Research Article Immunohistochemical Expression of Dual ...

Research ArticleImmunohistochemical Expression of Dual-Specificity ProteinPhosphatase 4 in Patients with Colorectal Adenocarcinoma

Jongmin Sim1 Kijong Yi1 Hyunsung Kim1 Hyein Ahn1 Yumin Chung1 Abdul Rehman1

Se Min Jang1 Kang Hong Lee2 Kiseok Jang1 and Seung Sam Paik1

1Department of Pathology College of Medicine Hanyang University Seoul 133-792 Republic of Korea2Department of Surgery College of Medicine Hanyang University Seoul 133-792 Republic of Korea

Correspondence should be addressed to Seung Sam Paik sspaikhanyangackr

Received 27 October 2014 Revised 30 December 2014 Accepted 31 December 2014

Academic Editor Vikram Kate

Copyright copy 2015 Jongmin Sim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The role of dual-specificity protein phosphatase 4 (DUSP4) appears to vary with the type of malignant tumors and is stillcontroversial The purpose of our study was to clarify the exact role of DUSP4 expression in colorectal adenocarcinoma Weconstructed tissue microarrays and investigated DUSP4 expression by immunohistochemistry DUSP4 was more frequentlyexpressed in adenocarcinomas and lymph nodedistant metastases compared to that in normal colorectal tissues and tubularadenomas (119875 lt 0001) Mean DUSP4 expression score was significantly higher in malignant tumors than in benign lesions(119875 lt 0001) DUSP4 expression was significantly correlated with older age (119875 = 0017) male gender (119875 = 0036) larger tumorsize (119875 = 0014) nonmucinous tumor type (119875 = 0023) and higher T stage (119875 = 0040) Kaplan-Meier survival curves revealed asignificant effect of DUSP4 expression on both overall survival and disease-free survival in AJCC stage I (119875 = 0008 and 119875 = 0003resp log-rank test) and male gender (119875 = 0017 and 119875 = 0049 resp log-rank test) DUSP4 protein is frequently upregulatedin colorectal adenocarcinoma and may play an important role in carcinogenesis and cancer progression and may be a marker ofadverse prognosis

1 Introduction

Colorectal adenocarcinoma is one of themost common typesof cancer and the second cause of cancer-related deaths inindustrialized countries [1ndash3] Despite marked advances inthe understanding of carcinogenesis and improvements indiagnostic and treatment modalities the specific therapeuticproblem still persists [4 5] Surgery cannot always preventthe recurrence of advanced colorectal adenocarcinoma andup to 25of colorectal adenocarcinoma patients present withliver metastasis at the time of initial diagnosis [3]There is noappropriate targeted therapy to improve the clinical outcomeof patients with colorectal adenocarcinoma [6] The molec-ular prognostic markers related to a prognosis would be ofgreat help for patients with colorectal adenocarcinoma [7]

Dual-specificity protein phosphatase 4 (DUSP4) alsoknown as mitogen-activated protein kinase phosphatases 2(MKP2) is a member of the dual specificity phosphatase

family which inactivates target kinases through dephos-phorylating phosphoserinethreonine and phosphotyrosineresidues within one substrate [8 9] DUSP4MKP2 islocated on chromosome 8p12-p11 [10] DUSP4 can specifi-cally dephosphorylate the mitogen-activated protein (MAP)kinases ERK12 p38 and JNK [11]These pathways drive pro-liferation differentiation apoptosis and inflammation [1213] DUSP4 expression is observed in various human cancersincluding breast cancer [14 15] colorectal cancer [8 9 16]pancreatic cancer [17] lung cancer [18] glioma [10] andmalignant melanoma [19] However whether DUSP4 acts asa tumor promoter or tumor suppressor is still controversialand the consensus has not been reached on the exact role ofDUSP4 expression in various human cancers

In the present study we investigated DUSP4 expres-sion immunohistochemically in a large series of colorectaladenocarcinoma and evaluated the association of DUSP4expression with clinicopathological variables as well as the

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2015 Article ID 283764 8 pageshttpdxdoiorg1011552015283764

2 Gastroenterology Research and Practice

impact of DUSP4 expression on survival in patients withcolorectal adenocarcinoma

2 Materials and Methods

21 Patients and Specimens A consecutive series of 439patients with colorectal adenocarcinoma was enrolled in thisstudy All cases were underwent operation at the Surgery ofHanyang University Hospital (Seoul South Korea) betweenJanuary 1991 and August 2001 There were 239 male and 200female patients The patient age ranged from 17 years to 85years (a mean age of 5762 years) Out of 439 cases tumorswere located in cecum (119899 = 16) ascending colon (119899 = 64)hepatic flexure (119899 = 9) transverse colon (119899 = 21) splenicflexure (119899 = 4) descending colon (119899 = 18) sigmoid colon(119899 = 95) and rectum (119899 = 212) The size of the tumor rangedfrom 03 to 15 cm (a mean size of 568 cm) Tumors consistedof 418 nonmucinous adenocarcinomas and 21 mucinousadenocarcinomas Mean follow-up interval was 590 years152 (346) patients died and 287 (654) patients remainedalive Six cases were stage 0 31 cases were stage I 154 caseswere stage II 231 cases were stage III and 17 cases werestage IV according to the American Joint Committee onCancer (AJCC) staging system In addition 23 samples ofnormal colorectal tissue 50 samples of tubular adenoma56 samples of lymph node metastasis and 53 samples ofdistantmetastasis were selected to evaluate the role of DUSP4expression in carcinogenesis and tumor progression Alltissue samples were formalin-fixed and paraffin embeddedPathologic reports hematoxylin-eosin stained slides andmedical records were reviewed to confirm the final diagnosisand detail clinicopathologic parameters including genderage tumor size tumor type tumor location T stage lymphnode metastasis AJCC stage Dukes stage differentiationlymphovascular invasion and patientsrsquo survival

22 TissueMicroarray Construction Weused amanual tissuemicroarrayer (Quick Ray Set Unitama Seoul South Korea)for tissue microarray construction As previously described[20] we selected areas rich in tumor cells without necrosis bylight microscopy of HampE stained slides We punched a tissuecylinder with a 2mm diameter from a previously markedlesion of each donor block and transferred to the recipientblock (Quick Ray Set Unitama Seoul South Korea) Eachtissue microarray was made up of 5 times 10 samples

23 Immunohistochemical Staining We used a polyclonalrabbit anti-DUSP4 antibody (ab72593 Abcam CambridgeUK) at 1 150 dilution 4 120583m sections were cut from tissuemicroarray block using Leica microtome and transferredto adhesive coated slides and deparaffinized The stainingwas performed using the Bond Max automated immunos-tainer (Vision Biosystems San Francisco CA USA) Beforestaining the heat-induced epitope retrieval was performedin Bond epitope retrieval solution Endogenous peroxidaseactivity was blocked using 03 hydrogen peroxide Theprimary antibodywas incubated for 30minutes and the slideswere incubated with postprimary reagent for 15 minutes atroom temperature The reactions were developed using aBond polymer refine detection kit and followed by color

development with 331015840-diaminobenzidine tetrahydrochlorideas a chromogen

24 Interpretation of Immunohistochemical Staining DUSP4expression was evaluated semiquantitatively by two indepen-dent pathologists (Hyunsung Kim and Seung Sam Paik) whowere blinded to the patientsrsquo clinical outcomeWe categorizedthe cytoplasmic DUSP4 expression in terms of both stainingintensity and extent as described previously [20] Stainingintensity was graded as negative (0) weak (1) moderate (2)and strong (3) and staining extent was graded as 0 (0)1ndash25 (1) 26ndash50 (2) 51ndash75 (3) and 76ndash100 (4) Theproduct of intensity grade and extent grade was used as thefinal staining score Thus the maximum combined scorewas 12 and the minimum score was 0 For the purpose ofstatistical analysis a cutoff value of 3 was adopted accordingto the receiver operating characteristic curve Therefore thesamples were finally classified as either negative (score 0ndash2)or positive (score 3ndash12) for DUSP4 expression

25 Statistical Analysis We performed statistical analysisusing the SPSS software version 190 (Chicago ILUSA) Chi-square test for linear trend Chi-square test for independenceand Mann-Whitney 119880 test were used to investigate theassociation between DUSP4 expression and clinicopatho-logical features including gender age tumor size tumorlocation tumor type AJCC stage Dukes stage T categoryN category differentiation and lymphovascular invasionSpearmanrsquos analysis was used to obtain correlation coefficientTheKaplan-Meiermethodwas used to determine overall sur-vival and disease-free survival Univariable survival analysiswas used to compare the survival rates of subgroups withthe log-rank test Multivariable survival analysis was usedto determine independent prognostic factors with the Coxproportional hazards regression model A difference of 119875 lt005 was considered statistically significant

3 Results

31 Patterns of DUSP4 Expression We evaluated DUSP4expression in 23 samples of normal colorectal tissue 50 sam-ples of tubular adenoma 439 samples of adenocarcinoma56 samples of lymph node metastasis and 53 samples ofdistant metastasis Various grades of cytoplasmic DUSP4expression were observed Representative photomicrographsof DUSP4 immunostaining in colorectal adenocarcinomaare shown in Figure 1 DUSP4 expression was positive in 2cases (87) of normal colorectal tissue and 2 cases (40) oftubular adenoma however DUSP4 expression was positivein 166 cases (378) of adenocarcinoma 19 cases (339)of lymph node metastasis and 32 cases (604) of distantmetastasis (Table 1) DUSP4 was more frequently expressedin malignant tumors compared to that in benign lesions(119875 lt 0001) Mean DUSP4 expression score was 056 innormal colorectal tissue 036 in tubular adenoma 258 inadenocarcinoma 210 in lymph node metastasis and 475in distant metastasis Mean DUSP4 expression score wassignificantly higher in malignant tumors than in benignlesions (119875 lt 0001 Kruskal-Wallis test) (Figure 2)

Gastroenterology Research and Practice 3

(a) (b)

(c) (d)

Figure 1 Representative microphotographs of DUSP4 immunostaining in colorectal adenocarcinoma (times200) (a) Negative (b) weak (c)moderate and (d) strong The tumor cells showed cytoplasmic DUSP4 staining

6

2

4

0NL TA CA LNM DM

P lt 0001

P lt 0001

Figure 2MeanDUSP4 expression score in normal colorectal tissue(NL) tubular adenoma (TA) adenocarcinoma (CA) lymph nodemetastasis (LNM) and distant metastasis (DM)

32 Correlation between DUSP4 Expression and Clinicopatho-logical Parameters We investigated the correlation betweenDUSP4 expression and clinicopathological parameters toassess the clinicopathological significance of DUSP4 expres-sion in colorectal adenocarcinoma DUSP4 expression wassignificantly correlated with older age (119875 = 0017) malegender (119875 = 0036) larger tumor size (119875 = 0014) non-mucinous tumor type (119875 = 0023) and higher T stage(119875 = 0040) (Table 2) However there was no correlation

Table 1 DUSP4 expression inNL TA CA LNM andDM (119899 = 621)

Tissuesamples

119899

DUSP4 expression

Negative ()(119899 = 400)

Positive ()(119899 = 221)

119875 valuedagger 119903119904

NL 23 21 (913) 2 (87)

lt0001 0218TA 50 48 (960) 2 (40)

CA 439 273 (622) 166 (378)

LNM 56 37 (661) 19 (339)

DM 53 21 (396) 32 (604)daggerChi-square test for linear trendDUSP4 dual specificity protein phosphatase 4 NL normal colorectal tissueTA tubular adenoma CA adenocarcinoma LNM lymph node metastasisDM distant metastasis 119903

119904 Spearmanrsquos rank correlation coefficient

with tumor location N category AJCC stage Dukes stagedifferentiation and lymphovascular invasion

33 Correlation between DUSP4 Expression and Overall Sur-vival and Disease-Free Survival The impact of DUSP4expression on survival in 439 patients with colorectal ade-nocarcinoma was evaluated We observed that patient agedifferentiation AJCC stage and vascular invasion show asignificant effect on overall and disease-free survival in the


Table 2 Correlation between DUSP4 expression and clinicopathological factors in colorectal adenocarcinomas (119899 = 439)

Factors 119899

DUSP4 expressionNegative ()(119899 = 273)

Positive ()(119899 = 166)

119875 value 119903119904

Age (years)Mean plusmn SD 439 5646 plusmn 1330 5951 plusmn 1133 0017dagger 0114

GenderMale 239 138 (577) 101 (423) 0036Dagger minus0100Female 200 135 (675) 65 (325)

Tumor locationColon 227 137 (604) 90 (396) 0412Dagger minus0039Rectum 212 136 (642) 76 (358)

Tumor sizeMean plusmn SD 439 545 plusmn 192 605 plusmn 224 0014dagger 0117

Tumor typeNonmucinous 418 255 (610) 163 (390) 0023Dagger minus0109Mucinous 21 18 (857) 3 (143)

T categoryTis T1 T2 46 35 (761) 11 (239) 0040Dagger 0098T3 T4 393 238 (606) 155 (394)

N categoryN0 192 123 (641) 69 (359)

0618lowast 0025N1 114 68 (596) 46 (404)N2 133 82 (617) 51 (383)

AJCC stage0 I II 191 122 (639) 69 (361) 0522Dagger 0031III IV 248 151 (609) 97 (391)

Dukes stageA B 187 118 (631) 69 (369) 0733Dagger 0016C D 252 155 (615) 97 (385)

DifferentiationWellModerately 347 220 (634) 127 (366) 0308Dagger 0049Poorly 92 53 (576) 39 (424)

Lymphatic invasionAbsent 186 120 (645) 66 (355) 0388Dagger 0041Present 253 153 (605) 100 (395)

Vascular invasionAbsent 429 266 (620) 163 (380) 0606Dagger minus0025Present 10 7 (700) 3 (300)

daggerMann-Whitney 119880 test DaggerChi-square test for independence lowastChi-square test for linear trendDUSP4 dual specificity protein phosphatase 4 SD standard deviation AJCCAmerican Joint Committee onCancer 119903


univariable and multivariable analyses (Table 3) There wasno significant correlation between DUSP4 expression andoverall survival (119875 = 0091 log-rank test) or disease-freesurvival (119875 = 0100 log-rank test) according to the Kaplan-Meier survival curves in all 439 patients with colorectaladenocarcinoma (Figures 3(a) and 3(b)) However Kaplan-Meier survival curves revealed a significant effect of DUSP4expression on both overall survival and disease-free survival

in AJCC stage I (119875 = 0008 and 119875 = 0003 resp log-ranktest) (Figures 3(c) and 3(d)) and male gender (119875 = 0017 and119875 = 0049 resp log-rank test) (Figures 3(e) and 3(f))

4 Discussion

In this study we investigated DUSP4 expression in 23 sam-ples of normal colorectal tissue 50 samples of tubular


Table 3 Effect of variables on overall survival and disease-free survival in colorectal adenocarcinomas (119899 = 439)

Variables Univariable analysisdagger Multivariable analysisdagger

HR (95 CI) 119875 value HR (95 CI) 119875 valueOverall survival

DUSP4 expression (negative versus positive) 1317 (0956ndash1816) 0092 1156 (0833ndash1604) 0386Patient age (lt58 yrs versus ge58 yrs) 1856 (1373ndash2508) lt0001 1694 (1216ndash2360) 0002Differentiation (low versus high) 2391 (1757ndash3253) lt0001 1678 (1183ndash2381) 0004AJCC stage (0 I II versus III IV) 3062 (2180ndash4300) lt0001 2729 (1873ndash3977) lt0001Vascular invasion (absent versus present) 3326 (1561ndash7090) 0002 3058 (1422ndash6574) 0004

Disease-free survivalDUSP4 expression (negative versus positive) 1268 (0955ndash1685) 0101 1147 (0859ndash1533) 0352Patient age (lt58 yrs versus ge58 yrs) 1491 (1150ndash1934) 0003 1363 (1022ndash1816) 0035Differentiation (low versus high) 2115 (1598ndash2798) lt0001 1527 (1113ndash2095) 0009AJCC stage (0 I II versus III IV) 3287 (2442ndash4425) lt0001 3122 (2236ndash4360) lt0001Vascular invasion (absent versus present) 2512 (1183ndash5334) 0016 2280 (1066ndash4879) 0034

daggerCox proportional hazards modelHR hazard ratio CI confidence interval DUSP4 dual specificity protein phosphatase 4 AJCC American Joint Committee on Cancer

adenoma 439 samples of adenocarcinoma 56 samples oflymph node metastasis and 53 samples of distant metastasisand evaluated the correlation between DUSP4 expressionand clinicopathological parameters and patient survivalin patients with colorectal adenocarcinoma DUSP4 wasmore frequently expressed in adenocarcinomas and lymphnodedistant metastases compared to that in normal colorec-tal tissues and tubular adenomas Mean DUSP4 expressionscore was significantly higher in malignant cases than inbenign cases DUSP4 expression was significantly correlatedwith older age male gender larger tumor size nonmucinoustumor type and higher T stage Kaplan-Meier survival curvesrevealed a significant effect of DUSP4 expression on bothoverall survival and disease-free survival in male gender andAJCC stage I patients

In colorectal cancer themitogen-activated protein kinase(MAPK) pathway is a commonly mutated pathway with 35ndash40 of patients having an activating mutation in KRAS and5ndash10 of patients having an activating mutation in BRAF[21 22] Previous analysis of the gene expression profile ofprimary tumors revealed that DUSP genes are among a setof genes specific for the BRAF mutated tumors [8] Dualspecificity protein phosphatases (DUSPs) are a heterogeneousgroup of phosphatases that can dephosphorylate phosphoty-rosine and phosphoserinephosphothreonine residues withinone substrate [16] DUSPs can be divided into seven sub-groups based on their sequence similarity [23] Among themdual specificity protein phosphatase 4 (DUSP4) is a memberof the inducible nuclear MKP group and specifically dephos-phorylates the mitogen-activated protein kinases ERK12p38 and JNK DUSP4 plays a crucial role in regulating thetumor-relevantMAPKpathways [9]The exact role ofDUSP4in cancer development and progression appears to vary withthe type of malignant tumors

Recently Saigusa et al [9] reported that decreased expres-sion of DUSP4 was related to metastases to liver and lung incolorectal cancer They investigated the association between

DUSP4 expression and clinical outcome in 212 patients withcolorectal cancer They described that decreased DUSP4expression was significantly related to advanced T categorylymphatic invasion vascular invasion advanced stage anddistant metastasis and increased DUSP4 expression was sig-nificantly associated with better prognosis They concludedthat DUSP4 expression was negatively correlated with factorsreflecting tumor progression including distant metastases incolorectal cancer and suggested that DUSP4 may act as atumor suppressor in colorectal cancer Some reports havealso described that DUSP4may play a tumor suppressor roleWaha et al [10] described that epigenetic downregulationof mitogen-activated protein kinase phosphatase MKP-2relieved its growth suppressive activity in glioma cells Chitaleet al [18] declared DUSP4 as a novel growth suppressor inEGFR-mutant lung adenocarcinoma Armes et al [24] foundthat DUSP4 is present in primary tumors but could be lost inearly onset and high-grade breast cancers

While several reports have revealed that DUSP4may playa role in promoting cancer progression Vriendt et al [8]demonstrated that patients with highDUSP4 expressionweresignificantly linked with a worse overall survival comparedto patients with low DUSP4 expression in colorectal cancerGroschl et al [16] showed that DUSP4 was frequently over-expressed in colorectal cancer with high frequent micro-satellite instability (MSI-H) compared to colorectal cancerwith microsatellite stable (MSS) and suggested that DUSP4may act as an important regulator of cell growth within theMAPK pathway and may cause enhanced cell growth inMSI-H colorectal cancer Liu et al [14] described that over-expression of DUSP4 may play an important role in promot-ing the epithelial-mesenchymal transition in breast cancerand suggested that DUSP4 may be a marker of adverseprognosis

In our study we found that DUSP4 was more frequentlyexpressed in cases of adenocarcinoma and lymph nodemetastasis compared to that in cases of normal colorectal


Ove

rall

surv

ival

()

0

100

50

0 50 200150100Follow-up period (month)

P = 0091

DUSP4-negativeDUSP4-positive

(a)

0

100

50


P = 0100

Dise

ase-

free s

urvi

val (

)


(b)

Ove

rall

surv

ival

()

0

100

50


P = 0008

AJCC stage I DUSP4-negativeAJCC stage I DUSP4-positive

(c)

0

100

50


P = 0003

Dise

ase-

free s

urvi

val (

)


(d)

Ove

rall

surv

ival

()

0

100

50


P = 0017

Male DUSP4-negativeMale DUSP4-positive

(e)

0

100

50


P = 0049

Dise

ase-

free s

urvi

val (

)


(f)

Figure 3 Cumulative overall and disease-free survival curves according to DUSP4 expression in all 439 patients with colorectaladenocarcinoma (a and b) AJCC stage I patients (c and d) and male gender (e and f) (Kaplan-Meier method with log-rank test)


tissue and tubular adenoma (119875 lt 0001) In addition DUSP4was more frequently expressed in cases of distant metastasiscompared to that in cases of adenocarcinoma and lymphnode metastasis (119875 lt 0001) These results suggest thatDUSP4may be involved in carcinogenesis and distant metas-tasis of colorectal cancer The clinicopathological correlationanalysis revealed that DUSP4 expression was significantlyassociated with tumor size (119875 = 0014) and higher T stage(119875 = 0040) These results suggest that DUSP4 may beinvolved in tumor progression of colorectal cancer In sur-vival analyses Kaplan-Meier survival curves revealed a sig-nificant effect of DUSP4 expression on both overall survivaland disease-free survival in AJCC stage I (119875 = 0008 and 119875 =0003 resp log-rank test) These results suggest that DUSP4may be a marker of adverse prognosis especially in patientswith colorectal cancer in early stage Our results suggest thatDUSP4 may play a role as a cancer promoter not as a tumorsuppressor in colorectal adenocarcinoma

In conclusion we investigated DUSP4 expression in alarge series of colorectal adenocarcinoma Our results weresimilar to the results of Vriendt et al [8] They demonstratedthat patients with high DUSP4 expression were significantlylinked with a worse overall survival compared to patientswith low DUSP4 expression However our results showedthe discrepancy with the results of Saigusa et al [9] Theyconcluded that DUSP4 expression was negatively correlatedwith factors reflecting tumor progression and suggested thatDUSP4 may act as a tumor suppressor The exact role ofDUSP4 should be further investigated in colorectal cancerand DUSP4 role as a potential novel therapeutic target forcolorectal cancer should be investigated in the further study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jongmin Sim and Kijong Yi are equal contributors to thiswork

Acknowledgment

This work was supported by the research fund of HanyangUniversity (HY-2014)

References

[1] C C Compton ldquoColorectal carcinoma diagnostic prognosticand molecular featuresrdquo Modern Pathology vol 16 no 4 pp376ndash388 2003

[2] A Jemal T Murray EWard et al ldquoCancer statistics 2005rdquoCAA Cancer Journal for Clinicians vol 55 no 1 pp 10ndash30 2005

[3] W Weng F Feng H Qin and Y Ma ldquoMolecular therapy ofcolorectal cancer progress and future directionsrdquo InternationalJournal of Cancer vol 136 no 3 pp 493ndash502 2015

[4] D M Parkin F Bray J Ferlay and P Pisani ldquoGlobal cancerstatistics 2002rdquo CA A Cancer Journal for Clinicians vol 55 no2 pp 74ndash108 2005

[5] J Walker and P Quirke ldquoPrognosis and response to therapy incolorectal cancerrdquo European Journal of Cancer vol 38 no 7 pp880ndash886 2002

[6] Y J Jun S M Jang H L Han K H Lee K-S Jang andS S Paik ldquoClinicopathologic signifcance of GULT1 expressionand its correlation with Apaf-1 in colorectal adenocarcinomasrdquoWorld Journal of Gastroenterology vol 17 no 14 pp 1866ndash18732011

[7] L T Soumaoro H Uetake T Higuchi Y Takagi M Enomotoand K Sugihara ldquoCyclooxygenase-2 expression a significantprognostic indicator for patients with colorectal cancerrdquo Clin-ical Cancer Research vol 10 no 24 pp 8465ndash8471 2004

[8] V deVriendtW deRoock A F diNarzo et al ldquoDUSP4 expres-sion identifies a subset of colorectal cancer tumors that differin MAPK activation regardless of the genotyperdquo Biomarkersvol 18 no 6 pp 516ndash524 2013

[9] S Saigusa Y Inoue K Tanaka et al ldquoDecreased expression ofDUSP4 is associated with liver and lungmetastases in colorectalcancerrdquoMedical Oncology vol 30 no 3 article 620 2013

[10] AWaha J FelsbergWHartmann et al ldquoEpigenetic downregu-lation of mitogen-activated protein kinase phosphatase MKP-2relieves its growth suppressive activity in glioma cellsrdquo CancerResearch vol 70 no 4 pp 1689ndash1699 2010

[11] S M Keyse ldquoDual-specificity MAP kinase phosphatases(MKPs) and cancerrdquo Cancer andMetastasis Reviews vol 27 no2 pp 253ndash261 2008

[12] W H Shen JWang JWu V B Zhurkin and Y Yin ldquoMitogen-activated protein kinase phosphatase 2 a novel transcriptiontarget of p53 in apoptosisrdquo Cancer Research vol 66 no 12 pp6033ndash6039 2006

[13] L Cadalbert C M Sloss P Cameron and R Plevin ldquoCon-ditional expression of MAP kinase phosphatase-2 protectsagainst genotoxic stress-induced apoptosis by binding andselective dephosphorylation of nuclear activated c-jun N-terminal kinaserdquo Cellular Signalling vol 17 no 10 pp 1254ndash1264 2005

[14] Y Liu F Du W Chen M Yao K Lv and P Fu ldquoKnockdownof dual specificity phosphatase 4 enhances the chemosensitivityof MCF-7 andMCF-7ADR breast cancer cells to doxorubicinrdquoExperimental Cell Research vol 319 no 20 pp 3140ndash3149 2013

[15] H-Y Wang Z Cheng and C C Malbon ldquoOverexpression ofmitogen-activated protein kinase phosphatases MKP1 MKP2in human breast cancerrdquo Cancer Letters vol 191 no 2 pp 229ndash237 2003

[16] B Groschl M Bettstetter C Giedl et al ldquoExpression of theMAP kinase phosphatase DUSP4 is associated with microsatel-lite instability in colorectal cancer (CRC) and causes increasedcell proliferationrdquo International Journal of Cancer vol 132 no7 pp 1537ndash1546 2013

[17] M T Yip-Schneider A Lin and M S Marshall ldquoPancreatictumor cells with mutant K-ras suppress ERK activity by MEK-dependent induction of MAP kinase phosphatase-2rdquo Biochem-ical and Biophysical Research Communications vol 280 no 4pp 992ndash997 2001

[18] D Chitale Y Gong B S Taylor et al ldquoAn integrated genomicanalysis of lung cancer reveals loss of DUSP4 in EGFR-mutanttumorsrdquo Oncogene vol 28 no 31 pp 2773ndash2783 2009

[19] J Teutschbein J M Haydn B Samans et al ldquoGene expressionanalysis after receptor tyrosine kinase activation reveals newpotential melanoma proteinsrdquo BMC Cancer vol 10 article 3862010


[20] S M Jang J M Sim H Han et al ldquoClinicopathologicalsignificance of CADM4expression in invasive ductal carcinomaof the breastrdquo Journal of Clinical Pathology vol 66 no 8 pp681ndash686 2013

[21] H-T Li Y-Y Lu Y-X An X Wang and Q-C Zhao ldquoKRASBRAF and PIK3CAmutations in human colorectal cancer rela-tionship with metastatic colorectal cancerrdquo Oncology Reportsvol 25 no 6 pp 1691ndash1697 2011

[22] C P Vaughn S D Zobell L V Furtado C L Baker and W SSamowitz ldquoFrequency of KRAS BRAF andNRASmutations incolorectal cancerrdquo Genes Chromosomes and Cancer vol 50 no5 pp 307ndash312 2011

[23] K I Patterson T Brummer PMOrsquoBrien andR J Daly ldquoDual-specificity phosphatases critical regulators with diverse cellulartargetsrdquo The Biochemical Journal vol 418 no 3 pp 475ndash4892009

[24] J E Armes F Hammet M de Silva et al ldquoCandidate tumor-suppressor genes on chromosome arm 8p in early-onset andhigh-grade breast cancersrdquo Oncogene vol 23 no 33 pp 5697ndash5702 2004

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


impact of DUSP4 expression on survival in patients withcolorectal adenocarcinoma

2 Materials and Methods

21 Patients and Specimens A consecutive series of 439patients with colorectal adenocarcinoma was enrolled in thisstudy All cases were underwent operation at the Surgery ofHanyang University Hospital (Seoul South Korea) betweenJanuary 1991 and August 2001 There were 239 male and 200female patients The patient age ranged from 17 years to 85years (a mean age of 5762 years) Out of 439 cases tumorswere located in cecum (119899 = 16) ascending colon (119899 = 64)hepatic flexure (119899 = 9) transverse colon (119899 = 21) splenicflexure (119899 = 4) descending colon (119899 = 18) sigmoid colon(119899 = 95) and rectum (119899 = 212) The size of the tumor rangedfrom 03 to 15 cm (a mean size of 568 cm) Tumors consistedof 418 nonmucinous adenocarcinomas and 21 mucinousadenocarcinomas Mean follow-up interval was 590 years152 (346) patients died and 287 (654) patients remainedalive Six cases were stage 0 31 cases were stage I 154 caseswere stage II 231 cases were stage III and 17 cases werestage IV according to the American Joint Committee onCancer (AJCC) staging system In addition 23 samples ofnormal colorectal tissue 50 samples of tubular adenoma56 samples of lymph node metastasis and 53 samples ofdistantmetastasis were selected to evaluate the role of DUSP4expression in carcinogenesis and tumor progression Alltissue samples were formalin-fixed and paraffin embeddedPathologic reports hematoxylin-eosin stained slides andmedical records were reviewed to confirm the final diagnosisand detail clinicopathologic parameters including genderage tumor size tumor type tumor location T stage lymphnode metastasis AJCC stage Dukes stage differentiationlymphovascular invasion and patientsrsquo survival

22 TissueMicroarray Construction Weused amanual tissuemicroarrayer (Quick Ray Set Unitama Seoul South Korea)for tissue microarray construction As previously described[20] we selected areas rich in tumor cells without necrosis bylight microscopy of HampE stained slides We punched a tissuecylinder with a 2mm diameter from a previously markedlesion of each donor block and transferred to the recipientblock (Quick Ray Set Unitama Seoul South Korea) Eachtissue microarray was made up of 5 times 10 samples

23 Immunohistochemical Staining We used a polyclonalrabbit anti-DUSP4 antibody (ab72593 Abcam CambridgeUK) at 1 150 dilution 4 120583m sections were cut from tissuemicroarray block using Leica microtome and transferredto adhesive coated slides and deparaffinized The stainingwas performed using the Bond Max automated immunos-tainer (Vision Biosystems San Francisco CA USA) Beforestaining the heat-induced epitope retrieval was performedin Bond epitope retrieval solution Endogenous peroxidaseactivity was blocked using 03 hydrogen peroxide Theprimary antibodywas incubated for 30minutes and the slideswere incubated with postprimary reagent for 15 minutes atroom temperature The reactions were developed using aBond polymer refine detection kit and followed by color

development with 331015840-diaminobenzidine tetrahydrochlorideas a chromogen

24 Interpretation of Immunohistochemical Staining DUSP4expression was evaluated semiquantitatively by two indepen-dent pathologists (Hyunsung Kim and Seung Sam Paik) whowere blinded to the patientsrsquo clinical outcomeWe categorizedthe cytoplasmic DUSP4 expression in terms of both stainingintensity and extent as described previously [20] Stainingintensity was graded as negative (0) weak (1) moderate (2)and strong (3) and staining extent was graded as 0 (0)1ndash25 (1) 26ndash50 (2) 51ndash75 (3) and 76ndash100 (4) Theproduct of intensity grade and extent grade was used as thefinal staining score Thus the maximum combined scorewas 12 and the minimum score was 0 For the purpose ofstatistical analysis a cutoff value of 3 was adopted accordingto the receiver operating characteristic curve Therefore thesamples were finally classified as either negative (score 0ndash2)or positive (score 3ndash12) for DUSP4 expression

25 Statistical Analysis We performed statistical analysisusing the SPSS software version 190 (Chicago ILUSA) Chi-square test for linear trend Chi-square test for independenceand Mann-Whitney 119880 test were used to investigate theassociation between DUSP4 expression and clinicopatho-logical features including gender age tumor size tumorlocation tumor type AJCC stage Dukes stage T categoryN category differentiation and lymphovascular invasionSpearmanrsquos analysis was used to obtain correlation coefficientTheKaplan-Meiermethodwas used to determine overall sur-vival and disease-free survival Univariable survival analysiswas used to compare the survival rates of subgroups withthe log-rank test Multivariable survival analysis was usedto determine independent prognostic factors with the Coxproportional hazards regression model A difference of 119875 lt005 was considered statistically significant

3 Results

31 Patterns of DUSP4 Expression We evaluated DUSP4expression in 23 samples of normal colorectal tissue 50 sam-ples of tubular adenoma 439 samples of adenocarcinoma56 samples of lymph node metastasis and 53 samples ofdistant metastasis Various grades of cytoplasmic DUSP4expression were observed Representative photomicrographsof DUSP4 immunostaining in colorectal adenocarcinomaare shown in Figure 1 DUSP4 expression was positive in 2cases (87) of normal colorectal tissue and 2 cases (40) oftubular adenoma however DUSP4 expression was positivein 166 cases (378) of adenocarcinoma 19 cases (339)of lymph node metastasis and 32 cases (604) of distantmetastasis (Table 1) DUSP4 was more frequently expressedin malignant tumors compared to that in benign lesions(119875 lt 0001) Mean DUSP4 expression score was 056 innormal colorectal tissue 036 in tubular adenoma 258 inadenocarcinoma 210 in lymph node metastasis and 475in distant metastasis Mean DUSP4 expression score wassignificantly higher in malignant tumors than in benignlesions (119875 lt 0001 Kruskal-Wallis test) (Figure 2)


(a) (b)

(c) (d)


6

2

4

0NL TA CA LNM DM

P lt 0001

P lt 0001




Tissuesamples

119899

DUSP4 expression

Negative ()(119899 = 400)

Positive ()(119899 = 221)

119875 valuedagger 119903119904

NL 23 21 (913) 2 (87)

lt0001 0218TA 50 48 (960) 2 (40)

CA 439 273 (622) 166 (378)

LNM 56 37 (661) 19 (339)







Factors 119899


Positive ()(119899 = 166)

119875 value 119903119904







N categoryN0 192 123 (641) 69 (359)

0618lowast 0025N1 114 68 (596) 46 (404)N2 133 82 (617) 51 (383)










4 Discussion
















Ove

rall

surv

ival

()

0

100

50


P = 0091


(a)

0

100

50


P = 0100

Dise

ase-

free s

urvi

val (

)


(b)

Ove

rall

surv

ival

()

0

100

50


P = 0008


(c)

0

100

50


P = 0003

Dise

ase-

free s

urvi

val (

)


(d)

Ove

rall

surv

ival

()

0

100

50


P = 0017


(e)

0

100

50


P = 0049

Dise

ase-

free s

urvi

val (

)


(f)









Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)


6

2

4

0NL TA CA LNM DM

P lt 0001

P lt 0001




Tissuesamples

119899

DUSP4 expression

Negative ()(119899 = 400)

Positive ()(119899 = 221)

119875 valuedagger 119903119904

NL 23 21 (913) 2 (87)

lt0001 0218TA 50 48 (960) 2 (40)

CA 439 273 (622) 166 (378)

LNM 56 37 (661) 19 (339)







Factors 119899


Positive ()(119899 = 166)

119875 value 119903119904







N categoryN0 192 123 (641) 69 (359)

0618lowast 0025N1 114 68 (596) 46 (404)N2 133 82 (617) 51 (383)










4 Discussion
















Ove

rall

surv

ival

()

0

100

50


P = 0091


(a)

0

100

50


P = 0100

Dise

ase-

free s

urvi

val (

)


(b)

Ove

rall

surv

ival

()

0

100

50


P = 0008


(c)

0

100

50


P = 0003

Dise

ase-

free s

urvi

val (

)


(d)

Ove

rall

surv

ival

()

0

100

50


P = 0017


(e)

0

100

50


P = 0049

Dise

ase-

free s

urvi

val (

)


(f)









Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Factors 119899


Positive ()(119899 = 166)

119875 value 119903119904







N categoryN0 192 123 (641) 69 (359)

0618lowast 0025N1 114 68 (596) 46 (404)N2 133 82 (617) 51 (383)










4 Discussion
















Ove

rall

surv

ival

()

0

100

50


P = 0091


(a)

0

100

50


P = 0100

Dise

ase-

free s

urvi

val (

)


(b)

Ove

rall

surv

ival

()

0

100

50


P = 0008


(c)

0

100

50


P = 0003

Dise

ase-

free s

urvi

val (

)


(d)

Ove

rall

surv

ival

()

0

100

50


P = 0017


(e)

0

100

50


P = 0049

Dise

ase-

free s

urvi

val (

)


(f)









Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Ove

rall

surv

ival

()

0

100

50


P = 0091


(a)

0

100

50


P = 0100

Dise

ase-

free s

urvi

val (

)


(b)

Ove

rall

surv

ival

()

0

100

50


P = 0008


(c)

0

100

50


P = 0003

Dise

ase-

free s

urvi

val (

)


(d)

Ove

rall

surv

ival

()

0

100

50


P = 0017


(e)

0

100

50


P = 0049

Dise

ase-

free s

urvi

val (

)


(f)









Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Immunohistochemical Expression of Dual ...

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