William A. McMillan

A Rapid, Fully Automated, Molecular-Based Assay Accurately Analyzes Sentinel Lymph Nodes for the Presence of Metastatic Breast Cancer

Objective:To develop a fully automated, rapid, molecular-based assay that accurately and objectively evaluates sentinel lymph nodes (SLN) from breast cancer patients. Summary Background Data:Intraoperative analysis for the presence of metastatic cancer in SLNs from breast cancer patients lacks sensitivity. Even with immunohistochemical staining (IHC) and time-consuming review, alarming discordance in the interpretation of SLN has been observed. Methods:A total of 43 potential markers were evaluated for the ability to accurately characterize lymph node specimens from breast cancer patients as compared with complete histologic analysis including IHC. Selected markers then underwent external validation on 90 independent SLN specimens using rapid, multiplex quantitative reverse transcription-polymerase chain reaction (QRT-PCR) assays. Finally, 18 SLNs were analyzed using a completely automated RNA isolation, reverse transcription, and quantitative PCR instrument (GeneXpert). Results:Following analysis of potential markers, promising markers were evaluated to establish relative level of expression cutoff values that maximized classification accuracy. A validation set of 90 SLNs from breast cancer patients was prospectively characterized using 4 markers individually or in combinations, and the results compared with histologic analysis. A 2-marker assay was found to be 97.8% accurate (94% sensitive, 100% specific) compared with histologic analysis. The fully automated GeneXpert instrument produced comparable and reproducible results in less than 35 minutes. Conclusions:A rapid, fully automated QRT-PCR assay definitively characterizes breast cancer SLN with accuracy equal to conventional pathology. This approach is superior to intraoperative SLN analysis and can provide standardized, objective results to assist in pathologic diagnosis.

Toward Next Generation Clinical Diagnostic Instruments: Scaling and New Processing Paradigms

Looking toward future clinical diagnostic instruments, there is little debate as to the features that need improvement over the current state-of-the-art. Increasing the speed and sensitivity of the assays, while reducing costs are clear goals. Recently, it has become possible to microminiaturize fluidic and sensing components using micromachining and precision injection molding. There has been a large amount of interest and effort in the area of miniaturization of such systems, yet not all of the properties of fluidics and sensing methods improve as they are drastically reduced in size. It is clear that implementing miniaturized diagnostic instruments is not a matter of simply “shrinking” their conventional counterparts, nor of automating existing manual procedures. What is required to harness the full potential of scaling technologies is the use of design methods that take into account scaling effects and the development of completely new processing approaches. Beginning with a general overview of the relevant scaling principles, sample preparation and detection approaches are addressed in this context.

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

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 assess a completely automated, cartridge-based version of the assay for accuracy, predictive value, and reproducibility. The triplex (two markers + control) QRT-PCR assay was incorporated into a single-use cartridge for point-of-care use on the GeneXpert system. Three academic centers participated equally. Twenty-nine positive lymph nodes and 30 negative lymph nodes were analyzed to establish classification rules. SLNs from 120 patients were subsequently analyzed by QRT-PCR and histology (including immunohistochemistry), and the predetermined decision rules were used to classify the SLNs; 112 SLN specimens produced an informative result by both QRT-PCR and histology. By histological analysis, 21 SLNs were positive and 91 SLNs were negative for metastasis. QRT-PCR characterization produced a classification with 100% sensitivity, 97.8% specificity, and 98.2% accuracy compared with histology (91.3% positive predictive value and 100% negative predictive value). Interlaboratory reproducibility analyses demonstrated that a 95% prediction interval for a new measurement (DeltaCt) ranged between 0.403 and 0.956. This fully automated QRT-PCR assay accurately characterizes breast cancer SLNs for the presence of metastasis. Furthermore, the assay is not dependent on subjective interpretation, is reproducible across three clinical environments, and is rapid enough to allow intraoperative decision making.

A new generation of PCR instruments and nucleic acid concentration systems

Publisher Summary The polymerase chain reaction (PCR) technique has clearly evolved into an important tool for researchers and clinicians. This has been afforded by the commercialization of robust and dependable instruments for thermal cycling and, recently, with homogenous fluorescence detection.. The state-of-the-art instruments that include real-time, homogeneous, fast thermal cycling, and quantitative detection capabilities still leave significant opportunities for improvements. Efforts to develop PCR on a chip or micromachined/miniaturized systems have shown some interesting capabilities, but still fall short of providing the types of results that surpass or even equal those of commercial systems. However, in the future the development of new nucleic acid systems based on some of the principles from such research devices will probably occur. This chapter describes the extension of previous work based on silicon micromachining that has shown equivalent and improved performance over commercial systems. Other improvements over commercial systems have been discovered. These include new graphical user interface, independent control of each reaction site, modularity, and rapid thermal cycling of large volumes. Ultimately, the chapter concludes with anticipation that one day all the processing and homogenous quantitative detection will occur in one low-cost disposable, integrated system, which will take PCR to the new level of utility.

Nucleic Acid Concentration and PCR for Diagnostic Applications

Sample preparation involving the extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface area to volume ratios. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50%, and concentration factors of about 10X using bacteriophage lambda DNA as the target. These results, when combined with rapid amplification and detection, confirm the viability of utilizing these components as elements of a compact system for the purification and detection of nucleic acids in applications such as clinical diagnostics, food quality control, and environmental monitoring.