Sensitive detection of squamous cells in bone marrow and blood of head and neck cancer patients by E48 reverse transcriptase-polymerase chain reaction.

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.