignant mesotheliomas displayed up to 25% of positive cells for PCNA expression. The remaining 24 specimens showed high percentages of positive cells ranging from 26% to 95%. All specimens of reactive hyperplasia had less than 25% of PCNA positive cells. The difference between malignant mesothelioma and mesothelial hyperplasia for PCNA immunoreactivity was statistically significant (p < 0.01). A positive relationship was also found between PCNA expression level and the overall survival of those affected by malignant mesothelioma (p = 0.0032). Our results suggest on important role for PCNA in differentiating diagnosis of mesothelial proliferations. It remains unclear whether PCNA expression truly correlates with the proliferation rate of the malignant mesotheliomas and with the overall survival of patients affected by this neoplasm.

p53 immunostaining in differential diagnosis of pleural mesothelial proliferations.

We evaluated tissues for the overexpression of p53 protein in 35 malignant mesotheliomas and 20 reactive pleural hyperplasias, obtained from open biopsies or pleurectomies. Of the 35 neoplasms investigated, 30 (85.7%) displayed positive nuclear staining for p53. No staining was seen in any specimens of hyperplasia. We found no significant statistical difference in p53 expression when we compared p53 overexpression in the different histological subtypes of mesotheliomas. Moreover, p53 overexpression did not correlate in a statistical manner with survival. We conclude that p53 overexpression is a frequent feature of pleural mesothelioma and is useful for routine differentiating between malignant and non-neoplastic mesothelial alterations. The reason for p53 overexpression in mesothelioma, however, remains to be determined.

Retinal pigment epithelium cells cultured on synthetic biodegradable polymers.

Alterations in the normal structure or functions of retinal pigment epithelium (RPE) can result in a number of ocular diseases. Implantation of RPE cells cultured on thin, biodegradable polymer films may provide a means of transplanting an organized sheet of RPE cells with distinct apical/basal characteristics for the restoration of normal RPE function. We have investigated the interactions of human RPE cells with different biodegradable polymer films to assess their suitability as substrates for RPE culture. Four biodegradable polymers were used: low molecular weight (MW) 50:50 poly(DL-lactic-co-glycolic acid) (PLGA); high MW 50:50 PLGA; 75:25 PLGA; and poly(L-lactic acid) (PLLA). Polymer film substrates were manufactured using a solvent casting technique. Human fetal RPE cells (10-16 weeks gestational) were plated on the polymer substrates and the cultures assessed with respect to cell attachment and proliferation. Histological and immunohistochemical studies were performed on the cells after 8 days in culture. RPE cells attached to all the polymers studied after 8 h in culture. After 8 h, 80.2 +/- 9.5% and 82.3 +/- 7.9% of the plated cells were attached to substrates of high MW 50:50 PLGA and 75:25 PLGA, respectively. The cells proliferated on all substrates, and there was about a threefold increase in cell number over the 8-day culture period on all the polymers studied. Immunohistochemistry after 8 days in culture demonstrated RPE cells labeled with a distinct reaction product for cytokeratin in the cell cytoplasm. All the polymers studied were suitable for RPE culture; however, high MW 50:50 PLGA and 75:25 PLGA proved to be the best in terms of manufacturing properties, cell attachment, and proliferation. These polymers can provide a suitable substrate for RPE cell culture and hold promise for the subretinal implantation of organized sheets of RPE cells.

Differential expression of Rb2/p130 and p107 in normal human tissues and in primary lung cancer.

Two proteins, p107 and pRb2/p130, which are structurally and functionally similar to the product of the retinoblastoma gene (pRb), were cloned by taking advantage of their ability to bind transforming proteins of DNA tumor viruses through a particular region called the "pocket domain." Like pRb, both proteins play a fundamental role in growth control. Using immunocytochemical techniques, we examined a variety of normal human tissues for the expression of pRb2/p130 and p107. Both proteins were expressed ubiquitously, although a different tissue distribution and/or level of expression were found in various organs. Terminally differentiated cells, such as neurons and skeletal muscle, showed high expression levels for Rb2/p130, whereas p107 was expressed at higher levels in other cell types such as epithelia of the breast and prostate. We then examined the expression pattern of Rb2/p130 in 158 specimens of human lung cancer and found an inverse correlation between the histological grading of the tumors, the development of metastasis, and the level of expression of Rb2/p130.

Manufacture and characterization of poly(alpha-hydroxy ester) thin films as temporary substrates for retinal pigment epithelium cells.

For many disorders of the retinal pigment epithelium (RPE) for which there are no effective treatments, transplantation of RPE cells may provide a viable means of restoring function. Using a solvent casting technique, we have manufactured thin films of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) 75:25 and 50:50. Non-porous, flexible films with controlled thickness as thin as 12 +/- 3 microns and reproducible surface morphologies and flexural properties were produced. Fetal human RPE cells were found to attach to these substrates when cultured in vitro. The films made using this technique may provide a means of transplanting allogeneic RPE cells as a therapy for a number of ocular diseases related to RPE dysfunction.

The RB2/p130 gene product is a nuclear protein whose phosphorylation is cell cycle regulated.

The Rb2/p130 protein has been shown to have a high sequence homology with the retinoblastoma gene product (pRb), one of the most well-characterized tumor suppressor genes, and with pRB-related p107, especially in their conserved pocket domains, which display a primary role in the function of these proteins. In this study, we report on the biochemical and immunocytochemical characterization of the Rb2/p130 protein, using a polyclonal antibody developed against its "spacer" region included in the pocket domain of the whole protein. We show that pRb2/p130 is a phosphoprotein located at the nuclear level and that its phosphorylation pathway can be dramatically reduced by phosphatase treatment. Moreover pRb2/p130 with p107, is one of the major targets of the E1A viral oncoprotein-associated kinase activity, showing a phosphorylation pattern which is modulated during the cell cycle, reaching a peak of activation at the onset of S-phase.

Biodegradation and tissue reaction to intravitreous biodegradable poly(D,L-lactic-co-glycolic)acid microspheres.

We studied the biodegradation of and the tissue reaction to microspheres of 50:50 poly(D,L-lactic-co-glycolic)acid (PLGA) (viscosity-average MW: 3000 d), injected intravitreous in rabbits. These microspheres are under investigation as injectable devices for intravitreous sustained drug delivery. The rate of intravitreous degradation of PLGA microspheres has not been well documented in the literature. Twenty two pigmented rabbits underwent gas vitrectomy in one eye: 19 eyes received 2.5 mg of PLGA microspheres in 1 ml of balanced salt solution (BSS) and 3 control eyes received 1 ml of BSS only. Slit lamp exam and indirect ophthalmoscopy were performed periodically from day 1 to 6 months after surgery. The eyes were enucleated and studied by light microscopy and immunohistochemistry at various time points. The electroretinogram (ERG) was recorded in a subgroup of rabbits before injection and after 1 and 6 months. The amount of microspheres in the vitreous cavity progressively decreased. At 6 months microspheres were found in 1/4 rabbits at indirect ophthalmoscopy and in 4/4 rabbits histopathologically. A mild localized, non progressive foreign body reaction was observed. The cell reaction was composed mostly of vimentin and glial fibrillary acidic protein positive cells which probably represent glial cells and fibroblasts. The choroid and retina were normal. The ERG showed no abnormalities. No clinical inflammatory signs were observed 4 days postoperatively and thereafter.