IGF2BP3 Associates with Proliferative Phenotype and Prognostic Features in B-Cell Acute Lymphoblastic Leukemia

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ IGF2BP3 Associa es wi h P oli e a i e Pheno ype and P ognos ic Fea u es in B-Cell Acu e Lymphoblas ic Leukemia © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. Published e sion Mäkinen, A u i; Nikkilä, A e; Haapaniemi, Teppo; Oksa, Lau a; Meh onen, Juha; Vänskä, Ma i; Heinäniemi, Me ja; Paa onen, Timo; Lohi, Olli Mäkinen, A., Nikkilä, A., Haapaniemi, T., Oksa, L., Meh onen, J., Vänskä, M., Heinäniemi, M., Paa onen, T., & Lohi, O. (2021). IGF2BP3 Associa es wi h P oli e a i e Pheno ype and P ognos ic Fea u es in B-Cell Acu e Lymphoblas ic Leukemia. Cance s, 13(7), A icle 1505. h ps://doi.o g/10.3390/cance s13071505 2021 cance s A icle IGF2BP3 Associa es wi h P oli e a i e Pheno ype and P ognos ic Fea u es in B-Cell Acu e Lymphoblas ic Leukemia A u i Mäkinen 1,2,* , A e Nikkilä 1, Teppo Haapaniemi 2,3, Lau a Oksa 1, Juha Meh onen 4, Ma i Vänskä 5, Me ja Heinäniemi 4, Timo Paa onen 2,6 and Olli Lohi 1,7   Ci a ion: Mäkinen, A.; Nikkilä, A.; Haapaniemi, T.; Oksa, L.; Meh onen, J.; Vänskä, M.; Heinäniemi, M.; Paa onen, T.; Lohi, O. IGF2BP3 Associa es wi h P oli e a i e Pheno ype and P ognos ic Fea u es in B-Cell Acu e Lymphoblas ic Leukemia. Cance s 2021,13, 1505. h ps://doi.o g/10.3390/ cance s13071505 Academic Edi o : Ajay P a ap Singh Recei ed: 13 Feb ua y 2021 Accep ed: 22 Ma ch 2021 Published: 25 Ma ch 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1Tampe e Cen e o Child, Adolescen and Ma e nal Heal h Resea ch, Facul y o Medicine and Heal h Technology, Tampe e Uni e si y, 33520 Tampe e, Finland; [email p o ec ed] (A.N.); [email p o ec ed] (L.O.); [email p o ec ed] (O.L.) 2Fimlab Labo a o ies, Depa men o Pa hology, Tampe e Uni e si y Hospi al, 33520 Tampe e, Finland; [email p o ec ed] (T.H.); [email p o ec ed] (T.P.) 3Depa men o Biological and En i onmen al Sciences, Uni e si y o Jy äskylä, 40014 Jy äskylä, Finland 4Ins i u e o Biomedicine, School o Medicine, Uni e si y o Eas e n Finland, 70211 Kuopio, Finland; [email p o ec ed] (J.M.); [email p o ec ed] (M.H.) 5Depa men o In e nal Medicine, Tampe e Uni e si y Hospi al, 33520 Tampe e, Finland; [email p o ec ed] 6Depa men o Pa hology, Facul y o Medicine and Heal h Technology, Tampe e Uni e si y, 33520 Tampe e, Finland 7Tays Cance Cen e, Tampe e Uni e si y Hospi al, 33520 Tampe e, Finland *Co espondence: [email p o ec ed] Simple Summa y: Al hough he p ognosis o acu e lymphoblas ic leukemia (ALL) has imp o ed signi ican ly du ing he pas decades, ALL emains a majo cause o pedia ic cance mo ali y, and mo e accu a e isk-s a i ica ion is equi ed. We in es iga ed IGF2BP3, which has p e iously been associa ed wi h agg essi e cance s, and ound high and sub ype-speci ic exp ession o IGF2BP3 in B-cell ALL, ha was associa ed wi h good ou come in high- isk pa ien s. Resul s sugges ha IGF2BP3 could be use ul o imp o e s a i ica ion and p ognosis o B-ALL. Abs ac : The onco e al p o ein insulin-like g ow h ac o 2 mRNA-binding p o ein 3 (IGF2BP3) belongs o a amily o RNA-binding p o eins in ol ed in localiza ion, s abili y, and ansla ional egula ion o a ge RNAs. IGF2BP3 is used as a diagnos ic and p ognos ic ma ke in se e al malignancies. Al hough he p ognosis o pedia ic B-cell acu e lymphoblas ic leukemia (B-ALL) has imp o ed, a subg oup o pa ien s exhibi s high- isk ea u es and su e om disease ecu ence. We sough o iden i y addi ional bioma ke s o imp o e diagnos ics, and we assessed exp ession o IGF2BP3 in a popula ion-based pedia ic coho o B-ALL using a issue mic oa ay pla o m. The majo i y o pedia ic B-ALL cases we e posi i e o IGF2BP3 immunohis ochemis y and we e associa ed wi h an inc eased p oli e a i e pheno ype and ac i a ed STAT5 signaling pa hway. Two la ge gene exp ession da a se s we e p obed o he exp ession o IGF2BP3— he highes le els we e seen among he B-cell lymphomas o a ge minal cen e o igin and well-es ablished (KMT2A- ea anged and ETV6-RUNX1) and no el sub ypes o B-ALL (e.g., NUTM1 and ETV6-RUNX1-like). A high mRNA o IGF2BP3 was associa ed wi h a p oli e a i e “me agene” signa u e and a high exp ession o CDK6 in B-ALL. A low exp ession po ended in e io su i al in a high- isk coho o pedia ic B-ALL. O e all, ou esul s show ha IGF2BP3 shows sub ype-speci ici y in exp ession and p o ides p ognos ic u ili y in high- isk B-ALL. Keywo ds: insulin-like g ow h ac o 2 mRNA-binding p o ein 3 (IGF2BP3); mRNA; pedia ic B-cell acu e lymphoblas ic leukemia; p ognosis; p oli e a ion; p o ein Cance s 2021,13, 1505. h ps://doi.o g/10.3390/cance s13071505 h ps://www.mdpi.com/jou nal/cance s Cance s 2021,13, 1505 2 o 12 1. In oduc ion Pedia ic B-cell acu e lymphoblas ic leukemia (B-ALL) is he mos common malignancy in childhood. Despi e he signi ican ly imp o ed p ognosis, a subg oup o pa ien s wi h ei he a poo he apy esponse o high- isk ea u es s ill o en expe ience a elapse. Be e diagnos ic ools a e needed o enhance ea men s a i ica ion and p ognosis, and o a oid o e ea men and ad e se long- e m side-e ec s [1–3]. Insulin-like g ow h ac o II mRNA-binding p o ein 3 (IGF2BP3), also known as he IGF2BP3 p o ein, is a 69 kDa p o ein ha localizes mos ly o he cy oplasm [ 4 , 5 ]. This onco e al RNA-binding p o ein is a membe o he IGF2BP- amily, which also includes IGF2BP1 and IGF2BP2 p o eins, and sha es 59–73% simila i y wi h he amino acid sequence wi h IGF2BP3 [ 6 , 7 ]. IGF2BP3 binds RNA molecules and ac s as a egula o o mRNA localiza ion and s abili y [ 7 , 8 ]. I is exp essed only a a low le el in mos adul issues, whe eas in mul iple human malignancies, i is o e exp essed [7,8]. Mu a ions o IGF2BP3 a e a e, bu he exp ession is dys egula ed a epigene ic, an- sc ip ional, and pos - ansc ip ional le els. A a cellula le el, IGF2BP3 d i es miRNA bio- genesis; in e cep s he cy oplasmic expo o mRNA; and egula es mRNA s abili y, deg a- da ion, and anspo a ion [ 7 ]. In gas oin es inal and u ogeni al malignancies, IGF2BP3 is highly exp essed, and is associa ed wi h cell adhesion, umo in asion, me as asis, and in e io ou comes [ 7 – 9 ]. IGF2BP3 exhibi s a s ong exp ession in lymphoid malig- nancies such as B cell lymphomas o a ge minal cen e o igin [ 10 , 11 ]. I is exp essed in Reed–S e nbe g cells and can be used as a supplemen a y diagnos ic ma ke in Hodgkin’s lymphoma [12–14] . Inc eased exp ession o IGF2BP3 is associa ed wi h p oli e a i e ea- u es in many solid umo s, man le cell lymphoma, and ch onic myeloid leukemia blas c isis [7,15,16], and p omo es cell su i al du ing ionizing adia ion in B-cells [17]. S oskus e al. [ 18 ] explo ed he exp ession o IGF2BP amily membe s in hema opoie ic issues and ALL by using iso o m-speci ic RT-qPCR. In heal hy s em o ma u e hema opoi- e ic cells, he exp ession o IGF2BP3 was ei he weak o absen in con as o IGF2BP2. The analysis o di e en ma u e cell popula ions demons a ed ha only CD19+ B-cells exp essed de ec able le els o IGF2BP3, in line wi h p e ious li e a u e [ 10 , 18 , 19 ]. Among B-ALL, he s onges exp ession was e iden in ETV6-RUNX1 and KMT2A- ea anged sub ypes. Liao e al. [ 16 ] and Palanichamy e al. [ 20 ] showed ha siRNA o CRISPR-Cas9- media ed he knockdown o IGF2BP3 educed p oli e a ion and inc eased apop osis in se e al cell lines (K562, RS4;11, and NALM6). While a g owing body o da a suppo s biological signi icance and p ognos ic u ili y o IGF2BP3 in di e en epi helial and so issue umo s, o da e, he e a e only wo s udies ha ha e explo ed i s exp ession in lymphoid leukemias [ 18 , 20 ], and no s udies ha ha e assessed exp ession a he p o ein le el. Hence, we in es iga ed he exp ession o IGF2BP3 ac oss hema ological malignancies and in a ephine biopsy sample coho o pedia ic B-ALL and co ela ed i s exp ession wi h cell p oli e a i e ea u es and pa ien su i al. 2. Ma e ials and Me hods 2.1. Pa ien Coho o Tissue Mic oa ay and Immunohis ochemis y The o malin- ixed and pa a in-embedded bone ma ow ephine biopsy samples o he pedia ic B-ALL pa ien s we e collec ed in o a issue mic oa ay (TMA) wi h 1.5 mm punches (see also [ 21 ]), and 4-mic ome e TMA sec ions we e used o immunohis o- chemis y. An appendix was used as a con ol ma e ial o he IGF2BP3 and CD19/Ki-67 immunos ainings. Immunohis ochemis y was pe o med using he Ven ana Benchma k Ul a ins umen . BCL6 and pSTAT5 (Y694) immunohis ochemis y was pe o med on whole issue sec ions using Ven ana Benchma k Classic [ 22 ]. The ollowing an ibodies we e used: IGF2BP3 (lo : 11085707, clone: 69.1, manu ac u e : Dako, San a Cla a, CA, USA, id: M3626, dilu ion: 1:100, species: mouse monoclonal, Ig class: IgG2a, kappa), CD19 (lo : 000085227, clone: EP169, manu ac u e : Cell Ma que, Rocklin, CA, USA, id: 119R-18, dilu ion: eady- o-use, species: abbi monoclonal, Ig class: IgG), Ki-67 (lo : F30644, clone: 30-9, manu ac u e : Ven ana, Tucson, AZ, USA, id: 790-4286, dilu ion: eady- o-use, species: Cance s 2021,13, 1505 3 o 12 abbi monoclonal, Ig class: IgG), BCL6 (lo : 48794, clone: LN22, id: PA0204, species: mouse monoclonal, manu ac u e : Leica Biosys ems, Newcas le, UK, dilu ion: 1:50), and pSTAT5 (Y694) (lo : GR208043, clone: E208, id: ab32364, manu ac u e : Abcam, Camb idge, UK, dilu ion: 1:50). Fo he IGF2BP3 and Ki-67 s ainings, we used he Op iView DAB de ec ion ki ; o he CD19 s ainings, he Ul aView Uni e sal Alkaline Phospha ase Red de ec ion ki ; and o BCL6 and pSTAT5 (Y694), he Ul a iew Uni e sal DAB de ec ion ki . All o he slides we e coun e s ained using hema oxylin. The exp ession o BCL6 and pSTAT5 was semiquan i a i ely g aded as nega i e when an igen was exp essed in unde 20% o leukemic blas s, and posi i e when exp essed in o e 20%. Clinical da a and he low cy- ome y da a (e.g., CD34 exp ession) we e e ie ed om pa ien hospi al eco ds ga he ed as desc ibed p e iously [ 21 ]. The low cy ome y esul s we e g aded as ei he nega i e o posi i e. 2.2. Image Analysis Slides we e scanned wi h Hamama su Nanozoome XR using 40 × magni ica ion. QuPa h so wa e ( e sion 0.2.3) [ 23 ] was used o de ec cy oplasmic IGF2BP3 posi i i y in TMA-sec ions om anno a ed a eas wi h leukemic cells. A pa hologis manually se de ec ion pa ame e s and h esholds using he cy oplasmic s aining o IGF2BP3 in ge minal cen e cells as a e e ence, and he nuclea s aining in ge minal cen e s and p oli e a ing epi helium as a e e ence o Ki-67 s aining. The s ain ec o s and in ensi y h esholds o he cell and an ibody de ec ion we e adjus ed acco ding o he ins uc ions o he QuPa h so wa e in isual con ol. Inadequa e samples we e emo ed om he analysis. A eas wi h a i ac s caused by comp ession o olding o he issue we e dis ega ded by se ing he p ope h eshold alues o backg ound in ensi y. Wi h he IGF2BP3 and CD19/Ki-67- double-s ained slides, s ain ec o s we e adjus ed o hema oxylin, 3,3 0 -diaminobenzidine (DAB), and alkaline phospha ase (AP) s aining using a ep esen a i e egion o in e es . Hema oxylin-s ained cells we e de ec ed using he cell de ec ion unc ion in he QuPa h, while he nuclea DAB o Ki-67-posi i e cells we e ecognized om he CD19-posi i e (AP) a eas. Single in ensi y h esholds o IGF2BP3, CD19, and Ki-67 we e used o assess he p opo ion o posi i e cells. 2.3. Mic oa ay and RNA-Sequencing Da a Se s Hemap is a mic oa ay gene exp ession da a se ha includes 6832 cance samples and 1304 B-ALL samples (662 pedia ic and 642 adul cases) [ 24 , 25 ]. The RNA-sequencing da a se om he PanALL s udy coho includes 1988 B-ALL cases (1234 pedia ic and 754 adul cases) [ 26 ]. Fo he su i al analyses, he TARGET da a se , which includes 155 cases o pedia ic high- isk B-ALL pa ien s, was e ie ed along wi h he ollowing clinical in o ma ion: e en s ( elapse, induc ion ailu e, dea h, and second malignancy), su i al, age, leukocy e coun , minimal esidual disease (MRD) a he end o induc ion (EOI), and he cy ogene ic sub ype [27,28]. 2.4. S a is ical Analysis The s a is ical analysis was conduc ed using IBM SPSS S a is ics ( e sion 26) and RS udio ( e sion 3.6.1). The Mann–Whi ney U es , K uskal–Wallis U es , chi-squa ed es , Fishe ’s exac , and log- ank es we e used o es he signi icance o he di e ences be ween g oups. All es s we e wo-sided, and p- alues unde 0.05 we e conside ed s a is ically signi ican . The ComplexHea map package in R was used o c ea e hea maps [ 29 ]. Cox p opo ional haza ds models we e i ed o su i al da a in o de o es ima e he haza d o indi idual isk ac o s. 3. Resul s 3.1. IGF2BP3 P o ein Is Widely Exp essed in Pedia ic B-ALL The IGF2BP3 p o ein has shown diagnos ic and p ognos ic u ili y in di e en malig- nancies [ 7 ]. To assess he exp ession o he IGF2BP3 p o ein in B-ALL, we employed a Cance s 2021,13, 1505 4 o 12 popula ion-based pedia ic coho o 83 B-ALL cases, and immunos ained he diagnos ic bone ma ow ephine biopsies embedded in a issue mic oa ay (TMA) wi h an an ibody agains IGF2BP3. The case summa y o he TMA samples is shown in Table 1. The ap- pendix was used as a posi i e con ol, and i was s ained posi i ely in he ge minal cen e s o he lymphoid ollicles, as expec ed (Figu e 1A) [ 10 ]. Posi i i y (>1%) o IGF2BP3 was de ec ed in 74 ou o 83 pa ien s (89%; Figu e 1B–D), while he p opo ion o posi i ely s ained leukemia cells anged om 1 o 100% (median 34%). IGF2BP3 exhibi ed a g anula s aining pa e n and was localized mos ly o he cy oplasm. Nega i e IGF2BP3 s aining was ound in 9 ou o 83 B-ALL cases (Figu e 1E). No exp ession o IGF2BP3 was ound in he emission bone ma ow specimens. Table 1. Case summa y o he issue mic oa ay (TMA) coho . Clinical Pa ame e Median (IQR) Age (yea s) 4.3 (2.7–9.7) WBC (x 10E9/l) 6.3 (2.7–29.2) MRD (%), EOI 0.01 (0.00–0.14) n(%) CNS disease 5 (6.0) To al 83 WHO Sub ype O he 32 (38.6) BCR-ABL1 1 (1.2) KMT2A- e 4 (4.8) ETV6-RUNX1 20 (24.1) Hype diploid 22 (26.5) Hypodiploid 1 (1.2) TCF3-PBX1 3 (3.6) EOI—end o induc ion; IQR—in e qua ile ange; KMT2A- e—KMT2A- ea anged; MRD—minimal esidual disease; WBC—whi e blood cell coun ; WHO—Wo ld Heal h O ganiza ion. Cance s 2021, 13, x FOR PEER REVIEW 5 o 13 Figu e 1. Immunohis ochemis y o insulin-like g ow h ac o II mRNA-binding p o ein 3 (IGF2BP3). (A) Appendix show- ing posi i i y (b own colo ) o IGF2BP3 in he ge minal cen e (dashed ci cle; 200× magni ica ion). (B) S ongly IGF2BP3- posi i e bone ma ow ephine biopsy o a B-cell acu e lymphoblas ic leukemia (B-ALL) pa ien (200× magni ica ion). (C) Pedia ic B-ALL case wi h a he e ogeneous pa e n o IGF2BP3 exp ession (200× magni ica ion). (D) Weakly IGF2BP3- posi i e B-ALL case wi h only singula posi i e cells isible (200× magni ica ion). (E) IGF2BP3-nega i e B-ALL case (200× magni ica ion). IGF2BP3 is no mally exp essed in ge minal cen e s, whe e he BCL6 p o ein is ac i e and associa ed wi h ge minal cen e - ype B-cell lymphomas [10]. We ecen ly showed ha he BCL6 p o ein is also exp essed in a ac ion o p ecu so B-ALL [22]. BCL6-posi i i y, CD34-nega i i y, and pSTAT5-nega i i y ha e been associa ed wi h a no el p e-B-cell e- cep o signaling sub ype o B-ALL [31]. Hence, we es ed he associa ion be ween IGF2BP3 and BCL6 p o eins and disco e ed ha he IGF2BP3 p o ein was signi ican ly lowe among he BCL6-posi i e cases (Mann–Whi ney U es ; p- alue = 0.003). Likewise, he mRNA exp ession o he p e-BCR “me agene” (see below), which is associa ed wi h BCL6- posi i i y [31], exhibi ed a signi ican ly lowe exp ession among he highes 10 h pe cen- ile o he IGF2BP3 exp essing pa ien s in he PanALL and Hemap da a se s (Mann–Whi - ney U; p- alue < 0.001). On he con a y, cases ha exhibi ed phospho yla ed STAT5 (pY694) p o ein o showed a high exp ession o he s em cell ma ke CD34 e idenced a highe - han-median le el o he IGF2BP3 p o ein (Figu e 2B–D). 3.2. Exp ession o Ki-67 Is Associa ed wi h High IGF2BP3 P o ein Exp ession A high exp ession o IGF2BP3 has been associa ed wi h p oli e a i e pheno ype in malignancies such as man le cell lymphoma [7,15]. We assessed whe he i is associa ed wi h cell p oli e a ion in B-ALL by co-s aining he ephine biopsy specimens wi h CD19, a ma ke o blas cells, and Ki-67, a well-es ablished ma ke o cell p oli e a ion [32,33]. O e all, he exp ession o Ki-67 was s ong in p oli e a ing cells o ge minal cen e s and he epi helium o appendix (Figu e S1A), and in CD19-posi i e cells o B-ALL samples (Figu e S1B,C; p opo ion o posi i e cells, median 95%, in e qua ile ange (IQR) 87– 98%). A highe - han-median le el o IGF2BP3 was signi ican ly associa ed wi h he ex- p ession o Ki-67 (Mann–Whi ney U es ; p- alue = 0.02; Figu e 2E). Figu e 1. Immunohis ochemis y o insulin-like g ow h ac o II mRNA-binding p o ein 3 (IGF2BP3). ( A ) Appendix showing posi i i y (b own colo ) o IGF2BP3 in he ge minal cen e (dashed ci cle; 200 × magni ica ion). ( B ) S ongly IGF2BP3-posi i e bone ma ow ephine biopsy o a B-cell acu e lymphoblas ic leukemia (B-ALL) pa ien (200 × magni ica ion). ( C ) Pedia ic B-ALL case wi h a he e ogeneous pa e n o IGF2BP3 exp ession (200 × magni ica ion). ( D ) Weakly IGF2BP3-posi i e B-ALL case wi h only singula posi i e cells isible (200 × magni ica ion). ( E )IGF2BP3-nega i e B-ALL case (200×magni ica ion). We classi ied he cases in o dis inc sub ypes acco ding o he WHO 2017 Classi i- ca ion o B-ALL [ 30 ]. Exp ession o IGF2BP3 p o ein was highes in he ETV6-RUNX1, “O he ”, KMT2A- ea anged, and hypodiploid sub ypes (Figu e 2A). The di e ence was Cance s 2021,13, 1505 5 o 12 s a is ically signi ican be ween ETV6-RUNX1 and o he sub ypes (Mann Whi ney U Tes p- alue = 0.04 ). The exp ession o IGF2BP3 p o ein did no co ela e wi h whi e blood cell coun (WBC), MRD a he end o induc ion (EOI), CNS disease, o exp ession o speci ic cell su ace ma ke s. Cance s 2021, 13, x FOR PEER REVIEW 6 o 13 Figu e 2. Immunopheno ype o a B-cell acu e lymphoblas ic leukemia (B-ALL) issue mic oa ay coho . (A) Exp ession o IGF2BP3 acco ding o he WHO classi ica ion o B-ALL. (B) Posi i i y o IGF2BP3 among cases wi h ei he a nega i e o posi i e exp ession o he BCL6 p o ein. (C) Posi i i y o IGF2BP3 in cases wi h nega i e o posi i e CD34. (D) Posi i i y o IGF2BP3 in cases wi h a nega i e o posi i e pSTAT5 (Y694). (E) Exp ession o Ki-67 among cases wi h ei he a low o high IGF2BP3 (median as a cu -o ). Do s depic ou lie s. p- alues o (B–E) Mann–Whi ney U es and (A) K uskal–Wallis es a e shown. 3.3. Exp ession o IGF2BP3 in Hema ological Malignancies and B-ALL IGF2BP3 is associa ed wi h a ious malignancies o a B-cell o igin, and pa icula ly wi h ge minal cen e lymphomas [10,11]. To ge a comp ehensi e pic u e ac oss hema o- logical umo s, we assessed IGF2BP3 mRNA le els in 6832 hema ological cance s ha in- cluded 24 di e en disease en i ies [24,25]. The median exp ession o he IGF2BP3 mRNA was he highes in B-ALL, Bu ki lymphoma, di use la ge B-cell lymphoma, ollicula lymphoma, man le cell lymphoma, and ju enile myelomonocy ic leukemia, while he Figu e 2. Immunopheno ype o a B-cell acu e lymphoblas ic leukemia (B-ALL) issue mic oa ay coho . ( A ) Exp ession o IGF2BP3 acco ding o he WHO classi ica ion o B-ALL. ( B ) Posi i i y o IGF2BP3 among cases wi h ei he a nega i e o posi i e exp ession o he BCL6 p o ein. ( C ) Posi i i y o IGF2BP3 in cases wi h nega i e o posi i e CD34. ( D ) Posi i i y o IGF2BP3 in cases wi h a nega i e o posi i e pSTAT5 (Y694). ( E ) Exp ession o Ki-67 among cases wi h ei he a low o high IGF2BP3 (median as a cu -o ). Do s depic ou lie s. p- alues o ( B – E ) Mann–Whi ney U es and ( A ) K uskal–Wallis es a e shown. IGF2BP3 is no mally exp essed in ge minal cen e s, whe e he BCL6 p o ein is ac i e and associa ed wi h ge minal cen e - ype B-cell lymphomas [ 10 ]. We ecen ly showed ha Cance s 2021,13, 1505 6 o 12 he BCL6 p o ein is also exp essed in a ac ion o p ecu so B-ALL [ 22 ]. BCL6-posi i i y, CD34-nega i i y, and pSTAT5-nega i i y ha e been associa ed wi h a no el p e-B-cell ecep o signaling sub ype o B-ALL [ 31 ]. Hence, we es ed he associa ion be ween IGF2BP3 and BCL6 p o eins and disco e ed ha he IGF2BP3 p o ein was signi ican ly lowe among he BCL6-posi i e cases (Mann–Whi ney U es ; p- alue = 0.003). Likewise, he mRNA exp ession o he p e-BCR “me agene” (see below), which is associa ed wi h BCL6-posi i i y [ 31 ], exhibi ed a signi ican ly lowe exp ession among he highes 10 h pe cen ile o he IGF2BP3 exp essing pa ien s in he PanALL and Hemap da a se s (Mann– Whi ney U; p- alue < 0.001). On he con a y, cases ha exhibi ed phospho yla ed STAT5 (pY694) p o ein o showed a high exp ession o he s em cell ma ke CD34 e idenced a highe - han-median le el o he IGF2BP3 p o ein (Figu e 2B–D). 3.2. Exp ession o Ki-67 Is Associa ed wi h High IGF2BP3 P o ein Exp ession A high exp ession o IGF2BP3 has been associa ed wi h p oli e a i e pheno ype in malignancies such as man le cell lymphoma [ 7 , 15 ]. We assessed whe he i is associa ed wi h cell p oli e a ion in B-ALL by co-s aining he ephine biopsy specimens wi h CD19, a ma ke o blas cells, and Ki-67, a well-es ablished ma ke o cell p oli e a ion [ 32 , 33 ]. O e all, he exp ession o Ki-67 was s ong in p oli e a ing cells o ge minal cen e s and he epi helium o appendix (Figu e S1A), and in CD19-posi i e cells o B-ALL samples (Figu e S1B,C; p opo ion o posi i e cells, median 95%, in e qua ile ange (IQR) 87–98%). A highe - han-median le el o IGF2BP3 was signi ican ly associa ed wi h he exp ession o Ki-67 (Mann–Whi ney U es ; p- alue = 0.02; Figu e 2E). 3.3. Exp ession o IGF2BP3 in Hema ological Malignancies and B-ALL IGF2BP3 is associa ed wi h a ious malignancies o a B-cell o igin, and pa icula ly wi h ge minal cen e lymphomas [ 10 , 11 ]. To ge a comp ehensi e pic u e ac oss hema- ological umo s, we assessed IGF2BP3 mRNA le els in 6832 hema ological cance s ha included 24 di e en disease en i ies [ 24 , 25 ]. The median exp ession o he IGF2BP3 mRNA was he highes in B-ALL, Bu ki lymphoma, di use la ge B-cell lymphoma, ollicula lym- phoma, man le cell lymphoma, and ju enile myelomonocy ic leukemia, while he lowes median exp essions we e obse ed in hai y cell leukemia, hepa osplenic T-cell lymphoma, and adul T-cell leukemia (Figu e 3A). The IGFBP3 mRNA was p esen in all sub ypes o B-ALL, wi h he highes exp ession in he KMT2A- ea anged and ETV6-RUNX1 sub ypes and he lowes in he TCF3-PBX1 and BCR-ABL1 sub ypes (Figu e 3B). Analysis o he PanALL da a se [ 26 ], which comp ises 1988 B-ALL cases, alida ed he indings, and also e ealed a s ong exp ession in no el sub ypes such as NUTM1- ea anged, PAX5-al e ed, ETV6-RUNX1-like, BCL2/MYC, and CRLF2 (Figu e 3C). Cance s 2021,13, 1505 7 o 12 Cance s 2021, 13, x FOR PEER REVIEW 8 o 13 Age 1.06 1.01–1.11 0.01 1.03 0.98–1.08 0.24 WBC 1 1.00–1.00 0.58 1 0.99–1.00 0.68 MRD a he EOI 1.02 0.97–1.08 0.47 0.99 0.94–1.05 0.79 CI—con idence in e al; EOI—end o induc ion he apy; HR—haza ds a io; MRD—minimal esidual disease; WBC— whi e blood cell coun a diagnosis. Figu e 3. Exp ession o IGF2BP3 ac oss di e en hema ological malignancies and sub ypes o B- ALL. (A) IGF2BP3 exp ession in he Hemap da a se in di e en hema ological malignancies (n = 6832) [24,25]. (B) IGF2BP3 exp ession in di e en cy ogene ic sub ypes o B-ALL (n = 1304) in he Hemap da a se [24,25]. (C) IGF2BP3 exp ession in di e en B-ALL sub ypes o B-ALL in he PanALL s udy coho (n = 1988) [26]. (D) P oli e a ion-associa ed “me agene” [33] exp ession in B- Figu e 3. Exp ession o IGF2BP3 ac oss di e en hema ological malignancies and sub ypes o B-ALL. ( A )IGF2BP3 ex- p ession in he Hemap da a se in di e en hema ological malignancies (n= 6832) [ 24 , 25 ]. ( B )IGF2BP3 exp ession in di e en cy ogene ic sub ypes o B-ALL (n= 1304) in he Hemap da a se [ 24 , 25 ]. ( C )IGF2BP3 exp ession in di e en B-ALL sub ypes o B-ALL in he PanALL s udy coho (n= 1988) [ 26 ]. ( D ) P oli e a ion-associa ed “me agene” [ 33 ] exp ession in B-ALL in he Hemap and PanALL da a se s (median as a cu -o o he IGF2BP3 exp ession g oups). AILT—angioimmunoblas ic T-cell lymphoma; ALCL—anaplas ic la ge cell lymphoma; AML—acu e myeloid leukemia; ATL—adul T-cell leukemia; B-ALL—B-cell lineage acu e lymphoblas ic leukemia; B-CLL—B-cell ch onic lymphocy ic leukemia; BCL2/MYC—BCL2/MYC- ea anged; BL—Bu ki lymphoma; CHL—classic Hodgkin lymphoma; CML—ch onic myeloid leukemia; CRLF2—CRLF2 (non-Ph-like); CTCL—cu aneous T-cell lymphoma; DLBCL—di use la ge B-cell lym- phoma; DUX4—DUX4- ea anged; ENKTL—ex anodal NK/T-cell lymphoma; FL— ollicula lymphoma; HCL—hai y cell leukemia; HLF—TCF3/TCF4-HLF; HSTCL—hepa osplenic T-cell lymphoma; iAMP21—in ach omosomal ampli ica ion o ch omosome 21; IKZF1 N159Y—IKZF1 missense al e a ion encoding p.Asn159Ty ; JMML—ju enile myelomonocy ic leukemia; KMT2A—KMT2A- ea anged; MALT—ex anodal ma ginal zone lymphoma o mucosa-associa ed lymphoid issue; MCL—man le cell lymphoma; MEF2D—MEF2D- ea anged; MM—mul iple myeloma; MZL—ma ginal zone lymphoma; n—numbe o cases; NLPHL—nodula lymphocy e p edominan Hodgkin lymphoma; NUTM1—NUTM1- ea anged; PAX5al —PAX5 al e a ions; PAX5 P80R—PAX5 p.P o80A g (P80R) al e a ion; Ph—Philadelphia ch omosome (BCR-ABL1); PTCL—pe iphe al T-cell lymphoma, no o he wise speci ied; T-ALL—T-cell lineage acu e lymphoblas ic leukemia; ZNF384—ZNF384- ea anged. Do s depic ou lie s. p- alues o ( D ) Mann–Whi ney U es and ( A – C ) K uskal– Wallis es a e shown. Cance s 2021,13, 1505 8 o 12 3.4. P oli e a i e “Me agene” Signa u e in B-ALL Recen ly, Giuliano e al. (2018) [ 33 ] desc ibed a p oli e a i e “me agene” (MKI67, PCNA,CCNB1,MCM2, and TOP2A) ha is co ela ed wi h cell p oli e a ion. Suppo ing ou ea lie obse a ions, he IGF2BP3 mRNA was signi ican ly associa ed wi h he p oli e - a i e “me agene” when assessed ac oss all hema ological malignancies (Mann–Whi ney U es ; p- alue < 0.001). When he analysis was es ic ed o he B-ALL cases, he “me agene” signa u e and he MKI67 mRNA showed ele a ed le els and we e signi ican ly associa ed wi h a highe - han-median exp ession o IGF2BP3 (Mann–Whi ney U es ; p- alue = 0.005 and p- alue = 0.04, espec i ely; Figu e 3D). A simila analysis in he PanALL da a se epli- ca ed he indings: he IGF2BP3 mRNA was signi ican ly associa ed wi h he high MKI67 mRNA (Mann–Whi ney U es , p- alue < 0.001) and he p oli e a ion-associa ed “me a- gene” wi h a disc e ized exp ession o IGF2BP3 (Mann–Whi ney U es , p- alue < 0.001 ; Figu e 3D; see also hea map in Figu e S2A–C). CDK6 and MYC oncop o eins ha e been epo ed as a ge s o he IGF2BP3 p o ein [ 20 ]. In he PanALL da a se , CDK6 was highe and MYC was lowe among cases wi h a highe - han-median IGF2BP3 mRNA (Mann–Whi ney U es p- alue < 0.001, Figu e S3A,B). In he Hemap da a se , CDK6 was s ongly exp essed among cases wi h a highe - han-median IGF2BP3 mRNA, whe eas he exp ession o MYC did no di e (Figu e S3C,D). 3.5. High IGF2BP3 mRNA Associa es wi h Fa o able Su i al in High-Risk B-ALL The p ognos ic alue o IGF2BP3 mRNA was e alua ed in he TARGET da a se ha included high- isk pedia ic B-ALL cases [ 27 , 28 ]. Highe - han-median IGF2BP3 mRNA showed a s a is ically signi ican associa ion wi h a o able e en - ee (EFS) and o e all su i al (OS; Figu e 4A,B). In a mul i a ia e analysis ha included age, whi e blood cell coun (WBC), and minimal esidual disease (MRD) a he EOI as co a ia es, highe - han- median IGF2BP3 mRNA exhibi ed a dec eased haza d a io o e en s (HR 0.46, 95% CI 0.31–0.68) and dea h (HR 0.50, 95% CI 0.31–0.81; Table 2). | | | ||| |||||||||||||||||| | | |||| || | || p = 0.00012 Log- ank 0.00 0.25 0.50 0.75 1.00 0 3 6 9 12 Time (yea s) E en - ee su i al | | IGF2BP3high IGF2BP3low A || |||| | | ||||||||||||||||||||||||||||||||||||| | |||| |||||| || | ||||||||| | || p < 0.0001 Log- ank 0.00 0.25 0.50 0.75 1.00 0 3 6 9 12 Time (yea s) O e all su i al | | IGF2BP3high IGF2BP3low B 78 42 22 19 1 77 21 9 3 0 - -Numbe a isk 78 62 45 25 1 77 36 22 13 0 - -Numbe a isk Figu e 4. Associa ion o IGF2BP3 exp ession a an mRNA le el on pa ien su i al. Kaplan–Meie su i al analysis o ( A ) e en - ee su i al and ( B ) o e all su i al in he high- isk B-ALL TARGET coho (n= 155) [ 27 , 28 ]. S a is ical signi icance was es ed using he log- ank es , while he median exp ession o IGF2BP3 was used as a cu -o o he wo di e en pa ien g oups.