Personalizing HER2-targeted therapy in metastatic breast cancer beyond HER2 status: what we have learned from clinical specimens

Abstract

HER2 is over-expressed in approximately 25% to 30% of human metastatic breast cancers, primarily due to gene amplification. There are currently two HER2-targeted therapies approved for clinical use, the monoclonal HER2 antibody trastuzumab and the EGFR/HER2 dual tyrosine kinase inhibitor lapatinib. Although both agents show clinical benefit in a subset of patients with metastatic breast cancer, many patients with HER2-over-expressing metastatic breast tumors do not respond to these agents. Furthermore, those who do show an initial response generally demonstrate disease progression, on average in less than one year. It has become clear that HER2 expression status alone does not adequately predict response to HER2-targeted therapy. Identification and clinical validation of molecular predictors of response to trastuzumab and lapatinib is critical for further personalizing treatment and improving clinical benefit for patients whose tumors over-express HER2. In this review, we discuss published data describing potential predictors of response or resistance to trastuzumab and lapatinib. While a discussion of the preclinical work is provided, the emphasis is placed on potential predictors that have been studied in clinical specimens such as tumor tissue or serum obtained from patients treated with HER2-targeted therapy. The present analysis and synthesis of the available literature therefore contribute towards an emerging knowledgebase to personalize breast cancer treatment taking into factors including but beyond HER2 expression.

Fig. (1)

BT474 and SKBR3 parental (Par) or BT474 Herceptin-resistant clones 1, 2, or 3 (BT-HRc1, c2, c3) or SKBR3 Herceptin-resistant pool 2 (SK-HRp2) were lysed for total protein, and immunoblotted for phosphorylated serine 32 inhibitor of kappa B alpha (p-Ser32 IkB alpha) (Cell Signaling; Denvers, MA) or actin (Santa Cruz Biotech; Santa Cruz, CA) as a loading control. Phosphorylation of IkB alpha on serine 32 promotes dissociation of IkB and nuclear factor kappa B (NF-kB), allowing NF-kB to localize to the nucleus and function as a transcription factor. Thus, phosphorylation of IkB alpha on serine 32 is associated with increased NF-kB activity. Herceptin-resistant cells showed increased phosphorylation of IkB alpha on serine 32, suggesting that NF-kB signaling may be increased in Herceptin (trastuzumab)-resistant cells.

Fig. (2)

BT474 Parental (BT-par) and BT474 Herceptin-resistant clones 1, 2, and 3 (BT-HRc1, c2, and c3) were treated with the NF-kB inhibitor curcumin at 12.5 or 25 μM or with ethanol (in which curcumin is dissolved) at a volume equal to that found in the highest dose of curcumin. After 72 hours (h) viable cells were counted by trypan blue exclusion. Each sample was done in duplicate or triplicate, and experiments were performed on two separate occasions for reproducibility. Values in graph represent the average of the two experiments, with error bars reflecting standard deviation between the two averages. Statistical significance was determined by student's t-test, and was considered significant at less than 0.05; *p<0.05, **p<0.005 for Herceptin-resistant versus parental cells. Trastuzumab-resistant cells were significantly more sensitive to curcumin than parental, trastuzumab-sensitive cells, suggesting that NF-kB inhibition may be an effective strategy in resistant cells.

Fig. (3)

BT474 Parental (BT-par) and BT474 Herceptin-resistant clones 1, 2, and 3 (BT-HRc1, c2, and c3) were plated in matrigel and treated with curcumin at 25 μM or with ethanol at the volume equal to that found in the dose of curcumin. Cells were maintained for 2 weeks. Representative photographs are shown, and were taken with an Olympus IX50 inverted microscope at 4X magnification. Experiments were done in duplicate and performed twice. Curcumin inhibited anchorage-independent growth of resistant cells to a greater degree than parental cells.

Fig. (4)

SKBR3 Parental (SK-par) and Herceptin-resistant pool 2 (SK-HRp2) were treated with curcumin at 12.5 or 25 μM or with ethanol (in which curcumin is dissolved) at a volume equal to that found in the highest dose of curcumin. After 72 hours (h) viable cells were counted by trypan blue exclusion. Each sample was done in duplicate or triplicate, and experiments were performed on three separate occasions for reproducibility. Values in graph represent the average of the three experiments, with error bars reflecting standard deviation between the three averages. Statistical significance was determined by student's t-test, and was considered significant at less than 0.05 for Herceptin-resistant versus parental cells. SKBR3 trastuzumab-resistant cells showed a trend of being more sensitive to curcumin than parental, trastuzumab-sensitive cells at the lower dose of 12.5 μM curcumin, but this was not statistically significant.

Fig. (5)

SKBR3 Parental (SK-par) and Herceptin-resistant pool 2 (SK-HRp2) were plated in matrigel and treated with curcumin at 25 μM or with ethanol at the volume equal to that found in the dose of curcumin. Cells were maintained for 2 weeks. Representative photographs are shown, and were taken with an Olympus IX50 inverted microscope at 4X magnification. Experiments were done in duplicate and performed twice. Curcumin appeared to inhibit anchorage-independent growth of resistant cells to a greater degree than parental cells.