A Prospective Multi-center Large-sample Study Of Intra-operative Genesearch^TM Bln Assay To Detect Breast Cancer Metastases In Sentinel Lymph Nodes

Objective:Sentinel lymph node (SLN) biopsy has become a standard procedure for early breast cancer patients with clinically negative axilla. The accurate and rapid intra-operative assessment of SLN has the advantage of enabling axillary lymph nodes dissetion to be performed immediately, thereby avoiding the morbidity, inconvenience and cost of a second, separate operation. Frozen section (FS) and touch imprint cytology (TIC) are traditional procedures for intra-operative assessments of SLN with low sensitivity to SLN micro-metastases. GeneSearchTM Breast Lymph Node (BLN) Assay is a real-time reverse-transcriptase polymerase chain reaction assay for the detection of nodal metastases larger than 0.2mm.The aim of this study is to evaluate the roles of BLN Assay as an intra-operative diagnostic method of SLN for breast cancer patient.Methods:Five hundred and forty-six consecutive patients from six centers were involved in this study, and 479 cases were valid according to the protocol. SLNs were identified and dissected, and then sectioned along the short axis into multiple blocks. The odd blocks were tested by BLN Assay intra-operatively and the even blocks were assessed by histopathologic examination post-operatively (4⁶μm thick step sectioning permanent HE slides were realized every 150μm and one block obtained 6 slides). In addition, the even blocks of 136 patients were prepared for FS and all blocks of 156 patients were evaluated by TIC intra-operatively. Results:In the patient basis analysis, the assay had the accuracy, sensitivity, specificity, posive predict value (PPV) and negative predict value (NPV) of 91.44%, 87.50%, 92.88%, 81.75%, 95.32%, respectively. While FS had the accuracy, sensitivity, specificity, PPV, NPV of 95.79 %, 84.48 %, 100.00%, 100.00%, 94.55%, and TIC of 92.31%, 75.00%, 99.11%, 97.06%, 90.98%, respectively. The sensitivity of BLN Assay was similar to that of FS (P=0.576) and was significantly higher than that of TIC (P =0.049).However, of 25 BLN Assay false-positive cases, four had non-sentinel axillary lymph node (nSLN) metastases; two had isolated tumor cells (ITC) in SLN; one had a posive TIC result and had ITC in SLN, and one had ITC in SLN and had nSLN metastases. After adjustment, the assay had the accuracy, sensitivity, specificity, PPV and NPV of 91.65%, 83.45%, 95.21%, 88.32%, 92.98%, respectively. While FS had the accuracy, sensitivity, specificity, PPV, NPV of 91.12%, 72.06%, 100.00%, 100.00%, 88.49%, respectively, and TIC of , 89.10%, 66.67%, 100.00%, 100.00%, 86.07%, respectively.The sensitivity of BLN Assay was higher than that of FS (P = 0.054), and was significantly higher than that of TIC (P=0.011).In the node basis analysis, the accuracy, sensitivity, specificity, PPV, and NPV were 93.02%, 85.56%, 94.57%, 76.62% and 96.92%, respectively, for BLN Assay, 97.17%, 84.88%, 100.00%, 100.00% and 96.63%, respectively, for FS, and 94.29%, 70.00%, 99.03%, 93.30% and 94.43%, respectively, for TIC. The sensitivity of BLN Assay was similar to that of FS (P=0.885) and was significantly higher than that of TIC (P =0.007).When assessing nodes with macro-metastases, the sensitivity BLN Assay was similar to that of FS (P=0.558) and was significantly higher than that of TIC (P=0.011). When assessing nodes with micro-metastases, BLN Assay had a higher sensitivity than FS (P=0.356) and TIC (P=0.094).In the patient basis analysis, there were no significant differences in the sensitities and the specificities of every center (P= 0.672; P= 0.628). After a learning curve of about 10 cases, the assay could be performed in a median time of about 35 min. Compared with the histopathologic evaluation, the assay would cost every patient 24.5% of the extra charge. However, it could save the time and decrease the workload of the pathologists and had a good cost/effectiveness ratio. The best Ct value for CK-19 was 31, and the sensitity and the specificity on this value were 85% and 94.5%, respectively. And the best Ct value for MG was 36, and the sensitity and the specificity on this value were 70% and 91.8%, respectively. A negative correlation was found between the Ct values for CK-19 or MG and the size or the type of metastases, and we could estimate the type of the metastases according to the Ct values. The Ct value for CK-19 lowers than 29 meant a SLN macro-metastases and the value of 29³1 meant a micro-metastases. The Ct value for MG lowers than 33 meant a SLN macro-metastases and the value of 33³6 meant a micro-metastases.Conclusion:GeneSearch^TM BLN Assay is an accurate and rapid intra-operative assay for assessing breast SLNs and it is suitable for application in general medical practice.