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 1 patients and 20 HLA-A2.1 1 healthy individuals. Tetramers specific for 3 HPV16 peptides (E7 11-20 , E7 82-90 and E7 86-93) , 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 1 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 E7 11-20 epitope compared to those with tumors negative for both markers. HPV16 E7 11-20 and HPV16 E7 86-93 specific T cells were expandable upon IVS with cognate peptide-pulsed DC and were reactive against peptide-pulsed targets or, in case of the E7 11-20 epitopespecific T cells, against HPV16 E7 expressing CaSki cell line. Thus, in patients with HPV16 1 SCCO, precursor T cells specific for E7 [11][12][13][14][15][16][17][18][19][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 1 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. ' 2005 Wiley-Liss, Inc.Key words: HPV; specific T cells; head and neck cancer; tetramer Squamous cell carcinomas (SCC) represent the most common type of head and neck cancer, accounting for 6% of all new cancer cases. 1 Typical risk factors include alcohol and nicotine consumption. However, a significant proportion of the patients do not have these risk factors, and recent studies have suggested an association of SCC to viral pathogens such as high risk (oncogenic) human papilloma virus (HPV) types, particularly HPV16 or 18. These viruses are frequently found in squamous carcinoma of the head and neck. 2-11 HPV16 infections have been observed in approximately one half of squamous cell carcinomas of the oropharynx (SCCO). 7,[10][11][12] The HPV16 1 SCCO have a good prognosis and are not associated with conventional risk factors, suggesting they may represent a separate tumor entity. 3,7,10,11 The HPV-derived oncoproteins E6 and E7 are mainly responsible for both the onset and maintenance of malignant transforma...

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 x 10(7) 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 microM for Saf and 8.5-13.6 microM for Che with no relationship to PKC activity or cisplatin sensitivity of the respective cell lines. Combinations of cisplatin (IC50 = 0.4-5.8 microg/ml) and either PKC inhibitor (5 microM Saf, 10 microM 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.

Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of squamous cell carcinoma of the head and neck (SCCHN). Monoclonal antibodies (mAbs) against EGFR are currently used for therapy of recurrent or metastatic disease; however, their mode of action is not completely understood. To investigate the immunological effects of anti-EGFR mAb, we generated a three-dimensional spheroid model of EGFR-expressing SCCHN and used this model to study the effect of anti-EGFR mAb on leukocyte migration toward tumors. Pretreatment with the blocking anti-EGFR mAb EMD 72000, its F(ab 0 )2 fragments or an EGFR tyrosine kinase inhibitor led to substantially increased leukocyte infiltration into EGFR overexpressing tumor spheroids, but not into those with low EGFR expression. Nonblocking anti-EGFR mAb or fibroblast-specific mAb did not affect leukocyte infiltration, suggesting that the observed increase in leukocyte infiltration depends on interference with EGFR activation. Using a human cytokine macroarray, we demonstrated that the blockade of EGFR by anti-EGFR mAb in EGFR-overexpressing SCCHN cells leads to differential expression of several cytokines and chemokines, including the chemokine MCP-1/CCL-2. The significant upregulation of MCP-1/CCL2 on exposure to anti-EGFR mAb was confirmed by quantitative PCR and enzyme-linked immunospot analyses. Moreover, blocking anti-MCP-1 antibody inhibited leukocyte migration toward tumor cells induced by anti-EGFR mAb, pointing to an important role of MCP-1/CCL2 in anti-EGFR mAb-induced leukocyte migration. Our findings demonstrate that anti-EGFR mAb induces leukocyte infiltration to tumor spheroids by upregulating chemokine expression. This novel mechanism for anti-EGFR mAb action may contribute to the antitumor effects of anti-EGFR mAb in vivo.

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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 Procoding allele. Altogether, aberrant p53 expression appears to be a common event in both CM and MuM. ' 2005 Wiley-Liss, Inc.Key words: melanoma; p53; immunohistochemistry; transcript analysis; LOH; LOT Abrogation of normal p53 function appears to be the most prevalent molecular alteration in human cancers, which allows tumor cells to survive, proliferate and continue to progress despite the accumulation of other alterations. Currently, p53 mutations are believed to contribute to the manifestation of nearly 50% of solid tumors. The evidence implicating sun exposure as the main cause of most types of skin cancer is persuasive. Exposure to both UV-A and UV-B can cause genetic changes in many biological systems. 1Typical UV-triggered mutations have been identified in protooncogenes and tumor suppressor genes in both melanoma and nonmelanoma skin cancer (NMSC). Mutations found in the p53 gene in human NMSC are mainly C to T and CC to TT transitions at dipyrimidine sequences, termed UV-B molecular signature mutations.2,3 In addition, UV-A caused mutations occur, e.g., T to G and G to T transversions. P53 mutations have also been detected in normal sun exposed skin as well as in actinic keratoses and Bowens disease, which are considered to be precancerous lesions of cutaneous squamous cell carcinoma, suggesting p53 as an important target for UV-induced mutations in the skin.4-6 Furthermore, up to 90% of xeroderma pigmentosum patients characterized by an highly increased risk to skin cancer due to impaired DNA-repair mechanisms show p53 mutations in their malignancies, and most of these mutations are typically UV-induced ones. 7With regard to cutaneous melanomas, some early studies have determined the p53 protein expression by immunohistochemistry based on the findings that wild-type p5...