1332 Evlution of the detection of 14 high-risk humn ppillomviruses with HPV 16 nd HPV 18 genotyping for cervicl cncer screening MEI-LU BIAN, JIAO-YING CHENG, LI MA, XIAO CONG, JUN LIU, YING CHEN nd XI CHEN Deprtment of Gynecology nd Obstetrics, Chin-Jpn Friendship Hospitl, Beijing 100029, P.R. Chin Received My 21, 2013; Accepted September 11, 2013 DOI: 10.3892/etm.2013.1309 Abstrct. The Americn Society for Colposcopy nd Cervicl Pthology (ASCCP) suggests tht women 30 yers old, with negtive cytopthologicl test but positive high-risk (HR) humn ppillomvirus (HPV) test should undergo HPV 16 nd HPV 18 genotyping. If this test is positive, immedite cervicl pthology is required. Therefore, the im of this study ws to evlute the effectiveness nd clinicl vlue of testing for 14 HR HPVs with HPV 16 nd HPV 18 genotyping for cervicl cncer (CC) screening. A totl of 424 femles from the Chin-Jpn Friendship Hospitl were selected nd rndomly divided into two groups (A nd B). All prticipnts underwent two different testing methods: the liquid-bsed cytology test (LCT) nd HPV DNA test. For the HPV DNA test, prticipnts in group A underwent the hybrid cpture II (HC-II) testing method while prticipnts in group B were tested using the quntittive polymerse chin rection (qpcr; HBRT H14) method. The sensitivity, specificity, positive predictive vlue nd negtive predictive vlue for the detection of cervicl intrepithelil neoplsi (CIN) grde II or greter using HBRT-H14 were 96.30, 78.17, 23.21 nd 99.68%, respectively. In Group B, compred with other HR HPV types, HPV 16 nd HPV 18 infection led to the incresed possibility of cervicl lesions grded CIN II or higher (8.11 nd 51.28%, respectively). A significnt difference in the rtes of CC nd CIN II or higher ws observed mong women who were i) infected with HPV 16 nd/or HPV 18, ii) infected with other HR HPV types nd iii) dignosed s negtive for HR HPV infection (χ 2 =93.976, P=0.0001). In conclusion, HBRT-H14 is pplicble for CC screening with the dvntge of genotyping for HPV 16 nd HPV 18, which my help to improve trige mngement for women with negtive cytology. Correspondence to: Dr Li M, Deprtment of Gynecology nd Obstetrics, Chin-Jpn Friendship Hospitl, 2 Yinghu Est Street, Choyng District, Beijing 100029, P.R. Chin E-mil: mli800809@gmil.com Key words: cervicl cncer screening, genotyping, cervicl intrepithelil lesions, humn ppillomvirus Introduction It hs been identified tht ~95-100% of the incidences of cervicl cncer (CC) re due to infection with the humn ppillomvirus (HPV) (1-3). There re 15 types of high-risk (HR) HPV tht my led to the development of cervicl intrepithelil neoplsi (CIN) nd CC (4,5). Over the pst 20 yers, vrious studies hve stted tht the detection of HPV my be used s n uxiliry tool for primry screening in CC prevention (6-10). Furthermore, the Interntionl Agency for Reserch on Cncer (IARC) hs proposed the use of HPV detection s screening option (11). Though 15 types of HR HPV hve been linked to CC, studies hve shown tht >50% of CC cses re cused by HPV 16, nd 10-15% re cused by HPV 18 (12,13). Thus HPV 16 nd HPV 18 my be detected in ~70% of CC cses (14). In ddition, HVP 18 cuses >35% of cervicl denocrcinoms, which re difficult to detect using cytologicl tests (12). Therefore the mnul of the Americn Society for Colposcopy nd Cervicl Pthology (ASCCP; 2006, 2009 nd 2012) suggests tht women 30 yers old, with negtive cytopthologicl test but positive HR HPV test should undergo HPV 16 nd HPV 18 genotyping. If this test is positive, immedite cervicl pthology is required. Ptients who re HPV 16/18 negtive, but who re HR HPV positive should undergo cytopthology nd HR HPV testing gin fter further 12 months (15,16). In this study, we evluted the effectiveness of rel-time PCR method from Hybribio to detect 14 high-risk HPV with HPV 16 nd HPV 18 genotyping kit (qpcr; HBRT-H14) for CC screening. Mterils nd methods Study popultion. Between August 15, 2011 nd November 10, 2011, totl of 424 femles, with men ge of 39.02±12.00 yers (rnge, 20-55 yers), underwent CC screening nd tretment in the Chin-Jpn Friendship Hospitl cervicl cncer tretment center (Beijing, Chin). None of the recruited ptients hd exhibited cute inflmmtion of the reproductive trct. Written informed consent ws obtined from ll ptients. In group A, hybrid cpture II (HC II) nd liquid-bsed cytology testing (LCT) were preformed; in group B, 14 ptients underwent HR HPV with HPV 16 nd HPV 18 genotyping using the quntittive polymerse chin rection (qpcr; HBRT H14) test nd LCT. For subjects who were HR HPV positive nd hd
BIAN et l: EVALUATION OF HBRT-H14 FOR CERVICAL CANCER SCREENING 1333 Tble I. HBRT-H14 detection result nd its ssocition with histopthologicl studies. HPV results Totl (n) Histopthologiclly negtive (n) CIN I (n) CIN II (n) CIN III (n) CA (n) HPV 16 (+) 34 13 2 11 5 3 HPV 18 (+) 5 4 0 0 0 1 Other (+) b 74 65 3 5 0 1 HPV DNA (-) 309 305 3 0 1 0 Totl 421 386 8 16 6 5 One subject ws both HPV 16 nd HPV 18 positive, nd ws demonstrted to hve inflmmtion ccording to the pthologicl test. b The HPV 16 positive group includes subjects with HPV 16 nd other HPV types, nd the HPV 18 positive group includes subjects with HPV 18 nd other HPV types; therefore, this group is non-hpv 16, non HPV 18 nd other HR HPV positive. HPV, humn ppillomvirus; CIN, cervicl intrepithelil neoplsi; CA, crcinom; HR, high risk. n LCT result tht ws t lest s severe s typicl squmous cells-undetermined significnce (ASC US); HPV 16 nd/or HPV 18 positive with negtive cytologicl results; or hd cytologicl test result tht ws t lest s severe s ASC US nd were HPV negtive, colposcopies were performed. Cytology test. The thin-lyer cytology specimens were obtined using SurePth (TriPth Imging, Becton Dickinson, Burlington, NC, USA), nd n experienced gynecologist evluted the histopthologicl slides. The Bethesd system (TBS) protocol, revised by the Interntionl Cncer Assocition in 2001, ws used s the stndrd for cytologicl dignosis (17). Squmous epithelil bnormlities include ASC [including ASC-US nd ASC but excluding high-grde squmous intrepithelil lesions (ASC-H)], low grde squmous intrepithelil lesion (LSIL), high-grde squmous intrepithelil lesion (HSIL) nd squmous cell crcinom (SCC). Detection of 14 HR HPVs with HPV 16 nd HPV 18 genotyping nd qpcr (HBRT-H14). HBRT-H14 qpcr is new PCR ssy tht detects 12 HR HPV genotypes (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 nd 68) with simultneous differentition between HPV 16 nd HPV 18. This method ws bsed on the multiplex qpcr ssy which utilizes 14 sets of specific primers nd four probe sets, designed in the E region of the HPV genome, with vrying fluorophores in order to obtin fluorescent detection signls. The chnnels of reporter dyes FAM, HEX, ROX nd Cy5 represent 12 HR HPV genotypes, HPV 16, HPV 18 nd the β-globin gene (internl control), respectively. Amplifiction ws performed ccording to the mnufcturer's instructions (Hybribio Biotechnology Ltd. Corp., Chozhou, Chin). HC-II for HPV detection. The HC-II test system (Qigen, Githersburg, MD, USA) ws used to detect HPV in the obtined smples (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 nd 68) s previously described (18). The 96-well HC-II is ble to detect up to 90 clinicl smples. Smples require 5,000 copies/ml of HR HPV in order to be detected. Pthologicl dignosis. A cohort tht were: HBRT-H14 positive; dignosed with ASC-US or bove; or HPV 16 nd/or HPV 18 positive in the HBRT-H14 ssy but cytologiclly negtive (n=122) underwent colposcopy testing nd cervicl biopsies. Multi-point biopsies were performed on the re of epithelil cells to which cetic cid hd been pplied nd on the unstined iodine test point. For the norml trnsformtion zone, four points (2,4,8,10) in totl were bioposies. Shermn et l (19) reported tht the cumultive incidence of CC ws 0.79% with negtive cytologicl nd HPV test results, nd ll smples were not deemed to be CIN or CC ccording to the histologicl tests. It ws ssumed tht 300 subjects with negtive results in the cytologicl nd HPV tests were norml without CIN or CC. Sttisticl nlysis. Histopthologicl results were considered to be the gold stndrd. SPSS 16.0 (SPSS, Inc., Chicgo, IL, USA) ws used to clculte the sensitivity, specificity, positive predictive vlue nd negtive predictive vlue of HBRT-H14 for CC screening. Comprisons mong groups were mde using the χ 2 test. P<0.05 ws considered to indicte sttisticlly significnt difference. Results Correltion between HBRT-H14 HPV nd histopthologicl test results. Using the HBRT-H14 HPV test nd the LCT screening method, 19 smples in 27 subjects with CIN II or bove were HPV 16 positive ccording to the HPV DNA test, ccounting for 70.37% (19/27), mong which four smples were cytologiclly negtive. One smple (3.70%, 1/27) ws identified to be HPV 18 positive with other HR HPV infection; six subjects tested positive for other HR HPV (non HPV 16 nd non-hpv 18), ccounting for 22.22% (6/27); nd one smple (3.70%, 1/27) gve negtive HR HPV result (Tble I). CIN II nd bove ws considered to be pthologiclly positive with CIN I nd below considered to be pthologiclly negtive. For the screening of CIN nd CC, the sensitivity of the HBRT H14 HPV test ws 96.30%, the specificity ws 78.17%, the positive predictive vlue ws 23.21% nd the negtive predictive vlue ws 99.68%. Nineteen smples in 34 subjects with HPV 16 positive were CIN II or bove (histopthology). However, four smples were HPV positive nd cytopthologiclly negtive. The subjects who were grded s CIN II or bove comprised one (20%, 1/5) who ws HPV 18 positive, six (8.11%,
1334 Tble II. HPV infection in women <30 nd 30 yers old. <30 yers old 30 yers old ----------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------- CIN grde HPV 16 (+) HPV 18 (+) Other (+) b Negtive Totl HPV 16 (+) HPV 18 (+) Other (+) b Negtive Totl CIN I 6 0 16 94 116 9 4 52 214 278 CIN II 2 0 2 0 4 17 1 4 1 23 Totl 8 0 18 94 120 26 5 56 215 301 One subject ws both HPV 16 nd HPV 18 positive, nd ws demonstrted to hve inflmmtion ccording to the pthologicl test. b The HPV 16 positive group includes subjects with HPV16 nd other HPV types, nd the HPV 18 positive group includes subjects with HPV 18 nd other HPV types; therefore, this group is non-hpv 16, non HPV 18 nd other HR HPV positive. HPV, humn ppillomvirus; CIN, cervicl intrepithelil neoplsi; HR, high risk. 6/74) who were other HR (non HPV16 nd non -HPV 18) positive nd one (0.32%, 1/309) who ws HR HPV negtive. The risk of cervicl precncer nd cncer demonstrted significnt difference between HPV 16 positive, HPV 18 positive, other HR HPV (non HPV 16 nd non-hpv 18) positive nd HR HPV negtive subjects (χ 2 =93.976, P=0.0001). Evlution of the clinicl vlue of the HBRT-H14 HPV test. In order to exmine whether the HBRT-H14 HPV test is only required in the CC screening of femles 30 yers old, the subjects were divided into two groups with 30 yers of ge s the borderline. Tble II shows tht the HR HPV infection rte ws 21.76% (26/120) in group 1 (femles <30 yers old), nd 28.57% (86/301) in group 2 (femles 30 yers old). There ws no sttisticl significnce between the two groups (χ 2 =2.09, P>0.05). The HPV 16 infection rtes [6.67%, (8/120) nd 8.64% (26/301)] lso showed no sttisticlly significnt difference between the two groups (χ 2 =2.09, P>0.05). In HPV 16 positive subjects, the percentge of ptients grded s CIN II or bove ws 25% (2/8) for group 1 nd 65.38% (17/26) for group 2; the difference ws deemed to be sttisticlly significnt (χ 2 =4.123, P<0.05). Due to the low detection rte of HPV 18, no sttisticl nlysis ws performed for this virus. The infection rtes of other HR HPV (non-hpv 16 nd non HPV 18) infections were 15.00% (18/120) nd 18.60% (56/301) in groups 1 nd 2, respectively (χ 2 =0.77, P>0.05); there ws no sttisticlly significnt difference between the two groups. The rtes of other HR HPV (non HPV 16 nd non HPV 18) infection with CIN II or bove re 11.11% (2/18) nd 7.14% (4/56) in groups 1 nd 2, respectively (χ 2 =0.0016, P>0.05); the difference between groups ws considered to hve no sttisticl significnce. Anlysis of cervicl cytologicl, HBRT-H14 nd histopthologicl test results. Of the 379 subjects considered to be norml or inflmed by the cervicl cytologicl method, 18, 5, nd 56 subjects, respectively, were tested to be HPV 16, HPV 18 nd other HR HPV (non-hpv 16 nd non HPV 18) positive. Histologicl results following the cervicl biopsy were tht smples from four of the HPV 16 positive ptients, one of the HPV 18 positive ptients nd none of the ptients positive for other HR HPV were grded s CIN II or bove. In the nine subjects dignosed with ASC-US using the cervicl cytologicl method, two were HPV 16 positive nd were grded CIN II ccording to the cervicl biopsy; the other seven subjects were both HPV 16 nd HPV 18 negtive, nd evluted s hving inflmmtion ccording to the biopsy. Using the cervicl cytologicl test, 17 subjects were dignosed with LSIL, of which two were HPV 16 positive nd grded CIN II by biopsy, nd 11 subjects were other HR HPV (non HPV 16 nd non HPV 18) positive, mong which four were grded CIN II. The remining four subjects were HR HPV negtive nd one ws confirmed to hve inflmmtion, three were confirmed to hve CIN I ccording to the biopsy results (Tble III). A grding of CIN II or bove ws mde in 19 of the 34 HPV 16 positive ptients, one of the 5 HPV 18 positive ptients, six of the 74 other HRHPV (non-hpv 16 nd non HPV 18) positive ptients nd one of the 309 HR HPV negtive subjects. The differences between groups were considered to be sttisticlly significnt (χ 2 =93.976, P=0.0001). In cytologiclly negtive subjects dignosed with ASC-US nd LSIL, the differences between the vrious types of HPV ssocited with the CC were deemed to be sttisticlly significnt (χ 2 =32.742, 9.535 nd 7.654, P=0.0001, 0.002 nd 0.022, respectively). Anlysis of HC-II HPV nd the cytologicl method of testing. Tble IV shows the correltion between the HC-II HPV nd histologicl results. CIN II nd bove ws considered to be pthologiclly positive nd CIN I nd below ws considered to be pthologiclly negtive. The sensitivity, specificity, positive predictive vlue nd negtive predictive vlue for the detection of CIN II nd bove using HC-II HPV nd cytologicl testing were 77.78, 79.35, 20.39 nd 98.13%, respectively. In terms of clinicl vlue for the detection of CIN nd CC, there ws no significnt difference between HBRT-H14 nd HC-II when used with cytologicl exmintion (P<0.05). Furthermore, the sensitivity nd the negtive predictive vlue were improved using HBRT-H14. Discussion Anlysis of 14 HR HPVs with the 16/18 genotyping rel-time PCR kit is bsed on multiplex qpcr ssy utilizing specific sets of mplifiction primers with vrying fluorophores in order to obtin fluorescent detection signls. qpcr is cpble of dispensing with the problems ssocited with electrophoresis nd the probbility of contmintion from trditionl PCR (20). PCR mplifiction nd detection re
BIAN et l: EVALUATION OF HBRT-H14 FOR CERVICAL CANCER SCREENING 1335 Tble III. Correltion between HR HPV, cervicl cytologicl nd histologicl results. Pthologicl results --------------------------------------------------------------------------------------------------------------------------------------------------------------------- HPV LCT Norml or inflmmtion CIN I CIN II CIN III CA n HPV 16 (+) Norml or inflmmtion 13 1 4 0 0 18 ASC-US 0 0 2 0 0 2 LSIL 0 0 0 2 0 2 HSIL 0 1 4 3 3 11 ASC-H 0 0 1 0 0 1 n 13 2 11 5 3 34 HPV 18 (+) Norml or inflmmtion 4 0 0 0 1 5 Other (+) b Norml or inflmmtion 56 0 0 0 0 56 ASC-US 4 0 0 0 0 4 LSIL 4 3 4 0 0 11 HSIL 1 0 1 0 1 3 n 65 3 5 0 1 74 HR HPV (-) Norml or inflmmtion 300 0 0 0 0 300 ASC-US 3 0 0 0 0 3 LSIL 1 3 0 0 0 4 HSIL 0 0 0 1 0 1 AGC 1 0 0 0 0 1 n 305 3 0 1 0 309 Totl N 386 8 16 6 5 421 One subject ws both HPV 16 nd HPV 18 positive, nd ws demonstrted to hve inflmmtion ccording to histologicl testing. b The HPV 16 positive group includes subjects with HPV 16 nd other HPV types, nd the HPV 18 positive group includes subjects with HPV 18 nd other HPV types; therefore, this group is non-hpv 16, non HPV 18 other HR HPV positive. HPV, humn ppillomvirus; LCT, liquid-bsed cytologicl technology; CIN, cervicl intrepithelil neoplsi; CA, crcinom; ASC-US, typicl squmous cells-undetermined significnce; LSIL, low-grde squmous intrepithelil lesions; HSIL, high-grde squmous intrepithelil lesions; ASC-H, typicl squmous cells-cnnot exclude HSIL; AGC, typicl glndulr cell; HR, high risk. Tble IV. Correltion between HC-II HPV test results nd cervicl histologicl results. Pthologicl results ------------------------------------------------------------------------------------------------------- HPV Negtive CIN I CIN II CIN III CA n HC-II (+) 77 5 11 5 5 103 HC-II (-) 312 3 5 1 0 321 Totl 389 8 16 6 5 424 HPV, humn ppillomvirus; CIN, cervicl intrepithelil neoplsi; CA, crcinom; HC-II, hybrid cpture-ii. conducted in the sme rection tube; multiple rection tubes use four different reporter dyes cpble of trcking different trgets. The rection mixture for ech smple includes cellulr β-globin gene which is cpble of fully monitoring the entire testing process from DNA extrction to signl detection. In this study, three β-globin negtive smples were reported mong 424 smples (0.7%), rendering these three subjects invlid. It ws possible tht the HPV copy number ws too low to be mplified due to too few cells being obtined. Flse negtive results in HPV DNA detection re likely to increse if this qulity monitoring option is lcking. Thus it is criticl to include qulity monitoring in HPV detection. In the nturl history of HPV infection, the mjority of infections re temporry, prticulrly mong young women. Only smll proportion re persistently infected by HR HPV, nd usully CC develops following 10 yers of continuous infection (21). Thus, the Food nd Drug Administrtion (FDA) pproved tht the testing of women 30 yers old my include HPV DNA detection s supplementry method of dignosis (22-24). In the present study, no sttisticlly significnt differences were observed in the incidence of HPV 16 infection nd of other HR HPV (excluding HPV 16 nd 18) infection between the ge groups (P>0.05). Sttisticl nlysis ws not performed for HPV 18 due to its low infection rte. However, there ws sttisticlly significnt difference between the two ge groups of HPV 16 positive femles (<30 nd 30 yers old), grded CIN II nd bove (P<0.05). Thus for women 30 yers old, HPV 16 infection hs very high risk. Song et l (25) considered the HPV DNA test to be vluble resource for CC screening, trige nd the mngement of ptients with ASC-US nd LSIL, nd post-surgicl followup for CIN nd CC. The clinicl dilemm is how to mnge femle ptients who re HR HPV positive but cytologiclly
1336 negtive. In the present study, sttisticlly significnt differences were identified mong the HPV 16 positive, HPV 18 positive, other HR HPV (excluding HPV 16 nd 18) positive nd HR HPV negtive femles with regrd to grding of CIN II nd bove. Thus, the risk fctors for CC my be listed s: HPV 16 positive > HPV 18 positive > other HR HPV type positive > HR HPV negtive. Our study hs shown tht the ptients my lso divided ccording to whether the type of HPV infection is HPV 16 or HPV 18. Colposcopies should be performed if the femles re demonstrted to be HVP 16 nd/or HPV 18 positive, wheres subjects who re HPV 16 nd HPV 18 negtive should be tested gin one yer lter (10,11). The guidelines from the ASCCP nd those of the Spnish CC screening nd prevention progrms suggest tht cytologiclly negtive but HR HPV positive subjects should continue to undergo HPV 16 nd HPV 18 genotyping (26). If the HPV 16 or HPV 18 tests re positive, exmintion by colposcopy is required. Therefore, the HBRT-H14 screening method tht detects 14 HR HPV types, including the specific detection of HPV 16/18 genotypes, is likely to sve time. Furthermore, HPV 18 infection relted to certin cervicl lesions is difficult to detect using cytologicl methods (for exmple cervicl denocrcinom). In our study, five cses were HPV 18 positive. These cses included one tht ws dignosed s inflmmtion by cervicl cytologicl testing but ws demonstrted to be cervicl denocrcinom by biopsy. Therefore, it is not sufficient to report the infection of HR HPVs without clssifiction; HPV 16 nd HPV 18 genotyping should lso be performed. Our study indicted with other HR-HPV, HPV 16 nd HPV 18 hd higher risk of cusing CC. The results of the current study were similr to those of Wong et l (27). Acknowledgements The uthors would like to thnk Longxu Xie nd Liejun Li for criticlly reviewing the mnuscript. References 1. de Villiers EM, Fuquet C, Broker TR, Bernrd HU nd zur Husen H: Clssifiction of ppillomviruses. 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