Metasin: An Intra-Operative Real-Time Quantitative Reverse Transcription Polymerase-Chain Reaction (RTqPCR) Assay to Detect Metastatic Breast Cancer in Sentinel Lymph Nodes
Abstract
The most important prognostic factor in breast cancer is the presence or absence of metastases in axillary lymph nodes. Frozen section and touch imprint cytology are
conventional intra-operative methods used in the detection of metastatic breast
cancer with varying sensitivities and specificities. The limitation of these methods led to the development of alternative molecular diagnostic tests, such as GeneSearch, a
commercial real-time quantitative Polymerase Chain Reaction (RT-qPCR) assay that allows for an intra-operative diagnosis of metastatic breast cancer. When the GeneSearch assay was discontinued, Metasin was developed as an in-house RT-qPCR
replacement assay. Metasin targets the epithelial cell marker cytokeratin 19 (CK19) and the breast marker mammaglobin (MGB) mRNA to confirm the presence or
absence of metastatic disease, whilst the reference gene porphobilinogen deaminase
(PBGD) acts as a positive control for the performance of the assay. The optimised assay can produce a result within 32 minutes allowing it to be used in the intra-operative setting to detect metastatic breast cancer in sentinel lymph nodes.
154 archived lymph node homogenates that were previously analysed by both
GeneSearch and histology in parallel were used to validate Metasin. Out of 154 cases,
148 showed concordance with both GeneSearch and Metasin with 111 cases being
negative and 37 cases being positive. There were six discordant cases, four in which only Metasin detected metastases and two in which only GeneSearch picked up
metastases. Out of the four Metasin-only positive cases, three were found to be
positive on histology after deeper levels were cut in the slices sent for histological assessment. Therefore, one case could not be shown histologically to be positive for
metastases. There were two cases that were missed by Metasin but picked up by
GeneSearch. One case was positive on histology and the second case negative for
histology. The error rate for Metasin was 3.89%. The sensitivity and specificity of the
Metasin assay were found to be 95% and 98% respectively, and the positive and
negative predictive values were 90% and 98% respectively. These results are
comparable to those of GeneSearch. Metasin had an assay time of less than 45
minutes and was operated by biomedical scientists. The results of the validation process were deemed acceptable for the assay to be run live and used in the clinical
setting. Metasin continues to provide breast cancer patients at Princess Alexandra Hospital with all the advantages that a molecular intra-operative diagnostic service provides.
Authors
Al-Ramadhani, SalmaCollections
- Theses [3919]