Background: Anti-HER2 therapy contributes to improvement of the clinical outcome in HER2-positive patients. However, current studies have reported the resistance to trastuzumab. To diagnose HER2 expression level, immunohistochemical (IHC) method with 3,3’-diaminobenzidine (IHC-DAB) staining is widely used, but is not quantitative and classifies tumors into only 4 categories. Additionally, the epitopes of trastuzumab and most antibodies used for IHC-DAB are different. These problems might induce a gap between diagnostics and therapeutic efficacy. To improve the method for cancer patients’ HER2 status, we developed a novel IHC method with trastuzumab conjugated with fluorescent nano-particles quantum dots (QDs) by single-particle imaging using tissue samples from patients with clinical information.

Methods: We prepared two kinds of QD-conjugated antibody complexes for IHC with QDs (IHC-QD). One was a QD-conjugated trastuzumab, the other was a QD-conjugated human IgG for control. We performed the several steps of antigen-antibody reaction by two kinds of QD-conjugated antibody probes for 37 human breast cancer specimens. The QD signal reflected HER2 expression level was observed by the optical system that consisted of an epi-fluorescent microscope with EM-CCD camera.

Results: We successfully obtained the images of only QDs signal by the image analyses, and calculated the number of QD particles in a cell and defined it as IHC-QD score that reflects the level of HER2 protein expression in cancer cells. In comparing IHC-QD scores with IHC-DAB scores, cases with a score of 0 and 1 by IHC-DAB had low scores of IHC-QD (0.01-0.67), being in narrow range. On the other hand, those with a score of 2 varied widely, with IHC-QD scores of 0.06 to 16.98, and those with a score of 3 also varied, from 0.18 to 18.81. In some of the sample with IHC-DAB score of 2 and 3, the binding of trastuzumab to these cancer cells is very weak although they are candidate for trastuzumab therapy in present HER2 diagnostic criteria. In comparison between FISH and IHC-QD scores, the straight-line approximation curve shows that both quantitative scores, FISH (0.96-8.76) and IHC-QD (0.01-18.81) are well correlated (R = 0.83, p<0.001). In analyzing the therapeutic efficacy in 14 metastatic breast cancers treated with trastuzumab according to IHC-QD or FISH or IHC-DAB scores, IHC-QD score was correlated with time to progression (TTP) more than FISH and IHC-DAB scores.

Table 1

Diagnostic methods Patient number Time to progression, median(range) P value 
IHC-DAB     0.207 
Score 2 6 (3-9)   
Score 3 11 12 (3-60)   
FISH     0.577 
Low score 12 (5-60)   
High score 9 (3-52)   
IHC-QD     0.094 
Low score 6 (3-60)   
High score 24 (3-52)   
Diagnostic methods Patient number Time to progression, median(range) P value 
IHC-DAB     0.207 
Score 2 6 (3-9)   
Score 3 11 12 (3-60)   
FISH     0.577 
Low score 12 (5-60)   
High score 9 (3-52)   
IHC-QD     0.094 
Low score 6 (3-60)   
High score 24 (3-52)   

Comparison between IHC-QD or FISH or IHC-DAB and the therapeutic efficacy with trastuzumab

Althogh the sample size was small, this result suggests that the IHC-QD score has a potential role as predictive factor for trastuzumab therapy.

Conclusion: We performed the precisely quantitative IHC using trastuzumab-conjugated QDs and single-particle imaging analysis, and propose using IHC-QD as a predictive factor for trastuzumab therapy. This new diagnostic method would be expected to contribute to the development of a therapeutic strategy.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-05-16.