Can vascular endothelial growth factor C expression be of use in predicting surgical stage or prognosis in vulvar cancer ?

Jou nal o Cance Resea ch & The apy O iginal esea ch Nybe g RH e al., J Cance Res The . 2017, 5(8):50-55 h p://dx.doi.o g/10.14312/2052-4994.2017-10 Can ascula endo helial g ow h ac o C exp ession be o use in p edic ing su gical s age o p ognosis in ul a cance ? Nybe g Rei a H1,, Lau ila Ma i a2, S a Synnö e1,3 and Mäenpää Johanna U1,4 1 Depa men o Gynecology and Obs e ics, Tampe e Uni e si y Hospi al, Tampe e, Finland 2 Depa men o Pa hology, Fimlab Labo a o ies Inc., Tampe e, Finland 3 Labo a o y o Cance Biology, BioMediTech, Uni e si y o Tampe e, Tampe e, Finland 4 Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland Abs ac In oduc ion: Nodal me as asis is a main p ognos ic ac o in ul a cance . Inc eased ascula endo helial g ow h ac o C (VEGF-C) exp ession has been associa ed wi h lymph node me as asis and poo p ognosis in many cance s. The aim o his e ospec i e s udy was o in es iga e VEGF-C exp ession pa e n in he in asi e edge o ul a cance and in sen inel lymph node me as asis, and i s associa ion wi h he s age and p ognosis. Me hods: Tumo and sen inel lymph node samples om 44 pa ien s we e e alua ed wi h immunohis ochemis y, and he esul s we e linked wi h he clinicopa hological da a. Resul s: Six y-se en pe cen o p ima y umo s and 76% o sen inel lymph node me as ases exp essed VEFG-C. Posi i e VEGF-C exp ession o he p ima y umo did no p edic su gical S age o sen inel lymph node in ol emen . The isk o elapse was no signi ican ly highe wi h VEGF-C exp essing umo s han wi h VEGF-C nega i e umo s (RR 2.55, 95% CI 0.66-9.90, p = 0.18). The isk o g oin ecu ence was signi ican ly lowe wi h VEGF-C posi i e han nega i e umo s (RR 0.36, 95% CI 0.16-0.79, p = 0.01). Su i al was simila in bo h g oups. No non-sen inel lymph node me as ases we e ound in case o nega i e VEGF-C exp ession in he sen inel lymph node me as asis, whe eas wi h posi i e VEGF-C exp ession hey we e ound in 5/13 (38%) o cases. Conclusions: Tumo al VEGF-C exp ession was no associa ed wi h highe su gical S age o poo e p ognosis in ul a cance . Howe e , absence o i s exp ession in sen inel lymph node me as asis migh indica e a low isk o non-sen inel lymph node me as ases. Keywo ds: ul a cance ; VEGF-C; sen inel lymph node; nodal me as asis; lympha ic sp ead; p ognosis; ecu ence Co esponding au ho : Rei a Nybe g, Depa men o Gynecology and Obs e ics, Tampe e Uni e si y Hospi al, Tampe e, Finland. Tel.: +358 50 384 7500; Email: [email p o ec ed] Recei ed 12 July 2017 Re ised 14 Augus 2017 Accep ed 22 Augus 2017 Published 31 Augus 2017 Ci a ion: Nybe g RH, Lau ila M, S a S, Mäenpää JU. Can ascula endo helial g ow h ac o C exp ession be o use in p edic ing su gical s age o p ognosis in ul a cance ? J Cance Res The . 2017; 5(8):50-55. DOI: 10.14312/2052-4994.2017-10 Copy igh : © 2017 Nybe g RH, e al. Published by NobleResea ch Publishe s. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Open Access An Open Access Publishe In oduc ion Nodal me as a ic in ol emen is he mos impo an p ognos ic ac o in ul a cance . Node-nega i e pa ien s ha e a 5-yea su i al a e o 70 - 98% bu hose wi h posi i e nodes only 12 - 41% [1]. Size o he p ima y umo , p esence o lympho ascula in asion and he dep h o in asion a e known o inc ease he isk o nodal me as asis [2–4], as well as he cen al loca ion o he p ima y umo [5]. Biologic p ognos ic a iables a e no as well known. Inc eased umo angiogenesis and al e ed essel cha ac e is ics a e sugges ed o lead in o a sho e disease- ee su i al [6]. Exp ession le els o ma ix me allop o einase-2 exp ession highe han 50% a e also an indica o o a lowe i e- yea su i al a e [7]. O e -exp ession o issue ma ix me allop o einases, ansmemb ane p o ein CD44 and i s iso o ms, h ombospondin-1, ascula endo helial g ow h ac o (VEGF) and some G2/M pa hway egula o s, as well as, loss o me as asis supp esso NM23-H1 gene, seem o p omo e local and me as a ic g ow h. Howe e , no all hese indings co ela e wi h clinical p ognosis [1, 8]. S udies in animal models and humans ha e shown ha lymphangiogenesis in a p ima y umo inc eases nodal me as asis [9]. E en be o e he me as asis ac ually akes place, he lymph nodes d aining s aigh om he umo – so called sen inel lymph nodes (SLNs) - unde go emodelling p ocesses including lymphangiogenesis, al e a ions in s uc u e, lympha ic low and immune cell composi ion, and inc eases in chemokine and cy okine p oduc ion, hus c ea ing a p eme as a ic niche [10]. Vascula endo helial g ow h ac o C (VEGF-C) sec e ed by he p ima y umo is he mos impo an lymphangiogenic ac o causing he emodelling. I al e s he lympha ic essels a ound he p ima y umo , inc eases he lympha ic low and causes expansion o lympha ic ne wo k in SLNs, all his p omo ing lympha ic sp ead [11]. 51 In many human cance s, o e -exp ession o VEGF-C by p ima y umo is associa ed wi h poo e p ognosis. I co ela es wi h sho e p og ession ee su i al (PFS), o e all su i al (OS) and lymph node me as asis in melanoma [12], and is a poo p ognos ic ac o in non- small-cell lung cance and adenoca cinoma o he lung [13, 14]. High le els o VEGF-C exp ession in gas ic cance issue imply wo se o e all p ognosis han low VEGF-C le els [15]. P ima y umo VEGF-C exp ession has been epo ed o co ela e wi h he possibili y o lymph node me as asis in lung, oesophageal, p os a e, hy oid and colo ec al cance s [11]. To ou knowledge, he in luence o VEGF-C on he clinical cou se o ul a cance has no been s udied, bu one epo o VEGF-C exp ession in 10 issue samples has been published [16]. The aim o his s udy was o explo e he p esence o VEGF-C exp ession in ul a cance (p ima y umo and SLN me as asis), and i s in luence on pa ien s’ su gical S age, isk o ecu ence and p ognosis. Ma e ials and me hods Pa ien s and issue samples Tissue samples om 44 ul a cance pa ien s ha had p e iously unde gone ul a su ge y and a SLN mapping be o e comple e lymph node dissec ion in Tampe e Uni e si y Hospi al we e used o his s udy. Unde a 4.5-yea amilia iza ion pe iod, a SLN mapping had been pe o med o all su gically ea ed ul a cance pa ien s, and has been desc ibed elsewhe e [17]. The specimens we e ob ained om he Tissue Biobank and Resea ch Se ices FinTiB (Fimlab Labo a o ies Inc., Tampe e, Finland). Fo y-six umo samples wi h ep esen a i e malignan g ow h as well as 17 me as a ic SLN samples we e a ailable o analysis. The clinicopa hological his o y and ollow-up da a o all pa ien s we e e ospec i ely collec ed om he hospi al eco ds. The his o y included he age a he ime o he su ge y, he da e o he su ge y, he si e o he p ima y umo , he ype o su ge y, he su gical S age o he disease, he his opa hology epo , he s a us o he SLN (posi i e o nega i e o me as asis) and o he egional lymph nodes, and whe he o no o he me as ases we e p esen . The ollow-up da a included also in o ma ion o a po en ial adju an ea men and i s du a ion, he obse a ion da e and loca ion(s) o a ecu ence, i any, he inal da e o he ollow-up, and he da e and cause o dea h, i i happened du ing he ollow-up pe iod. This da a was combined wi h he esul s o he VEGF-C immunos aining o he inal analysis. Immunohis ochemis y The VEGF-C p o ein exp ession in ul a umo s and SLN samples we e e alua ed by using immunohis ochemis y (IHC). Rep esen a i e samples om he in asi e edges o he p ima y umo s and SLN me as ases we e selec ed o he s udy by an expe ienced pa hologis (M.L.). 4 µm hick sec ions we e cu om pa a in-embedded issue blocks using a s anda d mic o ome. Fo IHC s aining, he slides we e hen depa a inized, ehyd a ed, and subsequen ly p e ea ed wi h a PT-Module (Lab Vision, F emon , CA) a 98°C o 15 min in 0.05 M T isHCl bu e , pH 9.0 con aining 0.001 M EDTA. The p ima y VEGF-C an ibody (Rabbi an i- VEGF-C; In i ogen, Cama illo, CA) was isualized wi h a Powe Vision + polyme ki (Leica Biosys ems Newcas le L d., Newcas le, UK) and diaminobenzidine as ch omogen (DABImmPac , Vec o labs, Bu lingame, CA). The issue sec ions we e coun e s ained wi h hema oxylin (Maye ’s hema oxylin, Oy FFChemicals Ab, Haukipudas, Finland). Human colon ca cinoma samples, known o ha e a s ong VEGF-C exp ession, we e used as a posi i e con ol. Nega i e con ols we e made by omi ing he p ima y VEGF-C an ibody om he p ocedu e. Analysis o he immunos aining Immunos ained sec ions we e scanned wi h an Ape io Scanscope XT (Ape io Technologies, Vis a, CA) and isually analysed on a compu e sc een. Two obse e s (R.N. and S.S.) assessed hem, blinded o he clinicopa hological da a o he pa ien s. In he i s ound, he assessmen s we e pe o med independen ly and he esul s hen pooled. I he assessmen s o wo obse e s we e con adic o y, he s aining was assessed again in consensus. In all issue samples, IHC s aining in ensi y was sco ed semiquan i a i ely as nega i e (no s aining a all), weak (some sca e ed s ained cells o ain mo e widesp ead s aining), mode a e (mo e abundan widesp ead s aining o ocal in ensi e s aining) o s ong (almos all cells in ensi ely s ained). Fo he s a is ical analysis, nega i e and weak s aining we e combined as “nega i e” and simila ly, mode a e and s ong s aining as “posi i e” s aining. S a is ical analysis The conco dance be ween wo obse e ’s assessmen s o he VEGF-C exp ession in he i s e alua ion ound was assessed using Cohen’s unweigh ed kappa es [18]. Associa ions be ween VEGF-C s aining and clinicopa hological pa ame e s we e analyzed using he Fishe ’s exac es , odds a io and ela i e isk. Disease- speci ic and p og ession ee su i al cu es we e calcula ed using Kaplan-Meie su i al analysis, and compa ed using he log ank es . A p- alue less han 0.05 was conside ed s a is ically signi ican . SPSS S a is ics o Windows ( e sion 19.0 eleased 2010, IBM Co p., A monk, NY, USA) was used in calcula ion o s a is ical analysis. E hical conside a ions The use o a chi ed issue specimens o IHC was app o ed by Val i a, Na ional Supe iso y Au ho i y o Wel a e and Heal h (6746/05.01.00.06/2009). The e ospec i e collec ion o pa ien da a om he hospi al eco ds was app o ed by he E hics Commi ee o Pi kanmaa Hospi al Dis ic (R09066). Resul s Pa ien and ollow-up da a The median age o pa ien s was 76 yea s ( ange 44-93). The median ollow-up ime was 39 mon hs ( ange 0.6 - 109 mon hs). Tumo cha ac e is ics wi h S age, adju an ea men and ollow-up da a a e p esen ed in Table 1. Du ing he ollow-up, one pa ien died o cance less han h ee weeks a e su ge y and wo pa ien s du ing he Nybe g RH e al., J Cance Res The . 2017, 5(8):50-55 52 adju an adio he apy be o e comple ion o he ea men (7%, 3/44). Thi een pa ien s (30%) had a ecu ence a e he comple ion o he ea men , 7 in he ul a and 6 ou side he ul a. Th ee ou o 7 pa ien s (43%) wi h ul a ecu ence we e sal aged by eope a ion and we e ali e a he end o he ollow-up, while all six pa ien s wi h ecu ences ou side ul a died o hei disease. A he end o he ollow-up, hal o he pa ien s we e s ill ali e. In e obse e ag eemen on IHC The in e obse e ag eemen s on he VEGF-C exp ession in p ima y umo and SLN samples we e subs an ial; Cohen’s unweigh ed kappa o conco dance in umo samples was 0.69 (95% CI 0.48-0.90) and in SLN samples 0.72 (95% CI 0.50-1.06). VEGF-C exp ession in p ima y umo s and SLN me as ases O 46 p ima y umo samples, only 7% (3/46) o he in asi e edges o ul a umo s did no exp ess any VEGF-C. The exp ession was weak in 26% (12/46). Thus, al oge he 15 umo s (33%) we e classi ied as VEGF-C nega i e (Figu e 1a). The s aining was mode a e o s ong in 53% (25/46) and 13% (6/46) o he umo edges, espec i ely, and a o al o 31 (67%) umo s classi ied as VEGF-C posi i e (Figu e 1b). The e was no di e ence in median age o pa ien s wi h ei he VEGF-C nega i e o VEGF-C posi i e umo s (75.5 s. 76 yea s, p = 1.00). The high- G ade umo s ended o exp ess VEGF-C mo e o en han he low-G ade umo s bu he di e ence was no s a is ically signi ican (p = 0.36), see Figu e 2. Fo one umo , he G ade was no a ailable. Table 1 Da a on disease and umo cha ac e is ics in all 44 pa ien s. Va iable De ini ion Numbe o pa ien s (pe cen age o all) FIGOa S age I 19 (43%) II 4 (9%) III 20 (46%) IV 1 (2%) His ology and G ade o he p ima y umo in ul a SCCb43 (98%) G ade 1 22 (50%) G ade 2 14 (32%) G ade 3 7 (16%) Anaplas ic ca cinoma 1 (2%) G ade 3 1 (2%) Sen inel node me as asis No 23 (52%) Yes 20 (46%) No SLN de ec ed 1 (2%) Pos ope a i e adju an ea men No adju an eamen 22 (50%) RTc21 (48%) Concu en CRTd1 (2%) Ali e a he end o he ollow-up Yes 22 (50%) No 22 (50%) Cause o dea h Vul a cance o ela ed 15 (34%) O he cause 7 (16%) aThe In e na ional Fede a ion o Gynecology and Obs e ics; bsquamous cell ca cinoma; c adia ion he apy; dchemo adio he apy Figu e 1 Examples o (a) a weak and (b) a s ong VEGF-C immunos aining in squamous cell ca cinoma o he ul a (magni ica ion x 20). Tumo al VEGF-C exp ession and su gical S age A he ime o he su ge y, 17 ou o 30 (57%) VEGF-C posi i e and 8 ou o 14 (57%) VEGF-C nega i e umo s we e ad anced (> FIGO S age I). The isk o mo e ad anced su gical S age was he same wi h VEGF-C posi i e and nega i e umo g oups (OR 0.98, 95% CI 0.27-3.53, p = 0.98). Also, he isk o ha ing SLN me as asis a he ime o su ge y did no signi ican ly di e be ween VEGF-C posi i e o nega i e umo s (47%, 14/30 and 46%, 6/13, espec i ely; OR 1.02, 95% CI 0.28-3.77, p = 0.98). Nybe g RH e al., J Cance Res The . 2017, 5(8):50-55 Figu e 2 VEGF-C exp ession in in asi e edges o ul a cance acco ding o he his ological G ade. 100 % 90 % 80 % 70 % 60 % 50 % 40 % 30 % 20 % 10 % 0 % VEGF-C exp ession nega i e VEGF-C exp ession posi i e G ade 1 G ade 2 G ade 3 (a) (b) 53 Figu e 5 P og ession- ee su i al analysis acco ding o VEGF-C exp ession o ul a umo s (log ank es p=0.19). Foo no e: — nega i e VEGF-C exp ession; --- posi i e VEGF-C exp ession; + censo ed VEGF-C exp ession in SLN me as asis The SLN me as ases we e VEGF-C nega i e in 24% (4/17) and VEGF-C posi i e in 76% (13/17) o he cases (Figu e 3). When he p ima y umo was VEGF-C posi i e, he SLN me as asis exp essed VEGF-C in 91% (10/11) o he cases as compa ed o 50% (3/6) o he SLN me as ases in he VEGF-C nega i e ul a umo s, bu he di e ence did no each a s a is ical signi icance (p = 0.099). Figu e 3 Examples o (a) a weak and (b) a s ong VEGF-C immunos aining in sen inel lymph node me as ases (magni ica ion x 20). When he SLN me as asis exp essed VEGF-C, in 5 cases ou o 13 (38%) me as a ic non-SLNs we e also ound. Howe e , in ou cases when he SLN me as asis was VEGF-C nega i e, no o he LN me as ases we e ound (0/4; OR 5.82, 95% CI 0.26-130.89, p = 0.267). The posi i e p edic i e alue o VEGF-C exp ession in he SLN me as asis in ela ion o he non-SLN me as ases was 38 % and he nega i e p edic i e alue 100%, bea ing in mind he small numbe o VEGF-C nega i e SLN me as ases. VEGF-C exp ession and he clinical cou se o he disease In p ima y umo s: Excluding h ee pa ien s ha died be o e he comple ion o he p ima y ea men , he pa ien s wi h VEGF-C posi i e p ima y umo s seemed o elapse mo e o en (39%, 11/28) du ing he ollow-up han he pa ien s wi h VEGF-C nega i e umo s (15%, 2/13), al hough he isk was no s a is ically signi ican (RR 2.55, 95% CI 0.66-9.90, p = 0.18). The VEGF-C posi i e umo s ecu ed mos ly in he ul a a ea (64%, 7/11) while he VEGF-C nega i e umo s ecu ed in he inguinal a ea (100%, 2/2). The isk o g oin ecu ence was signi ican ly lowe , when he umo exp essed VEGF-C (RR 0.36, 95% CI 0.16-0.79, p = 0.01). The disease-speci ic su i al (DSS) as a unc ion o he VEGF-C exp ession o he p ima y umo s is shown in Figu e 4. No di e ence was obse ed (Log ank es , p = 0.83). The e seemed o be a end owa ds a be e PFS in pa ien s wi h VEGF-C nega i e umo s when compa ed o VEGF-C posi i e umo s, see Figu e 5. Howe e , his end did no each a s a is ical signi icance (Log ank es , p = 0.19). Figu e 4 Disease-speci ic su i al analysis acco ding o VEGF-C exp ession o ul a umo s (log ank es p=0.83). Foo no e: — nega i e VEGF-C exp ession; --- posi i e VEGF-C exp ession; + censo ed In SLN me as ases: The e was no di e ence in he isk o ecu ence be ween pa ien s wi h VEGF-C posi i e and nega i e SLN me as ases (5/12 and 1/3, espec i ely, RR 1.25, 95 % CI 0.22-7.08, p = 0.80), espec i ely. The only ecu ence in he g oup wi h VEGF-C nega i e SLN me as asis appea ed in ul a a ea whe eas h ee ou o i e ecu ences (60%) in he g oup wi h VEGF-C posi i e SLN me as asis appea ed in inguinal a ea and wo Nybe g RH e al., J Cance Res The . 2017, 5(8):50-55 1.0 0.8 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 0.0 0 12 24 36 48 60 72 84 96 Follow-up ime (mon hs) 0 12 24 36 48 60 72 84 96 P og ession ee su i al (mon hs) Su i al p opo ionSu i al p opo ion (a) (b) 54 ecu ences (40%) in ul a a ea. These g oups we e oo small o s a is ical analysis. Discussion Acco ding o he esul s o his s udy, he p ima y umo s o mos ul a cance s exp ess VEGF-C on hei in asi e edges. The equency o exp ession ended o co ela e posi i ely wi h his ological G ade, bu he di e ence did no each s a is ical signi icance. VEGF-C was also exp essed in h ee qua e s o he SLN me as ases, mo e o en when he p ima y umo exp essed i , al hough he di e ence was again no signi ican . VEGF-C exp ession o he p ima y umo was no associa ed wi h highe su gical S age o isk o nodal me as asis, no did i ha e any s a is ically signi ican impac on DSS o PFS. A nega i e VEGF-C exp ession in a SLN me as asis could be a a ou able indica o o cance - ee non-SLNs. When conside ing he a i y o ul a cance , ou sample size o 44 umo s was ep esen a i e. Howe e , i was s ill no la ge enough o show s a is ical signi icance be ween di e en g oups e en when a end was obse ed. The s eng hs o his s udy we e a long ollow-up ime and i s clinical o ien a ion. Ou median ollow-up o 39 mon hs was long enough o elapses o become e iden . We chose o keep he IHC sco ing simple, bea ing in mind i s po en ial applica ion o clinical pa ien ca e. Ou inding o he equency o VEGF-C exp ession in ul a cance di e s om he only o he published epo by Jach e al. In hei much smalle popula ion, VEGF-C exp ession was obse ed only in 10% (1 ou o 10) o ul a squamous cell cance (SCC) cases. The ca cinoma specimens hey used o he IHC analysis we e indi idually selec ed [16]. Howe e , he au ho s did no speci y, which pa o he umo hese specimens ep esen ed no did hey ell he his ological G ades o ul a umo s – a ea u e ha in ou s udy seemed o e ec on he VEGF-C exp ession. When he exp ession o VEGF-C was s udied om 111 ce ical SCC samples by Gombos e al., i was ound o be he e ogeneous wi hin he umo s. The exp ession was signi ican ly highe in he ma ginal po ions o ca cinomas compa ed wi h he cen al egions [19]. We also ocused on he in asi e edge o ul a umo s and no iced he same phenomenon as Gombos e al. Fu he mo e; he semiquan i a i e sco ing sys em Jach e al. used o assess VEGF-C exp ession ook in o accoun he pe cen age o VEGF-C posi i e cells. I cen al pa s o he ul a umo s we e used o he immunos aining analysis, i migh ha e diminished hei sco es e en when he s aining was s ong. Acco ding o li e a u e, SCCs in many di e en o gans exp ess VEGF-C, i.e., umo s o o al ca i y [20], oesophageal cance [21] and ce ical cance [22]. The posi i e exp ession has been associa ed wi h poo e p ognosis and highe isk o lympha ic me as asis. Howe e , in ou s udy umo al VEGF-C exp ession did no p edic su gical S age o equency o he SLN in ol emen . We obse ed ha VEGF-C posi i e cance s ended o ecu mo e o en han VEGF-C nega i e cance s, and he e o e also PFS seemed o be mo e a o able in pa ien s wi h VEGF-C nega i e umo s, albei no signi ican ly. In ou s udy popula ion, 43% o pa ien s wi h a ul a ecu ence we e success ully sal aged, while all g oin ecu ences we e a al. The isk o g oin ecu ence was signi ican ly lowe in VEGF-C posi i e umo g oup han in VEGF-C nega i e umo g oup. Be e p ognosis o he local ecu ences compa ed o he g oin ecu ences migh pa ly explain why he posi i e umo al VEGF-C exp ession had no impac on he disease-speci ic su i al. The VEGF-C exp ession in SLN me as asis was no associa ed wi h he ecu ence a e. A he momen , he only known p ognos ic ac o o non-SLN me as asis in ul a cance in ela ion o cha ac e is ics o a SLN me as asis is he size o he me as asis. GROINSS-V s udy showed ha he isk o non- SLN me as asis inc eases wi h he size o SLN me as asis. No size cu -o exis ed below which chances o non-SLN me as ases would be close o ze o. The e o e, addi ional ea men was ecommended o all SLN posi i e pa ien s, bu i always inc eases side e ec s and lowe s quali y o li e [23]. Ou s udy sugges s ha a nega i e VEGF-C exp ession in SLN me as ases could ac as an indica o o cance - ee non-SLNs. Howe e , he small numbe o me as a ic SLN samples ( ou ) limi s d awing conclusions and his inding should be es ed in a la ge popula ion. I ep oducible, VEGF-C exp ession in SLN me as asis could se e as ano he p ognos ic ac o when conside ing addi ional ea men . Conclusion VEGF-C exp ession was equen in he in asi e edges o malignan ul a umo s and hei SLN me as ases. I e i ied in a la ge popula ion, he lack o VEGF exp ession in SLN me as asis may in he u u e p o e o be a use ul indica o o lowe isk o non-sen inel lymph node me as asis. O he wise, VEGF-C exp ession in p ima y umo s did no seem o unc ion as a help ul indica o o su gical S age o p ognosis in ul a cance . Acknowledgemen s The au ho s wan o hank P o . Jo ma Isola, MD, PhD, o his aluable ad ice and consul a ion in ela ion o IHC, Heini Huh ala, M.Sc. o he assis ance wi h he s a is ical analysis and AV consul an Mika Ma ikainen o his help wi h he digi al a wo k. Funding This s udy has been suppo ed by Resea ch G an s om Pi kanmaa Hospi al Dis ic 's Science Cen e (G an numbe 9H183), Compe i i e Resea ch Funding o Tampe e Uni e si y Hospi al (G an numbe 9L062) and Resea ch Founda ion o Obs e ics and Gynecology, Finland (Pe sonal G an o R.N.). 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