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Abstract. – OBJECTIVE: Reflux is the principalcomplication for patients after esophagectomy withgastric reconstruction. The aim of this study was toinvestigate the effect of the modified Nissen fundo-plication after resection of adenocarcinoma fromthe esophagogastric junction (AEG) on controllingthe reflux and the role of duodenogastroesophagealreflux (DGER) and cyclooxygenase-2 (COX-2) ex-pression level in the remnant esophagus.

PATIENTS AND METHODS: Sixty patients withAEG were randomly divided into two groups: (i)the conventional anastomosis group and (ii) theanti-reflux anastomosis group. Fifty esophagec-tomized patients were invited to participate inpostoperative follow-up after 6 to 12 months.Among those we had 29 cases in the convention-al anastomosis group and 21 in the anti-refluxanastomosis group. We used endoscopy, simul-taneous 24 hours esophageal pH and bilirubinmonitoring in this study. The COX-2 expressionlevel in the remnant esophagus was detected us-ing real-time PCR.

RESULTS: The reflux esophagitis prevalence inanti-reflux anastomosis group was comparableto that in the conventional group (p = 0.154). De-Meester score and fraction time of bilirubin abs>0.14 decreased more intensely in the anti-refluxanastomosis group (p < 0.05). The COX-2 expres-sion level in of anti-reflux anastomosis groupwas evidently lower than that in the conventionalanastomosis group (p = 0.022) while it was mean-ingfully higher compared to the normal controlgroup (p = 0.046). COX-2 up-regulation as well ashigh prevalence of esophagitis were observed insimultaneous acid reflux and DGER (p < 0.05).

CONCLUSIONS: Although modified fundoplica-tion following resection of AEG did not achievean optimal effect on controlling reflux, it was veryeffective in decreasing the reflux. COX-2 expres-sion monitoring can be considered as a possiblenew way to evaluate the impact of anti-refluxsurgery. DGER occurring in acidic environmentcould develop severe reflux esophagitis and up-regulate the COX-2 expression.

Corresponding Author: Wang JD, MD; e-mail: wangjd165@163.com

Key words:Nissen fundoplication, Acid reflux, DGER, AEG,

COX-2.

Introduction

Partial esophagectomy and proximal gastrecto-my are major surgical procedures for patients suf-fering from adenocarcinoma of esophagogastricjunction (AEG). In these cases, we have less than20%, five years survival rate1. Gastric and duode-nal reflux after esophagectomy with gastric con-duit reconstruction is a common problem and isoften considered an inevitable consequence of thistype of surgery. Reflux symptoms have beenfound in 60 to 80% of esophagectomy cases2 andproblems such as severe heartburn and regurgita-tion associated with this condition can createphysical discomfort as well as social complica-tions. Presently, the life quality after a surgical in-tervention has become paramount. There are gath-ering evidences indicating that gastroesophagealreflux almost occurs in all patients who underwentesophagectomy and in nearly all cases the conven-tional anastomotic orifice fails to prevent gastroe-sophageal reflux after the surgery3.

The prevalence of esophagitis and Barrett’s esoph-agus in the esophageal stump is respectively 45.9 to91.9% and 29.5 to 57.5% after the surgery. The devel-opment of Barrett’s esophagus might result in a newcarcinoma in patients who have achieved long-termsurvival after esophagectomy4.

Modified Nissen fundoplication shaped by thegastric fundus wrapping around the remnant oe-sophagus and conventional anastomosis afteresophagectomy may be considered as a powerfulanti-reflux manoeuvre in patients with esophageal

European Review for Medical and Pharmacological Sciences

Anti-reflux anastomosis following esophagectomyfor adenocarcinoma of the esophagogastricjunction: impact of duodenogastroesophagealreflux and expression of cyclooxygenase-2in the remnant esophagus

J.-D. WANG1, S.-Y. ZHU1, Y.-J. LU1, L.-Y. GAO2

1Department of Thoracic Surgery, First People’s Hospital in XuZhou City, XuZhou, China.2Department of Pathology, First People’s Hospital in XuZhou City, XuZhou, China.

2016; 20: 476-485

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carcinoma5. Its anti-reflux effect was demonstrat-ed by endoscopy and 24-hour pH monitoring6.Modified Nissen fundoplication is shaped by anadequate width and length of remnant gastric tubewrapping around the esophageal stump and anas-tomosis. However, the effectiveness of this im-proved surgical procedure in controlling gastroe-sophageal reflux is still unclear. To investigate theanti-reflux effect of this method and the impact ofDGER on reflux esophagitis we used endoscopy,simultaneous ambulatory 24 hours pH and spec-trometric bilirubin monitoring following the AEGresection.

Cyclooxygenase-2 (COX-2) is a rate-limitingenzyme in the conversion of arachidonic acid toprostaglandins pathway. COX-2 is involved inthe regulation of a broad range of cellularprocesses including proliferation, angiogenesisand resistance to apoptosis. According to the pri-or studies in this field, among many genes evalu-ated in the distal esophageal squamous mucosa,only COX-2 expression well correlated with theamount of esophageal acid exposure7. The in-creased expression levels seen in some genes,such as COX-2 and interleukin 8 (IL-8), in thedistal esophageal mucosa, were significantlylowered and returned to the level comparable tothat seen in the squamous mucosa of patientswithout reflux disease8,9. Based on these find-ings, using real-time PCR, the COX-2 expressionlevel in the remnant esophagus was measured inorder to verify whether our anti-reflux surgerycan change COX-2 expression in the remnantesophagus. We also evaluated the effects of acidreflux and DGER on COX-2 expression.

Patients and methods

PatientsFrom December 2011 to July 2013, sixty pa-

tients with adenocarcinoma of esophagogastricjunction (AEG) were chosen and randomly di-vided into two groups: (i) the conventional anas-tomosis group (modified Nissen fundoplicationadded to conventional anastomosis) and (ii) theanti-reflux anastomosis group. We had 39 malesand 21 females, with ages ranging from 40 to 76years (mean age was 60.2 years). The pro-opera-tive general condition, the size of the tumor andpathologic type were analogical between the twogroups. Fifty post-operative patients including 37males and 13 females, aging from 40 to 73 years(average age 61.6 years) took part in the follow-

up studies (6 to 12 months after operation). Thisincluded 29 patients in the conventional anasto-mosis group and 21 patients in the anti-refluxanastomosis group. All postoperative patients re-ceived endoscopy examination while thirty- sev-en patients underwent 24-hours pH and bilirubinmonitoring. The COX-2 expression level in theremnant esophagus was evaluated using real-time PCR.

Demographic and clinic pathological informa-tion such as age, gender, characteristics of AEGand the method of surgery were obtained frompatient’s records (Table I). The pathologic stageof AEG was determined according to the 7th edi-tion of the esophageal adenocarcinoma and gas-tric cancer TNM classification of the AmericanJoint Committee on Cancer (AJCC) and Unionfor International Cancer Control (UICC). Ac-cording to UICC, a tumor with an epicenter lo-cated within 5 cm from the EGJ with extensioninto EGJ and the distal esophagus (AEG type Iand II) was staged according to the esophagealadenocarcinoma staging scheme. Tumors with anepicenter greater than 5 cm from EGJ or thosewithin 5 cm of the EGJ without extension intoEGJ (AEG type III) were staged using the gastriccarcinoma staging scheme10. Twelve healthy vol-unteers with no reflux and reflux esophagitiswere selected as normal control for this study.These included ten males and two females withmean age of 45.6 years. They were selected us-ing 24-hour esophageal pH and bilirubin moni-toring and endoscopy. Written informed consentwas obtained from postoperative patients andvolunteers. This study was approved by HospitalEthics Committee.

Surgical procedure All patients underwent surgery via left thora-

cotomy and the esophagus and stomach weremobilized. The esophagus was mobilized to thelevel of inferior lung vein. The partial distalesophagus, gastroesophageal junction, and proxi-mal stomach were resected at 5 cm tumor-freemargin. Remaining parts of the stomach wereshaped into gastric tube 3 to 4 cm in width usinga linear stapling device (GIA6038S, Covidien,Saint Louis, MO, USA). The anastomosis wasperformed below the aortic arch. The conven-tional anastomosis was at the terminal side of theesophagogastric anastomosis using a circular sta-pling device (HuaSen 24#, ChangZhou MedicalEquipment Company, ChangZhou, China) andwas shaped on the apex of the gastric tube (Fig-

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Clinicopathologic Conventional Anti-reflux Totalcharacteristics anastomosis anastomosis

Gender (n)Male 21 16 37Female 8 5 13

Age (mean year) 62.3 58.5Siewert and Stein classification (n)Type I 4 3 7Type II 18 15 33Type III 7 3 10

Turmor size (cm) 1.5-5 1-4Histology (n)Papillary adenocarcinoma 12 7 19Tubular adenocarcinoma 7 9 16Mucinous adenocarcinoma 4 2 6Poorly differentiated adenocarcinoma 5 3 8Other types 1 0 1

Tumor stage (n)Ia 0 (0) 0 (0) 0IIb 0 (0) 1 (0) 1IIa 3 (1) 4 (1) 9IIb 7 (3) 7 (1) 18IIIa 9 (2) 5 (1) 17IIIb 2 (1) 1 (0) 4IIIc 1 (0) 0 (0) 1IV 0 (0) 0 (0) 0

Table I. Clinicopathologic data in patients with adenocarcinoma who underwent partial esophagectomy and proximal subtotalgastrectomy with the conventional anastomosis and the anti-reflux anastomosis.

The data in bracket was staged according to the gastric carcinoma staging scheme.

Figure 1. Schematic diagram of conventional anastomosis (A) and anti-reflux anastomosis (B to D). A, The conventionalesophagogastric anastomosis was the end to side esophagogastric anastomosis using a circular stapling device in apex of gas-tric tube and near to greater curvature of gastric tube. B, The esophagogastric anastomosis was performed in anterior wall ofthe stomach, which was in position 3 to 4 cm below the apex of gastric tube and near to greater curvature of the gastric tube.C, The gastric tube apex was drawn to left at the remnant esophagus. Four non-absorbable sutures were sewn from the gastricwall at side of the greater curvature of stomach to the gastric wall at side of lesser curvature of stomach. The upper two suturesincluded the terminal remnant esophagus wall. The third suture was only sewn at gastric wall between side of the greater cur-vature of stomach and lesser curvature of stomach. Fourth suture included anterior wall of the stomach below 1 cm anastomo-sis. D, The three sutures were tied tight to complete the modified Nissen fundoplication following the conventional esopha-gogastric anastomosis.

ure 1A). For the modified Nissen fundoplication(Figure 1B-D), the anastomosis was shaped onthe anterior wall of the stomach, which was in aposition 3 to 4 cm below the apex of the gastrictube and near to the greater curvature of the gas-tric tube. After conventional anastomosis, theapex of the gastric tube was drawn to the left sideof the remnant esophagus. This was placed in away that the gastric tube extended beyond thetwo sides of the remnant esophagus. The fundo-plication was sewn with four non-absorbable su-tures. The four sutures, which were sewn to gas-tric wall at the greater curvature side of gastrictube to gastric wall at lesser curvature of gastrictube side, passed across the terminal remnantesophagus and anastomosis. The first, secondand fourth sutures were sewn at the wall of ter-minal remnant esophagus and anterior wall of thestomach 1cm below anastomosis. The third su-ture was only sewn at the gastric wall from thegreater curvature to the lesser curvature of thegastric tube. All the sutures were placed 1 cmapart to close the wrap. The fundoplication wasperformed by drawing the lesser curvature ofstomach’s gastric tube side forward and right tothe terminal remnant esophagus and anastomosis,wrapping it around the terminal esophageal rem-nant 2 to 3 cm and the anastomosis and securingit to the gastric wall at side of the greater curva-ture of the stomach. The pyloroplasty was notperformed in the surgical procedure.

EndoscopySix months after the surgery, the remnant

esophagus mucosa was examined using a flexibleendoscope (Olympus GIF-Q260J; Olympus Opti-cal Co., Ltd., Tokyo, Japan). The severity of re-flux esophagitis observed during the endoscopywas graded according to Los Angeles (LA) clas-sification system11. Three biopsy specimens werecollected at 3, 9, 12 clock position of the remnantesophageal mucosa 3 cm above of esophageal-gastric anastomosis in postoperative patients andin the same region of distal esophageal mucosa 3cm above esophagogastric junction (Z-line) inhealthy volunteers.

Ambulatory 24 hours esophageal pH and bilirubin monitoring

Simultaneous esophageal pH and bilirubinmonitoring were performed for 24 hours. Anti-mony with single sensor for pH (Medtronic,Skovlunde, Denmark) was calibrated in buffersolutions of pH 1.07 and pH 7.01 before each

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study. Then, the fiber optic probe for bilirubinmonitoring (Medtronic) was calibrated with wa-ter. After the catheters had been passed transnasally, pH sensor and bilirubin monitoring werepositioned in the region of remnant esophagus 5cm above anastomotic orifice identified fluoro-scopically by the presence of the staple inesophagectomized patients and in the region ofdistal esophagus 5 cm above esophagogastricjunction (identified by endoscopy) in healthyvolunteers. The esophageal pH and bilirubinmonitoring was respectively recorded by aportable digital data recorder (Digitrapper MarkIII, Medtronic) and a portable optoelectronic datarecorder (Bilitec 2000, Medtronic). Data weredownloaded into a personal computer for furtheranalyses using Esophogram Reflux AnalysisSoftware (Medtronic). Elevated acid reflux wasdefined as DeMeester score above 14.7212 and el-evated duodenal gastro esophageal reflux(DGER), was defined as bilirubin absorbance ex-ceeding 0.14 for more than 1.8% of the monitor-ing time13.

Real-time PCRHistological biopsy specimens underwent

routine fixation. Total RNA from the biopsy sam-ples was extracted using High Pure FFPET RNAIsolation Kit according to the instructions provid-ed by the supplier (Roche Applied Science Co.,Basel, Switzerland). Total RNA samples (1 μg)were subjected to reverse transcription in orderto obtain complementary DNA (cDNA). The re-action was performed in 25 μl according to Tran-scriptor first strand cDNA synthesis kit (RocheApplied Science Co.). Real-time PCR measure-ment of COX-2 cDNA was performed on an ABIPRISM 7500 sequence detector (PE AppliedBiosystems, Foster City, CA, USA) with TaqManassay. The COX-2 primers and probe sequenceswere synthesized (PE Applied Biosystems) as de-scribed previously14 (Table II). The PCR reactionmixture consisted of 1200 nmol/of each primer,200 nmol/probe, 0.4 U of AmpliTaq Gold Poly-merase, 200 nmol/each dATP, dCTP, dGTP,dTTP, 3.5 mM MgCl2 and 1x Taqman Buffer Acontaining a reference dye, to a final volume of20 μl (all reagents from PE Applied Biosystems).Cycles conditions were 50°C for 2 min, 95°C for10 min, followed by 46 cycles at 95 °C for 15sand 60 °C for 1 min. TaqMan measurementsyield Ct values were inversely proportional to theamount of cDNA in the tube. A higher Ct valueindicated that more PCR cycles were required to

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reach the level of detection. Gene expression val-ues (relative mRNA levels) were communicatedas ratios (differences between the Ct values) ofCOX-2/β-actin in arbitrary units. An internal ref-erence gene (β-actin) provided a normalizationfactor for the amount of RNA isolated from aspecimen.

Statistical AnalysisAll statistical analyses were performed using

SPSS for Windows (Version 19.0; SPSS, Inc.,Chicago, IL, USA). The prevalence of refluxesophagitis and gastroesophageal reflux were an-alyzed by Chi Square test. The 24 hours pH andbilirubin monitoring parameters were comparedby Wilcoxon rank-sum test. The COX-2 expres-sion was estimated using Scheffe test. A p-valueof ≤0.05 was considered statistically significant.

Results

Using endoscopy examination, differentgrades of reflux esophagitis were observed in theconventional anastomosis as well as the anti-re-flux anastomosis groups (Figure 2). The refluxesophagitis occurrences were respectively 69%and 47.7% in the conventional anastomosis andthe anti-reflux anastomosis groups. Although re-flux esophagitis prevalences in the anti-refluxanastomosis group declined more intensely, dif-ferences between the groups were not statistical-ly significant (χ2=2.313, p=0.154).

The reflux patterns were classified into fourtypes: (i) neither acid reflux nor DGER, (ii) onlyacid reflux, (iii) only DGER and (iv) simultane-ous acid reflux and DGER by analysis of 24-hour

pH and bilirubin monitoring (Figure 3). The re-flux occurrences were respectively 85.2% and76.5% in the conventional anastomosis and anti-reflux anastomosis groups. No significant differ-ence in reflux occurrence was observed betweenthe two groups (χ2=0.343, p=0.434). The simulta-neous acid reflux and DGER were the main re-flux patterns in both groups. Higher refluxesophagitis occurrences (grade C and grade D)were observed patients with simultaneous acidreflux and DGER (χ2=4.723, p=0.043) (Figure4). DeMeester score and fraction time of biliru-bin abs >0.14 in the anti-reflux anastomosis

GenBank accession: NM-000962Forward primer of COX-2 5′-GCTCAAACATGAT-GTTTGCATTC-3′

Reverse primer of COX-25′-GCTGGCCCTCGCTTATGA-3′ TaqMan probe of COX-26FAM 5′-TGCCCAGCACTTCACGCATCAGTT-3′

TAMRA.GenBank accession: NM-001101Forward primer of β-actin5’-GAGCGCGGCTACAGCTT-3’Reverse primer of β-actin5’-TCCTTAATGTCACGCACGATTT-3’TaqMan probe of β-actin6FAM 5′-ACCACCACGGCCGAGCGG-3′TAMRA.

Table II. Primers and Probes of COX-2 and β-actin.

Figure 2. Prevalence of different grade of reflux esophagitisbetween the groups of conventional anastomosis and anti-re-flux anastomosis. There was no difference in prevalence ofreflux esophagitis between two groups (χ2=2.313, p=0.154).

Figure 3. Incidence of different types of reflux betweenthe groups of conventional anastomosis and anti-refluxanastomosis. No difference was observed in prevalence ofreflux between two groups (χ2=0.343, p=0.434).

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group were clearly dropped more than those inthe conventional anastomosis group and differ-ences were statistically significant (p<0.05)(Table III).

COX-2 expression level was lowest in the nor-mal control group (0.053±0.037). In patients un-derwent esophagectomy, the COX-2 gene expres-sion level was 0.126±0.085 for patients with nor-mal esophageal mucosa (n=20) and 0.147±0.112in patients with reflux esophagitis (n=30). TheCOX-2 expression levels in esophagectomized pa-tients with normal esophageal mucosa and refluxesophagitis were clearly higher compared to thatin the normal control (p=0.0314, 0.012) and therewas no significant difference between esophagec-tomized patients with normal esophageal mucosaand reflux esophagitis (p=0.1281). The COX-2expression levels in patients with simultaneousacid reflux and DGER (0.2301±0.0571) were evi-dently higher than that in only acid reflux

(0.0901±0.0546, p=0.004,) and control group(p=0.001). However, the difference was not signif-icant when compared to that in DGER only(p=0.142) (Figure 5).

COX-2 expression levels in the remnantesophagus mucosa are shown in Figure 6. TheCOX-2 expression level in anti-reflux anastomosisgroup was evidently lower compared to that in theconventional anastomosis group (0.192±0.148 vs.0.114±0.087, p=0.022), while it was significantlyhigher when compared to the normal controlgroup (0.114±0.087 vs. 0.053±0.037, p=0.046).

Discussion

Adenocarcinoma of the esophagogastric junc-tion (AEG), which occurs within 5 cm of theesophagogastric junction (EGJ), can be divided in-to three types according to Siewert and Stein clas-sification of AEG15. There are several possible ap-proaches for surgical resection of carcinoma of theesophagogastric junction. These included resectionof the proximal stomach and total gastrectomy, andproximal gastrectomy with partial esophagectomybeing the main surgical procedure for AEG. AEGpatients who underwent partial esophagectomy andproximal gastrectomy with gastric tube reconstruc-tion have a high risk of prolonged esophageal ex-posure to gastric acid and duodenal juice becausethe normal anti-reflux mechanisms (loweresophageal sphincter, angle of His, diaphragmaticsling) have been resected. Zang et al16 reported that60% of patients after surgery for AGE (cardiaccancer) had typical symptoms of gastroesophagealreflux and all patients with AGE suffered frompostoperative gastroesophageal reflux by 24-hourpH monitoring. Manual anastomosis, such as en-casing-in style and “scarf” style, could not de-crease gastroesophageal reflux. The height atwhich the anastomosis is shaped is considered to

Figure 4. Prevalence of various extents of reflux esophagitis indifferent reflux patterns. The higher occurrence of refluxesophagitis of grade C and grade D was observed in the patientswho had simultaneous acid reflux and DGER compared withthat in only DGER and only acid reflux (χ2=4.723, p=0.043).

Group Conventional Anti-reflux H pAnastomosis Anastomosis(n=19) (n=17)

x±SD x±SD

DeMeester score 132.72±83.01 56.18±48.18 2.758 0.0061

Fraction time of bilirubin abs>0.14 17.28±13.15 7.62±8.37 2.521 0.0118

Table III. Comparison of acid reflux and DGER between conventional and anti-reflux anastomosis groups.

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affect the extent of the reflux. More severe refluxesoccur on the lower anastomosis locations17,18 be-cause in lower anastomosis cases, a bigger part ofstomach is subjected to positive intra-abdominalpressure thus promoting greater reflux19. Sinceconventional anastomosis fails to prevent gastroe-sophageal reflux, many surgical techniques at-tempting to control reflux after esophagectomyhave been developed20-24. However, these surgicalprocedures are too complex and very challengingand are unsuitable for radical resection ofesophageal cancer and AEG. Hence, these im-proved surgical procedures have not been com-monly applied as standard techniques.

Treatment of patients with gastroesophagealreflux disease by Nissen fundoplication is a well-established surgical technique. Aly et al5 origi-nally reported that the modified Nissen fundopli-cation was added to conventional anastomosis inesophagectomized patients with esophageal can-cer and postoperative reflux symptoms, were re-duced in the majority of patients with this anti-reflux maneuver. Our previous study alsodemonstrated that this technique was effective incontrolling reflux6. In AEG patients when proxi-mal stomach must be resected, sufficient widthand length of remnant stomach is very importantbecause it should wrap around the terminal partof remnant esophagus and anastomotic orifice in

order to satisfy Nissen fundoplication. In ourcase series, the width of remnant gastric tube wasmore than 4 cm, and that the gastric tube apexovertopped 3 cm of anastomotic site on the ante-rior wall of the remnant stomach. In addition, thetumor, which was less than 4 cm in diameter, lo-cated in EGJ extension to distal esophagus andless gastric curvature (AEG type I and II) or inthe gastric fundus without extension to GEJ andgastric body (AEG type III). Hence, the locationand the size of tumor were important factors tosuccessfully complete the modified Nissen fun-doplication following resection of AEG.

Our data showed that various patterns of re-flux occurred in both groups and the simultane-ous acid reflux and DGER was the main refluxpattern. Although reflux pattern was comprisedof three types: only acid reflux, only DGER andsimultaneous acid reflux and DGER, it was pos-sible for patients with only DGER to have acidreflux due to the alkaline content in DGER neu-tralizing gastric acid. Previous findings demon-strated that most esophageal bilirubin exposuresoccurred in a pH ranging from 4 to 7 inesophagectomized patients25. Marshal et al26 de-scribed that esophageal mucosal injury was mini-mal in patients with isolated bile reflux. Vaezi etal27 argued that duodenal contents required anacid environment to produce more severe mucos-

Figure 5. COX-2 expression in the remnant esophagealmucosa in acid reflux and DGER profile in 36 patients in-cluding the conventional anastomosis and anti-reflux anas-tomosis groups. Boxes represent the range of standard devi-ation with the horizontal line representing mean value. Errorbars represent maximum and minimum values. The COX-2expression in simultaneous acid and DGER was evidentlyhigher than that in only acid reflux and control (p=0.004,p=0.001). Difference was not significant when compared tothat in only DGER (p=0.142).

Figure 6. COX-2 expression in the remnant esophageal mu-cosa among the conventional anastomosis, anti-reflux anasto-mosis and control groups. Boxes represent the range of stan-dard deviation with the horizontal line representing mean val-ue. Error bars represent maximum and minimum values. TheCOX-2 expression in the anti-reflux anastomosis group wasevidently lower than that in the conventional anastomosisgroup (p=0.022). COX-2 expression in the conventional anas-tomosis group significantly increased compared to the normalcontrol group (p=0.046).

al injury. Our findings showed that severeesophagitis (grade C and D reflux esophagitis)occurred only in DGER as well as simultaneousacid and DGER, rather than in acid reflux only.Furthermore, we observed higher occurrence ofsevere esophagitis in simultaneous acid andDGER. This suggested that DGER played a sig-nificant role in damaging the esophageal mucosaand the injury might be more severe when DGERwas exposed to acidic environment.

Combined ambulatory esophageal 24 hourspH and bilirubin monitoring could detect changesin acid reflux and DGER. Our results showed thatthe extent of acid reflux and DGER in the anti-re-flux anastomosis group was declined more in-tensely when compared to that in the convention-al anastomosis group. Nevertheless, the varyingdegrees of reflux still occurred in some patients inthe anti-reflux anastomosis group. It was suggest-ed that anti-reflux anastomosis surgical procedurecould be effective in controlling reflux; however,it could not prevent reflux across the anastomoticorifice. Therefore, the role of anti-reflux anasto-mosis in efficient control of reflux was not as op-timal as the treatment for gastroesophageal refluxdisease with Nissen fundoplication. Althoughproton pump inhibitors might decrease acid refluxin the esophagectomized patients, the use of theseinhibitors could cause a 3-fold increase in the riskof esophageal adenocarcinoma. This increase, inpart, might be due to an alteration in the refluxatecomposition created by these medications28. Animportant advantage of anti-reflux surgery is itsability to reduce all types of refluxes and this canbe explained by the fact that the gastroesophagealbarrier is mechanically constructed. Our previousstudy demonstrated that the pressure at anasto-motic site measured by esophageal manometrywas higher than that in the intrathoracic stomach6.The higher pressure at remnant esophagus (1 to 2cm above anastomotic site) has analogous effectof the low esophageal sphincter on controlling re-flux. However, the main problem associated withanti-reflux anastomosis is whether the scar inanastomotic orifice can influence the capability ofcontrolling reflux in the future. Hence, the anti-reflux outcome of the modified fundoplicationfollowing conventional anastomosis need to beinvestigated for long term.

There are indications that at the molecularlevel, some pathways associated with inflamma-tion and injuries are similar to those involved incarcinogenesis. Such mechanism involves thenuclear factor NF-κB pathway, in which activat-

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Anti-reflux anastomosis following esophagectomy for adenocarcinoma

ed COX-2 is a major downstream product. TheNF-κB is a transcription factor with regulatorycapacity on several genes involved in the inflam-mation response, and it is currently thought toplay a part in gene activation involved in cancerprogression29-31. Our study demonstrated that theCOX-2 expression was present in the remnantesophagus. The up-regulation of COX-2 wassimilar in presence of mucosa injury in the formof reflux esophagitis or absence of any visiblemucosal injury. Similar results have already beenreported8,32. This suggested that esophageal mu-cosa changes did not influence the COX-2 ex-pression in the remnant esophagus, and that theup-regulation of COX-2 might be the earlyepisode associated with reflux since it was pre-sent in patients without endoscopic evidence ofreflux-induced esophageal mucosal injury. COX-2 up-regulation was likely a more sensitive gas-troesophageal reflux indicator.

Several previous studies reported that acid expo-sure in the distal esophagus could lead to COX-2up-regulation in esophageal mucosa7,33. Other find-ings also demonstrated that bile acids stimulatedesophageal squamous cells and Barrett's epithelialcells to trigger the expression of COX-2 and causedoxidative stress34,35 and the ratio of hydrophobic tohydrophilic bile acids influence on COX-2 proteinexpression36. Our data also showed that higher levelof COX-2 expression was detected in the postoper-ative patients with DGER occurred in acidic envi-ronment, especially in simultaneous acid reflux andDGER. Our findings indicated that the synergeticrole in acid reflux and DGER, possibly contributedto COX-2 up-regulation. But other studies reportedthat the COX-2 up-regulation was induced by de-oxycholic acid was partially reversed by the addi-tion of curcumin37. It is a plausible idea that the ad-ditional DGER contents, including trypsin and oth-er digestive enzymes activated by gastric acid, caninduce COX-2 expression. Further studies in futuremay shed more light in this idea.

Earlier studies demonstrated that the increasedgene expression in the distal esophagus was nor-malized in patients with gastroesophageal refluxdisease following Nissen fundoplication8,9. One ofthe chief purposes of this study was to determinewhether the modified Nissen fundoplication follow-ing esophagogastrostomy might reduce the extentof reflux and would alter the COX-2 expressionlevel. COX-2 expression monitoring would evalu-ate the efficacy of anti-reflux anastomosis. Our datashowed that COX-2 expression level in the anti-re-flux group was significantly lower compared to that

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in the conventional anastomosis group. At the timethe reduced level of COX-2 expression did not re-turn to the level detected in the normal esophagealmucosa. This result indicated that a decrease in acidreflux and DGER exposing in the remnant esopha-gus could down-regulate COX-2 expression eventhough this modified anti-reflux surgery did notcompletely control the reflux. A message from ourstudy is that the monitoring of COX-2 expressionmight be a sensitive method to evaluate the effec-tiveness of anti-reflux surgery.

Conclusions

The modified Nissen fundoplication follow-ing resection AEG is effective in reducing reflux;however, it does not achieve an optimal effect oncontrolling the reflux. Monitoring COX-2 ex-pression level is likely a new method to evaluatethe impact of anti-reflux therapy. We reiteratethat DGER occurred in acidic environment in-duced an increase in COX-2 expression.

–––––––––––––––––––-––AcknowledgmentThe authors sincerely thank the departments of ClinicalLaboratory and Pathology in our hospital making contribu-tion to this study.

–––––––––––––––––––-––––Conflict of Interest

The Authors declare that there are no conflicts of interest.

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