Early-phase clinical trials suggested that everolimus may be an optional treatment for HR-positive metastatic breast cancers. A phase I study evaluated the pharmacokinetics and safety of everolimus plus letrozole in patients with metastatic breast cancer. In the study, seven out of 18 patients received combination therapy for more than 6 months. Prominent clinical toxicities were fatigue, stomatitis, diarrhea, anorexia, rash, and headache [20].

Additionally, a phase II study evaluated the safety and efficacy of fulvestrant combined with everolimus for patients with postmenopausal advanced breast cancer that was resistant to aromatase inhibitor. The median time to progression (TTP) in this study was 7.4 months, and the clinical benefit rate (CBR) was 49% [21]. In addition, 71% of the patients in this study received prior chemotherapy, 81% received prior tamoxifen therapy, and 26% received three or more types of endocrine therapy [21]. The most common adverse reactions were mucositis, weight loss, and rash. This study demonstrated that everolimus combined with fulvestrant is effective after aromatase inhibitor resistance in patients with heavily pretreated HR-positive breast cancer, and the toxicities were manageable [21].

The TAMRAD study is a randomized phase II study [9] on patients with HR-positive, HER2-negative metastatic breast cancer who were treated with prior aromatase inhibitor therapy. The purpose of the study was to evaluate the efficacy and safety of everolimus plus tamoxifen compared to tamoxifen alone. The results showed CBRs of 61% and 41% for the combination arm and the tamoxifen monotherapy arm, respectively, and this difference was statistically significant (p=0.04). The TTP was 4.5 months in patients treated with tamoxifen alone and 8.6 months in patients treated with everolimus plus tamoxifen. The prominent clinical toxicities reported in the combination arm were stomatitis, fatigue, rash, anorexia, and diarrhea. There was no difference in grade 3 or 4 adverse events between the two groups. Subgroup analysis of primary and secondary hormone resistance indicated that the median TTP was 14.8 months in patients with secondary resistance versus 5.4 months for patients with primary resistance. Similarly, patients with secondary resistance to aromatase inhibitors had a significantly higher CBR when treated with everolimus combined with tamoxifen (74%) than those treated with tamoxifen alone (48%). This study revealed that combination therapy with tamoxifen and everolimus increased the TTP, CBR, and OS when compared to tamoxifen monotherapy in patients with aromatase inhibitor-resistant postmenopausal metastatic breast cancer [9].

The promising results observed in the phase II studies of everolimus warranted further studies in patients with HR-positive, HER2-negative metastatic breast cancers, especially patients with de novo resistance to aromatase inhibitors. The BOLERO-2 study is a phase III study that compared everolimus combined with exemestane to placebo plus exemestane for patients with HR-positive HER2-negative breast cancer that is resistant to nonsteroidal aromatase inhibitor therapy. The results from the final analysis, after a median follow-up of 18 months, indicated that the primary end-point median PFS was 7.8 months in the everolimus plus exemestane arm versus 3.2 months in the placebo plus exemestane arm (p=0.0001). The CBR in the everolimus arm was significantly higher than that in the placebo arm (51.3% vs. 26.4%, p=0.0001) [7]. The median OS was 31.0 in patients treated with everolimus plus exemestane and 26.6 months in those treated with placebo plus exemestane; however, this difference was not statistically significant (p=0.1426) [8].

Tumors harboring mutations in genes encoding proteins involved in the PI3K/AKT/mTOR pathway may activate the PI3K enzyme. Therefore, such tumors are expected to be sensitive to everolimus and agents targeting this pathway. The results of several preclinical studies have suggested that genetic aberrations in the PI3K/AKT/mTOR pathway could predict the efficacy of mTOR inhibitors [2223]. However, data from clinical studies regarding the predictive capability of these genetic aberrations are contradictory. A retrospective study showed that patients with advanced breast cancer treated with inhibitors of the PI3K/AKT/mTOR pathway in combination with endocrine therapy, anti-HER2 therapy, or chemotherapy had a longer TTP compared to patients with wild-type tumors [24]. Baselga et al. [25] analyzed the relationship between the presence of mutations in exon 9 of PIK3CA and the efficacy of everolimus plus letrozole in a neoadjuvant trial. The results from the study indicated that the presence of these PIK3CA mutations provided an improved response to the combination of everolimus with letrozole [25]. However, in the TAMRAD trial, the researchers analyzed the relationship between mutations in the primary tumor tissue and the efficacy of everolimus, and they did not find any correlation between the presence of PI3K mutations and the response to everolimus [26]. Although they did find that everolimus was more effective in patients with low PI3K expression. In addition, patients with low levels of liver kinase B1 (LKB1), a known suppressor of mTOR, and high levels of phospho-4E binding protein, which is downstream of mTOR, received a greater benefit from everolimus treatment [26]. The BOLERO-2 study also failed to identify any specific gene mutations in the tumor tissue that were associated with a greater benefit from everolimus treatment [27]. Nevertheless, the investigators found that patients with no alteration or a single genetic alteration in PIK3CA/PTEN/CCND1 or FGFR1/2 received a greater PFS benefit from everolimus treatment [28]. In the BOLERO-2 study, mutation analysis of plasma cell-free DNA (cfDNA) suggested that there was no relationship between the PFS of patients treated with everolimus and the PIK3CA genotypes in cfDNA, which was consistent with previous tumor tissue DNA analysis. These results suggest that PIK3CA mutations, including H1047R, E545K, and E542K, cannot predict patient response to everolimus. In conclusion, the current evidence is insufficient to demonstrate that the PIK3CA genotype is an effective predictive biomarker for everolimus benefit [29].

Some PTEN gene variations, including germline and somatic mutations, can activate the PI3K enzyme. Preclinical models support the notion that cells with PTEN gene loss are more sensitive to PI3K/AKT inhibitors [3031]. However, the results from the TAMRAD trial showed that PTEN gene loss did not influence the response to everolimus [26]. In a similar analysis in the BOLERO-2 trial, PTEN gene status was not correlated with clinical outcome, which seems to confirm the results reported by the TAMRAD study [27].

In one study, a panel of breast cancer cell lines with HER2 amplification was sensitive to everolimus [32]. Another study showed that the combined presence of HER2 gene amplification along with PIK3CA mutation was highly predictive of sensitivity to an inhibitor of the PI3K/AKT/mTOR pathway (GDC-0941) [31]. Preclinical studies showed that the mTOR pathway is regulated by neurofibromin, a protein encoded by the NF1 gene, and several other studies demonstrated that cells containing mutations in the NF1 gene were highly sensitive to mTOR inhibitors, including everolimus and rapamycin [33]. A recent study explored the genomic alterations that confer extreme sensitivity to everolimus in 39 tumors of various types from patients who were treated with everolimus. The results showed that patients with mTOR, NF1, PIK3CA, TSC1, TSC2, and PIK3CG mutations could benefit from the mTOR inhibitor everolimus. Conversely, BAP1 and FGFR4 mutations were noted only in patients who did not receive a clinical benefit from everolimus. However, there were no breast cancer patients enrolled in this study [34]. In the BOLERO-2 study, researchers explored the correlation between ESR1 mutations, including Y537S and D538G, in cfDNA and sensitivity to exemestane and everolimus. Interestingly, these two activating mutations of the ESR1 gene appeared to have differential effects on sensitivity to everolimus. The results indicated that patients with the Y537S or D538G mutations had a poorer prognosis and shorter OS; however, the PFS of patients treated with everolimus was lower in those with the Y537S mutation than in those with wild-type ESR1, whereas the magnitudes of the PFS benefit of everolimus in patients with wild-type ESR1 and those with the D538G mutation were similar [35].

Preclinical studies in tumor cell lines indicated that high levels of phosphorylated AKT, glycogen synthase kinase 3 β, and tuberous sclerosis complex 2 are correlated with increased sensitivity to everolimus [36]. O'Reilly et al. [37] investigated potential predictive protein biomarkers for sensitivity to everolimus in human and animal studies, and univariate analysis showed that the levels of pS6, total S6, pS6/total S6, and phospho-AKT (pAKT) were significantly correlated with sensitivity to everolimus. Further analysis found that the combination of high pAKT and high p235-S6/total S6 levels could be a predictor of sensitivity to everolimus, whereas low pAKT and low p235-S6/total S6 levels could be a predictor of insensitivity to everolimus [37]. The TAMRAD trial found that everolimus was more effective in patients who had low PI3K expression, low LKB1 expression, and high phospho-4E binding protein expression; however, they did not find a relationship between the presence of PI3K, PTEN, and pAKT mutations and the efficacy of everolimus [26]. Baselga et al. [25] evaluated tumor core biopsies before treatment and on day 15 of treatment in patients treated with letrozole and either everolimus or placebo in a neoadjuvant trial of everolimus. The results indicated that patients treated with everolimus had a statistically significant decrease in Ki-67 and pS6 levels [25].