Do selective estrogen receptor modulators treat cervicalprecancer and cancer? time to pool data from relevant trials

Philip E. Castle

Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892-7234

Dear Editor,

Based on the nearly absolute etiologic link between carcino-genic human papillomavirus (HPV) and cervical cancer, twonew approaches for the prevention of cervical cancer haveemerged: (1) HPV vaccination for primary HPV preventionin younger women and (2) carcinogenic HPV detection forsecondary prevention via identifying and treating cervicalprecancer and early cancers. Both have demonstrated highdegrees of efficacy with maximum effectiveness guided by anunderstanding of the causal model and application of thesetechnologies in an age appropriate manner.1 Despite theadvent of these promising prevention tools, there is a realneed to develop nonsurgical methods for treating cervicalprecancerous lesions and even early cancer for three reasons.First, current HPV vaccines are prophylactic and do not treatpre-existing HPV infections.1 Second, surgical excision ofscreen-detected precancerous lesions, while a highly effica-cious (90–95%) treatment1 increases the risk of pretermdelivery and infant morbidity and mortality.2 Third, mostwomen who screen positive by a Pap and/or HPV do nothave clinically actionable disease, yet these women are at riskof cervical precancer and cancer in the future and must befollowed up intensively. In addition, in the low-resource set-tings, surgical methods are not commonly available and cryo-therapy, while moderately efficacious when its use is limitedto smaller lesions, causes significant watery discharge andrequires sexual abstinence for several weeks to permit healingof treated epithelium.

Despite the knowledge that HPV infection is the obligatecause of cervical cancer, targeted immunological approachesfor therapy such as therapeutic vaccines have been unsuccess-ful.3 Chung and Lambert4 present a novel approach of usingestrogen receptor antagonists (selective estrogen receptormodulators or SERMs) to treat cervical precancer and cancer.As noted by the authors, there are strongly supportive labora-tory5 and epidemiological data6,7 that estrogen plays contrib-uting role in cervical carcinogenesis. In this study, Chungand Lambert4 successfully treated 7 E6/E7 transgenic micethat spontaneously manifested cervical cancer with Raloxi-fene, an estrogen receptor antagonist that reduces the risk ofbreast cancer.

To investigate whether Raloxifene might reduce the inci-dence of cervical cancer and its immediate precursor, carci-noma in situ (CIS), previously unpublished data on theseoutcomes from two randomized clinical trials to evaluate twoSERMs, tamoxifen and raloxifene, for breast cancer riskreduction were analyzed. The NSABP (National Surgical Ad-

juvant Breast and Bowel Project), an NCI funded CooperativeGroup, has designed and conducted the two large breast can-cer prevention trials, Breast Cancer Prevention Trial P-1(BCPT-P1)8 and Study of Tamoxifen vs. Raloxifene (STAR).9

While both tamoxifen and raloxifene are estrogen receptorantagonists in breast tissue, raloxifene is an estrogen receptorantagonist whereas tamoxifen is an agonist in the humanreproductive tract (e.g., Tamoxifen increases the risk of endo-metrial cancer while raloxifene does not.).4,10,11 However, asshown in the Table 1, despite small numbers, there was noevidence that long-term raloxifene use reduces, or long-termtamoxifen use increases, the risk of cervical CIS and cancer(n.b., results were combined because SERMS were reportedto treat both in the mouse models, and CIS has a high riskof invading if left untreated12) (BCPT P-1: Placebo vs. Ta-moxifen, p ¼ 1.0, two-sided Fisher’s exact; STAR: Tamoxifenvs. Raloxifene, p ¼ 1.0, two-sided Fisher’s exact).

This post-hoc analysis was limited by small numbers. Datafrom other clinical trials of SERMs13,14 should be pooled toexamine the impact of SERMs on outcomes of cervical pre-cancer and cancer. Until evidence is forthcoming on the posi-tive effects of SERMs on cervical precancer and cancer inhumans, extreme caution in interpreting data from mousemodels is warranted. Despite the probable role of estrogensin cervical carcinogenesis, the protective effects of SERMsagainst cervical cancer cannot be inferred based on mousemodels. Importantly, the therapeutic effectiveness of SERMswould need to be exceeding high to be acceptable, given theefficacy of current methods of treatment for cervical intraepi-thelial neoplasia15 and early cervical cancer.16

AcknowledgementsDr. Castle acknowledges Dr. Joseph Costantino (University of Pittsburgh,Pittsburgh, PA, USA) for providing data from Breast Cancer Prevention

Table 1. Incidence of cervical cancer and carcinoma in situ in tworandomized trials, breast cancer prevention Trial P-1 (BCPT-P1)8 andStudy of Tamoxifen vs. Raloxifene (STAR)9

BCPTP-1 STAR

Placebo Tamoxifen Tamoxifen Raloxifene

Cervical cancer 1 3 3 0

Carcinomain situ (cervix)

3 2 4 8

Combined 4 5 7 8

N (subjects) 6707 6681 9872 9875

Person years 40,588 40,645 26,851 27,743

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International Journal of Cancer

IJC

Trial P-1 (BCPT-P1) and the Study of Tamoxifen vs. Raloxifene (STAR). Dr.Castle was supported by the Intramural Research Program of the NIH,National Cancer Institute and he reports no conflicts of interest.

Yours sincerely,Philip E. Castle

References1. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S.

Human papillomavirus and cervical cancer. Lancet 2007;370:890–907.

2. Arbyn M, Kyrgiou M, Simoens C, Raifu AO, Koliopoulos G, Martin-Hirsch P, Prendiville W, Paraskevaidis E. Perinatal mortality andother severe adverse pregnancy outcomes associated with treatmentof cervical intraepithelial neoplasia: meta-analysis. BMJ 2008;337:a1284. doi: 10.1136/bmj.a1284.:a1284.

3. Trimble CL, Frazer IH. Development of therapeutic HPV vaccines.Lancet Oncol 2009;10:975–80.

4. Chung SH, Lambert PF. Prevention and treatment of cervical cancerin mice using estrogen receptor antagonists. Proc Natl Acad Sci USA2009;106:19467–72.

5. Brake T, Lambert PF. Estrogen contributes to the onset, persistence,and malignant progression of cervical cancer in a humanpapillomavirus-transgenic mouse model. Proc Natl Acad Sci USA2005;102:2490–5.

6. Appleby P, Beral V, Berrington de GA, Colin D, Franceschi S,Goodhill A, Green J, Peto J, Plummer M, Sweetland S. Cervicalcancer and hormonal contraceptives: collaborative reanalysis ofindividual data for 16,573 women with cervical cancer and 35,509women without cervical cancer from 24 epidemiological studies.Lancet 2007;370:1609–21.

7. Reimers LL, Anderson WF, Rosenberg PS, Henson DE, Castle PE.Etiologic heterogeneity for cervical carcinoma by histopathologictype, using comparative age-period-cohort models. Cancer EpidemiolBiomarkers Prev 2009;18:792–800.

8. Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M,Cronin WM, Vogel V, Robidoux A, Dimitrov N, Atkins J, Daly M,Wieand S, et al. Tamoxifen for prevention of breast cancer: report ofthe National Surgical Adjuvant Breast and Bowel Project P-1 Study.J Natl Cancer Inst 1998;90:1371–88.

9. Vogel VG, Costantino JP, Wickerham DL, Cronin WM, CecchiniRS, Atkins JN, Bevers TB, Fehrenbacher L, Pajon ER, Jr, Wade JL,

III, Robidoux A, Margolese RG, et al. Effects of tamoxifen vsraloxifene on the risk of developing invasive breast cancer and otherdisease outcomes: the NSABP Study of Tamoxifen and Raloxifene(STAR) P-2 trial. JAMA 2006;295:2727–41.

10. Shang Y. Molecular mechanisms of oestrogen and SERMs inendometrial carcinogenesis. Nat Rev Cancer 2006;6:360–8.

11. Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M,Cronin WM, Vogel V, Robidoux A, Dimitrov N, Atkins J, Daly M,Wieand S, et al. Tamoxifen for prevention of breast cancer: report ofthe National Surgical Adjuvant Breast and Bowel Project P-1 Study.J Natl Cancer Inst 1998;90:1371–88.

12. McCredie MR, Sharples KJ, Paul C, Baranyai J, Medley G, JonesRW, Skegg DC. Natural history of cervical neoplasia and risk ofinvasive cancer in women with cervical intraepithelial neoplasia 3: aretrospective cohort study. Lancet Oncol 2008;9:425–34.

13. Clarke M, Coates AS, Darby SC, Davies C, Gelber RD, Godwin J,Goldhirsch A, Gray R, Peto R, Pritchard KI, Wood WC.Adjuvant chemotherapy in oestrogen-receptor-poor breast cancer:patient-level meta-analysis of randomised trials. Lancet 2008;371:29–40.

14. Cummings SR, Tice JA, Bauer S, Browner WS, Cuzick J, Ziv E,Vogel V, Shepherd J, Vachon C, Smith-Bindman R, Kerlikowske K.Prevention of breast cancer in postmenopausal women: approachesto estimating and reducing risk. J Natl Cancer Inst 2009;101:384–98.

15. Martin-Hirsch PPL, Paraskevaidis E, Kitchener HC. Surgery forcervical intraepithelial neoplasia. Cochrane Database of SystematicReviews 1999, Issue 3. Art. No.: CD001318. DOI:10.1002/14651858.CD001318.

16. Willmott LJ, Monk BJ. Cervical cancer therapy: current, future andanti-angiogensis targeted treatment. Expert Rev Anticancer Ther2009;9:895–903.

DOI: 10.1002/ijc.25393

History: Received 25 Jan 2010; Accepted 8 Apr 2010; Online15 Apr 2010Correspondence to: Philip E. Castle, Division of CancerEpidemiology and Genetics, National Cancer Institute, 6120Executive Blvd. Room 5026, MSC 7234, Bethesda, Maryland 20892-7234, USA, Tel.: 301-435-3976, Fax: 301-402-0916,E-mail: castlep@mail.nih.gov

Divergent expression of CD133 in different studieson HCT-116 cell line

Marica Gemei1,2, Peppino Mirabelli1,3, Rosa Di Noto1,3, Giuliana Fortunato1,3 and Luigi Del Vecchio1,2,3

1 CEINGE-Biotecnologie Avanzate, Napoli, Italy2 European School of Molecular Medicine, Napoli, Italy3 Dipartimento di Biochimica e Biotecnologie Mediche, Universita Federico II, Napoli, Italy

Dear Sir,

This letter aims at emphasizing the importance of using cer-tified continuous cell lines in cancer research. The editors’growing demand to submit articles analyzing already certifiedcell lines is a praiseworthy request as it may have a very posi-tive impact on the achievement of sound results.

Using human continuous cell lines is a key element incancer stem cell (CSC) studies. Considering the scarcity offresh samples and the consequent need to have access to awider sampling of available material for biochemical andin vivo assays, the use of cell line models able to recapitulate

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as faithfully as possible the properties of the tumor fromwhich they have been derived plays a pivotal role in scientificresearch. Furthermore, the use of CD133 antibodies and flowcytometry shows that the analysis of cell markers may be avery useful technique for splitting the putative stem cell frac-tion in cancer cell line specimens.1

Several articles have recently reported divergent dataabout CD133 expression on human colon cancer-derivedHCT-116 cell line.1–5 In fact, it has been observed that HCT-116 cells are described as either lacking2,3 or fully1,4,5 express-ing CD133 glycoprotein.

These results have spurred us to purchase two different cer-tified batches of HCT-116 cell line from American Type Cul-ture Collection (ATCC; Manassas, VA) and Deutsche Samm-lung von Mikroorganismen und Zellkulturen GmbH (DSMZ;Braunschweig, Germany) cell banks. As for cell line practice,we have complied with the criteria of the United KingdomCoordinating Committee on Cancer Research,6 moreover weused the well-characterized CD133 monoclonal antibodyAC133-1 from Miltenyi, which has been used to correctly iden-tify CSC in fresh colon carcinoma samples.7,8 Of note, in oneresearch study, it has been demonstrated that AC133 epitope ispresent on CSC but lost during differentiation.7

Both ATCC- and DSMZ-derived HCT-116 cells includedin our study turned out to be more than 90% CD133 posi-tive, as shown in Figure 1.

Some authors are aware that their findings are differentfrom data reported elsewhere.2 They usually claim thatinconsistent findings might be due to the presence, in vari-ous laboratories, of different HCT-116 clones showing di-vergent features probably acquired over long periods of cellculture.2

If on the one hand we do agree on the above-mentionedexplanation, on the other we believe it is worthwhile recallingthat cross-contamination between different cell lines can beconsidered as another cause of divergent data. Dirks et al.9

have recently evidenced the growing perception in scientificcommunity that cross-contamination of mammalian cell linesrepresents a major risk of generating discordant scientificdata. Freshney10 has also evidenced that 15–20% of cell linescurrently in use may be cross contaminated, which has led tothe production of articles, discussions at scientific meetingsas well as recommended procedures for anyone employingcell lines to assess in vitro aspects related to biologicalresearch.

For the above reasons, we are firmly convinced that theauthentication of cell lines upon submission of articlesrequired by Scientific Journals has to be fulfilled becausecross-contamination of cell lines is a widespread problemthat is likely to invalidate many experimental observationsand consequent clinical results in basic and oncologicresearch.

Figure 1. (a) Unlabelled negative control and labeled HCT-116 from ATCC. CD133 positive cells (on the right of the marker) were 94%. (b)

HCT-116 from DSMZ. CD133 positive cells were 90%.

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Yours sincerely,Marica Gemei

Peppino MirabelliRosa Di Noto

Giuliana FortunatoLuigi Del Vecchio

References1. Botchkina IL, Rowehl RA, Rivadeneira DE, Karpeh MS Jr, Crawford

H, Dufour A, Ju J, Wang Y, Leyfman Y, Botchkina GI. Phenotypicsubpopulations of metastatic colon cancer stem cells: genomicanalysis. Cancer Genomics Proteomics 2009;6:19–29.

2. Kai K, Nagano O, Sugihara E, Arima Y, Sampetrean O, Ishimoto T,Nakanishi M, Ueno NT, Iwase H, Saya H. Maintenance of HCT116colon cancer cell line conforms to a stochastic model but not acancer stem cell model. Cancer Sci 2009;100:2275–82.

3. Yi JM, Tsai HC, Glockner SC, Lin S, Ohm JE, Easwaran H, JamesCD, Costello JF, Riggins G, Eberhart CG, Laterra J, Vescovi AL,et al. Abnormal DNA methylation of CD133 in colorectal andglioblastoma tumors. Cancer Res 2008;68:8094–103.

4. Dittfeld C, Dietrich A, Peickert S, Hering S, Baumann M, Grade M,Ried T, Kunz-Schughart LA. CD133 expression is not selective fortumor-initiating or radioresistant cell populations in the CRC celllines HCT-116. Radiother Oncol 2009;92:353–61.

5. Song B, Wang Y, Xi Y, Kudo K, Bruheim S, Botchkina GI, Gavin E,Wan Y, Formentini A, Kornmann M, Fodstad O, Ju J. Mechanism

of chemoresistance mediated by miR-140 in human osteosarcomaand colon cancer cells. Oncogene 2009;28:4065–74.

6. United Kingdom Coordinating Committee on Cancer Researchguidelines for the use of cell lines in cancer research. Br J Cancer2000;82:1495–509.

7. Kemper K, Sprick MR, de Bree M, Scopelliti A, Vermeulen L, HoekM, Zeilstra J, Pals ST, Mehmet H, Stassi G, Medema JP. The AC133epitope, but not the CD133 protein, is lost upon cancer stem celldifferentiation. Cancer Res 2010;70:719–29.

8. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M,Peschle C, De Maria R. Identification and expansion of humancolon-cancer-initiating cells. Nature 2007;445:111–5.

9. Dirks WG, MacLeod RA, Nakamura Y, Kohara A, Reid Y, Milch H,Drexler HG, Mizusawa H. Cell line cross-contamination initiative: aninteractive reference database of STR profiles covering commoncancer cell lines. Int J Cancer 2010;126:303–4.

10. Freshney RI. Database of misidentified cell lines. Int J Cancer2010;1:302.

DOI: 10.1002/ijc.25406

History: Received 7 Apr 2010; Accepted 12 Apr 2010; Online 19Apr 2010

Correspondence to: Luigi Del Vecchio, CEINGE-BiotecnologieAvanzate, Via Comunale Margherita 482, 80145 Napoli, Italy,E-mail: delvecchio@dbbm.unina.it

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Int. J. Cancer: 128, 997–1000 (2011) VC 2010 UICC