Intraoperative qRT-PCR for detection of lymph node metastasis in head and neck cancer

Abstract

Purpose: Sentinel node biopsy (SNB) has been shown to accurately stage the regional lymphatics in oral carcinoma. However, intraoperative pathology is only moderately sensitive and final pathology takes several days to complete. The purpose of this study was to develop a rapid, automated, and quantitative real-time PCR (qRT-PCR) assay that can match final pathology in an intraoperative time frame.

Experimental design: Four hundred forty-eight grossly tumor-negative lymph nodes were evaluated for expression of 3 markers [PVA (pemphigus vulgaris antigen), PTHrP (parathyroid hormone-related protein), and TACSTD1 (tumor-associated calcium signal transducer 1)]. Conformity of metastasis detection by qRT-PCR was determined using hematoxylin and eosin and immunohistochemistry staining as the gold standard. PVA and TACSTD1 were then multiplexed with β-glucuronidase to develop a rapid, automated single-tube qRT-PCR assay using the Cepheid GeneXpert system. This assay was used to analyze 103 lymph nodes in an intraoperative time frame.

Results: Four hundred forty-two nodes produced an informative result for both qRT-PCR and pathologic examination. Concordance of qRT-PCR for individual markers with final pathology ranged from 93% to 98%. The best marker combination was TACSTD1 and PVA. A rapid, multiplex assay for TACSTD1 and PVA was developed on the Cepheid GeneXpert and demonstrated an excellent reproducibility and linearity. Analysis of 103 lymph nodes demonstrated 94.2% accuracy of this assay for identifying positive and negative nodes. The average time for each assay to yield results was 35 minutes.

Conclusions: A rapid, automated qRT-PCR assay can detect lymph node metastasis in head and neck cancer with high accuracy compared to pathologic analysis and may be more accurate than intraoperative pathology. Combined, SNB and rapid qRT-PCR could more appropriately guide surgical treatment of patients with head and neck cancer.

Figure 1

Pathologic and molecular tissue handling and sectioning protocol. Nodes were harvested at surgery and subjected to the sectioning process shown, for either histologic (H&E or IHC) or qRT-PCR analysis.

Figure 2

Distribution of relative expression (top) of PTHrP, PVA, and TACSTD1 in histologically positive and negative lymph nodes as determined by review of adjacent tissue sections (research pathology). The Y-axis shows relative expression as ΔC_T and the horizontal line indicates the most accurate cutoff determined from ROC curve analysis (bottom).

Figure 3

Marker combinations and correlation with lymph node classification according to research pathology. Green circles: negative nodes; red plus: positive node; blue circle: node with ITCs. Axes show ΔC_T.

Figure 4

Results of the triplex GeneXpert assay compared to research pathology (top) and overall consensus pathology (bottom). First, expression of TACSTD1 and PVA in positive (+) and negative (○) nodes; second, the risk score (probability of being positive calculated based on the TACSTD1 and PVA expression) for each node; and third, the horizontal line indicates the most accurate cutoff determined by ROC curve analysis.