Association between LAPTM4B gene polymorphism and susceptibility to and prognosis of diffuse large B cell lymphoma

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1 264 Assocition between LAPTM4B gene polymorphism nd susceptibility to nd prognosis of diffuse lrge B cell lymphom HUIRONG DING 1*, XIAOJING CHENG 2*, NING DING 3*, ZHIHUA TIAN 1, JUN ZHU 3, CHUNLIAN ZHOU 4, JING SHEN 1 nd YUQIN SONG 3 1 Centrl Lbortory; 2 Division of Gstrointestinl Cncer Trnsltionl Reserch Lbortory, Key Lbortory of Crcinogenesis nd Trnsltionl Reserch (Ministry of Eduction/Beijing); 3 Deprtment of Lymphom, Peking University Cncer Hospitl nd Institute; 4 Deprtment of Nosocomil Infection Prevention nd Control, Beijing Friendship Hospitl, Cpitl Medicl University, Beijing , P.R. Chin Received Jnury 12, 2016; Accepted July 14, 2017 DOI: /ol Abstrct. Lysosoml protein trnsmembrne 4β (LAPTM4B) is n oncogene tht is overexpressed in number of vrious types of humn cncer. There re two known lleles of LAPTM4B: LAPTM4B*1 nd LAPTM4B*2. The present study ssessed the ssocition between LAPTM4B polymorphisms nd the susceptibility to diffuse lrge B cell lymphom (DLBCL) nd its prognosis. LAPTM4B genotypes were determined using polymerse chin rection nlysis in 164 DLBCL nd 350 helthy control cses. The ssocition between LAPTM4B polymorphisms nd the risk of DLBCL ws nlyzed using unconditionl logistic regression. Differences in ptient survivl were clculted using Kpln Meier nlysis. The present study indicted no significnt differences (P>0.05) in the frequency of LAPTM4B*2 lleles between DLBCL cses (26.5%) nd controls (24.1%). The risk of DLBCL ws slightly incresed in cses with the LAPTM4B*1/2 genotype [odds rtio (OR)=1.160; 95% confidence intervl (CI)= ] or the LAPTM4B*2/2 genotype (OR=1.446; 95% CI= ) compred with those with the LAPTM4B*1/1 genotype. There ws no significnt ssocition between the presence of the Correspondence to: Professor Jing Shen, Centrl Lbortory, Key Lbortory of Crcinogenesis nd Trnsltionl Reserch (Ministry of Eduction/Beijing), Peking University Cncer Hospitl nd Institute, Fucheng 52, Hidin, Beijing , P.R. Chin E mil: shenjing69@sin.com Professor Yuqin Song, Deprtment of Lymphom, Peking University Cncer Hospitl nd Institute, Fucheng 52, Hidin, Beijing , P.R. Chin E mil: songyuqin622@sin.com * Contributed eqully Key words: diffuse lrge B cell lymphom, lysosoml protein trnsmembrne 4β, polymorphism, susceptibility, prognosis LAPTM4B*2 llele nd overll survivl (OS) nd disese free survivl (DFS) in ptients with DLBCL (P=0.399 nd 0.520, respectively). However, there ws tendency for ptients with LAPTM4B*2 nd Interntionl Prognostic Index (IPI) score 3 5 to hve longer OS nd DFS (P=0.126 nd 0.109, respectively). These findings suggest tht genetic polymorphisms of LAPTM4B is not risk fctor for the development of DLBCL, but the LAPTM4B*2 llele my better prognostic indictor in ptients with IPI score 3 5 in DLBCL. Introduction Diffuse lrge B cell lymphom (DLBCL) is the most common subtype of non Hodgkin lymphom (NHL), constituting up to 40% of ll cses globlly (1). DLBCL is highly heterogeneous disese. The stndrd front line therpy for DLBCL, which includes rituximb plus cyclophosphmide, doxorubicin, vincristine nd prednisone (R CHOP), hs improved the survivl rte of DLBCL ptients (2). However, ~33% of DLBCL ptients hve relpsed or refrctory type of disese, which ws reported by Sehn et l (2) in the province of British Columbi in 2005, nd the moleculr mechnism underlying DLBCL development remins to be fully understood (2 4). Although certin indictors my ssist in predicting prognosis in ptients with DLBCL, including the Interntionl Prognostic Index (IPI) score, MYC proto oncogene, nd tumor loction (5 7), the development of novel biomrkers for estimting the efficcy of therpeutic strtegies nd prognosis is required. LAPTM4B exists s two lleles: LAPTM4B*1 with one 19 bp segment (GenBnk ccession no. AY219176) nd LAPTM4B*2 with two tndem repet segments (GenBnk ccession no. AY219177) in the 5' untrnslted region of exon 1 (8). Previous studies hve demonstrted tht LAPTM4B polymorphisms were ssocited with susceptibility to multiple types of cncer, including lung, brest, gstric, colon, ovrin nd primry liver cncer (9 16), which suggested tht LAPTM4B*2 my be ssocited with significntly incresed risk of developing these types of cncer. LAPTM4B*2 ws lso ssocited with poor prognosis in ptients with heptocellulr,

2 DING et l: LAPTM4B GENE POLYMORPHISM IN DIFFUSE LARGE B-CELL LYMPHOMA 265 lung or endometril cncer (17 19). To the best of our knowledge, no study hs previously reported on the ssocition between LAPTM4B polymorphisms nd clinicl dt on DLBCL. The present study evluted whether LAPTM4B polymorphisms were ssocited with the susceptibility to nd prognosis of DLBCL. Mterils nd methods Ptients nd control cses. A totl of 164 ptients with DLBCL were enrolled (for the overll survivl nlysis, 35 cses were not included becuse of loss to follow up or ccepting non first line therpy), which included 81 mles nd 83 femles, men ge yers, with 2 individuls belonging to the LAPTM4B*1/3 genotype. The dignosis of the ptients ws confirmed by the Deprtment of Pthology (Peking University Cncer Hospitl nd Institute, Beijing, Chin) ccording to the World Helth Orgniztion clssifiction. Finl dignosis of ll ptients ws confirmed by pthologicl ssessment t the Beijing Cncer Hospitl, Peking University School of Oncology (Beijing, Chin), nd ll cses were collected between June 2007 nd December The Ann Arbor stging clssifiction system were used to determine the stge of these ptients (20). The dt for the 350 helthy control cses were quoted from the dt of Cheng et l (12), which included 225 mles nd 125 femles, men ge yers. The clinicl reserch protocol of the present study ws pproved by the Institutionl Review Bord (Peking University Cncer Hospitl nd Institute). The present study ws pproved by the Reserch nd Ethics Committee of Peking University School of Oncology. Ech ptient enrolled in the present study provided written informed consent for prticiption. DNA extrction. Blood smples were obtined from ll ptients with DLBCL prior to genetic nlysis. Genomic DNA ws extrcted from peripherl blood mononucler cells using blood genomic DNA extrction kit ccording to the mnufcturer's protocol (BioTeke Corportion, Beijing, Chin). The genomic DNA ws subsequently dissolved in Tris EDTA buffer nd stored t 80 C. Polymerse chin rection (PCR) nlysis. The genomic DNA (30 ng/20 µl) ws mplified using GoTq DNA polymerse (Promeg Corportion, Mdison, WI, USA) nd primers forwrd, 5' GCC GAC TAG GGG ACT GGC GGA 3' nd reverse, 5' CGA GAG CTC CGA GCT TCT GCC 3' which correspond to the nd bp of LAPTM4B, respectively (8). GAPDH ws used s the positive internl control in the present study, with the following primers: Forwrd, 5' GTC TGC CCT AAT TAT CAG GTC CA 3' nd reverse, 5' CCT GGC TCC TGG CAT CTC T 3'. PCR rection conditions were set using thermo cycler (Gene Cycler ; Bio Rd Lbortories, Inc., Hercules, CA, USA) s follows: Denturtion t 94 C for 2 min, followed by 35 cycles t 94 C for 30 sec, t 65 C for 30 sec, nd t 72 C for 30 sec. The lst cycle ws followed by uto extension t 72 C for 7 min. The mplified products were subsequently nlyzed using electrophoresis on 10% polycrylmide or 2% grose gel. Visuliztion ws performed using GelRed (Biotium, Hywrd, CA, USA). All smples re repeted by two independent PCR nlysis. The DNA frgments were purified using the AxyPrep DNA Gel Extrction kit ccording to the mnufcturer's protocol (Axygen Scientific, Inc., Union City, CA, USA). The purified products were sequenced using n ABI 3730XL Avnt Genetic nlyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Wlthm, MA, USA), ccording the mnufcturer's protocol. The sequences were subsequently nlyzed using Seqmn softwre DNASTAR version 5.2 (DNASTAR Inc., Mdison, WI, USA). Sttisticl nlysis. Sttisticl nlysis ws performed using SPSS 16.0 softwre (SPSS, Inc., Chicgo, IL, USA). The χ 2 test or the Fisher's exct test ws used to clculte genotype frequency (including Hrdy Weinberg equilibrium) nd other clinicl prmetric distributions between DLBCL nd control cses. Unconditionl logistic regression nlysis models were used to ssess the ssocition, djusted by ge nd sex, between different genotypes nd cncer risks. The clinicl chrcteristics nd response rte of the ptients were compred using the χ 2 test or the Fisher's exct test ccording to the different genotypes. The ssocition between LAPTM4B gene polymorphism nd overll survivl (OS) nd disese free survivl (DFS) ws evluted using Kpln Meier curves nd the log rnk test. All sttisticl tests were two sided. P<0.05 ws considered to indicte sttisticlly significnt difference. Results LAPTM4B genotypes in ptients with DLBCL. Using PCR nlysis, the present study identified four different LAPTM4B polymorphisms: LAPTM4B*1/1, LAPTM4B*2/2, LAPTM4B*1/2 nd LATPM4B*1/3. As indicted in Fig. 1, 204 bp frgment is encoded by LAPTM4B*1/1, nd 223 bp frgment is encoded by LAPTM4B*2/2. LAPTM4B*1/2 nd LAPTM4B*1/3 re heterozygous. The 204 bp nd 223 bp frgments were both detected in LAPTM4B*1/2. The 204 nd 242 bp frgments were observed in two individuls with LAPTM4B*1/3. The present study detected significnt difference in the distribution of DLBCL between mle nd femle ptients (P<0.002; Tble I). No significnt differences in llele frequency were identified between the DLBCL nd control cses (Tble II). In the 350 controls, the frequency of the LAPTM4B*2 llele ws 24.1%, wheres the frequency in ptients with DLBCL ws 26.5%. The distribution of LAPTM4B genotypes in control nd DLBCL cses re displyed in Tble III. The genotype frequencies for the polymorphism were in greement with the Hrdy Weinberg equilibrium (P=0.898). No significnt differences in the distribution of LAPTM4B*1/2 nd LAPTM4B*2/2 genotypes when compred with LATPM4B*1/1 were detected between the DLBCL nd control cses (P=0.462 nd P=0.368, respectively). The odds rtios of LATPM4B*1/2 nd LATPM4B*2/2 genotypes in ptients with DLBCL compred with ptients with LATPM4B*1/1 is fold (95% CI= ) nd fold (95% CI= ), respectively.

3 266 Figure 1. Schemtic digrm showing LAPTM4B lleles. (A) Sequencing chromtogrms of LAPTM4B lleles. LAPTM4B * 1 contins one copy of the 19 bp sequence. LAPTM4B * 2 contins two tndem repets of the 19 bp sequence, nd LAPTM4B * 3 contins three tndem repets of the 19 bp sequence. (B) LAPTM4B polymorphisms s verified by polymerse chin rection nlysis. Lnes 1 2, LAPTM4B * 1/1; lnes 3 4, LAPTM4B * 1/2; lnes 5 6, LAPTM4B*2/2; lnes 7 8, LAPTM4B * 1/3 genotype. LAPTM4B, lysosoml protein trnsmembrne 4β. Tble I. Distribution of ge nd sex in control nd DLBCL cses. Control cses, DLBCL cses, Chrcteristic n (n=350) n (n=162) P vlue Age > Sex Mle Femle Tble II. Distribution of LAPTM4B lleles in controls (n=350) nd DLBCL cses (n=162). Controls, DLBCL Alleles n (%) cses, n (%) OR (95% CI) LAPTM4B * (75.9) 238 (73.5) LAPTM4B * (24.1) 86 (26.5) ( ) Anlyzed by logistic regression nd nlysis, nd djusted for ge nd sex. CI, confidence intervl; LAPTM4B, lysosoml protein trnsmembrne 4β; DLBCL, diffuse lrge B cell lymphom; OR, Odds rtio. Anlyzed using χ 2 test. DLBCL, diffuse lrge B cell lymphom. LAPTM4B genotypes nd clinicopthologicl prmeters in DLBCL. The present study lso ssessed the distribution of clinicl prmeters, including ge nd sex, mong different LAPTM4B genotypes in ptients with DLBCL (Tble IV). There were no sttisticlly significnt ssocitions between the genotype distribution of LAPTM4B in ptients with DLBCL nd clinicopthologicl prmeters (Tble IV).

4 DING et l: LAPTM4B GENE POLYMORPHISM IN DIFFUSE LARGE B-CELL LYMPHOMA 267 Tble III. Distribution of LAPTM4B genotypes in controls (n=350) nd DLBCL cses (n=162). Genotypes Controls, n (%) DLBC cses, n (%) P vlue OR (95% CI) LAPTM4B * 1/1 199 (56.9) 87 (53.7) LAPTM4B * 1/2 133 (38.0) 64 (39.5) ( ) LAPTM4B * 2/2 18 (5.1) 11 (6.8) ( ) Anlyzed by logistic regression nd nlysis nd djusted for ge nd sex. CI, confidence intervl; LAPTM4B, lysosoml protein trnsmembrne 4β; DLBCL, diffuse lrge B cell lymphom; OR, Odds rtio. Tble IV. Assocition between the distribution of LAPTM4B genotypes nd clinicopthologicl prmeters in DLBCL cses. LAPTM4B genotypes Prmeters *1/1 *1/2 *2/2 P vlue Sex Mle Femle Age > B symptoms b Positive Negtive LDH Positive Negtive β 2 MG Positive Negtive Stge I II III IV Bulky mss 10 cm <10 cm Loclized Yes No No extr nodl c > Incidence site Lymph node Extr lymph IPI score Tble IV. Continued. LAPTM4B genotypes Prmeters *1/1 *1/2 *2/2 P vlue Moleculr subtypes GCB Non GCB Other Anlyzed using χ 2 test or Fisher's exct test. b B symptom includes unexplined fever/chills/weight loss, ftigue nd drenching night swets. c No extr nodl: the number nd type of extr nodl will influence DLBCL ptient prognosis. LAPTM4B, lysosoml protein trnsmembrne 4β; DLBCL, diffuse lrge B cell lymphom; LDH, lctte dehydrogense; MG, mcroglobulin; IPI, Interntionl Prognostic Index; GCB, germinl center B cell. Associtions between LATPM4B genotypes nd prognosis of ptients with DLBCL. In the present study, follow up dt rnging from months (men, 50.5 months) ws obtined for 129 ptients with DLBCL. At the end dte of the follow up, totl of 93 ptients survived nd 36 succumbed to disese. Survivl nlysis ws conducted in the 129 ptients to exmine the effect of LATPM4B polymorphism on the prognosis of ptients with DLBCL. Kpln Meier nlysis nd log rnk test indicted tht LAPTM4B * 2 ws not ssocited with decresed OS nd DFS (P=0.399 nd P=0.520, respectively). However, ptients with the LAPTM4B*2 genotype nd IPI score 3 5 (n=40) tend to exhibit longer durtions of OS nd DFS compred with ptients with the LAPTM4B*1 genotype (P=0.126 nd 0.109, respectively; Fig. 2). Discussion The present study demonstrted tht the presence of the LAPTM4B*2 llele ws not ssocited with mrkedly incresed risk of developing DLBCL compred with LAPTM4B*1. However, in ptients with IPI score 3 5, significntly incresed risk of developing DLBCL ws identified in ptients with the LAPTM4B*2/2 genotype compred with those tht exhibited the LAPTM4B*1/1 genotype. A totl of three tndem repets comprising 19 bp segments were detected in 2/164 of the ptients with DLBCL. Furthermore, ptients with DLBCL tht exhibited LAPTM4B*2 hd tendency to

5 268 Figure 2. Kpln Meier survivl nlysis of ptients with DLBCL with LAPTM4B * 1 nd LAPTM4B * 2 lleles. (A) Kpln Meier survivl curves indicting OS nd DFS in ptients with DLBCL nd LAPTM4B * 1 or LAPTM4B * 2 lleles. OS nd DFS were strtified by IPI scores: (B) 0 2 nd (C) 3 5. LAPTM4B, lysosoml protein trnsmembrne 4β; OS, overll survivl; DFS, disese free survivl; DLBCL, diffuse lrge B cell lymphom; IPI, interntionl prognostic index. hve incresed durtions of OS nd DFS compred with those with LAPTM4B*1, prticulrly those tht lso exhibited IPI score 3 5. To the best of our knowledge, the present study is the first to demonstrte, lbeit not sttisticlly, tht LAPTM4B*2 is more useful prognostic indictor for DLBCL compred with LAPTM4B*1. However, this finding is not consistent with the results of previous studies, including those ssessing heptocellulr crcinom, lung nd brest cncer (17,18,21). LAPTM4B hs two known protein isoforms: LAPTM4B 24 (226 ) nd LAPTM4B 35 (317 ) (22). Previous studies hve indicted tht LAPTM4B 35 is ble to ctivte the binding of phosphoinositide 3 kinse (PI3K)/protein kinse B (Akt) to p85α subunits, nd thereby fcilitte cncer cell multidrug resistnce nd inhibit poptosis (23). Liu et l (22,24) used polyclonl ntibody to demonstrte tht LAPTM4B 35 nd LAPTM4B 24 my differ in expression nd function in tissues nd multiple cell lines of heptocellulr crcinom. The blnce of LAPTM4B 35 nd LAPTM4B 24 my ffect mlignnt trnsformtion. Multiple studies hve reveled tht LAPTM4B 35 my prticipte in mlignnt trnsformtion nd tumor invsion (25 28). However, recent report demonstrted tht the LAPTM4B 24 isoform ws ble to stimulte mechnistic trget of rpmycin complex (mtorc)1 through vcuolr type H + ATPse vi the influx of leucine resulting from the binding of LAT1 4F2hc to lysosomes (29). LAPTM4B 24 my lso promote cell growth nd prolifertion nd regulte immune responses by decresing trnsforming growth fctor β1 production in humn regultory T cells (29,30). Although s forementioned there re two known isoforms of LAPTM4B, the present study suggested tht nother isoform my exist due to the LAPTM4B*2 llele. The 19 bp difference in the first exon of LAPTM4B between LAPTM4B*1

6 DING et l: LAPTM4B GENE POLYMORPHISM IN DIFFUSE LARGE B-CELL LYMPHOMA 269 nd LAPTM4B*2 my lter the open reding frme, thereby resulting in two different protein isoforms: LAPTM4B 35 nd LAPTM4B 40 (8,17). Previous studies hve demonstrted tht LAPTM4B polymorphisms were ssocited with n incresed risk of multiple types of cncer, including ovrin, brest nd gllbldder cncer (13,31,32). These findings suggest tht the 19 bp sequence my serve n importnt function in trnscriptionl regultion, or different protein isoform encoded by LAPTM4B*2 my ffect cncer cell function (8). Yng et l (17) indicted tht LAPTM4B*2 ws ssocited with tumor recurrence nd poor histopthologicl differentition, nd is n independent prognostic fctor in heptocellulr crcinom. Other studies hve reported similr results for lung cncer, nd endometril nd gllbldder crcinom (18,19,32). In the present study, the LAPTM4B*2 llele ws not ssocited with significntly incresed risk of developing DLBCL, nd there ws no significnt ssocition with survivl in ptients with DLBCL. However, there ws tendency for ptients with LAPTM4B*2 to hve improved OS nd DFS compred with ptients with LAPTM4B*1 in DLBCL, nd this pttern ws more evident in cses with IPI score 3 5. The 19 bp sequence my serve crucil role in trnscriptionl regultion, including binding with trnscription fctors, micrornas or non coding linker RNA in ptients with DLBCL, which discrimintes its function with tht of other types of cncer, including heptocellulr crcinom (15,17). Therefore, the different LAPTM4B protein isoforms my hve diverse functions in ptients with LAPTM4B*2 compred with those with LAPTM4B*1 in DLBCL. The results of the present study on LAPTM4B lleles in ptients with DLBCL my provide dditionl evidence tht different LAPTM4B protein isoforms could serve multiple functions. For exmple, it ws previously reported tht LAPTM4B 35 my ctivte the PI3 K/Akt signling pthwy, nd LAPTM4B 24 my ctivte mtorc1 (23,29). Identifying the isoform tht predomintes in the induction of the 19 bp sequence in vrious types of cncer should be investigted in further studies. The present study ssessed the ssocition between LAPTM4B polymorphisms nd prognosis of ptients with DLBCL. It ws indicted tht LAPTM4B*2 my be more useful prognostic indictor for DLBCL compred with LAPTM4B*1, prticulrly in cses with IPI score 3 5 compred with IPI score 0 2 (lthough this trend ws not sttisticlly significnt). IPI is crucil indictor for selecting the pproprite therpeutic strtegies in DLBCL. In DLBCL, ptients with IPI score 0 2 hve good prognosis with the rte of 5 yer survivl reching 80% (33). In the present study, the lck of ssocition between LAPTM4B*2 llele nd survivl of ptients with IPI score 0 2 my be due to the smll number of cses in the present study. 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