Research ArticleVimentin 3 the New Hope DifferentiatingRCC versus Oncocytoma

Melanie von Brandenstein1 Katharina Puetz1 Monika Schlosser1

Heike Loumlser1 Joachim P Kallinowski2 Daniel Goumldde3 Reinhard Buettner1

Stefan Stoumlrkel3 and Jochen W U Fries1

1 Institute of Pathology University Hospital of Cologne Kerpenerstraszlige 62 50924 Cologne Germany2Institute of General Visceral and Minimal Invasive Surgery Clinic Northwest Steinbacher Hohl 2-2660488 Frankfurt am Main Germany3Institute of Pathology Helios Clinic Wuppertal University Clinic Witten-Herdecke Heusnerstraszlige 40 42283 Wuppertal Germany

Correspondence should be addressed to Jochen W U Fries jochenfriesuni-koelnde

Received 24 October 2014 Accepted 3 March 2015

Academic Editor Claudio Letizia

Copyright copy 2015 Melanie von Brandenstein et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Vimentin is currently used to differentiate between malignant renal carcinomas and benign oncocytomas Recent reports showingVimentin positive oncocytomas seriously question the validity of this present diagnostic approach Vimentin 3 is a spliced variantand endswith a uniqueC-terminal ending after exon 7which differentiates it from the full length version that has 9 exonsThereforethe protein size is different the full length Vimentin version has a protein size of sim57 kDa and the truncated version of sim47 kDaWedesigned an antibody called Vim3 against the unique C-terminal ending of the Vimentin 3 variant Using immune histologyimmune fluorescence Western blot and qRT-PCR analysis a Vim3 overexpression was detectable exclusively in oncocytomamaking the detection of Vim3 a potential specific marker for benign kidney tumorsThis antibody is the first to clearly differentiatebenign oncocytoma and the mimicking eosinophilic variants of the RCCs This differentiation between malignant and benignRCCs is essential for operative planning follow-up therapy and patientsrsquo survival In the future the usage of Vimentin antibodiesin routine pathology has to be applied with care Consideration must be given to Vimentin specific binding epitopes otherwise amisdiagnosis of the patientsrsquo tumor samples may result

1 Introduction

An oncocyte is an epithelial cell characterized by an excessiveamount of mitochondria Hamperl named them in 1931 afterthe Greek word ldquoonkousthairdquo (to swell) and first describedthem as a distinct cell system consisting of large epithelialcells with irregular nuclei and finely granular acidophiliccytoplasm [1] The fundamental morphological nature ofoncocytes an abundance of mitochondria was firmly estab-lished by electron microscopy [2] Since then oncocytes havebeen detected in various organs (ie thyroid parathyroidand salivary glands) as well as in different tumors (ieoncocytomas Hurthle cell tumors of the thyroid oxyphilic

adenoma of parathyroid gland and Warthinrsquos tumor of sali-vary gland) (encyclopedia of Biol Chem 2004)

Renal oncocytomas initially identified by Zippel in1942 [3] have been regarded as predominantly benign renalneoplasms since the first study by Klein and Valensi [4]although occasional reports of malignant cases have beenreported [5] The major diagnostic problem is the differentialto other renal tumors (i) the eosinophilic or granular variantof clear cell renal carcinoma (RCC) and (ii) the chromo-phobe RCC Differential diagnosis currently uses immunehistology to differentiate malignant renal cell carcinomafrom oncocytoma For chromophobe carcinoma positivityfor claudin 8 and negativity for claudin 7 have been shown

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 368534 8 pageshttpdxdoiorg1011552015368534

2 Disease Markers

as the characteristic constellation [6] To differentiate thechromophobe and eosinophilic RCC from oncocytoma thepositivity for Vimentin a structural protein has been usedto identify the former [7] However a series of oncocytomashas recently been reported in which a Vimentin positivityhas been observed making the differentiation questionableparticularly in preoperative evaluation [8] Hes et al analyzed234 oncocytoma of which 73 were positive for Vimentinstaining [8] Vimentin is an intermediate-sized filament thatfunctions in cellular signal transduction structural integrityof cells and tissues and adhesion and migration [9] In2007 a spliced variant of Vimentin with a unique C-terminalending was detected by a working group at the Craig VenterInstitute (NHLBI Resequencing and Genotyping Service(RSG) N01-NV-48196 J Craig Venter Institute RockvilleMD 20850) and published online in PubMed (AccessionnumberACA061031) In 2011Thakkar et al [10] described thepresence of this variant in gliomas However no further anal-ysis or investigation regarding its role has been performed

Based on the knowledge that the spliced variant ofVimentin is 35 amino acids smaller than the full lengthvariant we compared both sequences with the detailed infor-mation of the Vimentin 3B4 antibody From the literatureit is known that the 3B4 Vimentin antibody detects the roddomain [11] which is a homologue to the truncated Vimentinvariant 3 (Vim3) rod domain Thus it seemed possible thatthe protein expression of Vimentin described in the literatureby immune histology resulted from the combined detectionnot only of the protein from full length but also of the splicedvariant of Vimentin namely Vim3

Most of the commercially available antibodies (clones 3B4and SP20) are against epitopes located in the rod domain ofVimentin (Figure 1)The clone V9 is directed against the tail-domain of Vimentin However for the detection of the trun-cated Vimentin variant 3 (Vim3) the Vim3 antibody is usedwhich is designed against the unique C-terminal ending

In case of renal tumors with eosinophilic apperancewhich mimic oncocytoma the differential diagnosis betweenRCCs and oncocytomas is based on a panel of differentantibodies In particular the presence or absence of Vimentinstaining of paraffinized tumor samples can be of great impor-tance for the differentiation between malignant and benigntumors

This diagnostic approach has to be reevaluated sincea spliced Vimentin isoform exists This is also detectablewith the currently used antibodies against the N-terminalsequence thus Vimentin positivity is no longer a diagnosticfeature per se of malignant RCCs Thus in this paper weanalyzed the presence ofVim3 versus the full lengthVimentinin RCCs especially the eosinophilic variant of RCCs versusoncocytomas

We designed primers which detect either the full lengthversion of Vimentin or its spliced variant Vim3 Afterperforming a qRT-PCR on paraffin embedded tissues of thedifferent RCCs and oncocytoma we could indeed show thatVim3 is the predominant variant in oncocytoma Further-more we designed an antibody exclusively detecting theunique C-terminus of Vim3

V9 antibody

V3 antibody 3B4 antibody SP20 antibody

The antibody

1 2 3 4 5 6 7 8 9 39984005998400

Vimentin FL sim57kDa Vimentin 3 sim47kDaNH2NH2

COOHCOOH

Figure 1 Differential location of the different antibodies againstVimentin The commercially available antibodies of the clones 3B4and SP20 were against the rod domain (slight blue) the antibodyclone V9 is against C-terminal ending of the full length variant(slight purple) and the truncated antibody against the unique C-terminal ending Vim3 is indicated with dark blue

This is the first report describing the presence and thestructural differences of Vim3 versus the full length Vimen-tin Our data present strong evidence that Vim3 is the isoformresponsible for the so-called Vimentin positive oncocytomasdescribed in the literature Furthermore we show that theV9 Vimentin antibody as well as antibodies detecting thefull length version of Vimentin cannot be used any longerfor differential diagnosis between RCCs and oncocytomasbecause these result in misdiagnoses with potentially graveconsequences for the patients involved

2 Materials and Methods

21 Antibody Design and Quantification The Vim3 anti-body was commercially designed (EZbiolab Inc) using theunique C-terminal ending of Vim3 as target (for detailedinformation please see patent by University of CologneBrandensteinFries patent number EP 131608762-1405) TheVim3 expression versus that of full length Vimentin (cloneV9) (Santa Cruz Heidelberg) was shown using immunehistology on paraffin embedded colon mucosa biopsies fromour pathology archive Western blot analysis (see below)of macrodissected material of cryptal epithelial cells andlymphoid cells was performed for further evaluation andproof of specificity of the newly designed antibody

22 Immune Fluorescence of ParaffinEmbedded Tissues 4 120583mthick paraffin embedded tissue sections were deparaffinizedby incubation for 1 x 10min in xylene followed by 1 x 5min100 ethanol and 1min 70 ethanol and then rinsed withdistilled waterThe slides were digested with Proteinase K for30min at room temperature After an incubation period in5PBSmilk for 30min the slideswere reincubated for 1 hourat room temperature with specific primary antibodies (Vim3)in 3 PBS milk Following washes with PBS the sectionswere incubated with a secondary FITC-anti-rabbit antibody(Santa Cruz) Subsequent to rinsing with PBS the slides werethen counterstained with DAPI mounting medium (nuclearstaining) and cover slipped

Disease Markers 3

23 Immune Histology of Paraffin Embedded Tissues Paraffinembedded tissue sections (4120583mthick)were deparaffinized byincubation for 2ndash5minutes in xylene followed by 2-3minutesin 100 ethanol and 1minute in 95 ethanol and then rinsedwith distilled water The slides were incubated with a specificserum blocker (anti-rabbit) for 30 minutes in order to avoidnonspecific binding After that incubation period the slideswere reincubated for 1 hour at room temperaturewith specificprimary antibodies (Vim3 EZBiolab Inc Carmel USAVimentin V9 Santa Cruz Heidelberg Germany AMACRand CD117 [12] Dako Hamburg Germany) Followingwashes with PBSndashTween 20 the sections were incubated witha secondary anti-rabbit antibody (Santa Cruz HeidelbergGermany) After rinsing with PBSndashTween 20 the slides werereincubated for 2 minutes in 95 ethanol followed by 2-3minutes in 100 methanol counterstained with HampE andcover slipped

The analyzed paraffin embedded tissue sections werefrom retrospective nephrectomies nevertheless we per-formed a blind study so any bias of the results could beexcluded

24 Oncocytic Tumors Since human materials were usedprocedures were followed as outlined in accordance with eth-ical standards formulated in the Declaration of Helsinki 1975with preapproval by the Ethics Committee at the UniversityHospital Cologne Germany (reference number 09-232)

25 Quantitative Real-Time PCR (qRT-PCR) The qRT-PCRwas performed as previously described [13 14]

For quantitative analysis 120573-actin was measured Allsamples were normalized to 120573-actin as the reference geneAll experiments were performed in triplicate Relative fluo-rescencewas calculated using theΔΔ-CTmethod as outlinedin User Bulletin 2 (PE Applied Biosystems Darmstadt Ger-many)The statistical significance of qPCR values at differenttime points was assessed by Studentrsquos paired 119905-test Table 2provides primer information

26 RNA-Extraction Paraffin Embedded Tissues and RT-PCR Formalin-fixed and paraffinized (FFPE) human tissuesamples from the archives of the Department of PathologyUniversity Hospital of Cologne Cologne Germany and theDepartment of Pathology Helios Clinic Wuppertal Uni-versity Clinic Witten-Herdecke Wuppertal Germany wereused

RNA extraction from FFPE tissue was performed accord-ing to the RNeasy FFPE kit (Qiagen Germany) RNA quan-tification was accomplished using NanoDrop technology

The cDNA was obtained from 250 ng of RNA usingrandom primers and SuperScript III reverse transcriptaseaccording to the manufacturerrsquos protocol (Invitrogen Darm-stadt Germany)

27 Statistical Analysis For statistical analysis the GraphPadPrism 5 program was used Analysis of variance (ANOVA)was performed and the significant differences were calculatedand indicated by stars (lowast119875 lt 005 lowastlowast119875 lt 001 and

lowastlowastlowast

119875 lt 0001) All differences without indication were notstatistically significant

28 Western Blot All Western blots were performed intriplicate as outlined in detail before (Gerstung et al [13])120573-actin served as loading control (Santa Cruz HeidelbergGermany) The Vimentin 3 antibody was used in a 1 500dilution and the V9 antibody (Santa Cruz) against fulllengthVimentinwas employed in 1 1000 as recommended bythe supplier Protein extraction from paraffinized tissue wasdone as described in Ikeda et al [15] The 4 120583m paraffinizedtissue samples were incubated in Xylol for 15 sec mixedand then centrifuged for 2min at full speed and at roomtemperature 100 ethanol was added to the pellet for 2minthen mixed and again centrifuged for 2min at full speedand at room temperature After carefully discarding thesupernate the pellet was air dried 50120583L of RIPA buffer wasadded incubated at 100∘C for 20min and then followed byan incubation period of 2 hours at 60∘C The samples weresubsequently centrifuged at full speed at 4∘C for 20min Thesupernate was then stored at minus80∘C until further use Proteinquantification was performed as previously described [13]

3 Results

31 Antibody Evaluation Since Vimentin is commonlyknown primarily as a mesenchymal marker we characterizedtheVim3 antibody using frozen sections of appendiceal tissuecontaining epithelial mesenchymal and lymphatic tissueelements As Figure 2 shows Vim3 was expressed in coloniccrypt epithelium particularly in the regeneratively active partof the crypt in mesenchymal cells and in lymphocytes AWestern blot was performed to verify the expected size ofthe Vim3 splice form being 47 kDa (Figure 2) while the fulllength molecule was predictably 57 kDa (data not shown)

We also established the Vim3 antibody binding pattern inrenal tissues The Vimentin full length molecule was evidentin different types of mesenchymal cells (such as fibroblastsand smooth muscle cells) and also in proximal tubule cells

32 mRNA Detection of Vimentin and Vim3 The full lengthmolecule of Vimentin is used as a marker to differentiatebenign oncocytomas expected to be negative from malig-nant renal cell carcinomas beingVimentin positive Our qRT-PCR evaluation of renal tumors confirmed this finding incases from the pathology archives while demonstrating thatfull length Vim3 was expressed in (Table 1) Onco (Oncocy-toma) RCC subtypes express lower levels of Vim3 mRNA

33 Protein Detection of Vim3 versus Full Length Vimentinin RCCs By immune histology on paraffinized tissue slicesfrom renal tumors full length Vimentin protein was found tobe strongly expressed in clear cell RCCs and papillary RCCsChromophobe RCCs showed a weak reactivity with theantibody while oncocytomas demonstrated no reactivity Incontrast Vim3 expression was strong in oncocytomas whileall three malignant RCCs subtypes were negative (Figure 4)

4 Disease Markers

Negative tissue

Crypt

Lymphoid tissue

(a)

Negative tissue Lymphoid tissue Crypt

Vim3

sim43kDa

sim47kDa

120573-actin

(b)

Figure 2 Evaluation of Vim3 antibody (a) Immune histology showing expression of Vim3 in colonic crypt epithelium and in lymphocytes(b) Western blot analysis after macrodissection of crypt epithelium and lymphocytes 120573-actin serves as loading control

Table 1 Tumor types and patient number

Patient number Diagnosis1ndash6 Normal kidney control7ndash22 Oncocytoma23ndash33 Chromophobe RCC34ndash44 Papillary RCC45ndash54 RCC55ndash60 Eosinophilic RCC

Consequently using immune fluorescence analyses ofthe different RCC subtypes and the oncocytoma a clearexpression of Vim3 was only detectable in oncocytomaThe oncocytoma mimicking variant of RCC namely theeosinophilic variant was negative for Vim3 (Figure 5)

34 Collision Tumor To further demonstrate the applicabil-ity of the new Vim3 antibody a collision tumor consisting oftwo different tumor subtypes was used Figure 6 shows theHampE staining of its papillary RCC differentiation Since thepatient suffered from pleural metastases it was important toidentify their origin Therefore we performed an immunefluorescence staining for Vimentin FL positive (V9) andfor Vim3 This indicated that the first tumor type with V9being positive and Vim3 being negative was the malignantcomponentThe histogenesis of the second tumor type foundin the tissue sample was questionable possibly being a ldquorealrdquooncocytoma After immune fluorescence for Vimentin FL(V9) as well as for Vim3 was performed only the Vim3staining showed positive areas indicating that the secondtumor type was indeed an oncocytoma

0

10

20

30

40Vim3 expression

RFU

RCC Onco CP Pap Eosino

lowastlowast lowastlowastlowastlowastlowast lowastlowastlowast

Figure 3 qRT-PCR analysis for Vim3 in oncocytoma versus clearcell renal cell carcinoma (RCC) chromophobe RCC (CP) papillaryRCC (Pap) and eosinophilic RCC (Eosino) 120573-actin was used asreference gene lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 and alldifferences without indication are not statistically significant

4 Discussion

In this paper we characterize a Vimentin splice isoformcalled Vimentin 3 (Vim3) as a potentially important struc-tural cellular protein Its unique structure leads to a 10 kDasmaller protein (Figure 1) which is more widely expressedthan its full length counterpart particularly in epithelial cellsand lymphocytes (Figure 2)

To study the importance ofVim3 for renal tubule cells fur-ther we analyzed Vim3 versus full length Vimentin expres-sion by qRT-PCR in renal tumors (Figure 3) Surprisinglywhile RCCs have high amounts of transcribed full lengthVimentin they are almost Vim3 negative In contrast thereverse is true for oncocytomas while their negativity for

Disease Markers 5

Table 2 Primers

Gene Sequence Annealing temp Cycles

120573-actin Forw 51015840-TTGGCAATGAGCGGTTCCGCTG-31015840Rev 51015840-TACACGTGTTTGCGGATGTCCAC-31015840 55∘C 40x

Vimentin full length Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-TCCAGCAGCTTCCTGTAGGTG-31015840 55∘C 40x

Vim3 Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-GAAATAAAATGCTTACCCCTCAG-31015840 55∘C 40x

HampE

Vim3

Vimentinfull length

RCC CP PapNormal kidney Oncocytoma

Figure 4 Immune histological analysis of the expression pattern between oncocytoma and RCC subtypes Full length Vimentin positivetumor cells are observed in clear cell and papillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise arenegative for full length Vimentin HampE staining of typical tumor morphology

full length Vimentin is not surprising (and being a criteriafor their identification) the levels for Vim3 are unexpectedlyhighThe papillary RCC subtype (Pap) has small mRNA levelof Vimentin full length and Vim3 detectable by qRT-PCR

Nevertheless due to some posttranscriptional modifica-tions the Vim3 signal is not detectable by immune histology(Figure 4) while a strong Vimentin (full length) stainingcan be easily detected The only positive signal regardingthe Vim3 was detectable in case of oncocytoma and theldquonormalrdquo tissue section was negative for Vim3 (Figure 4)

This result as well as the immune fluorescence results(Figure 5) identifies Vim3 as potential immune histologymarker for renal oncocytomas

Currently it is still common practice in routine pathologyto differentiate renal cell carcinomas from carcinomas ofhistogenetically different origins by using immune histologywith cytokeratins and Vimentin In particular Vimentinpositivity has been regarded as the major hallmark not onlyfor RCC but also for differentiating them from their benigncounterparts the oncocytomas Since Hes et al [8] reportedVimentin positivity in 73 of all tested oncocytomas thisdiagnostic approach has been questionable while its under-lying mechanism has been elusive

From our results we claim that by using an antibodyagainst the unique C-terminal sequence of Vim3 ldquorealrdquooncocytomas can be unequivocally identified

6 Disease Markers

Vim3

Vimentinfull length

RCCCPPap Eosino Oncocytoma

Figure 5 Immune fluorescence of oncocytoma and RCC subtypes Full length Vimentin positive tumor cells are observed in clear cell andpapillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise are negative for full length Vimentin HampE stainingof typical tumor morphology original magnification times400

Pap

ONCO

HampE AMACR CD117Vimentinfull length Vim3

Figure 6 Collision tumor with two different tumor subtypes Top row papillary RCC bottom row preliminary diagnosis oncocytomaHampE staining and immunohistological staining for AMACR and CD117 Vimentin FL and Vim3 were performed Questionable was thesecond tumor type unexpectedly found in the tissue sample Immune fluorescence staining for Vimentin FL positive (V9) was positive andthe immune fluorescence for Vim3 was negative in the papillary RCC component indicating the malignant tumor subtype whereas thesecond one is an oncocytoma (Vim3 positive)

Disease Markers 7

The exact nature and mechanism of the ldquoVimentinpositive oncocytomasrdquo require further clarification Ourresults indicate that these tumors have to be classified asan eosinophilic variant of clear cell RCCs Since their mor-phologic appearance on an HampE slide seems identical to aldquotruerdquo oncocytoma we performed an immune fluorescencefor Vim3 (Figure 5) This resulted in a clearly Vim3 negativeappearance of these tumors regarded as ldquotruerdquo oncocytomas

However the importance of this study for routine patho-logic diagnoses with respect to the mystery of ldquoVimentinpositive oncocytomasrdquo advocates our current explanation

To date an intracellular role of Vim3 has not beendefined while an intracellular role of the full length Vimentinmolecule has been described in the literature as an anchoringmolecule for the nucleus [16] Knowing the interaction ofits full length counterpart one may speculate about Vim3rsquosintracellular importance Since the N-terminal domain andthe rod domain have not changed binding partners such asankyrin [17] and interactions with plectin [18] should still bepossible In contrast the missing tail and the unique aminoacids of its C-terminal ending may result in differences inthe C-terminal interaction Currently the tail-domain hasbeen reported to be the binding and interactive site for F-actin [19] and lamin B [17] However since the major part ofthe C-terminus is absent in Vim3 and the exact interactionsites for both molecules are presently unknown furtherinvestigations have to be conducted in order to fully elucidatepotential interaction or its absence between Vim3 and otherstructural binding partnersThis protein differentiates benignoncocytoma from malignant RCC variants especially theeosinophilic RCC variant which mimics oncocytoma byimmune histology and immune fluorescence

To strengthen our interpretation we applied the Vim3antibody to a collision tumor (Figure 6) in which weidentified its metastatic component as belonging to thepapillary differentiation being Vim3 negative while the otherpart was identified as an oncocytoma based on its Vim3positivity CD117 expression is a hallmark in differentiat-ing oncocytoma and chromophobe RCC [20] In case ofthe examined collision tumor CD117 positivity could be aproblem since the chromophobe RCC is a malignant tumorand the oncocytoma is a benign one so the differentiationof the metastatic component of the collision tumor is stillquestionable (Figure 6)The further usage of an alpha-methylCoA racemase (AMACR) antibody can be used as well fordistinction between chromophobe RCC and oncocytoma[21] AMACR positivity is seen in papillary RCC [22] and canbe used as marker between primary andmetastatic RCC [21]

Figure 6 shows the two tumor types and a clear differenti-ation between the RCC subtypes namely the papillary part ofthe tumor and the benign part was possible due to the usageof our Vim3 antibody

In conclusion we present here a unique Vimentin iso-form Vim3 as a differential marker between malignantRCCs and oncocytoma We strongly believe that this cleardifferentiation between the benign and malignant kidneytumor types will be essential in the future for patientsrsquo therapyas well as operative planning follow-up therapy and patientsrsquosurvival

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Melanie von Brandenstein was supported by Koeln For-tune ProgramFaculty of Medicine University of Cologne arecipient of a postdoctoral fellowship and excellence clusterinitiative supported by University of Cologne and DFG Theauthors gratefully acknowledge the support by Susanne Sat-tler in immune histology Dr Heike Gobel for her help withthe antibody quantification and critical reading of the paperand Claudia Richter for her excellent technical assistance

References

[1] H Hamperl ldquoUber das Vorkommen von Onkocyten in ver-schiedenen Organen und ihren GeschwulstenmdashMundspei-cheldrusen Bauchspeicheldruse Epithelkorperchen Hypo-physe Schilddruse Eileiterrdquo Virchows Archiv fur PathologischeAnatomie und Physiologie und fur Klinische Medizin vol 298no 2 pp 327ndash375 1936

[2] R Kataoka Y Hyo T Hoshiya HMiyahara and TMatsunagaldquoUltrastructural study of mitochondria in oncocytesrdquo Ultra-structural Pathology vol 15 no 3 pp 231ndash239 1991

[3] J Zippel ldquoZur Kenntnis der Onkocytenrdquo Virchows Archiv vol308 no 2 pp 360ndash382 1942

[4] M J Klein and Q J Valensi ldquoProximal tubular adenomas ofkidney with so-called oncocytic features A clinicopathologicstudy of 13 cases of a rarely reported neoplasmrdquo Cancer vol 38no 2 pp 906ndash914 1976

[5] J D Oxley J Sullivan A Mitchelmore and D A GillattldquoMetastatic renal oncocytomardquo Journal of Clinical Pathologyvol 60 no 6 pp 720ndash722 2007

[6] A O Osunkoya C Cohen D Lawson M M Picken M BAmin and A N Young ldquoClaudin-7 and claudin-8 immuno-histochemical markers for the differential diagnosis of chro-mophobe renal cell carcinoma and renal oncocytomardquo HumanPathology vol 40 no 2 pp 206ndash210 2009

[7] R Waldherr and K Schwechheimer ldquoCo-expression of cytok-eratin and vimentin intermediate-sized filaments in renal cellcarcinomas comparative study of the intermediate-sized fila-ment distribution in renal cell carcinomas and normal humankidneyrdquo Virchows Archiv A vol 408 no 1 pp 15ndash27 1985

[8] O Hes M Michal N Kuroda et al ldquoVimentin reactivity inrenal oncocytoma immunohistochemical study of 234 casesrdquoArchives of Pathology and Laboratory Medicine vol 131 no 12pp 1782ndash1788 2007

[9] J Ivaska H-M Pallari J Nevo and J E Eriksson ldquoNovel func-tions of vimentin in cell adhesion migration and signalingrdquoExperimental Cell Research vol 313 no 10 pp 2050ndash2062 2007

[10] D Thakkar L Shervington and A Shervington ldquoProteomicstudies coupled with RNAi methodologies can shed furtherlight on the downstream effects of telomerase in gliomardquoCancerInvestigation vol 29 no 2 pp 113ndash122 2011

[11] M Malakoutikhah M J Gomara J A Gomez-Puerta R San-martı and I Haro ldquoThe use of chimeric vimentin citrullinatedpeptides for the diagnosis of rheumatoid arthritisrdquo Journal ofMedicinal Chemistry vol 54 no 21 pp 7486ndash7492 2011

8 Disease Markers

[12] P H Tan L Cheng N Rioux-Leclercq et al ldquoRenal tumorsdiagnostic and prognostic biomarkersrdquo The American Journalof Surgical Pathology vol 37 no 10 pp 1518ndash1531 2013

[13] M Gerstung T Roth H-P Dienes C Licht and J W U FriesldquoEndothelin-1 induces NF-120581B via two independent pathwaysin human renal tubular epithelial cellsrdquo American Journal ofNephrology vol 27 no 3 pp 294ndash300 2007

[14] M G von Brandenstein A N Abety R Depping et al ldquoA p38-p65 transcription complex induced by endothelin-1 mediatessignal transduction in cancer cellsrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1783 no 9 pp 1613ndash16222008

[15] K Ikeda T Monden T Kanoh et al ldquoExtraction and analysisof diagnostically useful proteins from formalin-fixed paraffin-embedded tissue sectionsrdquo Journal of Histochemistry and Cyto-chemistry vol 46 no 3 pp 397ndash403 1998

[16] A J Sarria J G Lieber S K Nordeen and R M Evans ldquoThepresence or absence of a vimentin-type intermediate filamentnetwork affects the shape of the nucleus in human SW-13 cellsrdquoJournal of Cell Science vol 107 no part 6 pp 1593ndash1607 1994

[17] S D Georgatos andG Blobel ldquoLamin B constitutes an interme-diate filament attachment site at the nuclear enveloperdquo Journalof Cell Biology vol 105 no 1 pp 117ndash125 1987

[18] R Spurny M Gregor M J Castanon and G WicheldquoPlectin deficiency affects precursor formation and dynamicsof vimentin networksrdquo Experimental Cell Research vol 314 no19 pp 3570ndash3580 2008

[19] O Esue A A Carson Y Tseng and D Wirtz ldquoA directinteraction between actin and vimentin filaments mediatedby the tail domain of vimentinrdquo The Journal of BiologicalChemistry vol 281 no 41 pp 30393ndash30399 2006

[20] S Kruger K Sotlar I Kausch and H-P Horny ldquoExpressionof KIT (CD117) in renal cell carcinoma and renal oncocytomardquoOncology vol 68 no 2-3 pp 269ndash275 2005

[21] F Lin R E Brown T Shen X J Yang and C SchuerchldquoImmunohistochemical detection of P504S in primary andmetastatic renal cell carcinomasrdquo Applied Immunohistochem-istry andMolecularMorphology vol 12 no 2 pp 153ndash159 2004

[22] V Molinie A Balaton S Rotman et al ldquoAlpha-methyl CoAracemase expression in renal cell carcinomasrdquo Human Pathol-ogy vol 37 no 6 pp 698ndash703 2006

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2 Disease Markers

as the characteristic constellation [6] To differentiate thechromophobe and eosinophilic RCC from oncocytoma thepositivity for Vimentin a structural protein has been usedto identify the former [7] However a series of oncocytomashas recently been reported in which a Vimentin positivityhas been observed making the differentiation questionableparticularly in preoperative evaluation [8] Hes et al analyzed234 oncocytoma of which 73 were positive for Vimentinstaining [8] Vimentin is an intermediate-sized filament thatfunctions in cellular signal transduction structural integrityof cells and tissues and adhesion and migration [9] In2007 a spliced variant of Vimentin with a unique C-terminalending was detected by a working group at the Craig VenterInstitute (NHLBI Resequencing and Genotyping Service(RSG) N01-NV-48196 J Craig Venter Institute RockvilleMD 20850) and published online in PubMed (AccessionnumberACA061031) In 2011Thakkar et al [10] described thepresence of this variant in gliomas However no further anal-ysis or investigation regarding its role has been performed

Based on the knowledge that the spliced variant ofVimentin is 35 amino acids smaller than the full lengthvariant we compared both sequences with the detailed infor-mation of the Vimentin 3B4 antibody From the literatureit is known that the 3B4 Vimentin antibody detects the roddomain [11] which is a homologue to the truncated Vimentinvariant 3 (Vim3) rod domain Thus it seemed possible thatthe protein expression of Vimentin described in the literatureby immune histology resulted from the combined detectionnot only of the protein from full length but also of the splicedvariant of Vimentin namely Vim3

Most of the commercially available antibodies (clones 3B4and SP20) are against epitopes located in the rod domain ofVimentin (Figure 1)The clone V9 is directed against the tail-domain of Vimentin However for the detection of the trun-cated Vimentin variant 3 (Vim3) the Vim3 antibody is usedwhich is designed against the unique C-terminal ending

In case of renal tumors with eosinophilic apperancewhich mimic oncocytoma the differential diagnosis betweenRCCs and oncocytomas is based on a panel of differentantibodies In particular the presence or absence of Vimentinstaining of paraffinized tumor samples can be of great impor-tance for the differentiation between malignant and benigntumors

This diagnostic approach has to be reevaluated sincea spliced Vimentin isoform exists This is also detectablewith the currently used antibodies against the N-terminalsequence thus Vimentin positivity is no longer a diagnosticfeature per se of malignant RCCs Thus in this paper weanalyzed the presence ofVim3 versus the full lengthVimentinin RCCs especially the eosinophilic variant of RCCs versusoncocytomas

We designed primers which detect either the full lengthversion of Vimentin or its spliced variant Vim3 Afterperforming a qRT-PCR on paraffin embedded tissues of thedifferent RCCs and oncocytoma we could indeed show thatVim3 is the predominant variant in oncocytoma Further-more we designed an antibody exclusively detecting theunique C-terminus of Vim3

V9 antibody

V3 antibody 3B4 antibody SP20 antibody

The antibody

1 2 3 4 5 6 7 8 9 39984005998400

Vimentin FL sim57kDa Vimentin 3 sim47kDaNH2NH2

COOHCOOH

Figure 1 Differential location of the different antibodies againstVimentin The commercially available antibodies of the clones 3B4and SP20 were against the rod domain (slight blue) the antibodyclone V9 is against C-terminal ending of the full length variant(slight purple) and the truncated antibody against the unique C-terminal ending Vim3 is indicated with dark blue

This is the first report describing the presence and thestructural differences of Vim3 versus the full length Vimen-tin Our data present strong evidence that Vim3 is the isoformresponsible for the so-called Vimentin positive oncocytomasdescribed in the literature Furthermore we show that theV9 Vimentin antibody as well as antibodies detecting thefull length version of Vimentin cannot be used any longerfor differential diagnosis between RCCs and oncocytomasbecause these result in misdiagnoses with potentially graveconsequences for the patients involved

2 Materials and Methods

21 Antibody Design and Quantification The Vim3 anti-body was commercially designed (EZbiolab Inc) using theunique C-terminal ending of Vim3 as target (for detailedinformation please see patent by University of CologneBrandensteinFries patent number EP 131608762-1405) TheVim3 expression versus that of full length Vimentin (cloneV9) (Santa Cruz Heidelberg) was shown using immunehistology on paraffin embedded colon mucosa biopsies fromour pathology archive Western blot analysis (see below)of macrodissected material of cryptal epithelial cells andlymphoid cells was performed for further evaluation andproof of specificity of the newly designed antibody

22 Immune Fluorescence of ParaffinEmbedded Tissues 4 120583mthick paraffin embedded tissue sections were deparaffinizedby incubation for 1 x 10min in xylene followed by 1 x 5min100 ethanol and 1min 70 ethanol and then rinsed withdistilled waterThe slides were digested with Proteinase K for30min at room temperature After an incubation period in5PBSmilk for 30min the slideswere reincubated for 1 hourat room temperature with specific primary antibodies (Vim3)in 3 PBS milk Following washes with PBS the sectionswere incubated with a secondary FITC-anti-rabbit antibody(Santa Cruz) Subsequent to rinsing with PBS the slides werethen counterstained with DAPI mounting medium (nuclearstaining) and cover slipped

Disease Markers 3

23 Immune Histology of Paraffin Embedded Tissues Paraffinembedded tissue sections (4120583mthick)were deparaffinized byincubation for 2ndash5minutes in xylene followed by 2-3minutesin 100 ethanol and 1minute in 95 ethanol and then rinsedwith distilled water The slides were incubated with a specificserum blocker (anti-rabbit) for 30 minutes in order to avoidnonspecific binding After that incubation period the slideswere reincubated for 1 hour at room temperaturewith specificprimary antibodies (Vim3 EZBiolab Inc Carmel USAVimentin V9 Santa Cruz Heidelberg Germany AMACRand CD117 [12] Dako Hamburg Germany) Followingwashes with PBSndashTween 20 the sections were incubated witha secondary anti-rabbit antibody (Santa Cruz HeidelbergGermany) After rinsing with PBSndashTween 20 the slides werereincubated for 2 minutes in 95 ethanol followed by 2-3minutes in 100 methanol counterstained with HampE andcover slipped

The analyzed paraffin embedded tissue sections werefrom retrospective nephrectomies nevertheless we per-formed a blind study so any bias of the results could beexcluded

24 Oncocytic Tumors Since human materials were usedprocedures were followed as outlined in accordance with eth-ical standards formulated in the Declaration of Helsinki 1975with preapproval by the Ethics Committee at the UniversityHospital Cologne Germany (reference number 09-232)

25 Quantitative Real-Time PCR (qRT-PCR) The qRT-PCRwas performed as previously described [13 14]

For quantitative analysis 120573-actin was measured Allsamples were normalized to 120573-actin as the reference geneAll experiments were performed in triplicate Relative fluo-rescencewas calculated using theΔΔ-CTmethod as outlinedin User Bulletin 2 (PE Applied Biosystems Darmstadt Ger-many)The statistical significance of qPCR values at differenttime points was assessed by Studentrsquos paired 119905-test Table 2provides primer information

26 RNA-Extraction Paraffin Embedded Tissues and RT-PCR Formalin-fixed and paraffinized (FFPE) human tissuesamples from the archives of the Department of PathologyUniversity Hospital of Cologne Cologne Germany and theDepartment of Pathology Helios Clinic Wuppertal Uni-versity Clinic Witten-Herdecke Wuppertal Germany wereused

RNA extraction from FFPE tissue was performed accord-ing to the RNeasy FFPE kit (Qiagen Germany) RNA quan-tification was accomplished using NanoDrop technology

The cDNA was obtained from 250 ng of RNA usingrandom primers and SuperScript III reverse transcriptaseaccording to the manufacturerrsquos protocol (Invitrogen Darm-stadt Germany)

27 Statistical Analysis For statistical analysis the GraphPadPrism 5 program was used Analysis of variance (ANOVA)was performed and the significant differences were calculatedand indicated by stars (lowast119875 lt 005 lowastlowast119875 lt 001 and

lowastlowastlowast

119875 lt 0001) All differences without indication were notstatistically significant

28 Western Blot All Western blots were performed intriplicate as outlined in detail before (Gerstung et al [13])120573-actin served as loading control (Santa Cruz HeidelbergGermany) The Vimentin 3 antibody was used in a 1 500dilution and the V9 antibody (Santa Cruz) against fulllengthVimentinwas employed in 1 1000 as recommended bythe supplier Protein extraction from paraffinized tissue wasdone as described in Ikeda et al [15] The 4 120583m paraffinizedtissue samples were incubated in Xylol for 15 sec mixedand then centrifuged for 2min at full speed and at roomtemperature 100 ethanol was added to the pellet for 2minthen mixed and again centrifuged for 2min at full speedand at room temperature After carefully discarding thesupernate the pellet was air dried 50120583L of RIPA buffer wasadded incubated at 100∘C for 20min and then followed byan incubation period of 2 hours at 60∘C The samples weresubsequently centrifuged at full speed at 4∘C for 20min Thesupernate was then stored at minus80∘C until further use Proteinquantification was performed as previously described [13]

3 Results

31 Antibody Evaluation Since Vimentin is commonlyknown primarily as a mesenchymal marker we characterizedtheVim3 antibody using frozen sections of appendiceal tissuecontaining epithelial mesenchymal and lymphatic tissueelements As Figure 2 shows Vim3 was expressed in coloniccrypt epithelium particularly in the regeneratively active partof the crypt in mesenchymal cells and in lymphocytes AWestern blot was performed to verify the expected size ofthe Vim3 splice form being 47 kDa (Figure 2) while the fulllength molecule was predictably 57 kDa (data not shown)

We also established the Vim3 antibody binding pattern inrenal tissues The Vimentin full length molecule was evidentin different types of mesenchymal cells (such as fibroblastsand smooth muscle cells) and also in proximal tubule cells

32 mRNA Detection of Vimentin and Vim3 The full lengthmolecule of Vimentin is used as a marker to differentiatebenign oncocytomas expected to be negative from malig-nant renal cell carcinomas beingVimentin positive Our qRT-PCR evaluation of renal tumors confirmed this finding incases from the pathology archives while demonstrating thatfull length Vim3 was expressed in (Table 1) Onco (Oncocy-toma) RCC subtypes express lower levels of Vim3 mRNA

33 Protein Detection of Vim3 versus Full Length Vimentinin RCCs By immune histology on paraffinized tissue slicesfrom renal tumors full length Vimentin protein was found tobe strongly expressed in clear cell RCCs and papillary RCCsChromophobe RCCs showed a weak reactivity with theantibody while oncocytomas demonstrated no reactivity Incontrast Vim3 expression was strong in oncocytomas whileall three malignant RCCs subtypes were negative (Figure 4)

4 Disease Markers

Negative tissue

Crypt

Lymphoid tissue

(a)

Negative tissue Lymphoid tissue Crypt

Vim3

sim43kDa

sim47kDa

120573-actin

(b)

Figure 2 Evaluation of Vim3 antibody (a) Immune histology showing expression of Vim3 in colonic crypt epithelium and in lymphocytes(b) Western blot analysis after macrodissection of crypt epithelium and lymphocytes 120573-actin serves as loading control

Table 1 Tumor types and patient number

Patient number Diagnosis1ndash6 Normal kidney control7ndash22 Oncocytoma23ndash33 Chromophobe RCC34ndash44 Papillary RCC45ndash54 RCC55ndash60 Eosinophilic RCC

Consequently using immune fluorescence analyses ofthe different RCC subtypes and the oncocytoma a clearexpression of Vim3 was only detectable in oncocytomaThe oncocytoma mimicking variant of RCC namely theeosinophilic variant was negative for Vim3 (Figure 5)

34 Collision Tumor To further demonstrate the applicabil-ity of the new Vim3 antibody a collision tumor consisting oftwo different tumor subtypes was used Figure 6 shows theHampE staining of its papillary RCC differentiation Since thepatient suffered from pleural metastases it was important toidentify their origin Therefore we performed an immunefluorescence staining for Vimentin FL positive (V9) andfor Vim3 This indicated that the first tumor type with V9being positive and Vim3 being negative was the malignantcomponentThe histogenesis of the second tumor type foundin the tissue sample was questionable possibly being a ldquorealrdquooncocytoma After immune fluorescence for Vimentin FL(V9) as well as for Vim3 was performed only the Vim3staining showed positive areas indicating that the secondtumor type was indeed an oncocytoma

0

10

20

30

40Vim3 expression

RFU

RCC Onco CP Pap Eosino

lowastlowast lowastlowastlowastlowastlowast lowastlowastlowast

Figure 3 qRT-PCR analysis for Vim3 in oncocytoma versus clearcell renal cell carcinoma (RCC) chromophobe RCC (CP) papillaryRCC (Pap) and eosinophilic RCC (Eosino) 120573-actin was used asreference gene lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 and alldifferences without indication are not statistically significant

4 Discussion

In this paper we characterize a Vimentin splice isoformcalled Vimentin 3 (Vim3) as a potentially important struc-tural cellular protein Its unique structure leads to a 10 kDasmaller protein (Figure 1) which is more widely expressedthan its full length counterpart particularly in epithelial cellsand lymphocytes (Figure 2)

To study the importance ofVim3 for renal tubule cells fur-ther we analyzed Vim3 versus full length Vimentin expres-sion by qRT-PCR in renal tumors (Figure 3) Surprisinglywhile RCCs have high amounts of transcribed full lengthVimentin they are almost Vim3 negative In contrast thereverse is true for oncocytomas while their negativity for

Disease Markers 5

Table 2 Primers

Gene Sequence Annealing temp Cycles

120573-actin Forw 51015840-TTGGCAATGAGCGGTTCCGCTG-31015840Rev 51015840-TACACGTGTTTGCGGATGTCCAC-31015840 55∘C 40x

Vimentin full length Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-TCCAGCAGCTTCCTGTAGGTG-31015840 55∘C 40x

Vim3 Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-GAAATAAAATGCTTACCCCTCAG-31015840 55∘C 40x

HampE

Vim3

Vimentinfull length

RCC CP PapNormal kidney Oncocytoma

Figure 4 Immune histological analysis of the expression pattern between oncocytoma and RCC subtypes Full length Vimentin positivetumor cells are observed in clear cell and papillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise arenegative for full length Vimentin HampE staining of typical tumor morphology

full length Vimentin is not surprising (and being a criteriafor their identification) the levels for Vim3 are unexpectedlyhighThe papillary RCC subtype (Pap) has small mRNA levelof Vimentin full length and Vim3 detectable by qRT-PCR

Nevertheless due to some posttranscriptional modifica-tions the Vim3 signal is not detectable by immune histology(Figure 4) while a strong Vimentin (full length) stainingcan be easily detected The only positive signal regardingthe Vim3 was detectable in case of oncocytoma and theldquonormalrdquo tissue section was negative for Vim3 (Figure 4)

This result as well as the immune fluorescence results(Figure 5) identifies Vim3 as potential immune histologymarker for renal oncocytomas

Currently it is still common practice in routine pathologyto differentiate renal cell carcinomas from carcinomas ofhistogenetically different origins by using immune histologywith cytokeratins and Vimentin In particular Vimentinpositivity has been regarded as the major hallmark not onlyfor RCC but also for differentiating them from their benigncounterparts the oncocytomas Since Hes et al [8] reportedVimentin positivity in 73 of all tested oncocytomas thisdiagnostic approach has been questionable while its under-lying mechanism has been elusive

From our results we claim that by using an antibodyagainst the unique C-terminal sequence of Vim3 ldquorealrdquooncocytomas can be unequivocally identified

6 Disease Markers

Vim3

Vimentinfull length

RCCCPPap Eosino Oncocytoma

Figure 5 Immune fluorescence of oncocytoma and RCC subtypes Full length Vimentin positive tumor cells are observed in clear cell andpapillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise are negative for full length Vimentin HampE stainingof typical tumor morphology original magnification times400

Pap

ONCO

HampE AMACR CD117Vimentinfull length Vim3

Figure 6 Collision tumor with two different tumor subtypes Top row papillary RCC bottom row preliminary diagnosis oncocytomaHampE staining and immunohistological staining for AMACR and CD117 Vimentin FL and Vim3 were performed Questionable was thesecond tumor type unexpectedly found in the tissue sample Immune fluorescence staining for Vimentin FL positive (V9) was positive andthe immune fluorescence for Vim3 was negative in the papillary RCC component indicating the malignant tumor subtype whereas thesecond one is an oncocytoma (Vim3 positive)

Disease Markers 7

The exact nature and mechanism of the ldquoVimentinpositive oncocytomasrdquo require further clarification Ourresults indicate that these tumors have to be classified asan eosinophilic variant of clear cell RCCs Since their mor-phologic appearance on an HampE slide seems identical to aldquotruerdquo oncocytoma we performed an immune fluorescencefor Vim3 (Figure 5) This resulted in a clearly Vim3 negativeappearance of these tumors regarded as ldquotruerdquo oncocytomas

However the importance of this study for routine patho-logic diagnoses with respect to the mystery of ldquoVimentinpositive oncocytomasrdquo advocates our current explanation

To date an intracellular role of Vim3 has not beendefined while an intracellular role of the full length Vimentinmolecule has been described in the literature as an anchoringmolecule for the nucleus [16] Knowing the interaction ofits full length counterpart one may speculate about Vim3rsquosintracellular importance Since the N-terminal domain andthe rod domain have not changed binding partners such asankyrin [17] and interactions with plectin [18] should still bepossible In contrast the missing tail and the unique aminoacids of its C-terminal ending may result in differences inthe C-terminal interaction Currently the tail-domain hasbeen reported to be the binding and interactive site for F-actin [19] and lamin B [17] However since the major part ofthe C-terminus is absent in Vim3 and the exact interactionsites for both molecules are presently unknown furtherinvestigations have to be conducted in order to fully elucidatepotential interaction or its absence between Vim3 and otherstructural binding partnersThis protein differentiates benignoncocytoma from malignant RCC variants especially theeosinophilic RCC variant which mimics oncocytoma byimmune histology and immune fluorescence

To strengthen our interpretation we applied the Vim3antibody to a collision tumor (Figure 6) in which weidentified its metastatic component as belonging to thepapillary differentiation being Vim3 negative while the otherpart was identified as an oncocytoma based on its Vim3positivity CD117 expression is a hallmark in differentiat-ing oncocytoma and chromophobe RCC [20] In case ofthe examined collision tumor CD117 positivity could be aproblem since the chromophobe RCC is a malignant tumorand the oncocytoma is a benign one so the differentiationof the metastatic component of the collision tumor is stillquestionable (Figure 6)The further usage of an alpha-methylCoA racemase (AMACR) antibody can be used as well fordistinction between chromophobe RCC and oncocytoma[21] AMACR positivity is seen in papillary RCC [22] and canbe used as marker between primary andmetastatic RCC [21]

Figure 6 shows the two tumor types and a clear differenti-ation between the RCC subtypes namely the papillary part ofthe tumor and the benign part was possible due to the usageof our Vim3 antibody

In conclusion we present here a unique Vimentin iso-form Vim3 as a differential marker between malignantRCCs and oncocytoma We strongly believe that this cleardifferentiation between the benign and malignant kidneytumor types will be essential in the future for patientsrsquo therapyas well as operative planning follow-up therapy and patientsrsquosurvival

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Melanie von Brandenstein was supported by Koeln For-tune ProgramFaculty of Medicine University of Cologne arecipient of a postdoctoral fellowship and excellence clusterinitiative supported by University of Cologne and DFG Theauthors gratefully acknowledge the support by Susanne Sat-tler in immune histology Dr Heike Gobel for her help withthe antibody quantification and critical reading of the paperand Claudia Richter for her excellent technical assistance

References

[1] H Hamperl ldquoUber das Vorkommen von Onkocyten in ver-schiedenen Organen und ihren GeschwulstenmdashMundspei-cheldrusen Bauchspeicheldruse Epithelkorperchen Hypo-physe Schilddruse Eileiterrdquo Virchows Archiv fur PathologischeAnatomie und Physiologie und fur Klinische Medizin vol 298no 2 pp 327ndash375 1936

[2] R Kataoka Y Hyo T Hoshiya HMiyahara and TMatsunagaldquoUltrastructural study of mitochondria in oncocytesrdquo Ultra-structural Pathology vol 15 no 3 pp 231ndash239 1991

[3] J Zippel ldquoZur Kenntnis der Onkocytenrdquo Virchows Archiv vol308 no 2 pp 360ndash382 1942

[4] M J Klein and Q J Valensi ldquoProximal tubular adenomas ofkidney with so-called oncocytic features A clinicopathologicstudy of 13 cases of a rarely reported neoplasmrdquo Cancer vol 38no 2 pp 906ndash914 1976

[5] J D Oxley J Sullivan A Mitchelmore and D A GillattldquoMetastatic renal oncocytomardquo Journal of Clinical Pathologyvol 60 no 6 pp 720ndash722 2007

[6] A O Osunkoya C Cohen D Lawson M M Picken M BAmin and A N Young ldquoClaudin-7 and claudin-8 immuno-histochemical markers for the differential diagnosis of chro-mophobe renal cell carcinoma and renal oncocytomardquo HumanPathology vol 40 no 2 pp 206ndash210 2009

[7] R Waldherr and K Schwechheimer ldquoCo-expression of cytok-eratin and vimentin intermediate-sized filaments in renal cellcarcinomas comparative study of the intermediate-sized fila-ment distribution in renal cell carcinomas and normal humankidneyrdquo Virchows Archiv A vol 408 no 1 pp 15ndash27 1985

[8] O Hes M Michal N Kuroda et al ldquoVimentin reactivity inrenal oncocytoma immunohistochemical study of 234 casesrdquoArchives of Pathology and Laboratory Medicine vol 131 no 12pp 1782ndash1788 2007

[9] J Ivaska H-M Pallari J Nevo and J E Eriksson ldquoNovel func-tions of vimentin in cell adhesion migration and signalingrdquoExperimental Cell Research vol 313 no 10 pp 2050ndash2062 2007

[10] D Thakkar L Shervington and A Shervington ldquoProteomicstudies coupled with RNAi methodologies can shed furtherlight on the downstream effects of telomerase in gliomardquoCancerInvestigation vol 29 no 2 pp 113ndash122 2011

[11] M Malakoutikhah M J Gomara J A Gomez-Puerta R San-martı and I Haro ldquoThe use of chimeric vimentin citrullinatedpeptides for the diagnosis of rheumatoid arthritisrdquo Journal ofMedicinal Chemistry vol 54 no 21 pp 7486ndash7492 2011

8 Disease Markers

[12] P H Tan L Cheng N Rioux-Leclercq et al ldquoRenal tumorsdiagnostic and prognostic biomarkersrdquo The American Journalof Surgical Pathology vol 37 no 10 pp 1518ndash1531 2013

[13] M Gerstung T Roth H-P Dienes C Licht and J W U FriesldquoEndothelin-1 induces NF-120581B via two independent pathwaysin human renal tubular epithelial cellsrdquo American Journal ofNephrology vol 27 no 3 pp 294ndash300 2007

[14] M G von Brandenstein A N Abety R Depping et al ldquoA p38-p65 transcription complex induced by endothelin-1 mediatessignal transduction in cancer cellsrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1783 no 9 pp 1613ndash16222008

[15] K Ikeda T Monden T Kanoh et al ldquoExtraction and analysisof diagnostically useful proteins from formalin-fixed paraffin-embedded tissue sectionsrdquo Journal of Histochemistry and Cyto-chemistry vol 46 no 3 pp 397ndash403 1998

[16] A J Sarria J G Lieber S K Nordeen and R M Evans ldquoThepresence or absence of a vimentin-type intermediate filamentnetwork affects the shape of the nucleus in human SW-13 cellsrdquoJournal of Cell Science vol 107 no part 6 pp 1593ndash1607 1994

[17] S D Georgatos andG Blobel ldquoLamin B constitutes an interme-diate filament attachment site at the nuclear enveloperdquo Journalof Cell Biology vol 105 no 1 pp 117ndash125 1987

[18] R Spurny M Gregor M J Castanon and G WicheldquoPlectin deficiency affects precursor formation and dynamicsof vimentin networksrdquo Experimental Cell Research vol 314 no19 pp 3570ndash3580 2008

[19] O Esue A A Carson Y Tseng and D Wirtz ldquoA directinteraction between actin and vimentin filaments mediatedby the tail domain of vimentinrdquo The Journal of BiologicalChemistry vol 281 no 41 pp 30393ndash30399 2006

[20] S Kruger K Sotlar I Kausch and H-P Horny ldquoExpressionof KIT (CD117) in renal cell carcinoma and renal oncocytomardquoOncology vol 68 no 2-3 pp 269ndash275 2005

[21] F Lin R E Brown T Shen X J Yang and C SchuerchldquoImmunohistochemical detection of P504S in primary andmetastatic renal cell carcinomasrdquo Applied Immunohistochem-istry andMolecularMorphology vol 12 no 2 pp 153ndash159 2004

[22] V Molinie A Balaton S Rotman et al ldquoAlpha-methyl CoAracemase expression in renal cell carcinomasrdquo Human Pathol-ogy vol 37 no 6 pp 698ndash703 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 3

23 Immune Histology of Paraffin Embedded Tissues Paraffinembedded tissue sections (4120583mthick)were deparaffinized byincubation for 2ndash5minutes in xylene followed by 2-3minutesin 100 ethanol and 1minute in 95 ethanol and then rinsedwith distilled water The slides were incubated with a specificserum blocker (anti-rabbit) for 30 minutes in order to avoidnonspecific binding After that incubation period the slideswere reincubated for 1 hour at room temperaturewith specificprimary antibodies (Vim3 EZBiolab Inc Carmel USAVimentin V9 Santa Cruz Heidelberg Germany AMACRand CD117 [12] Dako Hamburg Germany) Followingwashes with PBSndashTween 20 the sections were incubated witha secondary anti-rabbit antibody (Santa Cruz HeidelbergGermany) After rinsing with PBSndashTween 20 the slides werereincubated for 2 minutes in 95 ethanol followed by 2-3minutes in 100 methanol counterstained with HampE andcover slipped

The analyzed paraffin embedded tissue sections werefrom retrospective nephrectomies nevertheless we per-formed a blind study so any bias of the results could beexcluded

24 Oncocytic Tumors Since human materials were usedprocedures were followed as outlined in accordance with eth-ical standards formulated in the Declaration of Helsinki 1975with preapproval by the Ethics Committee at the UniversityHospital Cologne Germany (reference number 09-232)

25 Quantitative Real-Time PCR (qRT-PCR) The qRT-PCRwas performed as previously described [13 14]

For quantitative analysis 120573-actin was measured Allsamples were normalized to 120573-actin as the reference geneAll experiments were performed in triplicate Relative fluo-rescencewas calculated using theΔΔ-CTmethod as outlinedin User Bulletin 2 (PE Applied Biosystems Darmstadt Ger-many)The statistical significance of qPCR values at differenttime points was assessed by Studentrsquos paired 119905-test Table 2provides primer information

26 RNA-Extraction Paraffin Embedded Tissues and RT-PCR Formalin-fixed and paraffinized (FFPE) human tissuesamples from the archives of the Department of PathologyUniversity Hospital of Cologne Cologne Germany and theDepartment of Pathology Helios Clinic Wuppertal Uni-versity Clinic Witten-Herdecke Wuppertal Germany wereused

RNA extraction from FFPE tissue was performed accord-ing to the RNeasy FFPE kit (Qiagen Germany) RNA quan-tification was accomplished using NanoDrop technology

The cDNA was obtained from 250 ng of RNA usingrandom primers and SuperScript III reverse transcriptaseaccording to the manufacturerrsquos protocol (Invitrogen Darm-stadt Germany)

27 Statistical Analysis For statistical analysis the GraphPadPrism 5 program was used Analysis of variance (ANOVA)was performed and the significant differences were calculatedand indicated by stars (lowast119875 lt 005 lowastlowast119875 lt 001 and

lowastlowastlowast

119875 lt 0001) All differences without indication were notstatistically significant

28 Western Blot All Western blots were performed intriplicate as outlined in detail before (Gerstung et al [13])120573-actin served as loading control (Santa Cruz HeidelbergGermany) The Vimentin 3 antibody was used in a 1 500dilution and the V9 antibody (Santa Cruz) against fulllengthVimentinwas employed in 1 1000 as recommended bythe supplier Protein extraction from paraffinized tissue wasdone as described in Ikeda et al [15] The 4 120583m paraffinizedtissue samples were incubated in Xylol for 15 sec mixedand then centrifuged for 2min at full speed and at roomtemperature 100 ethanol was added to the pellet for 2minthen mixed and again centrifuged for 2min at full speedand at room temperature After carefully discarding thesupernate the pellet was air dried 50120583L of RIPA buffer wasadded incubated at 100∘C for 20min and then followed byan incubation period of 2 hours at 60∘C The samples weresubsequently centrifuged at full speed at 4∘C for 20min Thesupernate was then stored at minus80∘C until further use Proteinquantification was performed as previously described [13]

3 Results

31 Antibody Evaluation Since Vimentin is commonlyknown primarily as a mesenchymal marker we characterizedtheVim3 antibody using frozen sections of appendiceal tissuecontaining epithelial mesenchymal and lymphatic tissueelements As Figure 2 shows Vim3 was expressed in coloniccrypt epithelium particularly in the regeneratively active partof the crypt in mesenchymal cells and in lymphocytes AWestern blot was performed to verify the expected size ofthe Vim3 splice form being 47 kDa (Figure 2) while the fulllength molecule was predictably 57 kDa (data not shown)

We also established the Vim3 antibody binding pattern inrenal tissues The Vimentin full length molecule was evidentin different types of mesenchymal cells (such as fibroblastsand smooth muscle cells) and also in proximal tubule cells

32 mRNA Detection of Vimentin and Vim3 The full lengthmolecule of Vimentin is used as a marker to differentiatebenign oncocytomas expected to be negative from malig-nant renal cell carcinomas beingVimentin positive Our qRT-PCR evaluation of renal tumors confirmed this finding incases from the pathology archives while demonstrating thatfull length Vim3 was expressed in (Table 1) Onco (Oncocy-toma) RCC subtypes express lower levels of Vim3 mRNA

33 Protein Detection of Vim3 versus Full Length Vimentinin RCCs By immune histology on paraffinized tissue slicesfrom renal tumors full length Vimentin protein was found tobe strongly expressed in clear cell RCCs and papillary RCCsChromophobe RCCs showed a weak reactivity with theantibody while oncocytomas demonstrated no reactivity Incontrast Vim3 expression was strong in oncocytomas whileall three malignant RCCs subtypes were negative (Figure 4)

4 Disease Markers

Negative tissue

Crypt

Lymphoid tissue

(a)

Negative tissue Lymphoid tissue Crypt

Vim3

sim43kDa

sim47kDa

120573-actin

(b)

Figure 2 Evaluation of Vim3 antibody (a) Immune histology showing expression of Vim3 in colonic crypt epithelium and in lymphocytes(b) Western blot analysis after macrodissection of crypt epithelium and lymphocytes 120573-actin serves as loading control

Table 1 Tumor types and patient number

Patient number Diagnosis1ndash6 Normal kidney control7ndash22 Oncocytoma23ndash33 Chromophobe RCC34ndash44 Papillary RCC45ndash54 RCC55ndash60 Eosinophilic RCC

Consequently using immune fluorescence analyses ofthe different RCC subtypes and the oncocytoma a clearexpression of Vim3 was only detectable in oncocytomaThe oncocytoma mimicking variant of RCC namely theeosinophilic variant was negative for Vim3 (Figure 5)

34 Collision Tumor To further demonstrate the applicabil-ity of the new Vim3 antibody a collision tumor consisting oftwo different tumor subtypes was used Figure 6 shows theHampE staining of its papillary RCC differentiation Since thepatient suffered from pleural metastases it was important toidentify their origin Therefore we performed an immunefluorescence staining for Vimentin FL positive (V9) andfor Vim3 This indicated that the first tumor type with V9being positive and Vim3 being negative was the malignantcomponentThe histogenesis of the second tumor type foundin the tissue sample was questionable possibly being a ldquorealrdquooncocytoma After immune fluorescence for Vimentin FL(V9) as well as for Vim3 was performed only the Vim3staining showed positive areas indicating that the secondtumor type was indeed an oncocytoma

0

10

20

30

40Vim3 expression

RFU

RCC Onco CP Pap Eosino

lowastlowast lowastlowastlowastlowastlowast lowastlowastlowast

Figure 3 qRT-PCR analysis for Vim3 in oncocytoma versus clearcell renal cell carcinoma (RCC) chromophobe RCC (CP) papillaryRCC (Pap) and eosinophilic RCC (Eosino) 120573-actin was used asreference gene lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 and alldifferences without indication are not statistically significant

4 Discussion

In this paper we characterize a Vimentin splice isoformcalled Vimentin 3 (Vim3) as a potentially important struc-tural cellular protein Its unique structure leads to a 10 kDasmaller protein (Figure 1) which is more widely expressedthan its full length counterpart particularly in epithelial cellsand lymphocytes (Figure 2)

To study the importance ofVim3 for renal tubule cells fur-ther we analyzed Vim3 versus full length Vimentin expres-sion by qRT-PCR in renal tumors (Figure 3) Surprisinglywhile RCCs have high amounts of transcribed full lengthVimentin they are almost Vim3 negative In contrast thereverse is true for oncocytomas while their negativity for

Disease Markers 5

Table 2 Primers

Gene Sequence Annealing temp Cycles

120573-actin Forw 51015840-TTGGCAATGAGCGGTTCCGCTG-31015840Rev 51015840-TACACGTGTTTGCGGATGTCCAC-31015840 55∘C 40x

Vimentin full length Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-TCCAGCAGCTTCCTGTAGGTG-31015840 55∘C 40x

Vim3 Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-GAAATAAAATGCTTACCCCTCAG-31015840 55∘C 40x

HampE

Vim3

Vimentinfull length

RCC CP PapNormal kidney Oncocytoma

Figure 4 Immune histological analysis of the expression pattern between oncocytoma and RCC subtypes Full length Vimentin positivetumor cells are observed in clear cell and papillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise arenegative for full length Vimentin HampE staining of typical tumor morphology

full length Vimentin is not surprising (and being a criteriafor their identification) the levels for Vim3 are unexpectedlyhighThe papillary RCC subtype (Pap) has small mRNA levelof Vimentin full length and Vim3 detectable by qRT-PCR

Nevertheless due to some posttranscriptional modifica-tions the Vim3 signal is not detectable by immune histology(Figure 4) while a strong Vimentin (full length) stainingcan be easily detected The only positive signal regardingthe Vim3 was detectable in case of oncocytoma and theldquonormalrdquo tissue section was negative for Vim3 (Figure 4)

This result as well as the immune fluorescence results(Figure 5) identifies Vim3 as potential immune histologymarker for renal oncocytomas

Currently it is still common practice in routine pathologyto differentiate renal cell carcinomas from carcinomas ofhistogenetically different origins by using immune histologywith cytokeratins and Vimentin In particular Vimentinpositivity has been regarded as the major hallmark not onlyfor RCC but also for differentiating them from their benigncounterparts the oncocytomas Since Hes et al [8] reportedVimentin positivity in 73 of all tested oncocytomas thisdiagnostic approach has been questionable while its under-lying mechanism has been elusive

From our results we claim that by using an antibodyagainst the unique C-terminal sequence of Vim3 ldquorealrdquooncocytomas can be unequivocally identified

6 Disease Markers

Vim3

Vimentinfull length

RCCCPPap Eosino Oncocytoma

Figure 5 Immune fluorescence of oncocytoma and RCC subtypes Full length Vimentin positive tumor cells are observed in clear cell andpapillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise are negative for full length Vimentin HampE stainingof typical tumor morphology original magnification times400

Pap

ONCO

HampE AMACR CD117Vimentinfull length Vim3

Figure 6 Collision tumor with two different tumor subtypes Top row papillary RCC bottom row preliminary diagnosis oncocytomaHampE staining and immunohistological staining for AMACR and CD117 Vimentin FL and Vim3 were performed Questionable was thesecond tumor type unexpectedly found in the tissue sample Immune fluorescence staining for Vimentin FL positive (V9) was positive andthe immune fluorescence for Vim3 was negative in the papillary RCC component indicating the malignant tumor subtype whereas thesecond one is an oncocytoma (Vim3 positive)

Disease Markers 7

The exact nature and mechanism of the ldquoVimentinpositive oncocytomasrdquo require further clarification Ourresults indicate that these tumors have to be classified asan eosinophilic variant of clear cell RCCs Since their mor-phologic appearance on an HampE slide seems identical to aldquotruerdquo oncocytoma we performed an immune fluorescencefor Vim3 (Figure 5) This resulted in a clearly Vim3 negativeappearance of these tumors regarded as ldquotruerdquo oncocytomas

However the importance of this study for routine patho-logic diagnoses with respect to the mystery of ldquoVimentinpositive oncocytomasrdquo advocates our current explanation

To date an intracellular role of Vim3 has not beendefined while an intracellular role of the full length Vimentinmolecule has been described in the literature as an anchoringmolecule for the nucleus [16] Knowing the interaction ofits full length counterpart one may speculate about Vim3rsquosintracellular importance Since the N-terminal domain andthe rod domain have not changed binding partners such asankyrin [17] and interactions with plectin [18] should still bepossible In contrast the missing tail and the unique aminoacids of its C-terminal ending may result in differences inthe C-terminal interaction Currently the tail-domain hasbeen reported to be the binding and interactive site for F-actin [19] and lamin B [17] However since the major part ofthe C-terminus is absent in Vim3 and the exact interactionsites for both molecules are presently unknown furtherinvestigations have to be conducted in order to fully elucidatepotential interaction or its absence between Vim3 and otherstructural binding partnersThis protein differentiates benignoncocytoma from malignant RCC variants especially theeosinophilic RCC variant which mimics oncocytoma byimmune histology and immune fluorescence

To strengthen our interpretation we applied the Vim3antibody to a collision tumor (Figure 6) in which weidentified its metastatic component as belonging to thepapillary differentiation being Vim3 negative while the otherpart was identified as an oncocytoma based on its Vim3positivity CD117 expression is a hallmark in differentiat-ing oncocytoma and chromophobe RCC [20] In case ofthe examined collision tumor CD117 positivity could be aproblem since the chromophobe RCC is a malignant tumorand the oncocytoma is a benign one so the differentiationof the metastatic component of the collision tumor is stillquestionable (Figure 6)The further usage of an alpha-methylCoA racemase (AMACR) antibody can be used as well fordistinction between chromophobe RCC and oncocytoma[21] AMACR positivity is seen in papillary RCC [22] and canbe used as marker between primary andmetastatic RCC [21]

Figure 6 shows the two tumor types and a clear differenti-ation between the RCC subtypes namely the papillary part ofthe tumor and the benign part was possible due to the usageof our Vim3 antibody

In conclusion we present here a unique Vimentin iso-form Vim3 as a differential marker between malignantRCCs and oncocytoma We strongly believe that this cleardifferentiation between the benign and malignant kidneytumor types will be essential in the future for patientsrsquo therapyas well as operative planning follow-up therapy and patientsrsquosurvival

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Melanie von Brandenstein was supported by Koeln For-tune ProgramFaculty of Medicine University of Cologne arecipient of a postdoctoral fellowship and excellence clusterinitiative supported by University of Cologne and DFG Theauthors gratefully acknowledge the support by Susanne Sat-tler in immune histology Dr Heike Gobel for her help withthe antibody quantification and critical reading of the paperand Claudia Richter for her excellent technical assistance

References

[1] H Hamperl ldquoUber das Vorkommen von Onkocyten in ver-schiedenen Organen und ihren GeschwulstenmdashMundspei-cheldrusen Bauchspeicheldruse Epithelkorperchen Hypo-physe Schilddruse Eileiterrdquo Virchows Archiv fur PathologischeAnatomie und Physiologie und fur Klinische Medizin vol 298no 2 pp 327ndash375 1936

[2] R Kataoka Y Hyo T Hoshiya HMiyahara and TMatsunagaldquoUltrastructural study of mitochondria in oncocytesrdquo Ultra-structural Pathology vol 15 no 3 pp 231ndash239 1991

[3] J Zippel ldquoZur Kenntnis der Onkocytenrdquo Virchows Archiv vol308 no 2 pp 360ndash382 1942

[4] M J Klein and Q J Valensi ldquoProximal tubular adenomas ofkidney with so-called oncocytic features A clinicopathologicstudy of 13 cases of a rarely reported neoplasmrdquo Cancer vol 38no 2 pp 906ndash914 1976

[5] J D Oxley J Sullivan A Mitchelmore and D A GillattldquoMetastatic renal oncocytomardquo Journal of Clinical Pathologyvol 60 no 6 pp 720ndash722 2007

[6] A O Osunkoya C Cohen D Lawson M M Picken M BAmin and A N Young ldquoClaudin-7 and claudin-8 immuno-histochemical markers for the differential diagnosis of chro-mophobe renal cell carcinoma and renal oncocytomardquo HumanPathology vol 40 no 2 pp 206ndash210 2009

[7] R Waldherr and K Schwechheimer ldquoCo-expression of cytok-eratin and vimentin intermediate-sized filaments in renal cellcarcinomas comparative study of the intermediate-sized fila-ment distribution in renal cell carcinomas and normal humankidneyrdquo Virchows Archiv A vol 408 no 1 pp 15ndash27 1985

[8] O Hes M Michal N Kuroda et al ldquoVimentin reactivity inrenal oncocytoma immunohistochemical study of 234 casesrdquoArchives of Pathology and Laboratory Medicine vol 131 no 12pp 1782ndash1788 2007

[9] J Ivaska H-M Pallari J Nevo and J E Eriksson ldquoNovel func-tions of vimentin in cell adhesion migration and signalingrdquoExperimental Cell Research vol 313 no 10 pp 2050ndash2062 2007

[10] D Thakkar L Shervington and A Shervington ldquoProteomicstudies coupled with RNAi methodologies can shed furtherlight on the downstream effects of telomerase in gliomardquoCancerInvestigation vol 29 no 2 pp 113ndash122 2011

[11] M Malakoutikhah M J Gomara J A Gomez-Puerta R San-martı and I Haro ldquoThe use of chimeric vimentin citrullinatedpeptides for the diagnosis of rheumatoid arthritisrdquo Journal ofMedicinal Chemistry vol 54 no 21 pp 7486ndash7492 2011

8 Disease Markers

[12] P H Tan L Cheng N Rioux-Leclercq et al ldquoRenal tumorsdiagnostic and prognostic biomarkersrdquo The American Journalof Surgical Pathology vol 37 no 10 pp 1518ndash1531 2013

[13] M Gerstung T Roth H-P Dienes C Licht and J W U FriesldquoEndothelin-1 induces NF-120581B via two independent pathwaysin human renal tubular epithelial cellsrdquo American Journal ofNephrology vol 27 no 3 pp 294ndash300 2007

[14] M G von Brandenstein A N Abety R Depping et al ldquoA p38-p65 transcription complex induced by endothelin-1 mediatessignal transduction in cancer cellsrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1783 no 9 pp 1613ndash16222008

[15] K Ikeda T Monden T Kanoh et al ldquoExtraction and analysisof diagnostically useful proteins from formalin-fixed paraffin-embedded tissue sectionsrdquo Journal of Histochemistry and Cyto-chemistry vol 46 no 3 pp 397ndash403 1998

[16] A J Sarria J G Lieber S K Nordeen and R M Evans ldquoThepresence or absence of a vimentin-type intermediate filamentnetwork affects the shape of the nucleus in human SW-13 cellsrdquoJournal of Cell Science vol 107 no part 6 pp 1593ndash1607 1994

[17] S D Georgatos andG Blobel ldquoLamin B constitutes an interme-diate filament attachment site at the nuclear enveloperdquo Journalof Cell Biology vol 105 no 1 pp 117ndash125 1987

[18] R Spurny M Gregor M J Castanon and G WicheldquoPlectin deficiency affects precursor formation and dynamicsof vimentin networksrdquo Experimental Cell Research vol 314 no19 pp 3570ndash3580 2008

[19] O Esue A A Carson Y Tseng and D Wirtz ldquoA directinteraction between actin and vimentin filaments mediatedby the tail domain of vimentinrdquo The Journal of BiologicalChemistry vol 281 no 41 pp 30393ndash30399 2006

[20] S Kruger K Sotlar I Kausch and H-P Horny ldquoExpressionof KIT (CD117) in renal cell carcinoma and renal oncocytomardquoOncology vol 68 no 2-3 pp 269ndash275 2005

[21] F Lin R E Brown T Shen X J Yang and C SchuerchldquoImmunohistochemical detection of P504S in primary andmetastatic renal cell carcinomasrdquo Applied Immunohistochem-istry andMolecularMorphology vol 12 no 2 pp 153ndash159 2004

[22] V Molinie A Balaton S Rotman et al ldquoAlpha-methyl CoAracemase expression in renal cell carcinomasrdquo Human Pathol-ogy vol 37 no 6 pp 698ndash703 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Disease Markers

Negative tissue

Crypt

Lymphoid tissue

(a)

Negative tissue Lymphoid tissue Crypt

Vim3

sim43kDa

sim47kDa

120573-actin

(b)

Figure 2 Evaluation of Vim3 antibody (a) Immune histology showing expression of Vim3 in colonic crypt epithelium and in lymphocytes(b) Western blot analysis after macrodissection of crypt epithelium and lymphocytes 120573-actin serves as loading control

Table 1 Tumor types and patient number

Patient number Diagnosis1ndash6 Normal kidney control7ndash22 Oncocytoma23ndash33 Chromophobe RCC34ndash44 Papillary RCC45ndash54 RCC55ndash60 Eosinophilic RCC

Consequently using immune fluorescence analyses ofthe different RCC subtypes and the oncocytoma a clearexpression of Vim3 was only detectable in oncocytomaThe oncocytoma mimicking variant of RCC namely theeosinophilic variant was negative for Vim3 (Figure 5)

34 Collision Tumor To further demonstrate the applicabil-ity of the new Vim3 antibody a collision tumor consisting oftwo different tumor subtypes was used Figure 6 shows theHampE staining of its papillary RCC differentiation Since thepatient suffered from pleural metastases it was important toidentify their origin Therefore we performed an immunefluorescence staining for Vimentin FL positive (V9) andfor Vim3 This indicated that the first tumor type with V9being positive and Vim3 being negative was the malignantcomponentThe histogenesis of the second tumor type foundin the tissue sample was questionable possibly being a ldquorealrdquooncocytoma After immune fluorescence for Vimentin FL(V9) as well as for Vim3 was performed only the Vim3staining showed positive areas indicating that the secondtumor type was indeed an oncocytoma

0

10

20

30

40Vim3 expression

RFU

RCC Onco CP Pap Eosino

lowastlowast lowastlowastlowastlowastlowast lowastlowastlowast

Figure 3 qRT-PCR analysis for Vim3 in oncocytoma versus clearcell renal cell carcinoma (RCC) chromophobe RCC (CP) papillaryRCC (Pap) and eosinophilic RCC (Eosino) 120573-actin was used asreference gene lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 and alldifferences without indication are not statistically significant

4 Discussion

In this paper we characterize a Vimentin splice isoformcalled Vimentin 3 (Vim3) as a potentially important struc-tural cellular protein Its unique structure leads to a 10 kDasmaller protein (Figure 1) which is more widely expressedthan its full length counterpart particularly in epithelial cellsand lymphocytes (Figure 2)

To study the importance ofVim3 for renal tubule cells fur-ther we analyzed Vim3 versus full length Vimentin expres-sion by qRT-PCR in renal tumors (Figure 3) Surprisinglywhile RCCs have high amounts of transcribed full lengthVimentin they are almost Vim3 negative In contrast thereverse is true for oncocytomas while their negativity for

Disease Markers 5

Table 2 Primers

Gene Sequence Annealing temp Cycles

120573-actin Forw 51015840-TTGGCAATGAGCGGTTCCGCTG-31015840Rev 51015840-TACACGTGTTTGCGGATGTCCAC-31015840 55∘C 40x

Vimentin full length Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-TCCAGCAGCTTCCTGTAGGTG-31015840 55∘C 40x

Vim3 Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-GAAATAAAATGCTTACCCCTCAG-31015840 55∘C 40x

HampE

Vim3

Vimentinfull length

RCC CP PapNormal kidney Oncocytoma

Figure 4 Immune histological analysis of the expression pattern between oncocytoma and RCC subtypes Full length Vimentin positivetumor cells are observed in clear cell and papillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise arenegative for full length Vimentin HampE staining of typical tumor morphology

full length Vimentin is not surprising (and being a criteriafor their identification) the levels for Vim3 are unexpectedlyhighThe papillary RCC subtype (Pap) has small mRNA levelof Vimentin full length and Vim3 detectable by qRT-PCR

Nevertheless due to some posttranscriptional modifica-tions the Vim3 signal is not detectable by immune histology(Figure 4) while a strong Vimentin (full length) stainingcan be easily detected The only positive signal regardingthe Vim3 was detectable in case of oncocytoma and theldquonormalrdquo tissue section was negative for Vim3 (Figure 4)

This result as well as the immune fluorescence results(Figure 5) identifies Vim3 as potential immune histologymarker for renal oncocytomas

Currently it is still common practice in routine pathologyto differentiate renal cell carcinomas from carcinomas ofhistogenetically different origins by using immune histologywith cytokeratins and Vimentin In particular Vimentinpositivity has been regarded as the major hallmark not onlyfor RCC but also for differentiating them from their benigncounterparts the oncocytomas Since Hes et al [8] reportedVimentin positivity in 73 of all tested oncocytomas thisdiagnostic approach has been questionable while its under-lying mechanism has been elusive

From our results we claim that by using an antibodyagainst the unique C-terminal sequence of Vim3 ldquorealrdquooncocytomas can be unequivocally identified

6 Disease Markers

Vim3

Vimentinfull length

RCCCPPap Eosino Oncocytoma

Figure 5 Immune fluorescence of oncocytoma and RCC subtypes Full length Vimentin positive tumor cells are observed in clear cell andpapillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise are negative for full length Vimentin HampE stainingof typical tumor morphology original magnification times400

Pap

ONCO

HampE AMACR CD117Vimentinfull length Vim3

Figure 6 Collision tumor with two different tumor subtypes Top row papillary RCC bottom row preliminary diagnosis oncocytomaHampE staining and immunohistological staining for AMACR and CD117 Vimentin FL and Vim3 were performed Questionable was thesecond tumor type unexpectedly found in the tissue sample Immune fluorescence staining for Vimentin FL positive (V9) was positive andthe immune fluorescence for Vim3 was negative in the papillary RCC component indicating the malignant tumor subtype whereas thesecond one is an oncocytoma (Vim3 positive)

Disease Markers 7

The exact nature and mechanism of the ldquoVimentinpositive oncocytomasrdquo require further clarification Ourresults indicate that these tumors have to be classified asan eosinophilic variant of clear cell RCCs Since their mor-phologic appearance on an HampE slide seems identical to aldquotruerdquo oncocytoma we performed an immune fluorescencefor Vim3 (Figure 5) This resulted in a clearly Vim3 negativeappearance of these tumors regarded as ldquotruerdquo oncocytomas

However the importance of this study for routine patho-logic diagnoses with respect to the mystery of ldquoVimentinpositive oncocytomasrdquo advocates our current explanation

To date an intracellular role of Vim3 has not beendefined while an intracellular role of the full length Vimentinmolecule has been described in the literature as an anchoringmolecule for the nucleus [16] Knowing the interaction ofits full length counterpart one may speculate about Vim3rsquosintracellular importance Since the N-terminal domain andthe rod domain have not changed binding partners such asankyrin [17] and interactions with plectin [18] should still bepossible In contrast the missing tail and the unique aminoacids of its C-terminal ending may result in differences inthe C-terminal interaction Currently the tail-domain hasbeen reported to be the binding and interactive site for F-actin [19] and lamin B [17] However since the major part ofthe C-terminus is absent in Vim3 and the exact interactionsites for both molecules are presently unknown furtherinvestigations have to be conducted in order to fully elucidatepotential interaction or its absence between Vim3 and otherstructural binding partnersThis protein differentiates benignoncocytoma from malignant RCC variants especially theeosinophilic RCC variant which mimics oncocytoma byimmune histology and immune fluorescence

To strengthen our interpretation we applied the Vim3antibody to a collision tumor (Figure 6) in which weidentified its metastatic component as belonging to thepapillary differentiation being Vim3 negative while the otherpart was identified as an oncocytoma based on its Vim3positivity CD117 expression is a hallmark in differentiat-ing oncocytoma and chromophobe RCC [20] In case ofthe examined collision tumor CD117 positivity could be aproblem since the chromophobe RCC is a malignant tumorand the oncocytoma is a benign one so the differentiationof the metastatic component of the collision tumor is stillquestionable (Figure 6)The further usage of an alpha-methylCoA racemase (AMACR) antibody can be used as well fordistinction between chromophobe RCC and oncocytoma[21] AMACR positivity is seen in papillary RCC [22] and canbe used as marker between primary andmetastatic RCC [21]

Figure 6 shows the two tumor types and a clear differenti-ation between the RCC subtypes namely the papillary part ofthe tumor and the benign part was possible due to the usageof our Vim3 antibody

In conclusion we present here a unique Vimentin iso-form Vim3 as a differential marker between malignantRCCs and oncocytoma We strongly believe that this cleardifferentiation between the benign and malignant kidneytumor types will be essential in the future for patientsrsquo therapyas well as operative planning follow-up therapy and patientsrsquosurvival

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Melanie von Brandenstein was supported by Koeln For-tune ProgramFaculty of Medicine University of Cologne arecipient of a postdoctoral fellowship and excellence clusterinitiative supported by University of Cologne and DFG Theauthors gratefully acknowledge the support by Susanne Sat-tler in immune histology Dr Heike Gobel for her help withthe antibody quantification and critical reading of the paperand Claudia Richter for her excellent technical assistance

References

[1] H Hamperl ldquoUber das Vorkommen von Onkocyten in ver-schiedenen Organen und ihren GeschwulstenmdashMundspei-cheldrusen Bauchspeicheldruse Epithelkorperchen Hypo-physe Schilddruse Eileiterrdquo Virchows Archiv fur PathologischeAnatomie und Physiologie und fur Klinische Medizin vol 298no 2 pp 327ndash375 1936

[2] R Kataoka Y Hyo T Hoshiya HMiyahara and TMatsunagaldquoUltrastructural study of mitochondria in oncocytesrdquo Ultra-structural Pathology vol 15 no 3 pp 231ndash239 1991

[3] J Zippel ldquoZur Kenntnis der Onkocytenrdquo Virchows Archiv vol308 no 2 pp 360ndash382 1942

[4] M J Klein and Q J Valensi ldquoProximal tubular adenomas ofkidney with so-called oncocytic features A clinicopathologicstudy of 13 cases of a rarely reported neoplasmrdquo Cancer vol 38no 2 pp 906ndash914 1976

[5] J D Oxley J Sullivan A Mitchelmore and D A GillattldquoMetastatic renal oncocytomardquo Journal of Clinical Pathologyvol 60 no 6 pp 720ndash722 2007

[6] A O Osunkoya C Cohen D Lawson M M Picken M BAmin and A N Young ldquoClaudin-7 and claudin-8 immuno-histochemical markers for the differential diagnosis of chro-mophobe renal cell carcinoma and renal oncocytomardquo HumanPathology vol 40 no 2 pp 206ndash210 2009

[7] R Waldherr and K Schwechheimer ldquoCo-expression of cytok-eratin and vimentin intermediate-sized filaments in renal cellcarcinomas comparative study of the intermediate-sized fila-ment distribution in renal cell carcinomas and normal humankidneyrdquo Virchows Archiv A vol 408 no 1 pp 15ndash27 1985

[8] O Hes M Michal N Kuroda et al ldquoVimentin reactivity inrenal oncocytoma immunohistochemical study of 234 casesrdquoArchives of Pathology and Laboratory Medicine vol 131 no 12pp 1782ndash1788 2007

[9] J Ivaska H-M Pallari J Nevo and J E Eriksson ldquoNovel func-tions of vimentin in cell adhesion migration and signalingrdquoExperimental Cell Research vol 313 no 10 pp 2050ndash2062 2007

[10] D Thakkar L Shervington and A Shervington ldquoProteomicstudies coupled with RNAi methodologies can shed furtherlight on the downstream effects of telomerase in gliomardquoCancerInvestigation vol 29 no 2 pp 113ndash122 2011

[11] M Malakoutikhah M J Gomara J A Gomez-Puerta R San-martı and I Haro ldquoThe use of chimeric vimentin citrullinatedpeptides for the diagnosis of rheumatoid arthritisrdquo Journal ofMedicinal Chemistry vol 54 no 21 pp 7486ndash7492 2011

8 Disease Markers

[12] P H Tan L Cheng N Rioux-Leclercq et al ldquoRenal tumorsdiagnostic and prognostic biomarkersrdquo The American Journalof Surgical Pathology vol 37 no 10 pp 1518ndash1531 2013

[13] M Gerstung T Roth H-P Dienes C Licht and J W U FriesldquoEndothelin-1 induces NF-120581B via two independent pathwaysin human renal tubular epithelial cellsrdquo American Journal ofNephrology vol 27 no 3 pp 294ndash300 2007

[14] M G von Brandenstein A N Abety R Depping et al ldquoA p38-p65 transcription complex induced by endothelin-1 mediatessignal transduction in cancer cellsrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1783 no 9 pp 1613ndash16222008

[15] K Ikeda T Monden T Kanoh et al ldquoExtraction and analysisof diagnostically useful proteins from formalin-fixed paraffin-embedded tissue sectionsrdquo Journal of Histochemistry and Cyto-chemistry vol 46 no 3 pp 397ndash403 1998

[16] A J Sarria J G Lieber S K Nordeen and R M Evans ldquoThepresence or absence of a vimentin-type intermediate filamentnetwork affects the shape of the nucleus in human SW-13 cellsrdquoJournal of Cell Science vol 107 no part 6 pp 1593ndash1607 1994

[17] S D Georgatos andG Blobel ldquoLamin B constitutes an interme-diate filament attachment site at the nuclear enveloperdquo Journalof Cell Biology vol 105 no 1 pp 117ndash125 1987

[18] R Spurny M Gregor M J Castanon and G WicheldquoPlectin deficiency affects precursor formation and dynamicsof vimentin networksrdquo Experimental Cell Research vol 314 no19 pp 3570ndash3580 2008

[19] O Esue A A Carson Y Tseng and D Wirtz ldquoA directinteraction between actin and vimentin filaments mediatedby the tail domain of vimentinrdquo The Journal of BiologicalChemistry vol 281 no 41 pp 30393ndash30399 2006

[20] S Kruger K Sotlar I Kausch and H-P Horny ldquoExpressionof KIT (CD117) in renal cell carcinoma and renal oncocytomardquoOncology vol 68 no 2-3 pp 269ndash275 2005

[21] F Lin R E Brown T Shen X J Yang and C SchuerchldquoImmunohistochemical detection of P504S in primary andmetastatic renal cell carcinomasrdquo Applied Immunohistochem-istry andMolecularMorphology vol 12 no 2 pp 153ndash159 2004

[22] V Molinie A Balaton S Rotman et al ldquoAlpha-methyl CoAracemase expression in renal cell carcinomasrdquo Human Pathol-ogy vol 37 no 6 pp 698ndash703 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 5

Table 2 Primers

Gene Sequence Annealing temp Cycles

120573-actin Forw 51015840-TTGGCAATGAGCGGTTCCGCTG-31015840Rev 51015840-TACACGTGTTTGCGGATGTCCAC-31015840 55∘C 40x

Vimentin full length Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-TCCAGCAGCTTCCTGTAGGTG-31015840 55∘C 40x

Vim3 Forw 51015840-GAGAACTTTGCCGTTGAAGC-31015840Rev 51015840-GAAATAAAATGCTTACCCCTCAG-31015840 55∘C 40x

HampE

Vim3

Vimentinfull length

RCC CP PapNormal kidney Oncocytoma

Figure 4 Immune histological analysis of the expression pattern between oncocytoma and RCC subtypes Full length Vimentin positivetumor cells are observed in clear cell and papillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise arenegative for full length Vimentin HampE staining of typical tumor morphology

full length Vimentin is not surprising (and being a criteriafor their identification) the levels for Vim3 are unexpectedlyhighThe papillary RCC subtype (Pap) has small mRNA levelof Vimentin full length and Vim3 detectable by qRT-PCR

Nevertheless due to some posttranscriptional modifica-tions the Vim3 signal is not detectable by immune histology(Figure 4) while a strong Vimentin (full length) stainingcan be easily detected The only positive signal regardingthe Vim3 was detectable in case of oncocytoma and theldquonormalrdquo tissue section was negative for Vim3 (Figure 4)

This result as well as the immune fluorescence results(Figure 5) identifies Vim3 as potential immune histologymarker for renal oncocytomas

Currently it is still common practice in routine pathologyto differentiate renal cell carcinomas from carcinomas ofhistogenetically different origins by using immune histologywith cytokeratins and Vimentin In particular Vimentinpositivity has been regarded as the major hallmark not onlyfor RCC but also for differentiating them from their benigncounterparts the oncocytomas Since Hes et al [8] reportedVimentin positivity in 73 of all tested oncocytomas thisdiagnostic approach has been questionable while its under-lying mechanism has been elusive

From our results we claim that by using an antibodyagainst the unique C-terminal sequence of Vim3 ldquorealrdquooncocytomas can be unequivocally identified

6 Disease Markers

Vim3

Vimentinfull length

RCCCPPap Eosino Oncocytoma

Figure 5 Immune fluorescence of oncocytoma and RCC subtypes Full length Vimentin positive tumor cells are observed in clear cell andpapillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise are negative for full length Vimentin HampE stainingof typical tumor morphology original magnification times400

Pap

ONCO

HampE AMACR CD117Vimentinfull length Vim3

Figure 6 Collision tumor with two different tumor subtypes Top row papillary RCC bottom row preliminary diagnosis oncocytomaHampE staining and immunohistological staining for AMACR and CD117 Vimentin FL and Vim3 were performed Questionable was thesecond tumor type unexpectedly found in the tissue sample Immune fluorescence staining for Vimentin FL positive (V9) was positive andthe immune fluorescence for Vim3 was negative in the papillary RCC component indicating the malignant tumor subtype whereas thesecond one is an oncocytoma (Vim3 positive)

Disease Markers 7

The exact nature and mechanism of the ldquoVimentinpositive oncocytomasrdquo require further clarification Ourresults indicate that these tumors have to be classified asan eosinophilic variant of clear cell RCCs Since their mor-phologic appearance on an HampE slide seems identical to aldquotruerdquo oncocytoma we performed an immune fluorescencefor Vim3 (Figure 5) This resulted in a clearly Vim3 negativeappearance of these tumors regarded as ldquotruerdquo oncocytomas

However the importance of this study for routine patho-logic diagnoses with respect to the mystery of ldquoVimentinpositive oncocytomasrdquo advocates our current explanation

To date an intracellular role of Vim3 has not beendefined while an intracellular role of the full length Vimentinmolecule has been described in the literature as an anchoringmolecule for the nucleus [16] Knowing the interaction ofits full length counterpart one may speculate about Vim3rsquosintracellular importance Since the N-terminal domain andthe rod domain have not changed binding partners such asankyrin [17] and interactions with plectin [18] should still bepossible In contrast the missing tail and the unique aminoacids of its C-terminal ending may result in differences inthe C-terminal interaction Currently the tail-domain hasbeen reported to be the binding and interactive site for F-actin [19] and lamin B [17] However since the major part ofthe C-terminus is absent in Vim3 and the exact interactionsites for both molecules are presently unknown furtherinvestigations have to be conducted in order to fully elucidatepotential interaction or its absence between Vim3 and otherstructural binding partnersThis protein differentiates benignoncocytoma from malignant RCC variants especially theeosinophilic RCC variant which mimics oncocytoma byimmune histology and immune fluorescence

To strengthen our interpretation we applied the Vim3antibody to a collision tumor (Figure 6) in which weidentified its metastatic component as belonging to thepapillary differentiation being Vim3 negative while the otherpart was identified as an oncocytoma based on its Vim3positivity CD117 expression is a hallmark in differentiat-ing oncocytoma and chromophobe RCC [20] In case ofthe examined collision tumor CD117 positivity could be aproblem since the chromophobe RCC is a malignant tumorand the oncocytoma is a benign one so the differentiationof the metastatic component of the collision tumor is stillquestionable (Figure 6)The further usage of an alpha-methylCoA racemase (AMACR) antibody can be used as well fordistinction between chromophobe RCC and oncocytoma[21] AMACR positivity is seen in papillary RCC [22] and canbe used as marker between primary andmetastatic RCC [21]

Figure 6 shows the two tumor types and a clear differenti-ation between the RCC subtypes namely the papillary part ofthe tumor and the benign part was possible due to the usageof our Vim3 antibody

In conclusion we present here a unique Vimentin iso-form Vim3 as a differential marker between malignantRCCs and oncocytoma We strongly believe that this cleardifferentiation between the benign and malignant kidneytumor types will be essential in the future for patientsrsquo therapyas well as operative planning follow-up therapy and patientsrsquosurvival

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Melanie von Brandenstein was supported by Koeln For-tune ProgramFaculty of Medicine University of Cologne arecipient of a postdoctoral fellowship and excellence clusterinitiative supported by University of Cologne and DFG Theauthors gratefully acknowledge the support by Susanne Sat-tler in immune histology Dr Heike Gobel for her help withthe antibody quantification and critical reading of the paperand Claudia Richter for her excellent technical assistance

References

[1] H Hamperl ldquoUber das Vorkommen von Onkocyten in ver-schiedenen Organen und ihren GeschwulstenmdashMundspei-cheldrusen Bauchspeicheldruse Epithelkorperchen Hypo-physe Schilddruse Eileiterrdquo Virchows Archiv fur PathologischeAnatomie und Physiologie und fur Klinische Medizin vol 298no 2 pp 327ndash375 1936

[2] R Kataoka Y Hyo T Hoshiya HMiyahara and TMatsunagaldquoUltrastructural study of mitochondria in oncocytesrdquo Ultra-structural Pathology vol 15 no 3 pp 231ndash239 1991

[3] J Zippel ldquoZur Kenntnis der Onkocytenrdquo Virchows Archiv vol308 no 2 pp 360ndash382 1942

[4] M J Klein and Q J Valensi ldquoProximal tubular adenomas ofkidney with so-called oncocytic features A clinicopathologicstudy of 13 cases of a rarely reported neoplasmrdquo Cancer vol 38no 2 pp 906ndash914 1976

[5] J D Oxley J Sullivan A Mitchelmore and D A GillattldquoMetastatic renal oncocytomardquo Journal of Clinical Pathologyvol 60 no 6 pp 720ndash722 2007

[6] A O Osunkoya C Cohen D Lawson M M Picken M BAmin and A N Young ldquoClaudin-7 and claudin-8 immuno-histochemical markers for the differential diagnosis of chro-mophobe renal cell carcinoma and renal oncocytomardquo HumanPathology vol 40 no 2 pp 206ndash210 2009

[7] R Waldherr and K Schwechheimer ldquoCo-expression of cytok-eratin and vimentin intermediate-sized filaments in renal cellcarcinomas comparative study of the intermediate-sized fila-ment distribution in renal cell carcinomas and normal humankidneyrdquo Virchows Archiv A vol 408 no 1 pp 15ndash27 1985

[8] O Hes M Michal N Kuroda et al ldquoVimentin reactivity inrenal oncocytoma immunohistochemical study of 234 casesrdquoArchives of Pathology and Laboratory Medicine vol 131 no 12pp 1782ndash1788 2007

[9] J Ivaska H-M Pallari J Nevo and J E Eriksson ldquoNovel func-tions of vimentin in cell adhesion migration and signalingrdquoExperimental Cell Research vol 313 no 10 pp 2050ndash2062 2007

[10] D Thakkar L Shervington and A Shervington ldquoProteomicstudies coupled with RNAi methodologies can shed furtherlight on the downstream effects of telomerase in gliomardquoCancerInvestigation vol 29 no 2 pp 113ndash122 2011

[11] M Malakoutikhah M J Gomara J A Gomez-Puerta R San-martı and I Haro ldquoThe use of chimeric vimentin citrullinatedpeptides for the diagnosis of rheumatoid arthritisrdquo Journal ofMedicinal Chemistry vol 54 no 21 pp 7486ndash7492 2011

8 Disease Markers

[12] P H Tan L Cheng N Rioux-Leclercq et al ldquoRenal tumorsdiagnostic and prognostic biomarkersrdquo The American Journalof Surgical Pathology vol 37 no 10 pp 1518ndash1531 2013

[13] M Gerstung T Roth H-P Dienes C Licht and J W U FriesldquoEndothelin-1 induces NF-120581B via two independent pathwaysin human renal tubular epithelial cellsrdquo American Journal ofNephrology vol 27 no 3 pp 294ndash300 2007

[14] M G von Brandenstein A N Abety R Depping et al ldquoA p38-p65 transcription complex induced by endothelin-1 mediatessignal transduction in cancer cellsrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1783 no 9 pp 1613ndash16222008

[15] K Ikeda T Monden T Kanoh et al ldquoExtraction and analysisof diagnostically useful proteins from formalin-fixed paraffin-embedded tissue sectionsrdquo Journal of Histochemistry and Cyto-chemistry vol 46 no 3 pp 397ndash403 1998

[16] A J Sarria J G Lieber S K Nordeen and R M Evans ldquoThepresence or absence of a vimentin-type intermediate filamentnetwork affects the shape of the nucleus in human SW-13 cellsrdquoJournal of Cell Science vol 107 no part 6 pp 1593ndash1607 1994

[17] S D Georgatos andG Blobel ldquoLamin B constitutes an interme-diate filament attachment site at the nuclear enveloperdquo Journalof Cell Biology vol 105 no 1 pp 117ndash125 1987

[18] R Spurny M Gregor M J Castanon and G WicheldquoPlectin deficiency affects precursor formation and dynamicsof vimentin networksrdquo Experimental Cell Research vol 314 no19 pp 3570ndash3580 2008

[19] O Esue A A Carson Y Tseng and D Wirtz ldquoA directinteraction between actin and vimentin filaments mediatedby the tail domain of vimentinrdquo The Journal of BiologicalChemistry vol 281 no 41 pp 30393ndash30399 2006

[20] S Kruger K Sotlar I Kausch and H-P Horny ldquoExpressionof KIT (CD117) in renal cell carcinoma and renal oncocytomardquoOncology vol 68 no 2-3 pp 269ndash275 2005

[21] F Lin R E Brown T Shen X J Yang and C SchuerchldquoImmunohistochemical detection of P504S in primary andmetastatic renal cell carcinomasrdquo Applied Immunohistochem-istry andMolecularMorphology vol 12 no 2 pp 153ndash159 2004

[22] V Molinie A Balaton S Rotman et al ldquoAlpha-methyl CoAracemase expression in renal cell carcinomasrdquo Human Pathol-ogy vol 37 no 6 pp 698ndash703 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Disease Markers

Vim3

Vimentinfull length

RCCCPPap Eosino Oncocytoma

Figure 5 Immune fluorescence of oncocytoma and RCC subtypes Full length Vimentin positive tumor cells are observed in clear cell andpapillary RCCs while Vim3 positive cells are only found in oncocytoma which otherwise are negative for full length Vimentin HampE stainingof typical tumor morphology original magnification times400

Pap

ONCO

HampE AMACR CD117Vimentinfull length Vim3

Figure 6 Collision tumor with two different tumor subtypes Top row papillary RCC bottom row preliminary diagnosis oncocytomaHampE staining and immunohistological staining for AMACR and CD117 Vimentin FL and Vim3 were performed Questionable was thesecond tumor type unexpectedly found in the tissue sample Immune fluorescence staining for Vimentin FL positive (V9) was positive andthe immune fluorescence for Vim3 was negative in the papillary RCC component indicating the malignant tumor subtype whereas thesecond one is an oncocytoma (Vim3 positive)

Disease Markers 7

The exact nature and mechanism of the ldquoVimentinpositive oncocytomasrdquo require further clarification Ourresults indicate that these tumors have to be classified asan eosinophilic variant of clear cell RCCs Since their mor-phologic appearance on an HampE slide seems identical to aldquotruerdquo oncocytoma we performed an immune fluorescencefor Vim3 (Figure 5) This resulted in a clearly Vim3 negativeappearance of these tumors regarded as ldquotruerdquo oncocytomas

However the importance of this study for routine patho-logic diagnoses with respect to the mystery of ldquoVimentinpositive oncocytomasrdquo advocates our current explanation

To date an intracellular role of Vim3 has not beendefined while an intracellular role of the full length Vimentinmolecule has been described in the literature as an anchoringmolecule for the nucleus [16] Knowing the interaction ofits full length counterpart one may speculate about Vim3rsquosintracellular importance Since the N-terminal domain andthe rod domain have not changed binding partners such asankyrin [17] and interactions with plectin [18] should still bepossible In contrast the missing tail and the unique aminoacids of its C-terminal ending may result in differences inthe C-terminal interaction Currently the tail-domain hasbeen reported to be the binding and interactive site for F-actin [19] and lamin B [17] However since the major part ofthe C-terminus is absent in Vim3 and the exact interactionsites for both molecules are presently unknown furtherinvestigations have to be conducted in order to fully elucidatepotential interaction or its absence between Vim3 and otherstructural binding partnersThis protein differentiates benignoncocytoma from malignant RCC variants especially theeosinophilic RCC variant which mimics oncocytoma byimmune histology and immune fluorescence

To strengthen our interpretation we applied the Vim3antibody to a collision tumor (Figure 6) in which weidentified its metastatic component as belonging to thepapillary differentiation being Vim3 negative while the otherpart was identified as an oncocytoma based on its Vim3positivity CD117 expression is a hallmark in differentiat-ing oncocytoma and chromophobe RCC [20] In case ofthe examined collision tumor CD117 positivity could be aproblem since the chromophobe RCC is a malignant tumorand the oncocytoma is a benign one so the differentiationof the metastatic component of the collision tumor is stillquestionable (Figure 6)The further usage of an alpha-methylCoA racemase (AMACR) antibody can be used as well fordistinction between chromophobe RCC and oncocytoma[21] AMACR positivity is seen in papillary RCC [22] and canbe used as marker between primary andmetastatic RCC [21]

Figure 6 shows the two tumor types and a clear differenti-ation between the RCC subtypes namely the papillary part ofthe tumor and the benign part was possible due to the usageof our Vim3 antibody

In conclusion we present here a unique Vimentin iso-form Vim3 as a differential marker between malignantRCCs and oncocytoma We strongly believe that this cleardifferentiation between the benign and malignant kidneytumor types will be essential in the future for patientsrsquo therapyas well as operative planning follow-up therapy and patientsrsquosurvival

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Melanie von Brandenstein was supported by Koeln For-tune ProgramFaculty of Medicine University of Cologne arecipient of a postdoctoral fellowship and excellence clusterinitiative supported by University of Cologne and DFG Theauthors gratefully acknowledge the support by Susanne Sat-tler in immune histology Dr Heike Gobel for her help withthe antibody quantification and critical reading of the paperand Claudia Richter for her excellent technical assistance

References

[1] H Hamperl ldquoUber das Vorkommen von Onkocyten in ver-schiedenen Organen und ihren GeschwulstenmdashMundspei-cheldrusen Bauchspeicheldruse Epithelkorperchen Hypo-physe Schilddruse Eileiterrdquo Virchows Archiv fur PathologischeAnatomie und Physiologie und fur Klinische Medizin vol 298no 2 pp 327ndash375 1936

[2] R Kataoka Y Hyo T Hoshiya HMiyahara and TMatsunagaldquoUltrastructural study of mitochondria in oncocytesrdquo Ultra-structural Pathology vol 15 no 3 pp 231ndash239 1991

[3] J Zippel ldquoZur Kenntnis der Onkocytenrdquo Virchows Archiv vol308 no 2 pp 360ndash382 1942

[4] M J Klein and Q J Valensi ldquoProximal tubular adenomas ofkidney with so-called oncocytic features A clinicopathologicstudy of 13 cases of a rarely reported neoplasmrdquo Cancer vol 38no 2 pp 906ndash914 1976

[5] J D Oxley J Sullivan A Mitchelmore and D A GillattldquoMetastatic renal oncocytomardquo Journal of Clinical Pathologyvol 60 no 6 pp 720ndash722 2007

[6] A O Osunkoya C Cohen D Lawson M M Picken M BAmin and A N Young ldquoClaudin-7 and claudin-8 immuno-histochemical markers for the differential diagnosis of chro-mophobe renal cell carcinoma and renal oncocytomardquo HumanPathology vol 40 no 2 pp 206ndash210 2009

[7] R Waldherr and K Schwechheimer ldquoCo-expression of cytok-eratin and vimentin intermediate-sized filaments in renal cellcarcinomas comparative study of the intermediate-sized fila-ment distribution in renal cell carcinomas and normal humankidneyrdquo Virchows Archiv A vol 408 no 1 pp 15ndash27 1985

[8] O Hes M Michal N Kuroda et al ldquoVimentin reactivity inrenal oncocytoma immunohistochemical study of 234 casesrdquoArchives of Pathology and Laboratory Medicine vol 131 no 12pp 1782ndash1788 2007

[9] J Ivaska H-M Pallari J Nevo and J E Eriksson ldquoNovel func-tions of vimentin in cell adhesion migration and signalingrdquoExperimental Cell Research vol 313 no 10 pp 2050ndash2062 2007

[10] D Thakkar L Shervington and A Shervington ldquoProteomicstudies coupled with RNAi methodologies can shed furtherlight on the downstream effects of telomerase in gliomardquoCancerInvestigation vol 29 no 2 pp 113ndash122 2011

[11] M Malakoutikhah M J Gomara J A Gomez-Puerta R San-martı and I Haro ldquoThe use of chimeric vimentin citrullinatedpeptides for the diagnosis of rheumatoid arthritisrdquo Journal ofMedicinal Chemistry vol 54 no 21 pp 7486ndash7492 2011

8 Disease Markers

[12] P H Tan L Cheng N Rioux-Leclercq et al ldquoRenal tumorsdiagnostic and prognostic biomarkersrdquo The American Journalof Surgical Pathology vol 37 no 10 pp 1518ndash1531 2013

[13] M Gerstung T Roth H-P Dienes C Licht and J W U FriesldquoEndothelin-1 induces NF-120581B via two independent pathwaysin human renal tubular epithelial cellsrdquo American Journal ofNephrology vol 27 no 3 pp 294ndash300 2007

[14] M G von Brandenstein A N Abety R Depping et al ldquoA p38-p65 transcription complex induced by endothelin-1 mediatessignal transduction in cancer cellsrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1783 no 9 pp 1613ndash16222008

[15] K Ikeda T Monden T Kanoh et al ldquoExtraction and analysisof diagnostically useful proteins from formalin-fixed paraffin-embedded tissue sectionsrdquo Journal of Histochemistry and Cyto-chemistry vol 46 no 3 pp 397ndash403 1998

[16] A J Sarria J G Lieber S K Nordeen and R M Evans ldquoThepresence or absence of a vimentin-type intermediate filamentnetwork affects the shape of the nucleus in human SW-13 cellsrdquoJournal of Cell Science vol 107 no part 6 pp 1593ndash1607 1994

[17] S D Georgatos andG Blobel ldquoLamin B constitutes an interme-diate filament attachment site at the nuclear enveloperdquo Journalof Cell Biology vol 105 no 1 pp 117ndash125 1987

[18] R Spurny M Gregor M J Castanon and G WicheldquoPlectin deficiency affects precursor formation and dynamicsof vimentin networksrdquo Experimental Cell Research vol 314 no19 pp 3570ndash3580 2008

[19] O Esue A A Carson Y Tseng and D Wirtz ldquoA directinteraction between actin and vimentin filaments mediatedby the tail domain of vimentinrdquo The Journal of BiologicalChemistry vol 281 no 41 pp 30393ndash30399 2006

[20] S Kruger K Sotlar I Kausch and H-P Horny ldquoExpressionof KIT (CD117) in renal cell carcinoma and renal oncocytomardquoOncology vol 68 no 2-3 pp 269ndash275 2005

[21] F Lin R E Brown T Shen X J Yang and C SchuerchldquoImmunohistochemical detection of P504S in primary andmetastatic renal cell carcinomasrdquo Applied Immunohistochem-istry andMolecularMorphology vol 12 no 2 pp 153ndash159 2004

[22] V Molinie A Balaton S Rotman et al ldquoAlpha-methyl CoAracemase expression in renal cell carcinomasrdquo Human Pathol-ogy vol 37 no 6 pp 698ndash703 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 7

The exact nature and mechanism of the ldquoVimentinpositive oncocytomasrdquo require further clarification Ourresults indicate that these tumors have to be classified asan eosinophilic variant of clear cell RCCs Since their mor-phologic appearance on an HampE slide seems identical to aldquotruerdquo oncocytoma we performed an immune fluorescencefor Vim3 (Figure 5) This resulted in a clearly Vim3 negativeappearance of these tumors regarded as ldquotruerdquo oncocytomas

However the importance of this study for routine patho-logic diagnoses with respect to the mystery of ldquoVimentinpositive oncocytomasrdquo advocates our current explanation

To date an intracellular role of Vim3 has not beendefined while an intracellular role of the full length Vimentinmolecule has been described in the literature as an anchoringmolecule for the nucleus [16] Knowing the interaction ofits full length counterpart one may speculate about Vim3rsquosintracellular importance Since the N-terminal domain andthe rod domain have not changed binding partners such asankyrin [17] and interactions with plectin [18] should still bepossible In contrast the missing tail and the unique aminoacids of its C-terminal ending may result in differences inthe C-terminal interaction Currently the tail-domain hasbeen reported to be the binding and interactive site for F-actin [19] and lamin B [17] However since the major part ofthe C-terminus is absent in Vim3 and the exact interactionsites for both molecules are presently unknown furtherinvestigations have to be conducted in order to fully elucidatepotential interaction or its absence between Vim3 and otherstructural binding partnersThis protein differentiates benignoncocytoma from malignant RCC variants especially theeosinophilic RCC variant which mimics oncocytoma byimmune histology and immune fluorescence

To strengthen our interpretation we applied the Vim3antibody to a collision tumor (Figure 6) in which weidentified its metastatic component as belonging to thepapillary differentiation being Vim3 negative while the otherpart was identified as an oncocytoma based on its Vim3positivity CD117 expression is a hallmark in differentiat-ing oncocytoma and chromophobe RCC [20] In case ofthe examined collision tumor CD117 positivity could be aproblem since the chromophobe RCC is a malignant tumorand the oncocytoma is a benign one so the differentiationof the metastatic component of the collision tumor is stillquestionable (Figure 6)The further usage of an alpha-methylCoA racemase (AMACR) antibody can be used as well fordistinction between chromophobe RCC and oncocytoma[21] AMACR positivity is seen in papillary RCC [22] and canbe used as marker between primary andmetastatic RCC [21]

Figure 6 shows the two tumor types and a clear differenti-ation between the RCC subtypes namely the papillary part ofthe tumor and the benign part was possible due to the usageof our Vim3 antibody

In conclusion we present here a unique Vimentin iso-form Vim3 as a differential marker between malignantRCCs and oncocytoma We strongly believe that this cleardifferentiation between the benign and malignant kidneytumor types will be essential in the future for patientsrsquo therapyas well as operative planning follow-up therapy and patientsrsquosurvival

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Melanie von Brandenstein was supported by Koeln For-tune ProgramFaculty of Medicine University of Cologne arecipient of a postdoctoral fellowship and excellence clusterinitiative supported by University of Cologne and DFG Theauthors gratefully acknowledge the support by Susanne Sat-tler in immune histology Dr Heike Gobel for her help withthe antibody quantification and critical reading of the paperand Claudia Richter for her excellent technical assistance

References

[1] H Hamperl ldquoUber das Vorkommen von Onkocyten in ver-schiedenen Organen und ihren GeschwulstenmdashMundspei-cheldrusen Bauchspeicheldruse Epithelkorperchen Hypo-physe Schilddruse Eileiterrdquo Virchows Archiv fur PathologischeAnatomie und Physiologie und fur Klinische Medizin vol 298no 2 pp 327ndash375 1936

[2] R Kataoka Y Hyo T Hoshiya HMiyahara and TMatsunagaldquoUltrastructural study of mitochondria in oncocytesrdquo Ultra-structural Pathology vol 15 no 3 pp 231ndash239 1991

[3] J Zippel ldquoZur Kenntnis der Onkocytenrdquo Virchows Archiv vol308 no 2 pp 360ndash382 1942

[4] M J Klein and Q J Valensi ldquoProximal tubular adenomas ofkidney with so-called oncocytic features A clinicopathologicstudy of 13 cases of a rarely reported neoplasmrdquo Cancer vol 38no 2 pp 906ndash914 1976

[5] J D Oxley J Sullivan A Mitchelmore and D A GillattldquoMetastatic renal oncocytomardquo Journal of Clinical Pathologyvol 60 no 6 pp 720ndash722 2007

[6] A O Osunkoya C Cohen D Lawson M M Picken M BAmin and A N Young ldquoClaudin-7 and claudin-8 immuno-histochemical markers for the differential diagnosis of chro-mophobe renal cell carcinoma and renal oncocytomardquo HumanPathology vol 40 no 2 pp 206ndash210 2009

[7] R Waldherr and K Schwechheimer ldquoCo-expression of cytok-eratin and vimentin intermediate-sized filaments in renal cellcarcinomas comparative study of the intermediate-sized fila-ment distribution in renal cell carcinomas and normal humankidneyrdquo Virchows Archiv A vol 408 no 1 pp 15ndash27 1985

[8] O Hes M Michal N Kuroda et al ldquoVimentin reactivity inrenal oncocytoma immunohistochemical study of 234 casesrdquoArchives of Pathology and Laboratory Medicine vol 131 no 12pp 1782ndash1788 2007

[9] J Ivaska H-M Pallari J Nevo and J E Eriksson ldquoNovel func-tions of vimentin in cell adhesion migration and signalingrdquoExperimental Cell Research vol 313 no 10 pp 2050ndash2062 2007

[10] D Thakkar L Shervington and A Shervington ldquoProteomicstudies coupled with RNAi methodologies can shed furtherlight on the downstream effects of telomerase in gliomardquoCancerInvestigation vol 29 no 2 pp 113ndash122 2011

[11] M Malakoutikhah M J Gomara J A Gomez-Puerta R San-martı and I Haro ldquoThe use of chimeric vimentin citrullinatedpeptides for the diagnosis of rheumatoid arthritisrdquo Journal ofMedicinal Chemistry vol 54 no 21 pp 7486ndash7492 2011

8 Disease Markers

[12] P H Tan L Cheng N Rioux-Leclercq et al ldquoRenal tumorsdiagnostic and prognostic biomarkersrdquo The American Journalof Surgical Pathology vol 37 no 10 pp 1518ndash1531 2013

[13] M Gerstung T Roth H-P Dienes C Licht and J W U FriesldquoEndothelin-1 induces NF-120581B via two independent pathwaysin human renal tubular epithelial cellsrdquo American Journal ofNephrology vol 27 no 3 pp 294ndash300 2007

[14] M G von Brandenstein A N Abety R Depping et al ldquoA p38-p65 transcription complex induced by endothelin-1 mediatessignal transduction in cancer cellsrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1783 no 9 pp 1613ndash16222008

[15] K Ikeda T Monden T Kanoh et al ldquoExtraction and analysisof diagnostically useful proteins from formalin-fixed paraffin-embedded tissue sectionsrdquo Journal of Histochemistry and Cyto-chemistry vol 46 no 3 pp 397ndash403 1998

[16] A J Sarria J G Lieber S K Nordeen and R M Evans ldquoThepresence or absence of a vimentin-type intermediate filamentnetwork affects the shape of the nucleus in human SW-13 cellsrdquoJournal of Cell Science vol 107 no part 6 pp 1593ndash1607 1994

[17] S D Georgatos andG Blobel ldquoLamin B constitutes an interme-diate filament attachment site at the nuclear enveloperdquo Journalof Cell Biology vol 105 no 1 pp 117ndash125 1987

[18] R Spurny M Gregor M J Castanon and G WicheldquoPlectin deficiency affects precursor formation and dynamicsof vimentin networksrdquo Experimental Cell Research vol 314 no19 pp 3570ndash3580 2008

[19] O Esue A A Carson Y Tseng and D Wirtz ldquoA directinteraction between actin and vimentin filaments mediatedby the tail domain of vimentinrdquo The Journal of BiologicalChemistry vol 281 no 41 pp 30393ndash30399 2006

[20] S Kruger K Sotlar I Kausch and H-P Horny ldquoExpressionof KIT (CD117) in renal cell carcinoma and renal oncocytomardquoOncology vol 68 no 2-3 pp 269ndash275 2005

[21] F Lin R E Brown T Shen X J Yang and C SchuerchldquoImmunohistochemical detection of P504S in primary andmetastatic renal cell carcinomasrdquo Applied Immunohistochem-istry andMolecularMorphology vol 12 no 2 pp 153ndash159 2004

[22] V Molinie A Balaton S Rotman et al ldquoAlpha-methyl CoAracemase expression in renal cell carcinomasrdquo Human Pathol-ogy vol 37 no 6 pp 698ndash703 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Disease Markers

[12] P H Tan L Cheng N Rioux-Leclercq et al ldquoRenal tumorsdiagnostic and prognostic biomarkersrdquo The American Journalof Surgical Pathology vol 37 no 10 pp 1518ndash1531 2013

[13] M Gerstung T Roth H-P Dienes C Licht and J W U FriesldquoEndothelin-1 induces NF-120581B via two independent pathwaysin human renal tubular epithelial cellsrdquo American Journal ofNephrology vol 27 no 3 pp 294ndash300 2007

[14] M G von Brandenstein A N Abety R Depping et al ldquoA p38-p65 transcription complex induced by endothelin-1 mediatessignal transduction in cancer cellsrdquo Biochimica et BiophysicaActamdashMolecular Cell Research vol 1783 no 9 pp 1613ndash16222008

[15] K Ikeda T Monden T Kanoh et al ldquoExtraction and analysisof diagnostically useful proteins from formalin-fixed paraffin-embedded tissue sectionsrdquo Journal of Histochemistry and Cyto-chemistry vol 46 no 3 pp 397ndash403 1998

[16] A J Sarria J G Lieber S K Nordeen and R M Evans ldquoThepresence or absence of a vimentin-type intermediate filamentnetwork affects the shape of the nucleus in human SW-13 cellsrdquoJournal of Cell Science vol 107 no part 6 pp 1593ndash1607 1994

[17] S D Georgatos andG Blobel ldquoLamin B constitutes an interme-diate filament attachment site at the nuclear enveloperdquo Journalof Cell Biology vol 105 no 1 pp 117ndash125 1987

[18] R Spurny M Gregor M J Castanon and G WicheldquoPlectin deficiency affects precursor formation and dynamicsof vimentin networksrdquo Experimental Cell Research vol 314 no19 pp 3570ndash3580 2008

[19] O Esue A A Carson Y Tseng and D Wirtz ldquoA directinteraction between actin and vimentin filaments mediatedby the tail domain of vimentinrdquo The Journal of BiologicalChemistry vol 281 no 41 pp 30393ndash30399 2006

[20] S Kruger K Sotlar I Kausch and H-P Horny ldquoExpressionof KIT (CD117) in renal cell carcinoma and renal oncocytomardquoOncology vol 68 no 2-3 pp 269ndash275 2005

[21] F Lin R E Brown T Shen X J Yang and C SchuerchldquoImmunohistochemical detection of P504S in primary andmetastatic renal cell carcinomasrdquo Applied Immunohistochem-istry andMolecularMorphology vol 12 no 2 pp 153ndash159 2004

[22] V Molinie A Balaton S Rotman et al ldquoAlpha-methyl CoAracemase expression in renal cell carcinomasrdquo Human Pathol-ogy vol 37 no 6 pp 698ndash703 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom