Karin Beelen

Can predictive biomarkers in breast cancer guide adjuvant endocrine therapy

Personalized medicine for oestrogen receptor-α (ERα)-positive breast cancer requires predictive biomarkers for broad endocrine resistance as well as biomarkers capable of predicting resistance to a specific agent. In addition, biomarkers could be used to select patients that might benefit from the addition of treatments that do not target ERα. However, biomarker identification studies seem to be far from consistent and identified biomarkers seldom face an introduction into clinical practice. Importantly, most of the studies that seek to identify biomarkers have been performed using material from consecutive series of patients treated with tamoxifen (the most commonly prescribed ERα antagonist). Consequently, the predictive value of any biomarker identified is confounded by its prognostic value. Another important issue is the lack of differentiation between premenopausal and postmenopausal patients with breast cancer. The hormonal environment of a tumour in patients who are premenopausal is intrinsically distinct from those arising in postmenopausal women. Biomarkers of different biological mechanisms might enable the prediction of either broad endocrine resistance or resistance to a specific agent in each of these patient subtypes. Ultimately, improvements to study design are needed to establish the clinical validity of the most promising biomarkers to predict benefit from endocrine therapy.

PIK3CA mutations, phosphatase and tensin homolog, human epidermal growth factor receptor 2, and insulin-like growth factor 1 receptor and adjuvant tamoxifen resistance in postmenopausal breast cancer patients

IntroductionInhibitors of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway can overcome endocrine resistance in estrogen receptor (ER) α-positive breast cancer, but companion diagnostics indicating PI3K/AKT/mTOR activation and consequently endocrine resistance are lacking. PIK3CA mutations frequently occur in ERα-positive breast cancer and result in PI3K/AKT/mTOR activation in vitro. Nevertheless, the prognostic and treatment-predictive value of these mutations in ERα-positive breast cancer is contradictive. We tested the clinical validity of PIK3CA mutations and other canonic pathway drivers to predict intrinsic resistance to adjuvant tamoxifen. In addition, we tested the association between these drivers and downstream activated proteins.MethodsPrimary tumors from 563 ERα-positive postmenopausal patients, randomized between adjuvant tamoxifen (1 to 3 years) versus observation were recollected. PIK3CA hotspot mutations in exon 9 and exon 20 were assessed with Sequenom Mass Spectometry. Immunohistochemistry was performed for human epidermal growth factor receptor 2 (HER2), phosphatase and tensin homolog (PTEN), and insulin-like growth factor 1 receptor (IGF-1R). We tested the association between these molecular alterations and downstream activated proteins (like phospho-protein kinase B (p-AKT), phospho-mammalian target of rapamycin (p-mTOR), p-ERK1/2, and p-p70S6K). Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of canonic pathway drivers, by using Cox proportional hazard models, including a test for interaction.ResultsPIK3CA mutations (both exon 9 and exon 20) were associated with low tumor grade. An enrichment of PIK3CA exon 20 mutations was observed in progesterone receptor- positive tumors. PIK3CA exon 20 mutations were not associated with downstream-activated proteins. No significant interaction between PIK3CA mutations or any of the other canonic pathway drivers and tamoxifen-treatment benefit was found.ConclusionPIK3CA mutations do not have clinical validity to predict intrinsic resistance to adjuvant tamoxifen and may therefore be unsuitable as companion diagnostic for PI3K/AKT/mTOR inhibitors in ERα- positive, postmenopausal, early breast cancer patients.

Phosphorylated p-70S6K predicts tamoxifen resistance in postmenopausal breast cancer patients randomized between adjuvant tamoxifen versus no systemic treatment

IntroductionActivation of the phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways results in anti-estrogen resistance in vitro, but a biomarker with clinical validity to predict intrinsic resistance has not been identified. In metastatic breast cancer patients with previous exposure to endocrine therapy, the addition of a mammalian target of rapamycine (mTOR) inhibitor has been shown to be beneficial. Whether or not patients on adjuvant endocrine treatment might benefit from these drugs is currently unclear. A biomarker that predicts intrinsic resistance could potentially be used as companion diagnostic in this setting. We tested the clinical validity of different downstream-activated proteins in the PI3K and/or MAPK pathways to predict intrinsic tamoxifen resistance in postmenopausal primary breast cancer patients.MethodsWe recollected primary tumor tissue from patients who participated in a randomized trial of adjuvant tamoxifen (1–3 years) versus observation. After constructing a tissue micro-array, cores from 563 estrogen receptor α positive were immunostained for p-AKT(Thr308), p-AKT(Ser473), p-mTOR, p-p706SK and p-ERK1/2. Cox proportional hazard models for recurrence free interval were used to assess hazard ratios and interactions between these markers and tamoxifen treatment efficacy.ResultsInteractions were identified between tamoxifen and p-AKT(Thr308), p-mTOR, p-p70S6K and p-ERK1/2. Applying a conservative level of significance, p-p70S6K remained significantly associated with tamoxifen resistance. Patients with p-p70S6K negative tumors derived significant benefit from tamoxifen (HR 0.24, P < 0.0001), while patients whose tumor did express p-p70S6K did not (HR = 1.02, P =0.95), P for interaction 0.004. In systemically untreated breast cancer patients, p-p70S6K was associated with a decreased risk for recurrence.ConclusionsPatients whose tumor expresses p-p70S6K, as a marker of downstream PI3K and/or MAPK pathway activation, have a favorable prognosis, but do not benefit from adjuvant tamoxifen. A potential benefit from inhibitors of the PI3K/Akt/mTOR pathway in these patients needs to be further explored.

PI3K/AKT/mTOR pathway activation in primary and corresponding metastatic breast tumors after adjuvant endocrine therapy

Both preclinical and clinical data suggest that activation of the PI3K/AKT/mTOR pathway in response to hormonal therapy results in acquired endocrine therapy resistance. We evaluated differences in activation of the PI3K/AKT/mTOR pathway in estrogen receptor α (ERα) positive primary and corresponding metastatic breast cancer tissues using immunohistochemistry for downstream activated proteins, like phosphorylated mTOR (p‐mTOR), phosphorylated 4E Binding Protein 1 (p‐4EBP1) and phosphorylated p70S6K (p‐p70S6K). For p‐mTOR and p‐4EBP1, the proportion of immunostained tumor cells (0–100%) was scored. Cytoplasmic intensity (0–3) was assessed for p‐p70S6K. The difference between expression of these activated PI3K/AKT/mTOR proteins‐ in primary and metastatic tumor was calculated and tested for an association with adjuvant endocrine therapy. In patients who had received endocrine therapy (N = 34), p‐mTOR expression increased in metastatic tumor lesions compared to the primary tumor (median difference 45%), while in patients who had not received adjuvant endocrine therapy (N = 37), no difference was found. Similar results were observed for p‐4EBP1 and p‐p70S6K expression. In multivariate analyses, adjuvant endocrine therapy was significantly associated with an increase in p‐mTOR (p = 0.01), p‐4EBP1 (p = 0.03) and p‐p70S6K (p = 0.001), indicating that compensatory activation of the PI3K/AKT/mTOR pathway might indeed be a clinically relevant resistance mechanism resulting in acquired endocrine therapy resistance.

St. Gallen endocrine response classes predict recurrence rates over time

BACKGROUND In 2007 the St. Gallen consensus panel defined three endocrine response classes: highly endocrine responsive (ER-H), incomplete endocrine responsive (ER-I) and non-endocrine responsive tumours (ER-N). However, it is uncertain whether ER-I tumours are less responsive than ER-H tumours. We investigated whether recurrence rates vary over time between response classes. Additionally, we investigated the most predictive response class definition for tamoxifen benefit. PATIENTS AND METHODS We recollected tumours from 646 patients who participated in a randomized trial of adjuvant tamoxifen vs. OBSERVATION Estrogen receptor (ER), progesterone receptor (PgR), HER2 status and tumour grade were revised centrally. St. Gallen classes were evaluated for recurrence free interval (RFI). Change in hazards over time was assessed. Subsequently, 6 alternative response class definitions were compared to optimize the cut-off for PgR and ER. RESULTS Schoenfeld residuals indicate a failure of proportional hazards between the endocrine response groups (p = 0.0001). The HR for recurrence risk shifted over time with the ER-H group initially being at lower risk (HR ER-H vs. ER-I 0.5), but after six years the recurrence risk increased (HR 1.9). The cut-off values for ER and PgR that statistically best discriminated RFI in the first 4 years for lymph node positive patients were ER ≥ 50% and PgR ≥ 75%. CONCLUSION We demonstrated a marked variability in endocrine therapy benefit. Patients with ER-H tumours have a larger benefit during adjuvant tamoxifen and in the first years after accomplishing of the therapy, but suffer from late recurrences. This might have implications for optimal treatment duration.

Abstract PD4-12: Clustering of activated proteins of the PI3K and MAPK pathways distinguishes ER+/HER2- primary breast cancer patients with preferential tamoxifen benefit

Background Several (activated) proteins of the phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway have been analyzed for an association with adjuvant tamoxifen benefit in estrogen receptor-positive (ER+), HER2-negative (HER2-) breast cancer. Currently, no single protein has yielded convincing results allowing clinical implementation. Combining activated proteins could provide more robust predictive potential. We used unsupervised hierarchical clustering of 7 activated proteins downstream in these pathways, discriminated by expression profile, to analyze adjuvant tamoxifen benefit in ER+/HER2- postmenopausal breast cancer patients. Further, we developed a classification tool based on the generated heatmap groups by pathway activation status to categorize future patients into sensitive for or resistant to adjuvant tamoxifen. Methods Primary tumor blocks from 489 ER+/HER2- (stage I-III) postmenopausal patients previously randomized between tamoxifen (1 to 3 years) vs no adjuvant therapy (IKA trial 1982-1993; Beelen et al, Breast Cancer Res, 2014) were used for immunohistochemistry on tissue microarrays. Median follow-up of the patients without a recurrence event was 8.5 years. Scoring of PTEN, p-AKT(Thr308), p-AKT(Ser473) and p-p70S6K(Thr389) was by cytoplasmic intensity (0-3), p-4EBP1(Ser65) and pERK1/2(Thr202/204) by percentage of tumor cells with positive nuclear staining and p-S6RP(Ser235/236) by the percentage of tumor cells with positive membranous staining. Unsupervised hierarchical clustering was performed on tissue from 293 patients with informative data on all 7 proteins. Cox models were used to assess tamoxifen benefit (defined as the Hazard ratio (HR) of tamoxifen treatment vs no adjuvant therapy for recurrence-free interval) and to compare this benefit between subgroups generated by clustering as well as generated by the classification tool. Cox models included covariates (age, histological grade and subtype, tumor size, PR status) and were stratified for lymph node status. Results Two distinct groups were identified based on hierarchical clustering: one with preferentially activated pathway markers (A) and one with relatively no activation (N). Patients in group N derived significant benefit from tamoxifen (multivariable HR 0.28, 95% CI 0.14 − 0.56, p = 0.000324), while patients from group A had no benefit (multivariable HR 1.57, 95% CI 0.44 – 5.56, p  = 0.482), p for interaction 0.018. Contradictory, patients in group A had a better prognosis compared to those in group N (multivariable HR 0.17, 95% CI 0.04 − 0.7, p = 0.014). The classification tool was also successful in differentiating ER+/HER2- patients with or without tamoxifen benefit ( p for interaction = 0.016). Conclusions Hierarchical clustering of PI3K/MAPK pathway proteins is able to differentiate patients with adjuvant tamoxifen benefit and patients who would potentially need alternative treatment, for instance with inhibitors of the PI3K or MAPK pathway. The classification tool distinguishing patients with and without tamoxifen benefit should be validated in an independent cohort of postmenopausal patients with primary ER+/HER2- breast cancer. Citation Format: Kruger DT, Beelen K, Opdam M, Sanders J, van der Noort V, Boven E, Linn SC. Clustering of activated proteins of the PI3K and MAPK pathways distinguishes ER+/HER2- primary breast cancer patients with preferential tamoxifen benefit [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-12.

Hierarchical clustering of activated proteins in the PI3K and MAPK pathways in ER-positive, HER2-negative breast cancer with potential therapeutic consequences

BackgroundThe phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways are frequently activated in breast cancer which can result in antioestrogen resistance. Single protein markers failed to be introduced into clinical practice. We, therefore, aimed to find a better read-out of activation of the PI3K and MAPK pathways in ER+/HER2− breast cancer. Assessment of seven PI3K/MAPK proteins might better reflect pathway activation and distinguish patients without adjuvant tamoxifen benefit.MethodsTumour blocks were recollected from 293 primary postmenopausal ER+/HER2− breast cancer patients randomised between tamoxifen and no adjuvant therapy. PTEN, p-AKT(Thr308), p-AKT(Ser473), p-p70S6K, p-4EBP1, p-ERK1/2 and p-S6RP expression was assessed by immunohistochemistry followed by unsupervised hierarchical clustering. The primary endpoint was recurrence-free interval. Multivariate Cox models were used to assess tamoxifen benefit. A classification tool was developed based on protein expression profile.ResultsSubgroups were identified with preferentially activated (A) and preferentially not activated (N) proteins. Patients in group N derived significant benefit from tamoxifen (multivariate hazard ratio (HR) = 0.23, p = 0.000101), while patients from group A did not (multivariate HR = 1.37, p = 0.64), p for interaction 0.020. Our generated classification tool confirmed these results (p for interaction 0.024).ConclusionsHierarchical clustering of seven PI3K/MAPK proteins reflects pathway activation and can guide treatment decisions in primary ER+/HER2− postmenopausal breast cancer patients.

Abstract 5612: Cancer-immune interactions in luminal breast cancers:PI3KCAmutations, PI3K/AKT/mTOR activation and tumor-infiltrating lymphocytes

Introduction: Therapy resistance to adjuvant tamoxifen in estrogen receptor positive (ER+) breast cancer (BC) is related to activation of downstream proteins in the PI3K/AKT/mTOR pathway. However, clinical efficacy of mTOR inhibitors has so far been modest. Growing evidence shows that genomic make-up of cancer cells plays a crucial role in an anti-cancer immune response and this is reflected in the presence of tumor infiltrating immune cells. This begs the need for understanding the relationship between tumor-related immune activity, PIK3CA hot-spot mutations (PIK3CAm) and PI3K/AKT/mTOR pathway activation in ER+BC. Material and methods: The IKA trial recruited stage I to III postmenopausal BC patients (1982 till 1994), who were randomized to tamoxifen or no adjuvant therapy. Sequenom mass spectrometry-based genotyping was used for PIK3CAm assessment. Immune markers and phosphorylation status of proteins in the PI3K/AKT/mTOR pathway [p-AKT (Thr308 and 473), p-mTOR, p-ERK1/2, p-p-70S6K and p-4EBP1] were assessed by immunohistochemistry and scored by two pathologists. Expression of CD4, CD8 and FOXP3 was evaluated using automatic scoring by image-analysis software (SlidePath® or AxioVision®) and compared with manual scoring by two pathologists. Associations were assessed using multivariable logistic regression models, including as co-variables: age, tumor grade, lymph node status, tumor size, and progesterone receptor and HER2 status. Results: We included 563 ER+BC cases. PIK3CAm were found in 159 (32%) of the 486 tumors genotyped. On average, PI3K/AKT/mTOR downstream proteins and immune markers were scored in 409 tumors (range: 366 to 438). Stromal CD8 expression, but not CD4 or FOXP3, was significantly higher in PIK3CA mutated tumors (OR=1.11; 95%CI: 1.02-1.22). Stromal FOXP3 expression, but not CD4 or CD8, was significantly increased in tumors with activated proteins from the PI3K/AKT/mTOR pathway (except p-mTOR). The largest association was with p-4EBP1 (OR=1.34; 95%CI: 1.21-1.48) and smallest with p-70S6K (OR=1.15; 95%CI: 1.08-1.22). Conclusion: In our dataset of ER+BC, PIK3CAm are associated with higher level of CD8+ T cells. Tumors with activation of the PI3K/Akt/mTOR pathway tend to have more FOXP3+ T cells. Together, our results suggest that PIK3CA mutation/activation harbor an immune-related tumor microenvironment. Citation Format: Marcelo Sobral-Leite, Izhar Salomon, Mark Opdam, Karin Beelen, Ronald L. van Vlierberghe, Erik J. Blok, Hugo M. Horlings, Joyce Sanders, Koen Van de Vijver, Peter J. Kuppen, Sabine Linn, Marjanka K. Schmidt, Marleen Kok. Cancer-immune interactions in luminal breast cancers: PI3KCA mutations, PI3K/AKT/mTOR activation and tumor-infiltrating lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5612. doi:10.1158/1538-7445.AM2017-5612

Abstract OT2-01-11: Phase II of POSEIDON: A phase Ib / randomized phase II trial of tamoxifen plus taselisib or placebo in hormone receptor positive, HER2 negative, metastatic breast cancer patients with prior exposure to endocrine treatment

Background: The combination of PI3K-AKT-mTOR pathway inhibitors with endocrine therapy can improve clinical outcomes of hormone receptor positive (HR+) metastatic breast cancer (MBC) patients. Taselisib is a potent and selective PI3K inhibitor, with greater selectivity against mutant (MUT) PI3Kα isoforms than wild-type (WT) via a unique mechanism. Phase Ib data of POSEIDON with Taselisib + tamoxifen (TAM) demonstrated encouraging activity in patients with heavily pre-treated MBC, with an acceptable toxicity profile (Baird et al, ASCO 2016). The recommended phase II dose (RP2D) was Taselisib 4mg plus TAM 20mg, both administered on a daily continuous schedule. ctDNA monitoring may have value in drug development by (1) assessing predictive biomarkers to therapy, (2) providing an early indication of treatment response, and (3) shedding light on potential mechanisms of acquired drug resistance. In some patients included in phase Ib of POSEIDON, tumor response was preceded by a corresponding early change in plasma PIK3CA ctDNA levels. Methods: The phase II portion of the POSEIDON trial is a two-arm, randomized, double blind study of Taselisib plus TAM versus placebo (PLA) plus TAM in pre- and postmenopausal women with HR+/HER2- MBC. In the first part of the Phase II, 180 patients will be randomized (1:1) to receive continuous TAM with either Taselisib at the RP2D or PLA until disease progression, unacceptable toxicity or patient / physician decision. Crossover is allowed upon progressive disease in those patients receiving PLA plus TAM, after collection of tumor and blood samples for exploratory biomarker analysis. Stratification is based on menopausal status, histology [lobular breast cancer (LBC) vs. ductal/others], PIK3CA mutation (WT vs. exon 9 vs. exon 20), prior everolimus, timing of recurrence/progression after prior endocrine therapy, number of prior chemotherapy (CT) lines, and treatment center. After recruiting the initial 180 patients, trial will focus in LBC, until a total number of 110 patients with LBC are enrolled. Other key eligibility criteria include presence of measurable or evaluable disease (RECIST 1.1), prior progression to endocrine treatment, maximum of 5 prior CT lines in the metastatic setting, absence of diabetes under medical treatment, and absence of chronic inflammatory bowel disease. Primary endpoint is investigator-assessed PFS. Key secondary endpoints are PFS in LBC, objective response rate, clinical benefit rate, safety, and exploratory biomarker analysis (including ctDNA). The study has a 90% power at a two-sided log-rank test significance level of 0.2 to detect an HR of 0.64, which corresponds to an increase in median PFS from 4.5 months in the PLA plus TAM arm to 7 months in the Taselisib plus TAM arm. Enrollment to POSEIDON Phase II started in June 2016 (Clinicaltrials.gov NCT02285179). Citation Format: Oliveira M, Baird RD, van Rossum AGJ, Beelen K, Garcia-Corbacho J, Mandjes IAM, Vallier AL, van Werkhoven E, Garrigos L, Kumar S, van Tinteren H, Munoz S, Linossi C, Rosing H, Miquel JM, Schrier M, de Vries Schultink A, Saura C, Gallagher WM, Bernards R, Tabernero J, Cortes J, Caldas C, Linn SC. Phase II of POSEIDON: A phase Ib / randomized phase II trial of tamoxifen plus taselisib or placebo in hormone receptor positive, HER2 negative, metastatic breast cancer patients with prior exposure to endocrine treatment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-01-11.

Abstract OT1-1-02: PI3K-Akt-mTOR pathway analysis to obtain further insight in the efficacy of everolimus in combination with exemestane in metastatic, ER-positive breast cancer: A Dutch breast cancer research group (BOOG) study

Background In patients with hormone receptor-positive breast cancer, activation of the PI3K-Akt-mTOR pathway is associated with resistance against endocrine therapy. Previous research has shown that genetic aberrations in this pathway occur frequently, including mutation and/or amplification in PI3K subunits or PI3K effectors as well as loss of lipid phosphatases (Fu X, et al. The Breast; 2013). Central review of the BOLERO-2 randomized phase III trial in which patients refractory to a non-steroidal aromatase inhibitor were randomized between exemestane combined with the mTOR inhibitor everolimus versus exemestane and placebo has shown a progression-free survival (PFS) of, respectively, 11.0 and 4.1 months [hazard ratio = 0.38 (95% confidence interval 0.31-0.48; log-rank P Trial design/Aims This is a Dutch prospective, open-label, single-arm, investigator-initiated, multicenter trial in which approximately 30 hospitals will participate. A total of 175 patients will be included for baseline blood sampling and archival tumor tissue collection. From 50 patients, a fresh tumor biopsy is required at baseline and from 30 out of 50, another tumor biopsy will be collected upon progressive disease. Exploratory biomarker assessment includes immunohistochemistry (total and phosphorylated PI3K, AKT, mTOR, p70S6K and 4EBP1), tissue phosphoproteomics and circulating tumor DNA (mutations). The results of the biomarker analysis will be compared with clinicopathological characteristics and PFS. Eligibility Postmenopausal patients with hormone receptor-positive, HER2-negative, locally advanced or metastatic breast cancer, refractory to anastrozole or letrozole will be included. No previous treatment with exemestane or mTOR inhibitor for advanced disease is allowed and Informed consent must be signed before enrollment. Statistical methods Since the majority of the tests involve the use of new techniques, the study will be mainly explorative in design. The association between potential biomarkers and clinicopathological characteristics will be tested using Fisher exact test or the Mann-Whitney test. PFS curves will be drawn using the Kaplan-Meier method. PFS in association with potential biomarkers will be tested using Cox proportional hazard regression analysis. Present and target accrual Recently, the study has been opened for inclusion. A period of 2 years is planned for patient enrollment. Up to May 2014, two patients were included. ClinicalTrials.gov identifier NCT02109913 Financial support is received from Novartis, the Netherlands. Citation Format: Dinja T Kruger, Karin Beelen, Connie R Jimenez, Maurice PHM Jansen, Stefan Sleijfer, Sabine C Linn, Epie Boven. PI3K-Akt-mTOR pathway analysis to obtain further insight in the efficacy of everolimus in combination with exemestane in metastatic, ER-positive breast cancer: A Dutch breast cancer research group (BOOG) study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-02.