Non-Invasive Detection of Anastomotic Leakage Following Esophageal and Pancreatic Surgery by Urinary Analysis

Transcription

1 Originl Pper Received: October 3, 217 Accepted: Februry 22, 218 Published online: June 15, 218 Non-Invsive Detection of Anstomotic Lekge Following Esophgel nd Pncretic Surgery by Urinry Anlysis Victor D. Plt Nor vn Gl b Jmes A. Covington c Mtthew Nel d Tim G.J. de Meij e Donld L. vn der Peet Bbs Zonderhuis Geert Kzemier Nnne K.H. de Boer b Freek Dms Deprtment of Gstrointestinl surgery, VU University Medicl Center, Amsterdm, The Netherlnds; b Deprtment of Gstroenterology nd Heptology, VU University Medicl Center, Amsterdm, The Netherlnds; c School of Engineering, University of Wrwick, Coventry, UK; d Deprtment of Sttistics, University of Wrwick, Coventry, UK; e Deprtment of Peditric Gstroenterology nd Heptology, VU University Medicl Center, Amsterdm, The Netherlnds Keywords Pncretic surgery Esophgel surgery Anstomotic lekge Voltile orgnic compounds Field symmetric ion mobility spectrometry Abstrct Bckground: Esophgectomy or pncreticoduodenectomy is the stndrd surgicl pproch for ptients with tumors of the esophgus or pncretic hed. Postopertive mortlity is strongly correlted with the occurrence of nstomotic lekge (AL). Dely in dignosis leds to dely in tretment, which rtifies the need for development of novel nd ccurte non-invsive dignostic tests for detection of AL. Urinry voltile orgnic compounds (VOCs) reflect the metbolic sttus of n individul, which is ssocited with systemic immunologicl response. The im of this study ws to determine the dignostic ccurcy of urinry VOCs to detect AL fter esophgectomy or pncreticoduodenectomy. Methods: In the present study, urinry VOCs of 63 ptients fter esophgectomy (n = 31) or pncreticoduodenectomy (n = 32) were nlyzed by mens of field symmetric ion mobility spectrometry. AL ws defined ccording to interntionl study groups. Results: AL ws observed in 15 ptients (24%). Urinry VOCs of ptients with AL fter pncreticoduodenectomy could be distinguished from uncomplicted controls, re under the curve.85 (95% CI.76.93), sensitivity 76%, nd specificity 77%. However, this ws not observed following esophgectomy, re under the curve.51 (95% CI.37.65). Conclusion: In our study popultion AL following pncreticoduodenectomy could be discriminted from uncomplicted controls by mens of urinry VOC nlysis, NTC Introduction 218 The Author(s) Published by S. Krger AG, Bsel Anstomotic lekge (AL) is the most dreded nd frequent compliction fter intestinl surgery, leding to significntly incresed morbidity nd 3-dy mortlity [1]. According to the Dutch Institute for Clinicl Audit- E-Mil krger@krger.com The Author(s) Published by S. Krger AG, Bsel This rticle is licensed under the Cretive Commons Attribution- NonCommercil-NoDerivtives 4. Interntionl License (CC BY- NC-ND) ( Usge nd distribution for commercil purposes s well s ny distribution of modified mteril requires written permission. Victor D. Plt, BSc Deprtment of Gstrointestinl Surgery VU University Medicl Center De Boeleln 1117, ZH 7F2, NL 181 HV Amsterdm (The Netherlnds) E-Mil vumc.nl

2 ing, lekge occurs in 12.8% of pncreticoduodenectomies nd 2% of esophgectomies crried out in the Netherlnds [2]. In current prctice, AL is dignosed bsed on clinicl, biochemicl, nd rdiologicl findings. In the erly postopertive phse, clinicl prmeters hve been shown to be indecisive, requiring dditionl exmintion [3]. Primry biochemicl predictor is the cute phse C-rective protein (CRP). However, serum CRP lcks sensitivity nd specificity for mjor infectious complictions [4]. In ddition, incresed levels of mylse in drin fluid hve been shown to be predictive for esophgel [5] nd pncretic AL [6]. Nevertheless, erly drin removl is fvorble for the postopertive recovery, preventing secondry infection nd theoreticlly pncretitis [7]. This emphsizes the need for the development of novel, non-invsive biomrkers, with high ccurcy for predicting nd dignosing AL during the first postopertive dys. Since 2 BC, ncient physicins hve been dignosing ptients bsed on their own olfctory system [8]. Most gses, either odorous or non-odorous, re composed of voltile orgnic compounds (VOCs). These compounds hve severl origins: firstly, environmentl or exogenous; secondly, locl symptoms from the primry ffected disese site in disesed ptients which is often inflmmtion or cncer; nd thirdly, the systemic (immunologicl) response [8]. Assessment of VOCs in breth, feces, nd urine hs led to the identifiction of vriety of disese-specific smell prints in, for exmple, pulmonology, oncology, nd gstroenterology [9 14]. The im of this study ws to determine the dignostic ccurcy of nlysis of urinry VOCs to detect AL noninvsively fter mjor gstrointestinl surgery. Methods Study Design nd Prticipnts This study ws conducted in ccordnce with the World Medicl Assocition Declrtion of Helsinki. The locl institutionl ethicl review bord of the VU medicl center pproved the study protocol ( ). This project ws designed s single center prospective observtionl cohort study between Mrch 215 nd April 216. Eligible prticipnts were dignosed with (suspected) mlignncies of the esophgus, pncres, distl bile duct, mpull of Vter, or duodenum nd were scheduled for surgery (i.e., esophgectomy or pncreticoduodenectomy). Ptients were ged 18 9 yers nd hd n Americn Society of Anesthesiologists physicl sttus of 3 or lower. All prticipnts were cpble of understnding the study informtion, nd signed written informed consent. Ptient, surgery, nd pthology dt were prospectively registered. Procedure Ptients were recruited from the deprtment of surgery of the VU University medicl center (Amsterdm, The Netherlnds). They were dmitted to the hospitl 1 dy prior to surgery nd received stndrd preopertive tretment ccording to locl protocol. Ptients dignosed with esophgel cncer underwent the following procedures: Ivor Lewis, McKeown, or trnshitl esophgectomy. For pncretic cncer, pncreticoduodenectomy ws performed ccording to Whipple or with preservtion of the gstric pylorus (pylorus-preserving pncreticoduodenectomy [PPPD]). All procedures were performed under stndrdized ntibiotic prophylxis (cefuroxime/metronidzole). In cse of pncreticoduodenectomy, prophylctic drin ws plced t the pncreticojejunostomy. Postopertively, ll ptients were treted ccording to locl protocol, which consisted of dmittnce to the intensive cre unit, medium cre, or generl wrd. Drins were removed when drin mylse content ws within norml rnge on or fter postopertive dy 3. Ptients did not receive dditionl ntibiotics during the first 3 postopertive dys. Endpoints The primry endpoint of this study concerned clinicl postopertive AL. Lekge following esophgel surgery ws defined by the Esophgectomy Complictions Consensus Group s full thickness defect involving the esophgel nstomosis. Severity ws grded bsed on required tretment. Grde 1 ws locl defect only requiring medicl tretment with dietry modifiction. Grde 2 required interventionl but not surgicl therpy, nd grde 3 AL ws surgiclly treted [15]. Lekge of the pncreticojejunostomy ws defined s ny mesurble volume of drin fluid on or fter postopertive dy 3, with mylse content more thn 3 times the upper norml serum volume. The Interntionl Study Group on Pncretic Fistul grded lekge ccording to clinicl impct. A biochemicl lek (formerly grde A) hs by definition no clinicl impct nd ws therefore not listed s clinicl AL. Grde B requires chnge in therpeutic mngement of the expected postopertive pthwy. Grde C consists of AL-relted orgn filure or clinicl instbility such tht reopertion ws needed [16]. Furthermore, other postopertive complictions were prospectively registered. For this study, 3-dy morbidity ws determined nd ll complictions were detected using locl protocol, consisting of dily physicl nd routine biochemicl exmintions. When clinicl signs were indictive for AL, rdiologic or endoscopic imging ws requested to objectify. Smple Collection Urinry smples were collected in 4.5 ml continers (Cryopure ) on postopertive dy 1, 2, nd 3. All smples were obtined from urinry ctheters while mintining sterile system. Subsequently, smples were frozen t 8 C within 2 h of collection for simultneous btch nlysis. Field Asymmetric Ion Mobility Spectrometry of Urinry Gs Urinry gs nlysis ws performed using commercil field symmetric ion mobility spectrometry (FAIMS) unit (Lonestr, Owlstone, UK). FAIMS utilizes high electric fields to seprte different ionized chemicls bsed on their movement (clled mobility) in this high electric field. The unit ws fitted with n ATLAS smpling system (Owlstone UK), which hets the urine smple to 38 C nd controls the flow of clen ir over the top of the smple nd into the Lonestr instrument. The flow rte of the crrier gs 2 Plt et l.

3 Dispersion field, % Dispersion field, % Color version vilble online Compenstion voltge, V 3 6 b Ion unit Compenstion voltge, V (AU) Fig. 1. Urinry VOC dt generted using FAIMS from n AL ptient () nd n uncomplicted ptient (b) following pncreticoduodenectomy. FAIMS mesures positive nd negtive ion counts t rnge of compenstion voltges ( 6 to +6 V in 512 steps) nd electric field mgnitudes (clled the dispersion field, 1% in 51 steps) to generte 52,224 dt points per scn. Three scns re performed per smple. This figure shows the negtive ion counts for the finl scn. Distinct plumes in the imge correlte to the presence of distinct VOCs in the smple. VOCs, voltile orgnic compounds. (dry clen ir) over the smple ws 5 ml/min, with totl flow into the Lonestr of 2 L/min. The inlet of the Lonestr ws heted to 1 C to ensure tht no condenstion occurred. Inside the unit, hedspce VOCs re ionized using NI-63 rdition source nd pssed between 2 prllel pltes. Onto these pltes high-electric field is pplied, which ttrcts, repels, or does not ffect the ions. Ions tht touch one of the pltes lose their chrge nd re not detected. A compenstion voltge is dded to one of the pltes to counterct this movement. By scnning through rnge of compenstion voltges (+6 to 6 V in 512 steps), wide rnge of different mobilities were mesured. Furthermore, the mgnitude of the electric field is scnned through rnge of levels (clled the dispersion field), which ws incresed in 51 steps. Three scns of the compenstion voltge nd dispersion field strength were performed for ech smple. By mesuring positive nd negtive ions, totl of 156,672 dt points were collected per smple. Further detils of this nlysis hve been described previously [17]. Anlysis Smples were stored in the freezer for mximum of 12 months. Levels of VOCs strt to reduce fter 9 months of storge [18]. Smples were removed from the freezer to defrost t room temperture for mximum of 2 h. Before nlysis, the 4.5 ml of smple ws liquoted into 22 ml glss vil, plced in the Lonestr, nd heted to 38 C. All smples were mesured in triplicte. Before nd fter ech urine smple, smple of clen tp wter ws run 3 times to ensure tht the bseline response ws returned. Glss vils were snitized t the deprtment of microbiology fter ech smple. Sttisticl Anlysis Demogrphic nd disese specific dt will be tbulted. Urinry VOCs of ptients with AL were compred to control ptients who hd n uncomplicted postopertive course for both types of surgery. Dt of the 3 seprte dys were combined for both groups before comprison. IBM SPSS sttistics (version 23) ws used for stndrd sttisticl nlysis. All continuous vribles re expressed s medin nd rnge; percentges were clculted for dichotomous vribles. When pproprite, bseline chrcteristics were compred with n independent smple t test or Fisher s exct test. The FAIMS dtset ws nlyzed using our existing pipeline developed for previous studies (13). In brief, first denoising step ws pplied to the FAIMS dt, followed by dt compression lgorithm to reduce the dimensionlity of the dt. Ten-fold cross-vlidtion ws then used to ssess clssifier performnce. Figure 1 shows urinry VOC dt of ptient in the AL group nd n uncomplicted ptient following pncreticoduodenectomy. The displyed smples re closest to the men for their group (AL or uncomplicted) nd therefore representtive of the verge result. Dimensionlity reduction ws undertken using 2D wvelet trnsform (specificlly Dubechies D4 wvelets) nd ny resulting vribles with zero vrince were excluded. A 1-fold cross-vlidtion ws then performed, using 9% of the dt s trining set, nd the remining 1% s test set. Within ech fold, 64 informtive fetures were identified using Wilcoxon rnk-sum test on the trining set. These 64 fetures were then used to trin rndom forest clssifier nd predict the probbility of AL for the smples in the test set. A seprte initil cross-vlidtion ws used for model selection, compring sprse logistic regression, rdil SVM, liner SVM, nd stochstic grdient boosting. All models except liner SVM showed sttisticlly significnt predictive bility (t 95% significnce level) of AL fter pncreticoduodenectomy nd of these, rndom forest ws found to produce the best clssifiction results using re under the receiver opertor curve (AUROC) s metric. Non-Invsive Detection of AL 3

4 Tble 1. Summry of demogrphic nd clinicl chrcteristics of ptients undergoing esophgel surgery Chrcteristics Results AL (n = 9) Controls (n = 22) Gender Mle 9 (1) 16 (73) Femle () 6 (27) Age, yers 7 (4 81) 61 (46 79) Missing urine smples 3 (11) 1 (2) Loction of the nstomosis Cervicl 2 (22) 8 (36) Thorcic 7 (78) 14 (64) Type of crcinom Adenocrcinom 5 (56) 16 (73) Squmous cell crcinom 1 (11) 5 (23) Other 3 (33) 1 (5) Loction of tumor Upper third () () Middle third () () Lower third or gstroesophgel junction 9 (1) 22 (1) Neodjuvnt tretment Chemordiotherpy 9 (1) 2 (91) None () 2 (9) Lymph nodes resected 2 (13 32) 19 (6 38) Rdicl surgery 9 (1) 21 (95) Comorbidities Hypertension 5 (56) 9 (41) Hert filure 1 (11) 3 (14) Crdic rrhythmi 1 (11) () COPD 1 (11) 2 (9) Dibetes mellitus () 4 (18) Chronic kidney disese () 3 (14) Grding ccording to ECCG Grde 1 1 (11) Grde 2 1 (11) Grde 3 7 (78) Other postopertive complictions Respirtory filure 2 (22) 1 (5) Pneumoni 1 (11) 1 (5) Atril fibrilltion 1 (11) 2 (9) Other 3 (33) 3 (14) Hospitl sty, dys 31 (18 16) 8 (6 17) ICU sty, dys 19 (6 89) 1 ( 7) 3-Dy mortlity () () Dt re n (%) nd medin (rnge). AL, nstomotic lekge; COPD, chronic obstructive pulmonry disese; ECCG, Esophgectomy Complictions Consensus Group; ICU, intensive cre unit. Ptient Chrcteristics Between Mrch 215 nd April 216, 66 ptients were eligible for inclusion. Three ptients were excluded due to intropertive findings of irresectble tumors or distnt metstses. A totl of 63 ptients were included, undergoing esophgectomy (n = 31) or pncreticoduodenectomy (n = 32), 14 of whom developed AL (24%) postopertively. Urine smples were collected on the first 3 postopertive dys, resulting in totl of 184 smples s 5 smples were lost. Esophgectomy Thirty-one ptients undergoing esophgectomy were included, 25 men nd 6 women hd medin ge of 63 yers (4 81). Nine ptients (29%) developed postopertive AL nd medin time to clinicl presenttion ws 8 dys (interqurtile rnge 17.5). All ptients developed AL within the postopertive dmission, except for 1 delyed AL, which ws dignosed upon redmission fter 3 dys postopertively. Grde 1 nd 2 were seen in 1 ptient nd grde 3 ws observed in 7 ptients. A totl of 89 urine smples were collected, 3 missing smples in the lekge group nd 1 missing smple in the uncomplicted controls. Further subject chrcteristics re summrized in Tble 1. No sttisticlly significnt differences were present between groups. Urinry VOCs were not ble to distinguish AL from uncomplicted ptients, AUROC.51 (95% CIs.37.65). Results of the rndom forest nlysis for both types of surgery re displyed in Tble 2. Pncreticoduodenectomy A totl of 32 ptients underwent pncreticoduodenectomy 23 men nd 9 women who hd medin ge of 66 yers with rnge of yers. Postopertive lekge of the pncreticojejunostomy ws observed in 6 ptients (19%), consisting of 3 level B nd 3 level C AL, medin time to dignosis ws 5 dys (interqurtile rnge 14.25). In 1 cse, delyed AL ws dignosed upon redmission on 3 dys fter surgery. Lekge of the gstrojejunostomy or hepticojejunostomy ws not observed. A single smple ws not collected in the AL group, resulting in totl of 95 urine smples. Chrcteristics of AL ptients nd uncomplicted ptients re depicted in Tble 3, no sttisticlly significnt differences were found between groups. Further nlysis showed tht urinry VOCs were ble to distinguish AL from uncomplicted ptients following pncreticoduodenectomy with sensitivity of.76 (95% CI.5.93) nd specificity of.77 (95% CI.66.86). The AUROC ws.85 (95% CI.76.93) nd is displyed in Figure 2. For ech smple, the predicted probbility of AL ws determined. Figure 3 shows the difference in predictive probbility broken down by surgery type. The predictive probbility ws 4 Plt et l.

5 Tble 2. AUC, sensitivities, nd specificities of FAIMS technique for distinguishing AL from uncomplicted controls for both types of surgery Type of surgery AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) Pncreticoduodenectomy.85 (.76.93).76 (.5.93).77 (.66.86) p <.1 p <.1 p <.1 Esophgectomy.51 (.37.65).54 (.33.74).55 (.42.67) p =.88 p =.48 p =.48 p vlues re clculted by mens of the Mnn-Whitney U test for AUC, nd with the Fisher s exct test for sensitivity nd specificity. AUC, re under the curve; FAIMS, field symmetric ion mobility spectrometry; AL, nstomotic lekge. broken down by postopertive dy following pncreticoduodenectomy in Figure 4 nd reveled tht there does not pper to be tendency for individul postopertive dy smples to be more ssocited with AL. Smples collected on postopertive dy 3 ppered to be more consistently scored s low risk for AL compred to other postopertive dys. Discussion In the present pilot study, urinry VOCs of ptients undergoing pncreticoduodenectomy could predict AL correctly t n erly stge. Urine smples were collected during the first 3 postopertive dys, while the medin time to clinicl presenttion for pncretic AL ws bout 5 dys. Preclinicl detection could enble erly intervention (percutneous dringe or lprotomy), possibly leding to better outcome nd timely strt of djuvnt tretment. The results suggest tht non-invsive urine nlysis by mens of FAIMS cn be of vlue in the erly, preclinicl detection of AL following pncretic surgery. However, this ws not observed in ptients undergoing esophgectomy. This study is the first to our knowledge to use VOC nlysis to dignose postopertive AL. Lekge is frequently ccompnied by, or the result of, nstomotic ischemi. This cretes loclized biochemicl nd immunologicl chnges. These chnges consist of decresing glucose nd pyruvte levels nd incresing lctte nd glycerol levels. Medistinl microdilysis hs been shown to be promising method to detect these biochemicl chnges before the development of significnt clinicl symptoms [19]. Furthermore, during the inflmmtory response, other endogenous products re produced nd present themselves s VOCs. These VOCs defuse into the blood, re renlly excreted, nd cn be detected in urine smples [2, 21]. In current prctice, inflmmtion mrkers s serum CRP re frequently used to detect AL. Reserch hs shown tht postopertive CRP levels rise in response to the initil surgery nd show physiologicl pek pproximtely h postopertively. This hmpers the use of CRP in this phse. Urinry VOCs of ptients following esophgectomy could not predict AL. Medin time to clinicl presenttion of AL nd the leked content might hve been importnt fctors why the technique ppers to be ccurte following pncreticoduodenectomy but not esophgectomy. The medin time to clinicl presenttion of esophgel AL ws 8 dys, compred to 5 dys for pncretic AL. Therefore, the preclinicl biochemicl nd immunologicl chnges relted to AL were possibly not detectble during the first 3 postopertive dys following esophgectomy. Furthermore, pncretic AL is ssocited with lekge of pncretic enzymes. Incresed levels of pncretic enzymes in drin fluid hve shown to be predictive for pncretic AL (6). These enzymes might hve contributed to the ccurcy of the technique following pncretic surgery. In generl, 3 nlyticl methods re used to detect gs phse VOCs: gs chromtogrphy combined with mss spectrometry (GC-MS), electronic nose devices, nd IMS. Urinry VOC nlysis ws performed using FAIMS, which hs number of dvntges over other techniques. GC-MS is considered the primry method to detect individul VOCs. However, nlysis by mens of GC-MS is costly nd time consuming, mking it less fesible for the clinicl setting. Compred with trditionl electronic noses, using n rry of nnosensors nd pttern recognition, FAIMS hs the dvntge of higher sensitivity (i.e., prts per billion to prts per trillion) nd n bsence of sensor drift over time [22], due to the mesurement of physicl, not chemicl, properties of the voltile molecules. Non-Invsive Detection of AL 5

6 Tble 3. Summry of demogrphic nd clinicl chrcteristics of ptients undergoing pncretic surgery Chrcteristics AL (n = 6) Controls (n = 26) 1..8 Pncreticoduodenectomy (AUC =.85; 95% CI.76.93) Gender Mle 5 (83) 18 (69) Femle 1 (17) 8 (31) Age, yers 69 (58 78) 68 (45 82) Missing urine smples 1 (6) () Type of resection Pncreticoduodenectomy 3 (5) 2 (77) PPPD 3 (5) 6 (23) Primry tumor Pncres 3 (5) 14 (54) Ampull of Vter 1 (17) 4 (15) Duodenum 1 (17) 2 (8) Biliry () 5 (19) No mlignncy 1 (17) 1 (4) Lymph nodes resected 7 (4 14) 1 (3 26) Rdicl surgery 4 (67) 14 (54) Comorbidities Hypertension 3 (5) 15 (58) Hert filure 1 (17) 2 (8) Crdic rrhythmi () 4 (15) COPD () 4 (15) Dibetes mellitus 1 (17) 6 (23) Chronic kidney disese () () Grding ccording to ISGPF Grde A () Grde B 1 (17) Grde C 5 (83) Other postopertive complictions Gstropresis 2 (33) 3 (12) Chyle lekge 1 (17) 4 (15) Postopertive hemorrhge 1 (17) 2 (8) Pneumoni 1 (17) 1 (4) Other 2 (33) 7 (27) Hospitl sty, dys 48 (1 95) 11 (4 38) ICU sty, dys 4 (1 88) ( 11) 3-Dy mortlity () () Dt re n (%) nd medin (rnge). AL, nstomotic lekge; PPPD, pylorus-preserving pncreticoduodenectomy; COPD, chronic obstructive pulmonry disese; ISGPF, Interntionl Study Group on Pncretic Fistul; ICU, intensive cre unit. Sensitivity Specificity Fig. 2. Receiver opertor curve plots for AL vs. uncomplicted controls following pncreticoduodenectomy. AL, nstomotic lekge; AUC, re under the curve. In previous gstrointestinl reserch, feces nd exhled breth hve frequently been used for VOC nlysis [14, 23]. Metbolites of infectious processes in the gstrointestinl trct, such s AL, re dischrged in feces. Therefore, it could be hypothesized tht the nlysis of fecl VOCs is more pproprite to detect AL compred to urine. However, collection of feces ws considered less fesible, due to possible temporry impirment of bowel motility in ptients undergoing mjor gstrointestinl surgery during the erly postopertive phse. Furthermore, urine ws preferred over exhled breth, due to the costs nd logisticl chllenges of collection, storge, nd nlysis of exhled breth. The gretest limittion of this explortive study is the smll smple size. However, despite low numbers nd substntil heterogeneity within the study popultion, VOC nlysis by FAIMS llowed for discrimintion between AL nd uncomplicted ptients following pncreticoduodenectomy. Due to the smll cohort, it ws necessry to pool the dt cross dys for ech type of surgery when performing the nlysis. Since ech ptient in the study contributed multiple urine smples, nd ll smples from given ptient hve the sme AL sttus, this introduces potentil bis to the results: if the trining dt contins smples 1 nd 2 from ptient A, nd the test dt contins smple 3 from ptient A, then there is dnger tht insted of detecting VOC signture for AL, VOC signture for ech ptient is detected nd tht signture is used to ssign AL sttus to the smples in the test dt. However, if this effect were cusing erroneous positive results positive result would hve been seen for both the esophgel nd pncretic cohorts. Since positive result ws only observed for the pncretic cohort, it cn be inferred tht the bis introduced by this method is not sufficient to invlidte the positive result observed. Furthermore, the smll smple size prevented us from ssessing the potentil influence of comorbidities, other postopertive complictions, 6 Plt et l.

7 Predicted probbility of AL Disese sttus AL Control Color version vilble online Fig. 3. Anlysis of the urinry VOCs resulted in predicted probbility of AL for ech smple. This figure shows the predicted probbility of AL broken down by surgery type nd true AL sttus. AL, nstomotic lekge. Pncreticoduodenectomy Surgery Esophgectomy Predicted probbility of AL Disese sttus AL Control Color version vilble online Fig. 4. Predicted probbility of AL following pncreticoduodenectomy broken down by postopertive dy nd true AL sttus. AL, nstomotic lekge Postopertive dy vrying disese stges, nd types of cncer requiring neo-djuvnt chemotherpy or chemordiotherpy. Further reserch is justified to ddress these limittions nd should focus on improving the ccurcy of FAIMS nd use other nlyticl methods, such s GC-MS to identify individul VOC. In conclusion, in our study popultion, urinry VOC nlysis llowed for discrimintion between AL nd controls following pncreticoduodenectomy on ll 3 postopertive dys combined. VOC nlysis could not distinguish AL from controls following esophgectomy. Non-Invsive Detection of AL 7

8 References 1 Turrentine FE, Denlinger CE, Simpson VB, Grwood RA, Guerlin S, Agrwl A, et l: Morbidity, mortlity, cost, nd survivl estimtes of gstrointestinl nstomotic leks. J Am Coll Surg 215; 22: Dutch Institute for Clinicl Auditing. Annul Reports. 3 Strtmn J, Cuest MA, Gisbertz SS, Vn der Peet DL: Vlue of step-up dignosis pln: CRP nd CT-scn to dignose nd mnge postopertive complictions fter mjor bdominl surgery. Rev Esp Enferm Dig 214; 16: Strtmn J, Hrmsen AM, Cuest MA, Berkhof J, Jnsm EP, vn der Peet DL: Predictive vlue of C-rective protein for mjor complictions fter mjor bdominl surgery: systemtic review nd pooled-nlysis. PLoS One 215; 1:e Perry Y, Towe CW, Kwong J, Ho VP, Linden PA: Seril drin mylse cn ccurtely detect nstomotic lek fter esophgectomy nd my fcilitte erly dischrge. Ann Thorc Surg 215; 1: Reid-Lombrdo KM, Frnell MB, Cripp S, Brnett M, Mupin G, Bssi C, et l: Pncretic nstomotic lekge fter pncreticoduodenectomy in 1,57 ptients: report from the pncretic nstomotic lek study group. J Gstrointest Surg 27; 11: Ngkw Y, Mtsudo T, Hijikt Y, Kikuchi S, Bunso K, Suzuki Y, et l: Bcteril contmintion in scitic fluid is ssocited with the development of cliniclly relevnt pncretic fistul fter pncretoduodenectomy. Pncres 213; 42: de Groot EF, de Meij TG, Berkhout DJ, vn der Schee MP, de Boer NK: Fltogrphy: detection of gstrointestinl diseses by fecl gs nlysis. World J Gstrointest Phrmcol Ther 215; 6: Turner AP, Mgn N: Electronic noses nd disese dignostics. Nt Rev Microbiol 24; 2: Sonod H, Kohnoe S, Ymzto T, Stoh Y, Morizono G, Shikt K, et l: Colorectl cncer screening with odour mteril by cnine scent detection. Gut 211; 6: Horvth I, Lzr Z, Gyuli N, Kolli M, Losonczy G: Exhled biomrkers in lung cncer. Eur Respir J 29; 34: Buszewski B, Kesy M, Ligor T, Amnn A: Humn exhled ir nlytics: biomrkers of diseses. Biomed Chromtogr 27; 21: Arsrdnm RP, Westenbrink E, McFrlne MJ, Hrbord R, Chmbers S, O Connell N, et l: Differentiting coelic disese from irritble bowel syndrome by urinry voltile orgnic compound nlysis pilot study. PLoS One 214; 9:e Arsrdnm RP, McFrlne MJ, Ryn-Fisher C, Westenbrink E, Hodges P, Thoms MG, et l: Detection of colorectl cncer (CRC) by urinry voltile orgnic compound nlysis. PLoS One 214; 9:e Low DE, Alderson D, Cecconello I, Chng AC, Drling GE, D Journo XB, et l: Interntionl consensus on stndrdiztion of dt collection for complictions ssocited with esophgectomy: esophgectomy complictions consensus group (ECCG). Ann Surg 215; 262: Bssi C, Mrchegini G, Dervenis C, Srr M, Abu Hill M, Adhm M, et l: The 216 updte of the interntionl study group (ISGPS) definition nd grding of postopertive pncretic fistul: 11 Yers After. Surgery 217; 161: Covington JA, vn der Schee MP, Edge AS, Boyle B, Svge RS, Arsrdnm RP: The ppliction of FAIMS gs nlysis in medicl dignostics. Anlyst 215; 14: Esfhni S, Sgr NM, Kyrou I, Mozdik E, O'Connell N, Nwokolo C, et l: Vrition in gs nd voltile compound emissions from humn urine s it ges, mesured by n electronic nose. Biosensors (Bsel) 216; 6:pii:E4. 19 Ellebek M, Qvist N, Fristrup C, Mortensen MB: Medistinl microdilysis in the dignosis of erly nstomotic lekge fter resection for cncer of the esophgus nd gstroesophgel junction. Am J Surg 214; 28: Arsrdnm RP, Ouret N, Thoms MG, Qurishi N, Hetherington E, Nwokolo CU, et l: A novel tool for noninvsive dignosis nd trcking of ptients with inflmmtory bowel disese. Inflmm Bowel Dis 213; 19: Sethi S, Nnd R, Chkrborty T: Clinicl ppliction of voltile orgnic compound nlysis for detecting infectious diseses. Clin Microbiol Rev 213; 26: Covington JA, Wedlke L, Andreyev J, Ouret N, Thoms MG, Nwokolo CU, et l: The detection of ptients t risk of gstrointestinl toxicity during pelvic rdiotherpy by electronic nose nd FAIMS: pilot study. Sensors (Bsel) 212; 12: de Meij TG, Lrbi IB, vn der Schee MP, Lentferink YE, Pff T, Terhr Sive Droste JS, et l: Electronic nose cn discriminte colorectl crcinom nd dvnced denoms by fecl voltile biomrker nlysis: proof of principle study. Int J Cncer 214; 134: Plt et l.