Precision Medicine in Pediatric Oncology: Translating Genomic Discoveries Into Optimized Therapies Thai Hoa Tran1,2, Avanthi Tayi Shah3,4, and Mignon L

Published OnlineFirst June 9, 2017; DOI: 10.1158/1078-0432.CCR-16-0115

Review Clinical Cancer Research Precision Medicine in Pediatric Oncology: Translating Genomic Discoveries into Optimized Therapies Thai Hoa Tran1,2, Avanthi Tayi Shah3,4, and Mignon L. Loh3,4

Abstract

Survival of children with cancers has dramatically improved aberrant activation of signaling pathways, and epigenetic modi- over the past several decades. This success has been achieved fiers that can be targeted by novel agents. Thus, the recently through improvement of combined modalities in treatment described genomic and epigenetic landscapes of many child- approaches, intensification of cytotoxic chemotherapy for hood cancers have expanded the paradigm of precision med- those with high-risk disease, and refinement of risk stratifica- icine in the hopes of improving outcomes while minimizing tion incorporating novel biologic markers in addition to tra- toxicities. In this review, we will discuss the biologic rationale ditional clinical and histologic features. Advances in cancer for molecularly targeted therapies in genomically defined sub- genomics have shed important mechanistic insights on disease sets of pediatric leukemias, solid tumors, and brain tumors. biology and have identified "driver" genomic alterations, Clin Cancer Res; 23(18); 5329–38. 2017 AACR.

Introduction which may represent actionable therapeutic targets. In this review, we will discuss how genomic discoveries are being translated from Survival rates for children diagnosed with cancer have the bench into the clinic, resulting in the development of precision improved substantially over the past five decades. Today, long- medicine trials for specific subtypes of pediatric hematologic term survival is expected for approximately 80% of children malignancies, solid tumors, and brain tumors. Immunotherapeu- treated with contemporary therapies (1). These successes have tic approaches will be discussed separately. occurred as a result of multicentered, randomized clinical trials that have largely tested the efficacy of dose intensification of conventional cytotoxic chemotherapy and the implementation Precision Medicine Therapeutics in of enhanced supportive care. Nevertheless, childhood cancer Pediatric Hematologic Malignancies remains the leading cause of non–accident-related death in chil- Acute lymphoblastic leukemia dren, and for many cancers, further intensification of cytotoxic The treatment of Philadelphia chromosome–positive þ chemotherapy has failed to provide additional therapeutic benefit acute lymphoblastic leukemia (Ph ALL) represents the first and has instead resulted in increased treatment-related mortality paradigm of precision medicine principles in pediatric oncol- þ and undesirable long-term toxicity (2, 3). Therefore, innovative ogy. Comprising 3% of pediatric ALL, Ph ALL harbors the therapeutic approaches are urgently needed for patients with canonical t(9;22)(q34;q11) translocation, resulting in the þ high-risk (HR) disease. Meanwhile, advances in cancer genomics BCR–ABL1 oncoprotein. Ph ALL was previously associated have revolutionized the field of oncology and have become a with a dismal outcome despite intensive multiagent chemo- crucial component in modern risk stratification algorithms and therapy and required hematopoietic stem cell transplantation novel therapeutic avenues. The genomic and epigenetic land- (HSCT) in first remission (4). However, targeted inhibition scapes of many childhood cancers have been recently elucidated of the ABL1 kinase domain with imatinib in combination and provide important insights into genetic alterations, signal with an HR ALL cytotoxic chemotherapy backbone significantly þ transduction abnormalities, and epigenetic dysregulation, all of improved survival among children with Ph ALL, such that HSCT is no longer universally recommended in first remission (4). Recent large-scale genomic profiling studies have identified 1Department of Pediatrics, Centre Mere-Enfant, Centre Hospitalier de l'Uni- a new subtype of HR B-lineage ALL (B-ALL) in which this versite Laval, Quebec, Canada. 2Centre de Recherche du Centre Hospitalier paradigm of molecularly targeted therapy may be similarly Universitaire de Quebec, Universite Laval, Quebec, Canada. 3Department of efficacious. Philadelphia chromosome–like ALL (Ph-like ALL) Pediatrics, Benioff Children's Hospital, University of California, San Francisco, is characterized by a gene expression profile (GEP) similar to 4 þ San Francisco, California. Helen Diller Family Cancer Research Center, Univer- that of Ph ALL but lacks the hallmark BCR–ABL1 oncoprotein. sity of California, San Francisco, San Francisco, California. Patients with Ph-like ALL who exhibit adverse clinical features Corresponding Author: Mignon L. Loh, Benioff Children's Hospital, 1450 3rd face an exceptionally poor prognosis compared with patients Street, HD 284, San Francisco, CA 94143-0434. Phone: 415-577-5145; Fax: 415- with HR B-ALL without the Ph-like GEP when treated with 514-4996; E-mail: [email protected] modern chemotherapy regimens (5). The frequency of Ph-like doi: 10.1158/1078-0432.CCR-16-0115 ALL increases with age, ranging from 15% in children and 2017 American Association for Cancer Research. adolescents to over 20% in adults, with a peak among young

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adults between 21 and 39 years of age (6). A recent compre- chemotherapy backbone to establish their efficacy in Ph-like hensive genomic analysis of Ph-like ALL unraveled its hetero- ALL with ABL-class fusions or JAK/STAT pathway alterations, geneous genomic landscape characterized by a diverse range of respectively (Fig. 1; Table 1). mutations in genes that are crucial in B-cell development as Infant ALL represents another rare HR subset of B-ALL in which well as genetic alterations activating kinases and cytokine innovative approaches are needed to address the high rates receptor genes, rendering Ph-like ALL amenable to treatment of treatment failure and improve outcomes. Rearrangement of with tyrosine kinase inhibitor (TKI) therapy (7). Approximately the MLL gene at 11q23, now known as KMT2A, is a molecular half of Ph-like ALL patients harbor rearrangements of the hallmark of infant ALL that occurs in up to 75% of cases (10). cytokine receptor like factor 2 (CRLF2)gene,andconcomitant KMT2A-rearrangements (KMT2A-R) represent the most impor- activating JAK mutations are present in half of the CRLF2- tant negative prognostic factor in infant ALL, particularly for rearranged cases. The remainder of Ph-like ALL is characterized those less than 90 days old. Efforts to decipher the biology of by kinase-activating alterations in the ABL-class genes ABL1, infant ALL have identified potential therapeutic candidates to be ABL2, PDGFRB,andCSF1R (13%); JAK2 and EPOR rearrange- investigated in clinical trials, although the genomic landscape of ments (11%); other JAK/STAT pathway lesions, such as muta- infant ALL is characterized by the paucity of somatic mutations tions in IL7R or focal deletion of SH2B3 (13%); Ras pathway outside of KMT2A-R (11). AALL0631 was the first precision mutations (4%); and other rare kinase fusions, such as NTRK3 medicine trial in KMT2A-R infant ALL in which lestaurtinib, an and DGKH (<1%; ref. 6). A rapidly growing body of work from FLT3 inhibitor, was added to chemotherapy given the frequent in vitro assays and patient-derived xenografts has demonstrated FLT3 overexpression within this subtype (12). Unfortunately, the that the myriad of kinase alterations in Ph-like ALL converges to addition of lestaurtinib failed to improve survival outcomes activate three common downstream pathways: the JAK–STAT, relative to standard treatment in de novo cases. Similarly, chemo- ABL, and PI3K/AKT/mTOR signaling pathways, each of which is therapy combined with quizartinib, a second-generation FLT3 targetable with the relevant TKIs, providing compelling ratio- inhibitor, did not have significant clinical activity in the relapsed nale for testing the upfront efficacy of combinatorial TKI and setting (13). Alternatively, there is increasing evidence demon- conventional chemotherapy regimen (6, 8, 9). Specifically, the strating widespread epigenetic dysregulation in KMT2A-R ALL. Children's Oncology Group (COG) is currently testing the Therefore, the next generation of trials will test whether demethy- incorporation of dasatinib or ruxolitinib into a conventional lating/hypomethylating agents (e.g., decitabine, azacitidine, and

B-ALL T-ALL AML

Hyperdiploidy (25%) NOTCH mutations (50%) KMT2A-rearranged (20%) RAS ETV6–RUNX1 (25%) TAL1/LMO2 fusions (50%) mutations (20%) RUNX1–RUNX1T1 IKZF1 deletions (15%) TLX3 (12%) fusions (20%) FLT3-ITD CRLF2-rearranged (8%) mutations (10%) TLX1 fusions (10%) WT1 mutations (10%) Ph-like kinase fusions (7%) LYL1 CBFB–MYH11 KMT2A fusions (10%) (8%) -rearranged (6%) NPM1 mutations (8%) TCF3-rearranged (4%) ETP (10%) PML–RARα KMT2A (7%) DUX4-rearranged and/or -rearranged (5%) Monosomy 5 and 7 (6%) ERG deletions (4%) NUP214–ABL1 (5%) KIT mutations (5%) ZN384-rearranged (4%) PICALM–MLLT10 (5%) CEBPA mutations (5%) BCR–ABL1 (3%) NUP98 fusions (5%) MEF2D-rearranged (3%) IDH1/IDH2 mutations (4%) CSF3R iAMP21 (2%) mutations (2%) CBFA2T3–GLIS2 (2%) Hypodiploidy (1%) DEK–NUP214 (<1%) RBM15–MKL1 (<1%)

Figure 1. Recurrent cytogenetic and molecular abnormalities in pediatric hematologic malignancies. The frequency of different genomic alterations of pediatric B-ALL, T-lineage ALL (T-ALL), and acute myeloid leukemia (AML) is depicted. Of note, the cumulative incidence does not add up to 100%, as more than one alteration can occur concomitantly.

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Table 1. Precision medicine trials for different leukemia subtypes and their associated genomic alterations Leukemia subtype Common alterations Precision medicine approach Clinical trials PhþALL BCR–ABL1 Chemotherapy þ imatinib NCT03007147 – not yet recruiting Ph-like ALL ABL-class fusions (ABL1, Chemotherapy þ dasatinib NCT02883049 – recruiting ABL2, CSF1R,andPDGFRB) NCT02420717 – recruiting CRLF2 rearrangements and Chemotherapy þ ruxolitinib NCT02723994 – recruiting other JAK–STAT pathway NCT02420717 – recruiting lesions (JAK2 and EPOR rearrangements and/or IL7R and SH2B3 mutations) Infant ALL KMT2A rearrangements Chemotherapy þ azacitidine NCT02828358 – recruiting associated with FLT3 overexpression and epigenetic dysregulation Chemotherapy þ bortezomib þ vorinostat NCT02553460 – recruiting T-ALL Activated NF-kB pathway Chemotherapy þ bortezomib NCT02112916 – recruiting Relapsed ALL PI3K/AKT/mTOR pathway alterations Chemotherapy þ everolimus NCT01523977 – recruiting Chemotherapy þ temsirolimus NCT01614197 – recruiting Epigenetic dysregulation Chemotherapy þ decitabine þ vorinostat NCT01483690 – completed Chemotherapy þ panobinostat NCT01321346 – completed AML FLT3-ITD mutant Chemotherapy þ sorafenib NCT01371981 – recruiting PML–RARa Chemotherapy þ ATRA NCT02339740 – recruiting þ arsenic trioxide Relapsed AML with core-binding Chemotherapy þ dasatinib NCT02680951 – recruiting factor mutations Abbreviation: AML, acute myeloid leukemia.

DOT1L inhibitors) or histone deacetylation inhibitors (e.g., vor- following relapse are dismal (17). Genomic characterization of inostat, panobinostat, and bromodomain inhibitors) in combi- matched patient samples at diagnosis, remission, and relapse nation with an ALL chemotherapy backbone will improve out- has deepened our understanding of the intertwined and com- comes (14). plex interactions between clonal architecture, outgrowth and Unlike B-ALL, the heterogeneity of genetic alterations in T- emergence of mutations, and mechanisms governing therapy lineage ALL (T-ALL) has precluded the identification of prognostic resistance. Substantial genomic changes exist between diagnos- factors for risk stratification and the development of targeted tic and relapsed ALL samples, illustrating the dynamic clonal therapeutic regimens. Nevertheless, genome-wide analyses have evolution of leukemogenesis at recurrence (18). However, identified lesions that dysregulate key signaling pathways, some relapse-acquired mutations can often be detected at low levels of which may represent novel therapeutic targets. For instance, the at diagnosis, implying that ALL relapse can arise from the Notch signaling pathway is the most commonly deregulated enrichment of rare, preexisting subclones (18). This finding pathway in T-ALL (15). Inhibition of Notch signaling by g-secre- further suggests a paradigm shift in which subclonal mutations tase inhibitors (GSI) that block the second cleavage of Notch1 may not only play a "passenger" role but rather, "drive" disease and subsequent activation have been investigated in preclinical relapse. Recurrent genetic alterations associated with relapsed and early-phase studies. Unfortunately, the gastrointestinal tox- ALL include (i) activating somatic mutations in Ras pathway– icity associated with first-generation GSIs and their lack of efficacy related genes; (ii) somatic mutations in genes that confer in more common adult solid tumors have precluded further chemoresistance, such as NT5C2, CREBBP,andPRPS1;and development in T-ALL trials. Alternative Notch-inhibiting strate- (iii) epigenetic dysregulation (19). Collectively, these data gies utilizing newer GSIs and anti-Notch1 immunotherapies provide compelling rationale for the incorporation of MEK are currently being investigated in adult trials (15). Furthermore, inhibitors and epigenetic modifiers in future precision medi- T-ALL is characterized by frequent activation of the PI3K/AKT/ cine trials for relapsed ALL. mTOR pathway, mostly as a result of inactivating mutations in PTEN. Inhibitors of this pathway have demonstrated preclinical Acute myeloid leukemia efficacy, both alone and in combination with cytotoxic chemo- Pediatric acute myeloid leukemia (AML) is a genetically het- therapy (16). Bortezomib, a proteasome inhibitor, is the only erogeneous disease with historically dismal outcomes, although targeted therapy that has reached the threshold of testing in a first- the introduction of cytarabine and anthracycline-based intensive line phase III trial for de novo pediatric T-ALL. The inclusion of chemotherapy regimens now results in cure rates between 50% bortezomib is based on the observation that constitutive activa- and 65% (20). Intensification of conventional chemotherapy has tion of NF-kB often occurs in T-ALL blasts as a result of Notch or been maximized given the high frequency of treatment-related Akt activation. Furthermore, preclinical studies demonstrated that mortality in this patient population. Therefore, the development inhibition of NF-kB by bortezomib can enhance leukemia cell of novel therapeutic strategies relies on the knowledge derived sensitivity to other traditional chemotherapeutic agents and may from recent gene discovery studies. Approximately 75% of pedi- reverse steroid resistance, rendering it a promising target to atric AML cases harbor genetic aberrations that may represent incorporate into upfront clinical trials (15). critical molecular targets. The most common cytogenetic abnor- Despite survival rates approaching 90% in children with ALL malities in childhood AML are the t(8;21)(q22;q22) and inv(16) who are treated with modern chemotherapy regimens, approx- (p13.1q22) translocations involving core-binding factor (CBF- imately 15% to 20% of patients still relapse, and outcomes AML), KMT2A-rearranged AML, and t(15;17)(q22;q21). Another

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major subgroup is defined by activating mutations in genes such were identified as prognostic biomarkers (27, 28). Although as FLT3, KIT, and RAS, resulting in dysregulated signal transduc- some of these alterations have been incorporated into the tion (21). The first precision medicine paradigm in AML incor- International Neuroblastoma Risk Group (INRG) classification porated all-trans retinoic acid to target the pathognomonic PML– for risk group assignment (29), none of these findings have RARa fusion of acute promyelocytic leukemia. The next-genera- been translated into tailored therapies due to insufficient tion of AML-targeted therapy trials focused on the addition of understanding of the oncogenes or tumor suppressors at these FLT3 inhibitors to therapy regimens for patients with FLT3 muta- loci and their contribution to disease pathogenesis. Even for tions, which occur in 10% to 15% of pediatric AML and confer a MYCN, whose role in tumorigenesis is well understood (30), poor prognosis (21). The current COG phase III trial for de novo there are yet to be targeted approaches. This is attributable to AML, AAML1031, assesses the combinatorial effect of sorafenib, a difficulties in designing a small molecule that can bind its multi-TKI with activity against FLT3, and conventional chemo- helix–loop–helix structure (31). One approach is to target therapy for FLT3-mutant AML. Other potentially targetable muta- MAX, the heterodimerization partner of MYC, which is required tions that are now being investigated in early-phase clinical trials for DNA binding. Drugs targeting the MYC:MAX interaction include those involving KIT and components of the Ras pathway have demonstrated potential in vitro and in vivo (32). Another (22). A phase I study of dasatinib has been completed in children method is to target synthetic lethal interactions through CDK1, with imatinib-resistant KIT mutations, whereas small-molecule CDK2, or CHK1 inhibition (33–35). There are no active pedi- Ras-pathway inhibitors have not yet reached clinical trials despite atric trials with CDK1 or CDK2 inhibitors, but the COG will be promising results in preclinical models (23). The distinct epige- opening a phase I study with a CHK1 inhibitor. Drugs that netic profile of pediatric AML may further identify a subset of target mTOR, PI3K, Aurora kinase, and Akt decrease MYCN patients who may benefit from the addition of epigenetic modi- stabilization, providing an alternative therapeutic strategy (32, fiers. Recent studies testing the efficacy of hypomethylating 36, 37). Pediatric dosing for mTOR and PI3K/mTOR inhibitors agents, such as decitabine or azacitidine, in very HR or relapsed is available, although their utility in neuroblastoma has not AML patients have shown early efficacy and may warrant further been fully investigated. Aurora kinase and Akt inhibitors are investigation (24). currently in pediatric clinical phase testing. A final approach to targeting MYCN is through bromodomain and extra terminal MYC Precision Medicine Therapeutics in (BET) inhibition, as family genes are targets of these chromatin readers. Studies of BET inhibitors in neuroblastoma Pediatric Solid and Brain Tumors cell lines and mouse models have demonstrated cytotoxicity Neuroblastoma (38); however, these drugs have yet to make it into clinical trials The clinical presentation and survival rates of neuroblastoma due to concerns for off-target toxicities. patients vary greatly. Infants can present with tumors that regress Next-generation sequencing technologies have shed light on spontaneously. Children with localized disease have >90% sur- neuroblastoma heterogeneity at diagnosis and relapse (Fig. 2). vival with minimal chemotherapy, whereas those with widely Alterations most commonly occur in ALK at rates of 8% to 14% at metastatic disease have only 40% to 50% survival despite receiv- diagnosis and 26% to 43% at relapse (39). Clinical trials with ing intensive multimodal regimens. Tumors in older children and crizotinib, which is FDA approved for the treatment of ALK- adolescents are often chemoresistant, with a chronic, indolent rearranged non–small cell lung cancer, and early-phase clinical course (25). The association between MYCN amplification and trials with next-generation ALK inhibitors have generated excite- aggressive disease was first identified in the 1980s (26). Subse- ment among clinicians. Although RAS–MAPK alterations are quently, DNA ploidy, gains of 17q, and deletions of 1p or 11q rare at diagnosis, relapsed/refractory neuroblastoma specimens

Diagnosis Relapse

Other/ RAS–MAPK unknown ALK

Cell cycle Cell cycle ALK Figure 2. Relative frequency of genomic Other/unknown alterations in neuroblastoma at diagnosis compared with relapse. RAS–MAPK

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demonstrate enrichment for activating mutations in this pathway Table 2. Recurrent alterations in fusion-negative rhabdomyosarcomas are and susceptibility to MEK inhibition (40), offering another tar- listed as copy number gains/losses or mutations with associated amino acid change when published geted approach that is in clinical testing. Interestingly, the CDK4/ Gene Alteration Frequency CDK6 cell cycle–regulatory pathway is implicated in neuroblas- FGFR4 – CCND1 CDK4 fi Mutation 6% 9% toma pathogenesis through and ampli cation V550L, N535K, G528C CDKN2A or deletion, and the frequency of these alterations may IGF1R Copy number gain <1%–2% be increased in the relapsed setting (39). In xenograft models, IGF2 Mutation 1% dual CDK4/CDK6 inhibition induced cell-cycle arrest and senes- PDGFRA Mutation 1.4% cence (41). Furthermore, CDK4 inhibition sensitized MYCN- ERBB2 Mutation 1.4% MET amplified neuroblastoma cells to doxorubicin-mediated death Copy number gain 1% BRAF Mutation <1%–1% (42). Collectively, these discoveries underscore the potential V600E for precision medicine to augment conventional therapies NRAS Mutation 8%–12% for neuroblastoma and highlight the need for biopsy at the time G12A, Q61K, Q61H of relapse. KRAS Mutation 4%–6% G12A, G12C, G12D, G13D Sarcomas HRAS Mutation 3%–4% G13R, Q61K Sarcomas comprise <15% of pediatric malignancies (43) but NF1 Mutation 3.4% contribute largely to cancer-related morbidity and mortality. C1939_N1942FS, L18551, W784C These patients desperately need new therapeutic strategies yet PIK3CA Mutation 5%–6% have seen few advances over the decades. Recent studies have Q546, H1047R, E542K described the genomic landscape of Ewing sarcoma, rhabdomyo- CCND1 Mutation 1% sarcoma, and osteosarcoma (44–48). They have shed light on CCND2 Mutation 1% CDKN2A Copy number loss 2% sarcoma biology but have not yielded new treatments. This is due, MDM2 fi Copy number gain 8% in part, to the genomic pro le of pediatric sarcomas, which is TP53 Mutation 3.5% often characterized by gene rearrangements with a paucity of other A276D, C176F, P250L, L308V alterations. Canonical translocations are ideal candidates for CTNNB1 Mutation 2%–3.3% targeting, as they are drivers of oncogenesis only present in G34E, T41A, S45Y malignant cells. However, identifying tailored therapies has been MYOD1 Mutation <1% L122R challenging for at least two reasons. First, the chimeric protein is BCOR Mutation 6% often not easily druggable. For example, the Ewing sarcoma Copy number loss 1% translocation, EWS–FLI, does not form a fixed, three-dimensional FBXW7 Mutation 4.8% structure under physiologic conditions, impeding inhibitor R387P, R441G, R367P design (49). Only recently, a small-molecule, YK-4-279, was NOTE: Of note, frequency reported is across fusion-negative and fusion-positive identified; it is able to block EWS–FLI protein interactions and rhabdomyosarcomas. Data from Shukla et al. (93), Chen et al. (48), Shern et al. results in cell cytotoxicity (50). It has transitioned to phase I (47), and Kashi et al. (94). testing in relapsed/refractory patients (51). Second, targeting EWS–FLI, as well as the alveolar rhabdomyosarcoma PAX–FOXO fusion, is difficult due to an absence of inherent enzymatic activity. signaling pathways that affect growth, cell proliferation, and Unlike most oncogenic rearrangements that result in kinase survival, they pose an attractive target. However, clinical trials activation, these chimeric proteins activate and repress transcrip- with the FDA-approved agents imatinib, sorafenib, and sunitinib tional activity of a wide array of genes, and reversal of these gene did not induce significant responses in osteosarcoma (56–59). In signatures is quite complicated. Trabectedin, a synthetic alkaloid, addition, the anti–IGF-1R antibody cixutumumab, the anti-VEGF reversed the EWS–FLI signature in preclinical models; however, antibody bevacizumab, and the anti-HER2 antibody trastuzumab phase II testing did not demonstrate sufficient single-agent activity failed to demonstrate efficacy (60–62). Targeting the non–recep- (51). Phase Ib combination testing is currently recruiting. tor tyrosine kinase Src sheds light on another therapeutic avenue Incorporating precision medicine in the management of trans- for osteosarcoma patients, as it is implicated in the development location-negative sarcomas has posed a challenge as well. Patients of lung metastases through its activation of pathways necessary with fusion-negative rhabdomyosarcoma harbor alterations in for cell survival, migration, adhesion, and invasion. In vitro, multiple targetable pathways (Table 2). Unfortunately, these inhibition of Src resulted in decreased metastatic potential, but mutations occur at such a low rate that designing well-powered this was not replicated in vivo (63), highlighting that multiple clinical trials can be problematic. Even in the complex and pathways may be involved in the development of osteosarcoma unstable osteosarcoma genome, which harbors multiple struc- metastases. A clinical trial for patients with recurrent osteosarco- tural rearrangements and copy number changes, few targetable ma localized to the lung is currently underway. It is investigating alterations are recurrent across multiple patients. Although we the utility of saracatinib, an Src inhibitor, in patients who have have gained new insights into sarcoma oncogenesis through had a complete surgical resection of their tumor. Ewing sarcomas genomic profiling, there are still many obstacles to leverage them have recurrent abnormalities in cohesin complex genes, including into therapeutic strategies. TP53 alterations, CDKN2A deletions, and STAG2 mutations Despite these challenges, several candidates for targeted ther- (44–46). Synthetic lethality between cohesin complex mutations apies have been identified. IGF-1R, PDGFR, VEGFR, and HER-2 and PARP has been observed (64), thus prompting several have been observed to be overexpressed in osteosarcoma (52– trials investigating PARP inhibitors in combination with DNA- 55), and given their role in activating multiple downstream damaging agents in Ewing sarcoma.

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Central nervous system tumors loss of BRAF regulation and activation of the MAP kinase Recent advances in central nervous system (CNS) tumor pathway. The BRAFV600E mutation, which leads to constitu- sequencing are shifting the field from a focus on histopathologic tive activation of BRAF, is also found in low-grade gliomas. features to genetic identifiers. Rapid translation of molecular These findings have led to several clinical trials examining BRAF discoveries into informed therapies has gone hand in hand. inhibitors in the V600E-mutated patients. BRAF inhibitors are Recently, genomic profiling elucidated four subgroups, based on contraindicated in patients with BRAF fusions, as it results in molecular drivers of oncogenesis: WNT, SHH (Sonic Hedgehog), feedback loop–mediated upregulation of the pathway and group 3, and group 4. These subgroups have been incorporated accelerated tumor growth. Accordingly, MAP kinase inhibitors into the most recent 2016 World Health Organization (WHO) are under investigation in this cohort of patients. As our classification, which takes into account molecular markers in understanding of the molecular oncogenesis of CNS tumors addition to pathologic features (65), redefining the framework continues to grow, we are likely to identify new therapeutic in which CNS tumors were previously diagnosed. The WNT group strategies to enhance our current management of these patients. harbors somatic mutations in the CTNNB1 gene or, in more rare In light of these novel therapeutic avenues, the COG is instances, germline mutations in the APC tumor suppressor gene developing the pediatric counterpart of the adult NCI-MATCH (66). These alterations upregulate the WNT pathway, resulting in trial to enroll children and adolescents with relapsed/refractory tumorigenesis. No targeted therapies exist for these patients, but solid tumors, CNS tumors, and lymphomas (Fig. 3). In addi- because their overall survival approaches 90% (67), future studies tion to offering molecular-targeted therapy for these patients, may examine therapy deintensification. The SHH group contains the trial will also provide a rich source of genomic data for aberrations in the SHH pathway, most commonly in PTCH1 but future discovery. also in SMO, SUFU, and GLI2 (68). A phase I study of the FDA- approved SMO inhibitor vismodegib demonstrated activity in Precision Medicine in Pediatric Oncology: SHH medulloblastoma (69) and may offer a molecular-guided therapy for this subset of patients. It is important to note that New Horizons and Future Challenges tumors with abnormalities in SUFU or GLI2 are downstream of In addition to suggesting novel therapeutic targets, gene dis- SMO and, thus, resistant to vismodegib (70). Unfortunately, less covery studies enhance other aspects of the precision medicine is known about the specific genetic drivers in group 3 and group 4. algorithm, including cancer surveillance, assessment of inherited Group 3 tumors have the worst prognosis and a high frequency of susceptibility to therapy-related toxicity, and treatment response copy number abnormalities. They often have high MYC expres- monitoring. The Pediatric Cancer Genome Project identified that sion, and a subset is MYCN amplified. Group 4 tumors also 8.5% of children and adolescents with cancer had germline harbor copy number alterations and often have amplifications mutations in cancer predisposition genes in which family history in CDK6 and MYCN. As described above, there are challenges to failed to predict the presence of an underlying predisposition targeting MYC, but bromodomain inhibition has shown promise syndrome in most patients (78). The most commonly affected in preclinical models of medulloblastoma with MYCN amplifi- genes were TP53, APC, BRCA2, NF1, PMS2, RB1, and RUNX1.In cation (71). Palbociclib, a dual CDK4/CDK6 inhibitor is currently addition to several previously well-described predisposition syn- in phase I testing. dromes, such as Li-Fraumeni syndrome, new associations have Molecular features have also been integrated in the most recent been observed between germline mutations of TP53, PMS2, and WHO classification of posterior fossa and supratentorial ependy- RET mutations with Ewing sarcoma; APC and SDHB mutations momas given the wide histopathologic variation across tumors, with neuroblastoma; and a diverse range of mutations in APC, which results in lack of concordance even among experts (65, 72). VHL, CDH1, PTCH1, and SDHA with leukemia (78). Knowledge Posterior fossa ependymomas have been subdivided into two of these inherited predispositions has major implications on groups. Group A tumors lack recurrent genetic alterations but direct patient care and on disease surveillance as well as genetic have increased methylation of CpG islands, resulting in transcrip- counseling for patients and their families. Furthermore, several tional silencing of targets of polycomb repressive complex 2. In genome-wide association studies have identified polymorphisms vitro, drugs targeting the polycomb repressive complex 2 and in genes that predict susceptibility to common chemotherapy- DNA-demethylating agents impair proliferation of ependymoma related complications, such as polymorphisms in TPMT (79) and cells (73). Group B tumors have multiple chromosomal aberra- NUDT15, which are associated with abnormal thiopurine metab- tions, but none that are amenable to targeting (74). Supratentorial olism, resulting in severe myelosuppression (80); polymorph- ependymomas generally harbor two classes of gene rearrange- isms in GRIA1 associated with asparaginase allergy (81), whereas ments. The majority contains a fusion of c11orf95 and RELA, those in CPA2 are associated with asparaginase-induced pancre- whereas the remainder have fusions involving the transcriptional atitis (82); variants in ACP1, BMP7, and PROX1-AS1 that predis- coactivator YAP1 (75). Unfortunately, no targeted therapies exist pose to glucocorticoid-induced osteonecrosis (83, 84); poly- for these chimeric proteins. Because 75% percent of ependymo- morphisms in HAS3 predisposing to anthracycline cardiomyop- mas express aberrant ERBB2 and ERBB4 signaling (76), lapatinib, athy (85); polymorphisms in CEP72 associated with vincristine an ERBB inhibitor, has been the subject of active clinical inves- neuropathy (86); and variants in SLCO1B1 involved in metho- tigation (77). It has failed to demonstrate efficacy, but this may trexate clearance (87). Given that genetic testing for TMPT status reflect the need for higher levels of lapatinib in vivo and, thus, still to inform adjustments in mercaptopurine dosage has already holds promise. become standard practice in ALL, one could envision the imple- Pediatric low-grade gliomas are characterized by numerous mentation of a tailored pharmacogenomic panel relevant to a mutations and copy number alterations, including somatic specific chemotherapy regimen to individualize therapy based on alterations in BRAF. The most common alterations are translo- genetic risk factors that predict host toxicities. Finally, advances in cations in BRAF, which generate fusion proteins that result in the technologies to detect cancer cells have paved innovative ways

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Relapsed/refractory solid tumor, CNS tumor, or lymphoma

Biopsy at time of relapse

Actionable alteration identiﬁed

Matching study agent available

PI3K/mTOR MEK inhibitor BRAF inhibitor TRK inhibitor FGFR inhibitor ALK inhibitor EZH2 inhibitor PARP inhibitor inhibitor

Stable disease, partial response, complete response Progressive disease

Continue until progression Another actionable mutation identiﬁed

Progressive disease No Yes

Off study

Figure 3. Pediatric MATCH trial schema. to measure treatment response and ultimately predict risk of nologies into pragmatic, real-time, cost-effective clinical work- relapse. Childhood leukemia treatment response measured by flows for treatment decision making. In addition, new ethical minimal residual disease (MRD) has been identified as a powerful dilemmas have arisen due to the incidental detection of inherited independent prognostic factor (88). It has traditionally been susceptibility to cancer predisposition syndromes. Genomics- measured by flow cytometry or other molecular-based techni- driven targeted therapies will require a strong collaboration ques. However, sensitive next-generation sequencing–based MRD between basic scientists, molecular pathologists, bioinformati- monitoring is being tested prospectively in several pediatric cians, and oncologists to develop robust clinical trials to address oncology consortia for clinical relevance (89). Although MRD these issues in an effort to further improve patient survival. measurement has revolutionized the management of patients with leukemia, similar ultrasensitive biomarkers are essentially nonexistent for our solid tumor patients. Recently, several adult Conclusions studies demonstrated that detection of circulating tumor DNA Advances in cancer genomics have deepened our understand- (ctDNA) in the plasma can predict relapse earlier than can be seen ing of the disease biology, uncovered actionable genomic altera- on standard imaging studies. Very few investigations of the utility tions, and offered unique opportunities for precision medicine of ctDNA in pediatric solid tumors exist; however, preliminary approaches. Several molecular-targeted therapy trials are currently results using droplet digital PCR and next-generation sequencing under way in the hopes of balancing survival and toxicity for these are encouraging (90–92). In parallel, several challenges emerge HR patient populations. However, this exciting era of tailored from the new era of precision medicine, including intratumoral therapy also opens up new challenges. The next generation of genomic heterogeneity that makes it difficult to distinguish driver translational trials will aim: (i) to determine the optimal combi- from passenger mutations, lack of sustained response resulting nation between novel therapeutic agents and conventional cyto- from the emergence of acquired drug-resistant mutations and toxic drugs; (ii) to investigate the underlying mechanisms govern- escape pathways, and incorporation of genomic sequencing tech- ing therapy resistance as the emergence of drug-resistant

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mutations and compensatory feedback pathways can arise; and Grant Support (iii) to harness international collaboration to design statistically This work was supported by Alex's Lemonade Stand Foundation (to T.H. powered trials for rare, HR, genomically deﬁned entities. Hence, Tran and M.L. Loh), Leukemia & Lymphoma Society (6466-15; to M.L. Loh), St. precision medicine illustrates a novel treatment paradigm to Baldrick's Foundation (to A.T. Shah and M.L. Loh), NIH (1P50GM115279, P50 CA196519, U01CA176063, U10CA180886; to M.L. Loh), and Frank A. Cam- improve outcomes for children with cancer in this modern era pini Foundation. of multi-omics.

Disclosure of Potential Conﬂicts of Interest Received February 2, 2017; revised April 15, 2017; accepted June 6, 2017; No potential conﬂicts of interest were disclosed. published OnlineFirst June 9, 2017.

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5338 Clin Cancer Res; 23(18) September 15, 2017 Clinical Cancer Research

Precision Medicine in Pediatric Oncology: Translating Genomic Discoveries into Optimized Therapies

Thai Hoa Tran, Avanthi Tayi Shah and Mignon L. Loh

Clin Cancer Res 2017;23:5329-5338. Published OnlineFirst June 9, 2017.

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