multidimensionalclinomicsforprecisiontherapy of children ... · a fusion gene), which could be...

Personalized Medicine and Imaging

MultiDimensional ClinOmics for Precision Therapyof Children and Adolescent Young Adults withRelapsed and Refractory Cancer: A Report fromthe Center for Cancer ResearchWendy Chang1,2,3, Andrew S. Brohl1,4, Rajesh Patidar1, Sivasish Sindiri1, Jack F. Shern1,2,Jun S.Wei1, Young K. Song1, Marielle E. Yohe1,2, Berkley Gryder1, Shile Zhang1,Kathleen A. Calzone5, Nityashree Shivaprasad1, Xinyu Wen1, Thomas C. Badgett1,6,Markku Miettinen7, Kip R. Hartman8,9, James C. League-Pascual2,8, Toby N. Trahair10,Brigitte C.Widemann2, Melinda S. Merchant2, Rosandra N. Kaplan2, Jimmy C. Lin1, andJaved Khan1

Abstract

Purpose:We undertook a multidimensional clinical genomicsstudy of children and adolescent young adults with relapsed andrefractory cancers to determine the feasibility of genome-guidedprecision therapy.

Experimental Design: Patients with non-central nervous sys-tem solid tumors underwent a combination of whole exomesequencing (WES), whole transcriptome sequencing (WTS), andhigh-density single-nucleotide polymorphism array analysis ofthe tumor, with WES of matched germline DNA. Clinicallyactionable alterations were identified as a reportable germlinemutation, a diagnosis change, or a somatic event (including asingle nucleotide variant, an indel, an amplification, a deletion, ora fusion gene), which could be targeted with drugs in existingclinical trials or with FDA-approved drugs.

Results: Fifty-nine patients in 20 diagnostic categories wereenrolled from 2010 to 2014. Ages ranged from 7 months to

25 years old. Seventy-three percent of the patients had priorchemotherapy, and the tumors from these patients withrelapsed or refractory cancers had a higher mutationalburden than that reported in the literature. Thirty patients(51% of total) had clinically actionable mutations, of which24 (41%) had a mutation that was currently targetable in aclinical trial setting, 4 patients (7%) had a change indiagnosis, and 7 patients (12%) had a reportable germlinemutation.

Conclusions: We found a remarkably high number of clin-ically actionable mutations in 51% of the patients, and 12%with significant germline mutations. We demonstrated theclinical feasibility of next-generation sequencing in a diversepopulation of relapsed and refractory pediatric solid tumors.Clin Cancer Res; 22(15); 3810–20. �2016 AACR.

IntroductionWhile clinical genomic analysis of tumors has been increas-

ingly used to guide cancer care in adults, its efficacy in thepediatric setting is still under investigation. The genomic land-scape of pediatric cancers at diagnosis has been noted as havinga lower mutational burden than adult cancers (1–5). Currentstandard-of-care chemotherapy is particularly inadequate forthe 30%–40% of the pediatric solid tumor patients who havemetastatic, refractory, or relapsed disease. Given the need forimproved therapeutic strategies for these patients, we under-took a study to determine the utility and feasibility of perform-ing comprehensive genomic analyses to identify clinicallyactionable mutations in pediatric and young adult patientswith refractory or relapsed solid tumors.

Identification of somatic genomic events beyond single nucle-otide variations (SNV) increasingly plays a role in the diagnosisand prognosis of pediatric tumors. For example, the presence ofthe PAX3-FOXO1 fusion in rhabdomyosarcoma not only con-tributes the diagnosis of fusion-positive rhabdomyosarcoma, butalso imparts a poorer prognosis (6, 7). In neuroblastoma,MYCNamplification status places patients in the high-risk stage, which

1Oncogenomics Section, Genetics Branch, Center for CancerResearch, National Cancer Institute, NIH, Bethesda, Maryland. 2Pedi-atric Oncology Branch, Center for Cancer Research, National CancerInstitute, NIH, Bethesda, Maryland. 3Department of Pediatrics, Molec-ular Genetics, Columbia University Medical Center, New York, NewYork. 4Sarcoma Department, Moffitt Cancer Center, Tampa, Florida.5Genetics Branch, Center for Cancer Research, National Cancer Insti-tute,NIH,Bethesda,Maryland. 6PediatricHematology-Oncology,Ken-tuckyChildren'sHospital, Lexington, Kentucky. 7Laboratoryof Pathol-ogy, Center for Cancer Research, National Cancer Institute, Bethesda,Maryland. 8Walter Reed National Military Medical Center, Bethesda,Maryland. 9Uniformed Services University of the Health Sciences,Bethesda, Maryland. 10Centre for Children's Cancer and Blood Dis-orders, Sydney Children's Hospital, Randwick, New South Wales,Australia.

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

Corresponding Author: Javed Khan, Oncogenomics Section, Genetics Branch,Center for Cancer Research, National Cancer Institute, NIH, 37 Convent Drive,Room 2016B, Bethesda, MD 20892. Phone: 301-435-2937; Fax: 1-301-480-0314;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2717

�2016 American Association for Cancer Research.

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leads to a significant change in clinical management (8). Thepaucity of actionable mutations is a reflection of the low somaticmutational burden in pediatric cancers, which is in direct contrastto adult oncology where multiple reports have described a highpercentage of tumors with actionable mutations (9).

In this pilot study, we performed a multidimensional compre-hensive genomics analysis of patients referred to the PediatricOncology Branch (POB) of the National Cancer Institute's (NCI)Center for Cancer Research (CCR). This included whole exomesequencing (WES), whole transcriptome sequencing (WTS) of thetumor, WES of matched germline DNA, and high-density single-nucleotide polymorphism (SNP) array analysis of tumor. Ourgoal was to identify actionable genomic alterations defined as areportable germlinemutation, a change of diagnosis, or a somaticevent, which can be targetedwith drugs in existing clinical trials orwith drugs approved by the FDA. In particular, we determined theclinical utility of a multi-genomics platform to alter the manage-ment of children and young adults with relapsed and refractorycancers.

Patients and MethodsPatients

Patients with pediatric non-central nervous system (CNS) solidcancers that were undergoing biopsies for clinical indications ontreatment protocols at the POB, or at our collaborative institutes,were enrolled in our open-ended tumor profiling/specimenrepository protocol (NCT01109394). Written consent wasobtained from the patients or from their legal guardian if patientswere minors. Tumor tissue was only obtained if there was suffi-cient tissue available after clinical management needs of thepatientweremet. All patients hadmatchedwhole blood collected.The protocol was approved by the Institutional Review Board atthe NCI, at the NIH. Samples were deidentified after clinicalinformation and histologic diagnoses were compiled. Qualitycontrol genotyping for the samples was performed to ensure thematch of tumor/normal pairs.

Genome sequencing and analysisFor details, see Supplementary Methods.

Actionable mutationsWe defined actionable mutations as: (i) a reportable germline

mutation including nonsense or frameshift insertion or deletion(indel) of a cancer consensus gene or pathogenic or likely path-

ogenic mutation of an American College of Medical Genetics(ACMG) gene (11, 12), (ii) genomic alterations that changed thepatient's diagnosis, or (iii) a somatic event (including singlenucleotide variant (SNV), indel, amplification, deletion, or afusion gene), whichmay be targeted with FDA-approved medica-tions or drugs in existing clinical trials according to the NCI-adultMATCH Criteria (Supplementary Table S1). All actionable SNVsand indels reported in this article were confirmed first by visual-ization on an IGV viewer, and then by Sanger sequencing in aresearch setting. All actionable mutations for which clinical deci-sions weremade were validated in a Clinical Laboratory Improve-ment Amendments (CLIA)-certified laboratory.

ResultsPatient demographics

We present a unique cohort of 59 pediatric patients with solidtumors outside of the CNS, who were referred to the NIH from2010 to December 2014. A total of 64 children and adolescentyoung adults (AYA) with non-CNS solid tumors had sufficienttumor tissue and blood available for genomic analysis and wereconsented or enrolled onNCT01109394. Of these, 59 (92%) hadsuccessful complete multidimensional genomics performed onthe tumor and germlineDNA. Three patientswere excluded due topartial completion of the multi-omics studies. Two patients wereinitially sequenced using older poor quality NGS technology,resulting in insufficient material available for the newer sequenc-ing technologies. Patient demographics of this cohort are sum-marized in Supplementary Table S2. The median age at biopsywas 15 years, ranging from 7 months to 25 years. Gender distri-bution was 39% (n ¼ 23) female and 61% (n ¼ 36) male.Importantly, our cohort contained 20 different clinically aggres-sive solid tumor diagnoses reflecting the protocols and trials thatwere open at the POB during recruitment for this study. The mostcommon diagnoses in our cohort were Ewing sarcoma (n ¼ 10,20%), and neuroblastoma (n ¼ 10, 20%). Seventy-three percent(n ¼ 43) of the patients had received chemotherapy prior toenrolling in this study and were referred for clinical trials involv-ing novel agents. Finally, 15% (n ¼ 9) of the cases had multiplemetastases from the same time point and 8% (n¼ 5) of the casesincluded tumors from sequential time points (SupplementaryTable S2).

Multidimensional genomics platform: general somaticdiscoveries

On average, WES generated 86 million reads per sample to amedian depth of 68X (mean depth of 75X), and WTS had anaverage 227.6 million reads per sample (SupplementaryTable S2). We identified a total of 703 high confidence SNVsand 67 indels in our dataset (Supplementary Table S2) with amedian of 8 (range 0–387, average 22.2) somatic mutations perexome pair.

The number of somatic point mutations differed by diagnosis:malignantmelanoma had the highest number of somatic variantsper sample, while atypical teratoid rhabdoid tumors and fusiongene-driven cancers had the lowest number of SNVs per sample,similar to what has been described in the literature (Fig. 1; refs. 1,4, 5). In keeping with previous genomics studies of relapsedpediatric cancers by our group and others, mutations increasedwith respect to tumor time (Fig. 1; refs. 17–19). For neuroblas-toma, we found amedian somaticmutation relapse rate of 28.5 in

Translational Relevance

This study demonstrates the utility of a multidimensionalgenomics platform including whole exome sequencing(WES), whole transcriptome sequencing (WTS), and high-density single-nucleotide polymorphism array in the manage-ment of children and adolescent young adults with high risk,refractory, or relapsed cancers. We show that 51% of thepatients have clinically actionable mutations and 12% havesignificant reportable germlinemutations. Our study providesa roadmap for integrative genomics analysis as well as a robustbioinformatics and reporting pipeline for future precisiontherapy protocols for adults and children with cancers.

Multidimensional ClinOmics for Precision Therapy

www.aacrjournals.org Clin Cancer Res; 22(15) August 1, 2016 3811




the exome, which is about twice the median of 12–18 previouslyreported (2, 20). For Ewing sarcoma, we and others previouslyreported an average mutation rate of 6–7 somatic proteinaltering mutations per tumor (4, 21), whereas in our currentstudy with relapsed samples, we found approximately 3 timesthe rate with an average somatic protein altering mutation rateof 15. Of interest when a transcriptome filter with a variantallele frequency (VAF) of 10% was applied to somatic WESmutations, across the samples we found that 51% of the DNA-mutated genes were expressed at the transcript level. In 44% thesomatic variants were expressed, and in 7% only the referencealleles were expressed (Fig. 1). We and others have reported thesame phenomenon that approximately 50% of DNA somaticmutations are expressed in the RNA (22, 23).

Overall in our index cases, we found 125 somatic alterations inknown cancer consensus genes. This included 12 chimeric genes,54 nonsynonymous SNVs in 42 genes, 8 truncating or frameshift

mutations including stopgains or indels in 7 genes, 6 amplifica-tions in 2 genes, 4 homozygous gene deletions in 2 genes, and27 cases of loss of heterozygosity (LOH) in 5 genes (Fig. 2). Thesomatic SNVs were enriched for cancer pathways includingsignaling, transcription factors, splicing, DNA repair, and epi-genetic modifiers. As expected from the demographics of chil-dren and AYA patients seen at the POB, who were primarilydiagnosed with sarcomas, 26 (44%) of the patient's tumors hadgene fusions. All were identified with high confidence withexact breakpoint detection, placing WTS as an appropriatemethod for detection of these diagnostic fusion driver genesin the clinical setting (Fig. 2 and Supplementary Table S2).

Actionable genomic alterationsReportable germlinemutations. In the germline, 12 patients (20%)had nonsense or frameshift indels of cancer genes, with onepatient harboring a non-frameshift 2 base substitution, and an

Figure 1.Total and expressed SNVs in tumor samples. Red bars indicate expressed somatic SNVs, turquoise bars denote gene not expressed, and green show the referenceallele only was expressed. Horizontal dotted lines show the previously reported mean of total exomic SNVs by tumor diagnosis in literature. The purpletriangle represents a relapsed or refractory tumor sample. MM, malignant melanoma; TCC, transitional cell carcinoma; NET, neuroendocrine tumor; OS,osteosarcoma; FN-RMS, fusion-negative rhabdomyosarcoma; FP-RMS, fusion-positive rhabdomyosarcoma; US, undifferentiated sarcoma; DSRCT, desmoplasticsmall round cell tumor; MEC, myoepithelial carcinoma; CCS, clear cell sarcoma; SS, synovial sarcoma; MRT, malignant rhabdoid tumor; WT, Wilms tumor;ACC, adrenocortical carcinoma; ASPS, alveolar soft part sarcoma; MTC, medullary thyroid carcinoma; IMT, epithelioid inflammatory myofibroblastic sarcoma;RCC, renal cell carcinoma; PM, peritoneal mesothelioma.

Chang et al.

Clin Cancer Res; 22(15) August 1, 2016 Clinical Cancer Research3812




Figure 2.Landscape of pediatric and adolescent young adult solid tumors of index cases. At the top are the clinical characteristics, including change in diagnoses indicatedby a vertical arrow; the diagnostic abbreviations are the same as in Fig. 1 with the addition of EWLS for Ewing-like sarcoma. Fusion genes, somatic SNVs,copy number alterations in known cancer consensus genes, and germline alterations in cancer genes and American College of Medical Genetics (ACMG) genes arecolor coded as shown.






additional 5patientswith rare nonsynonymous SNVs.We foundatotal of 20 alterations in 18 genes in 16 patients (Fig. 2; Supple-mentary Table S2). All but one was validated by orthogonalsequencing methods. Overall 9 alterations in 8 genes (ATM,BRCA1, PMS2, PTEN, RET, TP53, TSC1, and TSC2) in 7 (12%)patients were considered reportable (Table 1). However, only fiveof these alterations would have been reported with strict adher-ence to the ACMG guidelines (Supplementary Table S1) (11, 12).In an adolescent patient with melanoma (NCI0072), we discov-ered a germline frameshift mutation in ATM, but interestingly,this patient's tumor had only one detected somatic alteration ofBRAF (V600E), which is a known driver mutation. A secondpatient with metastatic congenital melanoma (NCI0211) hadgermlinemutations in TSC1 and TSC2 and no somaticmutations.Although neither of these germline mutations alone was consid-ered reportable by ACMG guidelines because these are variants ofunknown significance, the mutation found in the TSC2 gene(T246A; see Table 1) is reported as a Human Gene MutationDatabase (HGMD) disease-causing mutation [CM087814 (24,25)]. Taken together, although the overall effect of these twomutations in combination was unknown, we considered themreportable. Our results indicate that children with melanomaswho have germline mutation have a low somatic burden com-pared with the highmutational burden seen in adult melanomas.

Two patients (NCI0152, and NCI0226) had germline TP53mutations, and had LOH with complete loss of the wild-typeallele in the tumor, rendering themutations likely pathogenic andreportable. The latter presented with a diagnosis of metastaticadrenocortical carcinoma, in which TP53 mutations are consid-ered the driver (26). Sequencing of a sibling and both parentsshowed this to be a de novo germline mutation (Table 1; Supple-mentary Fig. S2). The remaining ACMG reportable mutations

were found in a patient with neuroblastoma (NCI0010) that hadframeshift mutations in two cancer predisposition genes: BRCA1(ovarian and breast cancer) and PMS2 (Lynch syndrome andmismatch repair cancer syndrome; refs. 27, 28). Another patientwith a neuroendocrine tumor (NET) had a frameshift mutation inPTEN (R14fs) which is associated with hamartoma tumor syn-dromes. Thefinal patient withmedullary thyroid carcinomahad awell-described mutation in RET (M918T) associated with multi-ple endocrine neoplasia 2B (29). In summary, we report that 7 of59 (12%) patients with reportable germline mutations, which isconsiderably higher than the 2%–4% in expected incidentalfindings, but corresponds to a similar rate described in recentlypublished reports in germline mutations for both cancer predis-position genes in pediatric cancer and in WES of trios acrossclinical indications (30, 31).

Changes in diagnosis. Cancers of specific types have diagnosticgene expression signatures, as reported by our group and others(32). By hierarchical clustering of the 2,000 most highly differ-entially expressed genes, we found all the cancers clustered withtheir own subtype, with the exception of alveolar soft part sar-comas, which clustered with renal cell carcinoma samples (Sup-plementary Fig. S3). Interestingly, both of these tumors shared acommon oncogenic fusion gene, ASPSCR1-TFE3, indicating thatthe chimeric gene caused a dominant gene expression signature,which has important biologic implications. A tumor from asecond patient (NCI0108) who was initially referred to the NIHwith a diagnosis of neuroblastoma clusteredwith neuroendocrinetumors. A change in diagnosis was histologically confirmed afteranatomic pathology review.

Inspection of the fusion genes identified two unexpected diag-nostic fusions. One was a CIC-FOXO4 in a patient (NCI0165)

Table 1. Germline mutations in American College of Medical Genetics (ACMG) reportable genes and tumor suppressor genes identified in 7 patients

Sample Diagnosis Gene Mutation Disease Hotspot NotesReportable by StrictACMG Criteria

NCI0072 MM ATM p.Y380fs Ataxia-Telangiectasia and cancerpredisposition syndrome

No Frameshift insertion of tumorsuppressor gene

Yes

NCI0010 NB BRCA1 Q1313X Hereditary breast and ovarian cancersyndrome

Yes Pathogenic, reportable Yes

NCI0010 NB PMS2 p.K356fs Lynch syndrome and mismatch repaircancer syndrome

No Frameshift deletion of tumorsuppressor gene

Yes

NCINET2 NET PTEN p.R14fs PTEN Hamartoma tumor syndrome No Frameshift deletion of tumorsuppressor gene

Yes

NCI0228 MTC RET M918T Multiple endocrine neoplasia 2B Yes Pathogenic, reportable YesNCI0152 SS ! US TP53 R175H Li-Fraumeni syndrome Yes Patient tumor has LOH of

wild-type tp53 on otherallele

No

NCI0226 ACC TP53 A159K Li-Fraumeni syndrome Yes Tumor has LOH of wild-typetp53 on other allele, novel,2 base non-frameshiftsubstitution,c.358_359delGCinsTT

No

NCI0211 MM TSC1 p.S828R Tuberous sclerosis type 1,lymphangioleiomyomatosis, focalcortical dysplasia, and everolimussensitivity

No Nonsynonymous SNV,autosomal dominant, patientalso has a germline TSC2mutation

No

NCI0211 MM TSC2 p.T246A Tuberous sclerosis type 2, andlymphangioleiomyomatosis

Yes Nonsynonymous SNV,autosomal dominant, patientalso has a germline TSC1mutation

No

NOTE: Mutations were confirmed by direct visualization on an IGV viewer, and by Sanger sequencing.Abbreviations: ACC, adrenocortical carcinoma; MM, malignant melanoma; MTC, medullary thyroid carcinoma; NET, neuroendocrine tumor; RMS, rhabdo-myosarcoma; SS, synovial sarcoma; US, undifferentiated sarcoma; horizontal arrow indicates change in diagnosis.

Chang et al.





presenting to the NCI with a widely metastatic scalp Ewingsarcoma, whose diagnosis was changed to an Ewing-like sarcoma,as previously reported by our group (4). A second fusion wasfound in a patient presenting with a clear cell sarcoma of thekidney whose diagnosis was revised to an undifferentiated sar-coma following the identification of BCOR-CCNB3. This fusiongene has been reported in patients with Ewing-like and undiffer-entiated sarcomas (33). In addition to the presence of novelfusions, the absence of a diagnostic fusion gene resulted in achange in diagnosis, as seen in patientNCI0152, whowas initiallydiagnosed with a high-grade fibrous histiocytoma, which wasthen changed to a synovial sarcoma by histopathology after ahemipelvectomy. Our analysis revealed the absence of a patho-gnomonic synovial sarcoma SS18-SSX fusion gene. Instead, weidentified a germline TP53 (p.R175H) mutation. This mutationhas been previously described in a variety of sporadic tumors andwas foundas a germlinemutation in 2patientswithosteosarcoma(34, 35). The patient's diagnosis was subsequently changed to anundifferentiated sarcoma.

Linking mutation to drug.We next applied the NCI-Adult MATCHcriteria (Supplementary Table S1) tomatchmutations to drugs forpatients enrolled in our study.We found4 (12%)matched at level1, 13 (41%) at level 2, and 15 (47%) at level 3 (Table 2). Overallwe found a remarkably high drug-match of 32 genes (14 unique)in 24 (41%) patients in our population. This included alterationsin 14 genes (BRAF, ALK, RET, PIK3CA TSC1, TSC2, GNAQ,GNA11, CDKN2A, MYCN, PTEN, SMARCB1, STAG2, and IDH1)matching to 17 drugs or classes of drugs (36–42). Of note, two ofthese patients had germline mutations in TSC1 and TSC2(NCI0211), and RET (NCI0228) that would make them eligiblefor the NCI Adult-MATCH trial; as germline DNA is not currentlysequenced on their study, and it is not possible to distinguishgermline from somatic mutations if only the tumor DNA issequenced.We found that for the identification of these targetablemutations, the combination of WES and WTS was critical andconfirmatory in 22 of 32 (69%) matches where we required thesomatic driver variant to be expressed in the RNAseq experiments.SNP arrays in combination with WTS were used to match 9 of 32(28%) drugs in the cases of homozygous loss or amplification toresult in gene expression suppression or overexpression. Finally,in one case,WTS alone identified the actionable driver fusion geneRANBP2-ALK. For 14 of 32 (44%) of the matches, a pediatric trialis currently open, which would have enabled patients to bediverted to specific molecularly targeted therapy. For 16 of 32(50%) of the matches, there was an FDA-approved drug availablewith an adult indication.

Sequencing of multiple tumor samples per patient in relapsedand refractory pediatric tumors

In our cohort, we had 13 patients who had multiple biopsiesperformed. Of these patients, 9 samples were obtained frommultiple metastases from the same time point and 5 biopsieswere from different sequential time points (SupplementaryTable S2). We found that every tumor sample from the samepatient shared a set of common mutations (average 67.5%overlap; Fig. 3; Supplementary Table S2), demonstrating thatthese tumors shared a common ancestral clone in the patient.Eight of the 14 cases were fusion-positive sarcomas, and theidentical fusion was present in the matched samples. There werea significant number of nonoverlapping mutations in the

matched samples, and the majority of the driver mutations werecommon. The development of new mutations during tumorprogression has been previously reported by our group, wherewe showed that RAS pathway mutations were enriched for inthe relapsed setting, and that many of these mutations were notpresent in diagnostic samples, indicating tumor evolutionor selection during tumor progression (17). Similarly, in ourstudy, there was one notable example (NCI0167), who pre-sented with a refractory Ewing sarcoma lung metastasis, whichwas fully resected and found to have two likely driver muta-tions, an EWSR1-FLI1 fusion and a PIK3CA (p.D1017G) somat-ic mutation. After 16 months of treatment on a vaccine trial, thepatient relapsed in the lungs bilaterally, but both relapsedtumors lacked the PIK3CA mutation. However, the EWSR1-FLI1 fusion transcript in both metastases remained identical tothe initial fusion.

Responses to targeted therapy and development of resistanceWe present two vignettes to demonstrate the potential utility

of integrated genomic analysis of patient tumors at initialdiagnosis and relapse to guide therapy decisions. PatientNCI0244 was enrolled in our study at the time of a secondrelapse, at which time frozen tumor tissue from the first andsecond relapse was also available for genomic analysis (Sup-plementary Fig. S4A). The patient originally presented withabdominal distention and was found by laparotomy to havemesenteric and omental tumors. The patient was diagnosedwith epithelioid inflammatory myofibroblastic sarcoma (IMT),positive for ALK expression by IHC and a RANBP2-ALK fusiongene was confirmed by FISH (43). Crizotinib therapy wasinitiated. Follow-up imaging with PET/CT scans 8 months laterdocumented a complete metabolic and anatomic response.Crizotinib monotherapy was continued for another 6 months,at which time the patient relapsed and was switched to ceritinib(Supplementary Fig. S4A). WTS of both recurrent tumors dem-onstrated the presence of the RANBP2-ALK fusion. In addition,WTS and WES showed that both relapsed tumors acquired asecondary mutation in the ALK coding region, c.T3512C, p.I1171T (Supplementary Fig. S4A; Supplementary Table S2).Both the fusion gene and the ALK c.T3512C mutation wasconfirmed by RT-PCR and Sanger sequencing. Of note, thefusion gene was present by RT-PCR in a cell line derived fromthe tumor at initial diagnosis, but the ALK I1171T mutationwas absent by Sanger sequencing, indicating that mutationarose during therapy with crizotinib. This mutation has beenpreviously described to occur in the setting of post-treatmenttumor recurrence with resistance to the specific tyrosine kinaseinhibitor crizotinib (44, 45). Corresponding tumor shrinkageand a reduction in radiolabeled-2-fluoro-2-deoxy-D-glucose(FDG) uptake were documented one month after treatmentinitiation with ceritinib. Despite the initial rapid response, localdisease progression occurred after 2 months of treatment. Aliver metastasis taken post-ceritinib treatment contained thesame ALK mutation (c.T3512C) but no other somatic muta-tions were identified that could be implicated in the relapse.The patient passed away two years after initial commencementof crizotinib therapy.

In our second case, patient NCI0155 was initially diagnosedwith melanocytic neuroectodermal tumor, but the diagnosiswas changed to melanoma after histologic examination uponprogressive disease following cytotoxic chemotherapy. The






Table

2.Sum

maryofactiona

ble

mutations

inrelapsedan

drefractory

ped

iatric

solid

tumors

Sample

Diagno

sis

Gen

eStag

eModality

Mutation

AACha

nge

Leve

lDrug

Clin

ical

trial:

Ped

iatric

FDA-A

pprova

lin

adults

Exa

ctmutation

vs.ho

tspot

Referen

cepreclinical

data

forleve

l3

NCI0037

MM

BRAF

Relap

sed

WES/W

TS

NSSNV

p.V600E

1Vem

urafen

ib,

dab

rafenib

Yes

Yes

Exact

—

NCI0072

MM

BRAF

Diagno

stic

WES/W

TS

NSSNV

p.V600E

1Vem

urafen

ib,

dab

rafenib

Yes

Yes

Exact

—

NCI0215

MM

BRAF

Relap

sed

WES/W

TS

NSSNV

p.V600E

1Vem

urafen

ib,

dab

rafenib

Yes

Yes

Exact

—

NCI0155

MM

GNAQ

Relap

sed

WES/W

TS

NSSNV

p.Q20

9L

1Tem

sirolim

us,

tram

etinib,

vorino

stat

No

Yes

Exact

—

NCI0002

NB

ALK

—WES/W

TS

NSSNV

p.R1275

Q2a

Crizo

tinib

Yes

Yes

Exact

—

NCI0010

NB

ALK

Relap

sed

WES/W

TS

NSSNV

p.F1174

V2a

Crizo

tinib

Yes

Yes

Exact

—

NCI0017

NB

ALK

Relap

sed

WES/W

TS

NSSNV

p.F1174

L2a

Crizo

tinib

Yes

Yes

Exact

—

NCI0138

NB

ALK

Relap

sed

WES/W

TS

NSSNV

p.Y1278

S2a

Crizo

tinib

Yes

Yes

Exact

—

NCI024

4IM

TALK

Relap

sed

WTS

RANBP2-ALK

fusion

—2a

Crizo

tinib

No

Yes

Exact

—

NCI024

4IM

TALK

Relap

sed

WES/W

TS

NSSNV

p.I1171T

2aCeritinib

No

Yes

Exact

—

NCI0215

MM

GNA11

Relap

sed

WES/W

TS

NSSNV

p.S26

8F

2aTrametinib

No

Yes

——

NCI0041

EWS

IDH1

Relap

sed

WES/W

TS

NSSNV

p.R132C

2aIDH1Inhibitors

No

No

Exact

—

NCI0075

RMS

PIK3C

ARelap

sed

WES/W

TS

NSSNV

p.P104Q

2aPI3K/A

KT/m

TOR

Inhibitors

Yes

Yes

Exact

—

NCI0167

EWS

PIK3C

ARefractory

WES/W

TS

NSSNV

p.D1017G

2aPI3K/A

KT/m

TOR

Inhibitors

Yes

Yes

Exact

—

NCI0013

OS

PTE

NRelap

sed

WES/W

TS

Frameshiftdeletion

p.K80fs

2aPI3K/A

KT/m

TOR

Inhibitors

Yes

No

——

NCINET2

NET

PTE

N—

WES/W

TS

Germlinefram

eshift

deletion/somatic

LOH

p.R14fs

2aPI3K/A

KT/m

TOR

Inhibitors

Yes

No

——

NCI022

8MTC

RET

Relap

sed

WES/W

TS

GermlineSNV

p.M918T

2aVan

detan

ibYes

Yes

Exact

—

NCI0017

NB

CDKN2A

Relap

sed

SNPArray

/WTS

Homozygous

loss

—3

CDK4/6

Inhibitor

No

No

—36

NCI0071

EWS

CDKN2A

Relap

sed

SNPArray

/WTS

Homozygous

loss

—3

CDK4/6

Inhibitor

No

No

—36

NCINET2

NET

CDKN2A

—SNPArray

/WTS

Homozygous

loss

—3

CDK4/6

Inhibitor

No

No

—36

NCI0011

NB

MYCN

Relap

sed

SNPArray

/WTS

Amplifi

cation

—3

Bromodomain

inhibitors

No

No

—37

NCI0075

RMS

MYCN

Relap

sed

SNPArray

/WTS

Amplifi

cation

—3

Bromodomain

inhibitors

No

No

—37

NCI0102

NB

MYCN

—SNPArray

/WTS

Amplifi

cation

—3

Bromodomain

inhibitors

No

No

—37

NCI0136

NB

MYCN

Relap

sed

SNPArray

/WTS

Amplifi

cation

—3

Bromodomain

inhibitors

No

No

—37

NCI0138

NB

MYCN

Relap

sed

SNPArray

/WTS

Amplifi

cation

—3

Bromodomain

inhibitors

No

No

—37

NCI023

8WT

MYCN

Relap

sed

WES/W

TS

NSSNV

p.P44L

3Bromodomain

inhibitors

No

No

—37

,38

NCI0160

MRT

SMARCB1

—SNPArray

/WTS

Homozygous

loss

—3

EZH2Inhibitors

No

No

—39

,40

NCI025

0MRT

SMARCB1

Refractory

WES/W

TS

NSSNV

p.R40X

3EZH2Inhibitors

No

No

—39

,40

NCI0047

EWS

STAG2

Relap

sed

WES/W

TS

NSSNV

p.E984X

3PARPInhibitors

Yes

No

—41

NCI0150

EWS

STAG2

—WES/W

TS

NSSNV

p.R216X

3PARPInhibitors

Yes

No

Hotspot

41

NCI0211

MM

TSC1

Relap

sed

WES/W

TS

NSSNV

p.S828

R3

Eve

rolim

usNo

Yes

—42

NCI0211

MM

TSC2

Relap

sed

WES/W

TS

NSSNV

p.T24

6A

3Eve

rolim

usNo

Yes

—42

NOTE:S

NVswereco

nfirm

edbydirectvisualizationonan

IGVview

er,a

ndvalid

ationbySan

ger

seque

ncingorco

nfirm

ationCLIA-certified

laboratories.

Abbreviations:E

WS,E

wingsarcoma;

IMT,e

pithe

lioid

inflam

matory

myo

fibroblasticsarcoma;

MM,m

aligna

ntmelan

oma;

MRT,m

aligna

ntrhab

doid

tumor;MTC,m

edullary

thyroid

carcinoma;

NB,n

euroblastoma;

NET,

neuroen

docrinetumor;OS,o

steo

sarcoma;

RMS,rha

bdomyo

sarcoma;

WT,W

ilmstumor.

Chang et al.





primary tumor originated from the left frontotemporal scalpand left orbit and was metastatic to regional lymph nodes, duramater, liver, bone, and lung at diagnosis. WES and WTS of themetastatic tumor revealed a GNAQ (Q209L) mutation (presentin tumor DNA and RNA). The mutation, previously describedas a common driver mutation of uveal melanoma and anuncommon driver of cutaneous melanoma (46), was con-firmed in a CLIA-certified laboratory. The decision to treat withtrametinib, a MEK inhibitor approved for use in adult uvealmelanoma, was made at this time, and the patient was givenone month of bridging therapy with vorinostat while awaitingthe availability of trametinib. There was an initial mixedresponse to trametinib (Supplementary Fig. S4B). However,progressive disease of the primary tumor occurred within 5months after initiating therapy, and the patient was then takenoff trametinib therapy to receive adoptive cell therapy onanother clinical trial.

Our two clinical vignettes highlight the importance of multi-genomics analyses at initial presentation and disease progressionto identify driver mutations at diagnosis and during the emer-gence of resistant clones.

DiscussionIn this study, we developed a robust multidimensional

genomics platform to comprehensively interrogate the germ-line and cancer genome, to determine whether the managementof children with high risk, metastatic, refractory, or relapsedcancers can be considerably altered on the basis of these assays.

We used a combination of exome sequencing for both thetumor and matched normal tissue, high resolution copy num-ber analysis using SNP arrays, and whole transcriptomesequencing for the tumor. We defined actionable mutationsas a reportable germline mutation, a change in diagnosis, or asomatic or germline mutation that can be targeted with the useof existing drugs in clinical use.

We found a high percentage of reportable germline mutationsin 12%of patients, which underlies the importance of performinggermline WES. This high germline mutation rate is perhaps notsurprising given that these cancers occur at an early age, and areoften clinically aggressive. Our results are in accordance with arecent pediatric study which reported that 8.5% of children withcancer have pathogenic or likely pathogenic germline mutations(30). We were unable to confirm whether the majority of thegermline mutations in our patients were de novo or inherited, butour findings emphasize the importance of conducting futurelarge-scale familial cancer studies for all children presenting withcancer.

Pediatric cancers have been noted to have a generally quietgenomewith a low somatic burden in comparisonwith their adultcounterparts (4, 5, 20, 47). However, the majority of genomicstudies have been performed on diagnostic pretreatment tumorsamples. In this study we found, in keeping with two recentstudies in neuroblastoma, refractory or relapsed pediatric cancersnot only have an increased number of mutations but also containa higher percentage of actionable somatic mutations, comingclose to the number found in adult cancers at diagnosis (17,19, 47). Our findings also concur with another recently published

A B

Figure 3.Comparison of sSNVs in relapsed tumors and across metastases in the exome. A minimum threshold of 10 total reads, 3 variant reads, and a variant allele frequencyof �10% in the tumor DNA was used. Somatic SNVs common to the paired metastatic (A) or relapsed (B) tumors are shown in red bars, indicating acommon origin. Unique SNVs seen in each sample are shown in light blue bars.






study in the sequencing of relapsed cancers in youngpatients froma single center case series (48).

Overall, we found that the majority of patients had at least onemutation that was a previously described oncogenic driver. Fur-thermore,we founda total of 40 clinically actionablemutations in30 patients (51% of total), including germline findings, changesin diagnosis, and possible application of targeted therapy. Ofthese, the combination of WES with WTS was important foridentifying 67.5%, SNP arrays for 24.3%, and WTS alone for8.2%. Our WTS results showed that about one-half of all SNVsare not expressed in the transcriptome, and thus can be exclud-ed as a driver mutation. We and others have reported the samephenomenon that approximately 50% of DNA mutations areexpressed in the RNA (22, 23). The cause is usually that the RNAtranscript, and hence, the gene, is not expressed at the RNAlevel. In a small fraction of somatic mutations (7%) only thereference allele is expressed. This emphasizes the need for WTSas an important confirmatory assay for all samples, either bydemonstrating the expression of a somatic SNV, by showingloss of expression of a tumor suppressor, by displayingincreased gene expression levels with amplification, or byexhibiting a change in gene expression profiles. WTS is alsoa sensitive and specific method to identify diagnostic, novel,and druggable fusion genes.

We show here that the use of WES as part of a comprehensivemulti-omics platform allows for the detection of unexpectedactionable germline and somatic tumor mutations which maybe missed if panel sequencing or single-omics platforms areutilized. As the cost of sequencing drops, it will become feasibleto performWES for all patients with cancer, and many centers arerapidly moving to whole genome sequencing in combinationwith WTS for a more comprehensive analysis of the germline andcancer genome.

To identify targetable mutations, we used the criteria from thecurrently open National Cancer Institute Molecular Analysis forTherapy Choice (NCI-MATCH) tier system for adult patients. Thisclassification has been built to be flexible as additional medica-tions attain FDA approval, and new preclinical drugs emerge onthe experimental horizon. By using theNCIAdult-MATCHcriteriaas a reference for matching mutation to drug, 24 patients (41%)were considered to have a mutation that was currently targetablein a clinical trial. Of these, 12% matched at level 1 in which thegene variant is approved for selection of an approved drug (e.g.,BRAF V600E and vemurafenib). Forty-one percent matched atlevel 2a where the gene variant is an eligibility criterion for anongoing clinical trial. Themajority, 47%,matched at level 3wherepreclinical data in either in vivo or in vitromodels have previouslyprovided biologic evidence sufficient to support the use of avariant for treatment selection, but response to such therapy iscurrently unknown. All three levels of evidence are currently beingutilized in current clinical trials in adults with cancer; however,this is proposed to be tested using similar levels of evidence in apediatric MATCH, soon to open with the Children's OncologyGroup.

The Adult-MATCH trial does not perform germline evaluationand thus, the patient with a RET mutation (NCI0228; medullarythyroid carcinoma) would have been eligible for the trial, as itwould have been unknown if this mutation arose as a somaticevent or was germline. The actionability of the germline muta-tions in TSC1 and TSC2 (NCI0211; malignant melanoma) iscontroversial. In the setting of a congenital melanoma with a

low somatic mutational burden and no other actionable muta-tions, combined with predicted damaging germline mutations intwo TSC genes, in which one (TSC2), was reported as a causalmutation in HGMD (CM087814), we concluded this would beconsidered actionable.

Our study highlights the lack of drugs available for themajority of the somatic mutations that are detected in highconfidence but are mutations of unknown significance. Never-theless, 44% of the gene matches have a pediatric trial that iscurrently open, which could potentially enable precision ther-apy to be given to these identified patients. As many as 50% ofthe matches had an FDA-approved drug available with an adultindication, which can potentially be tested in future clinicaltrials.

On the basis of this pilot study, the CCR has established theClinOmics program to provide a multidimensional genomicsplatform to enable precision therapy trials in children and adultswith cancer enrolled on NCI trials (Supplementary Fig. S5). Theinfrastructure under the ClinOmics program will provide anumbrella protocol to identify actionable germline and somaticalterations in a patient within a CLIA-certified environment,whichwill be evaluated in germline genetics andmolecular tumorboards, and then reported into the electronic medical records. Inour feasibility study, our multi-omics analysis was performedusing fresh frozen tumors, which can cause potential problemsdue to normal tissue contamination. Ongoing clinical studiesincluding the NCI-MATCH trial and the planned ClinOmics trialwill utilize DNA and RNA macrodissected from formalin-fixedparaffin-embedded (FFPE) tissues verified by pathologists tocontain high tumor content. This, together with deep sequencing,will allow for the detection of subclonal mutations of 5% orlower.

Although the use of these comprehensive genomic analyses ofgermline and tumorwill likely identify actionable genomic altera-tions, single-agent targeted monotherapy may prolong survival,but is unlikely to be curative, as shown in our two clinicalvignettes. This underlies the importance of combination gene–drug matching, paired with conventional therapy, both at diag-nosis of high-risk metastatic cancers, as well in the refractory andrelapsed setting. Nevertheless, our findings demonstrate thatmultidimensional omics profiling in cancer is not only feasible,but alsowill likely havehighdiagnostic, therapeutic, and scientificyield for both adults and children with cancer.

Disclosure of Potential Conflicts of InterestM.S. Merchant is an employee of AstraZeneca. J.C. Lin is an employee

of Natera. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception anddesign:W.Chang, A. Brohl, J.S.Wei, T. Badgett,M.S.Merchant,J.C. Lin, J. KhanDevelopment of methodology: W. Chang, S. Sindiri, J.F. Shern, J.S. Wei,Y.K. Song, T. Badgett, J. KhanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Chang, Y.K. Song, M.E. Yohe, T. Badgett,M. Miettinen, K.R. Hartman, J.C. League-Pascual, T. Trahair, B.C. Widemann,M.S. Merchant, R.N. Kaplan, J. KhanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Chang, A. Brohl, R. Patidar, S. Sindiri, J.F. Shern,J.S.Wei, Y.K. Song, B.E.Gryder, S. Zhang, X.Wen,M.S.Merchant, J.C. Lin, J. KhanWriting, review, and/or revision of the manuscript: W. Chang, A. Brohl,R. Patidar, S. Sindiri, J.F. Shern, J.S. Wei, M.E. Yohe, B.E. Gryderm, K.A. Calzone,


Chang et al.




T. Badgett, K.R. Hartman, T. Trahair, B.C. Widemann, M.S. Merchant, J.C. Lin,J. KhanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W. Chang, R. Patidar, S. Sindiri, J.F. Shern,N. Shivaprasad, X. Wen, M. Miettinen, J.C. Lin, J. KhanStudy supervision: W. Chang, J.S. Wei, M.S. Merchant, J. KhanOther (data visualization): B.E. Gryder

AcknowledgmentsThe authors thank Paul S.Meltzer, Donna Bernstein, Hongling Liao, Amanda

Carbonell, ChohYeung, the Kaplan lab, JianbinHe, Li Chen, andBeverly Stalker

for their technical support and useful discussion. The authors also thank thePediatricOncology Branch clinical teamandpediatric oncology fellows for theircare of patients and sample procurement. This study utilized the high-perfor-mance computational capabilities of the Biowulf Linux cluster at the NIH(https://hpc.nih.gov/docs/userguide.html).

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.

ReceivedNovember10, 2015; revised January 28, 2016; accepted February 21,2016; published OnlineFirst March 18, 2016.

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Chang et al.




2016;22:3810-3820. Published OnlineFirst March 18, 2016.Clin Cancer Res Wendy Chang, Andrew S. Brohl, Rajesh Patidar, et al. Report from the Center for Cancer ResearchAdolescent Young Adults with Relapsed and Refractory Cancer: A MultiDimensional ClinOmics for Precision Therapy of Children and

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