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Detection and Characterization of Circulating Cell Free Tumor DNA in Cancer Patients with Malignant Solid Tumors

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Detection and Characterization of Circulating Cell Free Tumor DNA in Cancer Patients with Malignant Solid Tumors J Lab Med 2016; 40(5): 313–322 Molekulargenetische und zytogenetische Diagnostik/ Redaktion: H.G. Klein Molecular genetic and cytogenetic diagnosis Tanja Hinrichsen, Juliane K. Dworniczak, Oliver Wachter, Bernd Dworniczak and Barbara Dockhorn-Dworniczak* Detection and characterization of circulating cell free tumor DNA in cancer patients with malignant solid tumors. Liquid biopsy: a new tool in molecular pathology? Die Analyse und Charakterisierung von zellfreier Tumor-DNA in Patienten mit malignen soliden Tumorerkrankungen. Die „Flüssigbiopsie“: Ein neuer diagnostischer Ansatz in der Molekularpathologie? DOI 10.1515/labmed-2016-0035 The combination of molecular profiling of the tumor with Received May 18, 2016; accepted August 2, 2016; previously published ctDNA analytics by liquid biopsy is a promising step in the online September 22, 2016 advancement of precision medicine. Abstract: The term liquid biopsy comprises methods of Keywords: biomarker; cell-free circulating tumor DNA; blood-based analysis of nucleic acids, which are increas- colorectal cancer; ctDNA; dPCR; liquid biopsy; molecular ingly under discussion in oncology and personalized pathology; next-generation sequencing (NGS); non-small medicine, and are already applied in individual cases. cell lung cancer (NSCLC); tumor profiling; tumor resistance. The analysis of tumor markers, which in certain tumor diseases can be found as protein markers in vast amounts Zusammenfassung: Mit Liquid-Biopsy werden Verfahren in the blood, constitutes a primary form of liquid biopsy. der blutbasierten Analytik von Nukleinsäuren bezeichnet, Cell-free circulating DNA fragments in the blood (ctDNA), die zunehmend vor allem in der Onkologie und der per- which reflect the genetic profile of a tumor cell and are sonalisierten Medizin diskutiert werden und auch schon released in different ways by the tumor, represent a new in Einzelfällen angewendet werden. Eine ursprüngli- class of more specific and sensitive biomarkers that can che Form der Liquid Biopsy stellt die Bestimmung der be correlated with the dynamics of the tumor disease. New nach wie vor verwendeten Tumormarker dar, die als technologies based on PCR and sequencing techniques Proteinmarker in hohen Mengen im Blut bei bestimm- pave the way for diagnostic approaches to define molec- ten Tumorerkrankungen nachzuweisen sind. Zellfreie ular tumor characteristics, not only in tumor tissue but zirkulierende DNA-Abschnitte im Blut (ctDNA), die das also in the blood, by analyzing cell-free circulating DNA. genetische Profil einer Tumorzelle wiedergeben und auf unterschiedliche Weise vom Tumor freigesetzt werden, stellen eine neue Klasse sensitver und spezifischer Bio- *Correspondence: Barbara Dockhorn-Dworniczak, Zentrum für marker dar, die mit der Dynamik der Tumorerkrankung Humangenetik und Laboratoriumsdiagnostik (MVZ) – Molecular korreliert werden können. Neue Technologien basierend Oncology, Lochhamer Martinsried, Germany; und Zentrum für Pathologie Kempten-Allgäu, Kempten, Germany auf PCR- und Sequenzierverfahren sind die Wegbereiter E-Mail: [email protected] für diagnostische Ansätze molekulare Tumorcharakte- Tanja Hinrichsen and Oliver Wachter: Zentrum für Humangenetik ristika nicht nur am Tumorgewebe sondern auch im Blut und Laboratoriumsdiagnostik (MVZ) – Molecular Oncology, an der zellfreien zirkulierenden DNA zu bestimmen. Die Lochhamer, Martinsried, Germany Kombination des „molekularen Profilings“ am Tumor Juliane K. Dworniczak: Technische Universität München, Institut für Diagnostische und Interventionelle Radiologie, Munich, Germany mit der ctDNA Analytik in der Liquidbiopsy ist ein neuer Bernd Dworniczak: Westfälische Wilhelms-Universität Münster, vielversprechender Schritt in der Weiterentwicklung der Institut für Humangenetik, Münster, Germany „Precision Medicine“. 314 Hinrichsen et al.: Liquid biopsy in molecular pathology Schlüsselwörter: Biomarker; ctDNA; dPCR; Kolorektales mRNA and microRNA miRNA) in pathological processes Karzinom; Liquid Biopsy; Molekularpathologie; next- including malignant and benign tumor diseases as well generation sequencing (NGS); non-small cell lung cancer as inflammatory processes, stroke, trauma and sepsis [1, (NSCLC); Tumorprofiling; Tumorresistenz; zellfreie zirku- 8, 10]. Active, as well as passive processes are responsible lierende Tumor-DNA. for the release of DNA into the peripheral blood. During these processes nucleic acids are shed into the blood by apoptotic and necrotic cells. The concentration of tumor derived DNA is dependent on the turnover of tumor cells Introduction and may be different from one tumor entity to the other. CtDNA is a tiny proportion of the cell free DNA from tumor The earliest report of cell-free DNA in healthy individuals cells and may be different from one tumor entity to the dates from 1948. Mandel and Metais described the pres- other [11]. It is important to note that ctDNA is distinct ence of cell-free deoxyribonucleic acid (cfDNA) in human from circulating tumor cells which specifically refers to blood [1]. However, until recently the most common clini- intact cells [12]. However, circulating tumor cells in blood cal use was the evaluation of fetal DNA in the circulation are known to contribute to the release of ctDNA. Cell-free of pregnant women. Investigation of cell-free fetal DNA DNA (cfDNA) is also released by dying nonmalignant host (cffDNA) can now uncover germline fetal changes just cells, and this normal cfDNA dilutes the ctDNA in patients weeks after conception. The detection of aneuploidy has with cancer. become part of the standard prenatal assessment in high Depending on the extent of tumor disease and in par- risk women [2, 3]. Other studies have shown that cfDNA ticularly on the histological tumor type, the fraction of exists at a ready-state level in clinical scenarios such as ctDNA varies greatly, between 0.01% and more than 90% myocardial infarction, surgery, trauma and stroke. Cell- [13]. It has been estimated that a tumor of 100 g in weight free DNA may increase dramatically with cellular injury corresponds to 3 × 1010 tumor cells and up to 3.3% may or necrosis [4–7]. enter the blood every day [14]. The average size of ctDNA is In oncology, there are a variety of potential clinical 70–200 base pairs. During tumor development and tumor applications for non-invasive disease monitoring based on progression both tumor derived and normal wild-type cell tumor specific genetic alterations detectable in circulating free DNA will be released into the blood. The proportion of cell-free tumor DNA (ctDNA). The importance of ctDNA was ctDNA originating from the tumor cells varies depending recognized in 1994 as a result of the detection of mutated on the tumor stage and size, but may also be influenced RAS genes in patients with malignant hematological disease by the tumor environment; in particular in situations [8]. Microsatellite alterations were shown in cfDNA in head when tissue-damaging therapies such as surgery, chemo- and neck cancer patients [9]. Investigation of ctDNA in therapy, or radiotherapy have been applied [12]. The DNA cancer patients has recently increased, largely based on the fragment length might provide some information on the advances in genomic technologies with increasing sensitiv- origin of cfDNA [15, 16]. Although the clearance of cfDNA ity and specificity to detect mutant variants at a very low is not fully understood it is known that cfDNA has a short allelic level in complex mixtures of DNA. Deep sequencing half-life time of 15–20 min [17] and is cleared through the technologies, cold-PCR, droplet-digital PCR and BEAMing liver and kidneys [18]. (beads emulsion amplification and magnetics) were used It is known that cancer patients have higher cfDNA and reported to serve as feasible methods to detect tumor levels than healthy donors, however, the overall concen- specific genetic alterations in ctDNA from plasma samples. tration of cfDNA varies considerably in blood samples in Liquid biopsy has become one of the most exciting and con- both groups [19–23]. An average of 180 ng cfDNA per mL troversial research fields in precision medicine. of blood has been measured in cancer patients, whereas healthy individuals carry about 30 ng of cfDNA per mL of blood [24]. A prospective study of cell-free DNA levels Defining circulating cell free DNA in plasma samples and their impact in cancer patients showed considerable variation between the European and approaches to liquid biopsy Prospective Investigation into Cancer and Nutrition (EPIC) analysis centers [23]. It was discussed that this individual variation (Figure 1) was due to the type of population and the treat- There is an enormous research effort focused on under- ment of samples. Thus, the cfDNA concentrations alone standing the utility of free circulating nucleic acids (DNA, are not the only tool in a diagnostic setup including liquid Hinrichsen et al.: Liquid biopsy in molecular pathology 315 Figure 1: Quality and amount of total cell-free DNA and the included cell-free tumor DNA is influenced by a variety of conditions and characteristics. biopsies, and may not be predictive for the amount of Two main applications can be distinguished with ctDNA (Figure 1). The impact of the different mechanisms respect to the analysis of ctDNA. The first application is a involved in generating cell-free DNA and the release into targeted approach based either on the analysis of known the circulation needs further investigation, but will be genetic changes identified
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