Expression and activation of PI3K pathway proteins in breast cancer cells
To assess PI3K signaling activity in the panel of breast cancer cells used for the present investigation, the levels of phosphorylated forms of AKT, S6 protein kinase 1 and S6 (indicators of PI3K signaling activation), and the expression of PI3K catalytic subunit isoforms, PTEN, AKT isoforms and mTOR were examined (Figure ). The panel included ER-positive breast cancer cells with activating PIK3CA mutations (helical domain mutation: MCF7 and BT-483; kinase domain mutation: T47D), PTEN mutation (MDA-MB-415, ZR75-1 and CAMA-1), HER2 gene amplification (HCC1419) or wild-type PIK3CA and PTEN (HCC712, HCC1428, HCC1500 and MDA-MB-175), and ER-negative breast cancer cell lines with HER2 amplification (SK-BR-3), and wild-type PIK3CA and PTEN (HCC1806). The ER-negative MDA-MB-231 cell line is wild-type for PIK3CA and PTEN but harbors mutations in K-RAS and B-RAF.
Figure 1 Analysis of phosphatidylinositol-3-kinase pathway signaling in breast cancer cells. Cell lines were grown to subconfluency and placed in medium containing low (0.5%) fetal bovine serum overnight prior to preparation of cell lysates. Equal amounts (25 (more ...)
While the PI3K p110α and p110β catalytic subunits were present in all cell lines, the PI3K p110δ and p110γ catalytic subunits were significantly expressed only in ER-negative (SK-BR-3, HCC1806 and MDA-MB-231) cell lines. Akt1 and Akt2 were expressed in all tested breast cancer cell lines, but Akt3 was detectable only in MDA-MB-231 cells [9
]. Consistent with previous studies, high levels of p-Akt were present in cells with PIK3CA
kinase domain mutation (T47D), PTEN
mutation (MDA-MB-415, ZR75-1 and CAMA-1), HER2
amplification (HCC1419, SK-BR-3) [9
] and the heregulin-dependent MDA-MB-175 cell line. Phosphorylation of the PI3K downstream target S6 closely paralleled Akt phosphorylation.
These data indicate that mutations in PIK3CA and PTEN or amplification of HER2 are associated with PI3K pathway activation in breast cancer.
BGT226, BKM120 and RAD001 inhibit PI3K pathway signaling in breast cancer cells
There are at least four general subcategories of PI3K pathway inhibitors, based upon target specificity, that are currently in clinical use or in various phases of clinical testing. These include inhibitors of PI3K catalytic subunits; inhibitors of the Akt serine-threonine kinase; inhibitors of mTOR; and multi-targeted agents, which typically have dual-specificity PI3K and mTOR kinase inhibitors [12
]. This paper focuses on three of these four classes of agent: RAD001 (inhibitor against rapamycin-sensitive mTOR complex), BKM120 (inhibitor against PI3K catalytic isoforms) and BGT226 (dual inhibitor of PI3K/mTOR).
To illustrate the inhibitory activities of BGT226, BKM120 and RAD001 on PI3K pathway signaling, the phosphorylation levels of Akt (p-Akt) and S6 (p-S6) were assessed by western blotting in MDA-MB-231, MCF7, T47D, or HCC712 cell lines in the presence of increasing dose of drug. As expected, BGT226 and BKM120 inhibited the phosphorylation of both Akt and S6 in all tested lines (Figure ). BGT226 treatment produced almost complete inhibition of PI3K signaling at low nanomolar (50 nmol/l) concentrations, indicating a similar, or greater, potency compared with that of the dual PI3K/mTOR inhibitor BEZ235 [5
]. In contrast, significant inhibition of PI3K signaling following BKM120 treatment occurred in the mid-nanomolar to high-nanomolar concentration range (250 to 1,000 nmol/l) in most cell lines. In all cell lines, RAD001 treatment completely inhibited S6 phosphorylation at low nanomolar (5 nmol/l) concentrations, with the paradoxical increase in Akt phosphorylation MCF7 cells already noted by other investigators (Figure ) [14
Figure 2 BGT226, BKM120 and RAD001 inhibit phosphatidylinositol-3-kinase pathway signaling in breast cancer cells. Western blots showing effects of (a) BGT226, (b) BKM120 and (c) RAD001 dose escalation on p-Ser473 Akt (p-Akt) and p-Ser235/236 S6 (p-S6) in breast (more ...)
These data indicate that PI3K pathway inhibitors effectively suppressed their respective targets regardless of individual differences in PI3K pathway mutation status.
PIK3CA mutation sensitizes short-term estrogen-deprived ER-positive breast cancer cells to PI3K pathway inhibitors
To extend our previous observations regarding the sensitizing effect of estrogen deprivation on the apoptotic effect of PI3K pathway inhibitors in ER-positive breast cancer [5
], a larger panel of ER-positive breast cancer cell lines was examined that varied with respect to PIK3CA
mutation status (Figure ). Cells in the panel were acutely deprived of estrogen for 1 to 3 weeks prior to treatment with BGT226, BKM120 or RAD001 at concentrations that were found to be sufficient to abrogate pathway signaling (Figure to ). The MDA-MB-231 line served as a control for off-target inhibitor effects since this line does not undergo apoptosis when treated with the dual PI3K/mTOR inhibitor BEZ235 [5
] or combined siRNA knockdown of PIK3CA
Figure 3 PIK3CA mutation and phosphatase and tensin homolog loss confer sensitivity to phosphatidylinositol-3-kinase pathway inhibitors. PIK3CA mutation and phosphatase and tensin homolog (PTEN) loss confer sensitivity to phosphatidylinositol-3-kinase pathway (more ...)
Induction of apoptosis was measured by TUNEL assay after treatment with BGT226 (50 nmol/l), BKM120 (1 μmol/l) or RAD001 (30 nmol/l) (Figure to ). In the absence of estrogen, BGT226 treatment induced the highest levels of apoptosis, followed by BKM120, whereas RAD001 treatment produced only a modest increase in apoptosis in a few cell lines (Figure to ), suggesting this class of agent may be a relatively ineffective partner for endocrine therapy combinations. Importantly, we observed that the induction of high levels of apoptosis by both BGT226 and BKM120 was restricted to PIK3CA mutant lines (MCF7, T47D and BT-483) and the PTEN-negative MDA-MB-415 and ZR75-1 cell lines. BGT226 treatment also produced a significant but modest increase in apoptosis in the HCC1428 line (wild-type PIK3CA and PTEN) and the PIK3CB-amplified HCC712 cell line, compatible with this agent having the broadest inhibitory activity. Sensitivity to PI3K pathway inhibition and the presence of a pathway mutation, however, were not linked in all lines because PTEN mutant CAMA-1 cells were resistant to BGT226 and BKM120 (Figure ) despite effective inhibition of PI3K pathway signaling (data not shown). Interestingly, the absence of ERK1/2 phosphorylation (Figure ) in CAMA-1 argues against the activation of the ERK pathway as a mechanism of resistance. The effect of RAD001 on apoptosis was modest overall, but two of the three cell lines in which RAD001 induced apoptosis (MCF7, BT-483) contain PIK3CA helical domain mutations.
Taken together, these data indicate that dual PI3K/mTOR and PI3K isoform inhibitors are likely to produce the greatest effects in ER-positive breast cancer, particularly in tumors harboring PIK3CA mutation and, possibly, PTEN loss.
As a complementary approach for measuring relative drug sensitivity, the IC50 and LC50 values were calculated for all three inhibitors in the cell line panel under estrogen-deprived conditions (Table ). Consistent with TUNEL assay results, LC50 values in the low nanomolar per liter range were obtained in the PTEN-negative MDA-MB-415 and ZR75-1 lines and in the three PIK3CA mutant (MCF7, T47D, BT-483) cell lines. The LC50 values for BKM120 were higher than for BGT226, which is consistent with the higher concentration of BKM120 needed to inhibit PI3K signaling in cell lines (Figure ). As expected, BKM120-sensitive cell lines identified by TUNEL generally exhibited lower LC50 values. Although the LC50 value for RAD001 was attained in HCC1428 cells, we did not observe any induction of apoptosis by TUNEL assay (Figure ). Regardless, the data for IC50 and LC50 were mostly consistent with results obtained from TUNEL assays.
Determination of LC50 and IC50 values for BGT226, BKM120 and RAD001 in breast cancer cells
Estradiol inhibits BGT226 and BKM120 treatment-induced apoptosis but in a cell-line-dependent manner
We have previously shown that estradiol significantly suppressed the induction of apoptosis by inhibition of p110α and p110β by RNA interference or treatment with the dual PI3K/mTOR inhibitor BEZ235 in ER-positive MCF7, T47D and HCC712 cells [5
]. To determine whether estradiol broadly inhibits apoptosis induced by other PI3K inhibitors and in other ER-positive cell lines, the effect of BGT226 was compared in the presence and absence of estradiol. While estradiol suppressed BGT226-induced apoptosis in STED MCF7 and T47D cells, estradiol had no effect on PI3K inhibitor-induced apoptosis in BT-483, MDA-MB-415 and ZR75-1 cells (Figure ). Treatment with estradiol induced proliferation in these lines, however, suggesting that the ER was functional ([5
] and data not shown). Dose escalation of BGT226 (Figure ) and BKM120 (Figure ) in MCF7 and T47D cells demonstrated that inhibition of cell death by estradiol was progressively lost at higher PI3K inhibitor concentrations. The modest increase in apoptosis with RAD001 treatment in STED MCF7 cells (Figure ) was also suppressed by estradiol (data not shown).
Figure 4 Estrogen suppresses induction of apoptosis by phosphatidylinositol-3-kinase pathway inhibitors. (a) Estradiol suppresses induction of apoptosis by BGT226. Cells cultured without or with 10 nmol/l 17β-estradiol (E2) were treated with 10 nmol/l (more ...)
Overall, these data suggest estradiol-induced resistance is a shared characteristic across all three classes of PI3K pathway inhibitors tested, but there is marked heterogeneity in the inhibitory effect of estradiol across ER-positive breast cancer cell lines.
BGT226, BKM120 and RAD001 inhibit PI3K pathway signaling despite long-term estrogen deprivation
To model the effects of PI3K pathway inhibition in aromatase-inhibitor-resistant breast cancer cells, variants of the MCF7 and T47D lines were generated through LTED by over 9 months of culture in low-estrogen conditions (Figure ). ER upregulation and increased phosphorylation of Akt, S6 and the MAPK/ERKs (p-ERK) was observed in MCF7 LTED cells compared with the parental line. In the T47D LTED line, S6 and ERK phosphorylation, but not p-Akt, was higher than in parental T47D cells, and ER expression was downregulated to undetectable levels.
Figure 5 Inhibition of phosphatidylinositol-3-kinase pathway signaling in estrogen-independent long-term estrogen-deprived cells. (a) Western blot showing estrogen receptor (ER) expression and levels of p-Akt, p-S6 and p-ERK1/2 in estrogen-deprived parental MCF7 (more ...)
Both LTED lines were subsequently retreated with estradiol (10 nmol/l) for at least 4 months to determine whether estradiol re-exposure could reverse the signaling effects associated with LTED. In the resulting MCF7 revertant subline (MCF7 LTED-R), ER expression and levels of p-Akt, p-S6 and p-ERKs were downregulated to similar levels observed in the parental MCF7 cells, indicating that prolonged estradiol re-exposure reversed the effects of LTED on these proteins. In contrast, while S6 and ERK phosphorylation were downregulated by estradiol in T47D LTED-R cells, ER expression levels were not restored - at least not to a level detectable by western blot. The effect of the three PI3K pathway inhibitors on signal transduction demonstrated that the dose-response relationships for all three agents were similar to those observed in the parental MCF7 and T47D cell lines (Figure ). The sensitivity of the LTED lines to estradiol and fulvestrant was also determined. As expected, proliferation of MCF7 LTED and T47D LTED cells was not enhanced by increasing concentrations of estradiol (Figure ). Indeed the MCF7 LTED model was paradoxically inhibited by estradiol because 10 nmol/l treatment for >10 days inhibited growth and induced cell death [18
] (data not shown). Treatment of estrogen-deprived MCF7 LTED with the ER-selective inhibitor fulvestrant [20
] inhibited the growth of cells, demonstrating that ER remains functionally important for the growth of these cells despite the absence of supplemental estradiol. In contrast, treatment with estradiol or fulvestrant did not have significant effects on the growth of ER-negative T47D LTED cells (Figure ).
Long-term estrogen-deprived cells are resistant to the induction of apoptosis by low-dose PI3K pathway inhibitors
To determine the effect of LTED on PI3K drug sensitivity, we compared the ability of BGT226 and BKM120 to induce apoptosis in STED and LTED cell line pairs. In comparison with MCF7 and T47D STED cells, higher drug concentrations were required for both BGT226 (Figure ) and BKM120 (Figure ) to induce significant apoptosis under LTED conditions. The LC50 values for BGT226 in both LTED lines, and for BKM120 in T47D LTED cells, were consistent with resistance to apoptosis measured by TUNEL (Table ). At the highest doses of BKM120 and BGT226 tested, however, T47D LTED cells were more sensitive than STED T47D cells; this pattern was not replicated in MCF7 LTED cells, where resistance to BGT226 persisted at all of the doses tested.
Figure 6 Long-term estrogen-deprived cells are resistant to the induction of apoptosis by phosphatidylinositol-3-kinase pathway inhibitors. Estrogen-deprived cells were treated with the indicated concentrations of (a) BGT226 or (b) BKM120 and apoptosis was measured (more ...)
Despite resistance to the proliferative effects of estradiol, acute treatment with estradiol suppressed apoptosis induced by BGT226 and BKM120 treatment in MCF7 LTED cells -indicating that the survival effects of estradiol were decoupled from mitogenic effects (Figure ). In contrast, estradiol did not suppress BGT226-induced or BKM120-induced apoptosis in ER-negative T47D LTED cells.
Treatment with fulvestrant sensitizes MCF7 LTED cells to PI3K inhibition
To model options for patients with disease progression on aromatase inhibitor treatment, the effect of fulvestrant was studied in LTED lines. Fulvestrant alone did not promote apoptosis in STED cells or LTED cells (Figure ); fulvestrant strongly potentiated apoptosis when combined with BGT226, BKM120 and RAD001 treatment in MCF7 LTED cells, however, confirming that ligand-independent ER activity promoted PI3K inhibitor resistance (Figure ). In contrast, treatment with fulvestrant did not promote apoptosis in the ER-negative T47D LTED cells with any of the three agents tested.
Figure 7 Fulvestrant and estradiol treatment sensitize long-term estrogen-deprived cells to phosphatidylinositol-3-kinase inhibitors. Estrogen-deprived (a) MCF7 and MCF7 long-term estrogen deprivation (LTED) cells or (b) T47D and T47D LTED cells were treated with (more ...)
Taken together, these data suggest that fulvestrant may sensitize cells to the therapeutic effects of PI3K inhibitors under circumstances where resistance to estrogen deprivation is associated with ligand-independent ER activity.
Prolonged retreatment with estradiol re-sensitizes MCF7 LTED cells to PI3K inhibition
As an alternative to fulvestrant, breast cancer patients with advanced ER-positive aromatase-inhibitor-resistant disease can be treated with low-dose estradiol to induce tumor regression and, in some instances, resensitize the patients' tumor to estrogen deprivation therapy with an aromatase inhibitor [21
]. The MCF7 LTED line provides an in vitro
parallel of these clinical findings because, when these cells are re-exposed to estradiol, cell growth slows dramatically, followed by a period of recovery during which cell growth once again becomes estrogen dependent (MCF7 LTED-R) (data not shown).
To determine whether MCF7 LTED-R cells also recovered sensitivity to PI3K inhibition, the effects of BGT226, BKM120 and RAD001 treatment were compared between MCF7 LTED-R cells and MCF7 LTED cells (Figure ). Consistent with partial recovery of sensitivity to PI3K inhibition, lower doses of BGT226 were able to induce apoptosis in estrogen-deprived MCF7 LTED-R cells in comparison with MCF7 LTED cells (Figure ). In contrast, the levels of cell death with BKM120 (1 μmol/l) were similar in all three MCF7 cell line variants (Figure ) and sensitivity to RAD001 was lost in MCF7 LTED-R cells despite reintroduction of estrogen deprivation.
PIK3CA mutations are common in relapsed ER-positive breast cancer
The in vitro
studies described above suggested that a combination of fulvestrant and a PI3K pathway inhibitor may be an effective approach for aromatase-inhibitor-resistant advanced breast cancer, particularly in PI3KCA
mutant cases that are persistently ER-positive at relapse. Since PIK3CA
mutation has been reported to be associated with a more favorable prognosis [7
], however, it was unclear how many patients with ER-positive PIK3CA
mutant breast cancer would present with advanced disease. Fresh-frozen research biopsies were therefore obtained from 51 patients with recurrent or metastatic disease for PIK3CA
mutation testing (Table ). Their median age at initial cancer diagnosis was 53.4 (32.3 to 79.9) years. The median follow-up was 51.7 (0.9 to 256.7) months. Forty-three out of the 51 (84.3%) patients were deceased at the time of analysis. At initial diagnosis, 32 tumors were ER-positive, 17 tumors were ER-negative, and two tumors were of unknown status. Five out of the 32 ER-positive tumors changed to ER-negative status at recurrence.
Clinical characteristics of the 51 recurrent or metastatic breast cancers
mutation analysis was performed on the 27 ER-positive and 24 ER-negative recurrent specimens. We included both ER-positive and ER-negative cases to interrogate the relationship between PIK3CA
mutation and ER status in the recurrent disease population. A PIK3CA
mutation was identified in 16 of the 51 tumors (31.4%; eight in the helical domain, eight in the kinase domain), a prevalence similar to that observed in studies that examined primary breast cancer tissue [6
mutation was strongly associated with ER positivity (P
= 0.0076). Among the 27 ER-positive tumors, 13 (48%) were PIK3CA
mutant. In contrast, only three of the 24 ER-negative tumors were PIK3CA
mutant. ER expression was maintained in 13 out of 14 cases with PIK3CA
mutation (Table ). Consistent with previous reports [7
mutation was associated with a later relapse pattern (disease-free survival P
= 0.02, Figure ), with a trend for patients with PIK3CA
mutant disease exhibiting a lower mortality rate (overall survival P
= 0.06, Figure ). In an analysis restricted to patients with initially ER-positive disease, PIK3CA
mutant cases still relapsed later than nonmutant cases (disease-free survival P
= 0.02, Figure ). Survival after relapse in persistently ER-positive tumors (a potentially important endpoint for drug approval), however, was not different between PIK3CA
wild-type and mutant cases, although the very small sample size meant that only very large effects could have been detected (Figure ).
Figure 8 PIK3CA mutation associates with superior disease-free survival. (a) Kaplan-Meier curves for disease-free survival (DFS) based on PIK3CA mutation status in all patients. (b) Kaplan-Meier curves comparing overall survival (OS) in all patients with PIK3CA (more ...)
The primary aim of the present study was to assess the case for combined targeting of ER and PI3K pathway inhibition by examining an extended panel of ER-positive breast cancer cell lines using clinical grade PI3K and ER pathway inhibitors. Conclusions focused on the induction of apoptosis because the ability of PI3K inhibitors to induce cell death, rather than inhibit cell proliferation, is considered to be the best predictor of in vivo
anti-tumor response [17
]. The dual PI3K/mTOR inhibitor BGT226 generally produced the highest levels of apoptosis when combined with estrogen deprivation in sensitive cells, followed by the PI3K isoform selective inhibitor BKM120. In contrast, the level of apoptosis induced by the mTOR-selective inhibitor RAD001 in estrogen-deprived cells was modest by comparison, even in the most sensitive cells. Poor induction of apoptosis by RAD001 in estrogen-deprived ER-positive cells is consistent with the results of a randomized phase 2 trial (NCT00107016) that evaluated the efficacy of the aromatase inhibitor letrozole and RAD001 as neoadjuvant treatment for ER-positive breast cancer. Despite greater inhibition of tumor proliferation, the pathological complete response rate was not increased by RAD001 over that observed using letrozole alone - suggesting no clinically significant increase in cell death was achieved [23
]. Our data suggest that if tolerable at active doses, direct inhibitors of PI3K might be more effective in this setting.
The sensitizing effect of PIK3CA
mutation to the dual PI3K/mTOR inhibitor BEZ235 and to a selective Akt inhibitor in breast cancer cells has already been reported [9
]. These studies included few PIK3CA
wild-type ER-positive HER2-negative cells, however, and it was not clear how PIK3CA
mutation impacts PI3K inhibitor sensitivity in the setting of estrogen deprivation. Our data support the conclusion that PIK3CA
mutation confers sensitivity to PI3K pathway inhibitors in the setting of new agents in clinical development and that this differential effect is maintained under estrogen-deprived conditions. However, the impact of estradiol on PI3K pathway inhibitor activity in PIK3CA
mutant cells was not uniform. Estradiol suppressed apoptosis induced by BGT226 in MCF7 and T47D cells but not in BT-483 cells. The identification of additional biomarkers will probably therefore be necessary to fully predict the efficacy of PI3K/endocrine combination therapy in PIK3CA
mutant ER-positive tumors. Consistent with previous reports, the effect of PTEN
mutation on the sensitivity of ER-positive cells to PI3K inhibitors also appears complex [9
]. Whereas the PTEN-negative MDA-MB-415 and ZR75-1 lines were sensitive to both BGT226 and BKM120, the CAMA-1 line, which is PTEN mutant but does express low amounts of PTEN, was resistant to both inhibitors. The reasons for the inconsistent effects of PTEN deficiency on PI3K pathway inhibitor sensitivity in ER-positive cells will also require further study.
Estradiol is thought to prevent apoptosis through plasma-membrane-initiated or nongenomic signaling by the ER through activation of the PI3K and MAPK pathways [24
]. Consistent with these reports, our results indicate that transduction of the estradiol survival signal increases PI3K inhibitor dose requirements in some ER-positive breast cancer cells (for example, MCF7 and T47D cells) but not others (BT-483, MDA-MB-415 and ZR75-1 cells). Interestingly, our results also show that the anti-apoptotic activity of estradiol is preserved in breast cancer cells that do not require estradiol for proliferation as a consequence of prolonged estrogen deprivation (Figure ). The decoupling of the proliferative and anti-apoptotic effects of estrogen suggests that continuing estrogen deprivation in progressing patients and adding a PI3K inhibitor might be a strategy worth testing.
The optimal endocrine combination with PI3K inhibition in cells resistant to estrogen deprivation is a critical consideration since the overwhelming majority of patients with advanced breast cancer have already been treated with an aromatase inhibitor in the adjuvant setting. Treatment options include an anti-estrogen (such as the ER downregulator fulvestrant) [26
] or therapy with low-dose estradiol [21
]. We modeled these second-line approaches in contrasting LTED cell lines, one where ER expression was maintained and one where it was lost, in order to reflect the clinical observation that upon disease progression ER is downregulated in a proportion of cases [27
]. Both LTED lines were found to be relatively resistant to PI3K inhibitors compared with the parental lines, consistent with reports that acquiring the ability to grow in the absence of estrogen is associated with increased PI3K and MAPK signaling [29
]. The use of fulvestrant efficiently sensitized MCF7 LTED cells to both BKM120 and BGT226, however, consistent with a key role for ligand-independent ER activity in PI3K inhibitor resistance. The use of estradiol to revert the LTED phenotype, followed by re-institution of estrogen deprivation, is a viable alternative strategy; however, the restoration of sensitivity to PI3K inhibition with this approach appeared less profound than with fulvestrant treatment.
Taken together our data provide a rationale for combining estrogen deprivation with PI3K inhibitors for the treatment of PIK3CA mutant estrogen-dependent, ER-positive tumors and for the combination of fulvestrant with PI3K inhibitors in patients with ER-positive, aromatase-inhibitor-resistant disease. However, further studies will be necessary to effectively translate these preclinical data into the clinical setting. These studies could include additional preclinical modeling in PIK3CA wild-type estrogen-deprivation-resistant tumor lines to determine whether PIK3CA mutation is necessary in endocrine-resistant tumors to confer PI3K inhibitor sensitivity. In addition, incorporating biomarker (PIK3CA mutation, Ki67 tumor cell proliferation and cell death markers) analysis in early-phase PI3K inhibitor trials may aid in identifying patients most likely to benefit from these therapeutic agents.
To address the prevalence of the target population for a fulvestrant/PI3K inhibitor trial for second-line treatment of ER-positive PIK3CA
mutant relapsed disease, we analyzed 51 advanced disease biopsies from both ER-positive and ER-negative cases for PIK3CA
mutation and correlated findings with the clinical trajectory of the patients. While patients with ER-positive PIK3CA
mutant tumors tended to relapse later than patients with ER-negative or ER-positive PIK3CA
wild-type tumors, the PIK3CA
mutation prevalence in ER-positive relapsed disease was high (approximately 50%). These findings are consistent with those recently reported by Dupont Jensen and colleagues on an analysis of 104 paired primary and metastatic breast tumors [30
]. In this study, PIK3CA
mutation was detected in 53% of the metastatic tumors and 45% of the primary tumors, indicating an apparent net gain in PIK3CA
mutation in metastatic disease that was thought to be due to heterogeneity in the primary tumor. The high prevalence of PIK3CA
mutation in metastatic or recurrent breast cancer suggests that PI3K-pathway-targeted therapeutics will be clinically relevant in this setting. These data also indicate that analysis of the recurrent disease will be necessary for selection of patients based upon tumor PIK3CA