Henning Bier

Anti-(epidermal growth factor) receptor monoclonal antibodies for the induction of antibody-dependent cell-mediated cytotoxicity against squamous cell carcinoma lines of the head and neck

Abstract Squamous cell carcinomas of the head and neck (SCCHN) frequently display high levels of the epidermal growth factor receptor (EGFR). Since EGFR is expressed on the cell surface it may form a suitable target for anticancer therapy with anti-receptor monoclonal antibodies (mAb). Besides the interference with receptor/ligand interactions, binding of mAb to EGFR leads to immunoglobulin-coated tumour cells that may induce or enhance non-specific immune effector mechanisms like antibody-dependent cell-mediated cytotoxicity (ADCC). In established cell lines of SCCHN (UM-SCC 11B, 14C, 22B, and 8029 NA) we investigated the antitumour activity of allogeneic peripheral blood mononuclear cells (PBMC) in combination with rat (ICR 62), mouse (EMD 55900), and humanized (EMD 72000) anti-EGFR mAb. In addition, autologous PBMC were available for tumour line UD-SCC 4. The EGFR protein content of the tumour cell lines ranged between 170 fmol/mg protein and 8100 fmol/mg protein, and MCF-7 cells served as receptor-negative controls. PBMC activity against SCCHN targets was determined in 96-well microtitre-plate monolayer cultures by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after coincubation for 4 h, 24 h and 72 h at effector target ratios of 1:1, 5:1, 10:1 and 20:1. PBMC subpopulations were obtained by macrophage depletion (plastic adherence) or natural killer (NK) cell enrichment (magnetic bead negative selection). Prolonged time of exposure and increased effector:target ratios revealed marked antitumour activity of PBMC alone. This non-specific immune destruction was enhanced considerably by humanized and rat, but not mouse anti-EGFR mAb. Increased EGFR protein in tumour cells partly correlated with an intensification of ADCC but was accompanied by decreased primary PBMC cytotoxicity. The utilization of PBMC subpopulations suggested a mainly NK-cell-mediated ADCC, which appeared to benefit directly or indirectly, e.g. via the secretion of cytokines, from other PBMC components. In conclusion, humanized (EMD 72000) and rat (ICR 62) anti-EGFR mAb were able to generate strong antitumour ADCC in target monolayers of SCCHN.

Chemokine receptors in head and neck cancer: association with metastatic spread and regulation during chemotherapy.

Head and neck carcinomas are histologically and clinically heterogeneous. While squamous cell carcinomas (SCC) are characterized by lymphogenous spread, adenoid cystic carcinomas (ACC) disseminate preferentially hematogenously. To study cellular and molecular mechanisms of organ‐specific metastasis, we used SCC and ACC cell lines and tumor tissues, obtained from patients with primary or metastatic disease. Comprehensive analysis at the mRNA and protein level of human chemokine receptors showed that SCC and ACC cells exhibited distinct and nonrandom expression profiles for these receptors. SCC predominantly expressed receptors for chemokines homeostatically expressed in lymph nodes, including CC chemokine receptor (CCR) 7 and CXC chemokine receptor (CXCR)5. No difference in expression of chemokine receptors was seen in primary SCC and corresponding lymph node metastases. In contrast to SCC, ACC cells primarily expressed CXCR4. In chemotaxis assays, ACC cells were responsive to CXCL12, the ligand for CXCR4. Exposure of ACC cells to cisplatin resulted in upregulation of CXCR4 on the cell surface, which was repressed by the transcriptional inhibitor, α‐amanitin. Treatment of ACC cells with CXCL12 resulted in the activation of Akt and ERK1/2 pathways. Furthermore, CXCL12 suppressed the rate of apoptosis induced by cisplatin in ACC cells, suggesting that signaling via CXCR4 may be part of a tumor cell survival program. Discrimination of the chemokine receptor profile in SCC and ACC in vitro and in tissues provided insights into their distinct biologic and clinical characteristics as well as indications that chemokine receptors might serve as future therapeutic targets in these malignancies.

T cells specific for HPV16 E7 epitopes in patients with squamous cell carcinoma of the oropharynx

Squamous cell carcinomas of the oropharynx (SCCO) are often infected with oncogenic human papilloma virus (HPV) subtype 16. To determine the frequency of T cells specific for human leukocyte antigen (HLA)‐A2.1 restricted HPV16 E7 protein‐derived epitopes, tetramer analysis was performed using peripheral blood lymphocytes of 20 HLA‐A2.1+ patients and 20 HLA‐A2.1+ healthy individuals. Tetramers specific for 3 HPV16 peptides (E711–20, E782–90 and E786–93), an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were used in multicolor flow analysis. The HPV‐specific T‐cell frequencies were correlated with the HPV16 E7 and p16 status in tumor sections. In vitro stimulation (IVS) with autologous dendritic cells (DC) pulsed with HPV16 E7 epitopes was performed to demonstrate proliferation and antitumor activity of the HPV‐responsive T cells. Frequencies of CD8+ T cells specific for HPV16 E7 peptides were not significantly different in patients with SCCO relative to normal donors. However, patients with tumors expressing HPV16 E7 (60%) and p16 (50%) had an increased frequency (p < 0.05) of T cells specific for the E711–20 epitope compared to those with tumors negative for both markers. HPV16 E711–20 and HPV16 E786–93 specific T cells were expandable upon IVS with cognate peptide‐pulsed DC and were reactive against peptide‐pulsed targets or, in case of the E711–20 epitope‐specific T cells, against HPV16 E7 expressing CaSki cell line. Thus, in patients with HPV16+ SCCO, precursor T cells specific for E711–20 epitope are present (1/3,947) in the circulation, are responsive to stimulation with the cognate viral peptide and recognize in vitro HPV16 E7+ tumor cells. Further studies have to elucidate why those T cells are unable to eliminate the tumor in vivo and this might also allow for finding potential strategies that will increase the chances of developing a future HPV‐based vaccine in patients with SCCO.

Reliable detection of p53 aberrations in squamous cell carcinomas of the head and neck requires transcript analysis of the entire coding region.

Aberrations of the p53 tumor suppressor gene are common events in squamous cell carcinomas of the head and neck (SCCHN). However, reported frequencies range considerably, and the predictive value of aberrant p53 is continuing to be an issue of controversy. These inconsistencies are possibly caused by methodical limitations.

Targeting the immune system: novel therapeutic approaches in squamous cell carcinoma of the head and neck

Despite advances in surgery, radiotherapy, and chemotherapy, the overall survival rates for patients with squamous cell carcinoma of the head and neck (SCCHN) have not changed over the last decades. Clearly, novel therapeutic strategies are needed for this cancer, which is highly immunosuppressive. Therefore, biologic therapies able to induce and/or up-regulate antitumor immune responses could represent a complementary approach to conventional treatments. Because patients with SCCHN are frequently immunocompromised due to the elimination or dysfunction of critical effector cells of the immune system, it might be necessary to restore these immune functions to allow for the generation of more effective antitumor host responses. Simultaneously, to prevent tumor escape, it might be necessary to alter attributes of the malignant cells. The present review summarizes recent advances in the field of immunotherapy of SCCHN, including techniques of nonspecific immune stimulation, the use of monoclonal antibodies, advances in adoptive immunotherapy and genetic engineering, as well as anticancer vaccines. These biologic therapies, alone or in combination with conventional treatment, are likely to develop into useful future treatment options for patients with SCCHN.

Comprehensive analysis of the p53 status in mucosal and cutaneous melanomas

The abrogation of the function of the “gatekeeper of the genome”, p53, is the most prevalent molecular alteration in solid human tumors. Regarding melanomas the involvement of p53 alterations is discussed controversially to date. In order to evaluate the status of p53 in detail, primary tumors and metastases of 63 sporadic cutaneous (CM) and mucosal (MuM) melanomas were examined by immunohistochemistry and sequence analysis of the entire coding region of the p53 transcript, i.e., exons 2 to 11. In addition, loss of heterozygosity (LOH) and loss of allele‐specific transcription (LOT) were determined. Accumulation of the p53 protein occurred in most of the CM and MuM specimens (71% and 58%, respectively). In contrast, protein stabilizing p53 mutations were observed in 14% of the CM and no mutation was found in MuM specimens. Two of the aberrations located outside the core domain. LOH was detected in 22% CM and 58% MuM, and LOT in 25% of the CM specimens. The genotype distribution at the polymorphic p53 codon 72 in melanoma patients differed significantly from control subjects. The calculation of odds ratios (OR) and 95% confidence intervals (CI) indicated an increased risk for developing cutaneous melanomas in individuals carrying the Pro‐coding allele. Altogether, aberrant p53 expression appears to be a common event in both CM and MuM.

Antitumor activity of protein kinase C inhibitors and cisplatin in human head and neck squamous cell carcinoma lines

Protein kinase C (PKC) plays a pivotal role in signal transduction involved in the control of cell proliferation, differentiation and apoptosis. Interference with such signaling pathways may result in altered tumor cell response to antineoplastic drugs. We investigated the effects of two selective PKC inhibitors as single agents and in combination with cisplatin in cell lines derived from squamous cell carcinomas of the head and neck (SCCHN). Safingol (Saf) is directed against the regulatory domain, whereas chelerythrine (Che) interacts with the catalytic domain of PKC. In six SCCHN cell lines (UM-SCC 11B, 14A, 14C and 22B, 8029NA, and a 5-fold cisplatin-resistant subline 8029DDP). PKC activities ranged between 1 and 158 IU/1×107 cells, and they were inversely proportional to the amount of cellular epidermal growth factor receptor. Using the colorimetric MTT assay, PKC inhibitors Saf and Che showed comparable dose-dependent growth inhibition. The 50% inhibitory concentrations (IC50) were between 3.8–8.6 μ M for Saf and 8.5–13.6 μ M for Che with no relationship to PKC activity or cisplatin sensitivity of the respective cell lines. Combinations of cisplatin (IC50 = 0.4–5.8 μ g/ml) and either PKC inhibitor (5 μ M Saf, 10 μ M Che) led to a significant decrease of cisplatin IC50 values in most cell lines. However, comparison with theoretical additive dose–response curves showed additive rather than synergistic effects for both PKC inhibitors.

Serum level and tissue expression of c-erbB-1 and c-erbB-2 proto-oncogene products in patients with squamous cell carcinoma of the head and neck

The proto-oncogene products erbB-l (EGF-Receptor) and erbB-2 (HER-2/neu), distinct members of the epidermal growth factor receptor family, are frequently overexpressed in squamous cell carcinoma of the head and neck (SCCHN). The accumulation of these transmembrane proteins may lead to significant amounts of the respective extracellular receptor domains (ECD) that are shed from the tumour cell surface and enter blood circulation, thus representing potential serum tumour markers. For erbB-l and erbB-2, we determined the ECD serum levels with enzyme-linked immunosorbent assays and evaluated the protein expression in tumour tissue by immunohistochemistry. The present study included 49 patients (37 untreated, 12 recurrences) and the same number of age- and sex-matched healthy controls. In 24 patients ECD serum levels were determined before and 6 weeks after surgery. Mean ECD serum levels for erbB-1 and erbB-2 were 54.8+/-1.6 and 153.7+/-6.1 fmol/ml in cancer patients, and 54+/-1.5 and 147.9+/-4.5 fmol/ml in healthy controls, respectively. There was no significant difference between untreated and recurrent disease. Serum ECD follow-ups 6 weeks after surgery revealed a significant 12.3% decline of erbB-1 but no change of erbB-2 values. Immunohistochemistry showed strong staining for erbB-1 in 78% and erbB-2 in 47% of the SCCHN specimens. No correlation was detectable between receptor ECD serum levels and receptor tissue expression, tumour stage, and tumour differentiation. Hence, ECD serum levels of erbB-1 and erbB-2 are not considered to be valuable tumour markers in SCCHN.

Immunological characterization of missense mutations occurring within cytotoxic T cell‐defined p53 epitopes in HLA‐A*0201+ squamous cell carcinomas of the head and neck

Previous analyses of p53 in 40 HLA‐A*0201(HLA‐A2)+ squamous cell carcinomas of the head and neck (SCCHN) indicated that 6/13 p53 missense mutations that were detected, S149C, T150R, V157F, Y220C, Y220H and E271K, occurred within HLA‐A2‐restricted cytotoxic T lymphocyte (CTL)‐defined p53 epitopes. Of the 6, the p53 S149C, Y220C and Y220H peptides were immunogenic. Anti‐p53 mutant S149C and Y220H effector cells cross‐reacted against the parental wild type sequence (wt) p53 peptides, whereas anti‐p53 Y220C effector cells were specific for the mutant peptide, p53 Y220C cDNA‐transfected HLA‐A2+ SaOS cells, and an HLA‐A2+ SCCHN cell line naturally expressing the mutation. These results indicate that the p53 Y220C mutation can be processed and presented for CD8+ T cell recognition. Furthermore, using an autologous PBMC/tumor system, anti‐p53 Y220C peptide‐effector cells recognizing the autologous tumor could also be generated. Our analysis of p53 in 10 additional HLA‐A2+ SCCHN tumors detected the p53 Y220C in 2/10 tumors raising the overall frequency of the p53 Y220C mutation to 6/50 (12%) HLA‐A2+ SCCHN tumors. In contrast, independent of their HLA class I genotypes, the p53 Y220C mutation frequency for all human tumors analyzed to date is ∼1.5%. This unexpectedly high frequency of the p53 Y220C mutation in HLA‐A2+ SCCHN suggests that vaccines targeting this mutation would not only be expected to induce robust anti‐tumor immune responses in HLA‐A2+ subjects, but also be more widely applicable than previously envisioned for any given p53 missense mutation.

Analysis of BRCA1, TP53, and TSG101 germline mutations in German breast and/or ovarian cancer families

About 5%-10% of breast cancers are considered to be hereditary and associated with germline mutations of specific genes. As yet, the most frequently affected genes identified are BRCA1 and BRCA2, but also other genes such as TP53 are supposed to influence the predisposition toward breast cancer. In the present study, we analyzed patients of 19 German families with early onset breast cancer and/or a family history of breast and/or ovarian cancer for the presence of mutations in BRCA1 and TP53. In addition, we screened for germline mutations in the putative tumor suppressor gene TSG101. For this purpose we used direct sequence analysis of the entire coding regions for all three genes and, in the case of BRCA1, single-strand conformation polymorphism analysis and protein transcription-translation assays. We identified eight previously described polymorphisms and several aberrations in BRCA1: 1 unclassified missense mutation, 3 small protein truncating mutations, 1 novel pseudoexon, and 5 splicing variants. No mutation was detected in TP53. Analysis of TSG101 transcripts revealed an aberrant transcript in two breast cancer patients belonging to the same family, suggesting TSG101 as a predisposing gene in hereditary breast cancer.