Session 27 : Oncologie et thérapeutique anti-infectieuse
INTERACTION BETWEEN ERBB-2 (HER2/NEU) AND HUMAN BREAST CANCER RESISTANCE TO CHEMOTHERAPY
E.C.Martin, S. Liva, X. Sastre-Garau, C. Barbaroux, B. Bouchind’homme, V. Dieras, H. Magdelénat, P. de Cremoux*
Institut Curie and *Université Paris 7, 26 rue d’Ulm, 75231 Paris cedex, France.
CerbB-2 gene is amplified in approximately 30% of human breast cancers. Some in vitro data demonstrates that erbB2 amplification is associated with resistance to hormone and chemotherapy. More recently, two pilot studies demonstrates the role of cerbB-2 overexpression in resistance to adriamycin and taxanes.
We used a quantitative competitive PCR and an immunohistochemical assay on formalin fixed paraffin embedded biopsies from breast cancer and normal tissues. For the PCR assay, we have coamplified a neu target sequence and an internal standard corresponding to the same four base pair deleted sequence, with the same PCR efficiency. This PCR is normalized by coamplification of a reference gene and its own deleted internal standard. The amount of tumor cells and the immunostaining (CB11 antibody) were scored by the pathologist in each sample on two adjacent slides. This quantitative competitive PCR allowed us to determine accurately and reproducibly the average gene copy number (AGCN) of neu in tumor samples: 43 assays corresponding to nine different normal samples displayed less than 14% variation. 32 breast cancer patients were analyzed before treatment by adriamycin and /or taxane. Three groups of samples were observed: 9 samples with evident cerbB-2 amplification (AGCN>4) and overexpression; 12 samples non amplified and normally expressed; 11 samples slightly amplified (AGCN=3 or 4), but without any cerbB-2 overexpression. The reliability of both assays allows us to investigate this marker in prospective randomized clinical trials.
ACTIVATION OF ANTI-TUMOR CYTOTOXIC T LYMPHOCYTES BY NON-NATURAL LIGANDS: STRUCTURAL AND PHARMACOLOGICAL STUDIES
M. Ayyoub, B. Monsarrat, H. Mazarguil and J. E. Gairin .
Institut de Pharmacologie et Biologie Structurale, UPR 9062 CNRS, 205 route de Narbonne, 31400 Toulouse, France.
Tumor antigens are potential therapeutical agents for cancer immunotherapy and these antigens, under different forms, are currently assayed. MHC class I restricted antigenic peptides have been tested for in vivo vaccination trials. This approach could be severely limited by the rapid degradation of the peptide in biological fluids. The question we asked in the current study was: is it possible to design non-natural, peptidase-resistant analogues of MZ2-E that still efficiently activate tumor specitic CTLs ? To answer this question we used as a model the human tumor antigen MZ2-E (EADPT GHSY). MZ2-E, encoded by the MAGE- 1 gene in 40% of melanomas but not in normal tissus (testis excepted), is presented to cytotoxic T Iymphocytes (CTLs) by the major histocompatibility complex (MHC) class I molecule HLA-A 1. We studied: i) the degradation pathway of MZ2-E in human serum and ii) the interactions of MZ2-E with HLA-A 1 and its specific T cell receptors (TcR).
By HPLC-MS analysis, we found that MZ2-E was rapidly degraded (t1/2 < 30 min.) by aminopeptidase and dipeptidyl carboxypeptidase. Based on this data and on molecular modeling of MZ2-E-HLA-A1 interaction, we synthesiz.ed a series of analogues with minimal but efficient structural modifications with modified or non-natural amino acids. All the analogues modified at the two sensitive bonds (P1-P2 and P7-P8) were peptidase-resistant with t1/2 > 24 hours. Two analogues,
[Aib2, D-His7]- and [Aib2, NMe-Ser8]-MZ2-E showed HLA-A1 binding affinities comparable to the parent peptide. Importantly, [Aib2, NMe-Ser8]MZ2-E sensitized target cells to Iysis by two of three known anti-MZ2-E CTL clones. This analogue which is an efficient activator of anti-melanoma immune response might be considered as a potential therapeutical agent for anti-melanoma immunotherapy.
Part of this work was done in collaboration with Dr. B. Van den Eynde (Ludwig Institut for Cancer Research, Brussels, Belgium.). This work was supported in part by the Association pour la Recherche contre le Cancer and the Conseil Régional Midi-Pyrénées.
DNA-DEPENDENT PROTEIN KINASE ACTIVITY (DNA-PK) IS DETERMINANT IN THE RESPONSE OF NORMAL AND TUMORAL HUMAN PRIMARY B-LYMPHOCYTES TO NITROGEN MUSTARDS
C. Muller1, G. Christotodoulopoulos2, L. Panasci2 and B. Salles1
1Institut de Pharmacologie et de Biologie Structurale (CNRS UPR 9062), 205 route de Narbonne, 31077 Toulouse, France and 2The Lady Davis Institute for Medical Research, The Sir Mortimer B. Davis Jewish General H3755, Cote Ste-Catherine, Montreal, Quebec, H3T IE2, Canada.
DNA-PK is a nuclear serine/threonine kinase that plays a critical role in mammalian double strand break repair and lymphoid V(D)J recombination. Its regulatory sub-unit Ku (heterodimer of Ku70/Ku86) binds to DNA and recruits the kinase catalytic sub-unit, DNA-PKCS. In addition to DNA doublestrand breaking agents (ionizing radiation), DNA-PK mutant cells exhibit hypersensitivity to DNA-cross linking agents (nitrogen mustards, cisplatin) . The aim of our study was to investigate the role of DNA-PK in the response of chronic lymphocytic leukemia (CLL) lymphocytes to nitrogen mustard therapy. In a series of 34 patients with B-CLL either sensitive (n=16) or resistant to the nitrogen mustard chlorambucil (n=18), the kinase activity of the complex, (determined by its capacity to phophorylate a peptide substrate in vitro) is increased in the resistant samples as compared to the sensitive ones [24.4
± 2.6 a.u. (range 12.7 to 55.8 a.u.) versus 8.1 ± 2.8 a.u. (range 0.9 to 44.5 a.u.) respectively (p