Her2/neu analysis in formalin-fixed, paraffin-embedded breast carcinomas: comparison of immunohistochemistry and multiparameter DNA flow cytometry.
OBJECTIVE
The determination of Her2/neu over-expression has become necessary for the selection of breast cancer patients for Herceptin therapy. Immunohistochemistry in combination with FISH is currently the most used method for this detection. The objective of our study was to compare the use of multiparameter DNA flow cytometry (MP-FCM) for this assessment with immunohistochemistry (IHC). For this purpose the number of positive cells as well as the intensity of the immunofluorescence signal as determined by MP-FCM were compared with IHC scores.


METHODS
Single cell suspensions from 110 breast cancers were analysed on a DAKO Galaxy flow cytometer after simultaneous (immuno)labeling for cytokeratin, Her2/neu and DNA (propidium iodide). Next to ploidy and proliferative activity (S-phase fraction), the fraction of Her2/neu-positive epithelial cells was determined. In 25 cases the intensity of the immunofluorescence signal was also determined by MP-FCM in the total cytokeratin+ fraction as well as the cytokeratin+/Her2/neu+fraction. Immunohistochemistry for Her2/neu was done on paraffin sections of all cases using the same polyclonal Her2/neu antibody in a SABC-peroxidase procedure after heat-induced antigen retrieval.


RESULTS
Her2/neu overexpression as determined by IHC showed a score of 0 in 30%, 1 in 25%, 2 in 26% and 3 in 19% of the cases. There was a good correlation when comparing the fraction of Her2/neu-positive epithelial cells as determined by MP-FCM and semiquantitative IHC scores (r = 0.89, p < 0.0001). No correlation was found between the IHC-Scores and the intensity of the Her2/neu- staining in the total epithelial compartment as measured by MP-FCM. However, when restricting the intensity-determination to the Her2/neu-positive epithelial cells, a significant correlation was found between MP-FCM and IHC scores (r = 0.72, p < 0.0001).


CONCLUSIONS
The results of the current study show that MP-FCM can be a good alternative for the objective quantification of Her2/neu.
Leers
Mathie P G
MP
Atrium Medical Center Heerlen, Department of Pathology, P.O. Box 4446, 6401 CX Heerlen, The Netherlands. m.leers@cgozl.nl
Hoop
Jacobus G M
JG
Nap
Marius
M
D002288:Adenocarcinoma, Mucinous / Q000473:pathology; D000368:Aged; D014408:Biomarkers, Tumor / Q000032:analysis; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology* / Q000601:surgery; D018270:Carcinoma, Ductal, Breast / Q000473:pathology; D018275:Carcinoma, Lobular / Q000473:pathology; D002455:Cell Division; D004273:DNA, Neoplasm / Q000032:analysis*; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007633:Keratins / Q000032:analysis; D008875:Middle Aged; D011003:Ploidies; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D007633:Keratins; D018719:Erb-b2 Receptor Tyrosine Kinases