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Quantitative Molecular Detection of Minimal Residual Head and Neck Cancer in Lymph Node Aspirates¹

Departments of Otolaryngology/Head-Neck Surgery [E. J. C. N., R. G. A. W., F. D., C. R. L., G. B. S., R. H. B.], Pathology [L. H. J., B. B.], and Radiology [J. A. C.], Vrije Universiteit Medical Center, Amsterdam, 1007 MB Amsterdam, the Netherlands

Purpose: Staging of the clinically N₀ neck in patients with head and neck squamous cell carcinoma (HNSCC) using ultrasound-guided, fine needle aspiration cytology (USgFNAC) has a false-negative rate of 20% that might be caused by inaccurate cytology. Molecular analysis of aspirate residues might reduce the false-negative rate, and we therefore set up a quantitative reverse transcription-PCR (Q-RT-PCR) assay based on TaqMan technology using the squamous cell-specific antigen E48 (Ly-6D) as molecular marker.

Experimental design: The detection limit of the assay was determined in reconstruction experiments. The sensitivity of the assay was tested on cytological tumor-positive aspirate residues and the specificity on lymph node aspirate residues of noncancer controls. Subsequently, 235 lymph node aspirate residues of 64 HNSCC patients staged with USgFNAC were examined for the presence of E48 mRNA. E48 Q-RT-PCR results of the aspirated lymph nodes were compared with cytology and clinical outcome.

Results: The detection limit of E48 Q-RT-PCR was a single tumor cell in a background of 10⁶ peripheral blood mononuclear cells. From the 41 aspirates that were not evaluable at cytology, 24 (59%) could be diagnosed with E48 Q-RT-PCR. In the 191 aspirates that were tumor negative or not evaluable at cytology, 8 samples from 6 patients were E48 positive. These results were confirmed by histology or clinical outcome in 3 of 6 patients. E48 Q-RT-PCR showed an increase in sensitivity from 56 to 67% and an increase in frequency of reached diagnosis from 97 to 100% compared with cytology. The specificity decreased from 100 to 92%.

Conclusions: Real-time E48 Q-RT-PCR is an accurate technique for squamous cell detection in lymph node aspirates of HNSCC patients. The assay shows an increase in sensitivity and frequency of reached diagnosis compared with cytology. The test could be implemented routinely in USgFNAC to diagnose cases for which cytological examination is not conclusive.

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