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

ABSTRACT: 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.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 ?-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.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.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.

SUBMITTER: Ferris RL

PROVIDER: S-EPMC5270507 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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

Ferris Robert L RL Xi Liqiang L Seethala Raja R RR Chan Jon J Desai Shaun S Hoch Benjamin B Gooding William W Godfrey Tony E TE

Clinical cancer research : an official journal of the American Association for Cancer Research 20110225 7

<h4>Purpose</h4>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.<h4>Experimental design</h4>Four hundred forty-eight grossly tumor-negative lymph nodes were ...[more]

PMID: 21355082

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Project description:OBJECTIVES/HYPOTHESIS:Clinical staging of early head and neck squamous cell carcinoma (SCCHN) is often inaccurate, leading to elective neck dissection to detect the 30% of patients with micrometastatic disease. Sentinel node biopsy accurately stages the regional lymphatics, but intraoperative pathology is only moderately sensitive, and final pathology takes several days to complete. To facilitate immediate neck dissection where necessary, we have identified several promising marker genes of SCCHN metastasis and developed a rapid, accurate, and automated quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) assay for intraoperative use. STUDY DESIGN:Prospective tissue collection, retrospective pathologic correlation with qRT-PCR. METHODS:From a 40-gene marker screen, we quantified expression of 11 potential tumor genes using a test set of primary tumors (n = 32) and metastatic (n = 19) and benign (n = 10) lymph nodes. Eight patients' paired primary tumor and metastatic nodes were included. A validation set of 442 grossly tumor-negative nodes was evaluated for expression of the most promising markers, comparing metastasis detection by qRT-PCR with pathologic analysis (hematoxylin and eosin and immunohistochemistry). A novel multiplexed, automated, single-tube qRT-PCR assay was used to analyze more than 100 lymph nodes using a two-marker, 35-minute assay to determine its negative predictive value. RESULTS:Based on expression of 11 tumor-associated genes from the marker screen, the two most promising markers of SCCHN metastasis in the test set, pemphigus vulgaris antigen (PVA) and tumor-associated calcium signal transducer 1 (TACSTD1), also known as epithelial cell adhesion molecule (EpCAM), were selected. Development of a multiplexed qRT-PCR assay for the detection of metastasis compared favorably with pathologic analysis in the additional 442-node set. A rapid, multiplexed assay using PVA and TACSTD1 demonstrated excellent reproducibility, linearity, and accuracy (?96% negative predictive value) for identifying positive (n = 40) and negative (n = 62) nodes in a validation subset. CONCLUSIONS:Detection of metastatic SCCHN using multiplexed qRT-PCR can be rapid, accurate, and automated and may enable sentinel node biopsy to be used for intraoperative decision-making. PCR amplification of tumor marker genes is an effective method of intraoperative molecular staging of SCCHN and could more appropriately guide application of neck dissection in pN+ SCCHN patients, sparing 60% to 70% of pN0 patients from unnecessary neck dissection. This technique may also be used for identifying residual neck disease posttreatment, using outpatient fine-needle aspiration biopsy specimens.

Dataset Information

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

Publications

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

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