Induction by transforming growth factor-β1 of epithelial to mesenchymal transition is a rare event in vitro

INTRODUCTION: Transforming growth factor (TGF)-β1 is proposed to inhibit the growth of epithelial cells in early tumorigenesis, and to promote tumor cell motility and invasion in the later stages of carcinogenesis through the induction of an epithelial to mesenchymal transition (EMT). EMT is a multistep process that is characterized by changes in cell morphology and dissociation of cell–cell contacts. Although there is growing interest in TGF-β1-mediated EMT, the phenotype is limited to only a few murine cell lines and mouse models. METHODS: To identify alternative cell systems in which to study TGF-β1-induced EMT, 18 human and mouse established cell lines and cultures of two human primary epithelial cell types were screened for TGF-β1-induced EMT by analysis of cell morphology, and localization of zonula occludens-1, E-cadherin, and F-actin. Sensitivity to TGF-β1 was also determined by [(3)H]thymidine incorporation, flow cytometry, phosphorylation of Smad2, and total levels of Smad2 and Smad3 in these cell lines and in six additional cancer cell lines. RESULTS: TGF-β1 inhibited the growth of most nontransformed cells screened, but many of the cancer cell lines were insensitive to the growth inhibitory effects of TGF-β1. In contrast, TGF-β1 induced Smad2 phosphorylation in the majority of cell lines, including cell lines resistant to TGF-β1-mediated cell cycle arrest. Of the cell lines screened only two underwent TGF-β1-induced EMT. CONCLUSION: The results presented herein show that, although many cancer cell lines have lost sensitivity to the growth inhibitory effect of TGF-β1, most show evidence of TGF-β1 signal transduction, but only a few cell lines undergo TGF-β1-mediated EMT

Increased CpG methylation of the estrogen receptor gene in BRCA1-linked estrogen receptor-negative breast cancers

A distinctive feature of BRCA1-linked breast cancers is that they typically do not express estrogen receptor-alpha (ERalpha). Previous investigation suggests that methylation of CpGs within the ERa promoter mediates repression of gene expression in some ERalpha-negative breast cancers. To determine if methylation of CpGs within the ERalpha promoter is associated with BRCA1-linked breast cancers, we evaluated methylation in exon 1 of the ERalpha gene in 40 ERalpha-negative breast cancers, 20 of which were non BRCA1-linked and 20 BRCA1-linked. CpG methylation was evaluated by either methylation-sensitive restriction digest (HpaII), methylation-sensitive PCR (MSP), or direct sequencing of bisulfite-treated genomic DNA. Results from HpaII digests and MSP documented a high degree of methylation, the MSP data showing slightly higher methylation in the BRCA1-linked group. CpGs analysed by direct sequencing showed an overall average methylation of 25% among non BRCA1-linked cancers and 40% among BRCA1-linked cancers (P=0.0031). The most notable difference was found at five particular CpGs, each of which exhibited a greater than twofold increase in methylation in the BRCA1-linked group compared to the non BRCA1-linked group (P < 0.03 for each CpG). Methylation of certain critical CpGs may represent an important factor in transcriptional repression of the ERa gene in BRCA1-linked breast cancers

Abstract P6-07-03: Broken promise of liquid biopsy: Plasma DNA does not accurately reflect tumor DNA in metastatic breast cancer

Abstract Background: Circulating tumor DNA in plasma may present a minimally invasive approach to identify tumor-derived mutations that could be used to inform the selection of targeted therapies for individual patients, particularly in cases of metastatic disease where biopsy is often difficult. We hypothesized that plasma DNA will genetically reflect DNA derived from multiple tumors in patients with metastatic breast cancer. To test this hypothesis and assess the utility of plasma DNA obtained as a “liquid biopsy” for precision medicine, we sought to determine whether massively parallel sequencing of plasma DNA is a reliable surrogate for sequencing of DNA from tissue biopsies in patients with metastatic breast cancer. Methods: Blood samples were obtained from 7 patients with multiple advanced breast cancer lesions (recurrent breast and metastatic tumors), and tumor specimens were obtained thereafter by biopsy or surgical excision. DNA extracted from plasma, buffy coat of blood, and tumor tissues was used for probe-directed capture of all exons in 196 genes followed by massively parallel sequencing with an average coverage of 3000x for plasma DNA. Tumor and plasma DNA sequences were bioinformatically compared to buffy coat controls, and high-confidence somatic mutations were called. One patient with extensive metastatic disease was evaluated in further detail to study the contribution of different tumors to the overall plasma DNA pool. In this patient, 9 metastatic tumors were sampled in an axillary lymph node, heart, kidney (2), liver, omentum (3), and ovary by biopsy or at autopsy. Results: Mutations were found in plasma that were represented in one or more tumors in each patient. Three classes of mutations were discovered: 1) mutations overlapping between both plasma and tumors (e.g., TP53 p.R273C and SRC p.E527K); 2) mutations found in plasma but not tumors (e.g., AKT p.E17K and multiple known and novel ESR1 mutations); 3) mutations found in tumors but not plasma (e.g., PIK3CA p.H1047R, p.D350G, and p.N345K). The presence of mutations in each of these classes was validated in plasma and/or tumors using mutation-specific droplet digital PCR (ddPCR). In the patient with extensive metastatic disease, DNA sequencing revealed heterogeneity of tumor contribution to plasma DNA, with some tumors better represented than others. No correlation was found between tumor size (measured by CT scan) and mutational burden in plasma. Interestingly, a significant correlation was found between blood perfusion to the organ where the tumor resides and mutational burden in plasma, with the greatest tumor contribution coming from the heart metastasis (Pearson's r = 0.835, p=0.039). Conclusions: Plasma DNA sequencing adds a layer of depth to sequencing analysis of tumor biopsy samples, and serves to both confirm tumor-derived mutations as well as detect new mutations. However, plasma DNA profiling does not comprehensively reflect the mutational profiles of tumors in patients with metastatic breast cancer, and thus is unlikely to serve as a surrogate for tumor biopsy as a source of DNA for genetic profiling. Furthermore, plasma DNA contains many mutations not found in tumors, which will confound treatment decision-making and precision medicine.Background: Circulating tumor DNA in plasma may present a minimally invasive approach to identify tumor-derived mutations that could be used to inform the selection of targeted therapies for individual patients, particularly in cases of metastatic disease where biopsy is often difficult. We hypothesized that plasma DNA will genetically reflect DNA derived from multiple tumors in patients with metastatic breast cancer. To test this hypothesis and assess the utility of plasma DNA obtained as a “liquid biopsy” for precision medicine, we sought to determine whether massively parallel sequencing of plasma DNA is a reliable surrogate for sequencing of DNA from tissue biopsies in patients with metastatic breast cancer. Methods: Blood samples were obtained from 7 patients with multiple advanced breast cancer lesions (recurrent breast and metastatic tumors), and tumor specimens were obtained thereafter by biopsy or surgical excision. DNA extracted from plasma, buffy coat of blood, and tumor tissues was used for probe-directed capture of all exons in 196 genes followed by massively parallel sequencing with an average coverage of 3000x for plasma DNA. Tumor and plasma DNA sequences were bioinformatically compared to buffy coat controls, and high-confidence somatic mutations were called. One patient with extensive metastatic disease was evaluated in further detail to study the contribution of different tumors to the overall plasma DNA pool. In this patient, 9 metastatic tumors were sampled in an axillary lymph node, heart, kidney (2), liver, omentum (3), and ovary by biopsy or at autopsy. Results: Mutations were found in plasma that were represented in one or more tumors in each patient. Three classes of mutations were discovered: 1) mutations overlapping between both plasma and tumors (e.g., TP53 p.R273C and SRC p.E527K); 2) mutations found in plasma but not tumors (e.g., AKT p.E17K and multiple known and novel ESR1 mutations); 3) mutations found in tumors but not plasma (e.g., PIK3CA p.H1047R, p.D350G, and p.N345K). The presence of mutations in each of these classes was validated in plasma and/or tumors using mutation-specific droplet digital PCR (ddPCR). In the patient with extensive metastatic disease, DNA sequencing revealed heterogeneity of tumor contribution to plasma DNA, with some tumors better represented than others. No correlation was found between tumor size (measured by CT scan) and mutational burden in plasma. Interestingly, a significant correlation was found between blood perfusion to the organ where the tumor resides and mutational burden in plasma, with the greatest tumor contribution coming from the heart metastasis (Pearson's r = 0.835, p=0.039). Conclusions: Plasma DNA sequencing adds a layer of depth to sequencing analysis of tumor biopsy samples, and serves to both confirm tumor-derived mutations as well as detect new mutations. However, plasma DNA profiling does not comprehensively reflect the mutational profiles of tumors in patients with metastatic breast cancer, and thus is unlikely to serve as a surrogate for tumor biopsy as a source of DNA for genetic profiling. Furthermore, plasma DNA contains many mutations not found in tumors, which will confound treatment decision-making and precision medicine. Citation Format: Shee K, Chamberlin MD, Varn FS, Bean JR, Marotti JD, Wells WA, Trask HW, Hamilton JS, West RJ, Kaufman PA, Schwartz GN, Gemery JM, McNulty NJ, Tsapakos MJ, Barth RJ, Arrick BA, Gui J, Cheng C, Miller TW. Broken promise of liquid biopsy: Plasma DNA does not accurately reflect tumor DNA in metastatic breast cancer [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 P6-07-03.</jats:p