tp53 mutations in hypodiploid acute lymphoblastic leukemia

TP53 Mutations in Hypodiploid AcuteLymphoblastic Leukemia

Evan Q. Comeaux and Charles G. Mullighan

Departments of Pathology and the Hematological Malignancies Program, St. Jude Children’s ResearchHospital, Memphis, Tennessee 38105

Correspondence: [email protected]

Acute lymphoblastic leukemia (ALL) is an aggressive neoplasm of B- or T-lymphoid progen-itors and is the commonest childhood tumor. ALL comprises multiple subtypes character-ized by distinct genetic alterations, with stereotyped patterns of aneuploidy present in manycases. Although alterations of TP53 are common in many tumors, they are infrequent inALL, with the exception of two ALL subtypes associated with poor outcome: relapseddisease and ALL with hypodiploidy. TP53 alterations are present in almost all cases of ALLwith low hypodiploidy and are associated with alterations of the lymphoid transcriptionfactor IKZF2 and the tumor-suppressor gene loci CDKN2A and CDKN2B. Remarkably,more than half of TP53 mutations in low-hypodiploid ALL in children are present in non-tumor cells, indicating that low-hypodiploid ALL is a manifestation of Li–Fraumeni syn-drome. These findings have profound implications for our understanding of the geneticpathogenesis of hypodiploid ALL, suggesting that alteration of TP53 function may promotethe distinctive aneuploidy characteristic of hypodiploid ALL. Moreover, the identification ofhypodiploidy mandates offering testing for TP53 mutational status to patients and theirrelatives, with appropriate counseling and disease surveillance.

ACUTE LYMPHOBLASTIC LEUKEMIA

Acute lymphoblastic leukemia (ALL) is aneoplasm of B- or T-lineage lymphoid

progenitors (Hunger and Mullighan 2015).Proliferation of leukemia cells, or blasts, resultsin bone marrow failure and death caused byanemia, hemorrhage, and/or infection, and in-vasion of extramedullary sites, including thecentral nervous system (CNS). ContemporaryALL therapy involves administration of multi-ple cytotoxic chemotherapeutic agents in sever-al phases over several years, which is curativein .90% of children. However, relapse of ALL

occurs in up to 20% of children and in muchgreater frequency in adults and is often refrac-tory to further chemotherapy. Consequently,relapsed ALL is a leading cause of childhoodcancer death. Recent years have witnessed inten-sive effort to define the genetic basis of leuke-mogenesis and treatment failure in ALL.

ALL comprises multiple distinct subtypescharacterized by constellations of genetic alter-ations, including aneuploidy (gains or losses ofwhole chromosomes), chromosomal rearrange-ments, gains and losses of DNA, and sequencemutations (Mullighan 2013). Although ALLgenomes harbor, on average, a low number of

Editors: Guillermina Lozano and Arnold J. Levine

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mutations, multiple cellular pathways are mu-tated in the majority of ALL cases, including thetranscriptional regulation of lymphoid devel-opment, cell-cycle regulation, tumor suppres-sion, cytokine receptor and Ras signaling, andepigenetic regulation. These lesions are consid-ered to accumulate during leukemogenesis, inwhich a lymphoid progenitor acquires a found-ing lesion, commonly a chromosomal rear-rangement that deregulates an oncogene (oftena lymphoid transcription factor), or results information of a chimeric fusion gene with alteredfunction of the partner genes. These rearrange-ments commonly perturb lymphoid matura-tion, resulting in developmental arrest, activateproliferation, and/or result in epigenetic dereg-ulation. The nature of specific genes mutatedvaries significantly among different subtypesand has been reviewed in several overviews ofthe genetics of ALL.

These founding genetic alterations are usu-ally insufficient to establish the leukemic clone,and additional genetic alterations are ac-quired—commonly focal DNA deletions andsequence mutations that further perturb thesepathways. At the time a diagnosis of ALL ismade, patients typically harbor multiple genet-ically distinct subclones that share one or morelesions, including the initiating chromosomaltranslocation, and can show marked diversityin the number and range of genetic lesionsamong subclones. The majority of childrentreated with contemporary therapy, which in-cludes multiple cytotoxic chemotherapeuticagents given in rotating combinations over atleast 2 years coupled with prophylaxis of CNSrecurrence using intrathecal chemotherapyand/or CNS irradiation, are cured. However,up to 20% of children and a higher proportionof adults with ALL experience relapse that iscommonly incurable with chemotherapy. Se-quential genomic profiling studies of samplesobtained at diagnosis, remission, and relapsehave shown collapse of the multiclonal diversityat diagnosis and convergence to a single clonetypically present at low frequency at diagnosis,which harbors genetic alterations that promoteresistance to therapy. Additional genetic alter-ations are acquired, selected for, and predomi-

nate in the clone that emerges as dominant atrelapse. Commonly mutated pathways enrichedat relapse include tumor suppression (TP53),lymphoid development (IKZF1), glucocorti-coid metabolism (CREBBP, NR3C1), and thio-purine metabolism (NT5C2, PRPS1) (Mul-lighan et al. 2008b, 2011; Ma et al. 2015).Many of these insights were only made in thelast few years with the advent of genomic plat-forms to identify inherited and somatic geneticalterations throughout the genome. Collectively,hundreds of cases of childhood ALL have beensubjected towhole-genome, exome, and/or tran-scriptome sequencing. These approaches haveenabled a revised molecular taxonomy of acuteALL, by identifying new subtypes of ALL in casesthat previously lacked recurring chromosomalalterations identifiable on karyotypic analysis.These include cases with rearrangements of thecytokine receptor gene CRLF2 (cytokine recep-tor-like factor 2) (Russell et al. 2009), focal al-terations dysregulating expression of the ETSfamily transcription factor gene ERG (Mullighanet al. 2007b), and asubtypeof high-risk leukemiawith a diverse range of genetic alterations thatactivate cytokine receptor and kinase signalingknown as Ph-like ALL (Roberts et al. 2014).

TP53 ALTERATIONS IN ALL

Although TP53 mutations are one of the mostcommon somatic alterations in cancer, and arealso observed in acute myeloid leukemia, theyare relatively uncommon in ALL (Imamuraet al. 1994; Gump et al. 2001; Hof et al. 2011;Zhang et al. 2011; Chiaretti et al. 2013). Geneticalterations disrupting tumor-suppressor genesand genes regulating the cell cycle are commonin both B- and T-lineage ALL, but involve othergenes, such as CDKN2A/CDKN2B (encodingthe INK4/ARF family of tumor-suppressorgenes), RB1, and PTEN, particularly in T-ALL(Mullighan et al. 2007a; Gutierrez et al. 2009).The reasons underlying these differences in pat-terns of alterations in tumor suppressors amongtumor subtypes with relative sparing of TP53 inALL are unknown.

TP53 is frequently mutated in two contextsin ALL: relapsed and low-hypodiploid ALL.

E.Q. Comeaux and C.G. Mullighan

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TP53 mutations were first identified in relapsein T-ALL in 1994 (Hsiao et al. 1994) and werefound to be absent at diagnosis but presentin 28% of cases at relapse. These findings havebeen confirmed by multiple studies with dele-tions, mutations, and/or copy-neutral lossof heterozygosity (LOH) of chromosome 17p(leading to duplication of mutated TP53) pres-ent in up to one-third of relapsed ALL cases,which are also associated with a poor prognosis(Diccianni et al. 1994; Blau et al. 1997; Gumpet al. 2001; Hof et al. 2011; Ma et al. 2015).The mechanistic basis for treatment resistanceassociated with TP53 mutations in relapsedALL is unknown but does not appear to berelated to genomic instability, as this is not ev-ident in the majority of ALL genomes with TP53alterations.

The second context in which TP53 muta-tions are common is low-hypodiploid ALL(Holmfeldt et al. 2013). Hypodiploid ALL com-prises up to 5% of childhood ALL cases and isfurther stratified according to the severity ofaneuploidy, with several stereotyped patternsof chromosomal loss identified: near haploidy(24–31 chromosomes), low hypodiploidy (32–39 chromosomes), and high hypodiploidy(40–44 chromosomes). As described below,each of these is characterized by distinct geneticmutations uncommon in other forms of ALL,one of the most notable being TP53 mutationsin low-hypodiploid ALL.

HYPODIPLOID ALL

Previous cytogenetic analyses of hypodiploidALL cases subdivided tumors based on chro-mosome number and found natural groupingsof 24–30 and 32–41 chromosomes based onthe patterns of aneuploidy (Pui et al. 1990;Heerema et al. 1999; Harrison et al. 2004; Nach-man et al. 2007). These two subtypes of hypo-diploid ALL are associated with very poor out-come in the majority of studies. Less commonare hypodiploid cases with a higher modal chro-mosome number, either high-hyperdiploid cas-es with 42–44 chromosomes or near-diploidcases, which are frequently associated with theformation of dicentric chromosomes.

The pattern of aneuploidy within low-hy-podiploid ALL cases is not random, showingconsistency in the chromosomes that are com-monly lost and those that are rarely or neverlost. The chromosomes most frequently lostin low-hypodiploid cases are 2, 4, 7, 9, 15, and20 with 3, 12, 13, 16, and 17 being universallylost (Fig. 1). Chromosomes 5, 8, 14, 18, 19,22, X, and Y are rarely lost, whereas chromo-some 21 is universally retained. The lack of an-euploidy of chromosome 21 is further notablefor not being present in any form of acute leu-kemia, with gain commonly observed in bothALL with high hyperdiploidy, as well as hypo-diploidy.

Identification of hypodiploid ALL can bechallenging as these tumors have a tendency toduplicate their aneuploid genome, referred toas masking (Harrison et al. 2004; Carroll et al.2009). Masking has been observed in both low-hypodiploid and near-haploid cells, causingthem to present a hyperdiploid karyotype,which requires careful cytogenetic and struc-tural analysis to identify as masked hypodiploid.The patterns of chromosomal gain may sug-gest masking as typical high-hyperdiploid ALLhas trisomy of chromosomes, including 4, 10,14, 17, and 21, whereas masking typically resultsin tetrasomy of chromosomes that were diploidin the originating nonhypodiploid clone. Mask-ing may result in most cells in a patient having ahyperdiploid karyotype with no evidence of thenonmasked hypodiploid clone or, more com-monly, the presence of both masked and non-masked clones. This phenomenon may be sug-gested by analysis of the DNA index, which mayshow peaks representing both masked and non-masked clones, as well as analysis of genome-wide single-nucleotide polymorphism (SNP)data on microarray or genome sequencing anal-ysis, in which widespread LOH is observed indiploid chromosomes, indicating loss and sub-sequent reduplication. One of the few immortalhypodiploid cell lines, MHH-CALL-2, showsthis phenomenon (Tomeczkowski et al. 1995).This cell line was characterized as hyperdiploidwith a karyotype of 52 chromosomes. SNP arrayanalysis using the Illumina platform revealedthat the disomic chromosomes showed LOH

TP53 Mutations in Hypodiploid ALL

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(Aburawi et al. 2011). This contrasts with themuch lower frequency of copy-neutral chromo-somal LOH observed in high-hyperdiploid ALL(Paulsson et al. 2010). The distinction ofmasked hypodiploid and hyperdiploid ALL isof great clinical significance as hyperdiploidALL has a favorable prognosis, whereas hypo-diploid ALL has a substantially inferior out-come and is classified as a high-risk entity inmost risk classification algorithms (Pui et al.1987, 1990; Raimondi et al. 2003; Moormanet al. 2010).

THE GENETIC BASIS OF HYPODIPLOID ALL

Although hypodiploidy has been recognized formany years, the nature of any associated geno-mic alterations, the factors driving the genera-tion of aneuploidy, and the biologic basis ofpoor outcome have been poorly understood.

To address these questions, a collaborativestudy initiated by St. Jude Children’s ResearchHospital and the Children’s Oncology Groupperformed detailed genomic analysis of hypo-diploid ALL. One hundred and twenty-six tu-mors were studied by microarray profiling ofgene expression and DNA copy-number alter-ations, candidate gene sequencing, and whole-genome sequencing (WGS), including exomeand/or WGS of a subset of the tumors as partof the St. Jude Children’s Research Hospital–Washington University Pediatric Cancer Ge-nome project. These analyses provided the firstdetailed understanding of the genomic land-scape of this disease (Holmfeldt et al. 2013).

This study confirmed that the previouscytogenetic subclassification of hypodiploidALL was appropriate in that low-hypodiploid(32–39 chromosomes) and near-haploid (24–31 chromosomes) cases have distinct transcrip-tomic profiles and patterns of genetic alteration,and should be considered distinct diseases (Ta-ble 1). Specifically, near-haploid tumors have ahigh frequency of alterations activating Ras sig-naling, including NRAS- or KRAS-activatingsequence mutations in 18% of cases or dele-tions or mutations leading to loss of NF1 func-tion (44%). The NF1 mutations were notablefor their high frequency and the identification

of a recurrent deletion of exons 15–35 involvingthe Ras-GAP domain required for negative reg-ulation of Ras activity. Moreover, because ofaneuploidy involving chromosome 17, whichharbors NF1, the genetic alterations result inbiallelic inactivation of NF1. These Ras pathwayalterations were mutually exclusive, suggestingeach is sufficient to promote constitutive Rassignaling, cell growth, and proliferation.

Alterations of the Ikaros family of transcrip-tion factors was also a hallmark of hypodiploidALL. This family comprises multiple genes, in-cluding IKZF1 (Ikaros), IKZF2 (Helios), andIKZF3 (Aiolos), which encode zinc-finger-con-taining, DNA-binding transcription factorstemporally regulated during hematopoieticand lymphoid development (Cortes et al.1999). The most extensively studied is thefounding member, IKZF1 (Ikaros), which is re-quired for development of all lymphoid lineages

Table 1. Frequency of genetic lesions in hypodiploidALL

Gene

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CDKN2A/B 22.1 23.5 77.3PAX5 5.9 5.9 59.1FLT3 8.8 0 0NF1 44.1 5.9 4.5KRAS 2.9 0 9.1NRAS 14.7 0 18.2PAG1 10.3 2.9 0PTPN11 1.5 0 9.1Total Ras pathway 82.3 8.8 31.8IKZF1 4.4 2.9 9.1IKZF2 1.5 52.9 0IKZF3 13.2 2.9 0RB1 8.8 41.2 0TP53 2.9 91.2 4.5Histone cluster, 6p22 19.1 2.9 9.1

Numbers represent the percentage of cases with copy-

number alterations or sequence mutations within the

specified gene. Deletion of the CDKN2A/B locus is

commonly observed in numerous B-ALL subtypes. The

Ras pathway-activating mutations in near-haploid tumors

are typically found in other ALL subtypes at relapse—

NRAS, in particular. IKZF1 deletion, although recurrent

in many subtypes of B-ALL, is rare in hypodiploid ALL.

The TP53 alterations present in virtually all low-

hypodiploid cases are rarely seen in other ALL subtypes.



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(Georgopoulos et al. 1994) and is commonlymutated in other high-risk subtypes of ALL,particularly BCR-ABL1 (Philadelphia chromo-some, or Ph-positive) and Ph-like ALL (Mul-lighan et al. 2008a, 2009; Roberts et al. 2012,2014). In contrast, IKZF1 alterations were un-common in hypodiploid ALL, but near-haploidtumors showed recurrent homozygous loss ofIKZF3 (Aiolos) in 13% of cases compared with3% of low-hypodiploid cases.

The mutational profile of low-hypodiploidALL was characterized by a relatively low fre-quency of Ras pathway alterations (9% of cases),IKZF2 (Helios) alterations in 65% of cases, anda high frequency of TP53 alterations, as dis-cussed below. Alterations in the TP53 pathwaywere uncommon in near-haploid ALL (11%).Gene expression profiling by unsupervised hi-erarchical clustering and principal componentanalysis revealed discrete clustering of subtypesthat were independent of the pattern of aneu-ploidy, further illustrating the molecular differ-ence between near-haploid and low-hypodip-loid ALL. The role and cooperativity betweenthe alterations in Ras signaling, Ikaros-familyalterations, and TP53 alterations in leukemo-genesis and their potential relationship to thehighly stereotyped, severe aneuploidy charac-teristic of this disease remain poorly under-stood but are the subject of investigation. Inparticular, although there is now strong evidencesupporting a role of IKZF1 alterations in treat-ment resistance in B-ALL (Schjerven et al. 2013;Schwickert et al. 2014; Churchman et al. 2015),the role of IKZF2 and IKZF3 alterations in leu-kemogenesis has not been formally explored.

TP53 ALTERATIONS IN HYPODIPLOID ALL

A striking finding from this study was alterationof TP53 in 91% of childhood low-hypodiploidcases, which was present in only 8% (8/106) ofnon-low-hypodiploid cases. Of the cases withTP53 alteration, 97% were in the form ofsequence mutations that resulted in insertion-deletion or single-nucleotide variations withclustering at the proximal and distal regions ofthe DNA-binding domain, as well as in the nu-clear localization sequence (NLS) (Fig. 2). As in

the case of the NF1 and Ikaros family alterations,aneuploidy and loss of the nonmutated chro-mosome resulted in biallelic alteration ofTP53. A single case was found on reverse tran-scription and polymerase chain reaction (PCR)of tumor RNA to express no wild-type TP53,but it did express an aberrantly spliced isoformlacking exons 2–6. This case was not subjectedto WGS at the time of the original study (Holm-feldt et al. 2013), but subsequent sequencingidentified a focal deletion of TP53 resultingin this aberrant splicing that was not evidenton microarray-based DNA copy-number alter-ation analysis because of relatively poor coverageof the TP53 locus (Fig. 3) (Zhang et al. 2015).Thus, the previously high frequency of TP53alteration in low-hypodiploid ALL is an under-estimate, and most or all low-hypodiploid ALLcases likely have biallelic alterations of TP53.

Importantly, 43% of pediatric low-hypo-diploid ALL cases were found to have TP53 mu-tations in matched nontumor cells, suggestingthat these mutations may be inherited. More-over, many of the mutations identified in non-tumor cells had previously been identified inLi–Fraumeni syndrome (LFS), such as TP53p.Arg248Trp. For most cases, blood or bonemarrow obtained at remission was used as thesource of nontumor DNA; thus, the mutationsmay represent mutations that are either inher-ited or acquired de novo in the germline orhematopoietic lineage. However, several kin-dreds have been reported in which familialTP53 mutations are associated with hypodip-loid ALL and other malignancies. In this study,one childhood case of low-hypodiploid ALLharbored a p.Gly302fs mutation that was ho-mozygous in tumor samples and heterozygouson skin biopsy. This patient had a family historyof cancer, including glioblastoma multiformediagnosed in the father at age 31 years. Sequenc-ing of the father’s DNA revealed the identicalTP53 frameshift mutation. A second kindredhas been reported with five individuals withALL across three generations, several of whichwere low hypodiploid and each of which har-bored a germline TP53 p.Arg306X mutation(Powell et al. 2013). Thus, low-hypodiploidALL in children should be considered a mani-



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festation of LFS, and clinical testing and geneticcounseling should be offered to patients andtheir relatives following a diagnosis of low-hy-podiploid ALL.

The near-universal alteration of TP53 inlow-hypodiploid ALL was further confirmedin 10 of 11 (91%) adult cases that harboredmutation of TP53, all of which were located inthe DNA-binding domain. Whereas half ofthese mutations had previously been associatedwith LFS, the TP53 mutations were somatic inall adult hypodiploid ALL cases. The high fre-quency of TP53 mutations in low-hypodiploidALL has been confirmed in an independentstudy, in which 27 of 29 cases harbored aTP53 sequence mutation (Muhlbacher et al.2014). The majority of patients in this cohortwere adult, and germline mutational status wasnot reported.

The sequence mutations observed in bothchildhood and adult hypodiploid ALL showedclustering within the DNA-binding domain(Fig. 2). Three cases harbored frameshift mu-tations upstream of the DNA-binding domain

(Asp49fs, Ala88fs, and Gly108_Arg110fs),which are likely to eliminate protein expression.Truncating mutations at p.Arg306 were ob-served in four cases. Although germline andsomatic mutations at this residue have been de-scribed previously (Petitjean et al. 2007), muta-tions at this site are disproportionately morefrequent in hypodiploid ALL. While truncatingmutations of TP53 have been shown to result inloss-of-function, p.Arg306 is located distal tothe DNA-binding domain in a basic p.Lys305-Arg306 domain that forms part of the bipar-tite NLS required for nuclear localization ofTP53 (Liang and Clarke 1999, 2001). Analysisof a low-hypodiploid xenograft containing thep.Arg306� mutation identified elevated TP53expression (Holmfeldt et al. 2013). This muta-tion was also identified in a case of endome-trioid adenocarcinoma, in which the mutationwas present only within the serous componentof a mixed epithelial carcinoma, and was ac-companied by elevated TP53 expression in se-rous, but not endometrioid cells in the tumor(Sholl et al. 2012). Moreover, the p.Gly302fs

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Figure 3. Cryptic germline TP53 alterations in hypodiploid ALL. Germline DNA is shown in green and tumorDNA is shown in brown as a wiggle plot of whole-genome sequencing (WGS) read depth spanning the TP53gene shown on the University of California at Santa Cruz (UCSC) genome browser. One low-hypodiploid tumor(HYPO052) that was originally reported as an aberrantly spliced isoform was later found to contain an 8.7-kbfocal deletion of exon 2–exon 5 on deeper sequencing analysis (Holmfeldt et al. 2013). The original estimate of�90% of low-hypodiploid cases possessing TP53 alteration is likely an underestimation, with a higher numberof cases having germline mutations (data from Zhang et al. 2015).



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mutation identified in the kindred described inthis study was also accompanied by elevatedTP53 expression in the brain tumor sample.There is currently little direct knowledge regard-ing the functional consequence of these trun-cating mutations at or near the NLS on TP53intracellular localization, cell-cycle arrest, andapoptosis in response to DNA damage. Howev-er, the observation that missense TP53 muta-tions are associated with elevated TP53 proteinexpression in hypodiploid leukemic cells, andthe dearth of TP53 deletions without concom-itant sequence mutation in this disease, stronglysuggests a gain or change in TP53 function formany of the mutations. The high frequency ofTP53 alterations in low-hypodiploid ALL indi-cates that children with hypodiploid ALL andtheir families should be offered genetic counsel-ing and TP53 mutational testing. This also of-fers the opportunity for clinical surveillance ofchildren within families of known TP53 muta-tion carriers for early detection of low-hypodi-ploid ALL (Villani et al. 2011; McBride et al.2014).

Additional recurring genetic alterations in-volving cell-cycle regulatory genes and tumorsuppressors were also common in hypodiploidALL. Loss of RB1 was present in 41% of cases(14/34) compared with 9% (6/68) of near-hap-loid cases (Holmfeldt et al. 2013). Most caseswith RB1 alterations had concomitant TP53mutations, suggesting distinct and coopera-tive effects in leukemogenesis. Alterations ofCDKN2A/CDKN2B, encoding the INK4/ARFfamily of tumor suppressors (Sherr 2001), werepresent in 22.1% and 23.5% of near-haploidand low-hypodiploid cases, respectively. De-spite 62% of low-hypodiploid ALL cases con-taining either RB1 or CDKN2A/B alterations,only one case harbored concomitant TP53, RB1,and CDKN2A/B alterations, suggesting that theproducts of RB1 and CDKN2A/B are epistaticwithin the RB1 tumor-suppressor pathway inthis context (Fig. 4). Although RB1 is a well-known tumor suppressor that is often observedinactivated in many solid tumors (Lohmann1999; Deshpande and Hinds 2006; Chen et al.2014) and T-lineage ALL (Mullighan et al.2007a), it is otherwise uncommonly mutated

in B-ALL (Zhang et al. 2011; Schwab et al.2013).

Loss of TP53 in tumors is widely thought toinduce genomic instability (Hanel and Moll2012). Mutation of TP53 has also been associ-ated with aneuploidy and catastrophic DNA re-arrangements known as chromothripsis, as wasthe situation in a recent analysis of medulloblas-toma cases (Thompson and Compton 2010;Stephens et al. 2011; Rausch et al. 2012). How-ever, several observations suggest that TP53 al-terations in hypodiploid ALL do not result ingenome instability. First, low-hypodiploid ALLis not as severely aneuploid as near-haploidALL, in which TP53 is infrequently mutated,albeit with only one copy per cell. This suggeststhat mechanisms other than inactivation ofTP53 may be more responsible for the aneuploi-dy observed in hypodiploid ALL. Second, evi-dence of chromothripsis is lacking in low-hypo-diploid ALL genome sequences. Their genomesdo not contain clustered breakpoints or concen-trated structural rearrangements, which areconsidered a hallmark of chromosome instabil-ity (Jones and Jallepalli 2012). Overall, the ge-nomes of low-hypodiploid ALL are relativelystable, showing a consistent pattern of aneu-ploidy that is conserved at relapse. Xenograftsof low-hypodiploid ALL maintain this patternof aneuploidy and mutation even after succes-sive passages (Holmfeldt and Mullighan 2015).

MODELING OF HYPODIPLOID ALL

Currently, there are no preclinical models ofhypodiploid ALL. The hypodiploid ALL litera-ture consists largely of clinical case reports, pa-tient sample analysis, retrospective analysis ofclinical outcome, and in vitro study of the twohypodiploid cell lines NALM-16 and MHH-CALL-2, both of which are of the near-haploidsubtype (Pui et al. 1990; Ma et al. 1998; Heer-ema et al. 1999; Ramos et al. 2000; Das et al.2003; Raimondi et al. 2003; Harrison et al. 2004;Morrissette et al. 2006; Nachman et al. 2007;Aburawi et al. 2011; Safavi et al. 2013; Muhl-bacher et al. 2014; Mehta et al. 2015). The con-sensus of these studies is that hypodiploid ALLis associated with poor outcome. One exception



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is a recent report from the St. Jude Total TherapyXV study incorporating minimal residual dis-ease (MRD) monitoring and risk directed ther-apy, in which undetectable levels of leukemiccells in the bone marrow (MRD) early in ther-apy was associated with a favorable outcome(Mullighan et al. 2015). The lack of hypodiploidALL preclinical models has greatly limited ourability to assess the efficacy of new therapeuticagents and characterize the molecular pheno-type of both near-haploid and low-hypodiploidsubtypes.

The description of the genomic landscapeof 126 hypodiploid ALL patients has providedinsights into the genetic basis of this disease thatmay be exploited to improve outcome with tar-geted therapy. However, the diversity of geno-mic alterations, many of which have not beenstudied in lymphoid leukemogenesis, aneuploi-

dy, and lack of a known driving oncogene,result in challenges for the generation of faithfulmodels and testing of preclinical therapeuticapproaches. Moreover, the distinct genomic le-sions observed in each subtype mandate thegeneration of distinct engineered and patient-derived xenograft (PDX) mouse models.

PDX models may be the most clinically rel-evant for the testing of new therapeutics. One ofthe advantages of PDX models is the mainte-nance of the characteristic aneuploidy of thetumor, which is exceptionally challenging torecapitulate in genetically engineered mousemodels (GEMMs). Use of PDX models for pre-clinical testing of therapeutic agents should in-corporate tumors representing the diversity ofgenetic lesions found in hypodiploid patients asmuch as possible to both correlate the drugmechanism with the tumor genomic profile

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and define the extent of activity of the com-pound within each subtype. This requiresdetailed characterization of the phenotype, ge-nomic landscape, and signaling pathway activa-tion of each xenograft. The observation thatboth near-haploid and low-hypodiploid xeno-grafts show constitutive Ras activation and exvivo sensitivity to PI3K inhibitors suggests bothsubtypes may be dependent on Ras-mediatedgrowth mechanisms (Holmfeldt et al. 2013).Constitutive Ras activation was expected fornear-haploid tumors containing Ras-activatingmutations; however, it was surprising for low-hypodiploid tumors lacking such alterations.Targeted inhibition of PI3K/MAPK signalingmay, therefore, be a promising treatment to in-hibit the oncogenic effect of Ras. This dualblockade approach is being investigated in sever-al cancer types, including colorectal, melanoma,ovarian, and pancreatic tumors (Britten 2013).

GEMMs offer opportunities to study theinteraction of specific genetic lesions in the de-velopment of B-ALL through analysis of dis-crete complements of lesions within mice ofidentical genetic backgrounds. This will be ofgreat interest to compare the effects of alter-ations of the different Ikaros family memberson lymphoid maturation, tumor development,and chromatin remodeling but will likely re-quire directed deletion in the B-lymphoid line-age in view of the propensity of mice to developT-lymphoid neoplasms on perturbation ofIkzf1 (Winandy et al. 1995). The immunophe-notype of hypodiploid ALL typically observed isthat of a pre-B cell that lacks expression of amature B-cell receptor (Pui et al. 1990). It islikely that induction of B-ALL will dependon the stage of maturation at which Ikzf2/3alterations, and other lesions, are acquired, assuggested by immunophenotypic and anti-gen receptor recombination data indicative ofdifferent maturational states of near-haploidand low-hypodiploid ALL. Specifically, low-hy-podiploid tumors expressed significantly lessCD19 compared with near-haploid and near-diploid tumors, as well as much less frequentantigen receptor rearrangements (Holmfeldtet al. 2013). Gene set enrichment analysis re-vealed a pro-B-cell expression profile for low-

hypodiploid cells that was not seen in near-hap-loid cells, suggesting a less mature lymphoidprogenitor cell of origin for low-hypodiploidcompared with near-haploid cells. HypodiploidALL therefore, represents a biologically relevantmodel to study how loss of Ikzf2/3 affects B-celldevelopment and promotes persistent growthof immature B cells.

An important challenge is the modeling ofaneuploidy. There are few established models ofB-progenitor ALL, the majority being driven bypotent oncogenes (e.g., BCR-ABL1 and MLLrearrangement). The mechanism of inductionof the stereotyped aneuploidy and sequence ofacquisition relative to concomitant genetic al-terations are unknown, although it is likely thataneuploidy is an initiating event (Safavi et al.2013). The observation that many cases of low-hypodiploid ALL appear to result from inherit-ed TP53 mutation for which cells become ho-mozygous because of loss of one copy of chro-mosome 17 suggests that loss of TP53 functionmay be a necessary early event in the pathogen-esis of this disease. For those patients withoutinherited TP53 mutation, especially adult cases,it is not known at what stage of B-cell develop-ment the somatic TP53 mutations are acquiredthat facilitate hypodiploid ALL.

The modeling of hypodiploid TP53 alter-ations in GEMMs should consider the diversityof TP53 alterations, including mutations withindifferent regions of the gene that may affect pro-tein function, stability, and/or localization, aswell as loss of expression as a result of frameshiftmutation or aberrant RNA splicing. It is impor-tant to distinguish between altered p53 function(in the case of point mutations) versus com-plete loss of p53 (in the case of deletion or prox-imal frameshift mutations). Nearly all cases se-quenced were homozygous for the alteration ofTP53 because of aneuploidy. For mouse modelsto accurately represent TP53 alterations ob-served in hypodiploid ALL, both deletion andmutation of TP53 should be considered. A highpercentage of low-hypodiploid cases containedmissense mutations of TP53, 79% of which oc-curred within the DNA-binding domain (Fig.2). Several GEMMs currently exist that carrysome of these most common LFS mutations;



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however, no mouse has been created with theArg306� truncating mutation observed in fourhypodiploid cases that would allow for thestudy of the functional consequence of carbox-yl-terminus loss immediately after the DNA-binding domain.

A recent study on the homozygous deletionof TP53 in mouse pre-B cells using Mb1-Creexpression induced B-cell lymphoid tumorsharboring a variety of clonal translocationsthat appeared to be caused by dysfunctional re-pair of double-strand breaks caused by RAG (re-combination-activating gene) and AID (activa-tion-induced deaminase) activity (Rowh et al.2011). This illustrates the role TP53 plays in de-tecting and mediating repair of aberrant RAG(recombination-activating gene)/AID (activa-tion-induced deaminase) activity. Igh locustranslocations similar to those observed in thismodel have not been observed in hypodiploidALL patients; however, the focal deletions of NF1in some cases contained the heptamer footprintof RAG-mediated recombination. Consideringthat low-hypodiploid tumors consistently pre-sented with recurrent lesions in tumor-suppres-sor genes in addition to TP53, such as RB1,CDKN2A/B, and IKZF2, the comodeling of oth-er lesions with TP53 mutation may more accu-rately recapitulate this disease. Furthermore, itmay be that hypodiploid ALL transformationinitiates at an earlier stage of B-cell developmentthan that of pre-B, and conditional inductionof lesions at various stages should be investigat-ed. Although a majority of low-hypodiploidcases (62%) are presented with alteration in ei-ther RB1 or CDKN2A/B, in addition to TP53(Holmfeldt et al. 2013), the combination ofall three of these alterations was only observedin a single patient, suggesting that RB1 andCDKN2A/B inactivation are epistatic withinthe Rb1 tumor-suppressor pathway and are notnecessary to combine within a mouse model.

CONCLUSION

Compared with many types of cancer, however,ALL tends to have relatively few genomic alter-ations and is often characterized by oncogenicfusion genes (Zhang et al. 2011). Although

TP53 is one of the most well-known tumor-suppressor genes, it is relatively rare to findTP53 mutations in ALL at diagnosis. It was,therefore, quite surprising to discover thatlow-hypodiploid ALL cases harbor near-uni-versal TP53 alteration (Holmfeldt et al. 2013).This distinguishes low-hypodiploid ALL as amanifestation of LFS.

TP53 plays a critical role in protecting thecell from the transformative effects of DNAdamage by inducing cell-cycle arrest and apo-ptosis. Loss of TP53 function is associated withgenomic instability and the development ofaneuploidy in cancer (Thompson and Comp-ton 2010; Hanel and Moll 2012). However,there is insufficient evidence that loss of TP53is the cause of aneuploidy in hypodiploidALL as shown by the lack of TP53 lesions innear-haploid tumors. Furthermore, low-hypo-diploid cells do not display characteristicfeatures of genomic instability, such as chromo-somal translocations mediated by aberrantRAG/AID activity, which is a hallmark ofimpaired double-strand break response (Holm-feldt et al. 2013). So, although the TP53 alter-ation appears to be an essential component ofthe pathogenesis of low-hypodiploid ALL, thefunctional role of TP53 alterations in this dis-ease remains unclear.

Although multiple studies have reported anassociation among TP53 alterations with pooroutcome in ALL, remarkably few data exist re-garding the mechanistic basis of the role of suchalterations in leukemogenesis and drug resis-tance (Hof et al. 2011; Chiaretti et al. 2013).The recent discovery of near-universal TP53 al-teration in low-hypodiploid ALL has revealed akey role for this gene in leukemogenesis. There-fore, detailed studies of the role of loss of TP53function will likely provide unique insights intothe role of this gene in cancer development.

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