Designed ankyrin repeat proteins: a novel tool for testing epidermal growth factor receptor 2 expression in breast cancer.

Designed ankyrin repeat proteins are a novel class of specific binding molecules, which display increased thermodynamic stability, smaller size and at least equal target affinity compared to immunoglobulins, making them potentially powerful tools in diagnostic pathology and therapeutic oncology. Here, we investigated whether designed ankyrin repeat proteins can reliably identify the amplification status of the epidermal growth factor receptor 2 in breast cancer. Designed ankyrin repeat proteins specific for epidermal growth factor receptor 2 were tested in paraffin-embedded tissue sections. Detection using enzymatic biotinylation proved to be most specific and sensitive. The affinity of the designed ankyrin repeat proteins was found crucial, but for a picomolar binder no further gain was found by making it multivalent. The best designed ankyrin repeat protein, G3 (K(D) 90 pM) was compared on breast cancer tissue microarrays (n=792) to an FDA-approved rabbit monoclonal antibody against epidermal growth factor receptor 2 (clone 4B5; Ventana Medical Systems) and correlated with corresponding epidermal growth factor receptor 2 amplification status measured by fluorescent in situ hybridization. Amplification status and epidermal growth factor receptor 2 expression measured by designed ankyrin repeat protein and antibody correlated strongly with each other (P<0.0001 each), the correlation between designed ankyrin repeat protein and amplification status being the strongest (0.87 compared to 0.77 for the antibody, Kendall's tau-beta). Using a modified scoring system for the designed ankyrin repeat protein, we show that the designed ankyrin repeat protein detects a positive epidermal growth factor receptor 2 amplification status with similar sensitivity and significantly higher specificity than the antibody (P=0.0005). This study suggests that designed ankyrin repeat proteins provide a valuable alternative to antibodies for the detection of epidermal growth factor receptor 2 expression in breast cancer and adds further compelling evidence for the use of designed ankyrin repeat proteins in diagnostic pathology and therapeutic oncology.

NY-ESO-1 protein expression in primary breast carcinoma and metastases: correlation with CD8+ T-cell and CD79a+ plasmacytic/B-cell infiltration.

NY-ESO-1 is a cancer testis antigen expressed in various malignancies and testicular germ cells. Because of its capacity to induce specific humoral and cellular immunity in patients with NY-ESO-1-positive carcinomas, it represents a promising target for cancer immunotherapy. In breast cancer, NY-ESO-1-mRNA was reported in up to 42%, but protein expression has not been determined to larger extent. In the present tissue microarray-based study, primary breast cancers (n = 1,444), in situ lesion (n = 148), recurrences (n = 88), lymph node (n = 525) and distant metastases (n = 91) were studied for NY-ESO-1 expression by immunohistochemistry. NY-ESO-1-protein expression was compared with mRNA expression by real-time PCR. NY-ESO-1-protein was detected in 3.1% (4/128) in situ lesions and in 2.1% (28/1355) invasive breast cancer. There were 1.8% (9/493) NY-ESO-1-positive lymph node and 5.1% (4/78) positive distant metastases. NY-ESO-1 was more frequently expressed in grade 3 (4.9%) than in grade 2 (0.8%) and grade 1 (0.5%) carcinomas (p < 0.0001). Presence of tumor-infiltrating CD8+ T-cells correlated with NY-ESO-1 (p < 0.0001) on the tissue microarray. On randomly selected large sections, 4 out of 9 NY-ESO-1-positive tumors displayed a brisk infiltrate of CD79a+ plasmocytes/B-cells, but none of 10 NY-ESO-1-negative tumors (p < 0.05). NY-ESO-1-mRNA expression was detected in frozen samples of NY-ESO-1-protein positive (n = 6) and negative breast cancers (n = 8) and in normal testis. Comparison between mRNA and protein expression revealed that only breast cancers with NY-ESO-1-mRNA levels comparable or higher than testis expressed NY-ESO-1-protein. These findings suggest that NY-ESO-1-positive breast cancers represent a small subset of poorly differentiated tumors with evidence of cellular and humoral immune response.