The Project consists of one principal and two sub-projects. The former is on the determination of the angiogenic activity of human breast carcinoma, the first sub-project extends this determination to other solid tumours, namely: epithelial ovarian carcinoma, grade III-IV cerebral astrocitomas and Dukes stages B2-C colon-rectal adenocarcinomas. The second sub-project is the determination of levels of angiogenic peptides using biochemical methods.

1) Principal project on breast carcinoma

1) Introduction: Angiogenesis plays a key role in the processess of tumour progression and metastasis (1-4). The determination of the angiogenic activity (AA) is emerging as an important prognostic marker in operable breast cancer (2). Among the 13 studies up to now published 8/12 found a significant association between high angiogenic activity (i.e. high intratumoral microvessel density [IMD]) and poor relapse-free survival and 6/8 with poor overall survival. Most of the authors have documented a significant association between IMD and axillary lymph nodal and/or distant metastasis (reviewed in ref.2). AA has been up to now determined mainly using immunocytochemical methods (ICC) and specific antibodies to endothelial cells. With these methods important biological and clinical results have been obtained, however, an "optimal" method has not yet been found to evaluate AA in human neoplastic pathology.

2) Aims: To study in a series already collected of 200 cases operated of breast carcinoma, minimum follow-up of 3 years, with frozen and paraffin embedded pathological archivial material the following new methodological approaches:

2.1) To test the new monoclonal antibodies TEC-11 and E-9 (not commercially available) specific to proliferating/activated vascular endothelial cells (5,6). The possibility of using these antibodies to identify proliferating vessels from those which are quiescent may represent a methodological advantage to verify which between the total IMD (using the "pan-endothelial CD-31 monoclonal antibody)(7) and the number of proliferating/activated vessels correlates better with prognosis.

2.2) In the same series we will study the most important angiogenic peptides (i.e. bFGF and VEGF) using both ICC and molecular biology (MB) methods (8). The determination of the most important natural angiogenesis inhibitory way (i.e. p53/thrombospondin-1 system) will be determined by MB methods (9). The MB determinations of the system Flk-1/VEGF and of that p53/thrombospondin-1 are planned in collaboration with the Max-Planck Institute and the Laboratory of Immunology of the University of Chicago directed by Prof. Bouck.

2.3) The ongoing prospective randomized trial in operable breast cancer patients supported last year by A.I.R.C. grants, which stratified different adjuvant treatments on the basis of the IMD of the tumors, will continue (see enclosed protocol).

3) Methods:

- The Moab TEC-11 developed by Thorpe et al (5) is specific for endoglin, a protein selectively expressed by proliferating vascular endothelial cells. It reacts on fresh or frozen material only, using a conventional streptavidin-biotin-immunoperoxidase ICC technique.

- The Moab E-9 developed by Kumar et al (6) is specific for "activated" vascular endothelial cells. It reacts on fresh or frozen material only, using a conventional ICC technique.

- The Moab CD-31 is the most sensitive and specific "pan-endothelial" marker available (7).

The method of microvessels count and evaluation has been already published by us (7). Furthermore, a computerized image analysis system recently developed by us (10) will be used for comparison with the counts and evaluations performed by the two most expert pathologists in the field of our Unit.

- A specific antibody to VEGF will be supplied by Prof Harris, I.C.R.F. Univ of Oxford (see letter).

- The systems Flk-1/VEGF and p53/thrombospondin-1 will be determined by MB techniques using the methods published by the collaborators (8,9).

- Statistical methods. Univariate and multivariate log-logistic methods will be employed in order to compare the prognostic value of the different methods to study AA.

2) Sub-project on the determination of the AA in other solid tumours

1) Introduction: An important question is to verify whether the prognostic significance of AA is universal or restricted to specific neoplasia, such as breast cancer (2). Some "pilot" retrospective studies performed in small series of patients and with short follow-up times have investigated the prognostic role of AA, determined counting intratumoral microvessel density and ICC methods, in some human solid tumours: cutaneous malignant melanoma; non-small cell-lung cancer; prostatic cancer; head and neck squamous cell carcinoma; rectal adenocarcinoma; germinal tumours of the testis; epithelial carcinoma of the ovary; bladder cancer; soft-tissue sarcomas (reviewed in refs 11-12) and cerebral tumours of the infancy (13). Overall, even if no definitive conclusion can be drawn, it seems that the determination of AA may have potential clinical applications to predict prognosis.

We have already found that IMD correlates with metastasis in a series of 70 cases of head and neck squamous carcinoma (14).

2) Aims: 2.1) To determine IMD using the CD-31 Moab and a conventional method to assess microvessel density (7) in paraffin embedded material of patients operated of stages III-IV epithelial ovarian cancer (EOC) grade III-IV cerebral astrocitomas (CA) and Dukes stages B2-C colon-rectal adenocarcinomas (CRA). 2.2) To study also other biological markers of emerging importance such as: p53, bcl-2, bax, mdm-2, cell kinetics markers (MIB-1; D-E cyclines) for prognostic comparison. In fact, we will perform a multiparametric study by correlating the above markers with clinical outcome and response to conventional treatments and their joint prognostic/predictive effect in a multivariate log-logistic regression statistical analysis.

3) Methods: For a preliminary evaluation the above mentioned markers will be tested in 120 EOC, 110 CA and 200 CRA with paraffin-embedded pathological material and clinical follow-up available. As an initial step all the markers will be tested using ICC methods. By applying univariate and multivariate log-logistic regression statistical analysis the prognostic value of each biological marker and their joint effect will be compared with that of the conventional clinico-pathologic prognostic indicators specific for each of the tumour histotypes studied.

3) Sub-project on the biochemical determinations of angiogenic peptides

1) Introduction: Folkman et al (15) demonstrated that urinary levels of basic fibroblast growth factor (bFGF) give important clinical information on prognosis and the "status" of a wide spectrum of malignancies. The same group have also found that serum levels of bFGF predict clinical outcome of breast cancer patients (16).

2) Aims: We would prospectivelly evaluate the prognostic value of serum/urinary levels of bFGF in patients operated of the neoplastic diseases already mentioned. The prognostic value of bFGF will be compared with that of the ICC method using the CD-31 Moab and microvessel counts (7). Furthermore, we will evaluate in a prospective comparative study the prognostic significance of the microvessel counts using the E-9 Moab and ICC method and that of the ELISA assessment of E-9 Moab which detects "activated vessels" in the same neoplastic tissue.

3) Methods: Serum and urine levels of bFGF will be measured by using a commercial kit, based on the method of Watanabe et Al (17).

bFGF levels will be monitored at different times: before and after surgery, before and after adjuvant treatments when indicated, during follow-up and at the first tumour recurrence. bFGF levels will be correlated with clinical outcome and their prognostic significance will be compared with that of the ICC methods to detect IMD and the other prognostic indicators, using a multivariate statistical analysis.

References.

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• 3) Folkman, J., Watson, K., Ingber, D., and Hanahan, D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature, 339:58-61, 1989.
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• 10) Barbareschi, M., Weidner, N., Gasparini, G., Morelli, L., Eccher, C., Fina, P., Leonardi, E., Mauri, F., Bevilacqua, P., and Dalla Palma, P. Microvessel density quantification in breast carcinoma: A comparison between human counts and computer assisted image analysis. Appl. Immunohistochem., in press, 1995.
• 11) Gasparini, G. Quantification of intratumoral vascularisation predicts metastasis in human invasive solid tumours. Oncology Rep., 1:7-12, 1994.
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• 13) Li, V.W., Folkerth, R.D., Watanabe, H., Yu, C., Rupnick, M., Barnes, P., Scott, R.M., McL Black, P., Sallan, S.E., and Folkman, J. Microvessel count and cerebrospinal fluid basic fibroblast growth factor in children with brain tumours. Lancet, 344:82-86, 1994.
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