journal of international medical research-2014-yao-0300060514527060

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published online 14 May 2014Journal of International Medical Research Tingting Yao, Zhuna Wu, Yukun Liu, Qunxian Rao and Zhongqiu Lin

cervical cancerAldehyde dehydrogenase 1 (ALDH1) positivity correlates with poor prognosis in

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Clinical Note

Aldehyde dehydrogenase1 (ALDH1) positivitycorrelates with poor prognosis in cervical cancer

Tingting Yao 1,2, *, Zhuna Wu 3, *, Yukun Liu 1, *,Qunxian Rao 1 and Zhongqiu Lin 1

AbstractObjective: To analyse the associations between aldehyde dehydrogenase 1 (ALDH1) tumourimmunopositivity and disease-free survival in cervical carcinoma.Methods: ALDH1 immunohistochemistry was performed on formalin-fixed, paraffin wax-embedded cervical tumour tissue samples obtained from hospital archives. Data regardingdisease-free survival were obtained. Kaplan–Meier survival analyses and Cox proportional hazardsregression models were performed.Results: Patients with ALDH1-positive tumours ( n ¼ 31) had significantly shorter disease-free

survival than those with ALDH1-negative tumours ( n¼

167; 41.99 0.90 vs 53.64 2.67 months).ALDH1 positivity was associated with poor prognosis (relative risk 2.727; 95% confidence intervals1.253, 5.914).Conclusions: ALDH1 positivity is associated with poor prognosis of cervical carcinoma, and maybe an independent predictor of prognosis.

KeywordsCervical cancer, cancer stem cell, aldehyde dehydrogenase 1 (ALDH1), immunohistochemistry

Date received: 13 December 2013; accepted: 17 February 2014

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1 Department of Gynaecological Oncology, Sun Yat-senMemorial Hospital, Sun Yat-sen University, Guangzhou,Guangdong Province, China2 Key Laboratory of Malignant Tumour Gene Regulation

and Target Therapy of Guangdong Higher EducationInstitutes, Sun Yat-sen University, Guangzhou, GuangdongProvince, China

3 Department of Gynaecology and Obstetrics, The SecondAffiliated Hospital of Fujian Medical University, Quanzhou,Fujian Province, China

Corresponding author:Zhongqiu Lin, Department of Gynaecological Oncology,

Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107Yan Jiang West Road, Guangzhou 510120, China.Email: [email protected]

* Co-first authors, Tingting Yao, Zhuna Wu and Yukun Liucontributed equally to this study.

Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/ ) which permits non-commercial use, reproduction and

distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page(http://www.uk.sagepub.com/aboutus/openaccess.htm ).

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IntroductionCervical cancer is the third most commonmalignant tumour in Chinese women. 1

The widespread use of cervical cytology

screening has resulted in a substantialdecrease in disease incidence and mortality, 2

but the 5-year survival rate in patients withadvanced disease is 3.2–13%. 3 Recurrenceusually occurs within 2 years of diagnosis, 4

and the median survival time of recurringcases is only 7 months, due to resistance totraditional treatments. 5 Aldehyde dehydro-genase 1 (ALDH1) is a cancer stem-cellmarker, the presence of which stronglycorrelates with tumour malignancy andself-renewal properties of stem cells intumour types including breast cancer, hepa-toma, lung and colon cancer. 6–9

Our previous study used immunohisto-chemistry to evaluated ALDH1 in 109 sam-ples of cervical carcinoma and found thatthe presence of ALDH1 in tumour tissuewas signicantly related to lymph nodemetastasis and recurrence. 10 The aim of thepresent study was to analyse the relationshipbetween ALDH1 positivity and cervicalcancer prognosis.

Patients and methodsTissue samplesSamples of formalin-xed, paraffin wax-embedded cervical tumour tissue wereobtained from the archives of theDepartment of Gynaecology, Sun Yat-senMemorial Affiliated Hospital, Sun Yat-senUniversity, Guangzhou, China. Patientsunderwent treatment between October2003 and December 2007 for conrmedcervical cancer (including squamous cellcarcinoma and adenocarcinoma). Patientswith evidence of disease on chest X-radio-graph or magnetic resonance imaging(MRI) were excluded. Tumours werestaged according to 2009 InternationalFederation of Gynaecology and Obstetrics(FIGO) revised staging criteria. 11 Data

regarding treatment, myometrial invasion,vascular invasion and lymph node metasta-sis were obtained from medical records.

All patients provided written informed

consent for the use of their tissue samplesand the study was approved by the EthicsCommittee of Sun Yat-sen MemorialHospital, Sun Yat-sen University.

Follow upFollow up continued until April 2009.Patients were followed up every 3 monthsin the rst year, every 4 months in the secondyear, every 6 months for the next 3 years andannually thereafter. Each visit included apelvic examination, cytology and colpos-copy. Ultrasonography, MRI or computedtomography scan were performed if necessary.

Immunohistochemistry Tissue was cut into 3- mm sections andmounted on glass slides pretreated withpoly- L-lysine. Sections were boiled for15 min in citrate buffer (10 mM, pH 6.0),cooled in the same buffer, then incubated for10 min in 0.3% hydrogen peroxide to blockendogenous peroxidase activity. Slides wereincubated in mouse antihuman ALDH1antibody (1: 100 dilution; Santa CruzBiotechnology, Santa Cruz, CA, USA) over-night at 4 C in a humid chamber, andwashed three times at room temperature inphosphate buffered saline (PBS; 0.01 M, pH7.2; 3 min each wash). Sections were thenincubated with horseradish peroxidase-con- jugated antimouse secondary antibody(1 : 100 dilution; Zymed, San Francisco,CA, USA) for 60 min at room temperature,washed three times with PBS at room tem-perature (3 min each wash), then stainedusing the ABC Vectastain kit (Vectorlaboratories, Burlingame, CA, USA)according to the manufacturer’s instruc-tions. All assays included positive and

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negative controls (replacement of primaryantibody with mouse immunoglobulin G1used as negative control).Immunohistochemical staining of ALDH1

was classied as negative ( , no staining),weakly positive ( þ , light-brown or yellowcells) or positive ( þþ , brown staining). Forthe purpose of the study, ‘positive’ stainingincluded both weakly positive and positivestaining. All slides were evaluated by twoindependent pathologists.

Statistical analysesData were presented as mean SD.Kaplan–Meier analysis was used to estimatethe probability of survival, and log-rank testwas used to compare the survival differ-ences. Potential prognostic factors(including age, pathological type, clinicalstage, tumour differentiation, vessel inva-sion, deep myometrial invasion, lymph nodemetastasis, preoperative chemotherapy andALDH1 immunopositivity) were analysedby multivariate Cox proportional hazardsregression models and expressed as relativerisk (RR) with 95% condence intervals(CI). Statistical analyses were performedusing SPSS version 13.0 (SPSS Inc.,Chicago, IL, USA) for Windows .P -values < 0.05 were considered statisticallysignicant.

ResultsThe study included tumour tissue samplesfrom 198 patients with cervical cancer (meanage 44.6 8.7 years, age range 24–67 years).Patients were diagnosed with invasive squa-mous carcinoma ( n ¼ 155) or adenocarcin-oma ( n ¼ 43). A total of 112 patients hadundergone no preoperative radiotherapy orchemotherapy, four had undergone pre-operative radiotherapy and 82 had receivedpreoperative chemotherapy. Tumours werestage IB-1 ( n ¼ 53), stage IB-2 ( n ¼ 82), stageIIA ( n ¼ 49) and stage IIB ( n ¼ 14). A total

of 28 patients were lost to follow-up (follow-up rate 85.9%). Mean follow-up durationwas 35.13 15.78 months (range 11–77months).

Patients with ALDH1-positive tumours(n ¼ 31) had signicantly shorter disease-freesurvival than those whose tumours wereALDH1-negative ( n ¼ 167; 41.99 0.90 vs53.64 2.67 months; P < 0.05; Figure 1).

Lymph node metastasis (RR 5.445; 95%CI 2.014, 14.722) and ALDH1 positivity(RR 2.727; 95% CI 1.253, 5.914) wereassociated with poor prognosis.

DiscussionIn the current study of well-characterizedpatients with cervical carcinoma and long-term follow-up, ALDH1 positivity wasassociated with poor disease-free survival.ALDH1 plays a role in the biologicalsynthesis of retinoic acid and is involved inthe early differentiation of stem cells bymediating the oxidation of retinol into ret-inoic acid. 12 Retinoic acid is involved in celldifferentiation and proliferation via bindingto the retinoic acid receptor. 13

It is thought that cancer stem cells maybe the original cells in tumour metas-tases. 14 ALDH1 is a marker for the iden-tication of different types of cancer stemcell. 9,15,16 In bladder cancer, ALDH-1levels are higher in lymph node-positivepatients than in lymph node-negativepatients, 17 suggesting that ALDH-1-posi-tive cells promote invasion and metastasis.A clinical study indicated that breastcancer cells also express ALDH1. 18 Theprognosis of patients with breast cancerwho were ALDH1 positive was poor, andALDH1 positivity was closely correlatedwith tumour grade, estrogen receptor/pro-gestogen receptor status, overexpressionof the oncogene ERBB2 and formation of cytokeratin. 6,19

In conclusion, ALDH1 positivity is asso-ciated with poor prognosis in cervical

Yao et al. 3








carcinoma and may be an independentpredictor of prognosis.

Declaration of conflicting interest

The authors declare that there are no conicts of interest.

Funding

This study was supported by National NaturalScience Foundation of China (81101979 and30872743), Doctoral Programme of EducationalMinistry (20120171110097), GuangdongProvince Natural Scientic Grant(S2011040004639 and 2012020010913),Guangdong Province Medical Science

Technology Grant (B2011088), Yat-senScholarship for Young Scientists and Programfor training project Young Scientists in Sun Yat-sen University.

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2. Cuzick J, Myers O, Hunt WC et al. Apopulation-based evaluation of cervicalscreening in the United States: 2008–2011 .Cancer Epidemiol Biomarkers Prev 2014 Apr9. [Epub ahead of print].

3. Lai CH. Management of recurrent cervicalcancer. Chang Gung Med J 2004; 27: 711–717.

Figure 1. Kaplan–Meier analysis of disease-free survival in patients with cervical carcinoma, stratifiedaccording to the results of aldehyde dehydrogenase 1 (ALDH1) tumour immunohistochemistry (negative,n ¼ 31; positive, n ¼ 167). P < 0.05; log-rank test.

, ALDH1 negative;þ , þþ , ALDH1 positive; miss, ALDH1 negative patients lost to follow-up; þ , þþ miss,ALDH1 positive patients lost to follow-up.

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human pancreas modulates retinoic acidsignalling in mediating islet differentiationand survival. Diabetologia 2014; 57: 754–764.

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19. Tanei T, Morimoto K, Shimazu K, et al.Association of breast cancer stem cellsidentified by aldehyde dehydrogenase 1expression with resistance to sequentialPaclitaxel and epirubicin-based chemother-apy for breast cancers. Clin Cancer Res 2009;15: 4234–4241.

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