STUDY. Confocal Laser Scanning Microscopy vs 3-Dimensional Histologic Imaging in Basal Cell Carcinoma

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1 STUDY Confocl Lser Scnning Microscopy vs 3-Dimensionl Histologic Imging in Bsl Cell Crcinom Srh Ziefle; Deorh Schüle; Helmut Breuninger, MD; Wilfried Schippert, MD; Mtthis Moehrle, MD Ojective: To compre ex vivo confocl lser scnning microscopy (CLSM), which offers rpid imges without the need for tissue processing, vs 3-dimensionl histologic imging, the criterion stndrd tretment for sl cell crcinoms in high-risk res of the fce. Design: Single-center prospective tril. Setting: Dermtosurgicl unit of university hospitl. Ptients: Seventy-two consecutive surgiclly removed sl cell crcinoms were exmined using CLSM vs stndrd prffin-emedded 3-dimensionl histologic imging. Interventions: A totl of 312 imges, including 73 midsections, 196 lterl mrgins, 23 muffins, nd 20 red lof sections, were otined using CLSM. Immeditely fter surgery, the CLSM imges were evluted y the surgeon. The following dy, the 3-dimensionl histologic slides were evluted nd compred with the CLSM imges. Min Outcome Mesures: Dignostic ccurcy of ex vivo CLSM to detect tumor strnds of sl cell crcinoms nd the prcticlity of using CLSM vs 3-dimensionl histologic slides in microgrphic surgery. Results: The sensitivity of CLSM reched 94.0% in midsections, 73.7% in lterl mrgins, 80.0% in muffins, nd 80.0% in red lof sections. The CLSM imges were evluted y the surgeon within 7.5 minutes. Conclusions: Confocl lser scnning microscopy lcks high sensitivity to detect smll tumor strnds of sl cell crcinoms. In the future, CLSM my represent timesving nd less expensive lterntive to cryostt histopthologic exmintion. Arch Dermtol. 2010;146(8): Author Affilitions: Deprtment of Dermtology, University Hospitl, Eerhrd Krls University, Tüingen, Germny. BASAL CELL CARCINOMA (BCC) is the most common type of skin tumor in the world. 1 Principlly ffected re sunexposed ody surfces, predominntly the hed nd neck re. Excision y microgrphic surgery (using 3-dimensionl histologic imging) is the criterion stndrd tretment for BCCs in high-risk res of the fce, offering precise excision with miniml loss of surrounding helthy tissue. Surgicl removl of the tumor is followed y complete visuliztion of the 3-dimensionl excision mrgins vi the cryostt or prffin technique. For editoril comment see pge 909 In Mohs microgrphic surgery, the excised tumor specimens re sliced, pressed fltonthemicrotomeofcryostt, ndevluted using frozen sections. If tumor is identified t the deep mrgin periphery, the procedure is repeted with nother stge of mrking nd excision. Ech stge of removl nd exmintion tkes 20 to 45 minutes. In recent yers in Europe, 3-dimensionl histologic imging hs gined cceptnce s technique of choice using the Tüinger torte 2-5 or the muffin method. 6,7 For the Tüinger torte, nrrow lterl strip is excised verticlly round the full perimeter of the excised tissue, providing complete visuliztionoftheexcisedtumormrgins. For the muffin, the excised tumor se is plced in the sme lyer s the mrgins, providing complete 3-dimensionl view of the lterl excisionmrginsndtheseinsingleslide. All slides (Tüinger torte nd muffin) re emedded in prffin nd then stined with hemtoxylin-eosin. Prffin sections re ville within 20 hours. The procedure is repeted until tumor-free mrgins re otined. Ex vivo confocl lser scnning microscopy (CLSM) is newly developed procedure tht my represent n ttrctive lterntive to frozen histologic or prffin sections. 8,9 Immeditely fter excision of the 843 Downloded From: on 03/11/2018

2 A Tüinger torte B Muffin technique C Bred lof technique c c Figure 1. Techniques of tissue preprtion. A, Nrrow lterl strip excised verticlly 360 round the full perimeter of the tumor order (, the 0- to 6-o clock position;, the 6- to 12-o clock position) nd horizontl section tken from the ottom. B, Mrgins cn e folded lterlly to the horizontl plne. C, 1-mm sections of fresh tissue, here extrcted in 3 steps (,, nd c). Adpted from the study y Moehrle et l 10 with permission from Wiley-Blckwell, Oxford, Englnd. Figure 2. Confocl lser scnning microscopy mosic imge of nodulr sl cell crcinom fter incution of the specimen in 10% cetic cid for 90 seconds. Acetic cid incution efore confocl lser scnning microscopy imging induces compction of chromtin, which increses light cksctter nd renders nuclei detectle. 11 tumor, CLSM imges cn e evluted without the need for tissue processing. The ojective of this study ws to nlyze the potentil use of ex vivo CLSM for rpid visuliztion of excised BCC surgicl mrgins in high-risk res of the fce. METHODS EXCISION AND STAINING OF TISSUE Seventy-two consecutive surgiclly removed BCCs were exmined in the Deprtment of Dermtology, University Hospitl, Eerhrt Krls University in Tüingen, Germny. For 3-dimensionl histologic slides, the prffin technique ws used. The tumor specimens were further dissected using the following techniques: Tüinger torte, muffin, nd red lof (using 1-mm step sections) 10 (Figure 1). The Tüinger torte technique involves excision of the tumor nd clinicl sfety mrgin using verticl or even overhnging incision lines. From the specimen (ex vivo), nrrow lterl strip is excised verticlly 360 round the full perimeter of the tumor order, eginning with topogrphic mrk t the 12-o clock position reltive to the ody xis. The strip is then lid flt nd cut into pieces, which re plced in routine cssettes for horizontl fixtion. Susequently, horizontl section is tken from the ottom of the excised tumor nd is likewise prepred flt in histologic cssette. Routine overnight prffin fixtion is used. This method provides complete 3-dimensionl view of the excision mrgins. The muffin technique involves excision of the tumor nd clinicl sfety mrgin using verticl or even overhnging incision lines. After deep incisions t the 12-o clock nd 6-o clock positions reltive to the ody xis, nrrow lterl strip is cut without dissecting the se. Similr to peeling off muffin s pper mold, these mrgins cn e folded lterlly to horizontl plne. The specimen is plced in routine cssette for horizontl fixtion. The specimens, which re ville 20 hours lter, provide complete 3-dimensionl view of the excision mrgins nd the se on single slide. This technique my e used for specimens up to 2 cm in dimeter. The red lof technique involves excision of the tumor nd clinicl sfety mrgin using verticl or even overhnging incision lines. After topogrphic orienttion of the tumor, 1-mm sections of fresh tissue were extrcted nd plced in routine cssette for fixtion. The slides were ville 20 hours lter. All 72 tumor specimens were resected with sfety mrgin of 2 to 4 mm round the cliniclly visile tumor. As previously descried, 11 incution of the specimens in cetic cid efore CLSM imging induces compction of chromtin, which increses light cksctter nd renders nuclei right nd esily detectle. After testing different methods (eg, polyvinylpyrrolidone-iodine preprtions, eosin, methylene lue, nd toluidine lue) to improve stining of BCC specimens, toluidine lue yielded the most precise nd cler imges (Figure 2 nd Figure 3). For this study, tissue ws soked in 10% cetic cid for 90 seconds, stined in toluidine lue for 2 minutes, nd imged y CLSM. After imging, the specimens were plced in routine cssette, nd tissue ws prepred using the stndrd prffin method with hemtoxylin-eosin stining. CONFOCAL LASER SCANNING MICROSCOPY A modified version of commercilly ville confocl lser scnning microscope (VivScope 2500; Lucid Inc, Rochester, New 844 Downloded From: on 03/11/2018

3 solide Figure 3. Confocl lser scnning microscopy mosic imge of nodulr sl cell crcinom shown in Figure 2 fter incution of the specimen in 10% cetic cid for 90 seconds nd in toluidine lue for 2 minutes, providing etter contrst. Figure 5. Corresponding printed mosic imge of slide shown in Figure 6. The tumor detected y confocl lser scnning microscopy ws highlighted in red on the printout y the surgeon immeditely fter opertion. The next dy, the surgeon received corresponding hemtoxylin-eosin stined prffin sections nd highlighted the tumor islnd in green on the printout. Figure 4. Confocl lser scnning microscopy mosic imge of nodulr sl cell crcinom mrgin. York) ws used; this version ws designed for imging ex vivo excised fresh tissue. The design nd instrumenttion detils for this microscope were reported previously. 8,9,12 The illumintion wvelength is 830 nm from diode lser, with n illumintion power of less thn 16 mw t the tissue level. An 30 mgnifying wter immersion ojective lens (Lucid Stle View, Lucid Inc) with 0.9 numericl perture ws used, providing field of view of mm 2. The principle ws to otin totlly flt surfce on the cover glss of the inverse microscope without dmging the fresh tissue. For hndling lrge fresh specimens, cm plstic squre with 4 4-cm cvity in the middle filled with cellulr mteril s hydrophilic fom dressing (Curform Plus; Kendll Compny, Mnsfield, Msschusetts) ws constructed so the tissue could e pressed without dmging it. Another squre of plstic ws plced ove the cellulr mteril. The squre ws weighted with 1 to 6 cues of led (10-60 g totl). Adequte pressure ws pplied to the cellulr mteril to ring the excision mrgins into single flt plne. Illumintion ws utomticlly djusted to otin confocl imges. The depth hd to e mnully djusted. Becuse of the confocl field of view (0.25 mm 2 ), sequences of imges were stitched together to form confocl mosic. The imging of mm mosic took 150 seconds, nd the susequent stitching required out 30 seconds. All mosics could e oserved directly on the microscope disply using the zoom function. COMPARISON OF CONFOCAL MOSAICS WITH CONVENTIONAL HISTOPATHOLOGIC SLIDES Figure 6. Hemtoxylin-eosin stined histologic slide of the mrgin with nodulr sl cell crcinom outgrowths corresponding to the confocl lser scnning microscopy imges shown in Figure 4 nd Figure 5. Immeditely fter surgery, ll imged confocl mosics of n excised tumor specimen were evluted y the ttending surgeon (H.B., W.S., or M.M.) on the microscope disply. All 3 surgeons re experienced dermtopthologists with longstnding experience in evluting slides of skin tumors nd tumor mrgins in microgrphic surgery. They were fmilir with CLSM from their work on 2009 study. 7 Before performing tht study, ll 3 surgeons hd received forml introduction to interprettion nd evlution of CLSM imges. The imged mosics (Figure 4) were printed so the BCC nd suspicious res could e highlighted in red on the printout y the surgeon immeditely fter the opertion (Figure 5). The next dy, the surgeon red the corresponding hemtoxylin-eosin stined histologic slides (Figure 6); tumor islnds were highlighted in green (Figure 5). The study ws pproved y the ethics committee of the University Hospitl, Tüingen, Germny. RESULTS A totl of 312 imges, including 73 midsections, 196 lterl mrgins, 23 muffins, nd 20 red lof sections, were otined using CLSM from 72 surgiclly removed BCCs. These results re summrized in Tle 1. CLSM FOR RAPID EXAMINATION OF SURGICAL EXCISIONS On verge, the preprtion nd stining of tissue for CLSM took 4.5 minutes, including 0.5 minute for dissection of the surgicl specimen, 1.5 minutes for immersion in cetic cid, 2.0 minutes for toluidine stining, nd 0.5 minute for fixtion of tissue on the cover glss. Imging nd stitching of 143-mm 2 imges required 3.0 minutes t most. Ech mosic ws redy for evlution y the surgeon within 7.5 minutes. In contrst to previous study, 7 lrger imges of mm could e viewed, nd the zoom function rnged from to mm. 845 Downloded From: on 03/11/2018

4 SENSITIVITY AND SPECIFICITY OF BCC DETECTION The sensitivity nd specificity of BCC detection vried cross midsections, lterl mrgins, muffins, nd red lof sections. The overll sensitivity of CLSM ws 94.0% in midsections (Tle 2). Midsections hve high proility of showing lrge portions of BCC nests. The overll sensitivity of CLSM ws 73.7% in lterl mrgins with smller tumor strnds. The ccurcy of CLSM dignoses vried sed on the section type nd the oserver. These results re summrized in Tle 3. COMMENT Three-dimensionl histologic imging with frozen or prffin histologic exmintion is time-consuming nd expensive. It requires specilized equipment nd trined personnel. In contrst, CLSM my represent timesving nd less expensive lterntive for microgrphic surgery. With CLSM, imges of fresh excised tissue cn e otined nd evluted within 7.5 minutes. Tle 1. Confocl Lser Scnning Microscopy Results Among 312 Imges From 72 Surgiclly Removed Bsl Cell Crcinoms Sutype Midsections (n=73) Lterl Mrgins (n=196) Muffins (n=23) Bred Lof Sections (n=20) Nodulr Infiltrtive Mixed Superficil Tumor free Histologic slide not evlule In smll 13 nd lrge 7 recent studies, tumors nd their mrgins were evluted to ssess the reliility of CLSM compred with stndrd histologic exmintion. Suggested recommendtions nd technicl improvements were incorported into the present study. The use of midsections, lterl mrgins, muffins, nd red lof sections covers rod spectrum of techniques nd reflects the introduction of CLSM into dily surgicl routine. Immersion in cetic or citric cid rightens the morphologic structures of the nucleus in CLSM. 11,13 However, it is insufficient for identifiction of ll BCCs, ecuse smll nests of infiltrtive BCCs were often overlooked. 7,8,13 Toluidine lue is not considered 100% cncer specific ut hs een used in clinicl prctice for stining vrious crcinoms in vivo. 14 With this stining method, considerly higher contrst of tumor cells compred with norml dermis is otined, which likely explins the improved sensitivity herein compred with previous results. 7 The nucleus nd tumor structure re more esily identified. However, except for midsections, sensitivity herein for mrgins did not rech 90%, which would e required to replce the current method of stndrd histopthologic exmintion. Becuse of technicl improvements, complete mosic of 312 confocl imges cn e oserved directly on the microscope disply. For the first time, to our knowledge, we were le to integrte CLSM s stndrd procedure in dily surgicl routine. Imges were ville immeditely fter surgery. Therefore, we were le to evlute specimens t the edside of the ptient to decide immeditely out further tretment. Another new development in CLSM is zoom function, which llows closer view of the specimen up to field of view rnging from to mm 2.In the horizontl imging direction, the stitches overlpped y 1% to 2%, which ided in correct interprettion of the imges. However, in the verticl imging direction, the stitches overlpped y 8% to 10%, which mde Tle 2. Accurcy of Confocl Lser Scnning Microscopy in Detecting Bsl Cell Crcinoms Vrile Sensitivity Specificity Predictive Vlue % Negtive Predictive Vlue Flse Flse Negtive Midsections (n=69) Lterl mrgins (n=190) Muffins (n=23) Bred lof sections (n=19) The re of the confocl lser scnning microscopy imge did not correspond to the hemtoxylin-eosin stined slide ecuse of technicl rtifcts in 4 midsections, 6 lterl mrgins, nd 1 red lof. Tle 3. Accurcy of Confocl Lser Scnning Microscopy Dignoses Bsed on Rtes of the Individul Oservers Oserver Sensitivity Specificity Predictive Vlue % Negtive Predictive Vlue Flse Flse Negtive 1 (n=127) (n=62) (n=123) Downloded From: on 03/11/2018

5 correct evlution more difficult. Therefore, stitching qulity needs improvement. A complete mm mosic could e otined in less thn 3 minutes. Compred with previous studies 15,16 of CLSM in which t lest 9 minutes ws required, this is tremendous increse in imging speed. Another time-sving fctor ws the utomtic rightness nd contrst djustment efore imging. It ws chllenging to quickly otin flt surfce of fresh excised tissue without dmging it. In 2009 study, 17 tissue ws frozen nd cut for cryostt-sectioned mrgin control, with susequent cretion of CLSM imges. Tht procedure ws esier ut did not evlute the true outer surfce of the excisions. The histosurgicl flttening of the specimen incresed the risk of flse-positive results. With the tissue-mounting method used in this study, flt surfce ws otinle for lmost ll sizes nd thicknesses of fresh excised tissue nd voided the freezing step, which is undesirle t the edside. Asforementioned, llsurgeonshereinhdsimilrknowledge nd experience in evlution of norml histopthologic slides nd CLSM imges. However, the sensitivity nd specificity results of these slides nd imges vried considerly. Becuse of the immedicy of evlutions during or etween surgicl procedures, fctors such s time pressure, stress, or ftigue could hve influenced the results. Vrition in results mong the 3 surgeons my reflect the rel-life setting of the study. Unfortuntely, the 3 surgeons hd no further experience with CLSM etween the previous study 7 nd the present study, which mde interprettion of the first cses herein more difficult. A refresher introduction might hve een helpful. Future investigtions my focus on the ility of pthologists to evlute confocl imges. Digitl CLSM imges could e sent from the surgicl theter to pthologist trined in interprettion of CLSM imges, enling surgeons without specific dermtopthologic trining to perform rpid microgrphic surgicl procedures. There ws continuous improvement in hndling nd fixing the fresh tissue. This fctor might hve influenced the interoserver vriility. However, BCC identifiction on imges of microgrphic surgery specimens did not rech the desired rte. In recent studies, 15,17,18 fluorescent mrker ws used in CLSM, which fcilitted BCC identifiction on confocl imges nd resulted in high specificity. These studies were performed in lortory nd not in clinicl setting. Specific fluorescence could e used in comintion with the microscope used herein, offering fst imging nd correct interprettion directly in the operting theter. This would represent revolution in microgrphic surgery. In conclusion, CLSM lcks high sensitivity to detect smll tumor strnds of BCCs. Trining surgeons to red CLSM imges is essentil. For microgrphic surgery, CLSM my represent time-sving nd less expensive lterntive to cryostt histopthologic exmintion. Accepted for Puliction: Octoer 15, Correspondence: Mtthis Moehrle, MD, Deprtment of Dermtology, University Hospitl, Eerhrd Krls University, Lieermeisterstr 25, Tüingen, Germny (mtthis.moehrle@med.uni-tueingen.de). Author Contriutions: Ms Ziefle nd Dr Moehrle hd full ccess to ll the dt in the study nd tke responsiility for the integrity of the dt nd the ccurcy of the dt nlysis. Study concept nd design: Breuninger nd Moehrle. Acquisition of dt: Ziefle, Schüle, Breuninger, Schippert, nd Moehrle. Anlysis nd interprettion of dt: Ziefle, Schüle, nd Moehrle. Drfting of the mnuscript: Ziefle, Schüle, nd Moehrle. Criticl revision of the mnuscript for importnt intellectul content: Breuninger nd Schippert. Otined funding: Moehrle. Finncil Disclosure: None reported. Funding/Support: The study ws supported in prt y grnt from Mvig GmH. Role of the Sponsors: For the durtion of the study, Mvig GmH furnished the VivScope 2500 nd provided technicl support. Additionl Contriutions: Christoph Meisner, PhD, ssisted in the sttisticl evlution. Yvonne Frnke, Dipl Cult Mgmt, provided technicl support. REFERENCES 1. Jeml A, Siegel R, Wrd E, et l. Cncer sttistics, CA Cncer J Clin. 2008; 58(2): Breuninger H, Schumurg-Lever G. Control of excisionl mrgins y conventionl histopthologicl techniques in the tretment of skin tumours: n lterntive to Mohs technique. J Pthol. 1988;154(2): Breuninger H. Histologic control of excised tissue edges in the opertive tretment of sl-cell crcinoms. J Dermtol Surg Oncol. 1984;10(9): Breuninger H, Dietz K. Prediction of suclinicl tumor infiltrtion in sl cell crcinom. J Dermtol Surg Oncol. 1991;17(7): Moehrle M, Dietz K, Gre C, Breuninger H. Conventionl histology vs. threedimensionl histology in lentigo mlign melnom. Br J Dermtol. 2006;154 (3): Möhrle M, Breuninger H. The muffin technique: n lterntive to Mohs microgrphic surgery. J Dtsch Dermtol Ges. 2006;4(12): Schüle D, Breuninger H, Schippert W, Dietz K, Moehrle M. 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