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In recent years, the measurement of soluble CD44 levels in the circulation of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the detection of human cancer. The high CD44v6 expression in head and neck squamous cell carcinoma (HNSCC) would enable the use of soluble CD44v6 proteins present in the circulation of HNSCC patients as a marker of disease. In the present study, we determined CD44v6 plasma levels using a domain-specific ELISA in healthy volunteers, non-cancer patients, and HNSCC patients before and after surgical removal of the tumor. A difference between the CD44v6 plasma levels of HNSCC patients and controls could not be observed. Moreover, surgical removal of the tumor did not result in a reduction of the CD44v6 plasma level in the HNSCC patients. In addition, the spectrum of soluble v6-containing CD44 proteins present in the plasma of HNSCC patients and controls was determined by immunoprecipitation experiments, but again, tumor-related isoforms could not be distinguished in patient samples. Additional experiments to unravel the biological source of these circulating proteins indicated surprisingly that the v6-containing proteins present in the circulation of healthy individuals are only released in part, if at all, by activated lymphocytes or other nucleated blood cells. Most circulating CD44v6 proteins seem to be derived from the normal epithelial cell compartments, including breast cells, colon cells, and squamous cells. Taken together, these data do not support the use of soluble CD44v6 as a tumor marker in HNSCC or any other tumor type that has developed from tissues producing soluble isoforms.

In previous studies, we described the selective reactivity of monoclonal antibody E48 with normal squamous and transitional epithelia and their malignant counterparts and the capacity of monoclonal antibody E48 for selective tumor targeting in head and neck cancer patients. Cloning of the E48 encoding cDNA and elucidation of the gene structure enabled the selection of an intron-spanning primer set for the detection of circulating tumor cells in blood and bone marrow of head and neck cancer patients. Extensive optimizations led to a reproducible reverse transcriptase-PCR assay with an internal standard for RNA quality control and an external standard for sensitivity control. In reconstruction experiments, we were able to reach a reproducible sensitivity of one single tumor cell per 7 ml of blood (2 x 10(7) nucleated cells). When applying this method to patient material, we were able to detect positive signal in 35% of the bone marrow samples (0 of 2 stage II, 0 of 4 stage III, 4 of 11 stage IV, and 4 of 6 recurrences) and 10% of the blood samples (2 of 21) of patients with squamous cell carcinoma of the head and neck. The specificity of the method was demonstrated on 29 blood and bone marrow samples of noncancer controls, which were all negative. Our study shows the feasibility of E48 reverse transcriptase-PCR for the detection of squamous cells in nonsquamous tissues.

Monoclonal antibody (mAb) U36 was developed for the treatment of minimal residual disease of head and neck squamous cell carcinoma (HNSCC). The mAb-U36-defined antigen was characterized by cDNA cloning, and was shown to be identical to the keratinocyte-specific CD44 splice variant epican. The epitope recognized by mAb U36 was shown to be located in the v6 domain. Two amino acids within the epitope appeared to differ from the sequences that have been described in literature. The sequence of the epitope appeared to contain glutamic acid at position 367 and lysine at position 374, while valine and arginine respectively have been described before. Interestingly, another anti-CD44v6 antibody with possible clinical application, VFF18, recognizes an epitope in the same area. With respect to the applicability of these antibodies for tumor targeting, this variation might have an influence on antibody-antigen interaction and mAb accumulation in the tumor. Furthermore, this observation raised the question whether the different epitopes are related to the malignant behavior of tumor cells. In this paper we determine the relative affinity of mAb U36 for the variant epitope sequences by tumor cell binding assays using synthetic peptides for competition. The presence of glutamic acid instead of valine at position 367 caused strong competition. Further evaluation showed that the published valine variant does not exist in vivo, and is the result of a sequencing artefact. The effect of substitution of lysine for arginine at position 374 had no effect on the binding of mAb U36 to the cells. This amino acid variation was shown to be due to allelic polymorphism. There was no trend towards allelic imbalance in tumor cells as compared to normal cells.

The improvement of detection and eradication of minimal residual disease, to reduce local and distant relapse after primary therapy, is one of the major challenges in the management of squamous cell carcinoma of the head and neck (HNSCC). This paper describes perspectives arising from the use of monoclonal antibodies (MAbs) E48 and U36 directed against HNSCC-associated antigens, and the molecular characterization of these antigens. Novel strategies for the detection of minimal residual disease are outlined and comprise the use of immunocytochemistry in combination with automated image analysis, and the use of an E48-specific reverse transcriptase-polymerase chain reaction (RT-PCR) method. These methods have potential for the detection of single HNSCC cells in lymph nodes, bone marrow and peripheral blood, and may contribute to the better staging of head and neck cancer in the near future. Besides this, preclinical and clinical data on HNSCC targeting with radiolabelled MAbs E48 and U36 are summarized, illustrating the perspectives of systemic adjuvant radioimmunotherapy with these MAbs.