Prognostic and predictive impact of NOTCH1 in early breast cancer.

PURPOSE: Systemic therapy plays a major part in the cure of patients with early breast cancer (eBC). However, personalized treatment concepts are required to avoid potentially harmful overtreatment. Biomarkers are pivotal for individualized therapy. The Notch signalling pathway is widely considered as a suitable prognostic or predictive marker in eBC. This study aimed primarily at assessing the relationship between NOTCH1 mRNA expression levels and histopathological features of breast cancer tumors, as well as clinical characteristics of the correspondent eBC patients. As a secondary aim, we investigated the prognostic and predictive value of NOTCH1 by assessing possible associations between NOTCH1 mRNA expression and recurrence-free interval (RFI) and overall survival after five years of observation. PATIENTS AND METHODS: The relative NOTCH1 mRNA expression was determined in 414 tumour samples, using quantitative PCR in a prospective, multicenter cohort (Prognostic Assessment in Routine Application (PiA), 2009-2011, NCT01592825) of 1,270 female eBC patients. RESULTS: High NOTCH1 mRNA expression was detected in one-third of the tumours and was associated with negative hormone receptor status and high uPA/PAI-1 status. In addition, high NOTCH1 mRNA expression was found to be associated with more RFI related events (adjusted hazard ratio 2.1, 95% CI 1.077-4.118). Patients who received adjuvant chemotherapy and had high NOTCH1 mRNA expression in the tumour (n = 86) were three times more likely to have an RFI event (adjusted hazard ratio 3.1, 95% CI 1.321-7.245, p = 0.009). CONCLUSION: In this cohort, NOTCH1 mRNA expression had a prognostic and predictive impact. Tumours with high NOTCH1 mRNA expression may be less sensitive to cytotoxic treatment and downregulation of the Notch signalling pathway (e.g. by γ-secretase inhibitors) may be valuable for eBC therapy as an individualised treatment option.

PIK3CA-mutations in breast cancer.

PURPOSE: Phosphatidylinositide-3-kinase (PI3K) regulates proliferation and apoptosis; somatic PIK3CA-mutations may activate these processes. Aim of this study was to determine the prevalence of PIK3CA-mutations in a cohort of early stage breast cancer patients and the association to the course of disease. PATIENTS AND METHODS: From an unselected cohort of 1270 breast cancer patients (PiA, Prognostic Assessment in routine application, NCT01592825) 1123 tumours were tested for the three PIK3CA hotspot-mutations H1047R, E545K, and E542K by qPCR. Primary objectives were the prevalence of somatic PIK3CA-mutations and their association to tumour characteristics. Secondary objective was the association of PIK3CA-mutations to recurrence-free interval (RFI) and overall survival. RESULTS: PIK3CA-mutation rate was 26.7% (300 of 1123). PIK3CA-mutations were significantly more frequent in steroid hormone-receptor (SHR)-positive HER2-negative (31.4%), and G1 and G2 tumours (32.8%). Overall, we did not observe a significant association of PIK3CA-mutations to RFI. In SHR-positive BCs with PIK3CA-mutations, a strong trend for impaired  RFI was observed (adjusted HR 1.64, 95% CI 0.958-2.807), whilst in SHR-negative BCs PIK3CA-mutations were insignificantly associated with improved RFI (adjusted HR 0.49; 95% CI 0.152-1.597). Of note, we observed a significantly detrimental prognostic impact of PIK3CA-mutations on RFI in SHR-positive, HER2-negative BCs if only aromatase inhibitors were administered as adjuvant therapy (adjusted HR 4.44, 95% CI 1.385-13.920), whilst no impact was observed in tamoxifen treated patients. CONCLUSION: This cohort study speficies the overall mutation rate of PIK3CA in early breast cancer. The impact of PIK3CA-mutations on RFI and OS was heterogeneous. Our results suggest that estrogen deprivation failes to be active in case of PIK3CA-mutation.

Proteomic profiling of breast cancer metabolism identifies SHMT2 and ASCT2 as prognostic factors.

BACKGROUND: Breast cancer tumors are known to be highly heterogeneous and differences in their metabolic phenotypes, especially at protein level, are less well-understood. Profiling of metabolism-related proteins harbors the potential to establish new patient stratification regimes and biomarkers promoting individualized therapy. In our study, we aimed to examine the relationship between metabolism-associated protein expression profiles and clinicopathological characteristics in a large cohort of breast cancer patients. METHODS: Breast cancer specimens from 801 consecutive patients, diagnosed between 2009 and 2011, were investigated using reverse phase protein arrays (RPPA). Patients were treated in accordance with national guidelines in five certified German breast centers. To obtain quantitative expression data, 37 antibodies detecting proteins relevant to cancer metabolism, were applied. Hierarchical cluster analysis and individual target characterization were performed. Clustering results and individual protein expression patterns were associated with clinical data. The Kaplan-Meier method was used to estimate survival functions. Univariate and multivariate Cox regression models were applied to assess the impact of protein expression and other clinicopathological features on survival. RESULTS: We identified three metabolic clusters of breast cancer, which do not reflect the receptor-defined subtypes, but are significantly correlated with overall survival (OS, p ≤ 0.03) and recurrence-free survival (RFS, p ≤ 0.01). Furthermore, univariate and multivariate analysis of individual protein expression profiles demonstrated the central role of serine hydroxymethyltransferase 2 (SHMT2) and amino acid transporter ASCT2 (SLC1A5) as independent prognostic factors in breast cancer patients. High SHMT2 protein expression was significantly correlated with poor OS (hazard ratio (HR) = 1.53, 95% confidence interval (CI) = 1.10-2.12, p ≤ 0.01) and RFS (HR = 1.54, 95% CI = 1.16-2.04, p ≤ 0.01). High protein expression of ASCT2 was significantly correlated with poor RFS (HR = 1.31, 95% CI = 1.01-1.71, p ≤ 0.05). CONCLUSIONS: Our data confirm the heterogeneity of breast tumors at a functional proteomic level and dissects the relationship between metabolism-related proteins, pathological features and patient survival. These observations highlight the importance of SHMT2 and ASCT2 as valuable individual prognostic markers and potential targets for personalized breast cancer therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01592825 . Registered on 3 May 2012.

Prognostic Value of Tumour-Infiltrating Lymphocytes in an Unselected Cohort of Breast Cancer Patients.

Tumour-infiltrating lymphocytes (TILs) are considered to have prognostic and predictive value for patients with early breast cancer. We examined 1166 breast cancer patients from a prospective, multicentre cohort (Prognostic Assessment in Routine Application (PiA), n = 1270, NCT01592825) following recommendations from the International TILs Working Group. TIL quantification was performed using predefined groups and as a continuous variable in 10% increments. The primary objective was the distribution of TILs in different breast cancer types. The second objective was the association with the recurrence-free interval (RFI) and overall survival (OS). Stromal infiltration with more than 60% TILs appeared in 2% of hormone receptor (HR)-positive and HER2-negative tumours, in 9.8% of HER2-positive tumours (any HR) and 19.4% of triple-negative breast cancers (TNBCs). Each 10% increment was associated with an improvement in the prognosis in HER2-positive samples (RFI, hazard ratio 0.773, 95% CI 0.587-1.017; OS, hazard ratio 0.700, 95% CI 0.523-0.937). When defining exploratory cut-offs for TILs, the use of a 30% threshold for the HR-positive and HER2-negative group, a 20% threshold for the HER2 group and a 60% threshold for the TNBC group appeared to be the most suitable. TILs bore prognostic value, especially in HER2-positive breast cancer. For clinical use, additional research on the components of immune infiltration might be reasonable.

Identifying High-Risk Triple-Negative Breast Cancer Patients by Molecular Subtyping.

INTRODUCTION: Triple-negative breast cancer (TNBC) is considered the most aggressive type of breast cancer (BC) with limited options for therapy. TNBC is a heterogeneous disease and tumors have been classified into TNBC subtypes using gene expression profiling to distinguish basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal stem-like, luminal androgen receptor (LAR), and one nonclassifiable group (called unstable). OBJECTIVES: The aim of this study was to verify the clinical relevance of molecular subtyping of TNBCs to improve the individual indication of systemic therapy. PATIENTS AND METHODS: Molecular subtyping was performed in 124 (82%) of 152 TNBC tumors that were obtained from a prospective, multicenter cohort including 1,270 histopathologically confirmed invasive, nonmetastatic BCs (NCT01592825). Treatment was guideline-based. TNBC subtypes were correlated with recurrence-free interval (RFI) and overall survival (OS) after 5 years of observation. RESULTS: Using PAM50 analysis, 87% of the tumors were typed as basal with an inferior clinical outcome compared to patients with nonbasal tumors. Using the TNBCtype-6 classifier, we identified 23 (15%) of TNBCs as LAR subtype. After standard adjuvant or neoadjuvant chemotherapy, patients with LAR subtype showed the most events for 5-year RFI (66.7 vs. 80.6%) and the poorest probability of 5-year OS (60.0 vs. 84.4%) compared to patients with non-LAR disease (RFI: adjusted hazard ratio [aHR] = 1.87, 95% confidence interval [CI] 0.69-5.05, p = 0.211; OS: aHR = 2.74, 95% CI 1.06-7.10, p = 0.037). CONCLUSION: Molecular analysis and subtyping of TNBC may be relevant to identify patients with LAR subtype. These cancers seem to be less sensitive to conventional chemotherapy, and new treatment options, including androgen receptor-blocking agents and immune checkpoint inhibitors, have to be explored.

Specific allelic variants of SNPs in the MDM2 and MDMX genes are associated with earlier tumor onset and progression in Caucasian breast cancer patients.

BACKGROUND: Genetic factors play a substantial role in breast cancer etiology. Genes encoding proteins that have key functions in the DNA damage response, such as p53 and its inhibitors MDM2 and MDMX, are most likely candidates to harbor allelic variants that influence breast cancer susceptibility. The aim of our study was to comprehensively analyze the impact of SNPs in the TP53, MDM2, and MDMX genes in conjunction with TP53 mutational status regarding the onset and progression of breast cancer. METHODS: In specimen from 815 breast cancer patients, five SNPs within the selected genes were analyzed: TP53 - Arg72Pro (rs1042522), MDM2 - SNP285 (rs2279744), SNP309 (rs117039649); MDMX - SNP31826 (rs1563828), and SNP34091 (rs4245739). Classification of the tumors was evaluated by histomorphology. Subtyping according hormone receptor status, HER2-status and proliferation rate enabled provision of the clinico-pathological surrogate of intrinsic subtypes. RESULTS: The homozygous C-allele of MDM2 SNP285 was significantly associated with a younger age-at-diagnosis of 44.2 years, in contrast to G/G- and G/C-patients (62.4, 62.7 yrs., respectively; p = 0.0007; log-Rank-test). In contrast, there was no difference regarding the age-at-diagnosis for patients with the respective genotypes of MDM2 SNP309 (p = 0.799; log-Rank-test). In patients with estrogen receptor (ER)-positive and TP53-mutated tumors, however, the T/T-genotype of the MDM2 SNP309 was significantly associated with an earlier average age-at-diagnosis compared with T/G+G/G-patients (53.5 vs. 68.2 yrs; p = 0.002; log-Rank-test). In the triple-negative subgroup, the G/G-patients had an average age-at-diagnosis of 51 years compared with 63 years for SNP309T carriers (p = 0.004; log-Rank-test) indicating a susceptibility of the G/G genotype for the development of triple negative breast cancer. Patients with the A/A-genotype of MDMX SNP31826 with ER-negative tumors were diagnosed 11 years earlier compared with patients and ER-positive tumors (53.2 vs. 64.4 yrs; p = 0.025, log-Rank-test). Furthermore, in luminal B-like patients (HER2-independent) the C/C-genotype of MDMX SNP34091 was significantly correlated with a decreased event-free survival compared with the A/A-genotype (p < 0.001; log-Rank-test). CONCLUSIONS: We showed that SNPs in the MDM2 and MDMX genes affect at least in part the onset and progression of breast cancer dependent on the ER-status. Our findings provide further evidence for the distinct etiological pathways in ER-negative and ER-positive breast cancers.