PIcKing the Right Treatment for the Right Patient : anti-hormonal therapy resistance in breast cancer: PIK3CA related biomarkers and signaling pathways

Breast cancer is the most common type of cancer in women and second most common cancer worldwide. Most breast cancers are ER-positive (75-80%), for which anti-hormonal therapy is used. For ER-positive metastatic breast cancer (MBC), the objective response rate to anti-hormonal treatment is only 20-40%. This shows an urgent need for biomarkers which can identify patients who will or will not benefit from the therapy. As such, for those patients, unnecessary exposure to undesirable adverse events of (anti-hormonal) therapy can be avoided. Therefore, the aim of this thesis was to find biomarkers able to predict anti-hormonal treatment responsiveness or resistance in mainly advanced ER-positive breast cancer patients. To reach this goal different research approaches were followed. Since mutations in PIK3CA are the most prevalent mutations (up to 45%) in ER-positive breast cancers, the thesis was mainly focused on the relationship between PIK3CA genotype and PI3K pathway with treatment outcome. It was shown that PIK3CA mutations detected in primary breast tumors have a predictive value for aromatase inhibitors (AI) response in the advanced disease setting, but not for tamoxifen response nor for prognosis. Related to the PIK3CA genotype, it was demonstrated that high expression of LRG1 can be used as biomarker for AI treatment response, which upon neo-adjuvant AI therapy showed decreased levels in patients with clinical response. At the proteomic level, high MAPK1/3 phosphorylation levels in luminal breast cancer was shown to be related with PIK3CA exon specific mutations. This MAPK1/3 phosphorylation, especially when localized in the nuclei, has prognostic value in breast cancer. In an alternative approach, using ER-positive breast cancers with an inflammatory breast cancer phenotype, a metagene was constructed. This metagene, ABAT and STC2 were not prognostic. However, decreased expression of ABAT and STC2 were shown to be predictive for tamoxifen resistance in MBC. In the adjuvant setting, only low expression of ABAT was related to tamoxifen resistance. Finally, using cell free DNA (cfDNA) from liquid biopsies, tumor-specific mutations were explored as biomarkers for tamoxifen resistance in MBC patients. Mutations in PIK3CA, TP53, AKAP9, CREBBP and SMAD4 were observed in serum cfDNA taken at disease progression and these mutations, except for AKAP9, were also seen in the primary tumor. When further validated, all above biomarkers hopefully will guide us better to be able to pick the right treatment for the right breast cancer patient

Increased MAPK1/3 Phosphorylation in Luminal Breast Cancer Related with PIK3CA Hotspot Mutations and Prognosis

INTRODUCTION: While mutations in PIK3CA are most frequently (45%) detected in luminal breast cancer, downstream PI3K/AKT/mTOR pathway activation is predominantly observed in the basal subtype. The aim was to identify proteins activated in PIK3CA mutated luminal breast cancer and the clinical relevance of such a protein in breast cancer patients. MATERIALS AND METHODS: Expression levels of 171 signaling pathway (phospho-)proteins established by The Cancer Genome Atlas (TCGA) using reverse phase protein arrays (RPPA) were in silico examined in 361 breast cancers for their relation with PIK3CA status. MAPK1/3 phosphorylation was evaluated with immunohistochemistry on tissue microarrays (TMA) containing 721 primary breast cancer core biopsies to explore the relationship with metastasis-free survival. RESULTS: In silico analyses revealed increased phosphorylation of MAPK1/3, p38 and YAP, and decreased expression of p70S6K and 4E–BP1 in PIK3CA mutated compared to wild-type luminal breast cancer. Augmented MAPK1/3 phosphorylation was most significant, i.e. in luminal A for both PIK3CA exon 9 and 20 mutations and in luminal B for exon 9 mutations. In 290 adjuvant systemic therapy naïve lymph node negative (LNN) breast cancer patients with luminal cancer, high MAPK phosphorylation in nuclei (HR = 0.49; 95% CI, 0.25–0.95; P =.036) and in tumor cells (HR = 0.37; 95% CI, 0.18–0.79; P =.010) was related with favorable metastasis-free survival in multivariate analyses including traditional prognostic factors. CONCLUSION: Enhanced MAPK1/3 phosphorylation in luminal breast cancer is related to PIK3CA exon-specific mutations and correlated with favorable prognosis especially when located in the nuclei of tumor cells

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens

Decreased expression of ABAT and STC2 hallmarks ER-positive inflammatory breast cancer and endocrine therapy resistance in advanced disease

Background: Patients with Estrogen Receptor α-positive (ER+) Inflammatory Breast Cancer (IBC) are less responsive to endocrine therapy compared with ER+ non-IBC (nIBC) patients. The study of ER+ IBC samples might reveal biomarkers for endocrine resistant breast cancer. Materials & methods: Gene expression profiles of ER+ samples from 201 patients were explored for genes that discriminated between IBC and nIBC. Classifier genes were applied onto clinically annotated expression data from 947 patients with ER+ breast cancer and validated with RT-qPCR for 231 patients treated with first-line tamoxifen. Relationships with metastasis-free survival (MFS) and progression-free survival (PFS) following adjuvant and first-line endocrine treatment, respectively, were investigated using Cox regression analysis. Results: A metagene of six genes including the genes encoding for 4-aminobutyrate aminotransferase (ABAT) and Stanniocalcin-2 (STC2) were identified to distinguish 22 ER+ IBC from 43 ER+ nIBC patients and remained discriminatory in an independent series of 136 patients. The metagene and two genes were not prognostic in 517 (neo)adjuvant untreated lymph node-negative ER+ nIBC breast cancer patients. Only ABAT was related to outcome in 250 patients treated with adjuvant tamoxifen. Three independent series of in total 411 patients with advanced disease showed increased metagene scores and decreased expression of ABAT and STC2 to be correlated with poor first-line endocrine therapy outcome. The biomarkers remained predictive for first-line tamoxifen treatment outcome in multivariate analysis including traditional factors or published signatures. In an exploratory analysis, ABAT and STC2 protein expression levels had no relation with PFS after first-line tamoxifen. Conclusions: This study utilized ER+ IBC to identify a metagene including ABAT and STC2 as predictive biomarkers for endocrine therapy resistance