No abstract

The interpretation of the results obtained from immunomonitoring of clinical trials is a diYcult task due to the variety of methods and protocols available to detect vaccine-speciWc T-cell responses. This heterogeneity as well as the lack of standards has led to signiWcant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-speciWc T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pretested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-speciWc T-cells in each sample using tetramer staining and one functional assay. The results of the Wrst testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-speciWc CD8 + T-cells by tetramer staining. Analysis by ELISPOT was inXuenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the Wrst phase. 123The recommendations improved the number of antigenspeciWc T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identiWcation of distinct variables that inXuence the sensitivity of diVerent T-cell assays and to formally show that a deWned correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could deWne rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

The aims of tumor follow-up in head and neck cancer patients are (1) evaluation of therapeutic efficacy, (2) management of impairments, (3) detection of new tumor manifestations, and (4) psychosocial care. In general standardized 5-year-protocols are used for all such patients. However, it is questionable whether a rigid follow-up schedule is optimal for a very heterogeneous tumor population. Therefore 603 patients with sqamous cell carcinoma of the oral cavity, pharynx or larynx, or with cervical metastasis from an unknown primary site (CUP syndrome), who had been diagnosed and treated curatively by an operation with or without radiotherapy (n = 523) or just by radio(chemo)therapy (n = 80) between 1985 and 1994, and who had been followed-up regularly according to a standardized plan, were worked-up retrospectively. Data were evaluated for the manifestation and prognosis of curable new tumor manifestations as well as for tumor-specific factors likely to select groups which should be followed more or less intensively. Within a 5-year follow-up period new tumor growth was detected in 152/603 (25%) patients: 79 local and 31 regional recurrences, 18 systemic metastases and 24 second primary cancers. Where follow-up was extended beyond the 5th year, 168/603 (28%) patients presented a new tumor manifestation. One hundred and sixteen of the 152 (28%) patients had another operation with or without radiotherapy or had radio(chemo)therapy alone. So far 18/116 (14%) patients have survived their new tumor manifestation for more than 5 years and 30/116 for more than 2 years. Tumor-specific data on the initial tumors (T stage, N stage, site) did not indicate the risk of a new tumor manifestation, but 87% of patients who survived their new tumor manifestation for more than 2 years initially had T1 or T2 tumors and only 30% initially had N+ necks. Occurrence of distant metastasis or a second primary outside the head and neck region limited survival to < or = 2 years after detection. In terms of survival, follow-up efforts should therefore concentrate on detection of locoregional recurrence, particularly if an option for further curative local therapy exists. The limited success of detection of new tumor manifestations in terms of survival does not justify a reduction in tumor-follow-up examinations, since the benefit of the other efforts cannot be determined from survival figures.

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.