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

Home , Benign tumor, Molecular pathology

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 in the primary tumor together crucial to validate cfDNA as a biomarker. with a follow-up of the patient, or used as a screening However, an ever-growing number of reports con- technique alongside the early identification of resistance vincingly show the suitability of ctDNA to detect tumor- against therapeutic drugs. Many mutations causing resist- specific genetic alterations. Cell-free DNA includes coding ance are known and therefore patients can be tested for and non-coding genomic DNA. Tumor-specific gene these variations. mutations such as point mutations, deletions, insertions, The second application is a genome wide approach amplifications, translocations and methylation abnor- without the prior knowledge of specific changes in the malities may be present in ctDNA and, depending on the primary tumor. Whilst the targeted approach, due to its technical approach, detectable in liquid biopsies. high sensitivity, also allows analysis of minor tumor fractions, the genome wide application is limited to high tumor fractions (>10%) [26]. However, this genome wide screening approach will be the final goal and will help to Methods in ctDNA analysis identify patients before they fall ill. Besides next-generation sequencing (NGS), mutation Both normal cells as well as tumor cells steadily release specific real-time or end-point PCR methods are applied DNA fragments, which are present in the cell-free frac- for the targeted analysis of point mutations, deletions and tion of the blood. In most cases ctDNA is only a tiny frac- genomic rearrangements. tion of the complete cfDNA. Because of ongoing lysis of Due to the extremely high sequencing capacity, NGS the normal cells in the blood, the already tiny amount of enables a high diagnostic sensitivity even for the paral- ctDNA is even more diluted by cfDNA complicating the lel sequencing of entire gene panels. In addition parallel detection of biomarkers from the tumor. Therefore, a handling of samples is simple and can easily be auto- delay in processing the collected blood, agitation of the mated, making the processing more cost-effective. sample, as well as sample transport cause the additional However, the sensitivity of amplicon-based NGS is release of wild-type cfDNA from lyzed blood cells, and limited by the error rate of the DNA polymerase used, thereby influence the ratio between ct and cfDNA [25]. To which is generally 0.01% [27]. reduce continuous “contamination” of the specimen and On the other hand, techniques like Sanger sequenc- to facilitate comparisons of results from different labora- ing or pyrosequencing can only be used for patients that tories, pre-analytical procedures have to be standardized exhibit a high amount of ctDNA and therefore are not seriously in future. really applicable for the analysis of liquid biopsies. 316 Hinrichsen et al.: Liquid biopsy in molecular pathology

Deep sequencing-based approaches/ the same identifier. If a mutation is found in a UID family NGS-based methods with >95% the mutation is considered to be real. A lower percentage indicates a technical artifact. Besides techni- The NGS-based methods sensitivity depends primarily cal know-how; this method requires extensive bioinfor- on the sequencing depth. With 1000x vertical coverage of matic knowledge [31]. the target region-this depth can easily be obtained – the With further improvements in sensitivity, NGS tech- sensitivity is approximately 1%–5% mutation load in the nologies will become increasingly important in future clin- wild-type background. Despite enormous recent efforts, ical applications, so that even genome wide analyses with NGS-based methods frequently fail to detect biomarkers low tumor fractions will be possible and cost effective. in liquid biopsies of stage 1 patients purely because of lack of sensitivity and the small amount of ctDNA present in the blood of these patients. Quantitative PCR-based methods However, because of the enormous interest in the early detection of meaningful biomarkers numerous tech- Most relevant at the moment is the specific detection niques have been developed to reduce the detection limit. of a set of mutations known to be important and useful These technical developments are rapidly improving sen- for assessment of the patients’ disease. For this purpose sitivity, although even the most sophisticated techniques a couple of sensitive PCR based techniques have been are far from satisfactory for the routine detection of bio- developed, and constantly improved in recent years. markers e.g. in stage 1 patients. In most cases real-time PCR, or techniques based on The most promising technique at the moment was it, are used for targeted mutation detection. Thereby the recently published by Newman et al. [28], a capture-based relevant mutation is quantified via fluorescently labeled NGS ctDNA detection method called CAncer Personalized probes. Standard curves with known concentrations are Profiling by deep Sequencing (CAPP-Seq) designed espe- used to calculate the concentration of the target sequence. cially for detection of all known major classes of mutations Ideally a couple of housekeeping genes are used for nor- in non–small cell lung cancer (NSCLC). Capture-based NGS malization because PCR performance depends on the methods enrich for genomic regions before sequencing and amplification efficacy of the assays and sample quality. the capture library (a custom-derived collection of target- Real-time PCR is a very fast method for the targeted detec- specific oligonucleotides) can easily be adapted for any tion of mutations with a sensitivity of approximately 0.1% given cancer type. According to the authors, their CAPP Seq mutation load in a wild-type background. The sensitivity approach identified mutations in >95% of tumors with high of real-time PCR can also be increased e.g. by additional specificity and sensitivity down to approximately 0.02% [28, nested PCR methods, allele-specific PCR or by suppress- 29]. However, although ctDNA was detected in the majority ing the wild-type allele. This technology generally allows of patients with stage II–IV disease, biomarkers could be a report turnaround time of 1 day. identified in only 50% of patients with early-stage NSCLC Digital PCR (dPCR) was recently introduced as a (stage I). The same authors recently re-engineered their further improvement to real-time PCR. dPCR allows quan- approach by combining a digital error suppression (iDES) tification of identified mutations in a simple, sensitive technique to reduce background artifacts with a molecular and cost effective way. In contrast to real-time PCR, no barcoding strategy for the efficient recovery of cfDNA mol- standard curves, references or calibrators are needed for ecules. This combination of techniques resulted in a 15-fold the normalization of the data. The DNA-molecules in the improvement in respect to sensitivity [30]. This example probe are diluted and partitioned into hundreds or even shows that detection of unknown biomarkers within a millions of separate reaction chambers so that each con- screening procedure in stage 1 tumor patients is currently tains one or zero copies of the sequence of interest. As with at the technical limits of known methods, and that only a real-time PCR, the fluorescent signals indicating positive combination of bioinformatics and sophisticated molecular reactions to a specific mutation are set in ratio to the posi- techniques will solve this problem. tive reactions of the corresponding wild-type sequence. A further option is the Safe-SeqS (Safe-Sequencing By counting the number of“positive” partitions (in which System). The Safe-SeqS adds a unique identifier (UID) to the sequence is detected) after PCR versus “negative” every DNA template molecule. After amplification of the partitions (in which it is not), you can determine exactly templates a lot of clonal amplicons of the original mol- how many copies of a DNA molecule were in the original ecule are formed. If a mutation is present in the original sample. Positive reactions are labeled as “1” and negative molecule, it should turn up in all the other molecules with reactions as “0”, hence the term “digital” PCR [32]. Hinrichsen et al.: Liquid biopsy in molecular pathology 317

By using dPCR it is possible to reach a sensitiv- (carcinoembryonic antigen) are widely used in assessing ity of >0.01% mutation load in a wild-type background. therapeutic response, they lack specificity and may also However, this high sensitivity increases the risk of false- be elevated by circumstances unrelated to the disease [17, positive signals and values. Thus, the applied assays must 36–39]; molecular biomarkers facilitate tumor genotyping be checked thoroughly beforehand for their false-positive thus helping to categorize tumors for clinical decisions. rates. This technology generally allows a report turna- Thus, patients likely respond to various drugs can be iden- round time of 1 day. tified and therapies could be applied which are tailored to Different platforms are used for dPCR. They can be the genetic composition of the tumor. distinguished according to the applied methods. Drop- There is no question that the earlier biomarkers let-based digital PCR platforms utilize water-in-oil emul- can be detected the better it is for the treatment of the sions for the PCR reactions. Chip-based systems utilize patient. Nowadays most biomarkers are DNA based and micro-reaction chambers on their surfaces for the clonal consequentially the detection of tumor-specific genetic amplification. alterations in ctDNA leads to broad clinical applications to evaluate diagnosis and prognosis, and in particular to identify predictive biomarkers in blood. However, tumor tissue is the gold standard for inves- Clinical application of ctDNA tigational and clinical molecular pathology allowing the diagnostics verification of biomarkers, of which almost all are “driver mutations” representing “druggable” kinase alterations. The feasibility of “liquid biopsy” analyzing ctDNA for These kinases widely represent the basis for targeted early detection of cancer, real-time monitoring and thera- therapies. Agents such as trastuzumab, imatinib, cetuxi- peutic stratification of cancer patients is presented and mab, gefitinib and vemurafenib are kinase inhibitors used extensively discussed in a growing number of studies. in treatment of cancer patients with colorectal and lung Screening and early detection of cancer by analyz- cancer and those suffering from melanoma and gastrointes- ing ctDNA may be of particular interest in patients with a tinal stromal tumors. Since histopathological examination high risk of developing cancer [33, 34]. The greatest tech- is the first step in tumor diagnosis, tumor tissue material is nical challenge is the identification of very low amounts usually available. Although routine biopsy tumor samples of ctDNA within a background of cfDNA. Besides the nec- are usually formalin-fixed and paraffin embedded (FFPE) essary sensitivity, the choice of the cancer-specific gene they are suitable for cancer genome diagnostics. By apply- panel is essential to reveal reliable results. In one study of ing gene panel testing, a growing number of therapeutic 640 cancer patients, ctDNA alterations were identified in relevant biomarkers can be detected in the primary tumor 48%–73% of patients with localized cancer diseases (colo- material. Treatment relevant biomarkers such as EGFR rectal, gastroesophageal, pancreatic and breast cancer) alterations in lung cancer and KRAS, NRAS and BRAF [11]. These detection rates are not considered satisfactory mutations in colorectal cancer have to be known to be used for the early detection of cancer. Further large studies are for treatment decisions and can additionally be applied as required comparing patients with particular cancer types molecular biomarkers in tumor monitoring. to healthy individuals to define sensitivity and specificity, Several studies have shown that liquid biopsies may and finally to define an appropriate panel of genetic alter- be useful to monitor tumor burden [27, 40–43]. There is ations most likely associated with early cancer diseases. convincing evidence to include oncogenic molecular bio- However, cancer associated mutations can occur with markers based on ctDNA analysis during cancer patient increasing age even in persons who never develop cancer monitoring. In a study of patients with various cancer [35]. Thus, the detection of ctDNA carrying cancer associ- types it was shown that about 80% of patients with ated mutations has to be interpreted critically and does advanced disease had detectable ctDNA in blood [11]. In not yet prove an early cancer diagnosis. patients with localized disease up to 75% those with colo- In contrast, real-time tumor monitoring and molecu- rectal cancer patients had KRAS mutant DNA fragments in lar stratification has become an important object in the their plasma corresponding to the KRAS mutation in the treatment of cancer patients. The blood-based stratifica- primary tumor. It has also been shown that high ctDNA tion of targeted therapy may become one way to include levels were associated with decreased 2-year survival [11]. liquid biopsy into clinical routine diagnostics. Advances in molecular technology have improved the Although circulating biomarkers such as protein bio- analysis of even small amounts of tumor material to detect markers prostate cancer antigen (PSA), CA-19-9 and CA-125 cancer gene panels. If specific mutations are defined by 318 Hinrichsen et al.: Liquid biopsy in molecular pathology

Table 1: Currently utilized predictive molecular biomarkers, which treatment-naive NSCLCs that contain classic sensitizing may also be used for tumor monitoring in liquid biopsies. EGFR mutations. Treatment pressure may select growth enrichment of these minor T790M cell clones leading to Tumor type Gene treatment failure [56]. The most likely reason this muta- Colorectal Cancer KRAS tion in primary tumors may be missed is due to the low NRAS sensitivity of detection methods such as Sanger sequenc- BRAF ing. Choosing higher sensitivity methods may reveal more PIK3CA APC primary lung tumors carrying the resistance mutation in P53 addition to the classic sensitizing EGFR mutation. Recent Lung Cancer EGFR studies have shown that alternate methods reveal T790M KRAS mutations in up to 79% of EGFR-mutant primary NSCLC BRAF tumors [54, 55, 57, 58]. Although, applying more sensitive NRAS Melanoma BRAF detection methods such as NGS, dPCR or Beaming tech- NRAS nology will provide more insight into the molecular mech- KIT anisms of tumor development and tumor progression, GIST KIT heterogeneity is one reason treatment relevant mutations PDGFRa such as T790M are missed at initial diagnosis. It is still unclear, and under discussion, as to whether the analysis of ctDNA in liquid biopsies is suitable to characterize the evaluation of the primary tumor, these may be used for mutation status of a tumor in general. The total concentra- ctDNA analysis. In the future, cancer patients will increas- tion of ctDNA in the blood of cancer patients varies, and ingly have their tumors genetically analyzed to guide the presence of tumor-specific mutations corresponding treatment decisions. Thus, more genetic information will to minor tumor cell clones may be undetectable initially. be available from the primary tumor and may be utilized However, during tumor progression a growing clone with for ctDNA analysis in tumor monitoring (Table 1). rare, and in particular, resistance mutations can be iden- The use of molecular targeted therapies has advanced tified in liquid biopsies analyzing ctDNA. Improvements and has been shown to be successful by extended disease in analytical sensitivity and specificity allow tracking free survival, in particular in colorectal and lung cancer. of resistance mutations in tumor patient management. However, the greatest challenge of these treatment strate- Recently, the first ctDNA based test utilizing liquid biop- gies is therapy resistance due to “new” mutations devel- sies in EGFR gene testing was approved [59]. The use of oping under exposure to monoclonal antibodies and a TKI (IRESSA®) is permitted for EGFR mutation analysis tyrosine kinases (TKIs). For colorectal cancer, NSCLC of ctDNA in liquids. Furthermore, a third generation TKI resistance mutations are well known and can be identi- (Tagrisso®) has been licensed for patients carrying the fied within 12–14 months of starting treatment. Also in T790M mutation. Liquid biopsies are particularly recom- colorectal cancer, mutations in KRAS codon 12/13 and 61 mended for testing for T790M status due to the fact that as well as BRAF were known to be associated with therapy tumor tissue may not be available in progression. resistance to cetuximab or panitumumab. These genetic In a case of our own, a patient with a NSCLC carried a alterations may occur under therapy even in patients common EGFR mutation in exon 19. He developed resist- with wild-type RAS status in the primary tumor [44–47]. ance to first generation TKIs and relapsed. Re-biopsy at Similar treatment failure is known for patients with progression was not available thus a liquid biopsy was NSCLC. Cellular mechanisms of de novo and acquired analyzed for T790M. As shown in Figure 2, both mutations resistance to EGFR TKI therapy in NSCLC include various in T790M in exon 20, and in exon 19 of the EGFR gene were molecular abnormalities in tumor cells such as consti- detectable in the plasma fraction. tutive activation of transducers located downstream of Although tumor tissue may still be the gold standard for EGFR, overexpression of other tyrosine kinase recep- the detection of “druggable” targets, cancer liquid biopsy is tors, or in particular secondary EGFR mutations. Among a promising tool in tumor monitoring. A proposed workflow patients exhibiting acquired resistance, a secondary to analyze tumor tissue and plasma samples within a com- EGFR exon 20 mutation (T790M) occurs in about 50% of prehensive diagnostic procedure is given in Figure 3. Using cases and an amplification of MET has been detected in the mutation profile detected in the primary tumor mate- up to 20% of EGFR TKI-resistant tumors [48–55]. Minor rial as a “leading biomarker” the interpretation of results clones with the T790M mutation have been identified in from liquid biopsies may be easier, in particular when Hinrichsen et al.: Liquid biopsy in molecular pathology 319

Figure 2: Result of a liquid biopsy in a patient with NSCLC. (A) EGFR Exon 18–21 analysis of FFPE-tumor tissue with next-neneration sequencing (NGS) revealed a c.2235_2249del; p.(E746_A750del) in 11% of sequence reads at time of diagnosis. (B) At time of relapse plasma samples were analyzed for T790M-resistance mutation on ctDNA level applying dPCR. The deletion in Exon 19 was confirmed with NGS (data not shown).

negative results for the resistance mutations are obtained. tumor show different genetic profiles [60, 61]. One further The detection of a biomarker previously known from the promising advantage of utilizing liquid biopsy as a diag- primary tumor analysis, in a plasma sample during tumor nostic tool in cancer treatment procedures is the detection monitoring indicates usability of the plasma sample and of a mutational profile consisting of prognostic and predic- the obtained result. It may also be useful to monitor tumor tive biomarkers in tumor monitoring which may be unde- burden which is a central aspect in cancer patient manage- tectable in primary tumor tissue. ment that is typically assessed with imaging. Tumor progression is a dynamic process triggered by the molecular heterogeneity of the tumor cells. In analyzing tumor tissue Conclusions obtained from biopsies the genetic heterogeneity may be missed due to the limited material and the low number of Primary tumor diagnosis is based on histological examina- tumor cells. It is well known that different areas of the same tion and tumor typing using morphological and molecular 320 Hinrichsen et al.: Liquid biopsy in molecular pathology

clinical utility of liquid biopsy based on ctDNA analysis has to be addressed in future clinical trials. The early detection of cancer faces challenges of both sensitivity and specificity and reliable testing based on ctDNA is still far away. The proposition of companies and public press “cancer detection from a drop of blood” is still a vision for the future and distant from realty. In summary, the gold standard of tumor diagnosis is based on morphological and molecular examinations from tumor tissue material but liquid biopsies are increasingly becoming an auspicious amendment in the comprehensive diagnostic workflow management of tumor diseases. It remains a subject of speculation if and to what extend liquids may replace tumor biopsies in the future.

Figure 3: Proposed work-flow for molecular pathology in routine Author contributions: All the authors have accepted tumor diagnostics. Tumor diagnosis and classification is strongly responsibility for the entire content of this submitted based on histomorphology and immunhistochemistry. manuscript and approved submission. Molecular pathology is performed for the detection of diagnostic, prognostic and predictive biomarkers by panel sequencing. These Research funding: None declared. biomarkers may also be utilized in tumor monitoring and may be Employment or leadership: None declared. helpful as “leading mutations” in the early detection of secondary Honorarium: None declared. (resistance) mutations in analyzing plasma samples. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and characteristics. However, current treatment strategies in interpretation of data; in the writing of the report; or in the cancer patients are more and more determined by targeted decision to submit the report for publication. drugs based on the individual molecular biology of a tumor disease. Thus, molecular profiling of tumor material is an important task and a still growing field in tumor pathology. Tumor tissue acquisition becomes an increasingly chal- References lenging due to minimal invasive procedures with limited tumor material. For the primary diagnosis of tumors or 1. Mandel P, Metais P. Les acides nucléiques du plasma sanguin chez l’homme. C R Seances Soc Biol Fil 1948;142:241–3. metastases that are not easy to biopsy but need to be ana- 2. Papageorgiou EA, Karagrigoriou A, Tsaliki E, Velissariou V, Carter lyzed for molecular targets, liquid biopsy might be an alter- NP, Patsalis PC. Fetal-specific DNA methylation ratio permits non- native. This has been shown by promising results in lung invasive prenatal diagnosis of trisomy 21. Nat Med 2011;17:510–3. cancer patients regarding the detection of resistance muta- 3. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. tions such as T790M. However, although highly sensitive Noninvasive diagnosis of fetal aneuploidy by shotgun and specific methods are used to detect ctDNA it should be sequencing DNA from maternal blood. Proc Natl Acad Sci USA 2008;105:16266–71. emphasized that sensitivity is still a limitation. CtDNA con- 4. Beiter T, Fragasso A, Hudemann J, Nieß AM, Simon P. Short-term centrations may vary due to reasons such as pre-analytical treadmill running as a model for studying cell-free DNA kinetics in procedures and ctDNA release. Interpretation of genetic vivo. Clin Chem 2011;57:633–6. alterations detected exclusively in liquid biopsy may prove 5. Atamaniuk J, Kopecky C, Skoupy S, Säemann MD, Weichhart T. difficult. A known molecular profile of the primary tumor Apoptotic cell-free DNA promotes inflammation in haemodialysis patients. Nephrol Dial Transplant 2012;27:902–5. may be helpful in the interpretation of molecular data from 6. Antonatos D, Patsilinakos S, Spanodimos S, Korkonikitas P, liquids. This tissue related data provides useful informa- Tsigas D. Cell-free DNA levels as a prognostic marker in acute tion regarding the specificity in particular when using gene myocardial infarction. Ann N Y Acad Sci 2006;1075:278–81. panels. Despite the promising first results of liquid biopsy 7. Stroun M, Maurice P, Vasioukhin V, Lyautey J, Lederrey C, Lefort F, diagnostics there is a limited impact in clinical diagnos- et al. The origin and mechanism of circulating DNA. Ann N Y Acad tics. Detecting and tracking resistance mutations in lung Sci 2006;906:161–8. 8. Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun cancer is one approach but if possible biopsy material or M. Point mutations of the N-Ras gene in the blood plasma DNA of fluids (e.g. pleura) should be analyzed, in particular when patients with myelodysplastic syndrome or acute myelogenous ctDNA results from liquids do not show any mutations. The Lleukemia. Br J Haematol 1994;86:774–9. Hinrichsen et al.: Liquid biopsy in molecular pathology 321

9. Nawroz H, Koch W, Anker P, Stroun M, Sidransky D. Microsatel- 29. Chabon JJ, Simmons AD, Lovejoy AF, Esfahani MS, Newman AM, lite alterations in serum DNA of head and neck cancer patients. Haringsma HJ, et al. Circulating tumour DNA profiling reveals Nat Med 1996;2:1035–7. heterogeneity of EGFR inhibitor resistance mechanisms in lung 10. Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL. cancer patients. Nat Commun 2016;7:11815. Soluble normal and mutated DNA sequences from single-copy 30. Newman AM, Lovejoy AF, Klass DM, Kurtz DM, Chabon JJ, genes in human blood. Cancer Epidemiol Biomarkers Prev Scherer F, et al. Integrated digital error suppression for 1994;3:67–71. improved detection of circulating tumor DNA. Nat Biotechnol 11. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, 2016;34:547–55. et al. Detection of circulating tumor DNA in early-and late-stage 31. Kinde I, Wu J, Papadopoulos N, Kinzler KW, Vogelstein B. Detec- human malignancies. Sci Transl Med 2014;6:224ra24. tion and quantification of rare mutations with massively parallel 12. Schwarzenbach H, Hoon DSB, Pantel K. Cell-free nucleic acids as sequencing. Proc Natl Acad Sci USA 2011;108:9530–5. biomarkers in cancer patients. Nat Rev Cancer 2011;11:426–37. 32. Pekin D, Skhiri Y, Baret JC, Le Corre D, Mazutis L, Salem CB, et al. 13. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, et al. Quantitative and sensitive detection of rare mutations using Circulating mutant DNA to assess tumor dynamics. Nat Med droplet-based microfluidics. Lab Chip 2011;11:2156–66. 2008;14:985–90. 33. Yong E. Cancer biomarkers: written in blood. Nature 14. Diehl F, Li M, Dressman D, He Y, Shen D, Szabo S, et al. Detection 2014;511:524–6. and quantification of mutations in the plasma of patients with 34. Spellman PT, Gray JW. Detecting cancer by monitoring circulating colorectal tumors. Proc Natl Acad Sci USA 2005;102:16368–73. tumor DNA. Nat Med 2014;20:474–5. 15. Heitzer E, Auer M, Hoffmann EM, Pichler M, Gasch C, Ulz P, 35. Genovese G, Kähler AK, Handsaker RE, Lindberg J, Rose et al. Establishment of tumor-specific copy number altera- SA, Bakhoum SF, et al. Clonal hematopoiesis and blood- tions from plasma DNA of patients with cancer. Int J Cancer cancer risk inferred from blood DNA sequence. N Engl J Med 2013;133:346–56. 2014;371:2477–87. 16. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid 36. Mazzucchelli R, Colanzi P, Pomante R, Muzzonigro G, Montironi R. clearance of fetal DNA from maternal plasma. Am J Hum Genet Prostate tissue and serum markers. Adv Clin Path 2000;4:111–20. 1999;64:218–24. 37. Ballehaninna UK, Chamberlain RS. Serum CA 19-9 as a 17. Ruibal Morell A. CEA serum levels in non-neoplastic disease. Int biomarker for pancreatic cancer-A comprehensive review. Indian J Biol Markers 1992;7:160–6. J Surg Oncol 2011;2:88–100. 18. Elshimali YI, Khaddour H, Sarkissyan M, Wu Y, Vadgama JV. The 38. Wanebo HJ, Rao B, Pinsky CM, Hoffman RG, Stearns M, Schwartz clinical utilization of circulating cell free DNA (CCFDNA) in blood MK, et al. Preoperative carcinoembryonic antigen level as of cancer patients. Int J Mol Sci 2013;14:18925–58. a prognostic indicator in colorectal cancer. N Engl J Med 19. Allen D, Butt A, Cahill D, Wheeler M, Popert R, Swaminathan R. 1978;299:448–51. Role of cell-free plasma DNA as a diagnostic marker for prostate 39. Fleischhacker M, Schmidt B. Circulating nucleic acids cancer. Ann N Y Acad Sci 2004;1022:76–80. (CNAs) and cancer-A survey. Biochimica et Biophysica Acta 20. Schwarzenbach H, Stoehlmacher J, Pantel K, Goekkurt E. Detec- 2007;1775:181–232. tion and monitoring of cell-free DNA in blood of patients with 40. Tie J, Gibbs P. Sequencing circulating cell-free DNA: the potential colorectal cancer. Ann N Y Acad Sci 2008;1137:190–6. to refine precision cancer medicine. Clin Chem 2016;62:796–8. 21. Chun FK, Müller I, Lange I, Friedrich MG, Erbersdobler A, 41. Dawson S-J, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin S-F, Karakiewicz PI, et al. Circulating tumour-associated plasma DNA et al. Analysis of circulating tumor DNA to monitor metastatic represents an independent and informative predictor of prostate breast cancer. N Engl J Med 2013;368:1199–209. cancer. BJU Int 2006;98:544–8. 42. de Bono JS, Scher HI, Montgomery RB, Parker C, Miller MC, 22. Sunami E, Vu AT, Nguyen SL, Giuliano AE, Hoon DS. Tissing H, et al. Circulating tumor cells predict survival benefit Quantification of LINE1 in circulating DNA as a molecular from treatment in metastatic castration-resistant prostate can- biomarker of breast cancer. Ann N Y Acad Sci 2008;1137: cer. Clin Cancer Res 2008;14:6302–9. 171–4. 43. Diaz LA Jr, Williams RT, Wu J, Kinde I, Hecht JR, Berlin J, et al. 23. Gormally E, Hainaut P, Caboux E, Airoldi L, Autrup H, Malaveille The molecular evolution of acquired resistance to targeted EGFR C, et al. Amount of DNA in plasma and cancer risk: A prospective blockade in colorectal cancers. Nature 2012;486:537–40. study. Int J Cancer 2004;111:746–9. 44. Peters S, Adjei AA. MET: a promising anticancer therapeutic 24. Catarino R, Ferreira MM, Rodrigues H, Coelho A, Nogal A, Sousa target. Nat Rev Clin Oncol 2012;9:314–26. A, et al. Quantification of free circulating tumor DNA as a diag- 45. Heinemann V, Douillard JY, Ducreux M, Peeters M. Targeted nostic marker for breast cancer. DNA Cell Biol 2008;27:415–21. therapy in metastatic colorectal cancer – an example of person- 25. Brock G, Castellanos-Rizaldos E, Hu L, Coticchia C, Skog J. Liquid alised medicine in action. Cancer Treat Rev 2013;39:592–601. biopsy for cancer screening, patient stratification and monitor- 46. Sartore-Bianchi A, Di Nicolantonio F, Nichelatti M, Molinari F, ing. Transl Cancer Res 2015;4:280–90. De Dosso S, Saletti P, et al. Multi-determinants analysis of 26. Heitzer E, Ulz P, Geigl JB. Circulating tumor DNA as a liquid molecular alterations for predicting clinical benefit to EGFR- biopsy for cancer. Clin Chem. 2015;61:112–23. targeted monoclonal antibodies in colorectal cancer. PLoS One 27. Diaz LA, Bardelli A. Liquid biopsies: Genotyping circulating 2009;4:e7287. tumor DNA. J Clin Oncol 2014;32:579–86. 47. Valtorta E, Misale S, Sartore-Bianchi A, Nagtegaal ID, Paraf F, 28. Newman AM, Bratman S V, To J, Wynne JF, Eclov NC, Modlin L a, Lauricella C, et al. KRAS gene amplification in colorectal cancer et al. An ultrasensitive method for quantitating circulating tumor and impact on response to EGFR-targeted therapy. Int J Cancer DNA with broad patient coverage. Nat Med 2014;20:548–54. 2013;133:1259–65. 322 Hinrichsen et al.: Liquid biopsy in molecular pathology

48. Sequist LV, Waltman BA, Dias-Santagata D, Digumarthy S, 55. Querings S, Altmüller J, Ansén S, Zander T, Seidel D, Gabler F, Turke AB, Fidias P, et al. Genotypic and histological evolution of et al. Benchmarking of mutation diagnostics in clinical lung lung cancers acquiring resistance to EGFR inhibitors. Sci Transl cancer specimens. PLoS One 2011;6:e19601. Med 2011;3:75ra26. 56. Inukai M, Toyooka S, Ito S, Asano H, Ichihara S, Soh J, et al. 49. Turke AB, Zejnullahu K, Wu YL, Song Y, Dias-Santagata D, Lifshits Presence of epidermal growth factor receptor gene T790M E, et al. Preexistence and clonal selection of MET amplification mutation as a minor clone in non-small cell lung cancer. Cancer in EGFR mutant NSCLC. Cancer Cell 2010;17:77–88. Res 2006;66:7854–8. 50. Tokumo M, Toyooka S, Ichihara S, Ohashi K, Tsukuda K, 57. Rosell R, Molina-Vila MA, Taron M, Bertran-Alamillo J, Mayo Ichimura K, et al. Double mutation and gene copy number of C, Vergnenegre A, et al. EGFR compound mutants and survival EGFR in gefitinib refractory non-small-cell lung cancer. Lung on erlotinib in non-small cell lung cancer (NSCLC) patients (p) Cancer 2006;53:117–21. in the EURTAC study. ASCO Meet Abstr 2012;30(suppl; abstr 51. Sequist LV, Martins RG, Spigel D, Grunberg SM, Spira A, 7522). Jänne PA, et al. First-line gefitinib in patients with advanced 58. Fujita Y, Suda K, Kimura H, Matsumoto K, Arao T, Nagai T, et al. non-small-cell lung cancer harboring somatic EGFR mutations. Highly sensitive detection of EGFR T790M mutation using colony J Clin Oncol 2008;26:2442–9. hybridization predicts favorable prognosis of patients with 52. Arcila ME, Oxnard GR, Nafa K, Riely GJ, Solomon SB, lung cancer harboring activating EGFR mutation. J Thorac Oncol Zakowski MF, et al. Rebiopsy of lung cancer patients with 2012;7:1640–4. acquired resistance to EGFR inhibitors and enhanced detection 59. Douillard J-Y, Ostoros G, Cobo M, Ciuleanu T, Cole R, McWalter of the T790M mutation using a locked nucleic acid-based assay. G, et al. Gefitinib treatment in EGFR mutated caucasian NSCLC. J Clin Cancer Res 2011;17:1169–80. Thorac Oncol 2014;9:1345–53. 53. Jänne PA, Borras AM, Kuang Y, Rogers AM, Joshi VA, Liyanage H, 60. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, et al. A rapid and sensitive enzymatic method for epidermal Gronroos E, et al. Intratumor heterogeneity and branched growth factor receptor mutation screening. Clin Cancer Res evolution revealed by multiregion sequencing. N Engl J Med 2006;12(3 Pt 1):751–8. 2012;366:883–92. 54. Jurinke C, Oeth P, van den Boom D. MALDI-TOF mass spectrom- 61. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, etry: a versatile tool for high-performance DNA analysis. Mol Diaz LA Jr, LA, Kinzler KW. Cancer genome landscapes. Science Biotechnol 2004;26:147–64. 2013;339:1546–58.