Antiproliferative Effect of Lapatinib in HER2-Positive and HER2-Negative/HER3-High Breast Cancer: Results of the Presurgical Randomized MAPLE Trial (CRUK E/06/039)

Discussion

There is a need for novel trial designs more specifically adapted to the molecular era of targeted cancer medicine. Conventional adjuvant trials in early breast cancer with large numbers of unselected patients and traditional clinical endpoints, such as progression-free survival (PFS) or overall survival (OS), require large numbers, with prolonged follow-up, and may not be appropriate for taking forward targeted therapies aimed at increasingly small subsets of patients. Presurgical trials using surrogate markers of in vivo drug activity, such as the cell proliferation marker Ki67, have emerged as a useful setting in which to investigate biologic predictors of response (9). In addition, because such short-term (10–14 days) treatment exposure is of no proven clinical efficacy and study participation does not extend normal waiting time to surgery, an unselected patient population can be enrolled quickly in this clinical setting in an attempt to establish which tumors may exhibit the desired antiproliferative response. Furthermore, the randomization to a placebo is both permissible and acts as a valuable control for the biologic efficacy observed with the experimental therapy.

In the MAPLE study, we have confirmed that a short-term presurgical trial design in early breast cancer using an EGFR/HER2 TKI with a primary biologic antiproliferative endpoint is both feasible and safe, and that patients are willing to contribute sequential research biopsies. Our target patient recruitment was achieved in a reasonable time frame, with paired samples of adequate quality and cellularity obtained in 98% of patients. Because the primary endpoint was change in Ki67 and biomarker assessments were conducted as soon as paired samples were received, results were available shortly after recruitment was completed, thus illustrating how the presurgical trial design can provide a valuable tool to screen new agents in a fast and cost-effective manner.

Short-term lapatinib treatment was associated with a significant Ki67 downregulation. Importantly, there was no evidence of a change in Ki67 in the placebo control group, indicating that no protocol-related interventions affected the primary endpoint. The trial included 26 patients with a HER2⁺ tumor; 25 were HER2 3+ by IHC and were found to also demonstrate HER2 amplification by FISH. The only patient with a HER2 2+/FISH⁺ tumor was randomized to placebo. Although Ki67 reduction was greatest among HER2⁺ tumors (Fig. 2A), lapatinib also resulted in a statistically significant 27% Ki67 downregulation among HER2⁻ nonamplified tumors. Although lapatinib is currently used exclusively in HER2⁺ disease, MAPLE has shown that lapatinib may also have antiproliferative activity in a subset of HER2⁻ nonamplified breast cancers. Although Ki67 has not been validated as a surrogate endpoint in ER⁻ disease, the changes in Ki67 observed in patients with HER2⁻ breast cancer enrolled in MAPLE nonetheless indicate that this group is not entirely biologically refractory to lapatinib and are consistent with a beneficial effect of the drug. The challenge is, therefore, characterizing the sensitive subset in MAPLE as defined by an antiproliferative response. When considering individual cases (Fig. 2B), there was marked interpatient variability in Ki67 change, with 14% of HER2⁻ nonamplified lapatinib-treated tumors showing a >50% reduction in Ki67. We therefore investigated whether baseline biomarkers could identify these “Ki67 responders” (i.e., with a >50% Ki67 decrease), and demonstrated that among HER2⁻ tumors, the only independently significant baseline predictor for Ki67 response in a multivariable analysis was elevated levels of HER3 mRNA. These data are consistent with a recently reported smaller non–placebo-controlled study in 31 patients with HER2⁻ primary breast cancer where a similar >50% decrease in Ki67 was reported in four (14%) tumors, all of which expressed HER3 (16).

There has already been some evidence to suggest that HER2 inhibition may have a role in HER2-low breast cancer. Roughly 10% of patients enrolled in a trial of adjuvant chemotherapy alone or with trastuzumab revealed that some of the randomized patients were in fact neither HER2 3+ nor FISH amplified (17). These HER2⁻ patients enrolled in the phase III National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31 trial had significantly better disease-free survival with a relative risk of 0.40 (P = 0.026) when given trastuzumab after completing treatment for early breast cancer. These findings have provided the rationale for a large ongoing clinical trial, B-47. In addition, data from the Cancer and Leukemia Group B neoadjuvant study investigating the benefit of dual HER2 targeting suggest that intrinsic subtype is a strong driver of response to HER2-targeted therapy. Therefore, although HER2 overexpression or amplification may be an important predictor of response to HER2 targeted therapies, it may not be an obligate predictor (18).

In addition, there is a biologic rationale for expecting that in the setting of modest HER2 protein expression in nonamplified tumors, other molecular factors could determine an antiproliferative response to an intracellular TKI, such as levels of other HER family receptors that may heterodimerize with HER2 or levels of HER family ligands (19, 20). HER2 activation requires receptor dimerization; HER2 has no known ligand, and activation occurs either via ligand-independent HER2 homodimerization associated with high levels of receptor membrane expression such as that found in HER2 gene–amplified tumors or, alternatively, via ligand-dependent receptor heterodimerization (i.e., EGFR:HER2, or HER2:HER3; ref. 21). In contrast with the antibody trastuzumab that recognizes an epitope on the extracellular domain that is only exposed in the case of HER2 homodimers (21, 22), the TKI lapatinib inhibits the intracellular domains of EGFR and HER2, and as such may also target various HER2-containing heterodimers. HER3 has a docking site for the p85 subunit of PI3K and acts as the preferred heterodimerization partner for HER2. HER2–HER3 heterodimers are increasingly identified as key drivers of the PI3K cell survival pathway (23–26). Activation of HER3 and downstream PI3K has been shown to predict resistance to trastuzumab but sensitivity to lapatinib in vitro (27). In MAPLE, we have demonstrated that the HER2⁻ nonamplified tumors showing a Ki67 response with lapatinib tended to show elevated levels of both HER3 and HER2 (Fig. 4), raising the possibility that a subset of HER2⁻ breast cancers with significant HER2:HER3 dimerization and activated PI3K signaling may be sensitive to the antiproliferative effects of lapatinib. Novel imaging technologies such as FRET/FLIM (Förster resonance energy transfer/fluorescence lifetime imaging) that can detect the physical association between these two proteins could be a useful technology to investigate the relevance of various HER family heterodimers and lapatinib responsiveness (28, 29).

Whereas phosphorylated HER3 and AKT are useful readouts of the PI3K pathway in vitro, pAKT and pHER3 have proven more challenging to detect in patient samples, and baseline pAKT did not predict response to lapatinib in MAPLE. The chromatin-associated protein stathmin has been identified as a surrogate measure of PI3K signaling (15). In MAPLE, stathmin was significantly downregulated by lapatinib treatment in the HER2⁻ subset, whereas pAKT was not. However, a reduction in stathmin of similar magnitude was also observed in the placebo group, but was not statistically significant due to the small numbers in this group, indicating that further work is needed to confirm the effect of lapatinib on stathmin in the presurgical setting. More recent data indicate that stathmin is strongly related to proliferation, and this is likely to be detrimental to its use as a surrogate of PI3K signaling.

Not all HER2⁺ patients benefit from lapatinib, and in MAPLE only 37% of HER2⁺ tumors demonstrated a >50% Ki67 decrease, suggesting that additional biomarkers may improve the selection of patients with HER2⁺ breast cancer for lapatinib therapy. Among HER2⁺ cases, baseline epiregulin emerged as a significant predictor of response. Epiregulin is a ligand for EGFR and HER4, and high epiregulin may identify HER2⁺ tumors demonstrating high levels of HER2–EGFR or HER2–HER4 heterodimers that may preferentially benefit from lapatinib.

In conclusion, the short presurgical trial design is a feasible, safe, and useful setting to investigate candidate predictive biomarkers for antiproliferative response to novel therapeutic agents. Lapatinib demonstrated an antiproliferative response in a subset of HER2⁻ nonamplified primary breast tumors. Among these HER2⁻ tumors, high HER3 mRNA identified a subset of Ki67 responders, and there was a trend for combined increase in HER3 and HER2 mRNA expression to predict this antiproliferative response. It would, however, be necessary for further neoadjuvant trials to validate the use of HER2 TKIs in HER2⁻ patients before this is taken any further, with high HER2 and HER3 expression being examined as indicators of response. Among HER2⁺ tumors, high levels of the EGFR/HER4 ligand EREG identified the Ki67 responders. Although expression of HER family receptors, their ligands, or HER receptor hetero- or homodimerization status may merit further investigation, greater characterization of the responding HER2⁻ breast tumors is needed before clinical use can occur.