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Some patients with HR+ ABC show primary resistance to endocrine therapy, and in the
remainder, secondary resistance ultimately develops, representing a major clinical
problem. The biology of resistance to endocrine therapy is complex and still not completely
elucidated 23]. Preclinical evidence suggests that targeting the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian
target of rapamycin (mTOR) 24] or the cyclin D1-Cyclin-Dependent Kinases 4 and 6 (CDK4/6) pathway 25] might increase endocrine sensitivity. Based on this rationale, randomized clinical
trials have recently investigated whether combination therapies with biological agents
targeting these pathways would improve PFS or OS of patients with HR+ ABC 26],27].

The phase III BOLERO-2 trial randomized 724 postmenopausal patients with HR+ HER2–
ABC to receive everolimus, an mTOR inhibitor, and exemestane versus exemestane and
placebo. The primary end point, PFS, was shown to be significantly improved in patients
receiving everolimus compared to those receiving placebo according to both local (6.9
vs. 2.8 months; HR, 0.43; 95% CI, 0.35–0.54; P 0.001) and central assessment (10.6 vs. 4.1 months; HR, 0.36; 95% CI, 0.27–0.47;
P 0.001) 26]. However, the combination did not confer a statistically significant improvement
in OS (median OS: 31.0 months in the everolimus plus exemestane arm vs. 26.6 months
in the exemestane plus placebo arm; HR, 0.89; 95% CI, 0.73–1.10; P?=?0.1426) 28]. Given the remarkable PFS results, everolimus was approved by the Food and Drug Administration
for the treatment of postmenopausal women with HR+ HER2– ABC in combination with exemestane,
after failure of treatment with letrozole or anastrozole. However, the toxicity profile
of everolimus is far from ideal. Serious adverse events were higher in patients receiving
everolimus compared to those receiving placebo (55% and 33%, respectively) and a higher
proportion of patients discontinued everolimus because of adverse events compared
to placebo (29% vs. 5%) 28]. For this reason there is a great interest in identifying biomarkers of response
to screen patients who are likely to derive the largest benefit from this compound.

mTOR exists in two different complexes, mTORC1 and mTORC2. Everolimus targets mTORC1,
which signals via two major substrates, the p70 ribosomal protein S6-kinase (pS6)
and the eukaryotic initiation factor 4E binding protein 1 (4EBP1) 24]. The activity of mTORC1 is regulated by the serine/threonine kinase Akt, a downstream
effector of PI3-kinases. Activating mutations in the catalytic subunit of PI3-kinase
(PIK3CA) occur in around 40% of ER+ breast cancers 29], but the hypothesis that PIK3CA-mutated breast cancers would derive the largest benefit
from mTOR inhibitors was not confirmed in an exploratory analysis of the BOLERO-2
trial, although only a fraction of enrolled patients were included 30]. One explanation for these results might be that mutational status of PIK3CA does
not correlate with pathway activation. A seminal work of Loi et al. indeed demonstrated
that, in ER+ HER2? breast cancer, PIK3CA mutation surprisingly did not always result
in elevated downstream signaling, and correlated with relatively low mTORC1 signaling
31],32]. Reverse phase protein array data from the The Cancer Genome Atlas also confirmed
that phospho-AKT, phospho-pS6, and phospho-4EBP1, markers of PI3K pathway activation,
were not necessarily elevated in PIK3CA mutated luminal A breast cancer 33]. These data suggest that mutational status of PIK3CA should be combined with assessment
of downstream pathway activity to have a better prediction of everolimus benefit.
In support of this, Loi et al. 32] showed, in a dataset derived from patients enrolled in a randomized, double blind,
phase II neoadjuvant trial, that lower scores of a genomic signature of PIK3CA mutation
(PIK3CA-GS) were able to identify those patients with the largest relative decreases
in Ki67 (a surrogate marker of response) to letrozole/everolimus (R?=??0.43, P?=?0.008) compared with letrozole/placebo (R?=?0.07, P?=?0.58; interaction test P?=?0.02). However, in a second dataset from pre-surgical patients using everolimus
alone, the PIK3CA-GS was not correlated with relative change in Ki67 (R?=??0.11, P?=?0.37). In both datasets, changes in percentage of Ki67 decrease were not statistically
different between PIK3CA mutant and wild-type breast cancer 32]. Additionally, translational studies within the TAMRAD trial, a multicenter phase
II trial in which postmenopausal women with HR+ HER2– ABC previously treated with
AI were randomly allocated to receive tamoxifen plus everolimus (n?=?54) or tamoxifen
alone (n?=?57), have recently shown that the subgroups most likely to have an improvement
in TTP with tamoxifen plus everolimus therapy compared with tamoxifen alone were patients
with molecular evidence of PI3K pathway activation (i.e., high phospho-4EBP1, low
4EBP1) 34].

Another important point is that in the BOLERO-2 trial, PIK3CA mutational status was
assessed mainly on primary tumor tissues 30]. Studies indicate that discordance in PIK3CA mutational status between primary tumors
and metastases might occur 35]-37], suggesting that we should reassess molecular pathway alterations prior to starting
targeted treatment, either through analysis of metastatic tissue or, potentially,
‘liquid biopsies’. Indeed, the feasibility of assessing PIK3CA mutation in circulating
tumor cells 38]-41] and circulating free DNA 42]-44] has already been demonstrated.

To further assess the role of PIK3CA mutations in breast cancer, a mutational analysis
of PIK3CA/AKT1 and RAS/RAF was performed on 4,294 primary tumor samples from postmenopausal
patients with ER+ breast cancer who had been enrolled in the Tamoxifen Exemestane
Adjuvant Multinational phase III trial 29]. This study showed that PIK3CA mutations were more frequent in low-risk luminal breast
cancer and were associated with significantly improved 5-year distant relapse-free
survival in univariate analysis (HR, 0.76; 95% CI, 0.63–0.91; P?=?0.003) 29]. These results are consistent with previous findings that PIK3CA mutations are more
frequently seen in luminal A primary tumors and are associated with increased sensitivity
to endocrine therapy 31],33],45]. Mayer and Arteaga suggested, in an accompanying editorial 46], that the apparent discrepant role of PIK3CA mutations in early versus late ER+ breast
cancer might be explained by a predominant role of PIK3CA mutations in secondary endocrine
resistance 46]. Data supporting this hypothesis come from preclinical studies demonstrating an increased
pathway activation in long-term estrogen-deprived breast cancer cell lines 47] and from an exploratory subgroup analysis within the TAMRAD trial, which suggested
that the everolimus benefit was seen more in patients with secondary hormone resistance
than those with primary resistance 48].

In the absence of available biomarkers, clinical considerations regarding the population
of the BOLERO-2 trial should be taken into account when selecting patients for combination
therapy with everolimus. First, 84% of patients enrolled in the BOLERO-2 trial were
sensitive to prior endocrine therapy. Endocrine sensitivity was defined as at least
24 months of endocrine therapy before recurrence in the adjuvant setting or a response
or stabilization for at least 24 weeks of endocrine therapy for advanced disease 26]. Second, 84% of patients received everolimus as second line or more of therapy for
advanced disease 26]. Accordingly, in our opinion, the combination of exemestane and everolimus could
be considered an appropriate second line treatment option for patients who have demonstrated
benefit from first-line endocrine therapy.

Several PI3K inhibitors are in clinical development for patients with HR+ HER2– ABC
49]. Recently, the results from the FERGI study, a phase II randomized trial of the PI3K
inhibitor pictilisib plus fulvestrant versus fulvestrant plus placebo in patients
with ER+ AI-resistant ABC, have been presented. These data showed that the addition
of pictilisib to fulvestrant was associated with a non-statistically significant improvement
in PFS for the combination versus the control arm (6.2 vs. 3.8 months; HR, 0.77; 95%
CI, 0.50–1.19). The benefit from pictilisib was independent from PIK3CA mutational
status, while the subgroup of patients that seemed to benefit more from the addition
of pictilisib was that with ER+/PR+ tumors (PFS, 7.2 vs. 3.7 months in the combination
and control arm respectively; HR, 0.46; 95% CI, 0.27–0.78) 50].

Clinical trials are currently investigating the safety and efficacy of three CDK4/6
inhibitors, palbociclib, abemaciclib, and LEE011, for the treatment of HR+ HER2– ABC
25]. The compound with more mature clinical results is palbociclib. The phase II, randomized
PALOMA 1 trial was designed as a two-part study to evaluate palbociclib in combination
with letrozole versus letrozole alone for first line treatment of postmenopausal patients
with HR+ HER2– ABC. Part 1 of the study enrolled 66 unselected patients, while the
Part 2 enrolled 99 patients with tumors positively screened for cyclin D1 amplification
and/or loss of p16; the primary end point was PFS. The final analysis showed a statistically
significant improvement in PFS for the combination arm versus letrozole arm (20.2
vs. 10.2 months; HR, 0.488; 95% CI, 0.319–0.748; one-sided P?=?0.0004). When Part 1 and Part 2 were analyzed separately, treatment effects were
maintained (Part 1: HR, 0.299; 95% CI, 0.156–0.572; one-sided P??0.0001; Part 2: HR 0.508; 95% CI, 0.303, 0.853; one-sided P?=?0.0046) 27]. Side effects were mainly hematological, with grade 3/4 neutropenia and leucopenia
reported in 54% and 19% of patients receiving palbociclib, respectively 27]. At present, palbociclib is not registered for the treatment of breast cancer and
confirmatory results from ongoing phase III trials are eagerly awaited.

Numerous biological agents are currently being investigated in combination with endocrine
therapy for the treatment of postmenopausal patients with HR+ HER2– ABC, including
histone deacetylase, Akt, and IGF receptor inhibitors 51],52]. For the future clinical development of these agents, understanding which subgroup
of patients is more likely to benefit from the combination with endocrine therapy
is of critical importance. This might be achieved i) with sub-group analyses correlating
the clinical activity with the degree of response to prior endocrine therapies defined
according to standard criteria and ii) with the identification of biomarkers of response.