Molecular Assessment of HER2 to Identify Signatures Associated with Therapy Response in HER2-Positive Breast Cancer

Abstract

Trastuzumab, the prototype HER2-directed therapy, has markedly improved survival for women with HER2-positive breast cancers. However, only 40-60% of women with HER2-positive breast cancers achieve a complete pathological response to chemotherapy combined with HER2-directed therapy. The current diagnostic assays have poor positive-predictive accuracy in identifying therapy-responsive breast cancers. Here, we deployed quantitative single molecule localization microscopy to assess the molecular features of HER2 in a therapy-responsive setting. Using fluorescently labeled trastuzumab as a probe, we first compared the molecular features of HER2 in trastuzumab-sensitive (BT-474 and SK-BR-3) and trastuzumab-resistant (BT-474^R and JIMT-1) cultured cell lines. Trastuzumab-sensitive cells had significantly higher detected HER2 densities and clustering. We then evaluated HER2 in pre-treatment core biopsies from women with breast cancer undergoing neoadjuvant therapy. A complete pathological response was associated with a high detected HER2 density and significant HER2 clustering. These results established the nano-organization of HER2 as a potential signature of therapy-responsive disease.

Keywords: HER2; breast cancer; clustering; super-resolution microscopy; therapy response; trastuzumab.

Figure 1

SMLM images of HER2 detected in cultured breast cancer cell lines. (a). HER2 was detected using trastuzumab-AF647 (top) or pertuzumab-AF647 (bottom) in luminal B breast cancer cells BT-474 (trastuzumab-sensitive) and BT-474^R (trastuzumab-resistant). (b). HER2 was detected using trastuzumab-AF647 (top) or pertuzumab-AF647 (bottom) in HER2-overexpressing breast cancer cells SK-BR-3 (trastuzumab-sensitive) and JIMT-1 (trastuzumab-resistant). The localizations are colored based on the number of neighboring localizations within 70 nm (purple is least clustered and yellow is most clustered). Scale bars are 5 µm for full cells and 200 nm for insets. While the figure shows representative images, measurements were repeated three independent times, and 14–15 cells were imaged in each case.

Figure 2

Detected HER2 densities and clustering parameters in cultured luminal B breast cancer cell lines. Average detected HER2 density (a) and clustering parameters (b–e) obtained using detection with trastuzumab-AF647 in BT-474 (blue) and BT-474^R (red) cells. When trastuzumab was used as a probe, trastuzumab-sensitive cells (compared to trastuzumab-resistant cells) on average had a significantly higher detected density, fraction of HER2 molecules in clustered regions, and number of HER2 clusters per ROI. In total, 58 ROIs for BT-474 and 60 ROIs for BT-474^R were assessed. Clustering parameters were calculated based on 47 clustered ROIs for BT-474 and 51 clustered ROIs for BT-474^R cells. Average detected HER2 densities (f) and clustering parameters (g–j) obtained using detection with pertuzumab-AF647 in BT-474 (blue) and BT-474^R (red) cells. When pertuzumab was used as a probe, trastuzumab-sensitive and trastuzumab-resistant cells had comparable detected densities and clustering parameters. In total, 60 ROIs for BT-474 and 60 ROIs for BT-474^R were assessed. Clustering parameters were calculated based on 40 clustered ROIs for BT-474 and 47 clustered ROIs for BT-474^R cells. Dashed lines represent the mean, and all error bars represent SEM. Numerical details and statistics are provided in Supplementary Tables S1a and S2.

Figure 3

Detected HER2 densities and clustering parameters in cultured HER2 overexpressing breast cancer cell lines. Average detected HER2 density (a) and clustering parameters (b–e) obtained using detection with trastuzumab-AF647 in SK-BR-3 (blue) and JIMT-1 (red) cells. When trastuzumab was used as a probe, trastuzumab-sensitive cells (compared to trastuzumab-resistant cells) had a significantly higher detected density and an increase in HER2 clustering according to all clustering parameters. In total, 60 ROIs for SK-BR-3 and 59 ROIs for JIMT-1 were assessed. Clustering parameters were calculated based on 40 clustered ROIs for SK-BR-3 and 20 clustered ROIs for JIMT-1 cells. Average detected HER2 densities (f) and clustering parameters (g–j) obtained using detection with pertuzumab-AF647 in SK-BR-3 (blue) and JIMT-1 (red) cells. When pertuzumab was used as a probe, trastuzumab-sensitive cells (compared to trastuzumab-resistant cells) had significantly higher average detected density and number of HER2 clusters per ROI. In total, 55 ROIs for SK-BR-3 and 60 ROIs for JIMT-1 were assessed. Clustering parameters were calculated based on 26 clustered ROIs for SK-BR-3 and 17 clustered ROIs for JIMT-1 cells. Dashed lines represent the mean, and all error bars represent SEM. Numerical details and statistics are provided in Supplementary Tables S1b and S2.

Figure 4

Detected HER2 density and clustering parameters in human breast cancer core biopsies. (a). SMLM image of a HER2-positive cell from a core biopsy sample with HER2-enriched cell protrusions marked with arrows. Scale bar is 5 µm. (b) Average detected HER2 densities in patient specimens obtained using trastuzumab-AF647. P21, P23, and P25 (pCR, T0 N0 post-op) had significantly higher densities compared to P18 (RCB-I, T1aN0 post-op), P20 (RCB-II, T1b N1a post-op), and P26 (pCR, Tis N0 post-op). (c–f). Average HER2 clustering parameters in patient specimens obtained using trastuzumab-AF647. P21, P23, P25, P26 had significantly higher fractions of HER2 molecules in clustered regions and numbers of HER2 clusters per ROI compared to P18 and P20. Clustered ROIs were used for analysis: 17 ROIs for P18, 4 ROIs for P20, 61 ROIs for P21, 70 ROIs for P23, 9 ROIs for P25 S1, 21 ROIs for P25 S2, and 9 ROIs for P26. Red points indicate women with RCB and blue points indicate women with pCR. Dashed lines represent the mean, and all error bars represent SEM. Numerical details and statistics are provided in Supplementary Tables S4 and S5.