genomic ewsr1 fusion sequence as highly sensitive and...

Personalized Medicine and Imaging

Genomic EWSR1 Fusion Sequence as HighlySensitive and Dynamic Plasma Tumor Marker inEwing SarcomaManuela Krumbholz1, Julia Hellberg1, Benedikt Steif1, Tobias B€auerle2, Clarissa Gillmann2,Torsten Fritscher3, Abbas Agaimy4, Benjamin Frey5, Joerg Juengert1, Eva Wardelmann6,Wolfgang Hartmann6, Heribert Juergens7, Uta Dirksen7, and Markus Metzler1

Abstract

Purpose: The application of the tumor-specific genomic fusionsequence as noninvasive biomarker for therapy monitoring inEwing sarcoma (EwS) has been evaluated.

Experimental Design: EwS xenograft mousemodels were usedto explore detectability in small plasma volumes and correlationof genomic EWSR1-FLI1 copy numbers with tumor burden.Furthermore, 234 blood samples from 20 EwS patients wereanalyzedbefore andduringmultimodal treatment.EWSR1 fusionsequence levels in patients' plasma were quantified using dropletdigital PCR and compared with tumor volumes calculated fromMRI or CT imaging studies.

Results: Kinetics of EWSR1 fusion sequence copy numbers inthe plasma are correlated with changes of the tumor volume in

patients with localized and metastatic disease. The majority ofpatients showed a fast reduction of cell-free tumor DNA (ctDNA)during initial chemotherapy. Recurrence of increasing ctDNAlevels signalized relapse development.

Conclusions: Genomic fusion sequences represent promisingnoninvasive biomarkers for improved therapy monitoring inEwS. Until now, response assessment is largely based on MRIand CT imaging, implying restrictions on closely repeatedperformance and limitations on the differentiation between vitaltumor and reactive stromal tissue. Particularly in patientswith prognostic unfavorable disseminated disease, ctDNA is avaluable addition for the assessment of therapy response. ClinCancer Res; 22(17); 4356–65. �2016 AACR.

IntroductionEwing sarcoma (EwS) is an aggressive bone and soft-tissue

tumor occurring from birth to late adulthood with an overallincidence of 1 case per 1million.Half of all patients are diagnosedas adolescents; the median age at diagnosis is 15 years. Primarytumors are predominantly localized in the pelvis, chest wall, andlong tubular bones of the extremities. Main sites of metastasis arelung, bone, and bone marrow (1). Around 25% of patients arediagnosed with disseminated disease at multiple sites. Theirprognosis is particularly dismal with a 5-year overall survivalbetween 10% and 30% depending on additional risk factors(2, 3).

EwS belongs to the group of small round blue cell tumors inchildhood. Differentiation from other entities of this group hassubstantially benefited from comprehensive genetic and func-tional characterization, resulting in the availability of additionalimmunochemical markers and reliable molecular tests for detec-tion of the underlying EWSR1 gene rearrangement. Validation ofthe characteristic rearrangement of EWSR1 and a member of theETS family is now part of the standard diagnostic workup andclassification of EwS. Most prevalent are the fusion genes EWSR1-FLI1 (85%–90%) and EWSR1-ERG (�10%); rare, but recurrentvariants are also readily identified by RT-PCR (4–6).

Despite thebroad spectrumof clinical presentation at diagnosisand the variable phenotypic differentiation, initial therapy fol-lows a relatively uniform regimenof intensemultiagent inductionchemotherapy in current therapy trials. Assessment of therapyresponse during this intense treatment phase is largely based onfollow-up MRI and CT imaging studies of primary lesions atselected time points. In addition, the value of functional imagingfor staging and response assessment during induction therapy iscurrently addressed, e.g., in the ongoing EWING2008 trial byprospective evaluation of 18Fluoro-deoxyglucose PET/CT. Afterinitialmultiagent chemotherapy, local therapy, i.e., surgical resec-tion, irradiation, or combination of both, is mandatory fordefinitive disease control.

If tumor resection is feasible, histologic response, classified aspercentage of vital tumor cells after completion of inductiontherapy, is used to stratify for the intensity of the followingconsolidation therapy. However, complete resection and quan-tification of tumor necrosis are often not possible because of twocharacteristic EwS features. First, due to the typical involvement of

1Department of Pediatrics and Adolescent Medicine, University Hos-pital Erlangen, Erlangen, Germany. 2Department of Radiology, Uni-versityHospital Erlangen,Erlangen,Germany. 3DepartmentofNuclearMedicine, University Hospital Erlangen, Erlangen, Germany. 4Depart-ment of Pathology, University Hospital Erlangen, Erlangen, Germany.5Department of Radiation Oncology, University Hospital Erlangen,Erlangen, Germany. 6Department of Pathology, University HospitalMuenster, Muenster, Germany. 7Cooperative Ewing Sarcoma StudyGroup,DepartmentofPediatricHematologyandOncology,UniversityHospital Muenster, Muenster, Germany.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Manuela Krumbholz, University Hospital Erlangen,Loschgestrasse 15, 91054 Erlangen, Germany. Phone: 49-9131-85-40124; Fax:49-9131-85-34674; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-3028

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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bones of the pelvis, proximal extremities, thoracic wall, and spine,radical resection would cause inacceptable functional deficits andmutilating results. Second, therapy response is not easily recordedby volumetric reduction of the initial tumor size, because remo-deling of affected bones is a long-term process exceeding thepreoperative observation period. Therefore, it is often difficult todistinguish vital tumor from residual necrotic or inflammatorytissue by imaging studies alone.

Quantification of vital tumor burden would substantially sup-port the assessment of the actual therapy response and guidefurther treatment decisions. Because serum markers for EwS arecurrently lacking, our idea was to take advantage of the almostcomplete prevalence of characteristic chromosomal transloca-tions as potential therapy response marker in the form of circu-lating tumor DNA (ctDNA). Destruction of EwS tumor cellswould release DNA fragments including the EWSR1 fusion siteto the blood stream, where they could be quantified from serumsamples.

ctDNA has been proven to be a sensitive marker for tumorgrowth and therapeutic intervention in a number of solid tumors,virtually exclusively adult cancer with key driver gene mutationsidentified in various cancer types, mainly point mutations intumor-suppressor genes, e.g., TP53, APC, PTEN, and oncogenessuch as EGFR, KRAS, and PIK3CA. Targeted sequencing of theserecurrent mutations and quantification of mutated ctDNA inmetastatic colorectal and breast cancer showed clear correlationwith tumor burden, superior to standard biomarkers CEA and CA15-3, respectively (7–12).

The spectrum of malignancies in children and adolescents isvery different from the common cancer types in older age. Incontrast to the latter, their genomic landscape is generally—andparticularly in EwS—relatively silent with no recurrent additionalmutations, readily applicable as hot-spot targets for resequencingand tumor-associated ctDNA copy-number assessment (13–16).

However, the causal and recurrent chromosomal translocationsare theoretically even better-suitedmolecular DNAmarkers based

on their clonal homogeneity and the stability of chromosomaltranslocations during disease progression. High sensitivity anddynamics ofDNAmarkers designed fromgenomic fusion genes ofrecurrent translocations are well documented at their applicationin systemic diseases, wheremalignant cells are easily accessible bybone marrow aspiration (17). In contrast, there have been noapplications reported yet for the use of genomic fusion sequencesas noninvasive markers in patients with solid tumors.

Aim of this study was to develop a highly sensitive approach toquantify circulating tumor by genomic fusion DNA fragments asmolecular tumor marker in plasma samples. We tested determi-nants of ctDNA detection in xenotransplant mouse models andperformed a pilot study in pediatric EwS patients to prove itapplicability in routine medical care.

Materials and MethodsCell lines

Four EWSR1-FLI1–positive human EwS cell lines (A673,RD-ES, TC-71, and VH-64) were used for tumor generation inimmunodeficient mice. TC-71 and RD-ES cells were obtainedfrom the German Resource Center for Biologic Material (DSMZ);the A673 cell line was obtained from the American Type CultureCollection (ATCC); and the VH-64 cell linewas provided by Fransvan Valen in 2010 (18). Cell lines were authenticated by the cellline–specific genomic fusion sequence using single PCR directlybefore xenotransplantation (19). All cell lines were cultured inRPMI medium supplemented with 10% FBS, L-glutamine, andantibiotics at 37�C in 5% CO2 on collagen-coated flasks. A673and TC-71 cells express a fusion transcript consisting of EWSR1exon 1-7 and FLI1 exon 6-9 representing EwS type 1, whereas VH-64 and RD-ES cells express a fusion transcript of EWSR1 exon 1-7and FLI1 exon 5-9 representing EwS type 2 (20).

Xenograft mouse modelNMRI nu/nu mice and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG)

mice were purchased from Charles River Laboratories and bredand maintained under specific pathogen-free conditions in theanimal facilities of the University Hospital Erlangen. Mice weretreated in accordance with institutional and state guidelines. Allfurther described animal procedures have been approved by the"Regierung of Mittelfranken" and were conducted in accordancewith the guidelines of Federation of European Laboratory AnimalScience Associations.

Five- to 8-week-old immunodeficient mice were injected s.c.with 1 � 106EWSR1-FLI1–positive cells to model localized dis-ease or i.v. with 3� 106EWSR1-FLI1–positive cells to recapitulatedisseminated EwS. Mice were monitored daily for tumor forma-tion and symptoms of disease. EDTA-blood samples (100 mL)were taken once a week and centrifuged immediately at 600 g for15 minutes for plasma recovery. At the end of the study, plasmawas acquired from about 1 mL blood samples. Plasma sampleswere stored at –80�C until DNA isolation.

Volumes of subcutaneous tumors were calculated from calipermeasurements. Tumor burden in the i.v. injected, metastaticmouse model was evaluated postmortem by the number andsize of metastases. EWSR1-FLI1 fusion gene–specific single PCRwas used to confirm tumor tissue by the cell line–specific genomicEWSR1-FLI1 fusion site previously sequenced and deposited intheNCBIGenBankwith accession numbers JX266518, JX266520,JX266523, and JX266525.

Translational Relevance

Due to of the lack of tumor-associated serum markers,therapy monitoring in Ewing sarcoma is essentially based onimaging procedures that do not allow stratification upon earlychemotherapy response assessment. Prospective evaluation ofalternative, functional imaging techniques is difficult to con-duct in these young and often critically ill patients due totedious procedures frequently requiring sedation and cumu-lative high radiation doses. Hence, all patients are currentlytreated with the same intense chemotherapy regimen duringthe first 5 months.

Using tumor-associated genomic fusion sequences as non-invasive biomarkers will allow for closer therapy monitoringand might help to identify patients at risk earlier during thecourse of therapy. This may facilitate improved risk adapted,personalized therapy. Furthermore, quantification of circulat-ing tumor DNA in the follow-up period might help to detect arelapse on a molecular level. The approach described here isalso applicable to anymalignancy with recurrent chromosom-al translocation.

Circulating Tumor DNA in Ewing Sarcoma

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To visualize organ metastases ex vivo, resected samples werefixed with paraformaldehyde, embedded in agarose, and scannedon a preclinical ultra-high field MRI (7 Tesla, ClinScan; Bruker)using a T2-weighted spin-echo sequence (resolution: 50 � 50 �200 mm). For histologic examinations, resected tumors andmetastases were fixed with paraformaldehyde and embedded inparaffin. Sections (3 mm) were stained with hematoxylin andeosin and the antibodies CD99 (1:100 dilution; Dako clone12E7) and Ki-67 (1:100 dilution; Dako clone MiB1).

Patients and sample collectionTwenty EwSpatients (7 females and13males;median age, 14.6

years; range, 2–38 years) were accrued to our study. Nineteenindividuals had a new diagnosis of EwS and were enrolled to theEWING2008 trial (NCT00987636). Induction chemotherapyconsists of six cycles of vincristine, ifosfamide, doxorubicin, andetoposide (VIDE) in all patients followed by local therapy andconsolidation therapy depending on individual risk stratification.One patient, UPN6, was included at the time of first relapse andtreated in accordance with national guidelines. Histology andfusion transcript or FISH results were confirmed by a referencepathology review. Written informed consent was obtained fromall patients or their legal guardians in accordance with the Dec-laration of Helsinki. Clinical and molecular parameters are sum-marized in Table 1.

For each patient, depending on the individual's body weight, 2to 9 mL EDTA blood samples were collected at the time ofdiagnosis and during the course of chemotherapeutic treatment.Research blood specimens were always drawn at the time asroutine samples for clinical monitoring. We prospectively evalu-ated a total of 234 blood samples (21 pretreatment, 121 duringinduction therapy, 40 during consolidation therapy and surveil-lance, 52 after relapse diagnosis). Plasma was separated fromperipheral blood cells by centrifugation at 600 g for 15 minuteswithin 2 hours after blood was drawn and stored at –80�C.

The EWING2008 protocol includes scheduled assessment oftreatment response with MRI or CT scan and optional PET/CTafter the second and fifth VIDE blocks. In some cases, additional

studies were performed for specific clinical indications. Imagingdata fromPET/CTwere used to localize the ES lesions. The volumeof these lesionswas assessed retrospectively on contrast-enhancedMRI images either in transverse or coronal orientation usingdedicated segmentation software (Chimaera GmbH).

Identification of the patient-specific genomic EWSR1-FLI1 orEWSR1-ERG fusion sequences from primary tumor samples

DNA was isolated from fresh frozen cryopreserved biopsies orformalin-fixed paraffin-embedded tissues using the QIAampDNA Blood and Tissue Mini Kit (Qiagen GmbH) according tothe manufacturer's instructions.

Genomic EWSR1-FLI1 fusion sequences from cryopreservedtumor biopsies were identified by a nested multiplex long-rangePCR (MLR-PCR) assay described earlier (19). EWSR1-ERG fusionsequences were detected by an analogousMLR-PCR assay, includ-ing a reverse ERGprimer set (Supplementary Table S1). Formalin-fixed paraffin-embedded tissues are highly disadvantaged whenusing this originalmethod. To adjust for the higher fragmentationofDNA isolated from formalin-fixed paraffin-embedded tissues, alarger amount of template DNA is required, and additionalmultiplex-PCR primers were designed for amplification of shorterPCR products (Supplementary Table S1). All PCRs were per-formed using the AccuPrime Taq DNA Polymerase System (Invi-trogen) according to the manufacturer's instructions. PCR pro-ducts were purified using the QIAquick PCR Purification Kit(Qiagen) and sequenced by Eurofins Genomics.

Isolation and quantification of cell-free circulating DNAAfter thawing, plasma samples were centrifuged at 11,000 g for

3 minutes to remove residual cells or cell debris. Cell-free DNAwas isolated using the QIAamp Circulating Nucleic Acid Kit(Qiagen).

ctDNA was quantified by the detection of the cell line orpatient-specific EWSR1-FLI1 or EWSR1-ERG fusion genes usingbreakpoint spanning primers and probe sets (SupplementaryTable S2). Quantification was performed by droplet digital PCR(ddPCR) on a QX100 droplet generator and reader system (Bio-

Table 1. Patient characteristics and genomic breakpoint positions

Genomic breakpoint positiona

UPN Sex Age at diagnosis Tumor sample EWS Chr22: FLI1/ERG� Chr11:/Chr21:� Tumor localization Initial tumor volume (mL)

1 m 8.8 FFT 29,684,828 128,660,222 Chest wall 10902 f 19.5 FFPE 29,683,890 128,672,447 Thorax 4733 f 14.2 FFT 29,684,210 128,648,755 Tibia 314 f 10.4 FFT 29,683,363 128,647,160 Retroperitoneal 1255 m 17.7 FFT 29,687,600 128,677,198 Thorax 14956 m 10.8 FFT 29,683,162 39,758,924� Disseminated tumor 9.57 f 15.4 FFPE 29,684,719 128,670,750 Popliteal fossa n.a.8 f 9.7 FFT 29,684,818 128,667,067 Fibula 79 m 17.3 FFPE 29,684,642 128,657,758 Rib 76010 m 12.5 FFPE 29,684,492 128,678,099 Nuchal n.a.11 m 10.0 FFPE 29,684,308 128,649,485 Humerus 5012 m 17.6 FFT 29,683,472 128,670,953 Femur 36413 m 14.9 FFT 29,685,344 128,659,187 Os illium 26614 m 10.0 FFPE 29,684,045 128,678,890 Scapula 11615 m 20.9 FFT 29,683,597 128,669,317 Os illium 14316 m 15.3 FFT 29,683,670 128,675,070 Rib 15617 f 31.7 FFT 29,684,170 128,644,790 Scapula 28718 m 38.5 FFT 29,684,319 128,656,714 Os sacrum <20019 f 9.5 FFT 29,684,120 128,656,016 Disseminated tumor 40920 m 2.3 FFT 29,683,936 128,674,661 Paravertebral 9.8

Abbreviations: FFPE, formalin-fixed paraffin-embedded tissue; FFT, fresh frozen tissue; n.a., not applicable, primary tumor resection.aReferring to UCSC genebank built GRCh37/hg19.

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Rad). The ddPCR mixture was composed of 6 mL cell-free DNA,900 nmol/L of each primer, 250 nmol/L probe, 2x ddPCR Super-mix for Probes. The fluorescence signal after ddPCR was deter-mined on the QX100 droplet reader and analyzed with QuantaSoftware (Version 1.5.38). Cases with less than three follow-upsamples were quantified on a CFX96 real-time PCR detectionsystem with EvaGreen Supermix (Bio-Rad). To calculate theabsolute number of EWSR1-FLI1 or EWSR1-ERG copies, thefusion-specific probe signal was normalized to a signal of thesingle copy genes mouse ß-actin or human albumin and plasmavolume used for DNA extraction, respectively. The sensitivity ofthe ddPCR assay for the detection of ctDNA copies in plasma wasabout 0.1%.

ResultsQuantification of genomic EWSR1-FLI1 fusion sequences in theplasma of EwS xenograft mice

To evaluate whether tumor-specific genomic fusion sites arequantifiable in small plasma samples and can be used asmolecular serum markers for tumor growth monitoring inchildren, we first examined ctDNA levels in two EwS xenograftmouse models. Tumor size, tumor vitality, and the number ofmetastatic sites can be studied postmortem in mice, facilitatingevaluation of the correlation between ctDNA levels and tumorburden. Furthermore, the genomic EWSR1-FLI1 fusionsequence specific for each injected cell line is already knownfrom earlier work (19).

Four different EwS cell lines injected in NMRI nu/nu mice andinNSGmicewere analyzed inour preclinical study to estimate cellline and recipient-related effects of the ctDNA level in mouseplasma.

The tumor engraftment was different in NSG and NMRI nu/nuxenograft mouse models. After s.c. or i.v. injection, all NSG micedeveloped tumors independent of EwS cell line injected, at whichVH-64 and RD-ES cells, representing EwS type 2, showed adecelerated tumor engraftment (Supplementary Fig. S1A andS1B). After i.v. injection, tumors were preferentially located inthe liver, kidney, and lung (Supplementary Fig. S1C).

In contrast, NMRI nu/nu mice demonstrated a slower andoverall reduced tumor engraftment. Nearly all NMRI nu/nu micedeveloped tumors after s.c. injection of A673 and TC-71 cells, butno tumors were palpable after s.c. injection of VH-64 and RD-EScells (Supplementary Fig. S1A). After i.v. injection of A673 andTC-71 cells, 58% ofmice (14/24) presented withmetastases, only20% of mice (2/10) developed metastases after i.v. injection ofVH-64 cells, and no tumor growthwas observed after i.v. injectionof RD-ES cells (Supplementary Fig. S1B).Metastases developed inlung and bone similar to the metastatic pattern commonlyobserved in EwS patients; no additional organs were affected(Supplementary Fig. S1C).

Immunohistochemical studies confirmed tumor tissues as EwSby strong membranous CD99þ staining in both mouse modelswith highly active proliferation as more than 60% of tumor cellshad a positive Ki-67 signal (Supplementary Fig. S1C).

Plasma of xenograft mice was monitored weekly for the pres-ence of the tumor-specific fusion sequence. We could not detectany EWSR1-FLI1 copies in the plasma of s.c.-injected mice duringthe time of tumor growth. A positive EWSR1-FLI1 signal wasdetectable in 70% (14/20) of mice only at the end of the studywhen the localized tumor reached the maximum size of around1,000 mm3 (Fig. 1A). In median, 110 copies (range, 15–360)

EWSR1-FLI1per 1mLplasma comparedwith 1,700 copies (range,800–9,400) mouse-albumin per 1 mL plasma were detectable.

In contrast, i.v.-injected mice, modeling metastatic disease,showed positive EWSR1-FLI1 signals in median 8 days beforefirst symptoms of disease became visible (Fig. 1A). ctDNA copieswere detectable in all transplantedmice.Monitoring of the ctDNAlevel during the time of tumor development revealed an increaseof EWSR1-FLI1 copies with tumor progression (Fig. 1B). Micewith i.v.-injected VH64 or A673 cell were classified in minor,medium, and heavy infiltration based on the number of metas-tases observed.

The xenograftmousemodels confirmed the reproducible detec-tion of cell line–specific EWSR1-FLI1 copies in correlation withthe overall tumor burden from very small blood samples. Wetherefore continued to evaluate the clinical applicability of geno-mic fusion sites as molecular plasma marker in children andyoung adults treated for EwS.

Quantification of genomic fusion sequences in plasma samplesof EwS patients

Quantification of EWSR1-FLI1 or EWSR1-ERG fusion sites inthe patient's plasma requires the identification of the individualgenomic fusion sequence as a first step. In our present studycohort, we identified 19 patients with a EWSR1-FLI1 fusion geneand 1 patient with a EWSR1-ERG fusion sequence (UPN6). In 12EWSR1-FLI1–positive patients, the rearrangement resulted in thefusion of EWSR1 exon 7 to FLI1 exon 6, generating the mostfrequent type 1 fusion transcript (21). Four patients (UPN3, 4, 11,and 17) expressed the fusion transcript EWSR1 exon 1-7 fused toFLI1 exon 5-9, representing the second most frequent EwS fusiontype 2. Three patients showed infrequent fusion transcripts;EWSR1 exon 7 fused to FLI1 exon 8 (UPN10 and 14) and EWSR1exon 9 fused to FLI1 exon 8 (UPN5), respectively. The underlyingindividual genomic fusion sequences are shown in Supplemen-tary Fig. S2.

The patient-specific genomic EWSR1-FLI1 and EWSR1-ERGfusion sequences were used as templates for the design of fusiongene–specific primer probe sets.We first analyzed plasma samplesat the time of initial diagnosis and relapse expecting that thesesamples are most likely positive for ctDNA detection. Fourteenpatients had plasma samples at initial diagnosis, 2 patients hadplasma samples at initial diagnosis and relapse, and 1 patient hadtwo plasma samples at two different relapse times available. Wewere able to detect ctDNA copies in 18 of 20 plasma samples andobserved a consistent correlation between tumor volume andctDNA copy number (Fig. 2). Copy numbers of EWSR1-FLI orEWSR1-ERG normalized to the copy number of the single copygene albumin varied between 0.003 and 0.56. Two patients(UPN7 and 10) tested negative for EWSR1-FLI1 copies. Bothpatients had small localized tumors (3.2 mL in the poplitealfossa and 14 mL in the neck, respectively) that were resectedbefore first plasma samples had been taken.

To evaluate the use of genomic fusion sequences as serummarkers for therapy monitoring, we further analyzed plasmasamples during the course of chemotherapeutic treatment. A totalof 213 (median 7) follow-up samples were collected in 17patients. A summary of detectability of ctDNA copies and tumorvolume during the initial chemotherapy (VIDE1 – VIDE6) isshown in Fig. 3.

Two patients with initially resected tumors (UPN7 and 10) hadno detectable ctDNA copies in all follow-up samples analyzed,






confirming the high specificity of PCR-based quantification ofgenomic rearrangement; no false-positive results were generatedin the absence of specific template DNA. Fast reduction of thectDNA copy number after onset of chemotherapy could beobserved in most cases without initial tumor resection. At thebeginning of the second VIDE block, 60% of the patients (9/15)have no or only few EWSR1 fusion sites detectable. Of theremaining 6 patients, 3 patients were ctDNAnegative before VIDEblock three. All of these 12 patients had a good therapy response,and imaging confirmed significant tumor reduction (exemplarilysee Fig. 4 UPN1 and 20).

Three patients demonstrated a different ctDNA response pat-tern (Fig. 4). In patient UPN19 who had a disseminated tumorwith primary infiltration of the os ilium, single EWSR1-FLI1copies were still detectable at the third VIDE block. This patientrelapsed about 10 months after the initial diagnosis (7.5 mLmetastasis in the humerus) that could be discovered by anincrease of ctDNA copies in the patient's plasma. In the followingcourse of disease, the patient developed a local relapse in the os

ilium andmultiple lung metastases that could always be detectedby an increase of the ctDNA plasma levels (Fig. 5). In patientUPN5, ctDNA levels were detectable during the entire inductionchemotherapy (VIDE1–VIDE6). This patient also relapsed3yearsafter initial diagnosis. Despite the very small size of the lymphnodemetastasis (0.8 mL), an increase at the ctDNA level could bedetected at that time.

Patient UPN6 was included in our study at the time of firstrelapse. This patient initially showed a fast decrease of ctDNAcopy numbers, but 3 months later had a recurrence of EWSR1-ERG fusion sequences in the plasma, indicated by the small peakin the graph (Fig. 4). The patient developed a relapse 14 monthsafter treatment began. The relapse site at the second cervicalvertebra was difficult to identify by imaging studies, whereasincreasing EWSR1-ERG plasma DNA indicated the diseasereoccurrence.

Our present results show that the copy number of genomicfusion DNA fragments in the plasma of EwS patients correlateswith tumor burden and may indicate relapse development.

Figure 1.A, time to first symptoms of disease inrelation to EWSR1-FLI1 copies in theplasma of xenotransplanted mice.B, plasma ctDNA levels after i.v. injectionof VH64 and A673 cells in NSG mice.[VH64-injected mice were grouped asfollows:< 10metastases in lung andkidney(minor infiltration), 10–50 livermetastases, > 10 metastases in kidney andovary (medium infiltration), and > 50 livermetastases, > 10 metastases in kidney andovary (heavy infiltration). Mice withA673-injected cells were grouped:< 10 metastases in lung and liver (minorinfiltration), 100–500 livermetastases and< 10 lung metastases (mediuminfiltration), and > 500 liver metastases,> 20 lung metastases, and kidneymetastases (heavy infiltration).]

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DiscussionWe investigated the utility of the genomic EWSR1 fusion

sequence as a plasma marker in children and adolescents withEwS. ctDNA, small DNA fragments released from the tumor tissueto the blood streamby variousmechanisms, has been shown toberepresentative of the tumor genome of many cancer types andconstitutes a valuable additional source of tumor material, par-ticularly when incision biopsies are difficult to obtain fromcritically located tumors (22). The term Liquid Biopsy has beencoined to this approach andpictures thepotential of cell-freeDNAsamplingwithminimal invasiveness, enabling repeated samplingat multiple times during treatment, and the possibility of real-time monitoring.

Given the incidence and spectrum of malignant tumors inhumans, applications of ctDNA for the genetic assessment ofsolid tumors and monitoring during therapy have almost exclu-sively been explored in adult patients with epithelial tumors.

Initial studies in subjects with colorectal cancer undergoingsurgery or chemotherapy found that ctDNA measurementscould be used to monitor tumor dynamics. The ctDNAappeared to be a more reliable and sensitive indicator thanthe current standard biomarker (CEA) in this cohort of subjects(10). Dawson and colleagues further showed a greater dynamicrange, greater correlation with changes in tumor burden, andearlier measure of treatment response of ctDNA quantificationcompared with CA 15-3 or circulating tumor cells, followingthe clinical course of 20 patients with metastatic breast cancer(8). Meanwhile, many additional tumor types have been exam-ined for their representation in plasma samples, includingnon–small cell lung cancer, gastroesophageal, hepatocellular,pancreatic, bladder, prostate, ovary, melanoma, head, and neckcancers (23–25).

These cancer types share commonly acquired key driver genemutations, e.g., PIK3CA, TP53, EGFR, APC, KRAS, andNRAS. The

limited number of recurrent single-nucleotide changes at hot-spotmutation sites enabled the development of ready-made assays(23–26).

Figure 2.Concordance between ctDNA plasma levels and tumor volumes, calculatedby PET/CT, at initial diagnosis and relapse diagnosis. Dashed diagonaldisplays linear regression.

Figure 3.Comparison of ctDNA levels and tumor volume prior to each VIDE inductiontherapy block.






However, pediatric malignancies vary significantly from adultcancer with respect to incidence and typical tumor types andtherefore also exhibit an entirely different spectrum of cancer-associated mutations. A large proportion of entities are specifiedby the presence of recurrent fusion genes as a result of chromo-somal translocations. This is a common characteristic of tumors ofmesenchymal origin, but also increasingly identified in currenttumor sequencing data from epithelial neoplasia (27).

Both aspects, the lack of common hot-spot mutations and thepresence of distinct translocations, are particularly applicable toEwS. Three recent studies consistently described the genomiclandscape of EwS as relatively silent (13–15). In an analysis of27 cancer types of all age groups, EwS revealed the second lowestmedian frequency of nonsynonymousmutations, 10- to 100-foldbelow the frequency observed in other entities studied, e.g.,colorectal cancer, lung cancer, or melanoma (28). Contrary, thepresenceof oneof the characteristic fusiongenes involvingEWSR1and a partner gene of the ETS family is highly prevalent andtherefore became a diagnostic criterion in current guidelines (29).

Application of chromosomal translocations as biomarkers fortumor cells has several general advantages and disadvantagescompared with hot-spot single-nucleotide mutations more com-monly applied for ctDNA quantification.

Due to the combination of chromosomal material from dif-ferent chromosomes, the resulting fusion sequence is absolutelyunique and has a particularly distinctive nucleotide composition,conferring high sensitivity and specificity to probe sets spanningthe chromosomal breakpoint. Base calling in next-generationsequencing algorithms is less prone to false-negative and false-positive calls given the extended stretches of nucleotides differingfrom wild-type reference sequence. Because the underlying char-acteristic fusion gene is not only cancer associated but causativeand therefore stable during disease development, intratumorheterogeneity and clonal selection under treatment are not acritical issue in contrast to emerging clonal heterogeneity undertargeted therapy, e.g., with EGFR antibodies (30, 31).

However, each genomic fusion site is an individual sequencewithin large intronic gene segments requiring specific approachesto facilitate detection and sequencing (19). The patient-specificgenomic fusion site in our study has been identified from biopsymaterial used for the histologic diagnosis. In principle, identifi-cation of the fusion sequence is also feasible from plasma DNAusing appropriate sequence library enrichment strategies andnext-generation sequencing techniques (23, 32).

Stated previously, EwS is an overall rare disease in comparisonwith cancer types with high prevalence of hot-spot mutationsstudied for their utility as plasma biomarkers. We were uncertainat the beginning of the study, whether ctDNA assays would besufficiently sensitive to detect tumor kinetics in EwS andwanted toconfirm the technical feasibility, in particular dealing with smallsample volumes, before initiation of repeated blood samplingfrom children.We therefore decided to conduct an initial proof-of-concept study in preclinical EwS xenotransplant mouse model.Despite very small plasma volumes available, tumor-specific geno-mic fusion sequences were detectable in xenotransplanted mice inrelation to disease extent. Comparison of the ctDNA dynamicbetween subcutaneous and disseminated mouse model supportsthe assumption that not only the tumor size, but also the local-ization, respectively the access to blood circulation, is a determi-nant of ctDNA copy number. These results indicate that ctDNA canalso be used in preclinical mousemodels as plasma tumormarker

of EwS without the need to sacrifice animals at several time pointsto assess disease development or therapy response.

In clinical care of patients with EwS, there are two challengesthat could benefit from additional information on therapyresponse as determined by ctDNA. One aspect is the difficultyof radiographic assessment on the distinction between residual

Figure 4.Monitoring of ctDNA plasma levels and tumor volumes in 4 EwS patientsduring the treatment course. A, patients with fast reduction of ctDNA levelsduring the first two VIDE blocks. B, patients with relapse development,detected by increasing ctDNA levels. Asterisks display newly arisingmetastases at previously unaffected sites.

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tumor tissue, necrotic neoplastic cells, and reactive inflammatoryand stromal cells. In several of our cases, the discrepancy betweentumor volumes, calculated from imaging, and the detection ofctDNAwas attributable to the lack of definition of clear-cut tumormargins after onset of induction therapy. The second limitation ofthe current stratification is the fact that not all cases can undergosurgery for local disease control due to unfavorable or dissemi-nated tumor localization. Stratificationbasedon the percentage ofvital tumor cells after completion of induction therapy is thereforenot possible and relies on pretreatment parameters in those cases,ignoring the actual response on multiagent chemotherapyreceived until then.

DNA sequences specific for the primary tumor can be isolatedfrom blood samples either from circulating tumor cells or fromcirculating cell-free DNA. When circulating tumor cells weredirectly compared with tumor-specific cell-free DNA from thesame blood sample in larger cohorts of patients with diffuse largeB-cell lymphoma, quantitatively more tumor DNA was found inthe plasma (33, 34). Likewise, disease detection from plasma wasmore sensitive at the time points of overt disease, especially atrelapse, compared with the cellular blood fraction, although thisdisease has higher numbers of circulating tumor cells than sar-comas. Furthermore, measurement of disease from ctDNA, but

not from circulating tumor cells, was significantly correlated withtumor volume as measured by 18FDG PET/CT scan (33).

Circulating tumor cells in peripheral blood samples have beenfound qualitatively present in only one fifth of EwS patients atdiagnosis, most frequently in cases with large tumors (24),whereas ctDNA in our study was detectable in all patients beforetreatment start, except two cases (UPN7 and 10) with smallextraosseous tumors, resected before first blood samples weretaken. The ctDNA is the main source of tumor-associated DNA inour study, since sample management ensured immediate centri-fugation and therefore separation of cellular fraction. Further-more, disintegration of circulating tumor cells by sample han-dling and centrifugation is unlikely to contribute significantly tothe copy number of EWSR1-FLI1– or EWSR1-ERG–specific fusionfragments because the potential number of circulating tumor cellsin the blood sample is negligible compared with ctDNA frag-ments, given the average copy number at diagnosis.

The number of plasma ctDNA copies decreased with tumorregression and increased in case of relapse development. No false-positive ctDNA copies were detected in any of the samples testedat any time point. Relatively high ctDNA copy numbers wererecorded whenmetastases occurred at previously unaffected sites.A possible explanation is that micrometastatic lesions smaller

Figure 5.Monitoring of ctDNA plasma levels andtumor burden in patient UPN19. Newmetastases are marked by asterisks.Tumor volumes at initial diagnosis (A),remission (B), and relapsedevelopment (C–E) were measuredwith 18FDG PET and T1 after contrastmedium (CM). Arrows indicate tumorsites and tumor destruction.Calculation of tumor volumes is basedon segmentation analyses.






than a fewmillimeters are not detectable by PET/CT orMRI scans,but in aggregate maymake a large contribution to the total tumorburden (10).

Whether the ctDNA copy number is capable to differentiatelocalized metastases (UPN 5) from developing disseminatedmetastatic disease (UPN6 and 19) has to be subject of largerprospective studies, investigating ctDNA in combination withestablished clinical prognosis parameters. However, besides thepotential to monitor the disease burden during inductiontherapy in order to identify patients at high risk of treatmentfailure, follow-up samples during and after maintenance ther-apy may also prove instrumental in early detection of diseaserecurrence. At present, EwS relapse is an incurable disease(35, 36). A further and careful prospective validation of earlydetection of molecular relapse is required. If such detection isfeasible and would allow starting therapeutic intervention at anearly stage, this might lead to an improved survival after EwSrelapse.

The availability of a serum marker for disease monitoring inchildren is particularly beneficial. Its implementation will allevi-ate exposure to radiation, the need of sedation for performance ofradiographic studies in young children, and the constraints onfrequently therapy monitoring.

In the present study, we evaluated for the first time the use ofa genomic fusion sequence as stable, noninvasive tumor bio-marker in EwS. ctDNA containing the characteristic and caus-ative EWSR1-FLI1 and EWSR1-ERG rearrangements proved asuitable marker for monitoring tumor burden at diagnosis,response to therapy, and disease relapse, complementaryto regular imaging surveillance. The analogous approach isapplicable to any translocation-positive malignancy in bothpediatric and adult oncology.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Krumbholz, U. Dirksen, M. MetzlerDevelopment of methodology: M. Krumbholz, J. Hellberg, T. B€auerle,C. Gillmann, M. MetzlerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Krumbholz, J. Hellberg, T. B€auerle, C. Gillmann,T. Fritscher, A. Agaimy, B. Frey, J. Juengert, E. Wardelmann, W. Hartmann,U. Dirksen, M. MetzlerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Krumbholz, J. Hellberg, B. Steif, T. B€auerle,C. Gillmann, A. Agaimy, M. MetzlerWriting, review, and/or revision of themanuscript:M.Krumbholz, J.Hellberg,B. Steif, T. B€auerle, C. Gillmann, A. Agaimy, B. Frey, J. Juengert, E. Wardelmann,W. Hartmann, U. Dirksen, M. MetzlerAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): M. Krumbholz, B. Steif, T. B€auerle,E. Wardelmann, W. Hartmann, H. Juergens, M. MetzlerStudy supervision: M. Krumbholz, H. Juergens, M. Metzler

AcknowledgmentsThe authors thank Ursula Jacobs and Sabine Semper for excellent technical

assistance, as well as Perdita Weller, Tatjana Flamann, Susanne Jabar, DagmarClemens, and Petra Fischer for the storage and recovery of the patient's plasmaand tissue samples. They also thank Jennifer Lawlor for language editing.

Grant SupportThis research was supported by grants of the Madeleine Schickedanz Kin-

derkrebs-Stiftung, Schornsteinfeger helfen krebskranken Kindern, EUROEWING Consortium (EEC)—international clinical trials to improve survivalfrom EwS (grant agreement number 602856; to M. Metzler and U. Dirksen),German Cancer Aid (to U. Dirksen; DKH 108128), and the EraNet consortiumPrOspectiveVAalidationofBiomarkersinEwingSarcoma (PROVABES ERA-Net-TRANSCAN; 01KT1310).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 16, 2015; revised February 23, 2016; accepted April 14,2016; published OnlineFirst June 9, 2016.

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Correction

Correction:GenomicEWSR1FusionSequenceas Highly Sensitive and Dynamic PlasmaTumor Marker in Ewing Sarcoma

In this article (Clin Cancer Res 2016;22:4356–65), which was published in theSeptember 1, 2016, issue of Clinical Cancer Research (1), EURO EWING Consor-tium funding was incorrectly listed in the grant support statement. The fullstatement should read as follows: "This research was supported by grants of theMadeleine Schickedanz Kinderkrebs-Stiftung, Schornsteinfeger helfen krebskran-ken Kindern, German Cancer Aid to UD (DKH 108128) and the EraNetconsortium PrOspectiveVAalidationofBiomarkersinEwingSarcoma (PROVABESERA-Net-TRANSCAN (01KT1310)). This project has received funding from theEuropean Union's Seventh Framework Programme for research, techno-logical development and demonstration under grant agreement no 602856(to M. Metzler and U. Dirksen)." The authors regret this error.

Reference1. Krumbholz M, Hellberg J, Steif B, B€auerle T, Gillmann, Fritscher T, et al. Genomic EWSR1 fusion

sequence as highly sensitive and dynamic plasma tumormarker in ewing sarcoma. Clin Cancer Res2016;22:4356–65.

Published online January 16, 2017.doi: 10.1158/1078-0432.CCR-16-2685�2017 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 23(2) January 15, 2017610

2016;22:4356-4365. Published OnlineFirst June 9, 2016.Clin Cancer Res Manuela Krumbholz, Julia Hellberg, Benedikt Steif, et al. Dynamic Plasma Tumor Marker in Ewing Sarcoma

Fusion Sequence as Highly Sensitive andEWSR1Genomic

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