Ged Brady

Molecular analysis of circulating tumour cells—biology and biomarkers

Growing evidence for intratumour heterogeneity informs us that single-site biopsies fall short of revealing the complete genomic landscape of a tumour. With an expanding repertoire of targeted agents entering the clinic, screening tumours for genomic aberrations is increasingly important, as is interrogating the tumours for resistance mechanisms upon disease progression. Multiple biopsies separated spatially and temporally are impractical, uncomfortable for the patient and not without risk. Here, we describe how circulating tumour cells (CTCs), captured from a minimally invasive blood test—and readily amenable to serial sampling—have the potential to inform intratumour heterogeneity and tumour evolution, although it remains to be determined how useful this will be in the clinic. Technologies for detecting and isolating CTCs include the validated CellSearch® system, but other technologies are gaining prominence. We also discuss how recent CTC discoveries map to mechanisms of haematological spread, previously described in preclinical models, including evidence for epithelial–mesenchymal transition, collective cell migration and cells with tumour-initiating capacity within the circulation. Advances in single-cell molecular analysis are enhancing our ability to explore mechanisms of metastasis, and the combination of CTC and cell-free DNA assays are anticipated to provide invaluable blood-borne biomarkers for real-time patient monitoring and treatment stratification.

Tracking genomic cancer evolution for precision medicine: the lung TRACERx study.

TRACERx, a prospective study of patients with primary non-small cell lung cancer, aims to map the genomic landscape of lung cancer by tracking clonal heterogeneity and tumour evolution from diagnosis to relapse.

Application of Sequencing, Liquid Biopsies, and Patient-Derived Xenografts for Personalized Medicine in Melanoma

UNLABELLED Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell-derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. SIGNIFICANCE Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments.

Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.

EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer.

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene ‘hyperplastic discs’ and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT–PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.

Molecular analysis of circulating tumor cells identifies distinct copy-number profiles in patients with chemosensitive and chemorefractory small-cell lung cancer

In most patients with small-cell lung cancer (SCLC)—a metastatic, aggressive disease—the condition is initially chemosensitive but then relapses with acquired chemoresistance. In a minority of patients, however, relapse occurs within 3 months of initial treatment; in these cases, disease is defined as chemorefractory. The molecular mechanisms that differentiate chemosensitive from chemorefractory disease are currently unknown. To identify genetic features that distinguish chemosensitive from chemorefractory disease, we examined copy-number aberrations (CNAs) in circulating tumor cells (CTCs) from pretreatment SCLC blood samples. After analysis of 88 CTCs isolated from 13 patients (training set), we generated a CNA-based classifier that we validated in 18 additional patients (testing set, 112 CTC samples) and in six SCLC patient-derived CTC explant tumors. The classifier correctly assigned 83.3% of the cases as chemorefractory or chemosensitive. Furthermore, a significant difference was observed in progression-free survival (PFS) (Kaplan–Meier P value = 0.0166) between patients designated as chemorefractory or chemosensitive by using the baseline CNA classifier. Notably, CTC CNA profiles obtained at relapse from five patients with initially chemosensitive disease did not switch to a chemorefractory CNA profile, which suggests that the genetic basis for initial chemoresistance differs from that underlying acquired chemoresistance.

Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA

The application of precision medicine requires in-depth characterisation of a patients tumours and the dynamics of their responses to treatment. We used next-generation sequencing of cfDNA to monitor therapy responses of a metastatic vaginal mucosal melanoma and show that cfDNA can be used to monitor tumour evolution and subclonal responses to therapy even when biopsies are not available.

Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

BRAF mutation testing from circulating free DNA (cfDNA) using the amplification refractory mutation testing system (ARMS) holds potential as a surrogate for tumor mutation testing. Robust assay validation is needed to establish the optimal clinical matrix for measurement and cfDNA-specific mutation calling criteria. Plasma- and serum-derived cfDNA samples from 221 advanced melanoma patients were analyzed for BRAF c.1799T>A (p.V600E) mutation using ARMS in two stages in a blinded fashion. cfDNA-specific mutation calling criteria were defined in stage 1 and validated in stage 2. cfDNA concentrations in serum and plasma, and the sensitivities and specificities of BRAF mutation detection in these two clinical matrices were compared. Sensitivity of BRAF c.1799T>A (p.V600E) mutation detection in cfDNA was increased by using mutation calling criteria optimized for cfDNA (these criteria were adjusted from those used for archival tumor biopsies) without compromising specificity. Sensitivity of BRAF mutation detection in serum was 44% (95% CI, 35% to 53%) and in plasma 52% (95% CI, 43% to 61%). Specificity was 96% (95% CI, 90% to 99%) in both matrices. Serum contains significantly higher total cfDNA than plasma, whereas the proportion of tumor-derived mutant DNA was significantly higher in plasma. Using mutation calling criteria optimized for cfDNA improves sensitivity of BRAF c.1799T>A (p.V600E) mutation detection. The proportion of tumor-derived cfDNA in plasma was significantly higher than in serum.

SnapShot: Circulating Tumor Cells

Circulating tumor cells in the blood of patients are both signal flares for the existence of a tumor and harbingers of metastasis. With recent technological developments, these cells can be isolated and analyzed to provide insights into the biology of cancer spread and response to therapy and to offer new avenues for blood biomarker development.

Next-Generation Sequencing Analysis and Algorithms for PDX and CDX Models

Patient-derived xenograft (PDX) and circulating tumor cell–derived explant (CDX) models are powerful methods for the study of human disease. In cancer research, these methods have been applied to multiple questions, including the study of metastatic progression, genetic evolution, and therapeutic drug responses. As PDX and CDX models can recapitulate the highly heterogeneous characteristics of a patient tumor, as well as their response to chemotherapy, there is considerable interest in combining them with next-generation sequencing to monitor the genomic, transcriptional, and epigenetic changes that accompany oncogenesis. When used for this purpose, their reliability is highly dependent on being able to accurately distinguish between sequencing reads that originate from the host, and those that arise from the xenograft itself. Here, we demonstrate that failure to correctly identify contaminating host reads when analyzing DNA- and RNA-sequencing (DNA-Seq and RNA-Seq) data from PDX and CDX models is a major confounding factor that can lead to incorrect mutation calls and a failure to identify canonical mutation signatures associated with tumorigenicity. In addition, a highly sensitive algorithm and open source software tool for identifying and removing contaminating host sequences is described. Importantly, when applied to PDX and CDX models of melanoma, these data demonstrate its utility as a sensitive and selective tool for the correction of PDX- and CDX-derived whole-exome and RNA-Seq data. Implications: This study describes a sensitive method to identify contaminating host reads in xenograft and explant DNA- and RNA-Seq data and is applicable to other forms of deep sequencing. Mol Cancer Res; 15(8); 1012–6. ©2017 AACR.