Isolation and Molecular Profiling of Bone Marrow Micrometastases Identifies TWIST1 as a Marker of Early Tumor Relapse in Breast Cancer Patients (original) (raw)
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Breast Cancer Research, 2012
High TWIST1 mRNA expression is associated with poor prognosis in lymph node-negative and estrogen receptor-positive human breast cancer and is co-expressed with stromal as well as ECM related genes Abstract Introduction: The TWIST homolog 1 (TWIST1) is a transcription factor that induces epithelial to mesenchymal transition (EMT), a key process in metastasis. The purpose of this study was to investigate whether TWIST1 expression predicts disease progression in a large breast cancer cohort with long-term clinical follow-up, and to reveal the biology related to TWIST1 mediated disease progression. Methods: TWIST1 mRNA expression level was analyzed by quantitative real-time reverse polymerase chain reaction (RT-PCR) in 1,427 primary breast cancers. In uni-and multivariate analysis using Cox regression, TWIST1 mRNA expression level was associated with metastasis-free survival (MFS), disease-free survival (DFS) and overall survival (OS). Separate analyses in lymph node-negative patients (LNN, n = 778) who did not receive adjuvant systemic therapy, before and after stratification into estrogen receptor (ER)-positive (n = 552) and ER-negative (n = 226) disease, were also performed. The association of TWIST1 mRNA with survival endpoints was assessed using Kaplan-Meier analysis. Using gene expression arrays, genes showing a significant Spearman rank correlation with TWIST1 were used to identify overrepresented Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-annotated biological pathways.
Molecular Oncology, 2007
Keywords: DNA microarrays Breast cancer Micrometastases in bone marrow Molecular subtypes Clinical outcome A B S T R A C T Expression profiles of primary breast tumors were investigated in relation to disseminated tumor cells (DTCs) in bone marrow (BM) in order to increase our understanding of the dissemination process. Tumors were classified into five pre-defined molecular subtypes, and presence of DTC identified (at median 85 months follow-up) a subgroup of luminal A patients with particular poor outcome ( p ¼ 0.008). This was not apparent for other tumor subtypes. Gene expression profiles associated with DTC and with systemic relapse for luminal A patients were identified. This study suggests that DTC in BM differentially distinguishes clinical outcome in patients with luminal A type tumors and that DTC-associated gene expression analysis may identify genes of potential importance in tumor dissemination. (T. Sørlie). a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m w w w . e l s e v i e r . c o m / l o c a t e / m o l o n c 1574-7891/$ -see front matter ª
TWIST1Expression in Breast Cancer Cells Facilitates Bone Metastasis Formation
Journal of Bone and Mineral Research, 2014
The transcription factor TWIST1 induces epithelial-mesenchymal transition and/or escape to the oncogenic-induced failsafe program, facilitating the intravasation of breast cancer cells in the systemic circulation and their dissemination to the lungs. Its involvement in breast cancer bone metastasis is unknown. To address this question, human osteotropic MDA-MB-231/B02 breast cancer cells were stably transfected with a Tet-inducible vector encoding for TWIST1, whose expression was specifically repressed in the presence of doxycycline (dox). The intra-arterial inoculation of transfectants expressing TWIST1 in immunodeficient mice substantially increased the extent of osteolytic lesions in these animals, being 50% larger than that of animals bearing mock-transfected tumors, as determined by radiography. This difference was accompanied by a sharp reduction of the bone volume (indicating a higher bone destruction) and a twofold increase in the tumor volume compared with mice bearing mock-transfected tumors, as determined by histomorphometry. Importantly, the suppression of TWIST1 expression in MDA-MB-231/B02 cells in the presence of dox abolished the stimulatory effect of TWIST1 on bone metastasis formation in vivo. Additionally, examination of the bone marrow from untreated and dox-treated animals on day 7 after tumor cell inoculation, at which time there was no evidence of radiographic osteolytic lesions, revealed that the number of tumor cell colonies that were recovered from the bone marrow of untreated mice was dramatically increased compared with that of dox-fed animals. In vitro, TWIST1 expression promoted tumor cell invasion and enhanced microRNA 10b (miR-10b) expression, a proinvasive factor, but was dispensable for growth of tumor cells. In vivo, the repression of miR-10b substantially decreased the presence of TWIST1-expressing breast cancer cells in the bone marrow. Overall, these results establish that TWIST1 facilitates breast cancer bone metastasis formation through a mechanism dependent of miR-10b, which leads to increase tumor burden and bone destruction.
Cells, 2019
(1) Background: The aim of the study was to evaluate the prognostic significance of EMT-associated (TWIST1) and stem-cell (SC) transcript (CD24, CD44, ALDH1) quantification in EpCAM+ circulating tumor cells (CTCs) of early breast cancer patients. (2) Methods: 100 early stage breast cancer patients and 19 healthy donors were enrolled in the study. CD24, CD44, and ALDH1 transcripts of EpCAM + cells were quantified using a novel highly sensitive and specific quadraplex RT-qPCR, while TWIST1 transcripts were quantified by single RT-qPCR. All patients were followed up for more than 5 years. (3) Results: A significant positive correlation between overexpression of TWIST1 and CD24 −/low /CD44 high profile was found. Kaplan-Meier analysis revealed that the ER/PR-negative (HR-) patients and those patients with more than 3 positive lymph nodes that overexpressed TWIST1 in EpCAM + cells had a significant lower DFI (log rank test; p < 0.001, p < 0.001) and OS (log rank test; p = 0.006, p < 0.001). Univariate and multivariate analysis also revealed the prognostic value of TWIST1 overexpression and CD24 −/low /CD44 high and CD24 −/low /ALDH1 high profile for both DFI and OS. (4) Conclusions: Detection of TWIST1 overexpression and stem-cell (CD24, CD44, ALDH1) transcripts in EpCAM + CTCs provides prognostic information in early stage breast cancer patients.
IMMUNOHISTOCHEMICAL STUDY OF TWIST1 IN INVASIVE DUCT CARCINOMA OF THE BREAST
Twist1 is a key transcription factor, which confers tumor cells with cancer stem cell (CSC)-like characteristics and enhances epithelial-mesenchymal transition (EMT) in pathological conditions including tumor malignancy and metastasis. This study aimed to evaluate the expression patterns and clinical significance of Twist1 in invasive duct carcinoma of the breast. The cytoplasmic and nuclear expressions of Twist1 were examined in 70 tumor tissues by immunohistochemistry. The association between expression patterns of this marker and clinicopathologic parameters was analyzed separately. Twist1 was localized to the cytoplasm of tumor cells in (21.4%) of cases. It was localized to the nucleus of tumor cells in (48.6%) of cases. Increased cytoplasmic expression of Twist1 was associated with smaller tumor size (P = 0.011). Nuclear Twist1 was positively correlated with higher grade tumors (P = 0.002), lymph node status (>3 lymph nodes) (P = 0.049), poor Nottingham prognostic index (p=0.043), ER and PR negativity (p=0.048 and 0.016 respectively), Molecular subtypes (p=0.038) and distant metastasis (p=0.001). Increased nuclear expression of Twist1 had a critical role in worse prognosis in breast cancer. These findings suggest that nuclear, rather than cytoplasmic expression of Twist1 can be considered as a prognostic and therapeutic marker for targeted therapy of breast cancer patients.
TWIST1 Gene expression as a biomarker for predicting primary doxorubicin resistance in breast cancer
Balkan Journal of Medical Genetics, 2019
Doxorubicin is one of the most commonly used chemotherapeutic agents for adjuvant chemotherapy of breast cancer. In the studies focused on finding biomarkers to predict the response of the patients and tumors to the drugs used, the Twist transcription factor has been suggested as a candidate biomarker for predicting chemo-resistance of breast tumors. In this study, we aimed to investigate the relationship between TWIST transcription factor expression and the effectiveness of doxorubicin treatment on directly taken primary tumor samples from chemotherapy-naive breast cancer patients. Twenty-six primary breast tumor samples taken from 26 different breast cancer patients were included in this study. Adenosine triphosphate tumor chemo-sensitivity assay (ATP-TCA) has been used to determine tumor response to doxorubicin and real-time reverse-transcription polymerase chain reaction (RT-PCR) was used for analyzing the TWIST1 gene expression of tumors. There was a significant difference in T...
Oncogene, 2012
TWIST1 is a highly conserved basic helix-loop-helix transcription factor that promotes epithelial-mesenchymal transition (EMT). Its misregulation has been observed in various types of tumors. Using the MCF-10A-series of cell lines that recapitulate the early stages of breast cancer formation and EMT, we found TWIST1 to be upregulated during EMT and downregulated early in carcinogenesis. The TWIST1 3 0 UTR contains putative regulatory elements, including miRNA target sites and two cytoplasmic polyadenylation elements (CPE). We found that miR-580, CPEB1, and CPEB2 act as negative regulators of TWIST1 expression in a sequence-specific and additive/cooperative manner.
International Journal of Oncology, 2007
The incidence and prognostic relevance of bone marrow (BM) and leukapheresis (PBPC) tumor cell contamination (TCC) in breast cancer patients is still to be circumstantiated. We developed a new comprehensive gene expression panel to study cytokeratins (CK), maspin (MAS) and mammaglobin (MAM) as possible predictors of prognosis. Forty-eight patients undergoing high dose chemotherapy (HDCT) and PBPC support were enrolled and analyzed for TCC on 116 PBPC apheresis and 96 BM obtained at basal conditions. All of the patients were evaluated by reverse transcriptase nested PCR (RT-PCR) for MAM and MAS gene expression and by immunocytochemistry (ICC) and nested RT-PCR to evaluate CK expression. PBPC and BM frequency of CK-positive (+) cells was 12-13% by ICC and 71-73% by RT-PCR respectively. Sixty-seven percent of CK ICC + samples were MAM RT-PCR + and 89% of them were MAS RT-PCR +. PBPC and BM frequency of MAM + cells was 21% and 31% respectively, while for MAS + cells it was 48% and 52% respectively by RT-PCR. After 71 mo median FU, 16 patients (33%) relapsed and 14 (88%) had BM/PBPC TCC. No marker had an impact on overall survival (OS) but MAS expression on BM and MAM expression on PBPC correlated with a statistically significant improved (p=0.05) and worsened RFS (p=0.06) respectively. These data confirm the activity of MAM as a negative prognostic factor and show for the first time that MAS could work as a tumor suppressor gene even in a clinical setting, since it protects from recurrence.
Cancer Epidemiology, Biomarkers & Prevention, 2008
TWIST1, an antiapoptotic and prometastatic transcription factor, is overexpressed in many epithelial cancers including breast. Only little is known regarding the regulation of TWIST1 in these cancers. Recently, an increase in the TWIST1 promoter methylation has been shown in breast cancers. To correlate the percentage of TWIST1 promoter methylation to the protein levels, we analyzed simultaneously the methylation status as well as the mRNA and the percentage of cells expressing TWIST1 in normal breast tissue and 76 invasive breast cancers. We found that TWIST1 promoter methylation is significantly more prevalent in malignant compared with healthy breast tissue. Furthermore, the percentage of cells expressing TWIST1 was greater in breast malignancy compared with matched healthy tissue from the same patients. There was no correlation, however, between TWIST1 promoter methylation and TWIST1 protein or RNA expression. This indicates that although TWIST1 CpG methylation is useful as a bi...
Minimal residual disease (MRD) in the bone marrow in ER-α-positive primary breast cancer patients
Breast Cancer Research, 2005
Introduction Prognostic and predictive factors play important roles in the treatment of breast cancer. Genome-wide monitoring of gene expression using DNA microarrays makes it possible to study thousands of genes in a tumour sample in a single experiment. By looking for an association between the gene expression pattern and tumour behaviour, it should be possible to identify new prognostic and predictive factors. Method We used gene expression profiling using two different microarray platforms: one containing 25,000 oligonucleotide probes and one containing 18,000 cDNA probes. To obtain prognostic gene expression profiles, we isolated RNA from tumours from a series of 295 patients younger than 53 years presenting with stage I and II breast cancer treated at our institute between 1984 and 1993. The expression of 25,000 genes was assessed, and using various statistical approaches correlation of gene expression with distant metastasis-free probability and overall survival was assessed [1-3]. In addition, we started studies to obtain gene expression profiles predicting response to specific chemotherapy regimens. Within a single-institution, randomized phase II trial, patients with locally advanced breast cancer received six courses of either AC (n = 24) or AD (n = 24) containing neoadjuvant chemotherapy. Gene expression profiles for 18,000 genes were generated from core needle biopsies obtained before treatment and correlated with the response of the primary tumour to the chemotherapy administered [4]. Additionally, pretreatment gene expression profiles were compared with those in tumours remaining after chemotherapy. Results We previously identified a 70-gene expression profile associated with increased risk for developing distant metastases within 5 years [1,2]. More recently, we studied a Wound Signature in these same tumors [3]. By combining the 70-gene expression profile to subdivide the tumours into 'good prognosis' and 'poor prognosis' tumours, and the Wound signature to subdivide tumours into 'activated' and 'quiescent' tumours, subgroups of patients with markedly different prognosis can be identified. Additional gene expression signatures are being tested in this series of tumours to arrive at an optimal prognostic classifier and to obtain improved insight into breast cancer biology. In the study to identify predictive profiles, 10 (20%) of the 48 patients showed (near) pathological complete remission of the primary tumour after treatment [4]. No gene expression pattern correlating with response could be identified for all patients, or for the AC or AD treated groups separately. Conclusion Various gene expression profiles in breast cancer are associated with the propensity of the tumour to develop distant metastases. Gene expression profile predicting the response of primary breast carcinomas to AC or AD based neoadjuvant chemotherapy are most likely to be very subtle and cannot be detected when small series of patients are studied. Genetic tests derived from gene expression profiling studies are likely to become useful as prognostic and predictive tests to guide clinical decision making in the treatment of primary breast cancer. S2 Gene expression profiles and molecular classification to predict distant metastasis and tamoxifen-resistant breast cancer