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news and views Tumor signatures in the blood Michael R Speicher & Klaus Pantel npg © 2014 Nature America, Inc. All rights reserved. The goal of characterizing solid-tumor genomes with nothing more than a blood sample is now within reach. Taking biopsies to monitor primary tumors and metastases is often difficult for practical reasons, and cancer researchers have long searched for proxy measurements that would eliminate the need for tumor tissue samples1. Recent findings highlight the potential of three new approaches, all of which take advantage of the discovery that tumors shed parts of themselves into the blood. Analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) or tumor-derived exosomes could allow rapid monitoring of tumor genome evolution from a simple blood draw—an approach known as a ‘liquid biopsy’1 (Fig. 1). Two papers in this issue and one in Science Translational Medicine describe technologies for extracting molecular information about parent tumors from CTCs2, ctDNA3 and exosomes4, bringing the prospect of liquid biopsies a few steps closer to reality. Thanks to large-scale cancer genome sequencing projects, we now possess catalogs of genetic alterations that are present in a variety of human tumor types5. What we lack, however, are minimally invasive methods to monitor the presence of these genetic alterations in cancer patients. Serial biopsies of metastatic lesions are typically not practical because the metastatic tissue is often inaccessible or reachable only by invasive procedures. Moreover, different metastases in a single patient may harbor different genetic alterations6, and the purity and yield of biopsy samples are usually low. Writing in this issue, Lohr et al.2 sought to determine whether CTCs in the blood of prostate cancer patients can be used to analyze somatic single-nucleotide variants. Such projects Michael R. Speicher is at the Institute of Human Genetics, Medical University of Graz, Graz, Austria, and Klaus Pantel is at the Institute of Tumor Biology, University Medical Center Hamburg Eppendorf, Hamburg, Germany. e-mail: [email protected] or [email protected] face several major technical challenges. First, because a diploid cell has only 6.6 pg of DNA, which is insufficient for most types of detailed genomic analysis, the genome of single cells usually has to be amplified with a wholegenome amplification protocol (Fig. 1a). Lohr et al.2 achieved this using ‘multiple displacement’ amplification. However, the amplification success rate varied widely among single cells, as observed in previous studies. Another problem is that whole-genome amplification can introduce bias and polymerase errors, undermining the accuracy of variant calls. To overcome this, the authors developed a strategy for assessing the quality and uniformity of coverage in CTC whole genome amplification–derived libraries before performing in-depth, expensive sequencing analysis. Using low-pass (0.05× coverage) whole-genome sequencing and analysis of the correlation of single-base coverage across a chromosome, they were able to measure the level of amplification bias and accurately predict which single-cell CTC libraries were likely to yield robust data after in-depth (>100× coverage) whole-exome sequencing. However, even the libraries that passed the low-pass test showed non-uniform coverage after wholeexome sequencing. To address this issue, Lohr et al.2 combined data from independent CTC libraries, an approach called ‘census-based sequencing’ inspired by previous publications7. Indeed, combining multiple single CTC libraries markedly reduced the false-positive rate of called somatic single-nucleotide variants, and combining libraries from just three cells recovered >80% of the somatic variants previously identified in matched bulk tumor samples from the same patient. Using this optimized technique, the authors demonstrated that they could detect CTC mutations present early in tumor evolution or in the metastatic deposit. These founder or ‘trunk’ mutations are of high clinical relevance nature biotechnology volume 32 number 5 may 2014 because they represent potential therapeutic targets. They also identified panels of mutations in CTCs that did not overlap with those detected in the primary tumor or metastases. These nonoverlapping mutations are of interest as they may provide novel insights into the evolution of tumor genomes or predictive and prognostic biomarkers for clinical applications. In Science Translational Medicine, Bettegowda et al.3 start with cell-free ctDNA rather than CTCs as a source of information about tumorassociated somatic single-nucleotide variants and structural rearrangements in a large number of human tumor types (Fig. 1b). They found that the percentage of patients with detectable ctDNA varied substantially depending on tumor type, and within a single tumor type ctDNA was more likely to be detected in patients with more advanced disease. In patients with detectable ctDNA, the absolute number of mutant DNA fragments varied. The reasons for this variation are currently unknown, but mutant DNA abundance did correlate inversely with probability of two-year survival. To determine the sensitivity and specificity of ctDNA-based liquid biopsies, Bettegowda et al.3 focused on mutations in codons 12 and 13 of KRAS. Comparing results obtained using ctDNA or the primary tumor from the same patients, they achieved 99.2% specificity and 87.2% sensitivity with ctDNA. They also showed that ctDNA-based liquid biopsies can reveal the acquisition of mutations that likely contribute to resistance to targeted therapies. For example, mutations in EGFR pathway genes were detected in a substantial fraction of patients after but not before treatment with EGFR-blocking therapy. Surprisingly, many of these mutations were not observed in the traditional ‘hotspot region’ of codon 12 of the KRAS or NRAS gene but rather in codon 61. Notably, Bettegowda et al.3 sought to compare the quantities of mutant DNA in cell-free ctDNA and CTCs in the same patients. To this 441 npg 442 a CTCs CTC isolation WGA Cell lysis WTA c Au Marina Corral Spence/Nature Publishing Group b cfDNA end, they first identified genetic alterations in primary tumors by whole-genome sequencing. They then used PCR to detect these alterations in DNA isolated from the cell pellet (which contains CTCs but also white blood cells, platelets and other cellular fragments) and from the supernatant (which contains the ctDNA) obtained after centrifugation of blood. The analysis was limited to structural rearrangements because the background level of point mutations in PCR assays is too high to reliably detect tumor-associated somatic point mutations in rare CTCs. The results suggested that ctDNA is more sensitive than CTCs for detecting tumorassociated genetic rearrangements. However, this conclusion may depend on the method of isolating CTCs. Because CTCs are rare, constituting as few as 1 cell per 109 normal blood cells8, detection of tumor-associated genomic rearrangements from blood cell pellets without any enrichment for CTCs is challenging even with advanced quantitative PCR approaches. Thus, further studies using established CTC capture and enrichment systems will be needed to explore the relative sensitivity of assays based on ctDNA and CTCs. Also in this issue, Im et al.4 focus on a third type of material shed from tumors: exosomes (Fig. 1c). These small (50–100 nm) vesicles may represent a mobile source of information about the molecular makeup of the parental tumor, but it remains difficult to isolate and analyze them, especially in a high-throughput manner. Current isolation strategies require serial differential ultracentrifugation steps, size-based isolation or affinity purification with appropriate antibodies (e.g., markers like CD63 or EpCAM). For exosome analysis, traditional approaches like western blot and enyzme-linked immunosorbent assays require large amounts of material, which are often not available, and extensive processing, which hampers fast highthroughput analysis. To address these limitations of existing exosome isolation and analytical methods, Im et al.4 developed a strategy based on surface plasmon resonance that enables label-free, high-throughput characterization of proteins in exosomes. The approach uses a chip called a nano-plasmonic exosome (nPLEX) sensor, which consists of an array of periodic nanoholes patterned in a gold film. To enable specific capture of exosomes, the authors functionalized each array with affinity ligands specific for protein markers characteristic of exosomes (e.g., CD63). Exosome binding to the array changes the local refractive index of the nPLEX sensor to an extent proportional to target protein levels. Spectral shifts or changes in intensity are monitored to report both the concentration of exosomes and the abundance Exosomes © 2014 Nature America, Inc. All rights reserved. news and v i ews Figure 1 Minimally invasive approaches to monitor tumor genome evolution. (a) Circulating tumor cells (CTCs; blue) are isolated from total blood cells by cell separation systems. Cell lysis yields pure tumor DNA (gray) and RNA (black); for analysis of tumor-specific mutations, these are subjected to whole-genome or whole-transcriptome amplification, respectively (WGA, WTA). Proteins in CTCs can be analyzed by immunohistochemistry and, if viable, CTCs can be subjected to functional analyses8. (b) Cell-free DNA (cfDNA) is prepared from plasma by several centrifugation and filtration steps. The resulting DNA is a mixture of DNA fragments released from nonmalignant cells (brown) and from tumor cells (ctDNA; blue). Depending on the tumor stage, and especially during early disease, ctDNA may represent a minority of all cfDNA. cfDNA is analyzed by next-generation sequencing, which can reveal somatic copy number changes, single-nucleotide variants and rearrangements. (c) Exosomes can be efficiently captured from blood using the nPLEX assay and their molecular constituents analyzed; for example, proteins can be quantified by nPLEX, and RNA can be quantified or sequenced. of extra- and intravesicular proteins in each exosome. Although nPLEX and an enzymelinked immunosorbent assay reported similar expression levels for a panel of extravesicular markers, the nPLEX sensor was more sensitive, required smaller sample quantities and was faster. The nPLEX assay can detect as few as ~3,000 exosomes without labeling. The authors screened exosomes from different ovarian cancer cell lines to identify a set of protein markers capable of specifically detecting exosomes derived from ovarian cancer cells. Out of a panel of candidate markers, EpCAM and CD24 distinguished ovarian cancer–derived exosomes from vesicles originating from nonmalignant cells. In addition, in a small set of ovarian cancer patient samples, pre- and posttreatment changes in ascites exosomal EpCAM and CD24 abundance accurately classified patients as responding or not responding to therapy. Although Im et al.4 used the nPLEX assay to monitor exosomal proteins, it is also possible to release captured exosomes from the device and analyze their RNA content by quantitative real-time PCR. In this way exosomes can report on a subset of genomic alterations in the parental tumor. The technologies described in the three papers are important advances in developing liquid biopsies into a routine diagnostic method. Several key questions remain to be addressed. First, to ensure that the appropriate test is selected for each patient, we need more basic biological insight into the mechanisms by which CTCs, ctDNA and exosomes are released from different tumor types at different stages of disease. Whether release is altered by therapeutic interventions is also unknown. Related to this, many studies focus on patients with metastatic disease who are likely to have higher biomarker concentrations than patients with localized disease. More work is needed to validate the suitability of these technologies for primary diagnosis of cancer or for monitoring responses to adjuvant therapies in early-stage disease9. Second, amplification bias and sequencing artifacts are a concern for single-cell analysis in general. For example, because only approximately 12% of all CTCs studied by Lohr et al.2 passed the whole-genome amplification volume 32 number 5 may 2014 nature biotechnology npg © 2014 Nature America, Inc. All rights reserved. news and v i ews yield and library quality check, it was necessary to sequence multiple independent libraries and combine their results. This poses a problem for patients with low CTC numbers and precludes analysis of mutations that are unique to individual CTCs10. These mutations could lead to important insights into intrapatient tumor heterogeneity. For example, we recently performed targeted sequencing of 20 unique CTC mutations and found that 17 of them were also present in small subclones of the corresponding primary colorectal cancer or metastatic lesions from the same patients10. Third, regardless of the method chosen, it will be necessary to develop standardized protocols and to validate their clinical utility through multicenter clinical trials with defined endpoints, such as overall survival, before their integration into cancer diagnostics can be recommended. Independent of applications in cancer, the technologies reported in these studies may be useful in fields unrelated to liquid biopsy, such as minimally invasive prenatal tests to identify fetal aneuploidy and clinical genetics approaches for detecting suspected mosaicism. As such, they could eventually enhance patient care in a variety of clinical disciplines and further extend the reach of personalized medicine. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Pantel, K. & Alix-Panabieres, C. Cancer Res. 73, 6384–6388 (2013). 2. Lohr, J.G. et al. Nat. Biotechnol. 32, 479–484 (2014). 3. Bettegowda, C. et al. Sci. Transl. Med. 6, 224ra224 (2014). 4.Im, H. et al. Nat. Biotechnol. 32, 488–493 (2014). 5. Lawrence, M.S. et al. Nature 505, 495–501 (2014). 6. Gerlinger, M. et al. N. Engl. J. Med. 366, 883–892 (2012). 7. Hou, Y. et al. Cell 148, 873–885 (2012). 8. Pantel, K., Brakenhoff, R.H. & Brandt, B. Nat. Rev. Cancer 8, 329–340 (2008). 9. Wan, L., Pantel, K. & Kang, Y. Nat. Med. 19, 1450–1464 (2013). 10.Heitzer, E. et al. Cancer Res. 73, 2965–2975 (2013). Simply better glycoproteins Bernd Lepenies & Peter H Seeberger Reducing the glycan heterogeneity of recombinant proteins may improve the efficacy of biopharmaceuticals. Glycosylation is integral to more than half of eukaryotic proteins and influences many aspects of protein structure and function, including folding and stability, molecular recognition and immunogenicity1. Mammalian glycoproteins usually exist in many different glycoforms, and, in the context of protein therapeutics, this diversity can adversely affect drug potency and pharmacokinetics. In this issue, Meuris et al.2 present an elegant method to reduce the N-glycan heterogeneity of recombinant glycoproteins expressed in mammalian cells. By simplifying the N-glycosylation machinery, the authors expressed glycoproteins with smaller and more uniform glycan structures. This strategy opens the way for production of Bernd Lepenies & Peter H. Seeberger are at the Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Potsdam, Germany, and Peter H. Seeberger is also at the Freie Universität Berlin, Institute of Chemistry and Biochemistry, Berlin, Germany. e-mail: [email protected] or [email protected] therapeutic glycoproteins of higher quality and efficacy. Many of the protein drugs on the market are glycoproteins, including hormones, cytokines and monoclonal antibodies. Two aspects of glycosylation are particularly relevant to their therapeutic activity: first, glycoproteins should ideally display humanlike glycans; and second, glycosylation patterns should be as uniform as possible. Humanized glycosylation is desired because non-human glycans may elicit unwanted immune reactions. Currently, most therapeutic glycoproteins are produced in mammalian expression systems—mainly Chinese hamster ovary (CHO) cells. Glycans produced in non-human cell lines can have a terminal galactose-α-1,3-galactose epitope not found on human N-glycans. This modification influences the immunogenicity of glycoproteins and can cause anaphylactic reactions in patients. In addition, glycoproteins from CHO cells predominantly contain a non-human type of sialic acid that induces antibody responses and accumulates in human tissues. The issue of uniform glycosylation nature biotechnology volume 32 number 5 may 2014 patterns on therapeutic glycoproteins is a matter of regulatory concern because, although some degree of heterogeneity in biotherapeutics is often acceptable, it can result in batch-to-batch variations in efficacy or pharmacokinetics. Heterogeneity in glycans arises because glycosylation is not a template-driven process and is not under direct transcriptional control; instead, it is guided by the amino acid sequence of the protein and the accessibility of glycan-processing enzymes, such as glycosyltransferases and glycosidases, in the endoplasmic reticulum and the Golgi apparatus3. Simply removing N-glycosylation sites from proteins usually does not solve humanization or heterogeneity problems because glycans are often required for correct protein folding and biological activity. Several approaches to producing glycoproteins with uniform human-like glycans have been developed as alternatives to mammalian expression systems. Escherichia coli4, the yeast Pichia pastoris5, plants6 and insect cells7 have all been engineered to improve protein N-glycosylation. For example, expression of mammalian glycoprocessing enzymes in yeast5 is a promising approach, but further studies are needed to demonstrate the utility of a yeast production system for therapeutic glycoproteins. Glycoprotein expression in plants offers easy scalability and low production costs. Although there has been some recent progress toward expressing human-like glycan structures in plants6, further improvements in these systems are needed. In addition, chemoenzymatic8 and synthetic strategies from our own group and others9,10 have been used to generate N-glycan structures and glycoproteins displaying humanized N-glycosylation patterns. A recent example is the chemical synthesis of the glycosylated hormone erythropoietin9. However, to be competitive with biological systems, chemical synthesis methods require optimization with regard to speed, scalability and production processes. Therefore, despite these developments, production of biopharmaceuticals in mammalian cells, with their established pipelines and demonstrated scalability, remains an attractive option. The glycoengineering strategy developed by Meuris et al.2—which the authors call GlycoDelete—offers an interesting alternative as it achieves a balance between retaining essential N-glycan functions and reducing the complexity of N-glycosylation. To accomplish this, the authors manipulated the glycosylation machinery in the Golgi apparatus responsible for trimming nascent N-glycans and adding monosaccharide residues. They started with an existing human embryonic kidney cell line11 that lacks the enzyme 443