Quantitative evaluation of metastases in axillary lymph nodes of breast cancer

Abstract

We have established a highly sensitive and quantitative reverse transcriptase -polymerase chain reaction (RT -PCR) method to detect axillary lymph node metastases of breast cancer. Amplifying cytokeratin 19 (CK19) mRNA transcripts using real-time TaqMan PCR made it possible to quantify axillary metastatic burden. Metastases in 358 axillary lymph nodes obtained from 23 breast cancers of 22 patients were investigated by conventional haematoxylin and eosin (H&E) staining, immunohistochemical staining and quantitative RT -PCR assay. The detection rates of axillary lymph node metastasis using H&E staining, immunohistochemistry and RT -PCR assay were 4.5, 5.9 and 13.1%, respectively. RT -PCR assay was the most sensitive of these three methods for detecting lymph node metastases. Cytokeratin 19 mRNA expression values of both histologically and immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (Po0.0001), and those of histologically negative, but immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (Po0.0001). Furthermore, metastatic rates of sentinel nodes were higher than the rates of nonsentinel lymph nodes as measured by all three methods. These results indicate that quantitative RT -PCR assay is a sensitive and reliable method for detecting lymph node metastasis. Furthermore, quantification of metastases in sentinel lymph nodes by quantitative RT -PCR assay may be useful to assess the entire axillary burden of breast cancer patients.

Pathologic examination of the axillary sentinel lymph nodes in patients with early-stage breast carcinoma: current and resolving controversies on the basis of the European Institute of Oncology experience

“…Because a possible cause of false-positive identification of lymph node metastases by RT-PCR assays might be the illegitimate expression of low levels of target mRNAs by non-neoplastic cells, real-time quantitative RT-PCR (qRT-PCR) assays have been developed to correctly identify lymph node metastases according to a threshold level of mRNA expression [15,20,29,36,48]. Very recently, dedicated kits and instruments for "rapid" quantitative RT-PCR assays have also become commercially available for the intraoperative detection of SLN metastases from breast carcinoma.…”

Perioperative diagnosis of the positive axilla in breast cancer: A safe, time efficient algorithm

“…Secondly, intraoperative analysis of the sentinel node, via frozen sectioning, imprint cytology, immunohistochemistry (IHC) or more recently 'Genesearch' BLN (Biopsy Lymph node assay) real time RT-PCR assay, can provide an immediate diagnosis of the axilla and thus reduce the requirement for delayed axillary surgery [6][7][8][9].…”

Detection of occult carcinomatous diffusion in lymph nodes from head and neck squamous cell carcinoma using real-time RT–PCR detection of cytokeratin 19 mRNA

“…Previous studies using such markers in HNSCC have suggested a low specificity (Kawamata et al, 1999;Hamakawa et al, 2000a), particularly because of false-positive results associated with benign epithelial rests in lymph nodes found in 1.6% of cervical nodes, but only in lymph nodes from level I (Gnepp, 2000;Hamakawa et al, 2000b). The assay used in the present study, that is, RT -PCR detection of CK19 mRNA, has been developed in our laboratory for the detection of occult carcinomatous cells in mediastinal lymph nodes in non-small-cell lung carcinoma (Saintigny et al, 2005) and by other teams in various carcinomas (Van Trappen et al, 2001;Inokuchi et al, 2003;Favia et al, 2004). The primers and probe for CK19 mRNA detection were designed to avoid the detection of the two known pseudogenes (Rund et al, 1999;Van Trappen et al, 2001;Saintigny et al, 2005) and we used real-time RT -PCR with the Taqman chemistry.…”