A Quantitative Reverse Transcription-PCR Assay for Rapid, Automated Analysis of Breast Cancer Sentinel Lymph Nodes

Steven J. Hughes^*, Liqiang Xi, William E. Gooding, David J. Cole, Michael Mitas, John Metcalf^¶, Rohit Bhargava^||, David Dabbs^||, Jesus Ching^**, Lynn Kozma^**, William McMillan^** and Tony E. Godfrey

From the Departments of Surgery * and Pathology, ^|| University of Pittsburgh Medical Cancer, Pittsburgh, Pennsylvania; the Department of Pathology, Mount Sinai School of Medicine, New York, New York; the Biostatistics Facility, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; the Departments of Surgery and Pathology, ^¶ Medical University of South Carolina, Charleston, South Carolina; and Cepheid, ** Sunnyvale, California

We have previously reported that a quantitative reverse transcription(QRT)-PCR assay accurately analyzes sentinel lymph nodes (SLNs)from breast cancer patients. The aim of this study was to assessa completely automated, cartridge-based version of the assayfor accuracy, predictive value, and reproducibility. The triplex(two markers + control) QRT-PCR assay was incorporated intoa single-use cartridge for point-of-care use on the GeneXpertsystem. Three academic centers participated equally. Twenty-ninepositive lymph nodes and 30 negative lymph nodes were analyzedto establish classification rules. SLNs from 120 patients weresubsequently analyzed by QRT-PCR and histology (including immunohistochemistry),and the predetermined decision rules were used to classify theSLNs; 112 SLN specimens produced an informative result by bothQRT-PCR and histology. By histological analysis, 21 SLNs werepositive and 91 SLNs were negative for metastasis. QRT-PCR characterizationproduced a classification with 100% sensitivity, 97.8% specificity,and 98.2% accuracy compared with histology (91.3% positive predictivevalue and 100% negative predictive value). Interlaboratory reproducibilityanalyses demonstrated that a 95% prediction interval for a newmeasurement ( ${Delta}$ Ct) ranged between 0.403 and 0.956. This fullyautomated QRT-PCR assay accurately characterizes breast cancerSLNs for the presence of metastasis. Furthermore, the assayis not dependent on subjective interpretation, is reproducibleacross three clinical environments, and is rapid enough to allowintraoperative decision making.