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

Author Manuscript Published OnlineFirst on June 9, 2017; DOI: 10.1158/1078-0432.CCR-16-0115 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

Thai Hoa Tran1,2, Avanthi Tayi Shah3,4, Mignon L. Loh3,4

1Department of Pediatrics, Centre Mère-Enfant, Centre Hospitalier de l’Université Laval, Québec, QC, Canada 2Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec, QC, Canada 3Department of Pediatrics, Benioff Children’s Hospital, University of California, San Francisco, San Francisco, CA 4Helen Diller Family Cancer Research Center, University of California, San Francisco, San Francisco, CA

Running title: Precision Medicine in Pediatric Oncology

Keywords: leukemias and lymphomas, molecular diagnosis and prognosis, oncogene, tumor suppressor genes, and gene products as targets for therapy, Phase I-III trials, pediatric cancers

Funding support:

Alex’s Lemonade Stand Foundation: MLL, THT Leukemia Lymphoma Society: MLL (6466-15) St-Baldrick’s Foundation: MLL, ATS National Institutes of Health: MLL (1P50GM115279, P50 CA196519, U01CA176063, U10CA180886) Frank A. Campini Foundation

Correspondence:

Dr. Mignon Loh Loh Laboratory, Helen Diller Family Comprehensive Cancer Center, Box 3112 1450 3rd St, Room 284, San Francisco, CA 94158 E-mail: [email protected]

The authors have no conflicts of interest to declare.

Word count: 4356 words; Tables: 2; Figures: 3

Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 9, 2017; DOI: 10.1158/1078-0432.CCR-16-0115 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Survival of children with cancers has dramatically improved over the past several

decades. This success has been achieved through improvement of combined modalities

in treatment approaches, intensification of cytotoxic chemotherapy for those with high-

risk disease and refinement of risk stratification incorporating novel biologic markers in

addition to traditional clinical and histologic features. Advances in cancer genomics have

shed important mechanistic insights on disease biology and have identified “driver”

genomic alterations, aberrant activation of signaling pathways, and epigenetic modifiers

that can be targeted by novel agents. Thus, the recently described genomic and

epigenetic landscapes of many childhood cancers have expanded the paradigm of

precision medicine in the hopes of improving outcomes while minimizing toxicities. In

this review, we will discuss the biologic rationale for molecularly targeted therapies in

genomically-defined subsets of pediatric leukemias, solid tumors, and brain tumors.

Introduction

Survival rates for children diagnosed with cancer have improved substantially

over the last five decades. Today, long-term survival is expected for approximately 80%

of children treated with contemporary therapies (1). These successes have occurred as a

result of multi-centered, randomized clinical trials that have largely tested the efficacy of

dose intensification of conventional cytotoxic chemotherapy and the implementation of

enhanced supportive care. Nevertheless, childhood cancer remains the leading cause of

non-accident related death in children and for many cancers, further intensification of

cytotoxic chemotherapy has failed to provide additional therapeutic benefit and has

instead resulted in increased treatment-related mortality and undesirable long-term

toxicity (2, 3). Therefore, innovative therapeutic approaches are urgently needed for

patients with high-risk disease. Meanwhile, advances in cancer genomics have

revolutionized the field of oncology and have become a crucial component in modern

risk stratification algorithms and novel therapeutic avenues. The genomic and epigenetic

landscapes of many childhood cancers have been recently elucidated and provide

important insights into genetic alterations, signal transduction abnormalities and

epigenetic dysregulation, all of which may represent actionable therapeutic targets. In

this review, we will discuss how genomic discoveries are being translated from the bench

into the clinic, resulting in the development of precision medicine trials for specific

subtypes of pediatric hematologic malignancies, solid tumors and brain tumors.

Immunotherapeutic approaches will be discussed separately.

Precision medicine therapeutics in pediatric hematologic malignancies

Acute lymphoblastic leukemia

The treatment of Philadelphia-chromosome positive acute lymphoblastic

leukemia (Ph+ALL) represents the first paradigm of precision medicine principals in

pediatric oncology. Comprising 3% of pediatric ALL, Ph+ALL, harbors the canonical

t(9;22)(q34;q11) translocation, resulting in the BCR-ABL1 oncoprotein. Ph+ALL was

previously associated with a dismal outcome despite intensive multi-agent chemotherapy

and required hematopoietic stem cell transplantation (HSCT) in first remission (4).

However, targeted inhibition of the ABL1 kinase domain with imatinib in combination

with a high-risk (HR) ALL cytotoxic chemotherapy backbone significantly 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 a new subtype of HR B-lineage ALL in which this paradigm of molecularly

targeted therapy may be similarly efficacious. Philadelphia chromosome-like ALL (Ph-

like ALL) is characterized by a gene expression profile (GEP) similar to that of Ph+ALL

but lacks the hallmark BCR-ABL1 oncoprotein. Ph-like ALL patients who exhibit

adverse clinical features face an exceptionally poor prognosis compared to patients with

HR B-lineage ALL without the Ph-like GEP when treated with modern chemotherapy

regimens (5). The frequency of Ph-like ALL increases with age, ranging from 15% in

children and adolescents to over 20% in adults, with a peak among young adults between

21 to 39 years of age (6). A recent comprehensive genomic analysis of Ph-like ALL

unraveled its heterogeneous genomic landscape characterized by a diverse range of

mutations in genes that are crucial in B-cell development as well as genetic alterations

activating kinases and cytokine receptor genes, rendering Ph-like ALL amenable to

treatment with tyrosine kinase inhibitor (TKI) therapy (7). Approximately half of Ph-like

ALL patients harbor rearrangements of the cytokine receptor like factor 2 (CRLF2) gene,

and concomitant activating JAK mutations are present in half of the CRLF2-rearranged

cases. The remainder of Ph-like ALL is characterized by kinase-activating alterations in

the ABL-class genes ABL1, ABL2, PDGFRB, and CS1FR (13%), JAK2 and EPOR

rearrangements (11%), other JAK/STAT pathway lesions such as mutations in IL7R or

focal deletion of SH2B3 (13%), Ras-pathway mutations (4%), and other rare kinase

fusions such as NTRK3 and DGKH (<1%) (6). A rapidly growing body of work from in

vitro assays and patient-derived xenografts has demonstrated that the myriad of kinase

alterations in Ph-like ALL converges to activate three common downstream pathways:

the JAK-STAT, ABL, and PI3K-Akt-mTOR signaling pathways, each of which is

targetable with the relevant TKIs, providing compelling rationale for testing the upfront

efficacy of combinatorial TKI and conventional chemotherapy regimen (6, 8, 9).

Specifically, the Children’s Oncology Group (COG) is currently testing the incorporation

of dasatinib or ruxolitinib into a conventional chemotherapy backbone to establish their

efficacy in Ph-like ALL with ABL-class fusions or JAK/STAT pathway alterations,

respectively (Figure 1) (Table 1).

Infant ALL represents another rare high-risk subset of B-ALL in which

innovative approaches are needed to address the high rates of treatment failure and

improve outcomes. Rearrangement of the MLL gene at 11q23, now known as KMT2A, is

a molecular hallmark of infant ALL that occurs in up to 75% of cases (10). KMT2A-

rearrangements (KMT2A-R) represent the most important negative prognostic factor in

infant ALL, particularly for those less than 90 days old. Efforts to decipher the biology

of infant ALL have identified potential therapeutic candidates to be investigated in

clinical trials, although the genomic landscape of infant ALL is characterized by the

paucity of somatic mutations outside of KMT2A-R (11). AALL0631 was the first

precision medicine trial in KMT2A-R infant ALL in which lestaurtinib, a FLT3 inhibitor,

was added to chemotherapy given the frequent FLT3 overexpression within this subtype

(12). Unfortunately, the addition of lestaurtinib failed to improve survival outcomes

relative to standard treatment in de novo cases. Similarly, chemotherapy combined with

quizartinib, a second-generation FLT3 inhibitor, did not have significant clinical activity

in the relapsed setting (13). Alternatively, there is increasing evidence demonstrating

widespread epigenetic dysregulation in KMT2A-R ALL. Therefore, the next generation

of trials will test whether demethylating/hypomethylating agents (eg. decitabine,

azacitidine, DOT1L inhibitors) or histone deacetylation inhibitors (eg. vorinostat,

panobinostat, bromodomain inhibitors) in combination with an ALL chemotherapy

backbone will improve outcomes (14).

Unlike B-ALL, the heterogeneity of genetic alterations in T-lineage ALL (T-

ALL) has precluded the identification of prognostic factors for risk stratification and the

development of targeted therapeutic regimens. Nevertheless, genome-wide analyses have

identified lesions that dysregulate key signaling pathways, some of which may represent

novel therapeutic targets. For instance, the Notch signaling pathway is the most

commonly deregulated pathway in T-ALL (15). Notch signaling inhibition by gamma-

secretase inhibitors (GSIs) that block the second cleavage of Notch1 preventing its

activation, have been investigated in preclinical and early phase studies. Unfortunately,

the gastrointestinal toxicity associated with first-generation GSIs and their lack of

efficacy in more common adult solid tumors have precluded further development in T-

ALL trials. Alternative Notch-inhibiting strategies utilizing newer GSIs and anti-Notch1

immunotherapies are currently being investigated in adult trials (15). Furthermore, T-

ALL is characterized by frequent activation of the PI3K-Akt-mTOR pathway, mostly as a

result of inactivating mutations in PTEN. Inhibitors of this pathway have demonstrated

preclinical efficacy, both alone and in combination with cytotoxic chemotherapy (16).

Bortezomib, a proteasome inhibitor, is the only targeted therapy that has reached the

threshold of testing in a front line phase III trial for de novo pediatric T-ALL. The

inclusion of bortezomib is based on the observation that constitutive activation of NF-κB

often occurs in T-ALL blasts as a result of Notch or Akt activation. Furthermore,

preclinical studies demonstrated that inhibition of NF-κB by bortezomib can enhance

leukemia cell sensitivity to other traditional chemotherapeutic agents and may reverse

steroid resistance, rendering it a promising target to incorporate into upfront clinical trials

(15).

Despite survival rates approaching 90% in children with ALL who are treated

with modern chemotherapy regimens, approximately 15-20% of patients still relapse and

outcomes following relapse are dismal (17). Genomic characterization of matched patient

samples at diagnosis, remission, and relapse has deepened our understanding of the

intertwined and complex interactions between clonal architecture, outgrowth and

emergence of mutations, and mechanisms governing therapy resistance. Substantial

genomic changes exist between diagnostic and relapsed ALL samples, illustrating the

dynamic clonal evolution of leukemogenesis at recurrence (18). However, relapse-

acquired mutations can often be detected at low levels at diagnosis, implying that ALL

relapse can arise from the enrichment of rare, pre-existing subclones (18). This finding

further suggests a paradigm shift in which subclonal mutations may not only play a

“passenger” role, but rather may “drive” disease relapse. Recurrent genetic alterations

associated with relapsed ALL include: 1) activating somatic mutations in Ras pathway-

related genes; 2) somatic mutations in genes that confer chemo-resistance such as

NT5C2, CREBBP and PRPS1; and 3) epigenetic dysregulation (19). Collectively, these

data provide compelling rationale for the incorporation of MEK inhibitors and epigenetic

modifiers in future precision medicine trials for relapsed ALL.

Acute myeloid leukemia

Pediatric acute myeloid leukemia (AML) is a genetically heterogeneous disease

with historically dismal outcomes, though the introduction of cytarabine and

anthracycline-based intensive chemotherapy regimens now result in cure rates between

50 to 65% (20). Intensification of conventional chemotherapy has been maximized given

the high frequency of treatment-related mortality in this patient population. Therefore, the

development of novel therapeutic strategies relies on the knowledge derived from recent

gene discovery studies. Approximately 75% of pediatric AML cases harbor genetic

aberrations that may represent critical molecular targets. The most common cytogenetic

abnormalities in childhood AML are the t(8;21)(q22;q22) and inv(16)(p13.1q22)

translocations involving core-binding factor (CBF-AML), KMT2A-rearranged AML, and

t(15;17)(q22;q21). Another major subgroup is defined by activating mutations in genes

such as FLT3, KIT, and RAS, resulting in dysregulated signal transduction (21). The first

precision medicine paradigm in AML incorporated all-trans retinoic acid (ATRA) to

target the pathognomonic PML-RARα fusion of acute promyelocytic leukemia (APL).

The next-generation of AML targeted therapy trials focused on the addition of FLT3

inhibitors to therapy regimens for patients with FLT3 mutations, which occur in 10-15%

of pediatric AML and confer a poor prognosis (21). The current COG phase III trial for

de novo AML, AAML1031, assesses the combinatorial effect of sorafenib, a multi-TKI

with activity against FLT3, and conventional chemotherapy for FLT3-mutant AML.

Other potentially targetable mutations that are now being investigated in early phase

clinical trials include those involving KIT and components of the Ras pathway (22). A

phase I study of dasatinib has been completed in children with imatinib-resistant KIT

mutations, while small molecule Ras-pathway inhibitors have not yet reached clinical

trials despite promising results in preclinical models (23). The distinct epigenetic profile

of pediatric AML may further identify a subset of patients who may benefit from the

addition of epigenetic modifiers. Recent studies testing the efficacy of hypomethylating

agents such as decitabine or azacitidine in very high risk or relapsed AML patients have

showed early efficacy and may warrant further investigation (24).

Precision medicine therapeutics in pediatric solid and brain tumors

Neuroblastoma

The clinical presentation and survival rates of neuroblastoma patients vary

greatly. Infants can present with tumors that regress spontaneously. Children with

localized disease have >90% survival with minimal chemotherapy, while those with

widely metastatic disease only have 40-50% survival, despite receiving intensive

multimodal regimens. Tumors in older children and adolescents are often chemoresistant

with a chronic, indolent course (25). The association between MYCN amplification and

aggressive disease was first identified in the 1980s (26). Subsequently, DNA ploidy,

gains of 17q, and deletions of 1p or 11q were identified as prognostic biomarkers (27,

28). Although some of these alterations have been incorporated into the INRG

classification for risk group assignment (29), none of these findings have been translated

into tailored therapies, due to insufficient understanding of the oncogenes or tumor-

suppressors at these loci and their contribution to disease pathogenesis. Even for MYCN,

whose role in tumorigenesis is well-understood (30), there are yet to be targeted

approaches. This is attributable to difficulties in designing a small molecule that can bind

its helix-loop-helix structure (31). One approach is to target MAX, the

heterodimerization partner of MYC, which is required for DNA binding. Drugs targeting

the MYC:MAX interaction have demonstrated potential in vitro and in vivo (32).

Another method is to target synthetic lethal interactions through CDK1, CDK2, or CHK1

inhibition (33-35). There are no active pediatric trials with CDK1 or CDK2 inhibitors,

but the COG will be opening a Phase I study with a CHK1 inhibitor. Drugs that target

mTOR, PI3K, Aurora kinase, and Akt decrease MYCN stabilization, providing an

alternative therapeutic strategy (32, 36, 37). Pediatric dosing for mTOR and

PI3K/mTOR inhibitors is available, although their utility in neuroblastoma has not been

fully investigated. Aurora kinase and Akt inhibitors are currently in pediatric clinical

phase testing. A final approach to targeting MYCN is through bromodomain and extra

terminal (BET) inhibition, since MYC family genes are targets of these chromatin readers.

Studies of BET inhibitors in neuroblastoma cell lines and mouse models have

demonstrated cytotoxicity (38), however, these drugs have yet to make it into clinical

trials due to concerns for off-target toxicities.

Next-generation sequencing technologies have shed light on neuroblastoma

heterogeneity at diagnosis and relapse (Figure 2). Alterations most commonly occur in

ALK at rates of 8-14% at diagnosis and 26-43% at relapse (39). Clinical trials with

Crizotinib, which is FDA-approved for the treatment of ALK-rearranged non-small cell

lung cancer, and early phase clinical trials with next-generation ALK inhibitors have

generated excitement amongst clinicians. Although RAS-MAPK alterations are rare at

diagnosis, relapsed/refractory neuroblastoma specimens demonstrate enrichment for

activating mutations in this pathway, and susceptibility to MEK inhibition (40), offering

another targeted approach which is in clinical trial. Interestingly, the CDK4/CDK6 cell

cycle regulatory pathway is implicated in neuroblastoma pathogenesis through CCND1

and CDK4 amplification or CDKN2A deletion and the frequency of these alterations may

be increased in the relapsed setting (39). In xenograft models, dual CDK4/CDK6

inhibition induced cell cycle arrest and senescence (41). Furthermore, CDK4 inhibition

sensitized MYCN amplified neuroblastoma cells to doxorubicin-mediated death (42).

Collectively, these discoveries underscore the potential for precision medicine to

augment conventional therapies for neuroblastoma and highlight the need for biopsy at

the time of relapse.

Sarcomas

Sarcomas comprise <15% of pediatric malignancies (43), but contribute largely to

cancer-related morbidity and mortality. These patients desperately need new therapeutic

strategies, yet have seen few advances over the decades. Recent studies have described

the genomic landscape of Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma (44-

48). They have shed light on sarcoma biology, but have not yielded new treatments.

This is due, in part, to the genomic profile of pediatric sarcomas, which is often

characterized by gene rearrangements with a paucity of other alterations. Canonical

translocations are ideal candidates for targeting, as they are drivers of oncogenesis only

present in malignant cells. However, identifying tailored therapies has been challenging

for at least two reasons. First, the chimeric protein is often not easily druggable. For

example, the Ewing sarcoma translocation, EWS-FLI, does not form a fixed three-

dimensional structure under physiologic conditions, impeding inhibitor design (49). Only

recently, a small molecule, YK-4-279, was identified; it is able to block EWS-FLI protein

interactions and result in cell cytotoxicity (50). It has transitioned to phase I testing in

relapsed/refractory patients (51). Secondly, targeting EWS-FLI, as well as the alveolar

rhabdomyosarcoma PAX-FOXO fusion, is difficult due to an absence of inherent

enzymatic activity. Unlike most oncogenic rearrangements that result in kinase

activation, these chimeric proteins activate and repress transcriptional activity of a wide

array of genes and reversal of these gene signatures is quite complicated. Trabectedin, a

synthetic alkaloid, reversed the EWS-FLI signature in preclinical models, however, phase

II testing did not demonstrate sufficient single-agent activity (51). Phase Ib combination

testing is currently recruiting.

Incorporating precision medicine in the management of translocation-negative

sarcomas has posed a challenge, as well. Fusion-negative rhabdomyosarcoma patients

harbor alterations in multiple targetable pathways (Table 2). Unfortunately, these

mutations occur at such a low rate that designing well-powered clinical trials can be

problematic. Even in the complex and unstable osteosarcoma genome, which harbors

multiple structural rearrangements and copy number changes, few targetable alterations

are recurrent across multiple patients. Though we have gained new insights into sarcoma

oncogenesis through genomic profiling, there are still many obstacles to leverage them

into therapeutic strategies.

Despite these challenges, several candidates for targeted therapies have been

identified. IGF-1R, PDGFR, VEGFR, and HER-2 have been observed to be

overexpressed in osteosarcoma (52-55) and given their role in activating multiple

downstream signaling pathways that affect growth, cell proliferation, and survival, they

pose an attractive target. However, clinical trials with the FDA-approved agents

Imatinib, Sorafenib, and Sunitib did not induce significant responses in osteosarcoma

(56-59). Additionally, the anti-IGF-1R antibody Cixutumumab, the anti-VEGF antibody

Bevacizumab, and the anti-HER2 antibody Trastuzumab failed to demonstrate efficacy

(60-62). Targeting the non-receptor tyrosine kinase Src sheds light on another

therapeutic avenue for osteosarcoma patients, as it is implicated in the development of

lung metastases through its activation of pathways necessary for cell survival, migration,

adhesion, and invasion. In vitro, inhibition of Src resulted in decreased metastatic

potential, but this was not replicated in vivo (63), highlighting that multiple pathways

may be involved in the development of osteosarcoma metastases. A clinical trial for

patients with recurrent osteosarcoma localized to the lung is currently underway. It is

investigating the utility of Sarcatanib in patients who have had a complete surgical

resection of their tumor. Ewing sarcomas have recurrent abnormalities in cohesin

complex genes, including TP53 alterations, CDKN2A deletions, and STAG2 mutations

(44-46). Synthetic lethality between cohesin complex mutations and poly (ADP-ribose)

polymerase (PARP) has been observed (64), thus prompting several trials investigating

PARP inhibitors in combination with DNA-damaging agents in Ewing sarcoma.

CNS Tumors

Recent advances in CNS tumor sequencing are shifting the field from a focus on

histopathologic features to genetic identifiers. Rapid translation of molecular discoveries

into informed therapies has gone hand in hand. Recently, genomic profiling elucidated

four subgroups, based on molecular drivers of oncogenesis: WNT, SHH (Sonic

Hedgehog), group 3, and group 4. These subgroups have been incorporated into the most

recent 2016 WHO classification, which, takes into account molecular markers in addition

to pathologic features (65), redefining the framework in which CNS tumors were

previously diagnosed. The WNT group harbors somatic mutations in the CTNNB1 gene

or, in more rare instances, germline mutations in the APC tumor suppressor gene (66).

These alterations upregulate the WNT pathway, resulting in tumorigenesis. No targeted

therapies exist for these patients, but because their overall survival approaches 90% (67),

future studies may examine therapy de-intensification. The SHH group contain

aberrations in the SHH pathway, most commonly in PTCH1, but also in SMO, SUFU and

GLI2 (68). A phase I study of the FDA-approved SMO inhibitor Vismodegib

demonstrated activity in SHH medulloblastoma (69) and may offer a molecular-guided

therapy for this subset of patients. It is important to note that tumors with abnormalities

in SUFU or GLI2 are downstream of SMO and thus, resistant to Vismodegib (70).

Unfortunately, less is known about the specific genetic drivers in group 3 and group 4.

Group 3 tumors have the worst prognosis and a high frequency of copy number

abnormalities. They often have high MYC expression and a subset is MYCN amplified.

Group 4 tumors also harbor copy number alterations, and often have amplifications in

CDK6 and MYCN. As described above, there are challenges to targeting MYC, but

bromodomain inhibition has shown promise in preclinical models of medulloblastoma

with MYCN amplification (71). Palbociclib, a dual CDK4/CDK6 inhibitor is currently in

Phase I testing.

Molecular features have also been integrated in the most recent WHO

classification of posterior fossa and supratentorial ependymomas, given the wide

histopathologic variation across tumors, which results in lack of concordance even

amongst experts (65, 72). Posterior fossa ependymomas have been subdivided into two

groups. Group A tumors lack recurrent genetic alterations, but have increased

methylation of CpG islands, resulting in transcriptional silencing of targets of polycomb

repressive complex 2. In vitro, drugs targeting the polycomb repressive complex 2 and

DNA demethylating agents impair proliferation of ependymoma cells (73). Group B

tumors have multiple chromosomal aberrations, but none that are amenable to targeting

(74). Supratentorial ependymomas generally harbor 2 classes of gene rearrangements.

The majority contains a fusion of c11orf95 and RELA, while the remainder has fusions

involving the transcriptional co-activator YAP1 (75). Unfortunately, no targeted

therapies exist for these chimeric proteins. Because 75% percent of ependymomas

express aberrant ERBB2 and ERBB4 signaling (76), lapatanib, an ERBB inhibitor, has

been the subject of active clinical investigation (77). It has failed to demonstrate

efficacy, but this may reflect the need for higher levels of lapatanib in vivo and thus, still

holds promise.

Pediatric low grade gliomas are characterized by numerous mutations and copy

number alterations, including somatic alterations in BRAF. The most common alterations

are translocations in BRAF, which generate fusion proteins that results in loss of BRAF

regulation and activation of the MAP kinase pathway. The BRAFV600E mutation,

which leads to constitutive activation of BRAF, is also found in low grade gliomas.

These findings have led to several clinical trials examining BRAF inhibitors in the

V600E mutated patients. BRAF inhibitors are contraindicated in patients with BRAF

fusions, as it results in feedback loop-mediated upregulation of the pathway and

accelerated tumor growth. Accordingly, MAP kinase inhibitors are under investigation in

this cohort of patients. As our understanding of the molecular oncogenesis of CNS

tumors continues to grow, we are likely to identify new therapeutic strategies to enhance

our current management of these patients.

In light of these novel therapeutic avenues, the COG is developing the pediatric

counterpart of the adult NCI-MATCH trial to enroll children and adolescents with

relapsed/refractory solid tumors, CNS tumors and lymphomas (Figure 3). In addition to

offering molecular-targeted therapy for these patients, the trial will also provide a rich

source of genomic data for future discovery.

Precision medicine in pediatric oncology: new horizons and future challenges

In addition to suggesting novel therapeutic targets, gene discovery studies

enhance other aspects of the precision medicine algorithm including cancer surveillance,

assessment of inherited susceptibility to therapy-related toxicity, and treatment response

monitoring. The Pediatric Cancer Genome Project identified 8.5% of children and

adolescents with cancer had germline mutations in cancer-predisposition genes, in which

family history failed to predict the presence of an underlying predisposition syndrome in

most patients (78). The most commonly affected genes were TP53, APC, BRCA2, NF1,

PMS2, RB1 and RUNX1. In addition to several previously well-described predisposition

syndromes such as Li-Fraumeni syndrome, new associations have been observed between

germline mutations of TP53, PMS2 and RET mutations with Ewing’s sarcoma, APC and

SDHB mutations with neuroblastoma and a diverse range of mutations in APC, VHL,

CDH1, PTCH1, and SDHA with leukemia (78). Knowledge of these inherited

predispositions has major implications on direct patient care and on disease surveillance

aas well as genetic counseling for patients and their families. Furthermore, several

genome-wide association studies have identified polymorphisms in genes that predict

susceptibility to common chemotherapy-related complications, such as polymorphisms in

TPMT (79) and NUDT15, which are associated with abnormal thiopurine metabolism

resulting in severe myelosuppression (80), polymorphisms in GRIA1 associated with

asparaginase allergy (81) while those in CPA2 with asparaginase-induced pancreatitis

(82), variants in ACP1, BMP7 and PROX1-AS1 that predispose to glucocorticoid-induced

osteonecrosis (83, 84), polymorphisms in HAS3 predisposing to anthracycline

cardiomyopathy (85), polymorphisms in CEP72 associated with vincristine neuropathy

(86), and variants in SLCO1B1 involved in methotrexate clearance (87). Given that

genetic testing for TMPT status to inform adjustments in mercaptopurine dosage has

already become standard practice in ALL, one could envision the implementation of a

tailored pharmacogenomic panel relevant to a specific chemotherapy regimen to

individualize therapy based on genetic risk factors that predict host toxicities. Lastly,

advances in the technologies to detect cancer cells have paved innovative ways to

measure treatment response and ultimately predict risk of relapse. Childhood leukemia

treatment response measured by minimal residual disease (MRD) has been identified as a

powerful independent prognostic factor (88). It has traditionally been measured by flow

cytometry or other molecular-based techniques. However, sensitive next-generation

sequencing-based MRD monitoring are being tested prospectively in several pediatric

oncology consortia for clinical relevance (89). While MRD measurement has

revolutionized the management of leukemia patients, similar, ultra-sensitive biomarkers

are essentially non-existent for our solid tumor patients. Recently, several adult studies

demonstrated that detection of circulating tumor DNA (ctDNA) in the plasma can predict

relapse earlier than can be seen on standard imaging studies. Very few investigations of

the utility of ctDNA in pediatric solid tumors exist, however preliminary results using

droplet digitial PCR and next-generation sequencing are encouraging (90-92). In

parallel, several challenges emerge from the new era of precision medicine including

intratumoral genomic heterogeneity that makes it difficult to distinguish driver from

passager mutations, lack of sustained response resulting from the emergence of acquired

drug-resistant mutations and escape pathways, and incorporation of genomic sequencing

technologies into pragmatic, real-time, cost-effective clinical workflows for treatment

decision making. In addition, new ethical dilemmas have arisen due to the incidental

detection of inherited susceptibility to cancer predisposition syndromes. Genomics-

driven targeted therapies will require a strong collaboration between basic scientists,

molecular pathologists, bioinformaticians, and oncologists to develop robust clinical

trials to address these issues in an effort to further improve patient survival.

Conclusion

Advances in cancer genomics have deepened our understanding of the disease

biology, uncovered actionable genomic alterations and offered unique opportunities for

precision medicine approaches. Several molecular targeted therapy trials are currently

underway in the hopes of balancing survival and toxicity for these high-risk patient

populations. However, this exciting era of tailored therapy also opens up new challenges.

The next generation of translational trials will aim: 1) to determine the optimal

combination between novel therapeutic agents and conventional cytotoxic drugs; 2) to

investigate the underlying mechanisms governing therapy resistance as the emergence of

drug-resistant mutations and compensatory feedback pathways can arise; and 3) to

harness international collaboration to design statistically powered trials for rare, high-

risk, genomically-defined entities. Hence, precision medicine illustrates a novel treatment

paradigm to improve outcomes for children with cancer in this modern era of multi-

omics.

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

Figure 2: Relative frequency of genomic alterations in neuroblastoma at diagnosis compared to relapse

Figure 3: Pediatric MATCH trial schema

Table 1. Precision medicine trials for different leukemia subtypes and their associated genomic alterations Leukemia Common alterations Precision medicine Clinical trials subtype approach Ph+ALL BCR-ABL1 Chemotherapy + NCT03007147 – not yet Imatinib recruiting Ph-like ALL ABL-class fusions (ABL1, Chemotherapy + NCT02883049 – recruiting ABL2, CSF1R, PDGFRB) Dasatinib NCT02420717 – recruiting

CRLF2 rearrangements and Chemotherapy + NCT02723994 – recruiting other JAK-STAT pathway Ruxolitinib NCT02420717 – recruiting lesions (JAK2, EPOR rearrangements and/or IL7R, SH2B3 mutations) Infant ALL KMT2A rearrangements Chemotherapy + NCT02828358 – recruiting associated with FLT3 Azacitidine overexpression and epigenetic dysregulation NCT02553460 – recruiting Chemotherapy + Bortezomib + Vorinostat T-ALL Activated NF-κB pathway Chemotherapy + NCT02112916 – recruiting Bortezomib Relapsed ALL PI3K/AKT/mTOR pathway Chemotherapy + NCT01523977 – recruiting alterations Everolimus

Chemotherapy + NCT01614197 – recruiting Temsirolimus

Epigenetic dysregulation Chemotherapy + NCT01483690 – Decitabine + completed Vorinostat

Chemotherapy + NCT01321346 – Panobinostat completed

AML FLT3-ITD mutant Chemotherapy + NCT01371981 – recruiting Sorafenib

PML-RARα Chemotherapy + NCT02339740 – recruiting ATRA + arsenic trioxide

Relapsed AML with core- Chemotherapy + NCT02680951 – recruiting binding factor mutations Dasatinib

Table 2: Recurrent alterations in fusion-negative rhabdomyosarcomas are listed as copy number gains/losses or mutations with associated amino acid change when published. Of note, frequency reported is across fusion-negative and fusion-positive rhabdomyosarcomas. Data from Shukla et al. (93), Chen et al. (48), Shern et al. (47), and Kashi et al. (94).

Gene Alteration Frequency FGFR4 Mutation 6-9% V550L, N535K, G528C IGF1R Copy number gain <1-2%

IGF2 Mutation 1%

PDGFRA Mutation 1.4%

ERBB2 Mutation 1.4%

MET Copy number gain 1%

BRAF Mutation <1-1% V600E NRAS Mutation 8-12% G12A, Q61K, Q61H KRAS Mutation 4-6% G12A, G12C, G12D, G13D HRAS Mutation 3-4% G13R, Q61K NF1 Mutation 3.4% C1939_N1942FS, L18551, W784C PIK3CA Mutation 5-6% Q546, H1047R, E542K CCND1 Mutation 1%

CCND2 Mutation 1%

CDKN2A Copy number loss 2%

MDM2 Copy number gain 8%

TP53 Mutation 3.5% A276D, C176F, P250L, L308V

CTNNB1 Mutation 2-3.3% G34E, T41A, S45Y MYOD1 Mutation <1% L122R BCOR Mutation 6% Copy number loss 1% FBXW7 Mutation 4.8% R387P, R441G, R367P

Figure 1: B-ALL T-ALL AML

Hyperdiploidy (25%) NOTCH mutations (50%) KMT2A-rearranged (20%) ETV6-RUNX1 (25%) TAL1/LMO2 fusions (50%) RAS mutations (20%) RUNX1-RUNX1T1 (12%) IKZF1 deletions (15%) TLX3 fusions (20%) FLT3-ITD CRLF2-rearranged (8%) mutations (10%) TLX1 fusions (10%) WT1 mutations (10%) Ph-like kinase fusions (7%) LYL1 fusions (10%) CBFB-MYH11 (8%) KMT2A-rearranged (6%) NPM1 ETP (10%) mutations (8%) TCF3-rearranged (4%) PML-RAR (7%) KMT2A α 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 2:

Diagnosis Relapse

Other/ RAS-MAPK unknown ALK

Cell cycle Cell cycle ALK

Other/unknown

RAS-MAPK

Figure 3:

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

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

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

Clin Cancer Res Published OnlineFirst June 9, 2017.

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