mTOR Biology and the Growing Role for mTOR Inhibitors in the Management of Breast Cancer

Elisavet Paplomata, MD

Abstract

The PI3K/Akt/mTOR pathway stimulates protein synthesis, angiogenesis, and tumor proliferation and is an important target in multiple malignancies. Its activation has been implicated in resistance to endocrine agents, chemotherapy, and HER2-directed agents. The combination of PI3K/Akt/mTOR pathway inhibition with traditional or targeted agents may overcome this resistance.

Everolimus is approved for the treatment of advanced hormone receptor-positive breast cancer and is currently being evaluated in other breast cancer subtypes. Ongoing studies are also assessing biomarkers that will help define the patients who will derive the most benefit.

Introduction

The PI3K (phosphoinositide-3-kinase)/Akt (protein kinase B)/mTOR (mechanistic target of rapamycin) pathway responds to nutrient availability and growth factor tyrosine kinases. It plays a significant role in stimulating protein synthesis, angiogenesis and tumor proliferation, and inhibiting apoptosis.¹

The pathway starts with the PI3K unit, which comprises two subunits, the regulatory (p85) and the catalytic (p110) subunit; each has different isoforms and is encoded by its corresponding genes. The regulatory subunit is encoded by PIK3R1, PIK3R2, PIK3R3 and the p110α, p110β, and p110δ subunits are encoded by PIK3CA, PIK3CB and PIK3CD.² PI3K leads to the phosphorylation of Akt, a serine/threonine kinase, which activates the mechanistic (or mammalian) target of rapamycin (mTOR) by inhibiting the tuberous sclerosis complex proteins 1/2 (TSC1/2).^3-5 mTOR is a serine/threonine protein kinase that consists of complexes mTORC1 (mTOR Complex 1) and mTORC2 (mTOR Complex 2). mTORC1 is the main target of rapamycin analogs, while more recent data suggest that mTORC2 is also inhibited with sufficient dosing and exposure (Figure 1).^6,7

Everolimus is approved by the FDA (Food and Drug Administration) for the treatment of hormone receptor (HR)-positive breast cancer in postmenopausal women who had previously experienced disease progression or recurrence with a non-steroidal aromatase inhibitor, based on the BOLERO-2 trial.⁸

This review will focus on the current data of mTOR inhibitors in breast cancer. However, as the role of mTOR inhibition in breast cancer is evolving, ongoing studies are evaluating biomarkers that will identify the patients who will benefit.

The Role of mTOR Pathway in Breast Cancer

Aberrations of the PI3K/Akt/mTOR pathway have been implicated in the pathogenesis and growth of multiple malignancies. PIK3CA is the most common mutation found in breast cancer and is thought be oncogenic.^9-11

The mTOR pathway has been implicated in trastuzumab resistance in HER2-overexpressing breast cancer.^12,13 HER2-targeted therapy resistance has been associated with loss of PTEN, PI3K phosphorylation, and activation of mTOR.^14,15 mTOR inhibition can sensitize HER2-overexpressing breast cancer to HER2-directed therapy.¹⁶ In HR-positive breast cancer, the PI3K/Akt/mTOR signaling pathway can stimulate ERα (Estrogen Receptor Alpha) in both an estrogen-dependent and estrogen-independent manner^17,18; thus, activation of the mTOR pathway appears to be involved in endocrine resistance.^19-21 Preclinical data has demonstrated that inhibition of mTOR can restore hormone sensitivity, promote cell cycle arrest, and induce apoptosis when used in combination with endocrine therapy^.22-25 In triple negative breast cancer models, mTOR was also found to be activated and to mediate invasiveness and metastasis.^26,27

In conclusion, preclinical data demonstrate that activation of the mTOR pathway plays a role in the pathogenesis, promotion of invasiveness, and the development of resistance in various subtypes of breast cancer; this supports the further evaluation of mTOR inhibition in breast cancer, specifically in cancers that are resistant to standard therapies.

Clinical Data

Multiple clinical studies have evaluated the role of mTOR inhibition in breast cancer. Several landmark trials have shown its benefit especially in the HR-positive setting. Phase 3 clinical data have led to the approval of everolimus with exemestane in postmenopausal women with HR-positive, HER2-negative metastatic breast cancer.

HR-positive Breast Cancer

A phase 3 study that evaluated temsirolimus in combination with letrozole as a first-line treatment for HR-positive tumors (HORIZON) failed to show a significant improvement in progression-free survival (PFS) with mTOR inhibition, which was the primary endpoint and was closed for futility. The median PFS (9 and 8.9 months in the temsirolimus and placebo arm, respectively), objective response rate (ORR) (27% in both treatment groups), and clinical benefit rate (CBR) (17% and 19%, respectively) were similar in the temsirolimus and placebo arms, while more side effects were experienced by patients receiving temsirolimus.²⁸

The BOLERO-2 trial was a randomized, phase 3 trial of everolimus or placebo in combination with exemestane in postmenopausal women with advanced or metastatic HR-positive breast cancer; the eligible patients had disease relapse or progression on prior non-steroidal aromatase inhibitors (AI), such as anastrozole or letrozole.⁸ An interim analysis revealed that everolimus significantly improved the primary endpoint of PFS. The local assessment showed a PFS of 6.9 months in the treatment arm, versus 2.8 months in the placebo arm (HR = .43, P <.001); the central assessment showed a PFS of 10.6 months versus 4.1 months, respectively (HR = .36, P <.001).

An updated analysis with 18-month median follow-up showed continued benefit of mTOR inhibition versus placebo, 7.8 months versus 3.2 months, respectively (HR = .45, P <.0001) by investigator analysis, and 11 months versus 4.1 months, respectively (HR = .38, P <.0001) by central review.²⁹ The benefit was seen regardless of the presence of visceral metastases.³⁰ Although there were fewer deaths in the everolimus arm, compared to the placebo arm, the difference was not statistically significant.³¹ More adverse events were reported in the treatment arm and additionally, more patients in the everolimus arm discontinued treatment.^29,32,33 The biomarker analysis of BOLERO-2 reported that the efficacy of everolimus was independent of the genetic status of the tumors.³⁴

The TAMRAD trial was an open label, phase 2 study that evaluated everolimus plus tamoxifen in patients with HR-positive metastatic breast cancer. The CBR at 6 months, which was the primary endpoint, was significantly better with mTOR inhibition (CBR = 61% with everolimus versus 42% with tamoxifen alone, P = .045). Time to progression and risk of death were also superior in the everolimus group.³⁵

A neoadjuvant study by Baselga, et al evaluated everolimus with letrozole in postmenopausal women with operable HR-positive breast tumors.³⁶ Patients received letrozole and everolimus or letrozole and placebo for 4 months prior to definitive surgery. The primary endpoint, clinical response by palpation, was found to be significantly improved in the everolimus group. Everolimus also had a more significant effect on Ki67 down regulation. Tumors that harbor mutations in the PIK3CA exon 9 helical domain showed a relatively small antiproliferative response to letrozole alone but had a good response to the combination of everolimus and letrozole.^37,38

The HORIZON study data and the data reported with everolimus clearly contrast as noted above. This can be partly explained by the different patient population enrolled in these studies. Patients treated in the HORIZON trial were treated as first line for metastatic disease. About 60% were naïve to endocrine therapy.

The BOLERO-2 trial required prior exposure to a non-steroidal aromatase inhibitor and also required progression or recurrence during, within 1 month of metastatic treatment, or within 12 months of adjuvant treatment with these agents, thus implying aromatase inhibitor (AI) resistance.

The TAMRAD trial also showed that the mTOR inhibition benefitted patients with AI-resistant disease. Other possible explanations for the difference in results is the difference between everolimus and temsirolimus, agents with routes of administration, dosing and schedule. Finally, tumor biology and the presence of activating mutations might alter the efficacy of those agents, however not all studies have correlative data in order to draw a conclusion on tumor differences between trials.

HER2-amplified Breast Cancer

The possible role of PI3K pathway in mediating resistance to HER2-directed therapy was reported in a recent correlative study evaluating 504 tumor samples from patients that participated in the neoadjuvant GeparQuattro, GeparQuinto, and GeparSixto studies.³⁹ Overall, the rate of PIK3CA mutations in this HER2 amplified patient cohort was 21.4%. The presence of the mutation was associated with a significantly lower pathologic complete response (pCR) rate (19.4% versus 32.8% wild-type, P = .008). This was also shown in the HR-positive cohort (11.3% versus 27.5% wild-type, P = .011). Clinical outcomes were not significantly different based on the PIK3CA mutation status. These data are supported by the biomarker results of the Cleopatra study, which reported that median PFS was longer in patients with wild-type PIK3CA and patients with PIK3CA mutations had worse outcomes.⁴⁰

In early phase trials that accrued patients with HER2 amplified trastuzumab-resistant advanced breast cancer, the addition of everolimus to trastuzumab-based chemotherapy resulted in promising outcomes.^41,42 The overall response rate (ORR) was noted to be 44% when everolimus was added to paclitaxel plus trastuzumab and 19% when added to vinorelbine plus trastuzumab in 2 phase 1 trials.^41,42 A phase 2 study of everolimus in combination with paclitaxel and trastuzumab demonstrated an ORR of 21.8%, a CBR of 36.4%, a median PFS of 5.5 months, and an estimated overall survival (OS) of 18.1 months.⁴³

BOLERO-3 was a phase 3 study that evaluated everolimus (5 mg/day orally) in patients with advanced or metastatic HER2 overexpressing breast cancers resistant to trastuzumab.⁴⁴ Trastuzumab resistance was defined as having a recurrence within 12 months of adjuvant therapy or within five weeks of starting trastuzumab for metastatic disease. All patients had also been treated with a taxane and about half of the patients had HR-positive cancers. Enrolled patients received everolimus or placebo, plus vinorelbine and trastuzumab.

The trial showed that everolimus improved the PFS by local review, which was the primary endpoint, from 5.78 to 7 months, with an HR of 0.78 (P = .0067).⁴⁵ A central review and an adjudicated review, both of which were done retrospectively, adjusted the HR to 0.88 (95% CI 0.71—1.07) and 0.85 (95% CI 0.70–1.03), respectively. Subset analysis revealed that the positive signal with the addition of everolimus was noted in patients with HR-negative cancers, but not in HR-positive cancers, though this finding is only hypothesis-generating at this time. OS data from BOLERO-3 are currently still immature.

BOLERO-1 randomized patients with advanced HER2 overexpressing cancer to first-line paclitaxel, trastuzumab, and everolimus (10 mg/day orally), or placebo. The trial did not reach its primary endpoint of investigator-assessed improvement in PFS, even though there was a trend towards improved outcomes in patients with HR-negative cancer, this did not reach statistical significance.⁴⁶

The above studies indicate the need to further investigate the role of mTOR inhibition in HER2 amplified breast cancer, and especially in HR negative disease, a subset of patients where results are more promising.

Triple Negative Breast Cancer

Several clinical trials have evaluated mTOR inhibition in the triple negative setting.

A phase 2 neoadjuvant study in patients with triple negative breast cancer (TNBC) compared weekly paclitaxel for 12 weeks followed by FEC (fluouracil/epirubicin/cyclophosphamide), to the combination of everolimus (30 mg orally once a week) and paclitaxel, followed by FEC. The combination of paclitaxel and everolimus in this trial was well tolerated and more than 85% of patients completed treatment. Grade 3-4 toxicities included anemia, neutropenia, rash, and gastrointestinal side effects. However, this study used a weekly regimen of everolimus as opposed to the daily 5-10 mg oral regimen used in other studies. At 12 weeks, the response rates between the non-everolimus versus the everolimus arm were 29.6% versus 47.8% (P = .075), respectively, favoring everolimus, however the pCR rates were similar at 25.9% versus 30.4% (P = .76), respectively. The downregulation of mTOR by protein expression after 48 hours of treatment did not correlate with the response rates in the everolimus group.⁴⁷

The GeparQuinto trial combined everolimus with paclitaxel in TNBC patients in the neoadjuvant setting and found no difference in disease-free survival (DFS) (HR 0.997; P = .987) and OS (HR 1.11; P = .658) with the addition of everolimus.⁴⁸

A phase 2 study of neoadjuvant paclitaxel and cisplatin for twelve weeks with or without everolimus did not show any benefit in clinical response with the addition of everolimus and no significant improvement in pCR.⁴⁹

A phase 2 trial of carboplatin with daily oral everolimus in patients with TNBC demonstrated a 6-month CBR of 36% (95% CI 21.1 to 57.4%). Median PFS was 3 months (95% CI 1.6 — 4.6 months) and OS was 16.6 months (95% CI 7.3 months to not reached).50 These data demonstrate that the combination of everolimus with cytotoxic chemotherapy is possible and safe and may be worth exploring in future trials.

Adverse Events

The most common adverse events encountered with everolimus are stomatitis, rash, fatigue, diarrhea, cough, and hyperglycemia.^33,35,51,52 Stomatitis is one of the most common side effects, seen early during the course of treatment; it is very rarely life threatening and usually resolves with dose interruptions and reductions and supportive care.

Ongoing studies are evaluating steroid-based mouthwashes and pastes as a means to prevent everolimus-associated stomatitis. Fatigue was reported in about one third of patients in the BOLERO-2 trial and was easily managed with dose changes.⁵² Non-infectious pneumonitis is a less common side effect, seen in up to 20% of patients; it can be life threatening and therefore early detection and intervention are critical. Hyperglycemia and lipid abnormalities can be seen and managed supportively but can be clinically significant in elderly patients with risk factors for cardiac events.^52,53

Dose interruptions or reductions were required in 62% of patients receiving everolimus versus 12% in the placebo arm in the BOLERO-2 trial; additionally, the discontinuation rate was 26% in the treatment arm versus 9% in the placebo.⁵² The addition of everolimus to endocrine therapy is costly,⁵⁴ however, the cost of managing side effects of everolimus appears to be lower on average when compared to managing side effects of commonly used chemotherapy agents.⁵⁵

Biomarkers

Breast cancer is a heterogeneous disease and the identification of subsets of patients who will benefit from mTOR inhibition is essential, since the addition of PI3K/Akt/mTOR inhibitors may be associated with increased costs and toxicity.^52,54

Preclinical studies have demonstrated that elevated levels of phosphorylated S6 kinase (S6K), phosphorylated Akt, GSK3β, TSC2, and loss of inositol polyphosphate 4-phosphatase (INPP4B) can predict response to inhibitors of the PI3K/Akt/mTOR pathway.^56-61 Recently, it was reported that the presence of amplifications of AURKA and HER2 were associated with in vitro sensitivity to everolimus while PIKCA/PTEN mutations did not correlate.⁶² The presence of PIK3CA mutations, and specifically H1047R mutations, has also been associated with response.⁶³

However, clinical studies have failed to confirm this observation. Correlative studies from the TAMRAD trial revealed that PI3K mutations, PTEN, and pAKT did not have any affect in the response to mTOR inhibition; however, everolimus was more effective in patients with low PI3K expression, low LKB1, and high phospho-4E binding protein (p4-EBP1).⁶⁴ The BOLERO-2 trial failed to find biomarkers predictive of benefit from everolimus.⁶⁵

A clinical study by Ellard et al did not demonstrate an association between PTEN, HER2 and Akt levels and benefit from everolimus.⁶⁶

Resistance to mTOR Targeting

Resistance to mTOR inhibition is a great concern and can limit the use of mTOR inhibitors in clinical practice.⁶⁷ Multiple mechanisms can lead to resistance, including the increased activity of mTORC2 and insulin-like growth factor receptor-1 due to the inhibition of mTORC1, thus leading to the induction of Akt and Ser 473.^62,68 Other mechanisms include upregulation of the ERK/MAPK pathway, activation of the PIM kinases and MYC, PDK1 activation, dysregulation of p27 and production of VEGF.^69-73

The co-inhibition of PI3K/Akt and mTORC2 along with mTORC1 or the use of dual inhibitors can overcome these mechanisms of resistance. Several compounds with these properties are undergoing studies to evaluate their efficacy and safety in various settings. In addition, targeting angiogenesis and the mechanisms of autophagy and apoptosis may help overcome resistance.⁶⁷

Conclusion

Aberrant PI3K/Akt/mTOR activation by receptor tyrosine kinases (RTKs), ligand binding, or pathway mutations, are oncogenic and lead to cell survival and proliferation. Inhibitors of the mTOR pathway have a proven role in the management of multiple malignancies; in breast cancer, mTOR inhibitors may have a role in overcoming resistance to HER2-directed and endocrine therapy in breast cancer.

Everolimus is currently FDA approved for the treatment of HR-positive advanced breast cancer in postmenopausal women but is also the subject of undergoing studies in HER2-positive and triple negative disease.

As the role of mTOR inhibitors in breast cancer evolves, future efforts will focus not only on the evaluation of the efficacy of these agents but on identifying the patients who are more likely to benefit.

About the Authors:

Affiliations: Elisavet Paplomata, MD, Winship Cancer Institute of Emory University, Georgia Cancer Center for Excellence at Grady Memorial Hospital. Ruth O’Regan, MD, division of Hematology and Medical Oncology, University of Wisconsin.

Corresponding Author: Ruth M O’Regan, MD, Chief, division of Hematology and Medical Oncology, University of Wisconsin, 600 Highland Ave, K4/542 CSC, Madison, WI 53792; phone: 608-262-9638. E-mail: roregan@medicine.wisc.edu

Disclosures: The authors report no conflicts of interest.

References

1. Cantley LC. The phosphoinositide 3-kinase pathway. Science. 2002; 296(5573):1655-1657.
2. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006; 7(8): 606-619.
3. Tee AR, Fingar DC, Manning BD, et al. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc Natl Acad Sci U S A. 2002; 99(21):13571-13576.
4. Kenerson HL, Aicher LD, True LD, et al. Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res. 2002; 62(20):5645-5650.
5. Shioi T, McMullen JR, Kang PM, et al. Akt/protein kinase B promotes organ growth in transgenic mice. Mol Cell Biol. 2002; 22:2799-2809.
6. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell. 2006;22: 159-168.
7. Wander SA, Hennessy BT, Slingerland JM. Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy. J Clin Invest. 2011;121:1231-1241.
8. Baselga J, Campone M, Piccart M, et al. Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J Med. 2012;366:520-529.
9. Karakas B, Bachman KE, Park BH. Mutation of the PIK3CA oncogene in human cancers. Br J Cancer. 2006;94:455-459. 10. Bachman KE, Argani P, Samuels Y, et al. The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol Ther. 2004;3:772-775.
10. Isakoff SJ, Engelman JA, Irie HY, et al. Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cells. Cancer Res. 2005;65:10992-11000.
11. Berns K, Horlings HM, Hennessy BT, et al. A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell. 2007;12:395-402.
12. O’Brien NA, Browne BC, Chow L, et al. Activated phosphoinositide 3-kinase/AKT signaling confers resistance to trastuzumab but not lapatinib. Mol Cancer Ther. 2010;9:1489-1502.
13. Nagata Y, Lan KH, Zhou X, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell. 2004;6:117-127.
14. Wang L, Zhang Q, Zhang J, et al. PI3K pathway activation results in low efficacy of both trastuzumab and lapatinib. BMC Cancer. 2011;11:248.
15. O’Brien NA, McDonald K, Tong L, et al. Targeting PI3K/mTOR overcomes resistance to HER2-targeted therapy independent of feedback activation of AKT. Clin Cancer Res. 2014;20:3507-3520.
16. Sun M, Paciga JE, Feldman RI, et al. Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor alpha (ERalpha) via interaction between ERalpha and PI3K. Cancer Res. 2001;61:5985-5991.
17. Simoncini T, Hafezi-Moghadam A, Brazil DP, et al. Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature. 2000;407:538-541.
18. Campbell RA, Bhat-Nakshatri P, Patel NM, et al. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem. 2001;276:9817-9824.
19. Miller TW, Balko JM, Arteaga CL. Phosphatidylinositol 3-kinase and antiestrogen resistance in breast cancer. J Clin Oncol. 2011;29: 4452-4461.
20. Yamnik RL, Digilova A, Davis DC, et al. S6 kinase 1 regulates estrogen receptor alpha in control of breast cancer cell proliferation. J Biol Chem. 2009;284:6361-6369.
21. Chang SB, Miron P, Miron A, Iglehart JD. Rapamycin inhibits proliferation of estrogen-receptor-positive breast cancer cells. J Surg Res. 2007;138:37-44.
22. Ghayad SE, Bieche I, Vendrell JA, et al. mTOR inhibition reverses acquired endocrine therapy resistance of breast cancer cells at the cell proliferation and gene-expression levels. Cancer Sci. 2008;99: 1992-2003.
23. Beeram M, Tan QT, Tekmal RR, et al. Akt-induced endocrine therapy resistance is reversed by inhibition of mTOR signaling. Ann Oncol. 2007;18:1323-1328.
24. Boulay A, Rudloff J, Ye J, et al. Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer. Clin Cancer Res. 2005;11:5319-5328.
25. Khotskaya YB, Goverdhan A, Shen J, et al. S6K1 promotes invasiveness of breast cancer cells in a model of metastasis of triple-negative breast cancer. Am J Transl Res. 2014;6:361-376.
26. Montero JC, Esparis-Ogando A, Re-Louhau MF, et al. Active kinase profiling, genetic and pharmacological data define mTOR as an important common target in triple-negative breast cancer. Oncogene. 2014;33:148-156.
27. Wolff AC, Lazar AA, Bondarenko I, et al. Randomized phase 3 placebo-controlled trial of letrozole plus oral temsirolimus as first-line endocrine therapy in postmenopausal women with locally advanced or metastatic breast cancer. J Clin Oncol. 2013; 31: 195-202.
28. Yardley DA, Noguchi S, Pritchard KI, et al. Everolimus plus exemestane in postmenopausal patients with HR(+) breast cancer: BOLERO-2 final progression-free survival analysis. Adv Ther. 2013;30: 870-884.
29. Campone M, Bachelot T, Gnant M, et al. Effect of visceral metastases on the efficacy and safety of everolimus in postmenopausal women with advanced breast cancer: subgroup analysis from the BOLERO-2 study. Eur J Cancer. 2013;49: 2621-2632.
30. Piccart M, Hortobagyi GN, Campone M, et al. Everolimus plus exemestane for hormone-receptor-positive, human epidermal growth factor receptor-2-negative advanced breast cancer: overall survival results from BOLERO-2. Ann Oncol. 2014;25(12):2357-2362.
31. Piccart-Gebhart MJ, Pritchartd KI, Burris HA, et al. Everolimus for postmenopausal women with advanced breast cancer: updated results of the BOLERO-2 phase 3 trial. In ASCO Chicago: 2012. Abstract number: 559.
32. Pritchard KI, Burris HA, 3rd, Ito Y, et al. Safety and efficacy of everolimus with exemestane vs. exemestane alone in elderly patients with HER2-negative, hormone receptor-positive breast cancer in BOLERO-2. Clin Breast Cancer. 2013;13:421-432 e428.
33. Hortobagyi G, Piccart-Gebhart M, Rugo H, et al. Correlation of molecular alterations with efficacy of everolimus in hormone receptor—positive, HER2-negative advanced breast cancer: results from BOLERO-2. J Clin Oncol. 2013;31(Suppl.): Abstract LBA509.
34. Bachelot T, Bourgier C, Cropet C, et al. Randomized phase 2 trial of everolimus in combination with tamoxifen in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer with prior exposure to aromatase inhibitors: a GINECO study. J Clin Oncol. 2012;30:2718-2724.
35. Baselga J, Semiglazov V, van Dam P, et al. Phase 2 randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer. J Clin Oncol. 2009;27:2630-2637.
36. Dowsett M, Ebbs SR, Dixon JM, et al. Biomarker changes during neoadjuvant anastrozole, tamoxifen, or the combination: influence of hormonal status and HER-2 in breast cancer—a study from the IMPACT trialists. J Clin Oncol. 2005;23:2477-2492.
37. Dowsett M, Smith IE, Ebbs SR, et al. Prognostic value of Ki67 expression after short-term presurgical endocrine therapy for primary breast cancer. J Natl Cancer Inst. 2007;99:167-170.
38. Loibl S, von Minckwitz G, Schneeweiss A, et al. PIK3CA mutations are associated with lower rates of pathologic complete response to anti-human epidermal growth factor receptor 2 (HER2) therapy in primary HER2-overexpressing breast cancer. J Clin Oncol. 2014;32:3212-3220.
39. Baselga J, Cortes J, Im SA, et al. Biomarker analyses in CLEOPATRA: a phase 3, placebo-controlled study of pertuzumab in human epidermal growth factor receptor 2-positive, first-line metastatic breast cancer. J Clin Oncol. 2014;32:3753-3761.
40. Andre F, Campone M, O’Regan R, et al. Phase I study of everolimus plus weekly paclitaxel and trastuzumab in patients with metastatic breast cancer pretreated with trastuzumab. J Clin Oncol. 2010;28:5110-5115.
41. Jerusalem G, Fasolo A, Dieras V, et al. Phase I trial of oral mTOR inhibitor everolimus in combination with trastuzumab and vinorelbine in pre-treated patients with HER2-overexpressing metastatic breast cancer. Breast Cancer Res Treat. 2011;125:447-455.
42. Hurvitz SA, Dalenc F, Campone M, et al. A phase 2 study of everolimus combined with trastuzumab and paclitaxel in patients with HER2-overexpressing advanced breast cancer that progressed during prior trastuzumab and taxane therapy. Breast Cancer Res Treat. 2013;141:437-446.
43. O’Regan R, Ozguroglu M, Andre F, et al. Phase 3, randomized, double-blind, placebo-controlled multicenter trial of daily everolimus plus weekly trastuzumab and vinorelbine in trastuzumab- resistant, advanced breast cancer (BOLERO-3). J Clin Oncol. 2013;31(suppl; abstr 505).
44. Andre F, O’Regan R, Ozguroglu M, et al. Everolimus for women with trastuzumab-resistant, HER2-positive, advanced breast cancer (BOLERO-3): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Oncol. 2014;15:580-591.
45. Hurvitz SA, Andre F, Jiang Z, et al. Phase 3, randomized, double-blind, placebo-controlled multicenter trial of daily everolimus plus weekly trastuzumab and paclitaxel as first-line therapy in women with HER2+ advanced breast cancer: BOLERO-1. Presented at: 2014 SABCS; 2014; San Antonio, TX. Abstract: S6-01.
46. Gonzalez-Angulo AM, Akcakanat A, Liu S, et al. Open-label randomized clinical trial of standard neoadjuvant chemotherapy with paclitaxel followed by FEC versus the combination of paclitaxel and everolimus followed by FEC in women with triple receptor-negative breast cancerdagger. Ann Oncol. 2014;25:1122-1127.
47. von Minckwitz G, Loibl S, Untch M, et al. Survival after neoadjuvant chemotherapy with or without bevacizumab or everolimus for HER2-negative primary breast cancer (GBG 44 - GeparQuinto). Ann Oncol. 2014;25(12):2363-2372.
48. Mayer IA, Jovanovic B, Abramson VG, et al. A randomized phase 2 neoadjuvant study of cisplatin, paclitaxel with or without everolimus (an mTOR inhibitor) in patients with stage II/3 triple-negative breast cancer (TNBC). Cancer Res. 2013;73:PD1-6. Available at: http://www.abstracts2view.com/sabcs13/view.php?nu=SABCS13L_397.
49. Singh J, Novik Y, Stein S, et al. Phase 2 trial of everolimus and carboplatin combination in patients with triple negative metastatic breast cancer. Breast Cancer Res. 2014;16:R32.
50. Qiao L, Liang Y, Mira RR, et al. Mammalian target of rapamycin (mTOR) inhibitors and combined chemotherapy in breast cancer: a meta-analysis of randomized controlled trials. Int J Clin Exp Med. 2014;7:3333-3343.
51. Rugo HS, Pritchard KI, Gnant M, et al. Incidence and time course of everolimus-related adverse events in postmenopausal women with hormone receptor-positive advanced breast cancer: insights from BOLERO-2. Ann Oncol. 2014;25:808-815.
52. Paplomata E, Zelnak A, O’Regan R. Everolimus: side effect profile and management of toxicities in breast cancer. Breast Cancer Res Treat. 2013;140:453-462.
53. Diaby V, Adunlin G, Zeichner SB, et al. Cost-effectiveness analysis of everolimus plus exemestane versus exemestane alone for treatment of hormone receptor positive metastatic breast cancer. Breast Cancer Res Treat. 2014;147:433-441.
54. Campone M, Yang H, Faust E, et al. Cost of adverse events during treatment with everolimus plus exemestane or single-agent chemotherapy in patients with advanced breast cancer in Western Europe. J Med Econ. 2014;17(12):837-845.
55. Noh WC, Mondesire WH, Peng J, et al. Determinants of rapamycin sensitivity in breast cancer cells. Clin Cancer Res. 2004;10:1013-1023.
56. Noh WC, Kim YH, Kim MS, et al. Activation of the mTOR signaling pathway in breast cancer and its correlation with the clinicopathologic variables. Breast Cancer Res Treat. 2008;110:477-483.
57. O’Reilly T, McSheehy PM. Biomarker development for the clinical activity of the mTOR inhibitor everolimus (RAD001): processes, limitations, and further proposals. Transl Oncol. 2010;3:65-79.
58. Meric-Bernstam F, Akcakanat A, Chen H, et al. PIK3CA/PTEN mutations and Akt activation as markers of sensitivity to allosteric mTOR inhibitors. Clin Cancer Res. 2012;18:1777-1789.
59. Breuleux M, Klopfenstein M, Stephan C, et al. Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition. Mol Cancer Ther. 2009;8:742-753.
60. Bertucci MC, Mitchell CA. Phosphoinositide 3-kinase and INPP4B in human breast cancer. Ann N Y Acad Sci. 2013;1280:1-5.
61. Hurvitz SA, Kalous O, Conklin D, et al. In vitro activity of the mTOR inhibitor everolimus, in a large panel of breast cancer cell lines and analysis for predictors of response. Breast Cancer Res Treat. 2015;149:669-680.
62. Janku F, Wheler JJ, Naing A, et al. PIK3CA mutation H1047R is associated with response to PI3K/AKT/mTOR signaling pathway inhibitors in early-phase clinical trials. Cancer Res. 2013;73:276-284.
63. Treilleux I, Arnedos, M, Cropet, C, et al. (2013) Predictive markers of everolimus efficacy in hormone receptor positive (Hr+) metastatic breast cancer (Mbc): final results of the TAMRAD trial translational study. J Clin Oncol. 2013;31 (suppl; abstr 510).
64. Hortobagyi GN, Piccart-Gebhart M, Rugo HS, et al. Correlation of molecular alterations with efficacy of everolimus in hormone receptor—positive, HER2-negative advanced breast cancer: results from BOLERO-2. J Clin Oncol. 2013;31 (suppl; abstr LBA509).
65. Ellard SL, Clemons M, Gelmon KA, et al. Randomized phase II study comparing two schedules of everolimus in patients with recurrent/metastatic breast cancer: NCIC clinical trials group IND.163. J Clin Oncol. 2009;27:4536-4541.
66. Carew JS, Kelly KR, Nawrocki ST. Mechanisms of mTOR inhibitor resistance in cancer therapy. Target Oncol. 2011;6:17-27.
67. Chandarlapaty S. Negative feedback and adaptive resistance to the targeted therapy of cancer. Cancer Discov. 2012;2:311-319.
68. Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med. 2002;8:128-135.
69. Tan J, Lee PL, Li Z, et al. B55beta-associated PP2A complex controls PDK1-directed myc signaling and modulates rapamycin sensitivity in colorectal cancer. Cancer Cell. 2010;18:459-471.
70. Luo Y, Marx SO, Kiyokawa H, et al. Rapamycin resistance tied to defective regulation of p27Kip1. Mol Cell Biol. 1996;16:6744-6751.
71. Bihani T, Ezell SA, Ladd B, et al. Resistance to everolimus driven by epigenetic regulation of MYC in ER+ breast cancers. Oncotarget. 2015;6(4):2407-2420.
72. Beharry Z, Zemskova M, Mahajan S, et al. Novel benzylidene-thiazolidine-2,4-diones inhibit Pim protein kinase activity and induce cell cycle arrest in leukemia and prostate cancer cells. Mol Cancer Ther. 2009;8:1473-1483.