Phosphatidylinositol 3-kinase pathway activation in breast cancer brain metastases

The incidence of brain metastases (BMs) is approximately 15% among women newly diagnosed with metastatic breast cancer (BC) [1]. This figure likely underestimates the true incidence, as autopsy studies report a 30% incidence of BMs among women with advanced disease [2]. Current therapeutic interventions include corticosteroids, whole-brain radiotherapy, neurosurgical resection, stereotactic radiosurgery, and systemic chemotherapy [3]. Despite these treatment strategies, prognosis among patients with BCBMs remains poor, with a median overall survival of approximately 6 months [4, 5]. Although targeted agents show promise in the treatment of advanced extracranial BC, challenges in delivery of these agents to the central nervous system (CNS) include properties inherent to the blood barrier (that is, efflux mechanisms) and our incomplete understanding the biology underlying BCBMs. Moreover, optimal therapeutic targets within BCBM are largely unknown.

Previous studies indicate that the phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in the initiation and progression of human BC, and alterations in this pathway have been identified in approximately 50% of these tumors [6, 7]. PI3K pathway activation occurs in response to extracellular signals via either growth-factor receptor or integrin pathways. On its recruitment to the cellular membrane via receptor-mediated activation, the p110α catalytic subunit of PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) at the 3' position of the inositol ring, generating PIP3 [8]. PIP3 recruits phospholipid-binding domain containing proteins, particularly AKT, to the plasma membrane. Phosphorylated (p-)AKT, the primary downstream effector of PI3K signaling, moves from the cytoplasm to the nucleus to initiate its downstream effects. This cascade, including activation of the mammalian target of rapamycin (mTOR) and its downstream effectors, p70S6 kinase and 4E-binding protein-1, affects a number of cellular processes, including proliferation and motility, which clinically translate into endocrine and chemotherapy resistance and worse cancer-specific survival [6, 9‐12]. The PI3K/AKT pathway is negatively regulated by PTEN (phosphatase and tensin homologue), a lipid phosphatase that removes the 3-phosphate from PI(3,4)P2 and PI(3,4,5)P3, thus inactivating the signaling cascade [13]. Therefore, loss of PTEN contributes to the activation of the PI3K/AKT signaling cascade through inhibition of degradation of both PI(3,4)P2 and PI(3,4,5)P3.

To date, alterations and activation of the PI3K/AKT pathway are well established in the initiation and progression of extracranial human BC [6, 8‐10, 14, 15]. However, the contribution of this important signaling pathway to the pathogenesis of BCBMs has yet to be fully elucidated. This is of clinical importance as small-molecule inhibitors of the PI3K/AKT/mTOR pathway are in development and show promising activity in the treatment of primary brain tumors, suggesting sufficient blood-brain barrier penetration to elicit therapeutic effects [16, 17]. In this study, we quantitated the expression of the PI3K pathway biomarkers p-AKT, p-S6, and PTEN, and evaluated the prognostic implications, primarily overall survival (OS) and survival after BCBMs, of PI3K activation status in BCBMs. As secondary, exploratory end points, we evaluated the associations between PI3K pathway activation and time to distant recurrence (TTDR) and time to BCBM. Finally, similar analyses were also conducted among the subset of patients with triple-negative BCBM.

BCBMs represent one of the most challenging aspects in the clinical care of patients with advanced BC. Not only does intracranial recurrence limit survival, but associated symptoms also decrease functional status, limit independence, and negatively affect quality of life. No approved systemic therapies are available to treat patients with BCBMs, and it is unclear whether therapeutic targets, such as PI3K, differ between primary BC and BCBMs. In the present study, we explored the expression and prognostic implications of a panel of PI3K-pathway biomarkers, p-AKT, p-S6, and PTEN, in 52 BCBMs and 12 matched primary BCs. Our central goal was to improve our current understanding of the complex biology underlying BCBMs in hopes of guiding the future use of targeted agents to treat this aggressive disease. Our results show that the PI3K pathway is active in most BCBMs, regardless of IHC subtype; however, activation status does not appear to affect overall survival or survival after BCBMs in this cohort of patients. Interestingly, our secondary analyses indicate that the lack of PTEN expression may have prognostic value, independent of subtype. Moreover, among patients with aggressive TN BCBM, lack of PTEN expression may also be associated with worse overall survival.

Although alterations of the PI3K pathway are frequently observed in primary BC [25], different mechanisms of pathway activation exist [26]. Among them, activating PIK3CA mutations have been identified in about 15% to 30% of breast tumors and are more commonly associated with ER+ disease [27‐31]. Conversely, alternative mechanisms of PI3K pathway activation, such as loss of PTEN and loss of the tumor-suppressor inositol polyphosphate 4-phosphatase type II (INPP4B), are more commonly associated with basal-like BC [27, 32‐37]. Although our data indicate that PI3K pathway activation in BCBM is not entirely subtype specific, lack of PTEN expression was more commonly observed in the TN and basal-like subtypes when compared with the other tumor types. Given that brain metastases across subtypes were included in this study, multiple mechanisms of PI3K activation (that is, PIK3CA mutations, PTEN loss, and/or INPP4B loss) may be responsible for the high levels of PI3K pathway activation observed in this cohort. Future studies aimed at identifying subtype-specific mechanisms of PI3K activation are certainly warranted, both in primary BC and BCBM, to refine our current understanding of the biologic processes driving this disease process.

The role of PI3K-pathway activation as a prognostic and/or predictive biomarker is under investigation. Although our primary analyses did not reveal associations between PI3K-pathway activation and overall survival or survival after BCBM, several secondary analyses are worthy of discussion. Our exploratory analysis indicates PTEN may be prognostic, with lack of PTEN expression being associated with more rapid time to disease recurrence (across subtypes) and worse overall survival in the TN subset of patients [38]. Interestingly, of the three biomarkers evaluated in this study, PTEN showed the highest concordance (83%) between matched primary BC and BCBM. In the clinical setting, biologic specimens from brain metastases are not commonly available, as resection is generally reserved for solitary lesions, and biopsies are reserved for cases with equivocal radiographic findings. Given the high concordance of PTEN status between primary BCs and their BCBMs, PTEN status in primary breast tumors may also be prognostic, and potentially predictive of distant and CNS recurrence. Confirming these findings in a large, unselected cohort of patients with primary breast tumor tissue available for PTEN testing would certainly be of value.

We recognize that the data presented in this study have several limitations. First, all patients included in this study underwent a neurosurgical procedure, so the population studied here might not be representative of all patients with BCBMs. Second, the sample size in this study is small, but comparable to previously reported studies evaluating BCBM tissues [39]. Although this is the largest study evaluating PI3K activation in BCBM tissues to date, subset analyses should be interpreted with caution because of the small sample size and multiple comparisons. A laudable future goal would be to validate these findings in a larger cohort; however, the inherent difficulty of obtaining brain metastasis tissues remains an obstacle. Thus, the development of clinically annotated brain metastases and primary BC tissue repositories housing both paraffin-embedded and fresh, frozen tissues should be a priority among the scientific community.

Finally, we used an IHC definition to identify the intrinsic molecular subtypes based on ER, PR, and HER2 status. We recognize that considerable discordance may exist between subtype assignment by IHC biomarkers and molecular profiling [40]. However, similar results were observed when we evaluated publicly available gene-expression data in more than 800 tumors in which molecular profiling had been performed [19, 20]. Overall, this genomic analysis supports our IHC findings, in which PTEN expression was associated with time to distant and brain recurrence, basal-like tumors, and the development of BCBM.

In summary, results of this study indicate that the PI3K pathway is active in the majority of BCBMs across the spectrum of IHC subtypes. Although expression of the PI3K pathway did not correlate with OS and survival after BCBM, loss of PTEN may hold prognostic and/or predictive value among this group of very high risk patients. Presently, small-molecule inhibitors of the PI3K pathway are in clinical development to treat multiple malignancies, including BC [41, 42], and several cross the blood-brain barrier. Thus, inhibition of the PI3K pathway represents a promising therapeutic strategy for patients with BCBMs, with the ultimate goal of improving outcome and quality of life for patients diagnosed with this devastating disease.