ORIGINAL ARTICLE

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

M P G Leers¹, R H M G Schoffelen¹, J G M Hoop¹, P H M H Theunissen¹, J W A Oosterhuis², H vd Bijl², A Rahmy³, W Tan³, M Nap¹

¹ Department of Pathology, Atrium Medical Centre Heerlen, PO Box 4446, 6401 CX Heerlen, The Netherlands
² Department of Surgery, Atrium Medical Centre Heerlen
³ Department of Nuclear Medicine, Atrium Medical Centre Heerlen

Correspondence to:
Correspondence to:
Dr M P G Leers, Department of Pathology, Atrium Medical Centre Heerlen, PO Box 4446, 6401 CX Heerlen, The Netherlands;
m.leers{at}gozl.nl

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 non-SLNs 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.

Keywords: breast cancer; sentinel node; flow cytometry; double staining

Abbreviations: BSA, bovine serum albumin; CIRC, cytokeratin positive interstitial reticulum cells; FITC, fluorescein isothiocyanate; H&E, haematoxylin and eosin; IHC, immunohistochemistry; MP-FCM, multiparameter flow cytometry; PI, propidium iodide; RT-PCR, reverse transcriptase polymerase chain reaction; SLN, sentinel lymph node

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