A Quantitative Reverse Transcription-PCR Assay for Rapid, Automated Analysis of Breast Cancer Sentinel Lymph Nodes Steven J. Hughes, Liqiang Xi, William.

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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, Tony E. Godfrey The Journal of Molecular Diagnostics Volume 11, Issue 6, Pages 576-582 (November 2009) DOI: 10.2353/jmoldx.2009.090037 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Observed and predicted PIP and TACSTD1 relative expression from intralaboratory reproducibility experiments. Synthetic LN samples representing seven serial dilutions (four samples for each dilution) were sent to three participating laboratories. The objective was to assess assay reproducibility between and within laboratories. A and B: Observed target mRNA concentrations in ΔCt as determined by UPMC, MSSM, and MUSC laboratories. C: The estimated concentration-dilution curve based on a linear regression model. This model supports a separate function for each laboratory that differs by vertical distance and not by slope. D: The best fitting regression model for TACSTD1 permits a single concentration-dilution function for all laboratories. Dotted lines in C and D are 95% confidence bands for the expected ΔCt. The Journal of Molecular Diagnostics 2009 11, 576-582DOI: (10.2353/jmoldx.2009.090037) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: A total of 59 training nodes (29 positive and 30 negative) were used to train a risk probability classifier using expression of PIP and TACSTD1. Three equal probability contours were drawn to illustrate low (probability = 0.01), moderate (probability = 0.50), and high (probability = 0.99) probability that a lymph node is positive for micrometastasis. B: The same classifier was then plotted against the observed PIP and TACSTD1 in 115 sentinel lymph nodes. The plotting symbols show the final pathological results as negative (N = 91), positive (N = 21), or equivocal (N = 3). The Journal of Molecular Diagnostics 2009 11, 576-582DOI: (10.2353/jmoldx.2009.090037) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions