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Real-Time Gap Ligase Chain Reaction

A Rapid Semiquantitative Assay for Detecting p53 Mutation at Low Levels in Surgical Margins and Lymph Nodes from Resected Lung and Head and Neck Tumors

¹ Department of Otolaryngology-Head and Neck Surgery, and ² Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, and ³ National Cancer Institute-HIV Drug Resistance Program, Frederick, Maryland

Purpose: We have developed a real-time semiquantitative gap ligase chain reaction for detecting p53 point mutations at low level in a background of excess of wild-type DNA.

Experimental Design: This method was validated by direct comparison to a previously validated but cumbersome phage plaque hybridization assay. Forty-one surgical margins and lymph nodes from 10 cases of head and neck squamous cell carcinoma and lung carcinoma were tested for p53 mutant clones.

Results: Both methods detected p53 mutants in margins from 8 of the 10 cases, whereas standard pathology detected cancer cells in only 3 cases. Positive margins included tissue samples with a tumor/normal DNA ratio of up to 1:1000.

Conclusions: This novel molecular approach can be performed in <5 h facilitating intraoperative use for real-time surgical resection.

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