INVESTIGATION OF HLA CLASS-I DOWN-REGULATION IN BREAST CANCER BY RT PCR

INVESTIGATION OF HLA CLASS-I DOWN-REGULATION IN BREAST CANCER BY RT PCR. ^§G.L.Palmisano,^§M.P.Pistillo,^§P.Capanni,*L.Perdelli,*S.Salvi,*G.Nicolò,and^§G.B.Ferrar^§

Immuno-genetics Lab.,National Cancer Institute and Department of Oncology, Biology and Genetics c/o Advanced Biotecnology Center(CBA),University of Genoa.*Laboratory of Pathology, National Cancer Institute, Genova.

Downregulation of HLA class I antigen expression has been reported in a significant proportion of primary breast carcinomas suggesting an escape mechanism from CTL mediated lysis leading to tumor dissemination and metastasis. In our study we investigated the expression of HLA total class I (W6/32 mAb), a-chain (Q1/28 mAb) and b2-m (Namb-1 mAb) subunits, in 25 primary breast carcinomas, by conventional immuno-histochemistry (IHC) and by biochemistry (semiquantitative Western blot, WB). Three different HLA class I expression patterns could be observed either by IHC or WB: High, Low and Absent downregulation patterns.Because low expression levels of class I molecules in tumor cells may be due to limiting amounts of antigenic peptides that are required for proper assembly of class I a-chain and b2-microglobulin, analyses of HLA class-I RNA expression was examined in breast tumor patients showing Highand Absent expression patterns.HLA class I a-chain and b2-microglobulin gene expression levels were measured by RT-PCR using HLA-A, HLA-B, HLA-C and b2-microglobulin specific primers both in breast cancer tissues and breast normal tissues derived from the same patients in order to determine the basal level of gene expression for each patient.None (100%) of the patient tumor samples showed significant differences in expression the a-chain genes analysed in between High, Absent and Low patterns. In eight patients, all with the High down-regulation pattern, the b2-microglobulin gene specific PCR band was lost while the corresponding normal tissue counterpart showed a costitutive expression. Such result strongly suggests a b2-microglobulin gene defect at RNA level associated with breast tumor trasformation. The RT-PCR approach confirmed our WB analysis indicating that in these patients it was possible to address the downregulation of b2-microglobulin gene as a phenomenon which can occur at trascriptional level.Most importantly, the comparison between cancer and normal tissues belonging to the same patients helped us to betterdistinguish those patients showing a truly HLA class I downregulation (High pattern) from those patients having normal levels of HLA class I gene expression (Absent and Low patterns). These findings may contribute to understand the independent mechanisms underlying altered HLA phenotypes in breast cancer patients with important implication in developing more accurate and specific therapeutic approaches.