The$Metagenomic$Epidemiology$of$Childhood$Leukemia$ By$digitalassets.lib.berkeley.edu/etd/ucb/text/Francis_berkeley_0028E_14340.pdf · The$Metagenomic$Epidemiology$of$Childhood$Leukemia$

The$Metagenomic$Epidemiology$of$Childhood$Leukemia$$By$$

Stephen$Starko$Francis$$$$$

A$dissertation$submitted$in$partial$satisfaction$of$the$$$

requirements$for$the$degree$of$$$

Doctor$of$Philosophy$$in$$

Epidemiology$$

in$the$$

Graduate$Division$$

of$the$$

University$of$California,$Berkeley$$$$$$

Committee$in$charge:$$

Professor$Patricia$A.$Buffler,$Chair$(deceased)$Professor$Steve$Selvin,$Chair$Professor$Lee$W.$Riley$Professor$Fenyong$Liu$

$$

$Spring$2014$

The$Metagenomic$Epidemiology$of$Childhood$Leukemia$$

Copyright$2014$By$

Stephen$Starko$Francis$

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Abstract!

The!Metagenomic!Epidemiology!of!Childhood!Leukemia!

By!!

Stephen!Starko!Francis!

Doctor!of!Philosophy!in!Epidemiology!

University!of!California,!Berkeley!

Professor!Patricia!A.!Buffler,!Chair!(deceased)!

Professor!Steve!Selvin,!Chair!

!!The! etiology! of! childhood! acute! lymphoblastic! leukemia! (ALL),! the!most! common!childhood! cancer,! is! unknown.! ! A! role! for! infection! is! supported! by! considerable!research! over! the! last! twenty! years.! Despite! this! evidence! and! many! infection!related!hypotheses,!no!definitive!infectious!agent!has!been!identified.!Until!recently!broad!surveys!of!the!microbiome!were!not!possible.!Modern!developments!in!next!generation! sequencing! and! bioinformatics! however! now! allow! unbiased!investigation! into! the! astounding! diversity! of! organisms! that! inhabit! the! human!body.!!!This!dissertation!describes!the!first!comprehensive!analysis!of!tissue!from!children!with!childhood!leukemia!using!metagenomic!(the!study!of!nonKhuman!nucleic!acids)!and!epidemiologic!methods.!Metagenomes!in!pre!treatment!bone!marrow!from!the!Fallon,! NV! ALL! cluster! and! other! ALL! nonKclustered! cases! from! California! were!interrogated! for! known! and! novel! infections.! A! putative! and! potentially! novel!human! virus!was! found! in! the! Fallon! cases.! To! investigate! viruses! involved!more!broadly! in! childhood! ALL,! viromes! of! preKtreatment! bone! marrow! from! children!with!acute!myloid!leukemia!(AML)!and!from!children!with!ALL!(who!we!assumed!to!have!similar!background!infections)!were!compared.!In!children!with!ALL,!a!greater!intensity!of!viral!infection!as!well!as!a!significant!difference!in!the!expression!of!viral!packets! originating! from! double! stranded! DNA! viruses! was! observed.! ! Using! 16s!based! bacterial! metagenomics,! no! difference! was! observed! in! bacterial! diversity!between! the! groups! showing! the! absence! of! differential! contamination! and!suggesting!that!bacteria!do!not!play!a!role!in!ALL.!!These!results!suggest!a!possible!putative! causal! agent! in! the! Fallon! Nevada! leukemia! cluster! and! increased! viral!infection! driven! by! double! stranded! DNA! viruses! in! nonKclustered! children! with!ALL.!!

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These!data!contribute!to!an!etiologic!hypothesis!of!ALL!where!a!combination!of!preKnatal! and! postKnatal! events! lead! to! leukemia.! First,! an! underlying! genetic!predisposition,!combined!with!an!in!utero! infectious!exposure,!cause!chromosomal!anomalies!and!immune!dysregulation.!This!followed!by!postnatal!events,! including!toxic! and! infectious! exposures! that! further! modify! adaptive! immunity! lead! to! a!greater! intensity! of! infections.! ! This! feeds! the! loop! of! enhanced! bKcell! production!that! facilitates! virally! induced! selfK! mutation! caused! by! foreign! nucleic! acids!invading!pre!bKcells.!!Resulting!in!an!ABOBEC!arbitrated!hypermutation.!Eventually!the! loop! of! enhanced! yet! less! competent! bKcell! production,! RAG! mediated! over!mutation,! increased! infection! leading! to! pre! bKcell! viral! nucleic! acid! loading,! and!APOBEC! caused! self! mutation! remove! the! proliferative! and! apoptotic! cellular!checks.!!The!result!is!childhood!ALL.!!!!

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!!!!!!!!!

This!research!is!dedicated!to!the!memory!of!Professor!Patricia!A.!Buffler!She!was!an!incredible!epidemiologist,!the!greatest!mentor!and!an!amazing!woman!

I!miss!her! !

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!!!

Acknowledgments!!!

Forever!thanks!to!my!family!and!friends!for!your!unwavering!support!and!guidance!through!this!process!and!my!life.!You!are!all!the!best!I!could!ever!wish!for.!!!I!am!in!debt!to!the!California!Childhood!Leukemia!Study!for!the!support!and!resources!needed!to!complete!this!work.!Special!thanks!to!Joe!Wiemels,!Catherine!Metayer,!Amelia!Wallace,!Jake!Wendt,!Theresa!Saunders,!Pagan!Morris,!Alice!Kang,!Todd!Whitehead,!Monique!Does!and!Adam!De!Smith.!And!thanks!to!Blood!Systems!Research!Institute!and!Eric!Delwart!for!his!guidance.!Go!team!!!I!thank!my!committee!members!Steve!Selvin,!Lee!Riley!and!Fenyong!Liu!for!their!superb!guidance!and!mentorship,!and!the!Berkeley!Epidemiology!Department!and!the!School!of!Public!Health!for!providing!my!foundation.!!!I!especially!thank!the!children!and!families!that!volunteered!for!this!study,!without!their!participation!none!of!this!would!be!possible.!We!will!not!stop!until!we!solve!this!mystery.!! !

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Contents'Chapter'1-'A'new'frontier''' ' ' ' ' ' ' 1'! !

1.1!A!New!Frontier!!! ! ! ! ! ! ! ! 2!! !

1.2!Metagenomics! ! ! ! ! ! ! ! 2!! !

1.3!The!Human!Bacterial!Microbiome! ! ! ! ! 3!! !

1.4!The!Human!Virome! ! ! ! ! ! ! 4!! !

1.5!Microbial!Balance! ! ! ! ! ! ! 5!! !

1.6!Looking!Forward!! ! ! ! ! ! ! 6!!Chapter'2-The'Metagenome'of'Childhood'Leukemia' ' ' ' 7'! !

2.1!Introduction! ! ! ! ! ! ! ! 9!! !

2.2!Materials!and!Methods! ! ! ! ! ! ! 11!! ! 2.2.1:!Fallon!Viral!Discovery!! ! ! ! ! 11!! ! 2.2.2!ALL!vs.!AML!Particle!Isolation!454!Sequencing! ! 13!! ! 2.2.3!16s!Bacterial!Metagenomics! ! ! ! ! 14!! !

2.3!Results! ! ! ! ! ! ! ! ! 15!! ! 2.3.1!Fallon! ! ! ! ! ! ! ! 15!!!!!!!!!!!!!!!!!!!!!!!!!!!!2.3.2!Virus!Particle!Isolation!and!454!Sequencing!! ! 16!! ! 2.3.3!16s!Bacterial!Microbiome!! ! ! ! ! 16!!! 2.4!!Discussion!!!!!!!!!!! ! ! ! ! ! ! ! 17!!Chapter'3-'An'Etiologic'Framework'for'Acute'Lymphoblastic'Leukemia' 29'!

3.1!On!the!Trail! ! ! ! ! ! ! ! 30!!! 3.2!It!starts!from!the!beginning:!Pre]natal!Events!! ! ! 31!!! 3.3!Pre!and!Post!Natal!Immune!Development!! ! ! ! 31!!! 3.4!Post]natal!Events!! ! ! ! ! ! ! 32!!! 3.5!Future!Directions! ! ! ! ! ! ! 33!!References! ! ! ! ! ! ! ! ! ! 35!' ' ' ' ' ' ' ' ' ' ' '

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Chapter!1*!Metagenomic!Epidemiology!!! !

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1.1!A!New!Frontier!A! new! frontier! of! basic! human! biology! has! recently! opened.! Advances! in! next<generation!nucleic! acid! sequencing! technology! are! changing! our! understanding! of!what! constitutes! the! human! body! and! how! changes! ! in! the! genome! could! lead! to!disease.! !This!new!knowledge! is! fueling!a!paradigm!shift! from!the!dominant,!20th!century!view!that!viruses,!bacteria,!and!fungi!operated!alone!to!cause!disease!.!!Now,!we! are! beginning! to! view! a! more! complex! and! nuanced! interpretation! with!increased! importance! of! commensalism,! synergy,! and! balance! of! microbiota! in!human!health!(Honda!&!Littman,!2012).!This!paradigm!shift!affects!epidemiology.!!The!distribution!of!human!diseases! is!directly! linked!to!how!different!people!form!groups!and!interact!with!each!other!and!their!environment.! !Community!structure!has! been! long! recognized! as! a!major! factor! in! the! spread! of! traditional! infectious!diseases.! ! These! diseases! are! very! sensitive! to! the! configuration! of! the! social!network!of!the!population!into!which!they!are!introduced!(Keeling!&!Eames,!2005).!Moreover,! the! importance! of! social! networks! within! these! networks! have! been!documented! to!be! important! for! chronic! and! traditionally!non<infectious!diseases.!For! example,! obese! individuals! are!more! likely! to!have!other!obese! individuals! as!contacts! (Christakis! &! Fowler,! 2007).! Even! in! these! “non<infectious”! diseases!microorganisms!may!play! a! greater! role! than!previously! thought.!A! community! is!comprised! of! groups! of! ecosystems! any! number! of! which! can! potentially! be!disrupted!and!lead!to!disease!epidemics.!!An!important!feature!of!these!ecosystems!is! its!microbiome.! !Study!of! the!distribution,! relationship,!and!determinants!of! the!microbiome! structures! of! a! community! of! ecosystems! constitutes! metagenomic!epidemiology.!!As!our!ability!to!define!the!microbes!in!and!on!our!bodies!increases!so!will!our!ability!to!delineate!the!healthy!balance!of!the!microbiome.!!!This!will!lead!to!an! inhanced!definition!of! the! relative! ‘good’!microbes! from! ‘bad’.! !With!diligent!study,!robust!comparison,!and!population!level!data,!we!will!be!able!to!better!define!those! factors! that! influence! health,! disease,! and! community! structures..! Given! our!technical!power!today,!the!time!is!ripe!for!metagenomic!epidemiology.!!1.2!Metagenomics!The!scale!of!the!human!microbiome!is!almost!unimaginable.!It!is!estimated!that!for!every!human!cell!there!are!10!bacterial!cells!and!for!every!bacterial!cell!there!are!10!viral!particles(Clemente,!Ursell,!Parfrey,!&!Knight,!2012).!!Such!estimates!have!been!made! possible! by! technological! spill! over! tools! developed! from! human! genome!sequencing.! Next! generation! sequencing! technologies! designed! to! rapidly! and!accurately! sequence! the! genomes! of! human! communities! are! now! being! used! to!sequence!microbial!communities.!While! these! technologies!continue!to! improve! in!speed! and! cost,! the!massive! amount! of! data! generated! from! them!have! produced!immense!challenges!for!our!bioinformatics!capabilities.!Cluster!based!computing!is!essential! for! most! metagenomic! studies,! although! some! tool! kits! (QIIME! for!example)! are! bringing! 16s! based! bacterial! metagenomics! into! the! range! of! most!researchers.!!

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This!ability!to!sequence!all!nucleic!acids!in!a!given!sample!without!amplification!of!the! targeted! region! has! enabled! the! new! field! of! metagenomics.! Prior! to! these!technologies! culturing! or! PCR! based! amplification! was! required! to! study!microorganisms.! Next! generation! sequencing! now! enables! unbiased! identification!and!quantification!of!all!known!organisms!in!a!given!sample!be!it! from!the!human!body!or!the!environment.!!As!a!result,!a!flurry!of!environmental!microbiome!studies!is!afoot!ranging!from!the!ocean!floor!to!artic!ice!(Varin,!Lovejoy,!Jungblut,!Vincent,!&!Corbeil,!2012;!Yoshida,!Takaki,!Eitoku,!Nunoura,!&!Takai,!2013).!Metagenomics!has!enabled! us! to! identify! here<to<fore! unknown! microorganisms.! In! fact,! this! new!technology!has!enabled!the!discovery!of!nucleic!acid!sequences!in!the!human!body!and! in! environmental! niches! that! cannot! be! assigned! to! any! known! microbial!entities—bacteria,! viruses,! fungi,! protozoa,! or! helminthes.! We! call! these! entities!“dark!sequences”!analogous!to!the!“dark!matter”!which!astrophysicists!hope!could!explain!many!of!the!mysteries!of!the!universe.! ! !Not!only!are!there!new!sequences!but! the! quantity! of! these! is! vast.! In! fact!what! had!been! recognized! to! exist! in! the!microbial!world!until!20!years!ago!now!represents!but!a!small!microcosm!of!what!is!now!known!to!exist! today.! !And!surely! there!are!more! to!be!discovered.!What!are!they! and! how! do! they! affect! the! human! body! and! today’s! recognized! microbial!ecosystems!remain!to!be!determined.!!We!have!only!begun!to!scratch!the!surface.!!1.3!The!Human!Bacterial!Microbiome!:!The! Human! Microbiome! Project! (http://www.hmpdacc.org/)! has! contributed! a!strong!basis! for!understanding! ‘normal’!human!flora.!Recent!studies!show!that!the!core!microbiome!is!essential!in!metabolic!and!regulatory!processes!throughout!the!body!(Aagaard!et!al.,!2013).!Bacteria!are!required!for!basic!functions!ranging!from!digestion!of!lipids!and!vitamins!to!glucose!homeostasis.! !In!addition,!they!can!alter!host!gene!expression,!and!modify!drug!metabolism!(Clayton,!Baker,!Lindon,!Everett,!&! Nicholson,! 2009;! Devaraj,! Hemarajata,! &! Versalovic,! 2013;! Jones,! Martoni,!Ganopolsky,! Labbe,! &! Prakash,! 2014).! Bacteria! also! exist! as! communities! with!structure,!creating!an!ecosystem!that!synergistically!interacts!with!the!host.!We!are!just!beginning!to!understand!the!central!role!of!bacteria!in!the!human!gut.!!!It!is!now!clear! that! our! gut!microbiota! functions! like! a! previously! undescribed!organ.! ! Like!other!organs,!it!is!essential!for!health!and!a!factor!in!susceptibility!to!disease.!!!Most!research!in!this!arena!has!primarily!focused!on!the!gut!bacterial!flora!for!three!reasons.!First,!the!gut!is!a!most!rich!and!interactive!body!compartment!and!second,!sample! ascertainment! is! non<invasive.! Finally,! conducting! bacterial!metagenomics!through!16S!rRNA!sequencing!is!relatively!easy!due!to!shared!genes!across!bacteria!and! archaea.! In! this! regard,! recent! technological! advances! (Caporaso! et! al.,! 2011)!have! enabled! metagenomic! community! level! descriptions! for! as! little! as! $11! per!sample.! ! Such! feasibility! has! put! bacterial! metagenomics! into! the! hands! of!population<based!epidemiologic!studies.!!!Many!of! the!diseases!of! the!human! intestine! result! from!disruption!of! the! steady<state!microbial!population!structure!with!in!the!gut.!!!A!clear!link!exists!between!the!microbiome! and! host! immune! response! (Littman! &! Pamer,! 2011).! For! example,!

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inflammatory! bowel! disease! (IBD)! has! long! been! suspected! to! involve! gut!microbiota! as! some! patients! respond! to! antibiotics! and! fecal! transplants! (Brown,!2014).!!Indeed,!IBD!is!associated!with!mutations!within!bacterial!recognition!genes!(Hugot! et! al.,! 2001;! Ogura! et! al.,! 2001).! In! this! regard,! obesity! has! been! of! keen!interest.!Animal!studies!have!shown!that!transplantation!of!obese!mouse!microbiota!to!germ!free!mice!can!induce!obesity!(Turnbaugh!et!al.,!2006),!proving!that!certain!microbiota!aid!in!transfer!of!energy!to!the!host!and,!intriguingly,!that!the!microbiota!are! transmissible.! Human! twin! studies! have! also! provided! interesting! data.!Discordantly! obese! mono! and! dizygotic! twins! demonstrate! an! association! with!diminished!Bacteroidetes!prevalence!and! lower!overall!diversity! in! the!obese! twin!compared! to! the! non<obese! sibling! (Turnbaugh! et! al.,! 2009).! Transplanting! fecal!microbiota! from! an! obese!member! of! a! human! discordant! twin! pair! to! germ<free!mouse! caused! the! mouse! to! gain! significant! increase! in! body! mass! and!adiposity(Ridaura!et!al.,!2013).!Further,!antibiotic!use!during!the!first!6!months!of!life! is! associated! with! development! of! obesity! later! in! life! (Ajslev,! Andersen,!Gamborg,!Sorensen,!&!Jess,!2011),!suggesting!a!potentially!intervenable!target.!!!While!our!gut!microbiota!may!be!central!players! in!how!we!develop!disease,! they!may! also! be! a! reservoir! for! agents! that! cause! disease.! ! For! example,! our! gut!microflora!act!as!a!reservoir! for!the!horizontal! transfer!of!antimicrobial!resistance!genes(Penders,! Stobberingh,! Savelkoul,! &! Wolffs,! 2013),! a! finding! more! likely! to!occur!in!food!animals!given!low<dose!antibiotics!as!growth!promoters.!!!The!steady<state!intestinal!microbial!population!structure!is,!of!course,!maintained!by! what! is! introduced! into! the! intestine! as! food.! ! If! an! ingested! food! product! is!contaminated!with!a!human!pathogen!(e.g.!salmonella),!most!of!the!time,!the!normal!gut!flora!will!protect!the!host!against!such!a!pathogen.!!However,!a!large!inoculum!of!the!pathogen,!or!a!prior!exposure!of!the!intestine!to!an!antibiotic,!can!overcome!this!protective!microbial!barrier.!!Thus,!again,!it!is!not!only!the!pathogen!per,se!but!also!the!disruption!of! the!balance! in! the! intestinal!microbiota! that! serves!as!a!disease<causing!factor.!.!!The!association!of!prior!exposure!to!antibiotics!and!drug<resistant!salmonellosis!has!long!been!recognized!(Riley!et!al.,!1984),!but!now,!metagenomics!can!provide!a!biologic!explanation!for!this!epidemiologic!association.!!!!Targeted! study! of! the! gut!microbiome! has! provided! astounding! and! novel! insight!into!the!mechanistic!underpinnings!of!the!function!of!the!human!body.!!In!addition!to! the!gut!other!body!compartments!(Bogdanos,!Smyk,!&!Shoenfeld,!2013;!Chen!&!Tsao,! 2013;! B.! Ma,! Forney,! &! Ravel,! 2012)! and! other! organisms! are! proving! to!harbor!additional!fascinating!and!unique!properties.!!!1.4!The!Human!Virome! !The!totality!of!viruses!within!a!human,!the!“virome”,!is!a!fascinating!yet!challenging!(complex!and!expensive)!part!of!the!microbiome.!!The!evolution!of!viruses!has!likely!paralleled! all! cellular! life! since! the! formation! of! nucleic! acids! (Koonin! &! Martin,!2005).!!Their!diversity!is!staggering!and!it!has!been!postulated!that!viruses!are!the!most! genetically! diverse! group! of! organisms! on! the! planet! (Suttle,! 2005).! This!

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diversity! has! not! only! brought! forth! some! of! the! most! pathogenic! species! in!existence!but!has!also!made!studying!viral! communities!difficult!due! to!extremely!divergent! clades! of! viruses! that! share! no! genetic! similarity.! Recent! advances! in!metagenomics!have! for! the! first! time!allowed! for!a!wide! classification!of! all! know!viruses! in! a! given! sample! in! the! greater! virome.! ! Two!key! findings!have! emerged!from!these!studies.!First!we!are!full!of!both!viruses!of!bacteria!(phages)!and!viruses!of!humans,!and!second!we!are!just!beginning!to!understand!the!diversity!of!‘normal’!human!viral!flora.!!!It!is!somewhat!counterintuitive!to!accept!that!viruses!evolve!towards!avirulence!on!an! evolutionary! scale.! However,! in! maximizing! fitness,! pathogenicity! becomes!detrimental.! Nevertheless,! through!millions! of! generations! and! unstable! genomes,!many!viruses!have!“mistakenly”!ventured!into!extremely!pathogenic!mutations!that!lead! to! host! death.! Yet! pathogenicity! is! the! exception! rather! than! the! rule.! For!example!the!Anellovirus!family!(or!Torque!Teno!Virus<!TTV)!may!in!fact!be!the!most!common!human!virus,!yet!relatively! little! is!known!about! them!because! they!have!not! been! identified! as! causing! disease! in! humans.! ! Indeed!most! people! are! likely!infected!with!an!Anellovirus,!where!primary! infection!often!occurs!within!the! first!weeks! of! life.! There! is! also! evidence! that! constant! transmission! occurs! back! and!forth! in! adults! (Davidson! &! Shulman,! 2008;! TeKippe! et! al.,! 2012).! The! cloud! of!genotypes!that!make!up!TTV!is!highly!diverse!and!poorly!delineated.! It! is!possible!that,!akin! to!oncogenic!strains!of! largely!benign!human!papilloma!viruses,! specific!TTV!strains!may!be!associated!with!disease.!In!the!instance!of!these!highly!diverse!groups,!traditional!methods!of!identification!are!challenging.!!Yet!metagenomics!has!opened!the!door.!TTV!is!just!one!organism!of!many!known!to!science!that!is!highly!prevalent! yet! poorly! understood.! Strain<specific! differences! in! pathogenicity! have!been! recognized,! for! example,! between! serotypes! of! enteroviruses! or! strains! of!papillomaviruses;! some! cause! diarrhea! or! cancer! and! some! do! not.! To! assess! the!disease<causing! potential! of! TTV! and! many! yet<to<be! known! viruses! will! require!population<based!studies.!!1.5!Microbial!Balance!When!we! study! humans!we! are,! in! fact,! studying! groups! of!microbial! ecosystems!that! interact! positively! to! promote! health! and! negatively! to! promote! disease.! An!important! concept!when! considering! the! human!microbiome! is! that! of! neutrality.!The!human!body! consists! of! groups! of! ecosystems! that! have! evolved!over! tens! of!thousands! of! years! to! establish! a! stable! equilibrium! state.! ! It! is! when! this!equilibrium! state! is! disrupted! that! a! disease! occurs.! ! For! example,! antibiotic!exposure! (a! synthetic! product)! disrupts! the! fine! balance! that! existed! in! our!intestinal! microbiota! before! antibiotics.! ! This! resulting! “dysbiosis”! can! trigger!diseases!like!Clostridium,difficile!colitis.!!C.,diff!is!not!a!“bad”!bacterium.!!Indeed,!it!is!always!in!our!intestine.!!It!is!only!when!other!microbial!populations!are!eliminated!that!C.,diff!becomes!“bad”.!!Coprotransplantation!works!against!C.,diff!colitis!because!it! restores! the! balance.! ! A! disruption! of! the! “local”! intestinal!microbiota! can! also!affect!body!physiology!as!a!whole.!Other!complex!diseases!may!be!caused!by!!similar!

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disruptions!of!this!fine!balance!–!not!only!in!the!gut!with!bacteria!but!also!in!other!body!compartments!and!with!other!organisms.!!!The!dysbiosis!concept!can!also!be!extended!to!viruses.!A!primary!example!is!that!of!cytomeglavirus! (CMV),! where! prevalence! in! healthy! adults! ranges! from! 70<90%.!Infection! generally! occurs! early! in! life! with! minor! symptoms! and! otherwise!uninteresting! clinical! presentation.! Yet! life! long! latent! infection! ensues,! a! process!that!is!the!result!of!hundreds!of!thousands!of!years!of!co<evolution!of!humans!and!viruses! culminating! in!dynamic! and! intricate! immunologic! evasion! strategies.! In! a!healthy!balanced!individual,!CMV!causes!no!harm,!yet! in!an!immune!compromised!individual,! CMV! can! be! life! threatening.! ! Dysbiosis! resulting! in! CMV<caused!morbidity!can!be!triggered!by!a!number!of!factors;!some!are!known!such!as!HIV!and!immunosuppressive!therapies,!yet!we!suspect!many!others!are!unidentified.!Little!is!know!of!the!viral!interaction!between!CMV!and!other!common!viruses.!Yet!striking!epidemiologic! associations! have! been! drawn! between! CMV! and! a! borad! range! of!complex!diseases!from!brain!tumors!to!heart!disease!(Cobbs!et!al.,!2002;!Nieto!et!al.,!1996).! !For!the!first!time!we!now!have!the!ability!to!examine!such!relationships!in!terms!of!their!multifactorial!biological!causal!pathways.!!!1.6!Looking!Forward!The!microbiome!may!be!a!critical!component!of!the!epidemiology!of!many!diseases.!Genomic! changes! in! the! microbiome! are! measurable.! It! is! well! understood! how!pathogens! are! transmitted! between! individuals!within! communities.! Through! our!growing! understanding! of! the! metagenome,! it! is! clear! that! we! not! only! transmit!pathogens,! but!we! also! transmit! our! commensal! flora! as!well! (Unpublished!data).!!Through!direct!or! indirect! contact,!we!may!modify!disease! risk.! In!addition! to! the!dysbiosis! described! above,! additional! modification! may! occur! through! more!traditional!exposures:!!!Chemicals<! Immunotoxic!mechanisms! alter! host! immune! response! and! therefore!microbial!communities.!Stress<! A! psychologically<induced! response! that! can! be! immunosuppressive! and!alter!our!core!microbiome!or!risk!of!dysregulation.!!Contact<!Direct!or!indirect!contact!modification!of!microbiota!is!occurring!on!a!daily!basis.!!Timing!and! frequency!of! infection! <!Early!life!exposures!to!both!commensal!and!pathogenic!organisms!mold!our!immune!system!for!the!remainder!of!the!life!course.!!And!others….!!The! combined! ! ! tools! of! metagenomics! and! epidemiology! will! lend! insight! into!human! health! and! disease.! ! As! laboratory! and! bioinformatics! methods! become!streamlined,!defining!and!quantifying!known!organisms,!as!well! as! the!expanse!of!unknown!organisms! and! how! they! interact,!will! likely! become! a! critical! aspect! of!this! field.! Challenges! including! intra<subject! variation! and! temporal! stability! will!require!multiple! sampling! schemes.!Despite! these! challenges,!paired!with!detailed!genetic! predisposition,! environmental! and! social! exposures,! metagenomic!

! 7!

epidemiology! may! very! well! be! the! key! to! untangling! decades! of! cryptic! disease!associations.!We!are!excited!by!this!new!tool!in!our!epidemiologic!quiver.!!! !

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!!!!!!!

!!

Chapter!2*!The!Metagenome!of!Childhood!Leukemia!! !

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!!2.1!Introduction:!The! role! of! infection! in! the! etiology! of! acute! lymphoblastic! leukemia! (ALL)! in!childhood!has! been! the! focus! of! hypotheses! and! inquiries! for! decades.! This! study!represents! the! first! comprehensive! investigation! of! possible! etiologic! viruses! and!bacteria.!!Acute! lymphoblastic! leukemia! (ALL)! is! the! most! common! subtype! of! the! most!common!childhood!cancer.!!Within!common!ALL!(cALL),!a!pre!B<cell!ALL!occurring!between! the! ages! of! 2! and! 5,! the! presence! of! MLL<AF4! and! TEL<AML1! fusions!present! in! blood! spots! taken! at! birth! from! children! that! go! on! to! develop! this!common!type!of! leukemia!provides!unambiguous!molecular!evidence!for! initiation!in!utero,!(Maia!et!al.,!2003;!Wiemels!et!al.,!2002).!However!these!translocations!are!~100!times!more!common!than!disease!making!them!necessary,!but!not!sufficient!to!cause! disease(M.! Greaves,! 2006a).! Furthermore,! recent! studies! have! shown! a!profound!IL<10!deficit!in!children!that!later!develop!leukemia,!providing!evidence!of!immune!modification!at!birth! and! further! supporting! in<utero! initiation! (Chang!et!al.,! 2011).! Together,! these! observations! necessitate! a! post<natal! ‘second! hit’! for!development!of!disease.!!!A! strong! body! of! evidence! suggests! a! role! for! infection! in! the! etiology! of! ALL,!specifically!as!a!disease!initiating!‘second!hit’.!Two!main!infection!hypotheses!have!been!set! forth,!Greaves’!delayed! infection!model! (M.!Greaves,!2006b)!and!Kinlen’s!population!mixing!model! (Kinlen,! 1998).!While! both! of! these! hypotheses! explain!different! aspects! of! epidemiologic! observations,! the! models! are! not! mutually!exclusive.!!Greaves’!model!proposes!that!delayed!exposure!to!infectious!agents!leads!to!an!abnormal!immunologic!response!promoting!the!secondary!genetic!lesions!that!lead! to! disease.! Kinlen’s! model! proposes! that! leukemia! is! the! result! of! a! rare!response! to! common! infectious! agents.! The! observations! upon! which! these!hypotheses!were! built! have! since! been! greatly! expanded.!Most! notably,! it! is! now!clear!that!children!that!develop!leukemia!have!more!medically!diagnosed!infections!prior! to! the! diagnosis! of! leukemia! than! healthy! children! (Chang,! Tsai,! Tsai,! &!Wiemels,!2012;!Crouch!et!al.,!2012).!It! is!unclear!if!these!children!are!infected!at!a!greater! frequency! or! if! they! have! more! severe! infections! warranting! a! visit! to! a!physician.!!The!observation!of!decreased!IL<10!levels!at!birth!suggests!that!children!who!develop!leukemia!are!born!with!immunologic!defects!that!predispose!them!to!greater! and/or! more! severe! infections! throughout! their! life! course! prior! to!diagnosis.! Yet! a! central! question! is,! what! are! these! infections?! Is! the! infection! a!transforming! virus,! or! a! suite! of! infections! that! modify! the! immune! system! and!initiate! a! cascade! of! events! that! establish! a! pre<leukemic! clone?! Both! questions!remain!of!keen!interest.!!Both!PCR!and!immunologic!assays!have!been!used!to!investigate!specific!pathogens!including!bovine!leukemia!virus!(Bender!et!al.,!1988),!polyoma!viruses!(MacKenzie,!

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Perry,! Ford,! Jarrett,!&!Greaves,! 1999),! parvo!B19! (Heegaard,! Jensen,!Hornsleth,!&!Schmiegelow,! 1999;! Heegaard! &! Schmiegelow,! 2002;! Isa,! Priftakis,! Broliden,! &!Gustafsson,! 2004)! and! human! herpes! virus! (MacKenzie! et! al.,! 2001).! The! most!comprehensive!study!to!date!utilized!a!representative!difference!analysis!(RDA)!but!found!no!foreign!detectable!sequences!in!11!cALL!subjects!(MacKenzie!et!al.,!2006).!Yet!hybridization<based!RDA!has!many!potential!pitfalls!not!present!in!modern!deep!sequencing!based!analyses.!While!no!previous!study!has!identified!a!single!causative!agent,!all!fail!to!prove!the!contrary.!Investigating!multiple!infections!adds!additional!challenges.! Combining! epidemiologic! techniques! with! laboratory! methodologies!similar! to! those! presented! in! this! paper! provide! a! substantial! leap! in! current!knowledge!regarding!this!complex!mechanism.!!!Both!Kinlen!and!Greaves’!hypotheses!also!seek!to!explain!the!occurrence!of!clusters!of!childhood!leukemia.!The!significance!of!leukemia!clusters!remains!a!controversial!subject! (Goodman,!Naiman,!Goodman,!&!LaKind,!2012).!While!many!clusters!have!been!defined,!the!most!poignant!example!in!the!United!States!occurred!in!Fallon,!NV.!From! 1997<2001,! 17! children! were! diagnosed! with! childhood! leukemia! and! 9! of!these!cases!occurred!in!a!narrow!11<month!window!(Francis,!Selvin,!Yang,!Buffler,!&!Wiemels,!2012).!We!have!previously!conducted!a!spatial!analysis!of!the!cluster!cases!and! found! a! non! random! distribution! of! cases! around! Fallon,! rather! than! in! the!urban!center.!In!other!words,!cases!occurred!primarily!in!rural!areas!where!land!is!used!for!agricultural!purposes.!!One!such!use!is!flood<irrigated!farm!land.!Fallon!has!>50,000!hectares!of! such! land,! an! ideal!habitat! for!breeding!mosquitoes.!Given!an!extremely!high! infection!rate! in! this!area!with!other!vector<borne!pathogens,!such!as!West!Nile!Virus,!we!hypothesize!that!the!Fallon!cluster!may!have!been!the!result!of! an! unidentified! virus,! transmitted! by! a! mosquito! vector,! causing! the! so<called!‘second! hit’! event! for! children! living! around! Fallon,!who!were! already! at! risk! for!leukemia.! If! a! causal! virus! is! implicated! in! the! Fallon! cluster,! it! would! likely! be!unique! to! that! cluster,! or! to! leukemia! clusters! in! general! and! not! a! universal!etiologic!agent!of!ALL.!Therefore!we! also!investigated!the!more!prevalent!sporadic!cases!of!ALL.!!!To! investigate! the! ALL! infection! hypotheses! we! conducted! a! 4<part! study!comprising! the! most! comprehensive! viral! discovery! and! bacterial! quantification!project!in!ALL!to!date.!In!addition!to!comparing!clustered!to!non<clustered!subjects,!we! compared! non<clustered! ALL! cases! to! acute!myeloid! leukemia’s! (AML)! drawn!from!the!same!underlying!population!at!the!same!time.!We!selected!AML!subjects!as!a! comparison! group! because! AML! as! a! cancer! has! vastly! different! epidemiologic!patterns,!no!shared!genetic!risk!alleles,!and!an!early!differentiation!in!lineage!from!hematopoietic!stem!cells!yet!provides!and!estimate!of!infection!exposure!of!another!type! of! immune! suppressed! child! to! reduce! the! background! infection! ‘noise’.! !We!also!sought!to!identify!the!infection!burden!in!bone!marrow!of!children!with!ALL.!!

1< In<depth!virome!analysis!of!pretreatment!bone!marrow! from!2!subjects!from!Fallon!and!2!non<clustered!ALL!subjects!

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2< Comparison!of!all!viral!particles!in!pretreatment!bone!marrow!of!36!ALL!and!11!AML!subjects!using!particle!isolation!and!454!sequencing!!

3< Comparison!of!16s!bacterial!metagenomics!pretreatment!bone!marrow!of!73!ALL!and!28!AML!subjects.!!

!2.2!Materials!and!Methods:!!The! University! of! California! Institutional! Review! Board! and! all! participating!institutions! approved! this! study.! Informed! consent! was! obtained! from! all!participating!subjects.!!2.2.1:!Fallon!Viral!Discovery!!Sample!Selection:!!Two! pretreatment! bone! marrow! samples! were! obtained! from! the! Children’s!Oncology!Group!(COG).!The!eligibility!criteria!and!DNA!collection!procedures!have!been! previously! described! (O'Leary,! Krailo,! Anderson,! Reaman,! &! Children's!Oncology,!2008).!These!were!2!of!3!samples!collected!during!routine!COG!collection!as! part! of! the! Fallon! Nevada! leukemia! cluster! Center! for! Disease! Control!investigation.! ! These! samples! met! eligibility! criteria! for! admission! into! COG! and!were!not!collected!specifically!as!cluster!samples,!yet!were!involved!in!the!cluster.!!Two! additional! comparison! pretreatment! bone! marrow! specimens! were! selected!from!the!California!Childhood!Leukemia!Study!(CCLS).!The!CCLS!has!been!described!elsewhere!(X.!Ma,!Buffler,!Layefsky,!Does,!&!Reynolds,!2004).!Briefly,!the!CCLS!is!an!ongoing!case<control!study!in!California!that!commenced!in!1995.!!The!study!began!in!the!17!counties!surrounding!the!San!Francisco!Bay!Area,!and!now!encompasses!most!of!California! through!collaboration!with!35!pediatric!oncology!centers! in! the!state,! allowing! for! rapid! case! ascertainment! and! biospecimen! retrieval,! usually!within! 72! hours! from! diagnosis.! One! or! two! controls! are! recruited! using! birth!certificate! information! from! the! California! Department! of! Public! Health! Office! of!Vital! Records.! Controls! are!matched! on! age,! sex,! Hispanic! ethnicity,! and!maternal!race.!!!These!pretreatment!samples!were!matched!on!month!of!diagnosis!to!the!two!Fallon!samples!(1999<2000).!!These!samples!were!not!part!of!any!identified!cluster!within!the!CCLS.!!!RNA!extraction:!All! laboratory! equipment! and! reagents! were! thoroughly! cleaned! and!decontaminated! to! minimize! contamination! potential.! Ultra! clean! reagents! along!with! liberal! use! of!UV! and! chemical! treatment! (Bleach! solution! and!RNAse! away)!were! used! to! ensure! that! no! contaminating! sequences! entered! pre<extracted! or!post<extracted!samples.!!!!The!4!bone!marrow!specimens!were!extracted!using!the!same!protocol!in!the!same!decontaminated!hood!at!the!same!time.!!We!used!a!modified!protocol!for!trizol!RNA!extraction! with! back! extraction! for! DNA.! A! 250ul! aliquot! of! bone! marrow! was!

! 12!

suspended!in750uL!Trizol!LS.!After!incubating!for!5!minutes!at!room!temperature,!200uL!chloroform!was!added!and!shaken!vigorously!by!hand!for!15!seconds.!After!Incubating! for! 2<15! minutes! at! room! temperature! the! sample! was! centrifuge! at!12,000!xg!RCF!at!4°c!for!15!minutes.!The!aqueous!phase!was!then!removed!and!!!0.5ul!glycogen!was!added!and!mixed!with!pipette.!!500uL!of!100%!isopropanol!was!added! and! incubated! at! room! temperature! for! 10!minutes.! The! sample!was! then!centrifuged!at!12,000!xg!RCF!at!4°c!for!10!minutes.!The!supernatant!was!removed!and! the! RNA! pellet! was! washed! with! 1.0mL! 75%! ethanol! then! vortexed! and!centrifuged!at!7,500!xg!RCF,!4°c! for!5!minutes.!The!supernatant!was!removed!and!RNA!pellet! dried!using! vacuum.!The!RNA!pellet!was! then! re<suspend!30uL!RNase!free!water.!RNA!quantity!and!quality!was!analyzed!using!Aligent!Bioanalyzer.!!!Library!preparation!and!ribosomal!capture:!Prior!to!Illumina!library!preparation,!a!ribosomal!capture!was!performed!to!reduce!the!amount!of!human!ribosomal!transcripts!being!sequenced.!This!was!completed!to!increase! the! sensitivity! of! identifying! non<human! transcripts! through! physical!removal! of! ribosomal! sequences! prior! to! sequencing.! For! each! RNA! sample,!ribosomal! RNAs! were! depleted! using! Ribo<Zero™! Gold! Kits! (Epicenter)! &! the!depleted! RNA!was! confirmed! with! Bioanalyzer.! RNA<seq! libraries! were! prepared!using!PrepX!RNA<Seq!Library!kits!(WaferGen,!Inc)!according!to!the!manufacturer’s!protocol.! The! library! was! PCR! amplified! &! the! reaction! was! cleaned! with!AGENCOURT®!AMPURE®!XP!(Beckman).!!!Sequencing/Quality!Filtering:!Sequencing! was! preformed! on! an! Illumina! Hiseq2000.! Each! sample! generated!approximately!60!million!high!quality!100bp!paired!end!reads!per!sample.! !Those!reads!were! aggressively! filtered! using! both! Illumina’s! CASAVA! 1.8! ‘pass/no! pass’!system! then! were! further! quality! filtered! using! the! fastx! tool! kit! to! only! include!reads!with!a!minimum!phred!score!of!30!over!at!least!75%!of!the!read.!!!Bioinformatics:!!A! custom!bioinformatics!pipeline! was!developed! for!identification! of!known! and!unknown! non<human! nucleic!acids.! The! system,!named! DARK,!preforms!computational!human!subtraction,!contiguous!

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sequence!(contig)!assembly!and!sequence!identification!designed!for!use!on!SLURM!(https://computing.llnl.gov/linux/slurm/)! based! computer! clusters.! Briefly! after!aggressive! quality! filtering! DARK! iteratively! aligns! the! raw! 100bp! reads! using!BowTie2! (Langmead! &! Salzberg,! 2012)! to! 3! human! reference! genomes! and! the!Refseq! human! database.! All! human! reads! are! removed.! DARK! assembles! the! raw!non<human!100bp!reads!using!Trinity!(Haas!et!al.,!2013)! into! longer!contigs.!Both!the! raw! non<human! 100bp! reads! and! contigs! are! separately! queried! against!Genebank!(https://www.ncbi.nlm.nih.gov/genbank/)!and!the!refseq!virus!database!(https://www.ncbi.nlm.nih.gov/genbank/)!using!BLAST!(Altschul!et!al.,!1997).!This!is!accomplished!through!massively!parallel!computing!on!an!in<house!SLURM!based!cluster.!To!increase!our!specificity!at!the!cost!of!sensitivity!in!the!current!analyses,!we!chose!to!focus!on!contigs!assembled!by!Trinity.!By!using!contigs!instead!of!raw!100bp!sequences,!we!are!reducing!the!probability!of!sequencing!error!resulting! in!false! hit! by! requiring! support! for! every! base! pair! and! by! increasing! the! size! of!sequences! queried! in! blast.! Although! this! may! reduce! our! sensitivity,! this! is! a!conservative!approach.!!Analysis!of!Blast!files:!!We! used! a! combination! of! approaches! to! examine! the! resulting! BLAST! files!produced!by!our!DARK!pipeline.!We!designed!a!custom!database!to!parse!and!query!blast! files! by! subject! and! read.! This! enabled! filtering! of! hits! according! to! a! high!discriminatory!likelihood!and!collating!hits!by!subject.!We!utilized!MEGAN,!a!freely!available!package!that!complies!visualization!of!metagenomic!data(Huson,!Auch,!Qi,!&!Schuster,!2007).!!!2.2.2!ALL!vs.!AML!Particle!Isolation!454!Sequencing!Sample!Selection:!!All!samples!were!obtained!from!the!CCLS!(described!above).!We!selected!36!cALLs!(pre<B! cell!ALL!with!CD!10:19,! age! at!diagnosis!2<5.9! years)! and!11!AML’s!with! a!similar!age!distribution.! !All!samples!were!drawn!from!the!same!underlying!study!population! occurring! in! the! 17! counties! surrounding! the! San! Francisco! bay! area.!Pretreatment!bone!marrow!specimens!were!obtained! from! the!original!diagnostic!bone! marrow! aspirate.! The! existing! CCLS! study! allowed! for! rapid! case!ascertainment!of!both!biospecimens!and!epidemiologic!data.!!Viral!particle!purification:!!Bone!marrow!specimens!were! suspended! in!Hanks’! balanced! salt! solution! (Gibco!BRL).!Three!15,000!g!centrifugations!using!a!tabletop!microfuge!and!a0.45<um!filter!(Millipore)! to! remove! non<virus! sized! particles.! The! filtrate! was! treated! with! a!mixture!of!DNases!(Turbo!DNase!from!Ambion,!Baseline<ZERO!from!Epicen<!tre,!and!Benzonase! from! Novagen)! and! RNase! (Fermentas)! to! digest! all! nucleic! acids! not!protected! by! viral! capsids.! All! remaining! protected! viral! nucleic! acids! were! then!extracted!using!the!QIAamp!viral!RNA!and!DNA!extraction!kits!(Qiagen).!Viral!cDNA!synthesis! from! extracted! viral! RNA/DNA! was! performed! as! described! previously!(Victoria,!Kapoor,!Dupuis,!Schnurr,!&!Delwart,!2008).!!!

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!Bioinformatics:!After!sequencing,!raw!data!generated!from!a!Roche!454!sequencer!data!was!quality!filtered! for! sequencing! errors! and! haplopolymer! regions! then! converted! to! fasta!files!using! the! fastx! toolkit.! !The! resulting! fasta! files!were!parsed!and! trimmed!by!subject! based! on! the! unique! barcode! ligated! to! each! read.! Parsed! fasta! files!were!queries! using!BLAST! against! both! the!NT! and! viral! refseq! databases! using!BlastN!and!BlastX.!Recovered!nucleic!acids!that!were!assigned!a!virus!or!virus!family!based!on!E<values!tabulated!and!compared!between!groups.!The!E<Value!cutoff!for!calling!a!hit!to!a!virus!was!set!at!E<10!,!a!highly!stringent!cutoff.!The!number!of!viral!hits!was!normalized! by! subject! based! on! number! of! sequence! reads! recovered! from! each!sample,! the! number! of! viral! hits! was! simply! divided! by! the! total! recovered!sequences! for!a!given!sample.!Wilcox!rank!sum!tests!were!used! to!compare!mean!frequencies! of! called! viral! hits! between! ALL! and! AML! subjects.! These! were!graphically!displayed!using!95%!confidence! intervals.!All! statistical! analyses!were!preformed!in!R.!!!2.2.3!16s!Bacterial!Metagenomics!Sample!selection:!!All!samples!were!drawn!from!the!CCLS!study!base!and!biorepository!as!described!above.!We!analyzed!bone!marrow!from!73!cALLs!and!28!AMLs.!!!DNA!Extraction:!DNA! was! extracted! using! a! modified! TRIzol! LS! protocol.! During! initial! RNA!separation! described! above! any! remaining! aqueous! phase! was! removed,! 375uL!back!extraction!buffer!(4!M!guanidine!thiocyanate,!50!mM!sodium!citrate!and!1!M!Tris!pH!8.0)!was!added,!mixed!and!incubated!for!10!minutes!at!room!temperature.!The! samples! were! then! centrifuged! at! 12,000! xg! RCF! at! 4°c! for! 15!minutes.! The!aqueous!phase!was!removed!and!combined!with!0.5ul!glycogen!to!and!mixed!with!a!pipette.! DNA!was! precipitated!with!~260uL! 100%! isopropanol! (~80%! volume! of!aqueous! phase)! and! incubate! for! 5! minutes! at! room! temperature,! and! mixed!occasionally.!Then!the!samples!were!centrifuge!at!12,000g!RCF!at!4°c!for!5!minutes,!the! supernatant! was! removed! then! pellet! washed! with! 1.0mL! 75%! ethanol! and!incubated!at! room! temperature! for!15!minutes!while!mixed!occasionally.! Samples!were! then! centrifuged! at! 2,000g! RCF! at! 4°c! for! 5! minutes,! the! supernatant! was!removed! and! DNA! pellet! was! dried.! The! dry! pellet! was! re<suspend! in! 30uL! of!nuclease<free!water.!!!Bacterial!16s!PCR!and!sequencing!To!examine!the!diversity!and!prevalence!of!bacterial! infection!in!bone!marrow!we!performed!PCR!amplification!using!barcoded!primers!specific!to!the!16s!rRNA!gene!shared! by! all! bacteria,! we! modified! the! Caporaso! et! al.! protocol(Caporaso! et! al.,!2011).!Briefly,!custom!barcoded!primers!were!constructed!targeting!the!V4!region!of! the!16s!gene.! !A!unique!12<nucleotide!barcode!was!constructed!by!synthesizing!additional! nucleotides! to! existing! V4! primers.! One! hundred! unique! barcoded!

! 15!

primers!were!constructed.!Barcodes!were!optimized!for!error!reduction!(Caporaso!et!al.,!2010).!We!did!not!include!the!Illumina!sequencing!adapter!and!linker!on!our!primers.!Each!of!these!unique!primers!was!used!to!amplify!the!V4!region!of!16s!in!each! sample,! creating! a! uniquely! barcoded! amplicon.! The! PCR! was! preformed! in!triplicate.!!!Sample!pooling,!library!preparation!and!sequencing:!Samples! were! normalized! and! pooled! using! pico! green! in! triplicate.! All! samples!were!diluted! to! the! lowest! concentration! sample.!The!pool!was! then!prepared! for!sequencing! by! the! Berkeley! Functional! Genomics! core! facility,! where! an! Illumina!sequencing!pad,! linker!and!adapter!was! ligated! to!each!amplicon.!Sequencing!was!conducted!at!the!Berkeley!QB3!sequencing!core!facility.!An!Illumina!HiSeq2500!was!used!to!produce!100bp!paired!end!data.!!!16s!bioinformatics:!Sequence!pre<processing!and!OTU!analysis!was!performed!using!the!FastX!Toolkit!(http://hannonlab.cshl.edu/fastx_toolkit)!and!QIIME!(Caporaso!et!al.,!2010).!Quality!filtering!was!performed!where!a!minimum!30!quality!score!over!at!least!75%!of!the!sequence! read,!no!ambiguous!bases,! and!1!primer!mismatch!was!allowed.!Quality!sequences!were!then!parsed!by!barcode!and!binned!into!OTU’s.!!A!subsample!from!each!subject!of!20,000!sequences!was!used!for!OTU!construction!at!a!97%!sequence!similarity!cutoff!using!uclust.!Taxonomic!classification!was!performed!by!querying!the!Greengenes!database!(2011!release)!(DeSantis!et!al.,!2006).!!!!2.3!Results:!!!2.3.1!Fallon:!! !Approximately!299!million!100bp!paired!end!reads!were!recovered!that!passed!our!quality! filters.! DARK! then! removed! 217! million! reads! as! human! during! the! first!rounds! of! BowTie2! based! computational! subtraction,! leaving! 81! million! reads.!Those!raw!reads!were!denovo!assembled!into!165,072!contigs!using!Trinity.!!Using!BLAST!we!were! able! to! further! classify! 136,916! as! human! or! low! complexity.! Of!those! non<human! contigs! we! were! able! to! categorize! 4.2%! as! virus,! 23.4%! as!bacterial!using!both!Blastn!(Figure!1).!The!remaining!non<human!unknown!contigs,!the!dark!sequences,!made!up!the!remaining!72.5%!and!are!of!keen!interest!yet!not!explored!in!this!paper.!!!Of! the! identified! non<human! sequences,! both! the! Fallon! subjects! and! the! CCLS!subjects!share!many!of!the!same!viral!infections!(Figure!2).!Each!sample!contained!phages!belonging!to!the!Caudoviradae!and!Baculoviradae!families!and!are!likely!the!result! of! bacterial! infection! or! contamination! from! the! initial! lumbar! puncture! to!obtain! bone!marrow! aspirate! (see! 16s! section).! There! are!many! reads! that! hit! to!double!stranded!DNA!viruses,!specifically!herpes!viruses.! !All!4!samples!contained!strong! evidence! of! Cytomegalovirus! (CMV)! infection.! Human! endogenous!retroviruse! (HERV)! sequences! were! also! recovered! from! all! 4! subjects,! with! the!most!prevalent!HERV!species!identified!being!a!member!of!the!HERV!K!family.!!

! 16!

The!only!group!of!viruses!found!strictly!in!the!Fallon!subjects!and!not!the!sporadic!CCLS!cases!are!members!of!the!ssRNA!virus!family.!The!contigs!had!high!homology!bovine! viral! diarrheal! virus! (BVDV),! a! Flavivirus! and! common! pathogen! in! cattle.!!Although!these!reads!are!strong!hits!to!BVDV!there!is!evidence!in!the!raw!files!that!other!ssRNA!viruses!may!be!present.!!!2.3.2!Virus!Particle!Isolation!and!454!Sequencing!After! sequencing,! quality! filtering! and! parsing,! ~300<4000! reads! were! recovered!per!subject.!BlastN!and!tBlastX!results!noted!a!number!of!viral!hits!in!both!ALL!and!AML! subjects! and! significant! differences!were! observed!using!Wilcox! ranked! sum!test!between!the!groups!(Figures!3!&!4).!The!most!prevalent!viruses!were!members!of! the! double! stranded! (DS)! DNA! virus! family,! specifically! herpes! viruses.! All!subjects!had!evidence!of!DS!DNA!virus! infection.!A! statically! significant!difference!was!observed! for!DS!DNA!viruses!between!ALL!and!AML!subjects! (BlastN!p<value!0.0007,! tBlastX! p<vlaue=! 0.023).! HERVs!were! also! detected! in! both! groups,! and! a!marginally! significant! difference! was! noted! between! ALLs! and! AMLs! (BlastN! p<value=0.058,! tBlastX! p<value=0.143).! There! was! also! evidence! of! retrovirus!infection.!Yet!the!number!of!recovered!reads!was!low!and!misclassification!of!HERV!sequences!is!likely!and!there!was!no!observed!difference!between!groups!(BlastN!p<value=0.089).!Both!DS!RNA!and!single!stranded!(SS)!RNA!viruses!were!detected!in!low!levels!in!both!AML!and!ALL!subjects!though!there!was!no!significant!difference!between!the!groups!(DS<RNA!BlastN!p<value=!0.09,!DS<RNA!tBlastX!p<value=!0.158,!SS<RNA! BlastN! p<value=! 0.65,! SS<RNA! tBlastX! p<value=! 0.181).! ! Overall,! when!examining!mean!load!of!all!identified!viral!sequences!meeting!the!E<value!cutoff!we!observed!a! significant!difference!between!ALL!and!AML!subjects! (BlastN!p<value=!0.013,!tBlastX!p<value=!0.046).!!!2.3.3!16s!Bacterial!Microbiome!!After! sequencing,! 28,775,627! reads! passed! initial! Illumina! CASAVA! 1.8! quality!filtering.! ! After! barcode! parsing! and! rarefaction! we! utilized! 20,000! reads! per!subject.!Those!sub!sampled!reads!were!assigned!OTUs!in!the!QIIME!pipeline.!Figure!5!shows!the!bacterial!composition!at!the!class!level!for!each!group,!where!Figure!6!shows! individual! level! data.! The! most! common! bacterial! class! was!Gammaproteobacteria! (ALL=51.70%,! AML=49.37%),! followed! by!Betaproteobacteria! (ALL=23.34%,! AML=25.72)! and! Flavobacteria! (ALL=9.24%,!AML=10.98)! then! Alphaproteobacteria! (ALL=5.18%,! AML=4.27%)! being! the! last!class!above!5%!of!the!sample.!Additional!classes!of!bacteria!under!5%!are!detailed!in!Figures!5<7.! !When!examining!bacterial!classifications!at!the!most!granular!level!possible! without! misclassification,! by! genus,! the! most! common! bacteria! was!Pseudomonadaceae! (ALL=45.31%,! AML=42.56%),! followed! by! Comamonadaceae!(ALL=19.51,! AML=21.06%),! Weeksellaceae! (ALL=9.23%,! AML=10.96%),! and!Moraxellaceae!(ALL=!3.61%,!4.35%).!The!many!other!genus’s!representing!groups!less!than!4%!(detailed!in!supplemental!material).!!!

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No! evidence! of! differences! between! quantity! of! bacterial! classes! or! genus’s! using!Wilcoxon! sign<rank! test! while! adjusting! for! multiple! comparisons! was! observed.!There! is! no! evidence! for! a! single! bacterial! genera! present! differentially! between!ALL’s! and! AMLs! (Figure! 6! &! 7).! Furthermore,! when! examining! neighbor! joining!trees!produced!by!of!the!OTU’s!assigned!with!in!each!samples,!there!is!no!evidence!for!any!difference!between!ALL’s!and!AML’s!(Figure!8).!!!!2.4!Discussion:!!This! is! the! first! investigation! to! comprehensively! describe! the!non<human!nucleic!acids!present!in!pretreatment!bone!marrow!specimens!of!childhood!leukemia!using!new!!nucleic!acid!sequencing!tools.!This!study!was!designed!to!investigate!the!two!main!etiologic!hypotheses!postulated!by!Kinlen!and!Greaves!that!stipulate!the!role!of!infection!in!the!etiology!of!ALL.!We!examined!metagenomes!in!the!bone!marrow!of! subjects! from! a! defined! cluster! of! ALL! (Fallon),! non<clustered! ALL! subjects!(CCLS),!and!other!immunosuppressed!children!(AMLs).!!!This! metagenomic! investigation! demonstrates! that! children! who! have! either!lymphoid!or!myeloid!leukemia!harbor!diverse!viral!and!bacterial!communities!with!in!their!bone!marrow.!This!observation!should!come!as!no!surprise!as!bone!marrow!from! healthy! individuals! harbor! diverse! bacteria! and! viruses.! Nevertheless,! our!comparisons! show! clear! differences! in! the!microbiome! of! subjects!with! lymphoid!and!myeloid!leukemia.!There!are!3!main!findings!from!this!investigation:!

1< Subjects! from! the! Fallon! leukemia! cluster! were! infected! with!pestiviruses!not!found!in!the!non<clustered!subjects!examined.!

2< ALL!subjects!have!a!greater!infection!load,!specifically!of!Herpesviruses!than!AML!subjects!

3< No! detectable! differences! in! bacterial! communities! between! ALL! and!AML!subjects!were!detected.!

The! Fallon! leukemia! cluster! is! likely! the! most! significant! clustering! event! of!leukemia!known!to!the!medical!community.!Leading!infection!hypotheses!state!that!clusters! of! leukemia! may! be! the! result! of! a! transient! virus! spreading! through! a!community,! causing! leukemia! in! susceptible! children.! If! that! were! the! case,! we!would! expect! to! detect! the! causal! virus! in! cluster! cases! and! not! in! non<clustered,!non<related!leukemia!subjects.!It!is!that!prediction!and!our!subsequent!observation!of!a!ssRNA!virus,!definitely!a!pestivirus!likely!a!flavivirus,!in!the!Fallon!subjects!and!not!any!other!ALL!tested!that!deserves!further!consideration.!!A! flavivirus! or! other! vector! borne! virus! may! play! a! role! in! ALL.! The! flavivirus,!!related! to! BVDV,! causes! birth! defects! in! cattle(Lindberg,! 2003).! Related! viruses!show!the!ability! to! induce!genomic! instability.! !Also,! flaviviruses!can!be!spread!by!vector!means.!In!line!with!a!previous!hypothesis!set!forth!by!our!group!wherein!we!suggested!a!possible!vector!route!of!infection!!in!the!Fallon!leukemia!cluster(Francis!et!al.,!2012).!Taken!together,!previous!study!highlighting!the!abrupt!epidemic!curve!of!the!cluster!and!the!geographic!concordance!with!mosquito!breeding!habitat!and!captured!mosquitoes!along!with,!this!new!observation!suggesting!the!presence!of!a!virus!with!vector!borne!potential!in!the!bone!marrow!of!clustered!ALL!subjects!add!

! 18!

evidence!to!the!concept!of!a!vector!borne!virus!in!the!causal!chain!of!some!children!with! ALL.! Though,! our! ability! to! only! obtain! two! viable! samples! limits! inference!greatly.!Further!studies!of!additional!Fallon!samples!must!be!conducted.!!If!indeed!the!Fallon!cluster!was!the!result!of!vector!borne!agent!and!that!agent!was!unique!to!that!cluster,!or!clustered!cases!generally,!what!may!be!the!role!of!infection!for!the!majority!of!ALLs,!non<clustered!ALLs?!We!suspect!that!clusters!are!the!result!of! a! novel! infection! transiently! elevating! leukemia! risk,! where! other! potentially!common!infections!may!set! in!motion!the!immunologic!damage!that!generates!the!leukemic!clone.!Common!infections!may!pose!differential!leukemia!risk!by!timing!of!infection!as!set!forth!by!Greaves(M.!F.!Greaves!&!Alexander,!1993).!We!chose!AML!subjects!as!a!comparison!group!in!order!to!estimate! infection!exposure!of!another!type!of! immune!suppressed!child!to!reduce!the!background!infection! ‘noise’.!What!we!observed!was!a!striking!difference!between!ALLs!and!AMLs!in!terms!of!infection!load!with!any!virus,!and!more!specifically!a!statistically!significant!difference!in!the!amount!of!herpesvirus!detected!in!pretreatment!samples.!These!results!are!similar!to!a!previous!more!narrow!herpes!specific!study!showing!a!greater!infection!burden!of!CMV!in!B<cell!leukemia’s!(Hermouet!et!al.,!2003).!!! !Herpes!viruses!are!complex!viruses!with!highly!evolved!large!genomes!that!harbor!many!immune!evasion!genes,!most!of!which!are!poorly!understood!(Nicholas,!2000;!Vossen,! Westerhout,! Soderberg<Naucler,! &! Wiertz,! 2002).! ! Some! viruses! in! the!herpeviradae! are! nearly! ubiquitous! while! others! remain! relatively! rare.! Herpes!viruses!such!as!Kaposi!sarcoma!virus,!Epstein<Barr!virus,!and!cytomegalovirus!have!oncogenic! potential,! additionally! their! large! repertoire! of! immune! evasion! genes!suggests! that! immune! disregulation! is! a! critical! aspect! of! herpes! virus! life!cycles(Nicholas,!2000).!Herpesviruses!share!many!genes!across!species,!and!many!repeat!regions!exist!within!their!genomes(Davison,!2007).!These!repetitive!regions!are! thought! to! be! involved! non<redundantly! during! circularization,! yet! their!function! is! largely! unknown! (Davison,! 2007;! Nicoll,! Proenca,! &! Efstathiou,! 2012).!Related! to! these!repeats,!determining! the!exact!herpes!virus!where!our!recovered!sequences!originated!is!challenging.!Despite!these!challenges!it!is!clear!that!many!of!the! recovered! sequences! originate! from! cytomegalovirus! (CMV).! Human!herpesvirus<5! or! CMV! infects! 50<80%! of! adults! in! the! United! States! and! >90%!worldwide.! While! it! is! a! common! infection! with! typically! mild! symptoms!complications!can!occur!with!immune!suppression.!CMV!is!highly!evolved!to!remain!latent,! yet! if! that! latency! is!modified! by! immune! suppression! it! has! the! ability! to!cause! serious! harm! to! the! host.! Therefore! whether! the! measured! difference! in!herpesvirus!between!AMLs!and!ALLs! is! related! to! cause!or! is! simply! the! result! of!differential! immune! suppression! between! the! groups! is! a! valid! question! needing!further!investigation.!Yet,!evidence!exists!that!a!difference!in!herpesvirus!infection!may! exist! before! disease! initiation.! Maternal! infection! during! pregnancy,! as!measured! in!342! case!mothers! and!1,216! control!mothers! as!measured!by! serum!EBV!IgM!at!14!weeks!has!been!associated!(OR=!2.9,!p=0.0002)!with!childhood!ALL!(Lehtinen! et! al.,! 2003).! Additional! studies! of! the! timing! and! immune! response! to!herpes!virus!infection!and!its!role!in!childhood!leukemia!is!warranted.!!!

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Prior! evidence! of! the! existence! of! oncogenic! viruses!make! a! viral! agent! the!most!plausible! candidate! for! an! infectious! etiology! of! ALL,! yet! since! the! discovery! of! a!causal! role! of! Helicobacter! pylori! and! stomach! cancer! the! role! of! bacteria! as! an!etiologic! agent! in! cancer! has! gained! traction.! To! examine! if! a! candidate! bacterial!agent! could! be! identified! in! ALL! we! used! a! similar! theory! to! the! viral! discovery!comparison,! utilizing! AMLs! as! an! immune! suppressed! control! to! estimate! the!infection! exposure! history! of! an! immune! suppressed! child.! ! After! performing!barcoded!v4!16s!PCR!and!subsequent!analysis!we!found!no!difference!between!the!ALL!and!AML!groups.!While!this!similarity!suggests!that!there!is!no!etiologic!role!for!bacteria!in!leukemia,!there!may!be!clinical!relevance!to!the!description!of!the!type!of!bacteria!found!in!the!pretreatment!bone!marrow.!Yet,!inference!is!difficult.!Although!the! lumbar! area! was! sterilized! prior! to! the! bone! marrow! aspirate! sampling! any!bacterial!DNA!would!still!be!present.!We!would!therefore!be!unable!to!distinguish!between! skin! contamination! from! the! lumbar! puncture! and! true! bone! marrow!infection.!!!Despite! this! limitation! the! findings! from! this! 16s! study! are! valuable! for! drawing!inference!from!the!viral!discovery!portion!of!this!investigation.!During!the!analysis!phase!of!the!454!viral!discovery!data,!concern!was!raised!since!herpes!viruses!share!genes! and! repeat! regions! with! a! variety! of! bacteria! and! phages.! We! now! have!evidence! that! there!are!equal!bacterial!prevalence!and!diversity!between!ALL!and!AML! leukemia’s! suggesting! that! differential! contamination! has! not! occurred! and!that!our!measured!viral!differences!between!ALLs!and!AMLs!are!likely!not!the!result!of!bacterial!genes!recovered!from!phages!in!the!particle!isolation!viral!discovery.!!!There! are! significant! limitations! to! this! study.! ! The!most! in<depth! viral! discovery!work!was!conducted!on!2!Fallon!subjects!and!2!CCLS!subjects.!These!subjects!were!studies!with!the!greatest!sequencing!effort!and!therefore!are!subject!to!the!greatest!scrutiny.!Although,!different!viral!sequences!were!present!in!the!Fallon!subjects!it!is!difficult! to!draw! inference!due! to! the! small! sample.! Indeed,!with! randomly!drawn!subjects! it! would! not! be! unlikely! to! obtain! different! infection! patterns! that! may!converge!by!chance.!Yet! in! the!analysis!of! the!contigious!sequences! it! is!clear! that!the! Fallon! ALLs! share! most! of! the! same! viruses! with! the! CCLS! ALLs,! only! the!flavivirus! group! is! strictly! in! the! Fallon! ALLs.! The! particle! isolation! and! 454!sequencing! of! ALLs! and! AMLs! also! has! limitations.! The! statistical! power! was!reduced!by!the!small!sample,!thus!the!significant!results!obtained!are!a!testament!to!the!large!observed!difference!between!groups.!!The!lack!of!a!healthy!control!group!also! limits! inference,! although! a! proper! control! in! this! case! is! a! difficult! question.!Obtaining! bone! marrow! from! healthy! children! is! ethically! challenging,! yet! the!primary! reason! for! using! AMLs! as! a! comparison! is! that! we! believe! AMLs! better!represent!the!infection!exposure!history!of!an!immune!suppressed!child.!If!we!were!to! compare! the! virome! of! ALL! cases! to! healthy! controls!we!would! expect! to! find!many!more!infections!present!in!the!ALL’s!simply!due!to!the!result!of!an!ineffective!immune!system!at!the!time!the!samples!were!taken.!By!using!AMLs!as!controls!we!are! reducing! that! signal<to<noise! ratio! by! controlling! for! infections! common! to!immune!suppressed!children.!In!effect,!we!hope!that!this!comparison!will! improve!

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our! inference! by! helping! with! inherent! issues! of! temporality! when! searching! for!infections! in! diagnostic! samples.! Regardless,! having! a! healthy! control! population!would! greatly! improve! the! generalizability! of! this! study.! Finally! the! bacterial!analysis!highlights! some!of! the! sample! collection!difficulties.!With!new!and!highly!sensitive! metagenomic! techniques,! methods! of! sample! collection! that! eliminate!contamination! from! different! body! sites! are! challenging.! In! this! case,! the! lumbar!puncture! to!obtain!bone!marrow!undoubtedly! introduced!skin!microflora! into! the!bone!marrow!sample!making!the!skin!microbiota!and!the!bone!marrow!microbiota!indistinguishable.! We! are! unable! to! say! with! certainty! that! any! of! the! bacteria!measured!originated!in!bone!marrow.!Yet!the!observed!similarity!between!AML!and!ALL!subjects!strongly!suggests!that!contamination!was!equal!between!groups.!!!In!conclusion!we!have!completed!a!first!look!into!the!metagenome!of!children!with!leukemia.! Our! results! suggest! that! children! from! the! Fallon! leukemia! cluster!may!harbor!a!virus!that!other!children!with!leukemia!are!not!infected!with.!That!virus!is!likely! a! member! of! the! flavivirus! family! and! may! have! been! transmitted! by!mosquito.!Further! laboratory!studies!are!needed! to!determine! the!exact!virus!and!general!prevalence!in!leukemia!subjects!and!healthy!individuals.!Children!with!ALL!have!a!different!virome!than!children!with!AML.!!Children!with!ALL!have!more!viral!nucleic! acids! protected! by! capsids! in! bone! marrow! than! children! with! AML! and!most! of! that! difference! is! the! result! of! herpesviruses.! There! is! no! evidence! of! a!difference! in! the! bacterial! communities! of! bone!marrow! specimens! between! ALL!and!AML! subjects.! ! These! results! are! far! from! conclusive! yet! provide! the!window!into! the! metagenome! of! childhood! leukemia.! Clearly,! we! have! generated! more!questions!than!answers!and!detailed!follow!up!is!needed.!Is!the!ssRNA!virus!unique!to! Fallon?! Do! healthy! individuals! around! Fallon! harbor! the! same! virus?! Do! ALL!subjects!express!more!herpes!viruses!than!AMLs!in!a!replication!set?!Which!herpes!virus! specifically?! When! are! ALLs! infected! with! herpes! and! is! the! timing! and!prevalence!of!infection!different!than!healthy!children!and!AMLs?!!! !To!answer!these!questions!additional!metagenomic!studies!should!be!conducted!in!childhood! leukemia! and! healthy! subjects! to! define! the! timing,! prevalence! and!progression! of! infection! from! conception! into! adulthood.! These!mechanisms!may!indeed!be!fundamental!to!not!only!understanding!ALL,!but!also!understanding!both!health!and!disease.!!!!!!!!!!!!!!

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Figure! 1:! BLASTN! Results! of! High! Complexity! Non*Human! Contiguous!Sequences!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

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Figure!2:!Comparison!of!the!Virome!Between!Fallon!and!CCLS!Subjects.!!BLAST!results! from!Trinity! assembled! contiguous! sequences.!Red=!CCLS!Subject!1,!Blue=!CCLS! Subject! 2,! Green! =Fallon! Subject! 1,! Yellow=! Fallon! Subject! 2.!

!!!!!!!!!!!!!!!!!!!!

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Figure!3:!BLASTn!Viral!Metagenome!Comparison!ALL!vs.!AML!BLASTn!(E<Value!Cutoff=!E<10!)!classification!of!viral!capsids!sequenced.!!!

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ALL_HERV_bN

AML_HERV_bN

ALL_Retro_bN

AML_Retro_bN

ALL_DS_DNA_bN

AML_DS_DNA_bN

ALL_SS_DNA_bN

AML_SS_DNA_bN

ALL_DS_RNA_bN

AML_DS_RNA_bN

ALL_SS_RNA_bN

AML_SS_RNA_bN

ALL_Unk_bN

AML_Unk_bN

BLASTN--Viral Metagenome- Bone Marrow: ALL vs. AML

0

10

20

30

40

50

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Figure!4:!tBLASTx!Viral!Metagenome!Comparison!ALL!vs.!AML!tBLASTx!(E<Value!Cutoff=!E<10!)!classification!of!viral!capsids!sequenced.!!!

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ALL_HERV_TbX

AML_HERV_TbX

ALL_Retro_TbX

AML_Retro_TbX

ALL_DS_DNA_TbX

AML_DS_DNA_TbX

ALL_SS_DNA_TbX

AML_SS_DNA_TbX

ALL_DS_RNA_TbX

AML_DS_RNA_TbX

ALL_SS_RNA_TbX

AML_SS_RNA_TbX

ALL_Unk_TbX

AML_Unk_TbX

TBLASTX--Viral Metagenome- Bone Marrow: ALL vs. AML

0

20

40

60

80

100

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Figure!5:!Bacterial!16s!Classification!(Class)!of!Grouped!ALL’s!(73)!and!AML’s!(28)!

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Figure!6:!Bacterial!16s!Classification!(Class)!of!Individual!ALL’s!and!AML’s!For!legend!see!Figure!5!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

ALL! !

AML!

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Figure!7:!Heatmap!of!Bacterial!16s!Classification!(Class)!of!Individual!ALL’s!and!AML’s!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

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Figure!8:!Neighbor!Joining!Tree!of!16s!Defined!Genera!

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!!!!!!!

Chapter!3*!An!Etiologic!Framework!for!Acute!Lymphoblastic!Leukemia!

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3.1!On!the!Trail!!Despite! decades! of! major! advances! in! treatment,! acute! lymphoblastic! leukemia!(ALL)!remains!the!most!common!childhood!cancer.!!Although!clinical!resolution!can!be!obtained,!the!treatment!itself!and!the!long<term!effects!are!substantial!(Hudson!et! al.,! 2013;! Oeffinger! et! al.,! 2006).! Unfortunately! the! underlying! cause! of! ALL!remains! largely! a! mystery.! We! present! here! an! etiologic! framework! for! ALL,! a!combination!of! evidence! and!hypothesis! that!we!hope!will! guide! research! toward!the!understanding!of! the!cause!of!ALL.!Furthermore!we!hope! that! this! framework!will!identify!research!focused!on!etiologic!mechanisms!that!are!viable!intervention!targets!with!the!ultimate!goal!of!preventing!the!disease.!!!Hypothesis:!Here!it!is!hypothesized!that!a!combination!of!pre<natal!and!post<natal!events!lead!to!ALL!(see! figure).! !First,! an!underlying!genetic!predisposition,! combined!with!an, in,utero! infectious! exposure,! cause! chromosomal! anomalies! and! immune!dysregulation.! This! followed! by! postnatal! events,! including! toxic! and! infectious!exposures! that! further!modify! adaptive! immunity! leading! to!a! greater! intensity!of!infections.! !This!feeds!the!loop!of!enhanced!b<cell!production!that!facilitates!virally!induced! self<!mutation! caused! by! foreign! nucleic! acids! invading! pre! b<cells.! ! This!results!in!an!ABOBEC!arbitrated!hypermutation.!Eventually!the!loop!of!enhanced!yet!less!competent!b<cell!production,!RAG!mediated!over!mutation,!increased!infection!leading! to! pre! b<cell! viral! nucleic! acid! loading,! and! APOBEC! caused! self!mutation!remove!the!proliferative!and!apoptotic!cellular!checks.!!The!result!is!childhood!ALL.!!!

!

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!!3.2!It!starts!from!the!beginning:!Pre*natal!Events!Underlying! heritable! polymorphisms! in! IKZF1,, CEBPE, and, ARID5B, have! been!identified!in!childhood!ALL!(Trevino!et!al.,!2009).!Yet!a!low!concordance,!as!well!as!clear! evidence!of! a! single! clonal! origin! in! concordant! cases,! between!monozygotic!twins!nearly!ensures!a!non<heritable!exogenous!trigger!(M.!F.!Greaves!&!Alexander,!1993).!Some!subtypes!of!ALL,!including!the!most!common!subtype!(~24%)!with!an!TEL<AML1!(or!ETV6<RUNX<1)!translocation,!are!initiated!in!utero!as!demonstrated!by! the! presence! of! leukemic!B<cell! chromosomal! lesions! in! new!born! blood! spots!(NBS)!collected!directly!after!birth(Wiemels!et!al.,!1999).!Studies!have!shown!that!these!specific!lesions!at!birth,!are!!~100!times!more!prevalent!than!leukemia!itself,!therefore,!these!chromosomal!lesions!are!likely!a!necessary!but!not!sufficient!cause!of!ALL!(Mori!et!al.,!2002).! !This!observation!laid!the!foundation!for!Greaves!model!(M.!Greaves,!2006a;!M.!F.!Greaves,!1988)!which!proposed!a!secondary!postnatal!‘hit’!triggering!disease!in!susceptible!children.!!!Further,!recent!studies!have!shown!a!deficit!of!the!anti<inflammatory!cytokine!IL<10!in! NBS! at! birth! in! children! that! go! on! to! develop! leukemia! compared! to! healthy!controls! suggesting! that! immune! dysregulation! is! present! at! birth(Chang! et! al.,!2011).!Whether!this!deficit!is!due!to!other!genetic!lesions,!maternal!/fetal!immune!receptor! interactions!(such!as!KIR<HLA)!or!exogenous!exposures!(such!as! in!utero!infection)!is!unknown.!!!

!3.3!Pre!and!Post!Natal!Immune!Development!!This! underlying! damage! to! adaptive! immunity! may! result! in! errors! in! antigen!recognition.! For! instance! the! Tel<AML1! fusion! results! in! slowing! of! b<cell!differentiation.! During! maturation! b<cells! are! detained! in! a! critical! stage,! RAG!mediated!VDJ! recombination,! creating! additional! recombination! events.! During!B<cell! development! an! extraordinary! stochastic! shuffling!of! specific! immunoglobulin!(IGH)! or! T! cell! receptor! (TCR)! loci! occurs,! the! purpose! being! expansion! of! the!5immune! repertoire! by! randomly! creating! new! antigen, receptor! loci! that! could!recognize! novel! immunologic! challenges.! This! targeted! recombination! by! RAG!proteins! is! highly! regulated! because! of! the! inherent! danger! of! a!mutation! driven!process,! yet! the! TEL<AML1! fusion! appears! to! allow! for! enhanced! mutation! It! is!estimated!that!1x1011!B<cells!are!created!every!day,!yet!normally!only!1x109!survive!the!process,!this!may!be!drastically!different!in!TEL<AML.!These!errors!result!in!B<cells! that! would! have! sterile! antigen! recognition! and! fail! to! undergo! clonal!expansion! during! times! of! response! to! infection.! This! would! likely! facilitate!additional! B<cell! production! therefore! producing! a! greater! opportunity! for! RAG!mediated! VDJ! recombination.! An! increase! in! B<cell! production! and! RAG! activity!would!result!in!a!greater!chance!of!deleterious!RAG!mediated!mutations.!!

!!!!

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3.4!Post*natal!Events!Postnatal! events! associated! with! ALL! have! been! identified.! One! example! of! a!possible! postnatal! modifying! event! comes! from! a! recent! paper! suggesting! that!Cesarean!delivery!increases!risk!of!childhood!ALL;!possibly!by!virtue!of!differential!primary!population!of!the!human!microbiome!compared!to!children!born!by!vaginal!delivery.!This!change!may!lead!to!postnatal!endogenous!immune!modification!that!would! increase! susceptibility,! Indeed! it! is! becoming!widely! accepted! that! correct!priming! of! the! immune! system! early! in! life! is! critical! for! proper! immune!development.!!!Toxic! exposure!may! also! facilitate! dysregulation! of! adaptive! immune! response! in!ALL.!This!far<from<novel!concept!is!supported!by!decades!of!research!detailing!the!ability! of! immunotoxic! compounds! to! alter! immune! function! (Corsini! et! al.,! 2011;!Galloway! &! Handy,! 2003;! Gleichmann,! Kimber,! &! Purchase,! 1989;! Holladay! &!Smialowicz,!2000;!Voccia,!Blakley,!Brousseau,!&!Fournier,!1999;!Vojdani,!Ghoneum,!&!Brautbar,! 1992)! through!a! variety!of!mechanisms.!Extreme! toxic! exposures! can!result!in!death!of!immune!cells,!but!even!small!exposures!can!interrupt!immune!cell!signaling! and! cell! recognition! as! well! as! changes! in! cell! proliferation(Corsini,!Sokooti,!Galli,!Moretto,!&!Colosio,!2013).!Surprisingly!few!studies!have!documented!chemically! induced! immune! suppression! that! results! in! a! greater! number! of!infections! (Person!&!Whalen,!2010;!Phillips,!2000).!This! is! likely! the!result!of!non!overlapping! spheres! of! research! and! a! remarkably! wide! divide! between! the!infectious! disease! and! environmental! exposure! academic! communities.!Nevertheless!there!is!clear!evidence!of!the!immunotoxic!effects!of!many!chemicals.!!The!hypothesized!combined!effect!of!both!prenatal!immune!damage!and!exogenous!insults! (early! life! microbial! and! chemical)! to! immune! function! results! in! an!inadequate! immune! response.! ! This! causesfurther! immune! dysregulation! thereby!inducing!more! intense! and! higher! frequency! infections.! This! is! supported! by! two!separate!large!studies!in!the!United!Kingdom!and!Taiwan!have!shown!that!children!who!go!on!to!develop!leukemia!have!had!more!medically!diagnosed!infections!than!children!that!do!not!develop!leukemia!(Chang!et!al.,!2012;!Crouch!et!al.,!2012).!The!nature! of! these! infections! is! broad! with! many! groups! of! infections! having! been!associated.!!!!The! scope! of! the! human! virome! is! poorly! understood(Delwart,! 2013),! yet! we! do!know!that!humans!chronically!harbor!many!common!viruses.!!These!infections!may!play! critical! roles! in! the!development!of!ALL.!We! recently!observed!differences! in!viral! communities! in! children! with! ALL! during! a! comprehensive! series! of! viral!discovery!projects!identifying!viral!sequences!recovered!from!both!particle!isolation!plus! 454! sequencing! and! illumine! based!whole! transcriptome! viral! discovery.!We!found! more! identifiable! viruses! in! children! with! ALL! versus! with! acute! myeloid!leukemia’s! (AML)! (where! AML’s! have! no! suspected! infectious! etiology,! yet! are!immune! suppressed).! Intriguingly,! this! excess! of! infection! seems! to! be! driven! by!double! stranded! DNA! viruses! of! the! Herpes! family.! Additionally,! a! differential!expression! of! human! endogenous! retroviruses! (HERV)! was! also! observed,! where!

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ALL’s!are!producing!more!HERV’s!than!AML.!Intriguingly,!some!herpesviruses!have!been!shown! to!up!regulate!HERV!expression!making!detangling!of! the!herpes!and!HERV! observation! difficult.! Some! of! the! infections! discovered! are! common,! yet!under!times!of!immune!suppression,!can!become!over!active,!potentially!suggesting!reverse!causality.!Furthermore! there!are!many!unknown!sequences!actively!being!transcribed! within! leukemia! samples,! these! so! called! ‘dark! sequences’! ! require!further! study! to! understand.! But! a! single! causal! transforming! virus! has! yet! to! be!ruled!out!in!the!etiology!of!ALL.!!!With! a! normally! functioning! immune! system,! the! effect! of! these! viruses!would!be!clinically! insignificant.! But! in! the! case! of! children! with! compromised! immune!response,!we!hypothesize!that!the!constant!viral!challenge,!and!higher!infection!load!facilitates!up<regulation!of!innate!immune!response!in!an!attempt!to!fill!the!systemic!immunologic!gap!left!by!inadequate!humoral!immune!response.!This!constant!viral!infection!causes!high!viral!titers!that!result!in!acellular!loading!of!viral!nucleic!acids.!!Consequently,! the! presence! of! intracellular! foreign! nucleic! acids! stimulates!persistent!innate!immune!response.!The!signature!of!this!process!is!apolipoprotein!B! mRNA! editing! enzyme,! catalytic! polypeptide<like! (APOBEC)! mediated!hypermutation! (Alexandrov! et! al.,! 2013).! These! are! a! family! of! evolutionary!conserved! proteins! that! under! normal! circumstances! would! provide! some!protection!by!mutating!both!integrating!virus!and!intracellular!viral!assemblies.! In!addition!to!defending!against!foreign!viruses,!a!principal!function!of!APOBEC’s!is!to!not! only! fight! off! foreign! viruses! but! also! to! keep! HERV’s! in! check! (making! the!observance! of! up<regulated! HERVS! in! ALL! especially! interesting).! ! Yet! under!persistent! viral! challenge! and! inadequate! humoral! regulation! APOBEC<induced!somatic!hypermutation!which!likely!causes!collateral!human!genomic!damage!and,!ultimately,!ALL.!!!3.5!Future!Directions:!We! have! presented! a! framework! that! is! a! combination! of! molecular! and!epidemiologic! evidence! combined! with! scientific! conjecture.! This! proposed!mechanism! contains!many! gaps,! yet!we! see! these! gaps! as! opportunities! to! target!research! ! to! further!understand! the! etiologic!mechanisms!of!ALL.!Many!questions!remain:!<Is!a!specific!in,utero!infection!or!chemical!exposure!more!common!in!children!that!go!on!to!develop!leukemia?!<Do!children!who!go!on!to!develop!leukemia,!have! less! functional!B<cells?!Produce!more!B<cells!prior!to!onset!of!leukemia?!<Is!a!specific!virus!with!a!trophism!for!B<cells!present!in!all!ALL’s?!<Or!is!there!a!suite!of!viruses!with!a!tropism!for!B<cells!present!in!ALL’s?!<Do!these!viruses!infect!the!child!prior!to!the!initiation!of!disease?!!<Are!APOBEC!protiens!up!regulated!in!children!prior!to!developing!ALL?!<Are!other!self!mutation!pathways!involved!in!the!etiology!of!ALL?!<Are! germline! abnormalities! resulting! is! altered! immune! function! responsible! for!some!risk!of!ALL?!!!<Does!this!framework!apply!to!other!cancers?!

! 34!

!I!look!forward!to!answering!these!questions.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

! 35!

References!!

Aagaard,!K.,!Petrosino,!J.,!Keitel,!W.,!Watson,!M.,!Katancik,!J.,!Garcia,!N.,!.!.!.!Versalovic,!J.!(2013).!The!Human!Microbiome!Project!strategy!for!comprehensive!sampling!of!the!human!microbiome!and!why!it!matters.!Faseb,Journal,,27(3),!1012<1022.!doi:!Doi!10.1096/Fj.12<220806!

Ajslev,!T.!A.,!Andersen,!C.!S.,!Gamborg,!M.,!Sorensen,!T.!I.!A.,!&!Jess,!T.!(2011).!Childhood!overweight!after!establishment!of!the!gut!microbiota:!the!role!of!delivery!mode,!pre<pregnancy!weight!and!early!administration!of!antibiotics.!International,Journal,of,Obesity,,35(4),!522<529.!doi:!Doi!10.1038/Ijo.2011.27!

Alexandrov,!L.!B.,!Nik<Zainal,!S.,!Wedge,!D.!C.,!Aparicio,!S.!A.,!Behjati,!S.,!Biankin,!A.!V.,!.!.!.!Stratton,!M.!R.!(2013).!Signatures!of!mutational!processes!in!human!cancer.!Nature,,500(7463),!415<421.!doi:!10.1038/nature12477!

Altschul,!S.!F.,!Madden,!T.!L.,!Schaffer,!A.!A.,!Zhang,!J.,!Zhang,!Z.,!Miller,!W.,!&!Lipman,!D.!J.!(1997).!Gapped!BLAST!and!PSI<BLAST:!a!new!generation!of!protein!database!search!programs.!Nucleic,Acids,Res,,25(17),!3389<3402.!!

Bender,!A.!P.,!Robison,!L.!L.,!Kashmiri,!S.!V.!S.,!McClain,!K.!L.,!Woods,!W.!G.,!Smithson,!W.!A.,!.!.!.!Gibson,!R.!(1988).!No!Involvement!of!Bovine!Leukemia!Virus!in!Childhood!Acute!Lymphoblastic!Leukemia!and!Non<Hodgkin's!Lymphoma.!Cancer,Research,,48(10),!2919<2922.!!

Bogdanos,!D.!P.,!Smyk,!D.,!&!Shoenfeld,!Y.!(2013).!The!Microbiome!of!the!Lung.!Israel,Medical,Association,Journal,,15(12),!766<767.!!

Brown,!A.!C.!(2014).!Ulcerative!colitis,!Crohn's!disease!and!irritable!bowel!syndrome!patients!need!fecal!transplant!research!and!treatment.!J,Crohns,Colitis,,8(2),!179.!doi:!10.1016/j.crohns.2013.09.011!

Caporaso,!J.!G.,!Kuczynski,!J.,!Stombaugh,!J.,!Bittinger,!K.,!Bushman,!F.!D.,!Costello,!E.!K.,!.!.!.!Knight,!R.!(2010).!QIIME!allows!analysis!of!high<throughput!community!sequencing!data.!Nature,Methods,,7(5),!335<336.!doi:!Doi!10.1038/Nmeth.F.303!

Caporaso,!J.!G.,!Lauber,!C.!L.,!Walters,!W.!A.,!Berg<Lyons,!D.,!Lozupone,!C.!A.,!Turnbaugh,!P.!J.,!.!.!.!Knight,!R.!(2011).!Global!patterns!of!16S!rRNA!diversity!at!a!depth!of!millions!of!sequences!per!sample.!Proc,Natl,Acad,Sci,U,S,A,,108,Suppl,1,!4516<4522.!doi:!10.1073/pnas.1000080107!

Chang,!J.!S.,!Tsai,!C.!R.,!Tsai,!Y.!W.,!&!Wiemels,!J.!L.!(2012).!Medically!diagnosed!infections!and!risk!of!childhood!leukaemia:!a!population<based!case<control!study.!Int,J,Epidemiol,,41(4),!1050<1059.!doi:!dys113![pii]!

10.1093/ije/dys113!Chang,!J.!S.,!Zhou,!M.,!Buffler,!P.!A.,!Chokkalingam,!A.!P.,!Metayer,!C.,!&!Wiemels,!J.!L.!

(2011).!Profound!deficit!of!IL10!at!birth!in!children!who!develop!childhood!acute!lymphoblastic!leukemia.!Cancer,Epidemiol,Biomarkers,Prev,,20(8),!1736<1740.!doi:!10.1158/1055<9965.EPI<11<0162!

Chen,!Y.!E.,!&!Tsao,!H.!(2013).!The!skin!microbiome:!Current!perspectives!and!future!challenges.!Journal,of,the,American,Academy,of,Dermatology,,69(1),!143<+.!doi:!Doi!10.1016/J.Jaad.2013.01.016!

! 36!

Christakis,!N.!A.,!&!Fowler,!J.!H.!(2007).!The!Spread!of!Obesity!in!a!Large!Social!Network!over!32!Years.!New,England,Journal,of,Medicine,,357(4),!370<379.!doi:!doi:10.1056/NEJMsa066082!

Clayton,!T.!A.,!Baker,!D.,!Lindon,!J.!C.,!Everett,!J.!R.,!&!Nicholson,!J.!K.!(2009).!Pharmacometabonomic!identification!of!a!significant!host<microbiome!metabolic!interaction!affecting!human!drug!metabolism.!Proc,Natl,Acad,Sci,U,S,A,,106(34),!14728<14733.!doi:!Doi!10.1073/Pnas.0904489106!

Clemente,!J.!C.,!Ursell,!L.!K.,!Parfrey,!L.!W.,!&!Knight,!R.!(2012).!The!Impact!of!the!Gut!Microbiota!on!Human!Health:!An!Integrative!View.!Cell,,148(6),!1258<1270.!doi:!Doi!10.1016/J.Cell.2012.01.035!

Cobbs,!C.!S.,!Harkins,!L.,!Samanta,!M.,!Gillespie,!G.!Y.,!Bharara,!S.,!King,!P.!H.,!.!.!.!Britt,!W.!J.!(2002).!Human!cytomegalovirus!infection!and!expression!in!human!malignant!glioma.!Cancer,Res,,62(12),!3347<3350.!!

Corsini,!E.,!Oukka,!M.,!Pieters,!R.,!Kerkvliet,!N.!I.,!Ponce,!R.,!&!Germolec,!D.!R.!(2011).!Alterations!in!regulatory!T<cells:!rediscovered!pathways!in!immunotoxicology.!J,Immunotoxicol,,8(4),!251<257.!doi:!10.3109/1547691X.2011.598885!

Corsini,!E.,!Sokooti,!M.,!Galli,!C.!L.,!Moretto,!A.,!&!Colosio,!C.!(2013).!Pesticide!induced!immunotoxicity!in!humans:!A!comprehensive!review!of!the!existing!evidence.!Toxicology,,307,!123<135.!doi:!Doi!10.1016/J.Tox.2012.10.009!

Crouch,!S.,!Lightfoot,!T.,!Simpson,!J.,!Smith,!A.,!Ansell,!P.,!&!Roman,!E.!(2012).!Infectious!illness!in!children!subsequently!diagnosed!with!acute!lymphoblastic!leukemia:!modeling!the!trends!from!birth!to!diagnosis.!Am,J,Epidemiol,,176(5),!402<408.!doi:!10.1093/aje/kws180!

Davidson,!I.,!&!Shulman,!L.!M.!(2008).!Unraveling!the!puzzle!of!human!anellovirus!infections!by!comparison!with!avian!infections!with!the!chicken!anemia!virus.!Virus,Research,,137(1),!1<15.!doi:!Doi!10.1016/J.Virusres.2008.06.014!

Davison,!A.!J.!(2007).!Comparative!analysis!of!the!genomes.!In!A.!Arvin,!G.!Campadelli<Fiume,!E.!Mocarski,!P.!S.!Moore,!B.!Roizman,!R.!Whitley!&!K.!Yamanishi!(Eds.),!Human,Herpesviruses:,Biology,,Therapy,,and,Immunoprophylaxis.!Cambridge.!

Delwart,!E.!(2013).!A!roadmap!to!the!human!virome.!Plos,Pathogens,,9(2),!e1003146.!doi:!10.1371/journal.ppat.1003146!

DeSantis,!T.!Z.,!Hugenholtz,!P.,!Larsen,!N.,!Rojas,!M.,!Brodie,!E.!L.,!Keller,!K.,!.!.!.!Andersen,!G.!L.!(2006).!Greengenes,!a!chimera<checked!16S!rRNA!gene!database!and!workbench!compatible!with!ARB.!Applied,and,Environmental,Microbiology,,72(7),!5069<5072.!doi:!Doi!10.1128/Aem.03006<05!

Devaraj,!S.,!Hemarajata,!P.,!&!Versalovic,!J.!(2013).!The!Human!Gut!Microbiome!and!Body!Metabolism:!Implications!for!Obesity!and!Diabetes.!Clinical,Chemistry,,59(4),!617<628.!doi:!Doi!10.1373/Clinchem.2012.187617!

Francis,!S.!S.,!Selvin,!S.,!Yang,!W.,!Buffler,!P.!A.,!&!Wiemels,!J.!L.!(2012).!Unusual!space<time!patterning!of!the!Fallon,!Nevada!leukemia!cluster:!Evidence!of!an!infectious!etiology.!Chem,Biol,Interact,,196(3),!102<109.!doi:!10.1016/j.cbi.2011.02.019!

Galloway,!T.,!&!Handy,!R.!(2003).!Immunotoxicity!of!organophosphorous!pesticides.!Ecotoxicology,,12(1<4),!345<363.!!

! 37!

Gleichmann,!E.,!Kimber,!I.,!&!Purchase,!I.!F.!(1989).!Immunotoxicology:!suppressive!and!stimulatory!effects!of!drugs!and!environmental!chemicals!on!the!immune!system.!A!discussion.!Arch,Toxicol,,63(4),!257<273.!!

Goodman,!M.,!Naiman,!J.!S.,!Goodman,!D.,!&!LaKind,!J.!S.!(2012).!Cancer!clusters!in!the!USA:!what!do!the!last!twenty!years!of!state!and!federal!investigations!tell!us?!Crit,Rev,Toxicol,,42(6),!474<490.!doi:!10.3109/10408444.2012.675315!

Greaves,!M.!(2006a).!The!causation!of!childhood!leukemia:!a!paradox!of!progress?!Discov,Med,,6(31),!24<28.!!

Greaves,!M.!(2006b).!Infection,!immune!responses!and!the!aetiology!of!childhood!leukaemia.!Nat,Rev,Cancer,,6(3),!193<203.!doi:!nrc1816![pii]!

10.1038/nrc1816!Greaves,!M.!F.!(1988).!Speculations!on!the!cause!of!childhood!acute!lymphoblastic!

leukemia.!Leukemia,,2(2),!120<125.!!Greaves,!M.!F.,!&!Alexander,!F.!E.!(1993).!An!infectious!etiology!for!common!acute!

lymphoblastic!leukemia!in!childhood?!Leukemia,,7(3),!349<360.!!Haas,!B.!J.,!Papanicolaou,!A.,!Yassour,!M.,!Grabherr,!M.,!Blood,!P.!D.,!Bowden,!J.,!.!.!.!

Regev,!A.!(2013).!De!novo!transcript!sequence!reconstruction!from!RNA<seq!using!the!Trinity!platform!for!reference!generation!and!analysis.!Nat,Protoc,,8(8),!1494<1512.!doi:!10.1038/nprot.2013.084!

Heegaard,!E.!D.,!Jensen,!L.,!Hornsleth,!A.,!&!Schmiegelow,!K.!(1999).!The!role!of!parvovirus!B19!infection!in!childhood!acute!lymphoblastic!leukemia.!Pediatr,Hematol,Oncol,,16(4),!329<334.!doi:!10.1080/088800199277155!

66K0QAWA3QH64P0D![pii]!Heegaard,!E.!D.,!&!Schmiegelow,!K.!(2002).!Serologic!study!on!parvovirus!b19!

infection!in!childhood!acute!lymphoblastic!leukemia!during!chemotherapy:!clinical!and!hematologic!implications.!J,Pediatr,Hematol,Oncol,,24(5),!368<373.!!

Hermouet,!S.,!Sutton,!C.!A.,!Rose,!T.!M.,!Greenblatt,!R.!J.,!Corre,!I.,!Garand,!R.,!.!.!.!Casey,!J.!W.!(2003).!Qualitative!and!quantitative!analysis!of!human!herpesviruses!in!chronic!and!acute!B!cell!lymphocytic!leukemia!and!in!multiple!myeloma.!Leukemia,,17(1),!185<195.!doi:!10.1038/sj.leu.2402748!

Holladay,!S.!D.,!&!Smialowicz,!R.!J.!(2000).!Development!of!the!murine!and!human!immune!system:!differential!effects!of!immunotoxicants!depend!on!time!of!exposure.!Environ,Health,Perspect,,108,Suppl,3,!463<473.!!

Honda,!K.,!&!Littman,!D.!R.!(2012).!The!microbiome!in!infectious!disease!and!inflammation.!Annu,Rev,Immunol,,30,!759<795.!doi:!10.1146/annurev<immunol<020711<074937!

Hudson,!M.!M.,!Ness,!K.!K.,!Gurney,!J.!G.,!Mulrooney,!D.!A.,!Chemaitilly,!W.,!Krull,!K.!R.,!.!.!.!Robison,!L.!L.!(2013).!Clinical!ascertainment!of!health!outcomes!among!adults!treated!for!childhood!cancer.!JAMA,,309(22),!2371<2381.!doi:!10.1001/jama.2013.6296!

Hugot,!J.!P.,!Chamaillard,!M.,!Zouali,!H.,!Lesage,!S.,!Cezard,!J.!P.,!Belaiche,!J.,!.!.!.!Thomas,!G.!(2001).!Association!of!NOD2!leucine<rich!repeat!variants!with!susceptibility!to!Crohn's!disease.!Nature,,411(6837),!599<603.!doi:!Doi!10.1038/35079107!

! 38!

Huson,!D.!H.,!Auch,!A.!F.,!Qi,!J.,!&!Schuster,!S.!C.!(2007).!MEGAN!analysis!of!metagenomic!data.!Genome,Res,,17(3),!377<386.!doi:!10.1101/gr.5969107!

Isa,!A.,!Priftakis,!P.,!Broliden,!K.,!&!Gustafsson,!B.!(2004).!Human!parvovirus!B19!DNA!is!not!detected!in!Guthrie!cards!from!children!who!have!developed!acute!lymphoblastic!leukemia.!Pediatr,Blood,Cancer,,42(4),!357<360.!doi:!10.1002/pbc.20001!

Jones,!M.!L.,!Martoni,!C.!J.,!Ganopolsky,!J.!G.,!Labbe,!A.,!&!Prakash,!S.!(2014).!The!human!microbiome!and!bile!acid!metabolism:!dysbiosis,!dysmetabolism,!disease!and!intervention.!Expert,Opinion,on,Biological,Therapy,,14(4),!467<482.!doi:!Doi!10.1517/14712598.2014.880420!

Keeling,!M.!J.,!&!Eames,!K.!T.!(2005).!Networks!and!epidemic!models.!J,R,Soc,Interface,,2(4),!295<307.!doi:!L2L04UAFY5F3HYFF![pii]!

10.1098/rsif.2005.0051!Kinlen,!L.!J.!(1998).!Infection!and!childhood!leukemia.!Cancer,Causes,Control,,9(3),!

237<239.!!Koonin,!E.!V.,!&!Martin,!W.!(2005).!On!the!origin!of!genomes!and!cells!within!

inorganic!compartments.!Trends,Genet,,21(12),!647<654.!doi:!10.1016/j.tig.2005.09.006!

Langmead,!B.,!&!Salzberg,!S.!L.!(2012).!Fast!gapped<read!alignment!with!Bowtie!2.!Nature,Methods,,9(4),!357<359.!doi:!10.1038/nmeth.1923!

Lehtinen,!M.,!Koskela,!P.,!Ogmundsdottir,!H.!M.,!Bloigu,!A.,!Dillner,!J.,!Gudnadottir,!M.,!.!.!.!Lehtinen,!T.!(2003).!Maternal!herpesvirus!infections!and!risk!of!acute!lymphoblastic!leukemia!in!the!offspring.!Am,J,Epidemiol,,158(3),!207<213.!!

Lindberg,!A.!L.!(2003).!Bovine!viral!diarrhoea!virus!infections!and!its!control.!A!review.!Vet,Q,,25(1),!1<16.!doi:!10.1080/01652176.2003.9695140!

Littman,!D.!R.,!&!Pamer,!E.!G.!(2011).!Role!of!the!Commensal!Microbiota!in!Normal!and!Pathogenic!Host!Immune!Responses.!Cell,Host,&,Microbe,,10(4),!311<323.!doi:!Doi!10.1016/J.Chom.2011.10.004!

Ma,!B.,!Forney,!L.!J.,!&!Ravel,!J.!(2012).!Vaginal!Microbiome:!Rethinking!Health!and!Disease.!Annual,Review,of,Microbiology,,Vol,66,,66,!371<389.!doi:!Doi!10.1146/Annurev<Micro<092611<150157!

Ma,!X.,!Buffler,!P.!A.,!Layefsky,!M.,!Does,!M.!B.,!&!Reynolds,!P.!(2004).!Control!selection!strategies!in!case<control!studies!of!childhood!diseases.!Am,J,Epidemiol,,159(10),!915<921.!!

MacKenzie,!J.,!Gallagher,!A.,!Clayton,!R.!A.,!Perry,!J.,!Eden,!O.!B.,!Ford,!A.!M.,!.!.!.!Jarrett,!R.!F.!(2001).!Screening!for!herpesvirus!genomes!in!common!acute!lymphoblastic!leukemia.!Leukemia,,15(3),!415<421.!!

MacKenzie,!J.,!Greaves,!M.!F.,!Eden,!T.!O.,!Clayton,!R.!A.,!Perry,!J.,!Wilson,!K.!S.,!&!Jarrett,!R.!F.!(2006).!The!putative!role!of!transforming!viruses!in!childhood!acute!lymphoblastic!leukemia.!Haematologica,,91(2),!240<243.!!

MacKenzie,!J.,!Perry,!J.,!Ford,!A.!M.,!Jarrett,!R.!F.,!&!Greaves,!M.!(1999).!JC!and!BK!virus!sequences!are!not!detectable!in!leukaemic!samples!from!children!with!common!acute!lymphoblastic!leukaemia.!Br,J,Cancer,,81(5),!898<899.!doi:!10.1038/sj.bjc.6690783!

Maia,!A.!T.,!van!der!Velden,!V.!H.,!Harrison,!C.!J.,!Szczepanski,!T.,!Williams,!M.!D.,!Griffiths,!M.!J.,!.!.!.!Greaves,!M.!F.!(2003).!Prenatal!origin!of!hyperdiploid!acute!

! 39!

lymphoblastic!leukemia!in!identical!twins.!Leukemia,,17(11),!2202<2206.!doi:!10.1038/sj.leu.2403101!

Mori,!H.,!Colman,!S.!M.,!Xiao,!Z.,!Ford,!A.!M.,!Healy,!L.!E.,!Donaldson,!C.,!.!.!.!Greaves,!M.!(2002).!Chromosome!translocations!and!covert!leukemic!clones!are!generated!during!normal!fetal!development.!Proc,Natl,Acad,Sci,U,S,A,,99(12),!8242<8247.!doi:!10.1073/pnas.112218799!

Nicholas,!J.!(2000).!Evolutionary!aspects!of!oncogenic!herpesviruses.!Mol,Pathol,,53(5),!222<237.!!

Nicoll,!M.!P.,!Proenca,!J.!T.,!&!Efstathiou,!S.!(2012).!The!molecular!basis!of!herpes!simplex!virus!latency.!FEMS,Microbiol,Rev,,36(3),!684<705.!doi:!10.1111/j.1574<6976.2011.00320.x!

Nieto,!F.!J.,!Adam,!E.,!Sorlie,!P.,!Farzadegan,!H.,!Melnick,!J.!L.,!Comstock,!G.!W.,!&!Szklo,!M.!(1996).!Cohort!study!of!cytomegalovirus!infection!as!a!risk!factor!for!carotid!intimal<medial!thickening,!a!measure!of!subclinical!atherosclerosis.!Circulation,,94(5),!922<927.!!

O'Leary,!M.,!Krailo,!M.,!Anderson,!J.!R.,!Reaman,!G.!H.,!&!Children's!Oncology,!G.!(2008).!Progress!in!childhood!cancer:!50!years!of!research!collaboration,!a!report!from!the!Children's!Oncology!Group.!Semin,Oncol,,35(5),!484<493.!doi:!10.1053/j.seminoncol.2008.07.008!

Oeffinger,!K.!C.,!Mertens,!A.!C.,!Sklar,!C.!A.,!Kawashima,!T.,!Hudson,!M.!M.,!Meadows,!A.!T.,!.!.!.!Childhood!Cancer!Survivor,!S.!(2006).!Chronic!health!conditions!in!adult!survivors!of!childhood!cancer.!N,Engl,J,Med,,355(15),!1572<1582.!doi:!10.1056/NEJMsa060185!

Ogura,!Y.,!Bonen,!D.!K.,!Inohara,!N.,!Nicolae,!D.!L.,!Chen,!F.!F.,!Ramos,!R.,!.!.!.!Cho,!J.!H.!(2001).!A!frameshift!mutation!in!NOD2!associated!with!susceptibility!to!Crohn's!disease.!Nature,,411(6837),!603<606.!doi:!Doi!10.1038/35079114!

Penders,!J.,!Stobberingh,!E.!E.,!Savelkoul,!P.!H.!M.,!&!Wolffs,!F.!G.!(2013).!The!human!microbiome!as!a!reservoir!of!antimicrobial!resistance.!Frontiers,in,Microbiology,,4.!doi:!Artn!87!

Doi!10.3389/Fmicb.2013.00087!Person,!R.!J.,!&!Whalen,!M.!M.!(2010).!Effects!of!butyltin!exposures!on!MAP!kinase<

dependent!transcription!regulators!in!human!natural!killer!cells.!Toxicol,Mech,Methods,,20(5),!227<233.!doi:!10.3109/15376511003746034!

Phillips,!T.!M.!(2000).!Assessing!environmental!exposure!in!children:!immunotoxicology!screening.!J,Expo,Anal,Environ,Epidemiol,,10(6!Pt!2),!769<775.!!

Ridaura,!V.!K.,!Faith,!J.!J.,!Rey,!F.!E.,!Cheng,!J.,!Duncan,!A.!E.,!Kau,!A.!L.,!.!.!.!Gordon,!J.!I.!(2013).!Gut!microbiota!from!twins!discordant!for!obesity!modulate!metabolism!in!mice.!Science,,341(6150),!1241214.!doi:!10.1126/science.1241214!

Riley,!L.!W.,!Cohen,!M.!L.,!Seals,!J.!E.,!Blaser,!M.!J.,!Birkness,!K.!A.,!Hargrett,!N.!T.,!.!.!.!Feldman,!R.!A.!(1984).!Importance!of!host!factors!in!human!salmonellosis!caused!by!multiresistant!strains!of!Salmonella.!J,Infect,Dis,,149(6),!878<883.!!

Suttle,!C.!A.!(2005).!Viruses!in!the!sea.!Nature,,437(7057),!356<361.!doi:!10.1038/nature04160!

! 40!

TeKippe,!E.!M.,!Wylie,!K.!M.,!Deych,!E.,!Sodergren,!E.,!Weinstock,!G.,!&!Storch,!G.!A.!(2012).!Increased!Prevalence!of!Anellovirus!in!Pediatric!Patients!with!Fever.!Plos,One,,7(11).!doi:!ARTN!e50937!

DOI!10.1371/journal.pone.0050937!Trevino,!L.!R.,!Yang,!W.,!French,!D.,!Hunger,!S.!P.,!Carroll,!W.!L.,!Devidas,!M.,!.!.!.!

Relling,!M.!V.!(2009).!Germline!genomic!variants!associated!with!childhood!acute!lymphoblastic!leukemia.!Nat,Genet,,41(9),!1001<1005.!doi:!10.1038/ng.432!

Turnbaugh,!P.!J.,!Ley,!R.!E.,!Mahowald,!M.!A.,!Magrini,!V.,!Mardis,!E.!R.,!&!Gordon,!J.!I.!(2006).!An!obesity<associated!gut!microbiome!with!increased!capacity!for!energy!harvest.!Nature,,444(7122),!1027<1031.!doi:!Doi!10.1038/Nature05414!

Turnbaugh,!P.!J.,!Ridaura,!V.!K.,!Faith,!J.!J.,!Rey,!F.!E.,!Knight,!R.,!&!Gordon,!J.!I.!(2009).!The!Effect!of!Diet!on!the!Human!Gut!Microbiome:!A!Metagenomic!Analysis!in!Humanized!Gnotobiotic!Mice.!Science,Translational,Medicine,,1(6).!doi:!ARTN!6ra14!

DOI!10.1126/scitranslmed.3000322!Varin,!T.,!Lovejoy,!C.,!Jungblut,!A.!D.,!Vincent,!W.!F.,!&!Corbeil,!J.!(2012).!Metagenomic!

analysis!of!stress!genes!in!microbial!mat!communities!from!Antarctica!and!the!High!Arctic.!Appl,Environ,Microbiol,,78(2),!549<559.!doi:!10.1128/AEM.06354<11!

Victoria,!J.!G.,!Kapoor,!A.,!Dupuis,!K.,!Schnurr,!D.!P.,!&!Delwart,!E.!L.!(2008).!Rapid!identification!of!known!and!new!RNA!viruses!from!animal!tissues.!Plos,Pathogens,,4(9).!doi:!Artn!E1000163!

Doi!10.1371/Journal.Ppat.1000163!Voccia,!I.,!Blakley,!B.,!Brousseau,!P.,!&!Fournier,!M.!(1999).!Immunotoxicity!of!

pesticides:!a!review.!Toxicol,Ind,Health,,15(1<2),!119<132.!!Vojdani,!A.,!Ghoneum,!M.,!&!Brautbar,!N.!(1992).!Immune!alteration!associated!with!

exposure!to!toxic!chemicals.!Toxicol,Ind,Health,,8(5),!239<254.!!Vossen,!M.!T.,!Westerhout,!E.!M.,!Soderberg<Naucler,!C.,!&!Wiertz,!E.!J.!(2002).!Viral!

immune!evasion:!a!masterpiece!of!evolution.!Immunogenetics,,54(8),!527<542.!doi:!10.1007/s00251<002<0493<1!

Wiemels,!J.!L.,!Cazzaniga,!G.,!Daniotti,!M.,!Eden,!O.!B.,!Addison,!G.!M.,!Masera,!G.,!.!.!.!Greaves,!M.!F.!(1999).!Prenatal!origin!of!acute!lymphoblastic!leukaemia!in!children.!Lancet,,354(9189),!1499<1503.!!

Wiemels,!J.!L.,!Leonard,!B.!C.,!Wang,!Y.,!Segal,!M.!R.,!Hunger,!S.!P.,!Smith,!M.!T.,!.!.!.!Pine,!S.!R.!(2002).!Site<specific!translocation!and!evidence!of!postnatal!origin!of!the!t(1;19)!E2A<PBX1!fusion!in!childhood!acute!lymphoblastic!leukemia.!Proc,Natl,Acad,Sci,U,S,A,,99(23),!15101<15106.!doi:!10.1073/pnas.222481199!

Yoshida,!M.,!Takaki,!Y.,!Eitoku,!M.,!Nunoura,!T.,!&!Takai,!K.!(2013).!Metagenomic!analysis!of!viral!communities!in!(hado)pelagic!sediments.!PLoS,One,,8(2),!e57271.!doi:!10.1371/journal.pone.0057271!

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