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Background: Individual therapy efficiency of HER2−positive metastatic and pre-metastatic breast cancer patients varies significantly and spans from effectual responsiveness over acquired insensitivity to complete resistance from the outset. Thus no predictive information can be deduced from HER2 diagnostics so that molecular biomarkers indicative for sensitivity / resistance to Herceptin are needed to be identified. The HER2 related HER4-receptor has been shown to have ambivalent (pro-apoptotic or pro-proliferative) activity and consequently represents a prime candidate to affect HER2 activity under Herceptin treatment. We retrospectively analyzed potential her4 gene amplification and HER4 protein expression in HER2−positive, Herceptin treated patients. Patient's overall and recurrence free survival was evaluated as a function of HER2/HER4 expression. Methods: Using dual color Fluorescence in-situ Hybridization (FISH probes, Zytovision, Bremerhaven, Germany) and qPCR (LC480, Roche, Penzberg, Germany) we quantitatively investigated primary breast cancer tissues from nearly 50 (FISH) and 160 (PCR) patients who received Herceptin treatment. We quantified the her4 gene copy numbers and evaluated the protein expression profile of all four known HER4 isotypes (JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, JM-b/CYT2). Results: FISH analysis revealed a positive and independent prognostic marker in Herceptin treated breast cancer patients with respect to overall survival. Moreover by quantitative PCR analysis we found a significant variability of HER4 protein expression (JM-a/CYT1 and JM-a/CYT2; no JM-b isotypes) in HER2 positive breast cancer tissues, whereas HER2/HER4 positive patients show a significant better recurrence free survival compared to HER2 positive but HER4 negative patients (p = 0,003). Conclusions: HER4 has been demonstrated to potentially exert tumor suppressing activity and in turn to have a favourable impact on the course of breast cancer disease. We show here that HER4 expression prolongs in particular recurrence free survival of Herceptin treated patients which indicates a functional integration of HER4 into anti-HER2 targeting. Complementing functional studies allowing for isotype specific function of HER4 will elucidate the special role of this receptor tyrosine kinase in the context of Herceptin treatment and might facilitate individualized anti-ErbB-receptor targeting with higher efficiency. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-23.

The prolonged life span of chronic lymphocytic leukemia (CLL) cells in vivo is assumed to depend on the surrounding microenvironment since this biologic feature is lost in vitro. We studied here the molecular interactions between CLL cells and their surrounding stroma to identify factors that help CLL cells to resist apoptosis. Sorted CLL cells from 21 patients were cultured in vitro on allogenous, normal bone marrow stromal cells (BMSCs) in the presence/absence of CD40 ligand or in culture medium alone. Surface and mRNA expression of interaction molecules, cytokine production, and apoptosis rate was measured by flow cytometric, real-time PCR and standard immunologic assays. The interaction between CLL cells and BMSCs rescued CLL cells from apoptosis. BMSCs co-cultured with CLL cells showed a strong increase in IL-8 and IL-6 secretion and up-regulated the expression of ICAM-1 and CD40 mRNA. The mRNA expression of CXCL12 and VCAM1 remained unchanged. In turn, CLL cells in interaction with BMSCs significantly up-regulated the expression of CD18 and CD49d that are ligands for the critical adhesion molecules on BMSCs. As a validation of the in vitro data, we found a significant higher expression of CD49d on CLL cells in bone marrow aspirates compared to peripheral blood CLL cells in patient samples. Up-regulation of adhesion molecules and their ligands in CLL–BMSCs interaction along with the increased cytokine production of BMSCs indicate a strong effect of CLL cells on BMSCs in favor of their apoptosis resistance.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B-lymphocytes that manifests in a variety of clinical courses. The accumulation of CLL-cells is primarily caused by defective apoptosis; however, a higher proliferative capacity has also been found to correlate with poorer prognostic factors. Proliferating CLL-cells are confined to specialized structures called pseudofollicles, which contain CLL-cells, T-lymphocytes, and stromal cells. We established an in vitro model for pseudofollicles to characterize the behavior of CLL-cells in relation to clinical courses with different outcomes. Only CLL-cells from progressive clinical cases were inducible to proliferate by a combination of soluble CD40L/IL-2/IL-10 in co-culture with stromal cells. Proliferating CLL-cells showed a higher and more extensive expression of antigens, which are important in T-B-cell interactions such as CD40, MHC II, and adhesion molecules. IL-4 increased interferon regulatory factor-4 expression and induced a specific immunophenotype, which may imply plasmacytic differentiation. Furthermore, it was shown that co-cultured stromal cells protected CLL-cells from apoptosis. CLL-cells from clinically indolent cases had a far worse survival rate in medium than the cells from poor prognostic cases. Thus, we can assume that not only a different resistance to apoptosis, but also proliferation contributes to the progression of CLL resulting in bone marrow failure with thrombocytopenia and anemia.

The transcription factor AP-2 is encoded by a gene located on chromosome 6 near the HLA locus. Here we describe the genomic organization of the AP-2 gene including an initial characterization of the promoter. We have mapped two mRNA initiation sites, the entire exon-intron structure and located two polyadenylation sites. The mature AP-2 mRNA is spliced from 7 exons distributed over a region of 18 kb genomic DNA. A recently cloned inhibitory AP-2 protein is generated by alternative usage of a C-terminal exon. The proline-rich transactivation motif is encoded by a single exon within the N-terminal region in contrast to the complex DNA binding and dimerization motif which involves amino acid residues located on four different exons. The sites of mRNA initiation are located 220 and 271 bases upstream from the ATG translation start site. Although the promoter contains no canonical sequence motifs for basal transcription factors, such as TATA-, CCAAT- or SP-1 boxes, it mediates cell-type-specific expression of a CAT reporter gene in PA-1 human teratocarcinoma cells and is inactive in murine F9 teratocarcinoma cells. We demonstrate that the promoter of the AP-2 gene is subject to positive autoregulation by its own gene product. A consensus AP-2 binding site is located at position -622 with respect to the ATG. This site binds specifically to bacterially expressed AP-2 as well as to multiple proteins, including AP-2, present in PA-1 and HeLa cell nuclear extracts. A partial AP-2 promoter fragment including the AP-2 consensus binding site is approximately 5-fold transactivated by cotransfection of an AP-2 expression plasmid.

Combination of chemotherapeutic drugs with agents that induce cell differentiation is a possible means of improving cancer chemotherapy. To explore this approach we used 4 cell lines established from the human teratocarcinoma-derived cell line PA-1; 2 retinoic acid (RA)-sensitive lines compared to 2 RA-resistant lines transformed by an activated N-ras oncogene. Equal numbers of colony-forming cells were exposed for 72 hr to 10(-6)M RA and subsequently to a range of concentrations of cisplatinum, etoposide or bleomycin. Enhanced cytotoxicity of cisplatin and etoposide (3- to 5-fold) was observed in the N-ras-transformed cell lines compared to the non-transformed lines. Treatment with RA caused an increase in the cytotoxicity of all 3 drugs to the 2 RA-sensitive cell lines. In contrast, a reduction of cytotoxicity was observed in the 2 N-ras-transformed lines. Our results indicate that sensitivity to cytotoxic agents can be increased by RA in RA-sensitive cells, but the opposite effect is seen in N-ras transformed, RA-resistant cells. Therefore, a general rationale for combination therapy with RA and cytotoxic drugs cannot be inferred.

O44 Characterization of the proliferating chronic lymphocytic leukemia cells in an in vitro model for pseudofollicles M. Plander1,3 *, S. Seegers1, P. Ugocsai2, S. Schwarz1, E. Orso2, R. Knuchel4, J. Ivanyi3, G. Brochkoff1. 1Institute of Pathology, 2Institute of Clinical Chemistry, University Regensburg, Germany, 3County Hospital of Vas, Szombathely, Hungary, 4Institute of Pathology, University Aachen, Germany