Identification of mechanisms of resistance to treatment with abiraterone acetate or enzalutamide in patients with castration-resistant prostate cancer (CRPC).

BACKGROUND: Two androgen receptor (AR)-targeted therapies, enzalutamide and abiraterone acetate plus prednisone (abiraterone), have been approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). Many patients respond to these agents, but both de novo and acquired resistance are common. The authors characterized resistant phenotypes that emerge after treatment with abiraterone or enzalutamide. METHODS: Patients who received abiraterone or enzalutamide in the course of routine clinical care were consented for serial blood collection. A proprietary system (CellSearch) was used to enumerate and enrich circulating tumor cells (CTCs). RNA-sequencing (RNA-seq) was performed on pools of up to 10 epithelial cell adhesion molecule (EpCAM)-positive/CD45-negative CTCs. The impact of gene expression changes observed in CTCs between patients who responded or were resistant to abiraterone/enzalutamide therapies was further explored in a model cell line system. RESULTS: RNA-seq data from CTCs identified mutations commonly associated with CRPC as well as novel mutations, including several in the ligand-binding domain of AR that could facilitate escape from AR-targeted agents. Ingenuity pathway analysis of differentially regulated genes identified the transforming growth factor ß (TGFß) and cyclin D1 (CCND1) signaling pathways as significantly upregulated in drug-resistant CTCs. Transfection experiments using enzalutamide-sensitive and enzalutamide-resistant LNCaP cells confirmed the involvement of SMAD family member 3, a key mediator of the TGFß pathway, and of CCND1 in resistance to enzalutamide treatment. CONCLUSIONS: The current results indicate that RNA-seq of CTCs representing abiraterone and enzalutamide sensitive and resistant states can identify potential mechanisms of resistance. Therapies targeting the downstream signaling mediated by SMAD family member 3 (SMAD3) and CCND1, such as cyclin-dependent kinase 4/cyclin-dependent kinase 6 inhibitors, could provide new therapeutic options for the treatment of antiandrogen-resistant disease. Cancer 2018;124:1216-24. © 2017 American Cancer Society.

Genetic variation in radiation-induced cell death.

Radiation exposure through environmental, medical, and occupational settings is increasingly common. While radiation has harmful effects, it has utility in many applications such as radiotherapy for cancer. To increase the efficacy of radiation treatment and minimize its risks, a better understanding of the individual differences in radiosensitivity and the molecular basis of radiation response is needed. Here, we integrated human genetic and functional genomic approaches to study the response of human cells to radiation. We measured radiation-induced changes in gene expression and cell death in B cells from normal individuals. We found extensive individual variation in gene expression and cellular responses. To understand the genetic basis of this variation, we mapped the DNA sequence variants that influence expression response to radiation. We also identified radiation-responsive genes that regulate cell death; silencing of these genes by small interfering RNA led to an increase in radiation-induced cell death in human B cells, colorectal and prostate cancer cells. Together these results uncovered DNA variants that contribute to radiosensitivity and identified genes that can be targeted to increase the sensitivity of tumors to radiation.

Genetic analysis of radiation-induced changes in human gene expression.

Humans are exposed to radiation through the environment and in medical settings. To deal with radiation-induced damage, cells mount complex responses that rely on changes in gene expression. These gene expression responses differ greatly between individuals and contribute to individual differences in response to radiation. Here we identify regulators that influence expression levels of radiation-responsive genes. We treated radiation-induced changes in gene expression as quantitative phenotypes, and conducted genetic linkage and association studies to map their regulators. For more than 1,200 of these phenotypes there was significant evidence of linkage to specific chromosomal regions. Nearly all of the regulators act in trans to influence the expression of their target genes; there are very few cis-acting regulators. Some of the trans-acting regulators are transcription factors, but others are genes that were not known to have a regulatory function in radiation response. These results have implications for our basic and clinical understanding of how human cells respond to radiation.

ATM gene mutations result in both recessive and dominant expression phenotypes of genes and microRNAs.

The defining characteristic of recessive disorders is the absence of disease in heterozygous carriers of the mutant alleles. However, it has been recognized that recessive carriers may differ from noncarriers in some phenotypes. Here, we studied ataxia telangiectasia (AT), a classical recessive disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. We compared the gene and microRNA expression phenotypes of noncarriers, AT carriers who have one copy of the ATM mutations, and AT patients with two copies of ATM mutations. We found that some phenotypes are more similar between noncarriers and AT carriers compared to AT patients, as expected for a recessive disorder. However, for some expression phenotypes, AT carriers are more similar to the patients than to the noncarriers. Analysis of one of these expression phenotypes, TNFSF4 level, allowed us to uncover a regulatory pathway where ATM regulates TNFSF4 expression through MIRN125B (also known as miR-125b or miR125b) [corrected] In AT carriers and AT patients, this pathway is disrupted. As a result, the level of MIRN125B is lower and the level of its target gene, TNFSF4, is higher than in noncarriers. A decreased level of MIRN125B is associated with breast cancer, and an elevated level of TNFSF4 is associated with atherosclerosis. Thus, our findings provide a mechanistic suggestion for the increased risk of breast cancer and heart disease in AT carriers. By integrating molecular and computational analyses of gene and microRNA expression, we show the complex consequences of a human gene mutation.

Global gene expression profiling of circulating endothelial cells in patients with metastatic carcinomas.

Increased numbers of endothelial cells are observed in peripheral blood of cancer patients. These circulating endothelial cells (CECs) may contribute to the formation of blood vessels in the tumor or reflect vascular damage caused by treatment or tumor growth. Characterization of these cells may aid in the understanding of the angiogenic process and may provide biomarkers for treatment efficacy of angiogenesis inhibitors. To identify markers typical for CECs in cancer patients, we assessed global gene expression profiles of CD146 immunomagnetically enriched CECs from healthy donors and patients with metastatic breast, colorectal, prostate, lung, and renal cancer. From the generated gene profiles, a list of 61 marker genes for CEC detection was generated, and their expression was measured by real-time quantitative PCR in blood samples from 81 metastatic cancer patients and 55 healthy donors that were immunomagnetically enriched for CECs. A set of 34 genes, among which novel CEC-associated genes, such as THBD, BST1, TIE1, POSTN1, SELE, SORT1, and DTR, were identified that were expressed at higher levels in cancer patients compared with healthy donors. Expression of the VWF, DTR, CDH5, TIE, and IGFBP7 genes were found to discriminate between cancer patients and "healthy" donors with a receiver operating characteristic curve accuracy of 0.93. Assessment of the expression of these genes may provide biomarkers to evaluate treatment efficacy.

Global gene expression profiling of circulating tumor cells.

Metastases from primary tumors are responsible for most cancer deaths. It has been shown that circulating tumor cells (CTCs) can be detected in the peripheral blood of patients with a variety of metastatic cancers and that the presence of these cells is associated with poor clinical outcomes. Characterization of CTCs in metastatic cancer patients could provide additional information to augment management of the disease. Here, we describe a novel approach for the identification of molecular markers to detect and characterize CTCs in peripheral blood. Using an integrated platform to immunomagnetically isolate and immunofluorescently detect CTCs, we obtained blood containing > or = 100 CTCs from one metastatic colorectal, one metastatic prostate, and one metastatic breast cancer patient. Using the RNA extracted from the CTC-enriched portion of the sample and comparing it with the RNA extracted from the corresponding CTC-depleted portion, for the first time, global gene expression profiles from CTCs were generated and a list of cancer-specific, CTC-specific genes was obtained. Subsequently, samples immunomagnetically enriched for CTCs from 74 metastatic cancer patients and 50 normal donors were used to confirm by quantitative real-time reverse transcription-PCR CTC-specific expression of selected genes and to show that gene expression profiles for CTCs may be used to distinguish normal donors from advanced cancer patients as well as to differentiate among the three different metastatic cancers. Genes such as AGR2, S100A14, S100A16, FABP1, and others were found useful for detection of CTCs in peripheral blood of advanced cancer patients.

Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells.

BACKGROUND: Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive. RESULTS: In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most. MATERIALS AND METHODS: We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms. CONCLUSION: We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.

Isolation and characterization of circulating tumor cells in patients with metastatic colorectal cancer.

PURPOSE: Development of targeted therapeutic agents in colorectal cancer (CRC) is impeded by the lack of a noninvasive surrogate of drug effect. This pilot study evaluated the ability of immunomagnetic separation to isolate circulating tumor cells (CTCs) and of the fluorescent microscope system and flow cytometry to enumerate and characterize CTCs from patients with metastatic CRC. PATIENTS AND METHODS: Fifty patients with metastatic CRC contributed 50 mL of blood at treatment initiation and disease evaluation timepoints. Fresh tumor specimens were obtained from 17 patients for comparison of circulating and in situ tumor cell characteristics. Epithelial cells were magnetically isolated from whole blood targeting the antiepithelial cell adhesion molecule (EpCAM). Circulating tumor cells were defined as EpCAM isolated, cytokeratin positive, nuclear stain positive, and CD45 negative. Total RNA was isolated from EpCAM-enriched CTCs and multigene reverse-transcriptase polymerase chain reaction analyses were performed. RESULTS: The median number of CTCs detected by flow cytometry was 2/7.5 mL blood. Mean change in cell count was significantly different for patients with tumor progression versus nonprogression (+6.7 vs. +0.2/7.5 mL; P = 0.001). A correlation was noted between mean fluorescence intensity (flow cytometry) of cytokeratin in CTC and matched tumor specimens (r = 0.79, P = 0.06). Nearly 80% (15 of 19) of samples with >or= 2 CTCs expressed >or= 1 epithelial marker gene (CK19, CK20, carcinoembryonic antigen, or epidermal growth factor receptor). CONCLUSION: Isolating and characterizing CTCs from patients with metastatic CRC is feasible. Change in the CTC number might reflect clinical status, and flow cytometric and gene expression data suggest similarity of circulating and in situ tumor cells. Further evaluation of CTCs for pharmacodynamic and clinical monitoring in patients with CRC is warranted.

Microfluidics and circulating tumor cells.

Circulating tumor cells (CTCs) are shed from cancerous tumors, enter the circulatory system, and migrate to distant organs to form metastases that ultimately lead to the death of most patients with cancer. Identification and characterization of CTCs provides a means to study, monitor, and potentially interfere with the metastatic process. Isolation of CTCs from blood is challenging because CTCs are rare and possess characteristics that reflect the heterogeneity of cancers. Various methods have been developed to enrich CTCs from many millions of normal blood cells. Microfluidics offers an opportunity to create a next generation of superior CTC enrichment devices. This review focuses on various microfluidic approaches that have been applied to date to capture CTCs from the blood of patients with cancer.

Physiological stress induces the metastasis marker AGR2 in breast cancer cells.

As an approach to understanding the factors that activate expression of tumor progression genes, the role of physiological stress in the activation of a panel of tumor cell markers was investigated. These studies identify the developmental gene product, anterior gradient 2 (AGR2) as a cancer cell marker specifically up-regulated in response to depletion of serum and oxygen. AGR2 has been identified as a tumor marker in primary and secondary cancer lesions, and as a marker for detection of circulating tumor cells (CTCs). Elevated levels of AGR2 are known to increase the metastatic potential of cancer cells, but conditions leading to increased expression of AGR2 are not well understood. The present results identify novel physiological parameters likely to contribute to AGR2 induction in situ.

Identification of genes associated with adenovirus 12 tumorigenesis by microarray.

A total of 242 genes were shown to be differentially expressed between haplotypically matched tumorigenic adenovirus 12 (Ad12) and nontumorigenic Ad5-transformed cells using a microarray containing 8734 cDNAs. Eighty-seven of the differentially expressed genes have known roles that include signal transduction, cell growth and proliferation, transcription regulation, protease, and immune functions. The remaining differentially expressed genes are represented by EST cDNAs which have functions that are either completely unknown or proposed, based on sequence similarity to known genes. A subset of 22 differentially expressed genes from the microarray was further examined by Northern blot analyses to verify the identification of new genes associated with Ad12 tumorigenesis. Growth factor receptor binding protein 10 (Grb10) and protease nexin 1 (PN-1) were overexpressed in all of the tumorigenic Ad12-transformed cells examined, whereas expression of these genes was negligible in all of the nontumorigenic Ad5-transformed cells. By contrast, other genes including B cell translocation gene 2 (BTG2) were shown to be significantly up-regulated in Ad5-transformed cells as compared to Ad12-transformed cells.

Method for manufacturing whole-genome microarrays by rolling circle amplification.

Comparative genomic hybridization (CGH) to metaphase chromosomes is a method for genome-wide detection of chromosomal aberrations in DNA samples. Recent advances in microarray technology have improved CGH by replacing metaphase chromosomes with a collection of mapped genomic clones placed on glass slides. However, it is quite expensive and labor-intensive to prepare DNA from the genomic clones for use in constructing genomic microarrays. Here we used strand-displacement rolling circle amplification (RCA) to manufacture whole-genome microarrays by using a collection of about 4,500 mapped RPCI-11 BAC clones that cover the human genome at approximately a 1-Mb resolution. These genomic microarrays detected all major chromosomal aberrations in cancer cells lines and in cell lines with aneuploidy. In this article, we discuss the advantages of using RCA for the manufacturing of large genomic microarrays.