Prospective Multicenter Randomized Intermediate...

Research ArticleSee related commentary by Stoffel and Walsh, p. 755

Prospective Multicenter Randomized IntermediateBiomarker Study of Oral Contraceptive versus Depo-Proverafor Prevention of Endometrial Cancer in Women with LynchSyndrome

Karen H. Lu1, David S. Loose5, Melinda S. Yates1, Graciela M. Nogueras-Gonzalez2, Mark F. Munsell2,Lee-may Chen6, Henry Lynch8, Terri Cornelison9, Stephanie Boyd-Rogers1, Mary Rubin6, Molly S. Daniels1,3,Peggy Conrad7, Andrea Milbourne1, David M. Gershenson1, and Russell R. Broaddus4

AbstractWomen with Lynch syndrome have a 40% to 60% lifetime risk for developing endometrial cancer, a

cancer associated with estrogen imbalance. The molecular basis for endometrial-specific tumorigenesis is

unclear. Progestins inhibit estrogen-driven proliferation, and epidemiologic studies have shown that

progestin-containing oral contraceptives (OCP) reduce the risk of endometrial cancer by 50% in women

at general population risk. It is unknown whether they are effective in women with Lynch syndrome.

Asymptomatic women ages 25 to 50 with Lynch syndrome were randomized to receive the progestin

compounds Depo-Provera (depo-MPA) or OCP for three months. An endometrial biopsy and transvaginal

ultrasound were conducted before and after treatment. Endometrial proliferation was evaluated as the

primary endpoint. Histology and a panel of surrogate endpoint biomarkers were evaluated for each

endometrial biopsy as secondary endpoints. A total of 51 women were enrolled, and 46 completed

treatment. Two of the 51 women had complex hyperplasia with atypia at the baseline endometrial

biopsy and were excluded from the study. Overall, both depo-MPA and OCP induced a dramatic

decrease in endometrial epithelial proliferation and microscopic changes in the endometrium charac-

teristic of progestin action. Transvaginal ultrasound measurement of endometrial stripe was not a useful

measure of endometrial response or baseline hyperplasia. These results show that women with Lynch

syndrome do show an endometrial response to short-term exogenous progestins, suggesting that OCP

and depo-MPA may be reasonable chemopreventive agents in this high-risk patient population. Cancer

Prev Res; 6(8); 774–81. �2013 AACR.

IntroductionLynch syndrome, or hereditary nonpolyposis colorectal

cancer syndrome (HNPCC), is an autosomal-dominantinherited condition characterized by early-onset colon can-

cer and endometrial cancer. In women with Lynchsyndrome, risk of endometrial cancer equals or exceedsrisk of colon cancer. Women with Lynch syndrome genemutations have a 40% to 60% lifetime risk of developingendometrial cancer, compared with 3% for the generalpopulation (1, 2). Endometrial cancer tends to occur at anearlier age in women with Lynch syndrome (mean age ofdiagnosis–48 years; refs. 3, 4). In the general risk patientpopulation, endometrial cancer, especially endometrioid-type, is known to be an estrogen-driven malignancy. Pro-gesterone is well-known to antagonize the effects of estro-gen. Accordingly, the Cancer and Steroid Hormone Study(CASH) has shown that use of progestin-containing oralcontraceptive pills (OCP) reduces the risk of endometrialcancer by 50% inwomen at general population risk (5). It isunclear whetherOCP are effective in high-risk women, suchas women with Lynch syndrome.

The goal of this short-term 3-month phase II biomarkerendpoint study was to examine the effects of progestin-containing OCP or depo-medroxyprogesterone acetate(depo-MPA) on the endometrium of women with Lynch

Authors' Affiliations: Departments of 1Gynecologic Oncology and Repro-ductive Medicine, 2Biostatistics, 3Clinical Cancer Genetics Program, and4Pathology, TheUniversity of TexasMDAndersonCancerCenter; 5Depart-ment of Integrative Biology and Pharmacology, The University of TexasHealth Science Center, Houston, Texas; Departments of 6GynecologicOncology and 7Medicine, The University of California San Francisco, SanFrancisco, California; 8Department of Preventative Medicine, CreightonUniversity, Omaha, Nebraska; and 9Department of Gynecologic Oncology,Johns Hopkins University, Baltimore, Maryland

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Karen H. Lu, Department of Gynecologic Oncol-ogy and Reproductive Medicine, The University of Texas MD AndersonCancer Center, 1155 Hermann Pressler Dr., Unit 1362, Houston, TX 77030.Phone: 713-745-8902; Fax: 713-792-7586; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-13-0020

�2013 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 6(8) August 2013774

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syndrome. Given the substantial risk of endometrial cancerin these women, it is essential to determine whether pro-gestins are able to induce the characteristic microscopicchanges in the endometrium of Lynch syndrome women,similar to those observed in average-risk women. As a steptoward determining the chemopreventive potential of OCPand depo-MPA in this population, we examined the short-term effect of depo-MPA and OCP on the endometrium ofwomen with Lynch syndrome using markers of progestinresponsiveness, including pre- and posttreatment endome-trial histology, endometrial epithelial proliferation index,and a panel of endometrial gene expression biomarkers ofestrogen action. Gene expression biomarkers were selectedon the basis of previous microarray studies and targetedgene expression analysis identifying estrogen-responsivegenes in the endometrium, including several markers thatare differentially expressed in estrogen-related endometrialcancer (6–11).

Materials and MethodsPatientsThe Institutional Review Boards at MD Anderson Cancer

Center (Houston, TX), University of California, San Fran-cisco (San Francisco, CA), and Creighton University (Oma-ha, NE) approved the study (trial registration ID:NCT00033358). Women between the ages of 25 and 50with a known mutation in MLH1, MSH2, or MSH6 wereeligible. In addition,womenwhohadapersonal history of aLynch syndrome-associated cancer and who fulfilledAmsterdam criteria based on family history were eligible.Additional inclusion criteria included no prior hysterecto-my, no history of prior pelvic radiation, no chemotherapyfor 2 years, and no use of OCP or hormones for 4 monthsbefore initiation of study. Women also had to have nomedical contraindication to use of OCP or depo-MPA,including known or suspected pregnancy, undiagnosedvaginal bleeding, active thrombophlebitis or past historyof thromboembolic disorders or cerebral vascular disease,gallbladder disease, history of diabetes, coronary arterydisease, or a current tobacco smoker age of 35 years ormore.On day 5 to 10 of the menstrual cycle, corresponding to

the endometrial proliferative phase, a transvaginal ultra-sound (TVS) and then an endometrial biopsy using a 3 and4mm pipelle were conducted for each participant. Half thebiopsy was placed into formalin for routine histologicprocessing, pathologic assessment, and Ki67 immunohis-tochemistry. The remaining half was frozen under liquidnitrogen and used for subsequent quantitative real-timePCR (qRT-PCR) assays. The TVS measured the endometrialstripe and the size, morphology, and any abnormalities ofthe uterus and ovaries. Women were randomized to receiveOCP (LoOvral, 0.3 mg ethinyl estradiol and 0.3 mg nor-gestrel daily for 21 days followed by 7 days of placebo) or asingle dose of 150 mg depo-MPA. For women randomizedto the OCP arm, the second TVS and endometrial biopsywere conducted on day 4 to 10 after initiation of the fourthpack of study medication. For women randomized to thedepo-MPA arm, the second TVS and endometrial biopsy

were conducted on day 90 � 5 days. Women were consid-ered inevaluable if they missed more than 4 consecutivenonplacebo pills and/or missed a total of more than 10nonplacebo pills during a 4-month period.

AssaysAll endometrial biopsies were microscopically exam-

ined. The baseline biopsies were examined to confirmproliferative phase histology and to rule out the presenceof endometrial hyperplasia or carcinoma. Posttreatmentbiopsies were evaluated for response to progestin treat-ment. The response was considered good if the glandswere inactive or secretory and there was lack of epithelialcell mitotic figures, and if there was evidence of stromalpredecidualization, characterized by stromal cells withincreased eosinophilic cytoplasm and acquisition of epi-thelioid shape. A poor response was characterized bythe absence of stromal cell pseudo-decidualization, theabsence of inactive endometrial glands, or the presenceof proliferative-type endometrial epithelial glands withmitotic figures. The presence of endometrial hyperplasiaor carcinoma in the posttreatment biopsy was character-ized as a pathologic response. Ki67 immunohistochemis-try (MIB-1, Dako) was conducted as per manufacturer’sinstructions; its expression was determined by microscop-ically quantifying the percentage of endometrial epithelialcells with positively stained nuclei.

Estrogen is well-known to be a stimulator of endometrialproliferation and uterine growth. We have previously char-acterized a panel of genes that are expressed in the endo-metrium andmodulated by estrogen (Supplementary TableS1; refs. 6–14). Importantly, these genes are differentiallyexpressed in endometrial carcinoma with several showingsignificantly higher expression in endometrioid-type carci-nomas (estrogen-related) compared with nonendome-trioid carcinomas (not related to estrogen excess). Thispanel includes IGF-1, IGF-1R, IGF-2, EIG121 (a.k.a.KIAA1324), RALDH2 (a.k.a. ALDH1A2), sFRP1, sFRP4, andsurvivin (a.k.a. BIRC5). IGFBP1 was also analyzed as apositive control for tissue action of progesterone. All quan-titative real-time PCR assays for genes in this panel wereconducted from the frozen endometrial biopsy using anABI7700 instrument (Life Technologies). All assays werevalidated using standards comprising a synthetic oligonu-cleotide that corresponds exactly to the amplicon that wasmeasured in sample RNA to ensure linearity of signal and todetermine the lower limit of detection of each assay. Assaysmust have been able to detect a minimum of 1,000 mole-cules of target and were linear (r2 > 0.98) over a concen-tration range of at least 5 logs. Each sample was assayed inquadruplicate including a negative control that was minusthe reverse transcriptase. This negative control served tomeasure any contaminating DNA in the RNA samples. Alltranscripts were normalized using the transcripts 18SmRNA, b-actin (ACTB), and 36B4 (a.k.a. RPLP0). Assaysfor these 3 transcripts were conducted on each sample and"gNORM" was calculated as the geometric mean of thenormalizer transcripts.

Endometrial Cancer Prevention Using Progestins in HNPCC

www.aacrjournals.org Cancer Prev Res; 6(8) August 2013 775




Statistical analysisThe primary outcome was the change in Ki-67 expression

before and after treatment. We analyzed these changeswithin, as well as between, treatment groups. Secondaryendpoints included the change from pre- to posttreatmentin histology and endometrial thickness (as measured byTVS), as well as the estimation of the frequency of endo-metrial abnormalities in this patient population on presen-tation. Another secondary endpoint included the examina-tion of the change in expression of estrogen-induced genesmeasured by qRT-PCR.

Measurements of each potential surrogate endpoint bio-marker were taken before and after hormone treatment.Although the actual data values observed were used in theanalysis of the trial, for simplicity in the computation ofsample size we assumed only that the value for a givenbiomarker increased or decreased. If the treatment had noeffect, then the probability of a marker increasing (ordecreasing) was 0.50. This study was designed to have80% power to detect a change in this probability to 0.82within each treatment arm. Using an exact test (2-sided)with a significance level of 0.05, we needed 22 patients in atreatment arm to detect a change in the proportion ofpatients in that armwithmarkers increasing (or decreasing)from 0.50 to 0.82 with 80% power. To allow for an approx-imate 15% dropout rate, we enrolled 51 patients total onthis multicenter trial.

This was an exploratory study, and any biomarkersfound to be elevated or depressed was of interest. How-ever, as there were 9 potential biomarkers of specialinterest (Ki67 immunohistochemistry and qRT-PCR of8 estrogen-modulated genes), to achieve an overall type Ierror rate of 5% we tested each biomarker at the nominallevel of 0.05/9 ¼ 0.0056. The proportion of patients witha marker increasing (or decreasing) had to have been 0.86for us to be able to detect a statistically significant changein the proportion of patients with markers increasing (ordecreasing) from 0.50 and maintain an overall signifi-cance level of 0.05.

Paired plots showing pre- and posttreatment values (socalled "box plots") were examined for each biomarker.Paired t tests (or signed rank test) were used to comparethe biomarkers. In addition, to determine whether patternsof changes in the biomarkers were associated with treat-ment, we conducted a multivariate analysis of the data. Weexamined the correlation in the changes following treat-ment among the biomarkers using either Pearson or Spear-man correlations as appropriate.

ResultsA total of 51 women were enrolled, and their demo-

graphics are summarized in Table 1. There were nosignificant differences between the treatment arms forage, race, mutation status, BMI, parity, or gravity. Twen-ty-four women had an MLH1 mutation, 22 women hadan MSH2 mutation, and 2 women had an MSH6 muta-tion. In addition, 2 women fulfilled Amsterdam criteriaand had a history of colon cancer. One patient was

Amsterdam positive, had a history of a benign ovariantumor, and had previously undergone a unilateral sal-pingo-oophorectomy.

As shown in Fig. 1, both depo-MPA and OCP caused adramatic decrease in endometrial epithelial proliferation asmeasured by Ki-67–positive cells [depo-MPA mean pre-51.8%, mean post- 13.1% (P < 0.001) and OCP mean pre-48.3%, mean post- 3.1% (P < 0.001)]. When histology wasexamined (Table 2), 20 of 23 patients in the depo-MPAgroup and 22 of 23 patients in the OCP group showedinactive and/or secretory-type glands (Fig. 2A). Interesting-ly, the 3 patients who had a poor histologic response totreatmentwere all in the depo-MPA group, the arm inwhich

Table 1. Demographic and clinicalcharacteristics

Depo-MPA(N ¼ 25)

OCP(N ¼ 26) P

Age, yMean 36.8 38.0 0.495Range 25–48 25–48

Age at menarche, yMean 12.7 12.7 0.841Range 10–16 11–16

RaceWhite 20 23 0.244Black 0 1Hispanic 4 1Asian 1 0Native American 0 1

MutationMLH1 11 13 0.841MSH2 12 10MSH6 1 1Amsterdam þ coloncancer

1 1

Amsterdam þ benignovarian tumor

0 1

BMIMean 28.4 26.2 0.267Range 19.3–48.4 18.2–44.7

Gravity0 4 7 0.3311 2 02 7 43 6 104 4 25 1 36 1 0

Parity0 5 9 0.2091 6 12 9 83 4 74 1 1

Lu et al.

Cancer Prev Res; 6(8) August 2013 Cancer Prevention Research776




treatment compliance was not an issue (Fig. 2B). An addi-tional patient, who had a normal endometrial biopsy atbaseline and was randomized to receive OCP, was found tohave a focus of complex hyperplasia without atypia in abackground of inactive glands on her 3-month biopsy(pathologic response; Fig. 2C). Two of 51 patients hadbaseline endometrial abnormalities [3.9%, 95%confidenceinterval (CI) 0.5–13.5]. Both abnormalities were complex

atypical hyperplasia (Supplementary Fig. S1), and bothwere found to have grade 1 endometrioid endometrialcarcinoma in the subsequent hysterectomy.

Transvaginal ultrasound measurement of baselineendometrial thickness revealed a mean of 5.5 mm (range2.6–10.1) for the depo-MPA arm and a mean of 6.5 mm(range 2.0–19.0) (P ¼ NS) for the OCP arm. Despite thechanges in endometrial histology posttreatment, themean follow-up endometrial thickness was not signifi-cantly decreased, with the mean in the depo-MPA arm of4.5 mm (range 1.0–9.3) and 4.5 mm (range 2.0–10.0) inthe OCP arm (Table 2).

In addition to Ki-67, transcripts for 8 different estrogen-modulated genes were quantified (Fig. 3). We also ana-lyzed the endometrial expression of IGFBP1, which iswell-known to be induced by progesterone (15, 16), toverify tissue action of OCP and depo-MPA. IGFBP1 wassignificantly induced in the posttreatment endometrialbiopsies for both treatment arms (Fig. 3). For bothtreatment groups, the posttreatment endometrial biopsieshad significantly altered expression of IGF-1, IGF-2,sFRP1, sFRP4, and survivin transcripts. For the depo-MPAgroup, EIG121 was also decreased in the posttreatmentbiopsy. These results are consistent with the fact thatprogesterone typically antagonizes the biologic effects ofestrogen. For 2 of the 3 poor histologic responders (all 3in the depo-MPA group), endometrial tissue was availablefor molecular analyses. In contrast to patients with a goodhistologic response, the 2 poor responders showed ele-vated sFRP1, sFRP4, and survivin in the posttreatmentendometrial biopsies (Fig. 4).

DiscussionWhile women with Lynch syndrome have a substantial

lifetime risk for the development of endometrial cancer,studies regarding the efficacy of preventive strategies are few.

Figure1. Proliferation index (Ki-67) is significantly reducedfollowing short-term treatment with Depo-Provera (depo-MPA) or oral contraceptive (OCP). þ, P < 0.05 forcomparison of pre- and posttreatment.

Table 2. Histology and endometrial thickness

Depo-MPA(N ¼ 25)

OCP(N ¼ 26) P

Baseline biopsyProliferative 23 24 0.999Not Proliferative 1 1Pathologic 1 1

Follow-Up BiopsyGood 20 22 0.233a

Poor 3 0Pathologic 0 1No Biopsy 2 3

Baseline endometrial thickness (mm)Mean 5.5 6.5 0.189Range 2.6–10.1 2.0–19.0

Follow-up endometrial thickness (mm)Mean 4.5 4.5 0.933b

Range 1.0–9.3 2.0–10.0Change in Endometrial Thickness (mm)Mean 0.9 1.7 0.225b

Range �5.0–6.0 �1.0–5.0

aExcludes "No biopsy" group.bTwo patients on depo-MPA and 3 patients on OCP did nothave follow-up endometrial thickness.






We previously reported that surgical prevention with hys-terectomy and bilateral salpingo-oophorectomy is highlyeffective for the prevention of endometrial and ovariancancer in this high-risk population (17). Whether OCP or

depo-MPA are effective chemopreventive agents for womenwith Lynch syndrome, as they are for women in the generalpopulation, is unknown. As a step toward determining thechemopreventive potential of OCP and depo-MPA in thispopulation, we examined the short-term effect of depo-MPA and OCP on the endometrium of women with Lynchsyndrome using several endometrial tissue markers of pro-gestin-responsiveness. With each woman as her own con-trol, after 3 months of treatment we observed a significantdecrease in endometrial epithelial proliferation (Ki-67) inboth the depo-MPA andOCP arms.Histologically, 20 of the23women in the depo-MPA armand22of 23women in theOCP arm showed the presence of inactive and/or secretory-type glands. While the endpoint of this study was notefficacy, the significant response both with Ki-67 and his-tology suggests that both depo-MPA and OCP may bereasonable chemopreventive agents in this high-risk cohort.Interestingly, 3 women in the depo-MPA arm had a poorhistologic response, although all 3 showed a significantdecrease in Ki-67. Given the small numbers of nonrespon-ders overall, it would be difficult to conclude that OCPswere more effective than depo-MPA. In addition, longitu-dinal studies will be necessary to determine whether poorhistologic response to progestins such as depo-MPA canbe amarker for increased risk of endometrial cancer in womenwith Lynch syndrome. The 1 woman in the OCP arm whohad a focus of endometrial hyperplasia without atypia in abackground of inactive glands had a pretreatment biopsythat showed normal proliferative endometrium. It is pos-sible that this small focus of hyperplasia had been presentbefore treatment and was missed with pipelle sampling.After completion of the study, the patient chose to remainon OCP and had a dilation and curettage 4 months latershowing benign endometrium.

As a secondary endpoint, we found that the point esti-mate of endometrial abnormalities in women with Lynchsyndrome under age 50was 3.9% (95%CI, 0.5–13.5). Bothwomen were completely asymptomatic and had complexatypical hyperplasia on their baseline biopsy. Both went ontohave ahysterectomy, andbothwere found tohave grade 1endometrial cancer. Given that the median age of womenwas 36.8 in the depo-MPA arm and 37 in the OCP arm, thefinding of complex endometrial hyperplasia (CAH) andcancer in 2 asymptomatic women highlights that thesewomenare at extremelyhigh riskof developing endometrialcancer. The point estimate of endometrial hyperplasia/cancer in the general population is difficult to ascertain,but there are some data from large studies of hysterectomyspecimens derived frompatients with uterine prolapse.Onestudy of 372 women with hysterectomies for uterine pro-lapse (84% of patients older than 55 years of age) found 4cases (1.1%) of simple/complex hyperplasia with atypia orendometrial carcinoma (18). Another study of 644 similarhysterectomies from women with a mean age of 59.7 � 12years found 12 patients (1.9%)with incidental endometrialcomplex hyperplasia or carcinoma (19). In both of thesestudies, the patient age was significantly older than themedian age of 36.8–37 years for this study.

A

B

C

Figure 2. Photomicrographs of posttreatment endometrial biopsies fromwomen enrolled in this chemoprevention trial. A, endometrial biopsyshowing typical good response to progestin treatment. Endometrialglands are small, inactive appearing, and have no mitotic figures. Thestromal cells are showing predecidualized changes. H&E, �10. B,posttreatment endometrial biopsy from a nonresponder. Note thepresence of mitotic figures (arrows) in the endometrial glands. H&E,�20.C, posttreatment endometrial biopsy from a different nonresponder. Thecircled area shows a focus of complex endometrial hyperplasia, in whichthe endometrial glands are greatly enlarged and hyperplastic comparedwith adjacent smaller, inactive endometrial glands. H&E, �4.

Lu et al.





As another secondary endpoint, we examined the ultra-sound measurement of the endometrial stripe in womenbefore and after treatment. While 1 of the 2 women withbaseline CAHhad a thickened endometrial stripe (19mm),the other woman did not (7 mm). Of note, the patientwith the 19 mm stripe had had a TVS 6 weeks earlier(outside the study) in which the stripe was measured at 7mm. For the other women in the study, there was nosignificant difference with respect to the change in endo-metrial thickness. The 3 patients with poor response in thedepo-MPA treatment group had changes in endometrialthickness �3.0, 0, and 3.6 mm. The 1 patient with apathologic response in the follow-up biopsy (in the OCPtreatment group) had no change in endometrial thicknessfrom before therapy to after therapy. Therefore, measure-ment of endometrial stripe as a correlate of response toprogestin is not useful. In addition, consistent with otherpublished studies, ultrasoundmeasurement of endometrialstripe is not a sensitive screening method for detectingendometrial abnormalities.As an additional secondary endpoint, we examined a

panel of qRT-PCR biomarkers of estrogen action in theendometrium. First, we showed the feasibility of conduct-ing a number of transcript assays using endometrial tissue

obtained from a pipelle. Second, we found a statisticallysignificant decrease in the expression of EIG121, IGF-1,sFRP-1, sFRP4, and survivin in the posttreatment biopsies.Therefore, thesemolecular biomarkers canbemodulatedbyshort-term exogenous progestins (OCP and depo-MPA).Third, we found that the women who received depo-MPAwho had a poor histologic response (only 2 of 3 had tissueavailable for qRT-PCR) showed elevated posttreatmentsFRP1, sFRP4, and survivin compared with baseline biopsyvalues. The survivin protein inhibits apoptosis and isincreased in multiple tumor types, including endometrialcancers (14, 20–24). While both OCP and depo-MPAdecreased survivin in the endometrium, the 2 poor respon-ders showed an increase in survivin. Theproteins encodedbyRALDH2, sFRP1, and sFRP4 are all thought to act as "brakes"to inhibit physiologic estrogen-induced endometrial pro-liferation. In this way, normal endometrial growth inducedby estrogen is controlled. The enzyme encoded by RALDH2catalyzes the synthesis of retinoic acid, a known inhibitor ofuterine growth (7). Both sFRP1 and sFRP4 act as molecularantagonists to ligands in the Wnt signaling pathway (25),binding to Wnt7a to act as "brakes" to decrease Wnt-asso-ciated proliferation in the endometrium. In this study,sFRP1 and sFRP4 expression were downregulated by both

Figure 3. Treatment with Depo-Provera (depo-MPA) and oral contraceptives (OCP) alter gene expression of several biomarkers. �, P < 0.05 for pairedpre- and posttreatment groups. þ, P < 0.05 for a difference in gene expression changes between treatment with depo-MPA compared with OCP.






OCP and depo-MPA. Considering progesterone’s knownantagonism of estrogen action, we speculate that underconditions of endometrial quiescence inducedby long-termprogestin exposure, expression of such estrogen-regulatedgenes is turned off. The abnormally elevated posttreatmentlevels of sFRP1, sFRP4, and survivin observed in the 2nonresponders may represent biomarkers predictive of aneven greater increased risk of endometrial cancer develop-ment in these women, but this will require longitudinalstudies to verify. From Fig. 4, it can be seen that the baselineendometrial expression of these genes is quite variable, butthe vast majority of posttreatment biopsies have a narrowerrange of gene expression. This, plus our observations ofdifferent gene expression in the 2 nonresponders, intro-duces the concept of assessing tissue biomarkers followingsome type of exogenous stimulus/treatment rather than inuntreated tissues. In other words, expression of biomarkersin response to a stimulus may be more informative than abaseline measure of a tissue biomarker. We are particularlyintrigued whether molecular analysis of gene transcriptsafter a progestin challenge may be a clinically useful test toidentify which women with Lynch syndrome are at partic-ularly high risk for endometrial cancer.

This study also highlights the challenges in conductingphase II gynecologic chemoprevention trials in high-riskpopulations.We screenedover 700women to enroll 51overa 6-year period (data not shown). Primary reasons forexclusion included no identified Lynch syndrome muta-tion, not wanting to come off OCP before enrollment, notwilling to take depo-MPA, unwilling to undergo 2 endo-metrial biopsies, smoker and over the age of 35 years,planned pregnancy, prior or planned hysterectomy, highcholesterol, or not having flexibility to undergo baselineendometrial biopsy on days 5 to 10 of menstrual cycle. On

the basis of our experience from this trial, 3 factors arenecessary for the completion of phase II gynecologic che-moprevention trials—(i) steady commitment of theresearch staff at all sites for recruitment of participants, (ii)multidisciplinary and multi-institutional commitment andcooperation from geneticists, gastrointestinal, and gyneco-logic services, and (iii) sponsor (NCI) patience in keepingthe study open as long as there is steady accrual.

In conclusion, this phase II biomarker study found thatwomen with Lynch syndrome have a normal response toshort-term exogenous progestins, based on histology andproliferation indices, compared with previous reports inthe general population (26, 27). This suggests that oralcontraceptives and depo-MPA may be reasonable chemo-preventive agents for this high-risk cohort, as they are inwomen at general population risk (5). In addition, youngwomen with Lynch syndrome who are asymptomatichave a high baseline rate of complex atypical hyperplasiaand endometrial cancer. Finally, while requiring coordi-nated, multi-institutional efforts, we showed the capacityto complete a phase II chemoprevention study in womenwith Lynch syndrome.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.H. Lu, D.S. Loose, T. Cornelison, R.R.BroaddusDevelopment of methodology: D.S. Loose, L. ChenAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): K.H. Lu, D.S. Loose, L. Chen, H. Lynch, S. Boyd-Rogers, M. Rubin, M.S. Daniels, P. Conrad, A. Milbourne, D.M. Gershenson,R.R. BroaddusAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): K.H. Lu, D.S. Loose, M.S. Yates, G.M.

Figure 4. Antiproliferative geneexpression biomarkers measuredin endometrial biopsies pre- andposttreatment. sFRP4 expressionis decreased in the vast majority ofpatients receiving Depo-Provera(A) and oral contraceptive (B).However, 2 patients with poorhistologic response show anincrease in sFRP4. Survivinexpression is also decreased in thevast majority of patients followingtreatment with Depo-Provera (C) ororal contraceptives (D). However,2 patients with poor histologicresponse show an increase insurvivin.

Lu et al.





Nogueras-Gonzalez, M.F. Munsell, L. Chen, S. Boyd-Rogers, M. Rubin, R.R.BroaddusWriting, review, and/or revision of the manuscript: K.H. Lu, D.S. Loose,M.S. Yates, G.M. Nogueras-Gonzalez, M.F. Munsell, L. Chen, H. Lynch,S. Boyd-Rogers, M. Rubin, M.S. Daniels, D.M. Gershenson, R.R. BroaddusAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.S. Yates, H. Lynch, S. Boyd-RogersStudy supervision: K.H. Lu, L. Chen, T. Cornelison, S. Boyd-Rogers, R.R.Broaddus

Grant SupportThis work was supported by the National Cancer Institute (N01-CN-

05127 and P50CA098258).The costs of publication of this article were defrayed in part by the pay-

ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 16, 2013; revised March 22, 2013; accepted April 16,2013; published OnlineFirst May 2, 2013.

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2013;6:774-781. Published OnlineFirst May 2, 2013.Cancer Prev Res Karen H. Lu, David S. Loose, Melinda S. Yates, et al. Endometrial Cancer in Women with Lynch Syndromeof Oral Contraceptive versus Depo-Provera for Prevention of Prospective Multicenter Randomized Intermediate Biomarker Study

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