PI3K pathway activation results in low efficacy of both trastuzumab and lapatinib

Human epidermal growth factor receptor 2 (HER2) is the most crucial ErbB receptor tyrosine kinase (RTK) family member in breast cancer with overexpression in about one fourth of patients [1]. Since HER2 plays a key role in HER2-positive breast cancer, these patients usually have bad prognosis, and HER2-related target drugs have been the foundation of treatment. Trastuzumab, a HER2 monoclonal antibody against the extracellular domain of the molecule, has been a new standard in neo-adjuvant, adjuvant and palliative treatment of HER2-positive breast cancer [1–3]. However, trastuzumab mono-therapy shows a response rate of no more than 30% in palliative setting [4], and there is still a problem of primary or acquired resistance even with combination regimens. HER2-overexpressing breast cancer cells are dependent on or "addictive" to the Phosphatidylinositol-3-kinase (PI3K) pathway [5]. Published literatures showed that PI3K pathway activation is associated with primary resistance to trastuzumab, and trastuzumab exerts its antitumor effects only in the presence of a normal PI3K pathway [6–11].

PI3K pathway is one of the most important signaling pathways in cell, which is involved in many fundamental cellular processes, including proliferation, cell survival, motility and cell growth [12, 13]. Class IA PI3K, the most important member of the PI3K complex, is composed of a heterodimer with a p85 regulatory subunit and a p110 catalytic subunit (PIK3CA), residing downstream of multiple receptor kinase families including ErbB RTK family (EGFR, HER2, HER3, HER4) and transducing signals originating from them [12, 13]. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a phosphotase that converts membrane-associated phosphatidylinositol 3,4,5-triphosphate (PIP3) back to phosphatidylinositol 4,5-bisphosphate (PIP2) and negatively regulates signaling transduction of PI3K pathway [14, 15]. It is well known that dysregulation of PI3K pathway plays an important role in the development of malignancy, and the most common genetic alterations in this pathway are PIK3CA mutation and PTEN loss [16, 17], both of which can lead to constitutive activation of PI3K pathway and resistance to trastuzumab [7]. PTEN-related resistance to trastuzumab can be reversed by combined treatment with trastuzumab and the PI3K inhibitor LY294002 [18]. Therefore, PI3K pathway activation resulting from PIK3CA mutation and/or PTEN loss warrants further studies.

Up to now, little knowledge is available about the correlation between PI3K pathway status and efficacy and resistance of the other FDA-approved anti-HER2 agent, lapatinib. Laptinib, a dual tyrosine kinase inhibitor of EGFR and HER2, binds to the intracellular kinase domain [19]. It has no cross-resistance with trastuzumab since it is effective against breast cancer expressing p95HER2 [20], an active truncated form of HER2 and with HER2 epitope masked by mucin 4 [21]. Clinical data have shown the safety and efficacy of lapatinib alone and in combination with capecitabine, paclitaxel and letrozole and it is still effective in patients who have progressed on trastuzumab [22–24]. Therefore, the HER2 pathway is still an "addictive" oncogenic pathway in breast cancer pretreated with trastuzumab. However, several recent papers touching on PI3K pathway activation and lapatinib resistance conflicted with each other [18, 25–29], so we conducted this study to explore their correlation and the protocol was approved by the Fudan University Shanghai Cancer Center Institutional review board on June 30, 2008.

PI3K pathway activation is the most common signal transduction pathway alteration in malignancies, including breast cancer [30]. It mostly results from PIK3CA mutation or amplification, PTEN loss [16]. Since the patients enrolled in this study were HER2-positive and high-level PIK3CA amplification without mutation is rare in this subpopulation [31, 32], so only PIK3CA mutation and PTEN loss were determined for the samples in our study.

Our data showed that PIK3CA mutation occurred in 12.3% of the patients, lower than 8-40% reported in other studies [33–38]. There are several reasons. First, only two exons accounting for approximately 85% of all mutations were determined in our study [33]. Second, mutation correlates with an old age and this phenomenon was confirmed by our study [39]. However, the median age of our patients was 49.0 years, about 10 years younger than Caucasian counterparts [39]. Third, the mutation was reported to occur more frequently in HER2-negative patients [39, 40], however, all patients in our study were HER2-positive.

Regarding mutations in hot spots, two common mutation points, H1047R and E542K were also present in our patients with no mutation of E545K observed [37, 39]. As to mutations in non-hot spots, two new points, L540F and T1052A mutations were first reported based on our knowledge. An analysis of our data showed that the ratio of hot spots to non-hot spots was 2.5 to 1, which is consistent with other reports [39]. Since there were only a few patients with the new mutation, our result needed further confirmation by other studies. Therefore, it remains a question whether the new mutation in non-hot spots results in an activation of PI3K pathway. As in other studies, these patients were considered to have a mutated gene in the analysis [39].

PTEN is a tumor suppressor gene, and can be down-regulated or lost of expression via deletion (common in breast cancer), mutation (rare in breast cancer), or promoter DNA methylation [14, 41, 42]. Loss of PTEN expression results in activation of PI3K pathway leading to development of cancer [15, 41]. PTEN loss is present in about one third of breast cancer patients, ranging from 15% to 48% [43–46]. Our study showed that the incidence of PTEN loss was 31.6%, which is consistent with other reported results.

Previous reports suggested that PIK3CA mutation and PTEN loss were mutually exclusive [37]. However, in 4 patients with H1047R mutations in our study, 3 patients were also found to have no PTEN expression. This fact was previously reported by Perez-Tenorio et al in 2009 [40]. PI3K mutation was indicated to be associated with ER positivity, HER2 negativity and primary tumor size, which were not observed in our study [37, 39, 40].

An analysis of our data showed that patients with PI3K pathway activation had a statistically significant shorter median PFS than those with no PI3K pathway activation (4.5 versus 9.0 months, P = 0.013), confirming the reported conclusion that PI3K pathway activation can result in resistance to trastuzumab [6–10]. Based on the published preclinical studies, these patients should be sensitive to lapatinib, a drug with a different mechanism of action [47]. There were some clinical data demonstrating that lapatinib induced objective responses in patients who had failed trastuzumab [22]. However, all patients were treated with lapatinib and capecitabine in our study, and PI3K pathway activation was still correlated with a lower clinical benefit rate (36.4% versus 68.6%, P = 0.017) and a lower overall response rate (9.1% versus 31.4%, P = 0.05), which is consistent with results of a smaller study reported by Cizkova et al [48]. Campone [49] et al pointed out that acquisition of resistance is frequently linked to an uncoupling between upstream signals emanating from HER2 itself and downstream signals related to PI3K, AKT and/or MAPK. Two studies showed that both knockdown of PTEN and transfection of mutant PIK3CA can result in lapatinib resistance and the mTOR/PI3K inhibitor, NVP-BEZ235 can reverse the resistance [26, 50].

However, there are also a few converse opinions. Based on the experimental results, O'Brien et al showed that lapatinib could overcome trastuzumab resistance via continued deactivation of PI3K/AKT/mTOR signaling [51]. A Japanese clinical study recruiting 122 patients attempted to illustrate the relationship between PI3K pathway activation and efficacy of lapatinib, but PIK3CA mutation was only found in 3 tissue samples among all 29 analyzed samples [52]. Recently, Toi et al indicated that low PTEN could predict response to lapatinib in a small phase 2 neoadjuvant trial [53]. Therefore, a definite conclusion regarding the PI3K pathway status and anti-HER2 therapy cannot be drawn up to now, and our study justifies further research.

It remains controversial whether the two gene alterations have any prognostic value. Li et al suggested that PIK3CA mutation was a negative prognostic factor [35]. On the contrary, a larger sample size study and a Japanese study indicated that it was a positive prognostic factor [36, 39]. Barbareschi [54] et al reported that mutation in exon 20 usually indicated good prognosis, while the mutation in exon 9 often meant bad prognosis. Perez-Tenorio [40] et al suggested that the two gene alterations should be combined with S phase fraction to give an accurate prediction of prognosis. Recently, Dupont Jensen [55] et al showed that there is a discrepancy of PIK3CA mutation between primary and metastatic tumors, urging on a simultaneous detection of the two matched samples. For the prognostic value of PTEN, it is relatively uniform and most investigators thought that PTEN loss is a negative prognostic factor [56, 57]. Our data showed that it was statistically associated with clinical benefit rate (P = 0.021). Due to a relatively smaller sample size of our study, no significant correlations between PI3K pathway status and clinicopathological parameters were found.

In conclusion, PIK3CA mutation occurs more frequently in elder patients and the ratio of mutations in hot spots to non-hot spots is about 2.5 to 1 in HER2-positive breast cancer patients. PTEN loss is present in about one third of patients. PIK3CA mutation and PTEN loss were not mutually exclusive. PI3K pathway activation may lead to drug resistance to lapatinib as well as trastuzumab.