 Although there is a significant scientific rationale for targeting the PI3K pathway in breast cancer, research findings presented recently at the 2014 San Antonio Breast Cancer Symposium (SABCS) highlight the inherent complexities in using agents directed at this cell-signaling network. Given the extensive heterogeneity of breast cancer, the identification of patients most likely to respond to these drugs is becoming increasingly pressing and challenging.

These challenges were illustrated at SABCS in data from the phase II FERGI study, which evaluated the pan- PI3K inhibitor pictilisib (GDC-0941) in combination with fulvestrant in patients with metastatic breast cancer. The addition of pictilisib demonstrat- ed statistically significant progression-free survival (PFS) benefits only in the subset of patients with both estrogen receptor (ER)— and progester- one receptor (PR)–positive disease. Thus far, the most successful strategies in attacking the PI3K pathway have been aimed at the downstream mammalian target of rapamycin (mTOR). Yet, while mTOR inhibitors remain the only class of PI3K path- way-targeting agent to have attained regulatory approval, a number of drugs targeting PI3K and dual inhibitors of PI3K/mTOR are now in the late stages of clinical development in patients with breast cancer (Table 1).

The phosphatidylinositol-3 kinase pathway is the most highly dysregulated signaling network in breast cancer. It has also been implicated in development of resistance to other kinds of breast cancer therapy, most significantly endocrine therapy, human epidermal growth factor receptor 2 (HER2)—directed therapy, and chemotherapy, suggesting that agents directed at PI3K may be promising in counteracting drug resistance that limits current treatments. Mutations in the genes encoding the PI3K pathway are particularly common, making this an extremely attractive point for therapeutic intervention.

PI3Ks encompass a family of kinases that phosphorylate phosphoinositide lipids. Three classes and numerous subclasses have been identified, but the most relevant from an oncology perspective are class IA PI3Ks. Their kinase activity is switched on by receptor tyrosine kinases (RTKs) and, upon activation, they phosphorylate their phosphoinositide target, phosphatidylinositol-4,5-bisphosphate (PIP2), to form phosphatidylinositol-3,4,5-triphosphate (PIP3).

Ks. One of the best characterized is Akt, which in turn regulates the function of a broad range of proteins involved in integral cellular processes. A key downstream effector of Akt is mTOR, which is a master regulator of cell growth and metabolism. So important are these two proteins that the network is often referred to as the PI3K/Akt/mTOR pathway.

Cell survival, growth, proliferation, metabolism, motility, and adhesion are a small representation of the cellular functions controlled by PI3K and its downstream elements. PI3K and the phosphatase that opposes its action, phosphatase and tensin homolog (PTEN), are conductors of a cell-signaling symphony.

In breast cancer, aberrant activation of at least one component of this pathway can be observed in more than 70% of cases. Mutations in the genes that encode PI3K itself are common, observed in around 18% of patients. PI3K is a heterodimeric protein composed of a catalytic (p110) subunit and a regulatory (p50/p55/p85) subunit. Three isoforms of the catalytic subunit exist (a, β, and δ), which are encoded by three genes: PIK3CA, PIK3CB, and PIK3CD, respectively. The PIK3CA gene is particularly frequently altered in breast cancer (20%-25%). Mutations occur most often in the parts of the gene that encode the helical and kinase domains of the PI3K protein, and three “hotspots” have been identified—E542K, E545K, and H1047R&mdash;all of which produce a constitutively active kinase that drives oncogenesis.

The frequency of PIK3CA mutations varies considerably by type and subtype of breast cancer. PIK3CA mutations appear most common in patients with ER-positive or HER2-positive disease (30%-40%) and are much less prevalent in triple-negative breast cancer. Numerous other types of alterations in the PI3K/Akt/mTOR pathway have been implicated in the development of breast cancer, and substantial time and resources have been committed to developing drugs targeting almost every aspect of the pathway. As it stands now, everolimus (Afinitor) remains the only drug targeting this pathway that the FDA has approved in a breast cancer setting. The agency approved everolimus in 2012 as a treatment for postmenopausal women with advanced hormone receptor (HR)—positive, HER2-negative breast cancer in combination with exemestane after failure of treatment with the nonsteroidal aromatase inhibitors (AIs) letrozole or anastrozole.

Various inhibitors of the PI3K protein have now been developed, including pan-isoform inhibitors that target all class I PI3Ks, isoform-specific inhibitors, and, thanks to DNA sequence similarities between mTOR and PI3K, dual inhibitors of both enzymes.

The principal argument for pan-isoform inhibitors of PI3K is that most cancer cells express multiple isoforms of PI3K, which have redundant oncogenic activity. Although these agents continue to be evaluated in clinical trials, they have demonstrated modest activity to date, particularly as monotherapy.

The greatest enthusiasm has been for isoform-specific inhibitors; these agents should offer the advantage of less toxicity, thus allowing them to be tolerated at doses that could result in more complete and reliable inhibition of kinase activity. Dual PI3K/mTOR inhibitors also have shown impressive activity in breast cancer, with tolerable side effect profiles, and offer the advantage of a more complete blockade of the PI3K/Akt/mTOR pathway. Both isoform-specific PI3K inhibitors and dual PI3K/mTOR inhibitors are less advanced in clinical development. The most common adverse events observed with PI3K inhibitors as a class are gastrointestinal toxicity (including diarrhea, nausea, and vomiting), rash, hyperglycemia, and mood changes (anxiety, depression, hallucinations. and affective disorder). The latter have most frequently been reported with buparlisib (BKM120) and are thought to result from the ability of this drug to cross the blood-brain barrier. Rare cardiac events have also been reported with pictilisib.

While single-agent therapy with PI3K inhibitors has proved disappointing, combination therapy appears much more fruitful, and a wide variety of combinations are being clinically evaluated. In fact, evidence suggests that aberrations in the PI3K pathway may be a critical mechanism employed by tumors to enable them to become resistant to various different lines of established therapy in breast cancer, including endocrine therapy, HER2-targeted therapy, and chemotherapy. Thus, blocking the PI3K pathway in combination with other agents could prove extremely useful in counteracting drug resistance that often limits the utility of these standard breast cancer treatments. The most exciting recent developments have come from the combination of PI3K inhibitors and endocrine therapies.

The rationale supporting this combination stems from observations that PIK3CA mutations are significantly related to the expression of HRs—both ER and PR&mdash;and it is thought that PI3K signaling may stimulate transcriptional activation of these receptors. Thus, aberrant activation of the PI3K pathway may enable tumors to overcome repression of HR pathways by endocrine therapy. Since a high proportion of breast cancers express HRs, endocrine therapy represents an important class of breast cancer therapy and the development of resistance, a substantial challenge. So far, the only definitive clinical evidence for synergy between PI3K pathway-targeting agents and endocrine therapy comes from trials of mTOR inhibitors. The phase III BOLERO-2 study demonstrated the benefit of adding everolimus to an AI inhibitor in patients with HR-positive disease refractory to AI treatment.

PI3K inhibitors also are under investigation now in combination with endocrine therapies. Buparlisib is the most advanced in clinical development in this respect, and the results of the phase III BELLE trial are eagerly anticipated.

Swathes of data from early-phase clinical trials reported at SABCS for buparlisib and pictilisib, both pan-isoform inhibitors, and the PI3Ka-specific inhibitors taselisib (GDC-0032) and BYL719 showed the promising efficacy and tolerability of a number of these combinations (Table 2).

Unfortunately, the FERGI study, presented by Ian Krop, MD, PhD, a medical oncologist at the Dana- Farber Cancer Institute in Boston, demonstrated that there was no benefit from the combination of pictilisib and fulvestrant in patients with ER-positive, AI-resistant advanced/metastatic breast cancer.

With 8 therapies approved by the US Food and Drug Administration (FDA) for the treatment of patients with advanced renal cell carcinoma (RCC), practitioners are faced with the challenge of selecting the most appropriate therapies for their patients within this crowded therapeutic landscape.

However, an unplanned posthoc analysis showed that patients with tumors that were positive for both HRs did derive benefit from the combination, with a 56% reduction in the risk of PFS of 7.2 months with pictilisib versus 3.7 months for fulvestrant and placebo (P = .002). Further study is warranted.

Juric et al reported early results from a phase I study of BYL719 and letrozole in combination with LEE011, a cyclin-dependent kinase (CDK) 4/6 inhibitor, a combination considered worth exploring because activation of CDKs has also been implicated in resistance to AIs, and synergy between these agents has been observed in the preclinical setting. Results from the first two arms evaluating LEE011 in combination with letrozole and BYL719 in combination with letrozole demonstrated acceptable safety and preliminary efficacy. The study continues, and once the maximum tolerated dose or recommended phase II dose has been identified, patients will be enrolled in a third arm evaluating the triple combination.

Though clearly promising, the optimal strategy for targeting the PI3K pathway in breast cancer remains to be identified. What has become clear is that not all patients benefit, and methods for identifying those patients who are most likely to respond and the molecular mechanisms underlying those responses are urgently needed. This is particularly important since breast cancer is an extremely heterogeneous disease and activation of other pathways can ultimately lead to the development of resistance, which frequently occurs after treatment with PI3K inhibitors, as with other targeted therapies.

In preclinical studies, the biomarkers with the most significant potential to predict treatment efficacy and resistance include PIK3CA mutation and PTEN loss. In clinical studies, however, establishing the significance of PIK3CA mutations has proved challenging.

While some clinical trials have suggested that patients with PIK3CA mutations may derive more benefit from PI3K inhibitors than those without the mutations, the results of the FERGI study somewhat surprisingly demonstrated that PIK3CA mutation status had no effect on the efficacy of pictilisib when combined with fulvestrant.

Meanwhile, several studies have shown that loss of PTEN is a prominent mechanism of resistance to PI3K inhibition. A recent study published in Nature examined the genomic evolution of a patient with metastatic breast cancer with PIK3CA mutations who was treated with BYL719. The patient’s tumor initially responded well to BYL719 but ultimately developed resistance and metastases. The researchers sequenced material from 14 metastatic sites and compared the genetic profile with that of the pretreatment tumor sample. All of the samples that had become refractory to BYL719 had developed alterations that resulted in PTEN loss.

Tumors with PTEN deficiency have been shown to be more dependent on PI3Kβ activity; thus, combined inhibition with inhibitors of PI3Ka and PI3Kβ or use of pan-isoform inhibitors in these tumors may be more effective. Several preclinical studies looking at the antitumor effects of combining PI3Ka and PI3Kβ inhibitors in patients with PTEN loss demonstrated that this combination is synergistic and may reverse resistance.

In HER2-positive breast cancers or those with high levels of RTK signaling, resistance may occur through loss of feedback suppression on RTKs such as HER3 and insulin-like growth factor 1 receptor (IGF1R). Using inhibitors of these drugs in combination with PI3K inhibitors may be effective and, indeed, several phase I studies evaluating IGF1R and HER3 inhibitors in combination with BYL719 are ongoing.

			Ciruelos Gil EM. Targeting the PI3K/AKT/mTOR pathway in estrogen receptor-positive breast cancer [published online March 26, 2014]. Cancer Treat Rev. 2014;40(7):862-871.

			Grunt TW, Mariani GL. Novel approaches for molecular targeted therapy of breast cancer: interfering with PI3K/AKT/mTOR signaling. Curr Cancer Drug Targets. 2013;13(2):188-204.

			Juric D, Castel P, Griffith M, et al. Convergent loss of PTEN leads to clinical resistance to a PI(3)Ka inhibitor [published online November 17, 2014]. Nature. doi:10.1038/nature13948.

			Juric D, Hamilton E, Garcia Estévez L, et al. Phase Ib/II study of LEE011 and BYL719 and letrozole in ER+, HER2- breast cancer: safety, prelim¬inary efficacy and molecular analysis. Presented at: 2014 San Antonio Breast Cancer Symposium; December 9-13, 2014; San Antonio, TX. Abstract P5-19-24.

			Krop I, Johnston S, Mayer IA, et al. The FERGI phase II study of the PI3K inhibitor pictilisib (GDC-0941) plus fulvestrant vs fulvestrant plus placebo in patients with ER+, aromatase inhibitor (AI)-resistant advanced or metastatic breast cancer—part I results. Presented at: 2014 San Antonio Breast Cancer Symposium; December 9-13, 2014.; San Antonio, TX. Abstract S2-02.

			Lauring J, Park BH, Wolff AC. The phosphoinositide-3-kinase-Akt-mTOR pathway as a therapeutic target in breast cancer. J Natl Compr Canc Netw. 2013;11(6):670-678.

			Paplomata E, O’Regan R. The PI3K/AKT/mTOR pathway in breast cancer: targets, trials and biomarkers. Ther Adv Med Oncol. 2014;6(4):154-166.

Articles

Although there is a significant scientific rationale for targeting the PI3K pathway in breast cancer, research findings presented recently at the 2014 San Antonio Breast Cancer Symposium (SABCS) highlight the inherent complexities in using agents directed at this cell-signaling network.

SABCS Studies Highlight Promise and Complexity in Targeting PI3K in Breast Cancer

		Genomic analyses have shed new light on the molecular drivers of HER activity that can activate cancer-causing pathways. Examples are illustrated above.

		AR indicates amphiregulin; EGF, epidermal growth factor; HER, human epidermal growth factor receptor; HRG, heregulin; TGFα, transforming growth factor alpha; TK, tyrosine kinase.

		Source: Recreated from Chang JC. Dual HER2 blockade: with and without chemotherapy. Presented at: 29th Annual Miami Breast Cancer Conference; March 14-17, 2012; Miami Beach, FL.

At least 20% of patients with breast cancer have tumors that demonstrate amplification of the gene encoding the human epidermal growth factor receptor (HER)-2 protein, a receptor tyrosine kinase. The development of a monoclonal antibody targeting HER2, trastuzumab (Herceptin), has had a huge impact on the survival of this population of patients.

gene amplification guides clinical decision-making in breast cancer, and HER2-targeted drugs are only approved for use in patients designated HER2-positive. Yet new research suggests that these drugs may actually have much broader applications, benefiting patients who are not designated HER2-positive by routine testing, with far-reaching implications not just for the diagnosis and treatment of breast cancer, but for the understanding of the molecular drivers of cancers as a whole and the development of targeted therapies in general.

In 1998, trastuzumab became the first HER2-targeted therapy to be approved by the FDA for the treatment of advanced, metastatic, HER2-overexpressing breast cancer in combination with chemotherapy or as a single agent, based on pivotal clinical trials showing overall response rates of 45% and 14%, respectively. This was followed in 2006 by approval to treat early-stage HER2-positive breast cancer in combination with a chemotherapeutic regimen containing doxorubicin, cyclophosphamide, and paclitaxel, following studies demonstrating greater than 50% reduction in the risk of recurrence, second primary cancer, or death among patients treated with trastuzumab plus chemotherapy, compared with chemotherapy alone.

		Table 1. Approved HER2-Targeting Therapies for Breast Cancer

					• Adjuvant treatment with chemotherapy or as single agent after multimodality therapy

					• MBC with chemotherapy in first line or as single agent after chemotherapy

					• Advanced BC or MBC with capecitabine after prior therapy including trastuzumab

					• Postmenopausal women with HR+ MBC in combination with letrozole

					• MBC in combination with trastuzumab and docetaxel in patients with no prior anti-HER2 or chemotherapy for metastatic disease

					• MBC as single agent after prior trastuzumab and taxane-based therapy, or disease recurrence within 6 months of adjuvant therapy

Herceptin also is indicated for HER2+ gastric cancer.

		BC indicates breast cancer; HER2+, patients overexpressing HER2 based on FDA-approved testing; HR+, hormone receptor-positive; MBC, metastatic breast cancer.

In the years that followed its approval, trastuzumab has been joined by several other FDA-approved HER2-targeted agents (

). The clinical trials that were instrumental in gaining approval for these HER2-targeted agents indicated that their benefits were restricted to patients whose tumors exhibited overexpression of the HER2 protein or amplification of the 

gene. As such, administration of these drugs is dependent on the demonstration of a patient’s HER2-positive status, typically using the standard FDA-approved assaysâŽ¯fluorescence in situ hybridization, to evaluate the number of 

 genes in a cancer cell (gene amplification), and immunohistochemistry, to examine the level of HER2 protein present on the cancer cell surface (protein overexpression) (

). A third method, chromogenic in situ hybridization, also has been approved to evaluate gene expression.

A hint of controversy surrounding the use of HER2-targeted agents solely in HER2-positive patients first emerged in 2008 (Paik et al, 2008), when re-evaluation of tumors previously thought to be HER2-positive showed that many were in fact HER2-negative. This finding posed a significant conundrum, since patients who were posited to be unresponsive to HER2 treatment did, in fact, appear to benefit from trastuzumab treatment in the same way as their HER2-positive counterparts.

A number of studies are now being conducted to confirm the findings of these initial reports, challenging the notion that HER2-targeted therapies only work in HER2-positive breast cancer patients. These studies indicate that some patients whose tumors are HER2-negative, based on the standard FDA-approved assays, and who would therefore be ineligible for HER2-targeted therapy, may in fact be highly sensitive to this type of therapy. While the approved indications for HER2 therapy currently remain unchanged, one study quantifies the potential future benefit to expanding treatment to certain HER2-negative patients, suggesting that we may be able to help significantly more patients with breast cancer. Importantly, researchers are also providing possible molecular explanations for these findings.

				IHC HER2 protein expression of 1+ or 0

		CEP indicates chromosome enumeration probe; FISH, fluorescence in situ hybridization; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry.

 Hammond ME. Pitfalls and solutions in HER2/ER/PR testing. Presented at: 11th International Congress on the Future of Breast Cancer; July 26-28, 2012; Coronado, CA.

Molecular Mechanism #1: HER2-Activating Mutations

Researchers from the Siteman Cancer Center and The Genome Institute at Washington University in St. Louis, Missouri, analyzed data from eight breast cancer genome-sequencing studies (a total of 1500 patients) to further examine HER2 status. They identified a group of patients who did not have 

 gene amplification (thus giving them a HER2-negative designation, meaning they would not receive HER2-targeted therapy according to current guidelines) but whose tumors displayed mutations in the 

 gene that resulted in excess activity of the HER2 protein. They then went on to show that these activating mutations (G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C) rendered the tumors susceptible to the approved HER2 agents trastuzumab and lapatinib (Tykerb), as well as neratinib, an inhibitor of HER2 and the epidermal growth factor receptor (EGFR).

All of the activating mutations described in this study were sensitive to neratinib. Importantly, however, a nonactivating mutation, L755S, was discovered in 25% of patients with somatic mutations; it likely drives resistance to lapatinib, and was also sensitive to neratinib. This suggests that neratinib could be an important agent in helping to overcome resistance to other HER2-targeted agents.

Lead author, Ron Bose, MD, PhD, said in an email interview that the findings about the L755S mutation prompted investigators to make changes to a phase II trial of lapatinib with trastuzumab in HER2-negative patients with stage IV breast cancer that had been launched to examine their 

 genes for mutations and assess their response to anti-HER2 therapy. Now, neratinib has replaced lapatinib (NCT01670877). Bose and colleagues concluded that these newly identified mutations represent “an alternative mechanism to activate HER2 in breast cancer” and valid drug targets for breast cancer treatment. It is estimated that these mutations could be responsible for tumor growth in approximately 1.6% of cases, translating into some 4000 cases of breast cancer per year in the United States alone.

Molecular Mechanism #2: HER2-Positive Stem Cells

The cancer stem cell hypothesis is based on a growing body of evidence that suggests that tumor growth is initiated and maintained by a group of cells with similar properties to stem cells. Cancer stem cells (CSCs) are thought to be important drivers of metastasis, recurrence, and resistance to traditional cancer therapies such as chemotherapy.

Researchers at the University of Michigan Comprehensive Cancer Center in Ann Arbor and others have previously shown that the HER2 protein is an important regulator of CSCs in patients with HER2-positive breast cancer, driving self-renewal. More recently, the same research team used breast cancer cell lines, mouse xenograft models, and human breast cancer tissue samples to show that the same was also true in HER2-negative breast cancers. Since the proportion of CSCs within the entire tumor cell population is small (between 1% and 5%), the level of HER2 expression still falls below the threshold of assays used to determine a cancer patient’s HER2 status; however, because the CSCs overexpress HER2, they are still effectively targeted by HER2-directed therapies. This provides another explanation for the finding that HER2-negative patients respond to HER2-directed therapy; indeed, some of the clinical efficacy observed with trastuzumab may in fact relate to its ability to target the CSC population.

Another interesting finding from this study was that an increased level of HER2 expression was observed in bone metastases (a common site of breast cancer metastasis) in both mouse xenograft models and breast cancer patients. This phenomenon was not associated with 

 gene amplification, but instead seemed to be regulated by the bone microenvironment. Thus, patients with advanced metastatic cancer may appear to be HER2-negative by conventional testing methods, but may still respond to HER2-targeted therapies as a result of HER2 expression regulated by the bone microenvironment. Importantly, however, appropriate timing of trastuzumab administration was vital. When trastuzumab was administered to mice when bone metastases were small, a significant decrease in the growth of breast cancer cells was observed, while treatment that was initiated after palpable tumors had become established was limited in its efficacy to tumors with 

 gene amplification. These results suggest that, if administered to breast cancer patients before bone metastases develop, trastuzumab could help to prevent the spread of tumors.

	The role of HER2-directed therapies in targeting the CSC population is being further explored in a large, randomized clinical trial (NCT01424865) and could drive an entirely new paradigm for the development of adjuvant therapies focusing on the prevention of tumor recurrence by targeting CSCs.

						Afatinib, formerly known as BIBW 2992, is an oral, irreversible, small-molecule inhibitor of the ErbB tyrosine kinase family, which includes the HER receptors. It has shown activity as monotherapy in a phase II study of patients with HER2- positive metastatic breast cancer who progressed on trastuzumab, with 46% of 41 participating patients (19 patients) achieving a clinical benefit rate. Afatinib is being evaluated in several breast cancer trials, particularly a pivotal phase III trial in combination with vinorelbine versus trastuzumab plus vinorelbine. The studies are part of the broader LUX clinical trial program involving afatinib, which is pending FDA approval in non-small cell lung cancer.

						NCT01125566, NCT01271725, NCT01441596

						Neratinib, formerly known as HKI-272, is an oral, irreversible, receptor tyrosine kinase inhibitor that targets HER2 and HER1 (also known as the epidermal growth factor receptor [EGFR]). In an open-label, multicenter phase II trial of patients with HER2-positive metastatic breast cancer, response rates of >50% were observed with single-agent neratinib in trastuzumab-naïve patients and >20% in trastuzumab-pretreated patients. In a phase II study of patients previously treated with trastuzumab and a taxane, a combination of neratinib and capecitabine showed an overall response rate of 50%. Neratinib is also being evaluated in a phase II trial involving 29 patients whose tumors are HER2-negative in conventional testing but carry 

						NCT01670877, NCT00741260, NCT00398567, NCT00445458

						NeuVax, previously called E75, is a peptide vaccine derived from the extracellular domain of the HER2 protein and combined with the granulocyte macrophage colony-stimulating factor sargramostim (Leukine) to boost the immune system so that HER2-expressing cells are destroyed. The vaccine is aimed at preventing or delaying breast cancer recurrence in women with low levels of HER2 overexpression who would be considered HER2-negative, and are disease-free after standard therapies that include surgery, chemotherapy, and radiation. After phase I/II trials indicated a lower rate of recurrence among women who received the vaccine, investigators have launched the phase III PRESENT trial. Researchers are seeking to enroll 700 women. NCT01479244

New research suggests that HER2-targeted drugs may actually have much broader applications, benefiting patients who are not designated HER2-positive by routine testing.

By Jane de Lartigue, PhD

Articles

SABCS Studies Highlight Promise and Complexity in Targeting PI3K in Breast Cancer

Revisiting HER2: Recent Research Shakes Views on Famed Marker