Vanja de Weerd

Four miRNAs associated with aggressiveness of lymph node-negative, estrogen receptor-positive human breast cancer

In this study, we quantified 249 mature micro-RNA (miRNA) transcripts in estrogen receptor-positive (ER+) primary breast tumors of patients with lymph node-negative (LNN) disease to identify miRNAs associated with metastatic capability. In addition, the prognostic value of the candidate miRNAs was determined in ER−/LNN breast cancer. Unsupervised analysis in a prescreening set of 38 patients identified three subgroups predominantly driven by three miRNA signatures: an ER-driven luminal B-associated miRNA signature, a stromal miRNA signature, and an overexpressed miRNA cluster located on chromosome 19q23, but these intrinsic miRNA signatures were not associated with tumor aggressiveness. Supervised analysis in the initial subset and subsequent analysis in additional tumors significantly linked four miRNAs (miR-7, miR-128a, miR-210, and miR-516–3p) to ER+/LNN breast cancer aggressiveness (n = 147) and one miRNA (miR-210) to metastatic capability in ER−/LNN breast cancer (n = 114) and in the clinically important triple-negative subgroup (n = 69) (all P < 0.05). Bioinformatic analysis coupled miR-210 to hypoxia/VEGF signaling, miR-7 and miR-516–3p to cell cycle progression and chromosomal instability, and miR-128a to cytokine signaling. In conclusion, our work connects four miRNAs to breast cancer progression and to several distinct biological processes involved therein.

mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients

Purpose: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer. Experimental Design: CTCs were isolated with the epithelial cell adhesion molecule–based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors. Results: We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels. Conclusions: Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs. Clin Cancer Res; 17(11); 3600–18. ©2011 AACR.

MicroRNA-30c expression level is an independent predictor of clinical benefit of endocrine therapy in advanced estrogen receptor positive breast cancer

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression and which have been implicated in cancer. We evaluated whether five candidate predictive miRNAs, derived from a pilot study in which 249 miRNAs were assayed, were associated with clinical benefit of tamoxifen therapy in advanced breast cancer. These five miRNAs were measured in an independent series of 246 estrogen receptor (ER)-positive primary breast tumors of patients who received tamoxifen for advanced disease by quantitative Real Time PCR. Univariate analysis showed that higher expression levels of hsa-miR-30a-3p, hsa-miR-30c, and hsa-miR-182 were significantly associated with benefit of tamoxifen treatment and with longer PFS (all P-values <0.01). In multivariate analysis, corrected for the traditional predictive factors, only hsa-miRNA-30c was an independent predictor (P-value <0.01). Finally, in an attempt to understand the biology connected to this miRNA, Global testing pathway analysis showed an association of hsa-miRNA-30c expression with HER and RAC1 signaling pathways. We identified hsa-miRNA-30c as an independent predictor for clinical benefit of tamoxifen therapy in patients with advanced breast cancer. Assessment of tumor levels and connected pathways could be helpful to improve treatment strategies.

Which Cyclin E Prevails as Prognostic Marker for Breast Cancer? Results from a Retrospective Study Involving 635 Lymph Node–Negative Breast Cancer Patients

Purpose: To evaluate the prognostic value of cyclin E with a quantitative method for lymph node–negative primary breast cancer patients. Patients and Methods: mRNA transcripts of full-length and splice variants of cyclin E1 (CCNE1) and cyclin E2 (CCNE2) were measured by real-time PCR in frozen tumor samples from 635 lymph node–negative breast cancer patients who had not received neoadjuvant or adjuvant systemic therapy. Results: None of the PCR assays designed for the specific splice variants of the cyclins gave additional prognosis-related information compared with the common assays able to detect all variants. In Cox multivariate analysis, corrected for the traditional prognostic factors, high levels of cyclin E were independently associated with a short distant metastasis-free survival [hazard ratio (HR), 3.40; P < 0.001 for CCNE1 and HR, 1.76; P < 0.001 for CCNE2, respectively]. After dichotomizing the tumors at the median level of 70% tumor cells, the multivariate analysis showed particularly strong results for CCNE1 in the group of 433 patients with stroma-enriched primary tumors (HR, 5.12; P < 0.001). In these tumors, the worst prognosis was found for patients with estrogen receptor–negative tumors expressing high CCNE1 (HR, 9.89; P < 0.001) and for patients with small (T1) tumors expressing high CCNE1 (HR, 8.47; P < 0.001). Conclusion: Our study shows that both CCNE1 and CCNE2 qualify as independent prognostic markers for lymph node–negative breast cancer patients, and that CCNE1 may provide additional information for specific subgroups of patients.

Clinical significance of the nuclear receptor co-regulator DC-SCRIPT in breast cancer: an independent retrospective validation study

IntroductionIn this study we aimed to validate the prognostic value of DC-SCRIPT mRNA expression in a large independent breast cancer cohort. In addition, since DC-SCRIPT is a transcriptional co-regulator of nuclear receptors, we explored its prognostic value in relation to estrogen-receptor-α (ESR1) and -β (ESR2) and evaluated its predictive value for response to tamoxifen treatment.MethodsDC-SCRIPT mRNA levels were measured by real-time PCR in 1,505 primary invasive breast cancers and associated with outcome (disease-free survival (DFS), metastasis-free survival (MFS) and overall survival (OS)) using univariate and multivariable Cox regression analysis. Logistic and Cox regressions were used to associate DC-SCRIPT levels with clinical benefit and progression-free survival (PFS) for 296 patients treated with first-line systemic tamoxifen for advanced disease.ResultsIn univariate and multivariable analysis higher DC-SCRIPT levels were associated with a favorable outcome for both the entire cohort and patients with lymph node-negative (LNN) disease that did not receive adjuvant therapy (DFS, MFS and OS; all, P < 0.001). This association was most pronounced in small (pT1) tumors, in ESR1-positive tumors and in tumors with low ESR2 expression. For first-line endocrine therapy for advanced disease no predictive association was seen with clinical benefit or PFS.ConclusionsThis study provides a higher level of evidence that DC-SCRIPT is indeed an independent, pure prognostic, factor for primary breast cancer and shows that DC-SCRIPT mRNA expression is most informative for either ESR1-positive and/or ESR2-low pT1 tumors.

Molecular characteristics of circulating tumor cells resemble the liver metastasis more closely than the primary tumor in metastatic colorectal cancer

Background CTCs are a promising alternative for metastatic tissue biopsies for use in precision medicine approaches. We investigated to what extent the molecular characteristics of circulating tumor cells (CTCs) resemble the liver metastasis and/or the primary tumor from patients with metastatic colorectal cancer (mCRC). Results The CTC profiles were concordant with the liver metastasis in 17/23 patients (74%) and with the primary tumor in 13 patients (57%). The CTCs better resembled the liver metastasis in 13 patients (57%), and the primary tumor in five patients (22%). The strength of the correlations was not associated with clinical parameters. Nine genes (CDH1, CDH17, CDX1, CEACAM5, FABP1, FCGBP, IGFBP3, IGFBP4, and MAPT) displayed significant differential expressions, all of which were downregulated, in CTCs compared to the tissues in the 23 patients. Patients and Methods Patients were retrospectively selected from a prospective study. Using the CellSearch System, CTCs were enumerated and isolated just prior to liver metastasectomy. A panel of 25 CTC-specific genes was measured by RT-qPCR in matching CTCs, primary tumors, and liver metastases. Spearman correlation coefficients were calculated and considered as continuous variables with r=1 representing absolute concordance and r= -1 representing absolute discordance. A cut-off of r>0.1 was applied in order to consider profiles to be concordant. Conclusions In the majority of the patients, CTCs reflected the molecular characteristics of metastatic cells better than the primary tumors. Genes involved in cell adhesion and epithelial-to-mesenchymal transition were downregulated in the CTCs. Our results support the use of CTC characterization as a liquid biopsy for precision medicine.

Integrative Analysis of Genomics and Proteomics Data on Clinical Breast Cancer Tissue Specimens Extracted with Acid Guanidinium Thiocyanate–Phenol–Chloroform

Acid guanidinium thiocyanate, phenol, and chloroform extraction (AGPC) is a commonly used procedure to extract RNA from fresh frozen tissues and cell lines. In addition, DNA and proteins can be recovered, which makes AGPC an attractive source for integrative analysis on tissues of which little material is available, such as clinical specimens. Despite this potential, AGPC has only scarcely been used for proteomic analysis, mainly due to difficulties in extracting proteins. We have used a quantitative mass spectrometry method to show that proteins can readily be recovered from AGPC extracted tissues with high recovery and repeatability, which allows this method to be used for global proteomic profiling. Protein expression data for a selected number of clinically relevant markers, of which transcript and protein levels are known to be correlated, were in agreement with genomic and transcriptomic data obtained from the same AGPC lysate. Furthermore, global proteomic profiling successfully discriminated breast tumor tissues according to their clinical subtype. Lastly, a reference gene set of differentially expressed transcripts was strongly enriched in the differentially abundant proteins in our cohort. AGPC lysates are therefore well suited for comparative protein and integrative analyses.

Progressive APOBEC3B mRNA expression in distant breast cancer metastases

Background APOBEC3B was recently identified as a gain-of-function enzymatic source of mutagenesis, which may offer novel therapeutic options with molecules that specifically target this enzyme. In primary breast cancer, APOBEC3B mRNA is deregulated in a substantial proportion of cases and its expression is associated with poor prognosis. However, its expression in breast cancer metastases, which are the main causes of breast cancer-related death, remained to be elucidated. Patients and methods RNA was isolated from 55 primary breast cancers and paired metastases, including regional lymph node (N = 20) and distant metastases (N = 35). APOBEC3B mRNA levels were measured by RT-qPCR. Expression levels of the primary tumors and corresponding metastases were compared, including subgroup analysis by estrogen receptor (ER/ESR1) status. Results Overall, APOBEC3B mRNA levels of distant metastases were significantly higher as compared to the corresponding primary breast tumor (P = 0.0015), an effect that was not seen for loco-regional lymph node metastases (P = 0.23). Subgroup analysis by ER-status showed that increased APOBEC3B levels in distant metastases were restricted to metastases arising from ER-positive primary breast cancers (P = 0.002). However, regarding ER-negative primary tumors, only loco-regional lymph node metastases showed increased APOBEC3B expression when compared to the corresponding primary tumor (P = 0.028). Conclusion APOBEC3B mRNA levels are significantly higher in breast cancer metastases as compared to the corresponding ER-positive primary tumors. This suggests a potential role for APOBEC3B in luminal breast cancer progression, and consequently, a promising role for anti-APOBEC3B therapies in advanced stages of this frequent form of breast cancer.

MicroRNA expression in pre-treatment plasma of patients with benign breast diseases and breast cancer

Background MicroRNAs (miRs) are small RNA molecules, influencing messenger RNA (mRNA) expression and translation, and are readily detectable in blood. Some have been reported as potential breast cancer biomarkers. This study aimed to identify and validate miRs indicative of breast cancer. Results Based on the discovery and literature, 18 potentially informative miRs were quantified in the validation cohort. Irrespective of patient and tumour characteristics, hsa-miR-652-5p was significantly upregulated in the malignant compared to benign patients (1.26 fold, P = 0.005) and therefore validated as potential biomarker. In the validation cohort literature-based hsa-let-7b levels were higher in malignant patients as well (1.53 fold, P = 0.011). Two miRs differentiated benign wildtype from benign BRCA1 mutation carriers and an additional 8 miRs differentiated metastastic (n = 8) from non-metastatic (n = 41) cases in the validation cohort. Methods Pre-treatment plasma samples were collected of patients with benign breast disease and breast cancer and divided over a discovery (n = 31) and validation (n = 84) cohort. From the discovery cohort miRs differentially expressed between benign and malignant cases were identified using a 2,000-miR microarray. Literature-based miRs differentiating benign from malignant disease were added. Using RT-qPCR, their expression was investigated in a validation cohort consisting of pre-treatment benign, malignant and metastatic samples. Additionally, benign and malignant cases were compared to benign and malignant cases of BRCA1-mutation carriers. Conclusions Plasma microRNA levels differed between patients with and without breast cancer, between benign disease from wildtype and BRCA1-mutation carriers and between breast cancer with and without metastases. Hsa-miR-652-5p was validated as a potential biomarker for breast cancer.

Prospects of targeting the gastrin releasing peptide receptor and somatostatin receptor 2 for nuclear imaging and therapy in metastatic breast cancer

Background The gastrin releasing peptide receptor (GRPR) and the somatostatin receptor 2 (SSTR2) are overexpressed on primary breast cancer (BC), making them ideal candidates for receptor-mediated nuclear imaging and therapy. The aim of this study was to determine whether these receptors are also suitable targets for metastatic BC. Methods mRNA expression of human BC samples were studied by in vitro autoradiography and associated with radioligand binding. Next, GRPR and SSTR2 mRNA levels of 60 paired primary BCs and metastases from different sites were measured by quantitative reverse transcriptase polymerase chain reaction. Receptor mRNA expression levels were associated with clinico-pathological factors and expression levels of primary tumors and corresponding metastases were compared. Results Binding of GRPR and SSTR radioligands to tumor tissue correlated significantly with receptor mRNA expression. High GRPR and SSTR2 mRNA levels were associated with estrogen receptor (ESR1)-positive tumors (p<0.001 for both receptors). There was no significant difference in GRPR mRNA expression of primary tumors versus paired metastases. Regarding SSTR2 mRNA expression, there was also no significant difference in the majority of cases, apart from liver and ovarian metastases which showed a significantly lower expression compared to the corresponding primary tumors (p = 0.02 and p = 0.03, respectively). Conclusion Targeting the GRPR and SSTR2 for nuclear imaging and/or treatment has the potential to improve BC care in primary as well as metastatic disease.