Urothelial carcinoma is the most common malignancy of

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1 Comprtive Evlution of nd ImmunoCyt/uCyt Assys in Atypicl Urine Cytology Mri E. Vergr-Lluri, MD; Eugeni Hu, BS; Jin-Yu Ro, MD; Mry Levin, BA; Sophi K. Apple, MD; Ned A. Motmed, MD Context. Detection of urothelil crcinom y urine cytology cn e chllenging. Recently, hs een studied s mrker to improve detection of urothelil crcinom. is n ssy trgeting expression of topoisomerse II- nd the minichromosome mintennce protein-2 nd is used to ssist in dignoses of gynecologic specimens. Ojective. To evlute the utility of nd ucyt in typicl urine cytology. Design. Sixty-eight specimens with dignosis of typicl urine cytology, concurrent ucyt testing, nd surgicl iopsy follow-up were included. Slides were restined with. ws recorded s positive when nucler stining ws seen in t lest one morphologiclly typicl urothelil cell. The ucyt ws scored s positive if t lest one morphologiclly typicl urothelil cell showed positive fluorescence stining. Thirteen cses (19%) hd enign histologic dignoses, 18 (26%) hd lowgrde ppillry urothelil crcinom, nd 37 (54%) hd high-grde urothelil crcinom.. The overll sensitivity ws 85% for, 85% for ucyt, nd 93% for the comintion of the 2 ssys. The overll specificity ws 69% for, 31% for ucyt, nd 23% for the comintion of the 2 tests. In predicting high-grde urothelil crcinom, sensitivity ws 92% for, 86% for ucyt, nd 92% for oth tests. In predicting low-grde ppillry urothelil crcinom, sensitivity ws est with the comintion of the 2 tests t 94%. Conclusion. hs superior specificity to ucyt. The comintion of the 2 tests yielded high sensitivity not only for high-grde urothelil crcinom ut lso for lowgrde ppillry urothelil crcinom. (Arch Pthol L Med. 2014;138: ; doi: /rp OA) Urothelil crcinom is the most common mlignncy of the lower urinry trct. In 2013, n estimted new cses of urinry ldder cncer were nticipted, with n estimted deths from disese. 1 High-grde urothelil crcinoms re often multifocl with high recurrence rte nd risks for progression to muscle-invsive disese. Constnt surveillnce for urinry ldder cncer is necessry once primry dignosis is mde. 2 5 Cytology is often the test tht recognizes high-grde urothelil crcinom, whether in the surveillnce or primry dignostic Accepted for puliction Novemer 12, From the Deprtment of Pthology & Lortory Medicine, Dvid Geffen School of Medicine, University of Cliforni, Los Angeles. Dr Vergr-Lluri nd Ms Hu re now with the Deprtment of Pthology & Lortory Medicine, University of Southern Cliforni, Los Angeles. The uthors hve no relevnt finncil interest in the products or compnies descried in this rticle. The study ws fully supported y Trnsltionl Reserch Fund grnt from the Deprtment of Pthology & Lortory Medicine, University of Cliforni, Los Angeles. This work ws not supported, either finncilly or in ny other mnner, y the BD Compny, the supplier of the ntiody. Presented in prt t the 102nd Annul Meeting of the United Sttes nd Cndin Acdemy of Pthology; Mrch 5, 2013; Bltimore, Mrylnd. Reprints: Ned A. Motmed, MD, Deprtment of Pthology & Lortory Medicine, Dvid Geffen School of Medicine, University of Cliforni, Le Conte Ave, Box , 1P-241 CHS, Los Angeles, CA , (e-mil: nmotmed@mednet.ucl.edu). settings. It lso complements cystoscopy, which is est for detecting low-grde urothelil crcinom, nd iopsy, the reference stndrd. Becuse oth cystoscopy nd iopsy re invsive nd expensive procedures, cytology is usully the first specimen received nd reviewed ecuse it is sfe nd inexpensive method of nlysis. 6,7 However, ecuse of the nture of the specimen, cells re often degenerted or rective to other conditions, such s infection, inflmmtion, or stones, mking dignosis of lesions difficult or, t lest, chllenging. 6,7 Severl reports hve een pulished out the utility of in the gynecologic, cytologic nd histologic, dignostic settings During the pst severl yers, literture hs emerged regrding the vlue of minichromosome mintennce protein 2 (MCM2) nd minichromosome mintennce protein 5 (MCM5) s immunossys in urothelil crcinom A short puliction in the Lncet in 1999, y Stoeer K et l 13 reported on the possile use of MCM5 s noninvsive, urinry, cytologic, immunofluorometric ssy for the detection of urothelil cncers. High expression levels of MCM2 hve lso een shown to e of prognostic vlue in predicting urothelil tumor recurrence, prticulrly in T1 tumor evlution. 11,12 MCM2 nd MCM5 re prt of highly conserved fmily of proteins responsile for the unwinding of the DNA helix during initition of repliction in eukryotic cells. The dysregultion of these proteins is key chrcteristic of chnges ssocited with urothelil neoplsi. 14 These findings ppered promising nd inspired further scrutiny of similrly well-estlished Arch Pthol L Med Vol 138, Septemer 2014 & ucyt in Urine Cytology Vergr-Lluri et l 1215

2 Tle 1. Summry of nd ucyt in Ptients With Urine Cytology Dignosis of Atypi nd Benign Histopthology Follow-up Dignoses Cse No. Age, y/sex Specimen ucyt Comined nd ucyt Intervl Between Cytology nd Histology Dignoses, d Histology Dignosis 1 61/M VU FP FP FP 210 Benign 2 58/M IR N N N 131 Benign 3 72/M IR N FP FP 95 Benign 4 45/F IR N N N 121 Benign 5 79/F CU N FP FP 22 Benign 6 82/M VU N FP FP 46 Benign 7 76/M IR N FP FP 181 Benign 8 74/M VU FP FP FP 90 Benign 9 74/M VU FP FP FP 90 Benign 10 86/F VU N FP FP 76 Benign 11 64/M VU N N N 266 Benign 12 76/M VU FP N FP 392 Benign 13 86/M VU N FP FP 98 Benign Arevitions: CU, ctheterized urine; FP, flse-positive; IR, ldder irrigtion; N, negtive; VU, voided urine. ntiody, BD Dignostics, Burlington, North Crolin. immunohistochemicl ntiody, (BD Dignostics- TriPth, Burlington, North Crolin), which detects oth MCM2 nd topoisomerse II- protein (TOP2A). To ssist with the evlution of cses dignosed s typicl urothelil cells (AUC), severl djunct mrkers, such s CK20, Ki67, nd UroVysion nd ImmunoCyt/uCyt (Scimedx Corportion, Denville, New Jersey), hve een investigted s possile ncillry tests y other investigtors. 11,15 18 In our institution, ucyt is commonly used ncillry test. 17 It identifies 3 iomrkers, 2 for the mucin glycoprotein nd one for the glycosylted form of crcinoemryonic ntigen commonly found in ldder cncer cells Recently, the utility of the ntiody cocktil in urinry cytology s n ncillry test to id in the strtifiction of AUCs ws introduced. 22 Tht ntiody cocktil trgeted MCM2 nd TOP2A, mrkers tht re highly expressed in hyperctive sl/prsl cells, s well s in cncer cells during the errnt induction of S phse. 9,23 The current study ws designed to compre ucyt, one of the widely used ncillry tests t our institution, to the utility of in typicl urinry cytology. An dditionl gol ws to ssess the use of oth ssys in comintion to further improve the detection of urothelil crcinom in AUCs. MATERIALS AND METHODS Cse Selection This study ws pproved y the Institutionl Review Bord of the University of Cliforni, Los Angeles (IRB ). This retrospective study investigted ptients specimens from Jnury 2006 to Decemer 2009 tht fit inclusion criteri. Criteri for inclusion comprised cses with cytologic dignoses of AUCs tht Tle 2. Summry of nd ucyt in Ptients With Urine Cytology Dignosis of Atypi nd Histopthology Follow-up Dignosis of Low-Grde Urothelil Crcinom (LGUC) Cse No. Age, y/sex Specimen ucyt Comined nd ucyt Intervl Between Cytology nd Histology Dignoses, d Histology Dignosis 1 64/M VU FN P P 657 LGUC 2 69/M IR P P P 191 LGUC 3 20/M VU FN P P 147 LGUC 4 81/F IR FN P P 1 LGUC 5 66/M VU P P P 15 LGUC 6 29/F VU P P P 14 LGUC 7 29/F IR P P P 701 LGUC 8 69/M UB FN P P 27 LGUC 9 83/M IR P P P 247 LGUC 10 66/M VU P P P 102 LGUC 11 76/M VU P P P 32 LGUC 12 77/M VU P P P 136 LGUC 13 77/M VU P P P 269 LGUC 14 73/M VU P FN P 111 LGUC 15 88/M VU P FN P 25 LGUC 16 78/F VU FN FN FN 908 LGUC 17 78/M NT P P P 11 LGUC 18 69/M VU P P P 145 LGUC Arevitions: FN, flse-negtive; IR, ldder irrigtion; NT, nephrostomy tue; P, positive; UB, ureterl rush; VU, voided urine. ntiody, BD Dignostics, Burlington, North Crolin Arch Pthol L Med Vol 138, Septemer 2014 & ucyt in Urine Cytology Vergr-Lluri et l

3 Tle 3. Summry of nd ucyt in Ptients With Urine Cytology Dignosis of Atypi nd Histopthology Follow-up Dignosis of High-Grde Urothelil Crcinom No. Age, y/sex Specimen ucyt Comined nd ucyt Intervl Between Cytology nd Histology Dignoses, d Histology Dignosis 1 69/M IR P P P 65 HGCIS 2 67/M RPW P P P 147 HGCIS 3 67/M RPW P P P 398 HGCIS 4 67/M RPW P P P 140 HGCIS 5 67/M IR P P P 129 HGCIS 6 96/M VU P P P 28 HGCIS 7 67/M VU P P P 49 HGCIS 8 85/M VU P P P 1 HGCIS 9 60/M VU P P P 13 HGCIS 10 73/M IR P P P 27 HGCIS 11 73/M RPW P P P 301 HGCIS 12 80/M VU P P P 60 HGCIS 13 71/F IR P P P 26 HGIUC 14 82/M VU P P P 293 HGIUC 15 61/F VU P P P 21 HGIUC 16 82/F IR P P P 398 HGIUC 17 79/M IR P P P 26 HGIUC 18 85/F VU P FN P 798 HGIUC 19 74/M VU FN FN FN 42 HGIUC 20 77/M VU P P P 269 HGPUC 21 84/F VU P P P 196 HGPUC 22 77/M VU P P P 40 HGPUC 23 73/M VU P P P 111 HGPUC 24 73/F RPW P P P 35 HGPUC 25 68/M VU P P P 18 HGPUC 26 62/F VU P P P 42 HGPUC 27 62/M VU P P P 101 HGPUC 28 75/M VU P P P 14 HGPUC 29 68/F RPW P P P 18 HGPUC 30 83/M VU P P P 19 HGPUC 31 61/M VU P P P 13 HGPUC 32 88/M VU P P P 22 HGPUC 33 65/M VU P P P 80 HGPUC 34 80/M VU P P P 63 HGPUC 35 71/M IR FN FN FN 126 HGPUC 36 79/F IR P FN P 368 HGPUC 37 56/M VU FN FN FN 309 HGPUC Arevitions: FN, flse negtive; HGCIS, high-grde crcinom in situ; HGIUC, high-grde invsive urothelil crcinom; HGPUC, high-grde ppillry urothelil crcinom; IR, ldder irrigtion; P, positive; RPW, renl pelvic wsh; VU, voided urine. ntiody, BD Dignostics, Burlington, North Crolin. hd concurrent ucyt stining with conclusive results nd definitive follow-up surgicl iopsies, the ltter of which ws treted s the reference stndrd. The pertinent ptients demogrphic dt, including the intervls etween the cytologic nd histopthologic dignoses, were recorded nd re displyed in Tles 1 through 3. Cytology The AUCs were defined s cytology cses tht fell short of definitive dignosis of urothelil crcinom; tht is, they contined some, ut not ll, of the dignostic fetures. The stndrd dignostic fetures used for urothelil crcinom re mny or single urothelil cells with drk hyperchromtic nuclei with corse chromtin, 24,25 incresed nucler to cytoplsmic rtios, nisonucleosis, nd irregulr nucler memrnes. 6,7,26 ucyt Immunofluorescence Stining Slide preprtion for ucyt immunofluorescence stining ws performed within 7 dys of smple collection, ccording to the procedure pulished in the mnufcturer s instruction sheet. Tht protocol hs lso een descried in seprte puliction y Sullivn et l. 17 A positive score ws ssigned when 1 of the 3 iomrkers (2 for the mucin glycoprotein nd 1 for the glycosylted form of crcinoemryonic ntigen) ws detected in cell with red or green fluorescence. Both green nd red fluorescent cells my e seen in single positive smple (Figure 1) Scoring of ucyt test results ws performed in ccordnce with the mnufcturer s instructions, which scores smple s positive if t lest one morphologiclly AUC hd the specified fluorescent rection. 27 Immunostining The ntiody cocktil ( Immunohistochemistry), SureDetect detection regents kit, nd SureDetect SiH cell control slides were otined from BD Dignostics TriPth. The stining procedure ws performed mnully following the pulished procedure in the mnufcturer s product insert. Archived ThinPrep (Hologic, Bedford, Msschusetts) Ppnicolou-stined slides were immersed for week to remove the glss coverslips. Removl of the mounting medi ws performed under stndrd protocol using xylene rinses. The slides were then rehydrted with series of grduted ethnol dilutions. Destining ws then ccomplished in 1% hydrochloric cid to 70% ethnol for 5 minutes, followed y wshing with phosphte-uffered sline solution. Susequently, the slides were restined with using mnul immunohistochemistry techniques nd positive nd negtive controls (SiH cells, Americn Type Culture Collection, Mnsss, Virgini). The primry ntiody of ( cocktil of mouse monoclonl nti- MCM2 nd nti-top2a) ws dispensed onto slides, incuted for Arch Pthol L Med Vol 138, Septemer 2014 & ucyt in Urine Cytology Vergr-Lluri et l 1217

4 Figure 1. The ucyt immunofluorescence ssy from mlignnt urine cytology specimen demonstrting positive stining. One ntiody is directed ginst glycosylted crcinoemryonic ntigen leled with Texs Red (red). The other ntiodies re ginst mucin glycoproteins, LD10 nd M344, leled with fluorescein (green). The presence of t lest one typicl cell with either red or green fluorescence ws considered positive for mlignncy (originl mgnifiction 3100). 30 minutes, nd rinsed off. Miniml rtifcts nd/or loss of cellulrity were encountered ecuse of destining nd restining. scoring ws recorded s positive if t lest one morphologiclly AUC displyed positive nucler stining. This protocol ws lso performed in the puliction y Motmed et l. 22 Exmples of the stins of AUCs nd their corresponding hemtoxylin-eosin stined histologic imges of low-grde ppillry urothelil crcinom (LGUC; Figure 2, A nd B) nd high-grde urothelil crcinom (HGUC; Figures 3, A nd B) re shown. Study Design The ucyt nd results were tulted in 3 ctegories: enign, LGUC, nd HGUC in Tles 1 through 3, respectively. The HGUC ctegory included high-grde, in situ, nd invsive crcinoms in oth ppillry nd flt lesions, s designted in Tle 3. Using the histopthology dignoses s the gold stndrd, the nd ucyt findings were recorded s true-positive, truenegtive, flse-positive, nd flse-negtive. In ech ctegory, the comintion of the test results ws lso dded to ech respective tle. For the result, the cse ws tulted s positive when t lest one mrker ws positive ( or ucyt) nd ws scored s negtive when oth mrkers displyed no immunorectivity. Assy Performnce nd Sttisticl Anlysis Assy performnce ws clculted for overll (Tle 4), enign (Tle 5), LGUC (Tle 6), nd HGUC (Tle 7) ctegories. 28 The dt in the enign ctegory (Tle 5) were used in overll s well s in individul ctegories (LGUC nd HGUC), s shown in Tles 6 nd 7. A 2-tiled, pired t test ws employed to compre the designted ctegories using the lists of the individul ptients s indicted in Tles 4 through 7. A P vlue of.05 or less ws considered significnt difference etween the groups when compred. The SPSS sttisticl softwre (version 21, IBM Corportion, Armonk, New York) nd Office Excel (2010 version, Microsoft Corportion, Redmond, Wshington) were used for dt tultion nd sttisticl nlyses. RESULTS Sixty-eight ptients with AUC were included in the study, with medin ge of 73 yers (rnge, yers). There were 52 men (76%) nd 16 women (24%), with mle to femle rtio of 3:1. Ptients presented in oth surveillnce nd primry dignostic clinicl settings. For histologic iopsies on follow-up, 19% of the cses were enign (n ¼ 13), 26% (n ¼ 18) were LGUC, nd 54% (n ¼ 37) were HGUC. The intervl rnges etween cytology nd histology dignoses were 1 to 908 dys, with medin of 92.5 dys. Among the 68 specimens, there were 43 voided urine (63%) specimens, 16 ldder irrigtions (24%), nd 6 renl pelvic wshings (9%). The remining 3 specimens (4%) were otined from ctheterized urine, ureterl rushing, nd nephrostomy tue. The pertinent demogrphic informtion nd smple sources for ll ptients re detiled in Tles 1 through 3. ws positive in 47 of 68 cses (69%) with iopsy dignosis of urothelil crcinoms (LGUC nd HGUC), leding to sensitivity of 85% (47 of 55), which ws the sme for ucyt (Tle 4). The specificity for ws 69% (9 of 13), wheres it ws 31% (4 of 13) for ucyt. When, the sensitivity incresed to 93% (51 of 55), wheres specificity decresed to 23% (3 of 13). The positive predictive vlue ws 92% (47 of 51) for, 84% (47 of 56) for ucyt, nd 84% (51 of 61) for comintion of the 2 tests. The negtive predictive vlue ws 53% (9 of 17) for, 33% (4 of 12) for ucyt, nd 43% (3 of 7) for comintion of the tests. The 2-til, pired t test etween the ssys reveled no sttisticlly significnt difference. The exct vlues for the performnce prmeters nd the sttisticl P vlues re recorded in Tle 4 for the overll ctegories. Benign Ctegory There were 13 ptients (19%) with enign lesions, 3 women (23%) nd 10 men (77%) with medin ge of 74 yers (Tle 1). The specimens were sumitted s voided urine (n ¼ 8; 62%), ldder irrigtion (n ¼ 4; 31%), nd ctheterized urine (n ¼ 1; 8%) smples. The histopthology dignoses were chronic cystitis (cses 1 nd 4; 15%), rective typi (cses 3 nd 5 12; 69%), denuded mucos (cse 13; 8%), nd nephrogenic denom (cse 2; 8%), s shown in Tle 1. The intervl rnges etween cytology nd histology dignoses were 1 to 392 dys, with medin of 98 dys. nd ucyt ssys hd 4 nd 9 flse-positive results, yielding specificity of 69% nd 31%, respectively (Tle 5). The t test P vlues for versus ucyt nd for versus the test results were.05 nd.01, respectively, wheres for ucyt versus the test results the P vlue ws.34 (Tle 5). The significnt differences in this ctegory were due to the higher flsepositive results from the ucyt ssy. In this ctegory, 8 of the 13 ptients (62%) hd history of cille Clmette-Guérin therpy for ldder cncer, 3 (23%) hd rchytherpy for prostte cncer, nd 2 (15%) did not hve ny history of tretment. LGUC Ctegory Of the 68, there were 18 ptients (26%) with LGUC, 4 women (22%) nd 14 men (78%), with medin ge of 71 yers (Tle 2). The specimens were sumitted s voided urine (n ¼ 12; 67%), ldder irrigtion (n ¼ 4; 22%), ureterl rush (n ¼ 1; 6%), nd nephrostomy tue (n ¼ 1; 6%) smples. The histopthology dignoses were uniformly LGUCs for ll 18 ptients (100%; Tle 2). The intervl rnges etween cytology nd histology dignoses were 1 to 908 dys, with medin of 124 dys. In this ctegory, the nd ucyt ssys hd 13 nd 15 true-positive results, 1218 Arch Pthol L Med Vol 138, Septemer 2014 & ucyt in Urine Cytology Vergr-Lluri et l

5 Figure 2. An exmple (cse 14; Tle 2) of typicl urine cytology showing positive rection with in cluster of typicl cells (A) nd histology follow-up of low-grde ppillry urothelil crcinom (B). In this cse, concurrent immunohistochemicl stin exhiits intense nucler positivity in more thn one typicl cell (originl mgnifiction 340 [A]; hemtoxylin-eosin, originl mgnifiction 320 [B]). Figure 3. An exmple (cse 2; Tle 3) of typicl urine cytology showing positive rection with (A) nd histology follow-up of high-grde urothelil crcinom in-situ (B). In this cse, concurrent immunohistochemicl stin exhiits intense nucler positivity in more thn one typicl cell (originl mgnifiction 3100; hemtoxylin-eosin, originl mgnifiction 340). yielding sensitivity of 72% nd 83%, respectively. The comintion of the 2 tests yielded 94% sensitivity (17 of 18; Tle 6). The positive predictive vlue ws 76% (13 of 17) for, 63% for ucyt (15 of 24) nd for the comintion of the 2 tests (17 of 27). Specificity ws 69% (9 of 13) for ProEx C, 31% (4 of 13) for ucyt, nd 23% (3 of 13) when. Negtive predictive vlue ws 64% (9 of 14) for, 57% (4 of 7) for ucyt, nd 75% (3 of 4) for comintion of 2 tests. The 2-til, pired t test etween the ssys reveled no sttisticlly significnt difference (Tle 6). In this ctegory, 6 of the 18 ptients (33%) hd history of cille Clmette-Guérin therpy, 1 (6%) hd history of tretment with mitomycin, nd 1 (6%) hd history of lser fulgurtion for ldder cncer. Ten ptients (56%) did not hve ny history of tretment. HGUC Ctegory There were 37 ptients (54%) with HGUC, 8 women (22%) nd 29 men (78%), with medin ge of 73 yers (Tle 3). The specimens were sumitted s voided urine (n ¼ 23), ldder irrigtion (n ¼ 8), nd renl pelvic wsh (n ¼ 6) smples. The histopthology dignoses were high-grde crcinom in situ (cses 1 12; 32%), high-grde invsive crcinom (cses 13 19; 19%), nd high-grde ppillry urothelil crcinom (cses 20 37; 49%), s shown in Tle 3. The intervl rnges etween cytology nd histology dignoses were 1 to 798 dys, with medin of 60 dys. nd ucyt ssys hd 34 nd 32 true-positive results, yielding sensitivity of 92% nd 86%, respectively. The comintion of the 2 ssys yielded 92% sensitivity (34 of 37; Tle 7). Specificity ws 69% (9 of 13) for, 31% (4 of 13) for ucyt, nd 23% (3 of 13) for comintion of the 2 tests. Positive predictive vlue ws 89% (34 of 38) for ProEx C, 78% (32 of 41) for ucyt, nd 77% (34 of 44) for the comintion of the 2 tests. NPV ws 75% for, 44% for ucyt, nd 50% when. The t test P vlues for versus ucyt nd versus the tests results were.03 nd.01, respectively; wheres for ucyt versus the tests results, the P vlue ws.26 (Tle 7). Agin, the significnt differences in this ctegory were Arch Pthol L Med Vol 138, Septemer 2014 & ucyt in Urine Cytology Vergr-Lluri et l 1219

6 Tle 4. Performnce Mesure, n ¼ 68 Overll Assy Performnces ucyt Comined True positives, No True negtives, No Flse negtives, No Flse positives, No Sensitivity, % Specificity, % PPV, % NPV, % P vlue c ProExC.58 versus ucyt P vlue c.36 versus P vlue c ucyt versus.07 Arevitions: NPV, negtive predictive vlue; PPV, positive predictive vlue. ntiody, BD Dignostics, Burlington, North Crolin. c P vlue s determined y 2-til, pired t test. due to the higher flse-positive rections of the ucyt ssy. In this ctegory, 13 of the 37 ptients (35%) hd history of cille Clmette-Guérin therpy, 1 (3%) hd interferon therpy, 1 (3%) hd intrvesiculr chemotherpy, nd 1 (3%) ptient hd mitomycin therpy for ldder cncer. Two ptients (5%) hd chemotherpy for colon cncer, nd 19 ptients (51%) did not hve ny history of tretment. COMMENT To our knowledge, this is the second study investigting the vlue of the ntiody cocktil s n ncillry tool in urinry cytologic evlution, nd it is the first to evlute its performnce in comprison to nother ncillry urine cytology test, ucyt. The 2 ncillry tests used on exfolitive urothelil cells re UroVysion nd ucyt. The former is more commonly used. These 2 ssys re generlly nlyzed y others, rther thn Tle 5. Performnce Mesure, n ¼ 13 Assy Performnce in Benign Ctegory ucyt Comined True positives, No. 0 0 True negtives, No Flse negtives, No Flse positives, No Sensitivity, % N/A N/A N/A Specificity, % PPV, % N/A N/A N/A NPV, % N/A N/A N/A P vlue c.05 versus ucyt P vlue c.01 versus P vlue c ucyt versus.34 Arevitions: N/A, not pplicle; NPV, negtive predictive vlue; PPV, positive predictive vlue. ntiody, BD Dignostics, Burlington, North Crolin. c P vlue s determined y 2-til, pired t test. Tle 6. Performnce Mesure, n ¼ 31 Assy Performnce in Low-Grde Ctegory ucyt Comined True positives, No True negtives, No. 9 c 4 c 3 c Flse negtives, No Flse positives, No. 4 c 9 c 10 c Sensitivity, % Specificity, % PPV, % NPV, % P vlue d ProExC.90 versus ucyt P vlue d.36 versus P vlue d ucyt versus.26 Arevitions: NPV, negtive predictive vlue; PPV, positive predictive vlue. ntiody, BD Dignostics, Burlington, North Crolin. c Dt imported from Tle 5. d P vlue s determined y 2-til, pired t test. y the primry cytotechnologists or y cytopthologists, imposing disdvntge for the 2 ssys. UroVysion is multitrget multicolor fluorescence in situ hyridiztion ssy tht exmines the normlities of 4 chromosomes tht commonly occur in urothelil crcinoms. 29 A metnlysis study performed y Hjdink 30 in 2008 showed sensitivity of 72% nd specificity of 83% for the FISH ssy. 29 However, tht test is n expensive nd timeconsuming ssy nd hs its own pitflls. 31 Comprison of nd UroVysion cn e the suject of nother study. The focus of the current investigtion ws to compre ucyt with the ssy. The first pulished study 22 using in urinry cytology presented quite fvorle test chrcteristics for in defining definitive enign nd mlignnt Tle 7. Performnce Mesure, n ¼ 50 Assy Performnce in High-Grde Ctegory ucyt Comined True positives, No True negtives, No. 9 c 4 c 3 c Flse negtives, No Flse positives, No. 4 c 9 c 10 c Sensitivity, % Specificity, % PPV, % NPV, % P vlue d ProExC.03 versus ucyt P vlue d.01 versus P vlue d ucyt versus.26 Arevitions: NPV, negtive predictive vlue; PPV, positive predictive vlue. ntiody, BD Dignostics, Burlington, North Crolin. c Dt imported from Tle 5. d P vlue s determined y 2-til, pired t test Arch Pthol L Med Vol 138, Septemer 2014 & ucyt in Urine Cytology Vergr-Lluri et l

7 cytologic specimens for predicting oth LGUC nd HGUC. In prticulr, the study showed tht ws most useful in strtifying the dignosis when the cytology ws nondefinitive (ie, AUC). Therefore, this study focused solely on the cses with cytology dignosis of AUC. Following the previous study, our current series highlights severl importnt points regrding in the urine cytology setting. First, the ssy performnce prmeters were comprle to those previously pulished. 22 In prticulr, y segregting LGUC cses from HGUC cses, s we did in this report, displyed lower sensitivity in detecting LGUC (72%; Tle 6) thn in detecting HGUC (92%, Tle 7), s lluded to in the previous puliction. 22 A recently pulished study, 32 using histologic sections of the urothelil neoplsi, hs demonstrted tht the stining involves the full thickness of the cncerous epithelium in HGUC, wheres the rection is focl in LGUC nd the positive cells my not rech the surfce for exfolition into the urine smples. This oservtion my ccount for the lower sensitivity of the ssy in LGUC. Overll, oth ssys showed sensitivity of 85% (47 of 55) for identifying urothelil crcinoms, with more fvorle PPV for ProEx C (92%; 47 of 51) thn for ucyt t 84% (47 of 56; Tle 4). In ddition, it ppers tht ucyt hs significntly lower specificity thn did (31% [4 of 13] nd 69% [9 of 13], respectively). Specificity nd the issue of flse-positive testing hs lwys een mjor criticism for urinry ncillry tests. 31 Although ssy specificity for ucyt hs een reported to e nywhere from 62% to 84.2%, 33 the specificity does decrese to 49% in AUC settings. 34 The specificity of ucyt ws even lower in our current study (31%; 4 of 13) likely ecuse of the use of the definitive histologic dignosis s the gold stndrd, rther thn cystoscopy, softer reference stndrd used y Odisho et l. 34 In contrst, t 69% (9 of 13), our study shows tht demonstrted higher specificity thn ucyt did. Tken together, lthough oth ssys hd the sme overll sensitivity, hd much etter specificity. Second, is more fvorle ncillry test thn ucyt ecuse urinry cytology slides will e exmined longside the stin y the sme cytotechnologists nd pthologists, requiring no specil expertise or dditionl certifiction. Finlizing ucyt report entils using fluorescence microscopy nd otining dditionl certifiction to estlish competency in test interprettion; such n ctivity is preferly undertken with the screening skills of proficient cytotechnologist nd the susequent review of specilly trined cytopthologist. Thus, overll, the immunofluorescence ucyt ssy is more cumersome nd more costly to perform thn the ssy. Therefore, ProEx C is more user-friendly ncillry test thn ucyt for cytotechnologists nd pthologists. Third, in cses of LGUC lone, the comintion of the 2 tests resulted in significnt improvement in the ssys sensitivity (Tle 6). Cytology lone hs low sensitivity, rnging from 5% to 18% in detecting LGUCs 33 ; however, when, nd ucyt ncillry tests mrkedly improved the sensitivity (94%; 17 of 18) in the current series. Although LGUC is generlly considered n re of poor performnce in urinry cytology, mny would rgue tht cystoscopic exmintion nd iopsy/resection re etter methods for estlishing the dignosis of LGUC thn re positive ncillry mrkers. 35 On relted note, the clinicl consequences of positive ncillry mrkers in detecting HGUC is much greter in the lredy-chllenging dignostic setting of AUC. Indeed, we note n impressive sensitivity of 92% (34 of 37) in detecting HGUCs using lone or in comintion with ucyt (Tle 7). In summry, when the 2 ssys re, the overll, s well s the ctegorized ssy, sensitivity will rise to 92% or greter (Tles 4, 6, nd 7). However, the poor specificity of the ucyt ssy (31%; 4 of 13) mkes the comintion of the 2 ssys (23%; 3 of 13) even less desirle (Tles 4 nd 5). 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