Jesus Ching

Molecular Staging of Cervical Lymph Nodes in Squamous Cell Carcinoma of the Head and Neck

Clinical staging of cervical lymph nodes from patients with squamous cell carcinoma of the head and neck (SCCHN) has only 50% accuracy compared with definitive pathologic assessment. Consequently, both clinically positive and clinically negative patients frequently undergo neck dissections that may not be necessary. To address this potential overtreatment, sentinel lymph node (SLN) biopsy is currently being evaluated to provide better staging of the neck. However, to fully realize the potential improvement in patient care afforded by the SLN procedure, a rapid and accurate SLN analysis is necessary. We used quantitative reverse transcription-PCR (QRT-PCR) to screen 40 potential markers for their ability to detect SCCHN metastases to cervical lymph nodes. Seven markers were identified with good characteristics for identifying metastatic disease, and these were validated using a set of 26 primary tumors, 19 histologically positive lymph nodes, and 21 benign nodes from patients without cancer. Four markers discriminated between positive and benign nodes with accuracy >97% but only one marker, pemphigus vulgaris antigen (PVA), discriminated with 100% accuracy in both the observed data and a statistical bootstrap analysis. A rapid QRT-PCR assay for PVA was then developed and incorporated into a prototype instrument capable of performing fully automated RNA isolation and QRT-PCR. The automated analysis with PVA provided perfect discrimination between histologically positive and benign lymph nodes and correctly identified two lymph nodes with micrometastatic tumor deposits. These assays were completed (from tissue to result) in approximately 30 minutes, thus demonstrating the feasibility of intraoperative staging of SCCHN SLNs by QRT-PCR.

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.

Simulation of microfluidic pumping in a genomic DNA blood-processing cassette

Microfluidic cassettes that perform integrated biological sample preparation and DNA analysis require fluidic control and transport mechanisms built into the device. In this study, pneumatically actuated diaphragm pumps and valves were employed to achieve precise fluidic manipulation and enabled the execution of several sample-processing steps within a single cassette. However, the design of the microfluidic cassette to accomplish this multi-step fluidic protocol required a complex three-dimensional fluid path through valves, bends, various sized passageways and a porous filter for cell capture. In order to understand the fluidic behavior in such a device, measurements were taken of the pneumatic pressure delivered to the diaphragm pump as it pushed sample through the complicated fluidic pathway. Simultaneously monitored were the resulting volumetric flow rate, and the corresponding pre- and post-filter fluid pressures. The data enabled the construction of a model that simulated the fluidic action through the device using established fluid mechanics theory that closely matched flow rate and pressure data. The ability to simulate the behavior of diaphragm pumping and resulting fluidic movements in complex microfluidic devices provides a greater comprehension of this phenomenon and a useful tool in the application to future devices for biochemical analysis.

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.

Real-time PCR Analysis on Nucleic Acids Purified from Plasma Using a Silicon Chip

Results are presented demonstrating the application of a micromachined chip for the purification of viral DNA or RNA from human plasma for rapid, real-time, quantitative PCR analysis.