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

In breast cancer and other malignancies, involvement of regional lymph nodes is a strong prognostic indicator and greatly influences staging and clinical management.1–4 One benefit from the implementation of sentinel lymph node biopsy (SLNB) techniques is the identification of metastatic foci of cancer in 10% to 15% of patients that would have been previously staged as node-negative (pN0) by conventional methods.5,6 This improved sensitivity is attributed to both the addition of immunohistochemical staining (IHC) and to an increase in sampling volume.5,6 Many of these additional positive nodes contain only micrometastatic foci of tumor. However, the clinical significance of micrometastatic disease identified by SLNB techniques is highly controversial.7–11 Nonetheless, SLNB techniques are now widely used in breast cancer and melanoma and are being applied with increasing frequency to other tumors, including colorectal, oropharyngeal, prostate, lung, and other solid organ cancers.12–15 Another clear advantage to the SLNB technique is that in the majority of breast cancer patients, SLNB safely avoids axillary lymph node dissection (ALND) and the associated morbidity when the SLN is negative for metastatic disease.16 However, rapid, frozen-section analysis of SLN for metastasis is only 50% to 70% sensitive for the detection of metastasis compared with the permanent histologic sections and IHC of the same lymph node,17,18 and complete analysis of the SLNB specimen currently requires extensive preparation and time-consuming review.8 Even in experienced hands, 10% of SLNB specimens are later found to contain metastases.19 As a result, these patients have required a second surgical procedure to complete the ALND. This is clearly an undesirable algorithm for the patient, the healthcare provider, and the healthcare payer, and this dilemma has contributed to a new controversy; if the SLNB is positive, does a completion ALND confer a therapeutic or staging benefit to the patient? Until ongoing multicenter trials determine whether completing the ALND when the SLNB is positive for metastasis benefits the patient, it is clear that the time required to accurately evaluate SLNB specimens has significant implications.20 It must also be recognized that, even with adequate time, the accurate histologic analysis of lymph nodes for metastatic disease is challenging, and discordance in the interpretation of these materials is a well-known clinical problem. Indeed, a recent study found alarming disparity among pathologists in the analysis of SLNB specimens.21 Furthermore, protocols for SLNB specimen analysis vary widely between healthcare centers.22 Thus, current methods of lymph node analysis clearly lack standardization, are dependent in part on subjective criteria, and are subject to human error. We and others have shown that a real-time, quantitative RT-PCR (QRT-PCR) analysis of lymph nodes can be more accurate than conventional histologic analysis in predicting prognosis for solid organ malignancies,23,24 and we have previously reported our development of a rapid QRT-PCR procedure that can be completed (including RNA isolation) in less than 25 minutes.25 Importantly, a revolutionary tool for molecular-based assays called the GeneXpert (Cepheid, Sunnyvale, CA) is in the final stages of development. This instrument fully automates sample preparation, RNA isolation and purification, and QRT-PCR in a single-use, cartridge-based format that removes the major obstacles limiting routine use of molecular-based assays. Thus, we aimed to determine if a rapid, fully automated, internally controlled QRT-PCR assay performed by a prototype GeneXpert instrument could produce equivalent results to histologic techniques of SLNB analysis, including IHC. This assay could produce significant benefits to patients and healthcare providers by definitively analyzing lymph nodes using objective criteria in a time frame that allows intraoperative use, reduces patient psychologic distress, and improves standardization between healthcare centers.