Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

Abstract

Aim: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. Methods: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 µm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100 000 cells were analysed on the flow cytometer.Results: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple nonSLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. Conclusion: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.

Immunolocalization and Cell Expression of Lung Resistance-related Protein (LRP) in Normal and Tumoral Human Respiratory Cells

“…However, the rate of LRP-positive cells evaluated by flow cytometry is higher than that previously reported for LRP expression evaluated by immunohistochemistry (Dingemans et al 1996;Volm et al 1997;Harada et al 2003;Raybarova et al 2004). This difference can probably be explained by a higher sensitivity of flow cytometry analysis compared with immunohistochemistry (Leers et al 2002). Similarly, LRP was detected in bronchial epithelial cells in our study as in other studies (Nooter et al 1995), suggesting a role of LRP in the cell detoxification process for cells localized at the air-tissue interface (Dingemans et al 1996;Izquierdo et al 1996a) where detoxification mechanisms are particularly well developed.…”

Multidrug resistance-associated protein (MRP1) is overexpressed in DNA aneuploid carcinomatous cells in non-small cell lung cancer (NSCLC)

“…6,23 This difference can probably be explained by a higher sensitivity of flow cytometry analysis compared to immunohistochemistry. 24 This biparametric analysis demonstrated that the MRP ratio, i.e., the MRP1 expression at the individual cell level, was significantly higher in DNA aneuploid cells than in DNA diploid cells. These results were confirmed by analysis of a selected group of tumors containing more than 80% of carcinomatous cells.…”

The clinical significance of sentinel lymph node micrometastasis in breast cancer

“…The addition of the aforementioned PCR based methods can significantly increase the apparent 'false-negative' rate of conventional analysis [22]. Multi-parameter flow cytometry, applied to permanently fixed sections, has also been shown to detect MM within SLNs [23]. Adjuncts to the conventional human assessment of specimens have been shown to be effective.…”