MODELS FOR EARLY CHEMOPREVENTION TRIALS IN BREAST CANCER

CANCER CHEMOPREVENTION

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MODELS FOR EARLY CHEMOPREVENTION TRIALS IN BREAST CANCER Carol J. Fabian, MD, Bruce F. Kimler, PhD, Richard M. Elledge, MD, William E. Grizzle, MD, PhD, Samuel W. Beenken, MD, and John H. Ward, MD

Ckemoprevention is the interruption or reversal of the neoplastic process before the clinical emergence of invasive disease.6' Because chemoprevention is generally used in healthy subjects without measurable breast cancer, the delineation of a nontoxic drug dose that will modulate precancerous changes in the breast presents methodologic and ethical challenges in performance of phase I and phase I1 chemoprevention trials. Despite these challenges, new and efficient methodologies for clinical testing must be developed that do not use cancer incidence as an endpoint. An estimated $60 million will be spent on the NSABPsponsored Phase I11 Breast Cancer Prevention Trial comparing 5 years of tamoxifen use to placebo in more than 13,000 high-risk women.87The planned follow-up trial of tamoxifen versus raloxifene in high-risk postmenopausal women may accrue 20,000 women at even greater expense.3h The National Cancer Institute (NCI) has identified more than 30 promising chemoprevention compounds from preclinical testing.57The financial and human resources are not available to initially estimate efficacy of these compounds by demonstrating a decreased cancer incidence. How do we then select the very best drugs for phase I11 comparative prevenFrom the Division of Clinical Oncology, Department of Internal Medicine (CJF), and Department of Radiation Oncology (BFK), University of Kansas Medical Center, Kansas City, Kansas; Division of Medical Oncology, University of Texas Health Science Center, San Antonio, Texas (RME); Departments of Pathology (WEG) and Surgery (SWB), University of Alabama at Birmingham, Birmingham, Alabama; and Division of Hematology/Oncology, University of Utah Medical Center, Salt Lake City, Utah (JHW)

HEMATOLOGY/ONCOLOGY CLINICS OF NORTH AMERICA

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VOLUME 12 NUMBER 5 OCTOBER 1998

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-Initiation

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Promotion

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Progression

Proliferation Differentiation DNA Repair Apoptosis

Mutation Methylation

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Normal-Hyperplasia

Dysplasia -In

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Invasion

Angiogenesis Cell Adhesion Proteases Situ

-,lnvasive

Figure 1. Step-wise alterations that characterize neoplastic progression, and the physiologic processes that may be assessed by biomarkers.

tion studies? Although potential chemopreventive activity of some drugs such as tamoxifen has been identified through adjuvant chemotherapeutic trials, there is high interest in compounds with even fewer side effects. These compounds, even if they have not been demonstrated to have activity in established cancer, might be efficacious if initiated earlier in the neoplastic process (Fig. 1).The proposed solution60has been to use biochemical, molecular, and morphologic tissue markers both to help establish a potentially effective but nontoxic dose range in phase I trials and to monitor response in placebo-controlled phase I1 trials, reserving the endpoint of cancer incidence for large phase I11 trials (Table 1). BIOMARKERS USED IN CHEMOPREVENTION TRIALS Three types of biomarkers have been identified as important to the conduct of early prevention trials: biochemical activity markers (BAMs), risk biomarkers (RBs), and surrogate endpoint biomarkers (SEBs). Biochemical activity markers are quantitative measures of drug activity and useful for mechanistic dose-response determinations. They are often highly reflective of drug class and the proposed mechanism of action but may or may not indicate reversal of the precancerous process at the Table 1. CANCER CHEMOPREVENTION TRIALS Phase

Cohort

la Ib

Normal High risk or cancer High risk High risk

I1 111 BAM

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Markers BAMS SEBs

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SEBs SEBs optional

biochemical/drug activity marker; SEB

No. Subjects

Time (yrs)

cost ($ millions)

50-1 00 50-100

0.5-1 1

0.5 1

100-200 thousands =

2-3 5-1 0

surrogate endpoint biomarker.

2-3

>50

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tissue level. Biochemical activity markers are often used for dose range finding in phase I trials and compliance monitoring in phase I1 and I11 trials. An example of a biochemical activity marker is urine polyamines, which is used to monitor activity of the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO). Risk biomarkers vary considerably in latency and strength of linkage to invasive cancer. They are used for cohort screening and, if reversible and associated with short latency, they may be considered for possible use as response indicators. Risk markers used as indicators of efficacy are termed surrogate endpoint biomarkers (SEBs). Examples of risk biomarkers suitable only for cohort screening would be BRCAl or BRCA2 genetic mutations. Morphologic changes of atypical hyperplasia, lobular carcinoma in situ (LCIS), or ductal carcinoma in situ (DCIS) are examples of risk biomarkers that might also be used as substitutes for invasive cancer development. Potential SEBs are often identified from preclinical and clinical cancer biology studies, demonstrated as feasible and relevant in phase I1 studies, and validated in phase I11 studies. To be validated, a potential SEB must show evidence of modulation in phase I1 studies, and this modulation must be linked to decreased cancer incidence in phase I11 studies. The validated SEB may then be used as a substitute or surrogate for cancer incidence in future phase I1 studies using similar cohorts and classes of drug or intervention. Ideal properties38,42, 58, 59 of an SEB are the following: biologically plausible differentially expressed in normal and high-risk tissue statistically associated with cancer or precancer in prospective studies present in a reasonable proportion of at-risk population to be studied easy to sample may be quantitated potentially reversible with successful chemoprevention modulation of SEB associated with altered cancer risk expression not seriously affected by normal physiologic processes The SEB development and validation process is summarized in Figure 2. SELECTION OF SURROGATE ENDPOINT BIOMARKERS FOR BREAST CANCER CHEMOPREVENTION TRIALS Morphologic Surrogates

Use of surrogate endpoint biomarkers to estimate response to chemoprevention agents is controversial."' At the present time, there is no

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Differential prevalence in high vs low risk groups

4 Expression correlated with increased risk in prospective studies

3.

Modulated expression in Phase I I prevention trials

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Modulation correlated with decreased cancer risk in Phase 111 prevention trials Figure 2. Process of development and validation of surrogate endpoint biomarkers.

ideal validated SEB for breast chemoprevention trials; that is, none meet all the criteria listed in the previous section and have gone through the process shown in Figure 2. Nevertheless, morphologic changes of in situ cancer and hyperplasia with or without atypia meet most critical requirements of biologic plausibility, statistical linkage to cancer, and potential reversibility. Morphologic changes of DCIS, LCIS, atypical intraductal hyperplasia, and hyperplasia without atypia are clearly associated with a heightened short-term risk of breast cancer (0.36% per year for hyperplasia to 2.5% per year for DCIS in the first 10 to 15 years after diagnosis).47, 90*92, 130, 132 DCIS has a relatively short latency period and is highly linked with later invasive cancer development. HER-2/neu and other growth factors including vascular endothelial growth factor (VEGF) are frequently overexpressed in high-grade DCIS? 16, 46, 99, 130, 139 making it an ideal model for the evaluation of HER-2/neu monoclonal antibody therapies, tyrosine kinase inhibitors, and anti-angiogenesis strategies. Likewise, estrogen receptor (ER) and progesterone receptor (PGR) are often overexpressed in low-grade DCIS, making it a good model for studying the therapeutic potential of antihormonal strategies such as antiestrogens, selective estrogen receptor modulators (SERMs), progestins, aromatase inhibitors, and so 134 DCIS is generally thought to require a treatment intervention, which limits the duration of studies that may utilize it as a response b i ~ m a r k e r .lol, ~ ~lI3, , 119 Furthermore, studies are needed to determine whether morphologic or morphometric changes associated with DCIS may be modulated with a short interval treatment, although reduction in in-situ cancer incidence was demonstrable with prolonged tamoxifen admini~tration.~~ A small focus of DCIS detected through mammographic screening is likely to be unicentric.68,114 Thus, if histologic evidence of DCIS is used as a response marker, tissue should be resampled in the same immediate area as with the original biopsy. Differentiating reactive from drug-induced changes in very short duration trials may be problematic.

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LCIS and hyperplasia with or without atypia do not require a treatment intervention and are often diffusely d i s t r i b ~ t e d .47,~ 7~1, ,77, 89, 90, 92, Iol Thus, morphologic changes associated with hyperplasia and LCIS may be more suitable response indicators in intermediate and long-term chemoprevention trials. Similar to the situation for DCIS, there is little current data indicating that changes associated with LCIS or hyperplasia are readily reversible with short to intermediate length chemopreventive drug applications, although information from phase I1 studies will soon be available. Unfortunately, morphologic markers are descriptive, not quantitative, and there is disagreement among pathologists on histologic and cytologic subcategories of benign disease.27,91, 92, Io5, 112, lZ4,lZ9 To quantify morphologic changes in histologic sections and cytology preparations, evaluation of nuclear morphometric features by image analysis is being exp10red.l~Nuclear and nucleolar area, nucleolar frequency, and texture features (chromatin pattern) have been shown to have prognostic discriminatory value in breast cancer141;however, there is overlap between histologic and cytologic subcategories of benign disease and between benign and malignant disease, particularly between large cell benign lesions and well-differentiated lobular and ductal lesions.66,141 In addition, there may be difficulties with quality control of samples or analysis resulting from staining or sectioning techniques or overriding nuclei.8, Bacus Laboratories have developed a high-resolution imaging system and software program (Bliss) that can analyze multiple features of cytologic and histologic preneoplasia. Values are expressed as a Z score of standard deviation from a specific normal setting. The software includes a common scale for different tissue types, adjustments for variance of normal tissue, and links to statistical language. Trials are underway that will correlate change in nuclear morphometry measurements with change in histopathology and cytopathology. Nuclear morphometry has been correlated with change in histology in a single-arm, highgrade cervical dysplasia chemoprevention trial,14 but it has yet to be validated in breast precancerous lesions. Several semiquantitative systems have been developed. Lagi0s,6~, 70 Silverstein et Tavassoli and Man,130and others have developed grading systems for DCIS that depend primarily on grade and the presence of necrosis. None of these systems were devised to evaluate a chemoprevention field effect. To this end, Lagios70has utilized the standard Bloom Richardson grading system to develop an index that will attempt to evaluate grade in heterogeneous tissue sections in which benign disease as well as in situ and invasive cancer will be present. Four areas of the tissue blocks of the initial and post-treatment specimens are evaluated at fixed intervals. Ten fields per biopsy specimen are evaluated, and a weighted nuclear grade based on the percentage of

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each grade in the field will be assigned to each field. A nuclear grade index score (10-30) is then constructed for each biopsy (M. D. Lagios, personal communication).Masood et a178,79 have developed a semiquantitative system for classifying precancerous and cancerous cytology specimens in which five morphologic features are assessed separately and given a point score from 1 to 5. Molecular Surrogates Molecular biomarkers indicative of perturbed physiologic processes, which may be present before morphologic changes, may be more amenable to quantitation in small tissue specimens by means of immunohistochemistry. Feasibility of molecular SEBs is being explored in phase 11 trials. Examples include elevated proliferation indices such as Ki-67 and proliferating cell nuclear antigen (PCNA)@, 117, 140; ER overexpressionM, 65; markers of oncogene overexpression such as Her-2/neu and epidermal growth factor receptor (EGFR)6,135*138; altered levelsof insulin-like growth factor receptor (IGFR), serum insulin-like growth factor (IGF-1), and the latter’s binding protein (IGFBP-3)20, 73, 95, lo3,139, 147; indicators of apoptotic imbalance including an increased bcl-2/bax ratio’, lo,13; markers of disordered cell signaling such as p53 protein accumulation or nuclear exclusion25,81.83, 116, 125,. altered levels of p16, p21, p27, cyclin D1, and cyclin EZ6, 56, 62, 86, 100, 104, 108. 120, 128, 145., alteration of differentiation signals such as cmyc and related proteins7,49, 51; loss of differentiation markers such as transforming growth factor beta (TGFB)-I1 receptor and retinoic acid receptor55, 149; cell adhesion alteration such as CD-44 isoform expression and E-cadherin 43; alteration of angiogenesis proteins such as VEGF overexpression16,41 and thrombospondin I loss12, 115,143; and methylation abnormalities.”, 43 None of the preceding molecular markers are specific for cancer or precancerous conditions; nor are they yet validated as SEBs, although alterations in all of these biomarkers have been associated with a portion of precancerous lesions.3,6, 24, 43. 52. 63. 72. 81, 93, 115, 120, 131, 135, 138, 144 Furthermore, expression of many of the molecular biomarkers may vary with physiologic conditions such as phase of menstrual cycle, menopause status, and age.20,75,88, lo2,lo6,118, Expression may also vary with fixation technique and antibody used for detection as well as antigen retrieval methods.45Finally, it is not known whether ”normalization” of expression of these markers even in precancerous lesions (eg, decrease inKi67) is necessarily associated with an improvement in morphologic features or decreased cancer incidence. Consequently, none of these alterations are validated as surrogate endpoint biomarkers, although modulation of the proliferation marker PCNA has been noted in a phase I study of DFMO in women with cervical intraepithelial neoplasia,5O and a decrease in Ki-67 expression has been associated with short-term tamoxifen administration.21 1367

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Serum biomarkers have particular appeal because they do not require repeated breast tissue sampling. IGF-1 and IGFBP-3 are close to validation as SEBs useful in trials of selective estrogen receptor modulators (SERMs) and retinoids in premenopausal women. IGF is a potent epithelial mitogen that can synergize with estrogen to stimulate growth of human breast cancer cellslZ6and prevent apoptosis even in the presence of chemotherapy agents.139An increase in IGF-1 and/or the ratio of IGF-1 to IGFBP-3 has been noted in premenopausal women at increased risk of breast cancer.17,48* 53, 54, 142 A decrease in IGF-1 was associated with a concomitant decrease in contralateral breast cancer incidence in premenopausal women treated with fenretinide.23, 37, 133 Tamoxifen has also been noted to decrease IGF-1 when given as an adjuvant to patients with early-stage breast cancer97,98; however, although modulation of serum levels in premenopausal women is associated with a decreased cancer incidence for at least some drug classes, it has not yet been demonstrated that this directly correlates with change in tissue morphology. Increased breast density is associated with increased breast cancer risk,ls, Io7 is measured via noninvasive techniques, and is theoretically reversible. It is not clear if breast density can be modulated in intermediate-length phase I1 trials. Spicer et have demonstrated decreased breast density following ovarian hormonal blockade and a low-dose replacement therapy in premenopausal women; however, a recent case control study of breast density in women receiving adjuvant tamoxifen failed to show any decrease in the treated group,127and no change in breast density was noted after several years of 4-HPR ingestion in a placebo-controlled trial.19Breast density is currently being further evaluated as a potential noninvasive SEB in randomized, placebo-controlled prevention trials.67

DESCRIPTION OF CURRENT PHASE I BREAST CANCER CHEMOPREVENTION TRIAL MODEL

There is considerable variation in the design of phase I trials that seek to define the nontoxic or minimally toxic dose of a potential chemoprevention agent associated with modulation of biochemical activity of markers specific to the drug class and/or of potential SEBs. If a candidate agent/drug has not been administered previously to humans, a traditional phase I finding study is often first performed in normal volunteers to determine a nontoxic to mildly toxic dose range. Pharmacokinetics and pharmacodynamics studies are performed, and a biochemical activity marker that is generally reflective of drug class and putative mechanism of action may be assayed as well to provide a rough measure of dose response.59Once a tolerable dose range has been

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established, some phase I trials have utilized a short-term DCIS model, in which women who have been diagnosed with DCIS or small invasive cancer by core biopsy receive one of several doses of the chemopreventive agent for a few weeks between the diagnostic biopsy and definitive surgery (Fig. 3). An attribute of this model is that women are not subjected to invasive procedures not medically indicated. Modulation of direct or indirect measures of proliferation (eg, Ki-67, PCNA, EGFR expression) as opposed to modulation of morphology may be used as evidence of activity and thus as an indication to proceed to phase I1 trialsh’ Agents that are ideal for a short interval trial in patients with incompletely resected DCIS are those that are likely to have efficacy in invasive or metastatic disease and as such are essentially chemotherapy agents. These would include orally administered hormonal therapies such as antiestrogens, selective estrogen receptor modulators (SEWS), and aromatase inhibitors, as well as parentally administered anti-angiogenesis inhibitors and antibodies targeting growth factor. Although the DCIS model allows examination of a relatively large amount of formalinfixed tissue containing a known direct precursor lesion of invasive breast cancer, tissue heterogeneity and biomarker change induced by prior biopsy can make analysis of drug-induced effects in re-excision samples difficult. A short-term DCIS model (see Fig. 3) is being used by the University of Kansas Medical Center and a consortium of contributing institutions to investigate the selective estrogen receptor modulator LY353381-HCl (SEW-3). Women undergoing a stereotactic or ultrasound guided needle biopsy are approached about possible participation at the time of biopsy or shortly after biopsy. All samples are fixed in formalin, and Parallel, Non-Randomized 1 4-,No-Treatment, - Control Arm ------

Dose

Figure 3. Ductal carcinoma in situ (DCIS) model for phase I chemoprevention trials. Eligible lesions include low to intermediate grade ductal carcinoma in situ and minimally invasive cancers.

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sections are processed for proliferation and tumor suppressor and oncogene expression markers in addition to tumor grade. Women found to have low to intermediate grade DCIS or a small non-high grade invasive tumor (likely to be ER positive) are invited to participate. All women willing to participate are randomized to one of three doses of SEW-3. Women not willing to receive drug but willing to participate are registered on a no-treatment "control" arm. Although change in histologic grade and many biomarkers are being assayed, the main endpoint in this short interval trial is change in Ki-67 expression. Although seemingly straightforward, this trial design is problematic in several areas. The majority of women will be peri- or postmenopausa1,28,80 and many will be on hormone replacement therapy (HRT) at the time their DCIS is diagnosed. Standard practice in many areas is to discontinue HRT at diagnosis. In postmenopausal women, it is possible that discontinuation of HRT may be enough to change a proliferation marker. Thus, it may be best to exclude women who discontinue HRT from trials with proliferation indices as the main efficacy endpoint in dose level determination. For premenopausal women, proliferation indices in the luteal phase of the cycle will generally be higher than in the follicular phase.96Consequently, if biopsy takes place in a different portion of the cycle than re-excision, proliferation indices may be altered, even in the absence of drug. Is it realistic to require premenopausal women with DCIS or invasive cancer to delay re-excision until they are in the same phase of their menstrual cycle as the original biopsy? If the answer is "no," premenopausal women may also need to be excluded from trials with proliferation indices as the main efficacy endpoint. A significant theoretic question is whether DCIS is the optimal model in which to identify the lowest effective chemoprevention (versus a chemotherapeutic) dose. Will a dose identified in phase I as sufficient to reduce proliferation or modulate morphology in tissue containing DCIS or invasive disease be the same dose needed in phase I1 and phase I11 trials, in which the majority of subjects do not exhibit cancer; or would a lower dose suffice? Is then the DCIS model the best for phase I or would one of the other models that use hyperplasia as the index lesion be more appropriate even though another procedure would be required to measure the effect of the intervention? These difficult questions have yet to be resolved.

DESCRIPTION OF CURRENT PHASE I1 BREAST CANCER CHEMOPREVENTION TRIAL MODELS

Phase I1 chemoprevention trials measure efficacy of the nontoxic or minimally toxic drug dose identified in phase I. The randomized phase

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I1 trial design minimizes both the number of subjects and follow-up time required by measuring differences in SEB expression instead of breast cancer development between the experimental and control (usually a placebo) groups. Biomarkers are assayed before and after the intervention. The main statistical endpoint is then the pre-post treatment biomarker change in the experimental group versus pre-post treatment change in the control group. Phase I1 chemoprevention trials generally last 3 to 12 months but may last as long as 3 to 5 years.59In addition to efficacy, phase I1 trials evaluate potential toxicity that may result from chronic drug ingestion. A double-blind, placebo-control design is necessary because biomarkers may change over time even without intervening treatment owing to physiologic variation (phase of menstrual cycle, menopause), concomitant hormone ingestion, or sampling and measurement variance. A randomized, double-blind, placebo-controlled design also helps accurately define the incidence of toxicities due to chemopreventive drug versus those due to illness or concomitant medications. A serious rate-limiting factor in the development of new chemopreventive agents for breast cancer is the lack of a validated model for phase I1 testing that may be readily utilized by a group of investigators located at different sites to permit rapid accrual. A satisfactory model should (1)provide the ability to reliably and repeatedly sample precancerous tissue that contains the SEBs of interest; (2) accomplish the sampling with a minimum of subject discomfort, which in turn will increase the acceptance rate; (3) require no out-of-pocket expense for the subject; and (4) utilize only validated S E B S . ~ ~ At the present time, four models are being used for phase I1 testing of agents: (1) a short-term DCIS model similar to that described for phase I trials in which women with incompletely resected DCIS with or without an associated small invasive tumor are randomized to receive drug or placebo in the interval between core biopsy and the definitive surgical procedure; (2) a core biopsy hyperplasia model in which women undergo a core biopsy of a palpable or mammographically defined area, and those with hyperplasia are randomized to drug or placebo and then undergo biopsy again; and two intermediate fine needle aspiration (FNA) hyperplasia models in which high-risk women without cancer but with cytologic evidence of hyperplasia detected by (3) random fourquadrant aspiration or (4) random periareolar aspiration are treated with drug or placebo and then undergo aspiration again. A potential model is the use of nipple aspirate fluid from high-risk women in which cytologic characteristics or a variety of biochemical markers might be m~nitored.’~, Io9 Each model has an associated set of attributes and limitations. Although core needle biopsy generally provides more tissue than

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does FNA, in the postmenopausal woman much of the sample of a random core biopsy may be fat. A core biopsy directed at a mammographic lesion may yield less fat, but it samples only one area of the breast and may not be representative of diffuse processes occurring in the breast tissue. Morbidity may also be greater with repeated core biopsies than with repeated FNAs. The random FNA model obtains tissue from multiple areas of both breasts and as such may be a more reliable method of sampling field defects versus focal lesions. FNA is inexpensive, minimally invasive, and well tolerated, and thus repeated sampling over a prolonged period is feasible. Random FNA theoretically allows testing drugs earlier in the process of neoplastic progression (see Fig. 1).Drawbacks to aspiration-based models include the minimal tissue acquired for study, high dependence on operator expertise for adequate material, and uncertainty over whether different precancerous subgroups may be reliably distinguished by cytologic analysis.'24,13' In a prospective study using filtration rather than a smear technique: however, and employing predefined cytologic criteria,150 Fabian et a131,32 have demonstrated that cytologic evidence of hyperplasia with atypia obtained by random periareolar FNA predicts later development/detection of breast cancer. Nipple aspiration atypical cytology has shown a prospective association with increased breast cancer Nipple aspiration is potentially the least uncomfortable for the subject, but even in experienced hands up to 40% of samples are a c e l l ~ l a r . ~ "Sauter ~~~"~'~~ et have demonstrated that nipple aspirate fluid may be obtained in over 90% of women, making this a potentially excellent model in which to develop future biochemical and molecular SEBs.

Histology-Based Models Phase I1 DCIS Model

Some of the earliest Phase I1 chemoprevention trials used the shortterm DCIS model (Fig. 4). At the University of Alabama at Birmingham, a phase I1 study was initiated in 1994 in which patients with DCIS were randomized to a 2-week course of treatment with DFMO versus placebo. Core biopsies were obtained before and after treatment. Within each specimen, areas of benign epithelium, DCIS, and invasive tumor were assessed separately for grade, proliferation markers, transforming growth factor alpha (TGFa), p53, Her-2/neu, EGFR, p53, bcl-2, and nuclear morphometry. After 30 months, only 26 subjects were accrued, even with the addition of two other collaborating institutions part way

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Figure 4. DCIS model for phase II chemopreventiontrials.

through the study. Accrual has been discontinued pending the results of interim analysis. Several key methodologic issues were identified in the AlabamaBirmingham trial. First, on-site training and follow-up of all personnel involved in a study are essential to guarantee that pre- and post-treatment specimens are handled in the same manner, and that tissue processing and fixation are equivalent at all sites5, 45 Despite explicit verbal and written instructions, off-site pathology laboratories initially tended to process and fix specimens by familiar techniques rather than those specified in the protocol. Honoring agreements to provide tissue blocks to a designated central site is also important. Utilization of only shipped unstained slides for immunohistochemistry assays may result in an underestimation of immunopositivity of some antigens.74 Second, the interval between biopsies must be long enough to avoid reactive changes in the re-excision sample. Because biomarker changes were noted in re-excision specimens from women receiving placebo as well as those receiving DFMO, the necessity of a placebo-controlled arm is obvious. Third, if the DCIS model is to be used in phase I1 trials, a consortium will be necessary to meet accrual goals. To date, all efforts to utilize the DCIS model in predominately single-institution phase I1 trials have failed to meet accrual goals (50 subjects in each arm) within the 2-year time frame desired by the NCI Chemoprevention Branch. The most important reasons are the relative scarcity of DCIS (approximately 25,000 cases diagnosed year in the United States), the narrow interval between diagnosis and definitive surgery in which treatment must be implemented, and the multiple treatment options available for women with DCIS. Only 10% of patients undergoing stereotactic biopsy because of an abnormal mammogram can be expected to harbor malignancy, and only a fraction of these are willing to enter a phase I1 trial.

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The most efficient approach in recruitment is to wait until after the diagnosis is established; however, the biopsy specimen may not be correctly processed in this scenario, and if patients are not approached until after the diagnosis, they may easily become overwhelmed with decisions regarding trial participation in addition to definitive treatment options. In an attempt to include all eligible DCIS patients in the preceding phase I1 chemoprevention trial, collaborating investigators at the University of Kansas Medical Center approached most patients undergoing stereotactic core biopsy the day of or immediately after biopsy, before the results were known. There was almost universal agreement to participate in the chemoprevention trial, but only 2 of 71 consecutive potential subjects met both histologic and protocol eligibility criteria. This underscores the need to simplify protocol eligibility requirements and use a consortium for accrual. Phase II Directed Core Biopsy Model

The directed core biopsy model is being used at the University of Texas at San Antonio for phase I1 evaluation of tamoxifen. This model (Fig. 5) applies to a wide variety of agents that might have activity in early as well as later stages of neoplastic development. Patients without cancer but with hyperplasia discovered after diagnostic biopsy of a mammographic abnormality are offered entry in this trial. They are randomized either to observation with an annual mammogram or to 1 year of tamoxifen. At the end of 1 year, all subjects undergo a repeat image-guided core needle biopsy of the previously targeted area. If proliferative changes persist and subjects were originally randomized to the observation group, they are then offered tamoxifen, and imageguided core needle biopsy is repeated again in one additional year. The study began accrual in 1994. Ten percent of all patients undergoing

m

1 A

Figure 5. Directed core biopsy hyperplasia model for phase II chemoprevention trials.

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biopsy exhibited hyperplasia without malignancy, and 30% of all eligible women (approximately 3% of all individuals undergoing biopsy) have to date agreed to participate in the study. In 4 years, 55 patients have been entered and 24 have complet-tudy. Several key methodologic issues have been identified: (1)Accrual is highly dependent on a multidisciplinary team approach including the evaluating physician, radiologist, and pathologist. (2) Pre- and post-treatment specimens must be handled, processed, and analyzed in a similar manner. The same individual should perform the first and second biopsy. Central review by a single pathologist helps maintain homogeneous and reproducible diagnostic criteria. (3) The area of prior biopsy can be located best if titanium clips are placed in the breast. (4) Patients with initial excisional biopsies should not be studied, because follow-up core biopsies often contain inadequate material. Specimen adequacy has been excellent, with greater than 95% of the specimens containing adequate material. Potential limitations include the cost of the second image-guided core needle biopsy, which is not standard of care, and the difficulty with performing a third biopsy if a crossover design is employed.

Cytology-Based Models Phase I! Four-Quadrant FNA Model

at the University of Utah reported the ability In 1990, Ward et to detect proliferative breast disease using four-quadrant FNA. Cytology preparations were filtered, not smeared. The prevalence of proliferative disease in first-degree relatives of breast cancer victims was more common than in matched controls.76,lZ1 With this information, these investigators initiated in 1991 a randomized phase I1 trial in women with FNA evidence of hyperplasia with or without atypia (Fig. 6). These women were randomized to receive either 3 years of tamoxifen or 3 years of placebo. Repeat fine needle aspirates were performed at yearly intervals. The only surrogate marker measured in this trial was the presence or absence of proliferative breast disease. Four hundred and fifty-three women with prior breast cancer or with at least one first-degree relative with breast cancer were screened by FNA, and 126 had proliferative breast disease. Sixty women who did not have proliferative breast disease at initial screening underwent aspiration again at least 1 year after the first screening. Fifty-four remained negative. Four of the six who were positive on retesting were then enrolled in the trial. A total of 103 of these women were enrolled, and the last woman will complete treatment in the fall of 1998. Thus, approximately 20% of women eligible on

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Random Approach ~

Random Peri-Areolar Approach

A

A

Figure 6. Fine-needle aspiration (FNA) hyperplasia model for phase II chemoprevention trials. Fine-needle aspiration can be performed either by a random four-quadrant or periareolar approach to sample the terminal ducts. Hyperplasia includes cases both with and without additional biomarker abnormalities.

the basis of epidemiologic criteria were enrolled in the study in 4 years. Thirty-eight women have withdrawn from the study because of side effects. Eighteen of these women were taking tamoxifen and 20 were taking placebo, emphasizing the need for a placebo arm. The side effects of FNA were minimal; there were no drop-outs owing to the FNA procedure itself. Phase II Random Periareolar FNA Model

In 1989, a prospective randomized periareolar FNA study in highrisk women (ages 30-60) was initiated at the University of Kansas Medical Center. The purpose was to determine, using breast morphologic and molecular makers, which women at increased epidemiologic risk on the basis of family history or prior cancerous or precancerous breast disease were at high short-interval risk for the development of breast cancer.29* 30, 33, 34 FNA was performed under local anesthesia with buffered lidocaine. Aspirate material from both breasts was pooled, and aliquots were studied for morphology as well as DNA ploidy (image analysis), ER, EGFR, p53, and HER-2/neu e x p r e s ~ i o nCytology .~~ preps were filtered, not smeared, and cytospins for p53 and HER-2/neu were acetone, not formalin, fixed. In the prospective FNA risk assessment study, women initially had aspiration done twice approximately 6 months apart and then were followed for cancer development. Women were also offered the option of repeated aspirations at 1- to 3-year intervals. Subject acceptance was excellent, with 80% returning for a second aspiration and some women returning for as many as 5 to 10 aspirations. Less than 1%of women had insufficient cells for cytologic analysis, but approximately 25% of women had insufficient cells for

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one or more other tests attempted. The proportion of specimens with insufficient cells was highly dependent on cytologic characterization. Later development or detection of invasive breast cancer or DCIS was most strongly predicted by cytologic evidence of hyperplasia with atypia and secondarily by multiple biomarker overexpression/abnormality. p53 and EGFR were the biomarkers most frequently overexpressed, and in a univariate analysis these markers as well as hyperplasia without atypia were predictive of later cancer de~elopment.~', 32 On the basis of these findings, a phase I1 randomized double-blind study of DFMO versus placebo was initiated in high-risk women with evidence of epithelial hyperplasia plus at least one additional marker (cytologic atypia, DNA aneuploidy, or overexpressed p53 or EGFR) in their fine needle aspirate. Selection of these biomarkers was justified, because women meeting these criteria demonstrated a significantly higher risk of breast cancer development than did women not meeting the criteria (Fig. 7). All premenopausal women initially undergo aspiration in the follicular phase of the menstrual cycle (documented by serum hormone levels) and then undergo aspiration again in the same phase after 6 months of drug. Postmenopausal women on hormone replacement must continue on their HRT regimen until study completion. In addition to morphology, cytology index (Masood), nuclear morphometry, p53, EGFR, and ploidy, change in serum IGF-I and IGFBP-3 are also being monitored along with a potential noninvasive marker, breast density. Urine polyamines are also monitored as a biochemical activity marker. The primary endpoint is change in morphology subcategory (hyperplasia with atypia, hyperplasia without atypia, nonproliferative

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Months from Initial Aspiration Figure 7. Subsequent cancer detection. Hazard function for detection and development of DCIS or invasive breast cancer as a function of time after initial fine-needle aspiration. Subjects are grouped according to whether they meet the study criteria of having epithelial hyperplasia plus one additional abnormality (cytologic atypia, DNA aneuploidy, or overexpression of p53 or EGFR) in their fine-needle aspirate.

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cytology) and cytology index. Enrollment of 100 to 120 eligible subjects is anticipated. The study opened in June 1997, and 60 subjects have been entered in 12 months. Approximately 15% of all high-risk women undergoing aspiration are both eligible and willing to enter the trial. Completion of accrual is anticipated in early 1999. A second randomized phase I1 FNA study utilizing women who are both BRCAl or BRCA2 mutation carriers and who have hyperplasia observed in breast FNA will open in mid to late 19 8. This is a cooperative effort between the University of Kansas Medic Center, Creighton University Cancer Center, and Baylor University/P Research. This study, although placebo-controlled, will allow all subjects to eventually receive the potentially active agent by utilizing a crossover design. Several methodologic issues have been identified in the FNA models: (1) It is necessary to screen a large number of high-risk women to obtain the 15% to 20% eligible and willing to enter a randomized, placebo-controlled study. (2) Funding to support cohort screening is not available from either NCI-sponsored contracts or from third-party carriers. (3) A cohesive, multidisciplinary team and tightly controlled procedures for subject selection, tissue processing, and analysis are necessary to minimize variance. (4) An appropriate accrual target must be identified that will accommodate sampling, processing, and interpretive variance. The extent of this variance is in turn largely being defined by these early phase I1 FNA trials.

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GENERAL RECOMMENDATIONS DERIVED FROM EARLY EXPERIENCE WITH EARLY PHASE I AND II CHEMOPREVENTION TRIALS

A multidisciplinary group consisting of basic scientists and a wide variety of translational scientists and clinicians, working closely together, is needed for rapid biomarker and model development. Multi-institutional consortia may be needed for rapid accrual for phase I and I1 trials. To minimize variance, technology transfer and standardization should be accomplished throughout the consortium prior to trial initiation. Funds must be made available for this to occur effectively. Even with a consortium effort, accrual will remain a significant problem unless adequate resources are made available for cohort screening, which is often outside of current medical practice and is inadequately covered as a part of phase I and phase I1 trial allocations. SUMMARY

Several models are being explored for use in the phase I and phase I1 evaluation of breast cancer chemoprevention agents. The short-term

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DCIS/small invasive cancer model is probably best used in late phase I trials in conjunction with agents likely to have activity in the progression phase of neoplastic development in addition to activity in earlier phases. The core biopsy or FNA hyperplasia models may be best used with drugs that are likely to have activity primarily in the promotion phase of neoplastic development and that are suitable for longer duration trials lasting several months to years. Morphology currently is the key surrogate endpoint biomarker for assessing efficacy in phase I1 trials. Other biomarkers that may undergo modulation will have to be validated, in that modulation will have to be shown to be directly related to decreased cancer risk in subsequent phase I11 trials. Only then can they be considered as validated surrogate endpoint biomarkers and used as stand-alone efficacy markers in phase I1 trials. Despite accrual challenges and technologic hurdles, interest in phase I and phase I1 chemoprevention trials is high. References 1. Allan DJ, Howell A, Roberts SA, et al: Reduction in apoptosis relative to mitosis in histologically normal epithelium accompanies fibrocystic change and carcinoma of the premenopausal human breast. J Pathol 1672532,1992 2. Allred DC, Clark GM, Molina R, et al: Overexpression of HER-2/neu and its relationship with other prognostic factors change during the progression of in situ to invasive breast cancer. Hum Pathol 233976979, 1992 3. Allred DC, OConnell P, Fuqua SAW Biomarkers of early breast neoplasia. J Cell Biochem 17G:125-131, 1993 4. Diagnostic cytopathology by membrane filter. Application Bulletin 100R, Gelman Sciences, Ann Arbor, 1984, pp 11-16 5. Arnold MM, Srivastava S, Fredenburgh J, et al: Effect of fixation and tissue processing on immunohistochemical demonstration on specific antigens. Biotech Histochem 71~224-230,1996 6. Athanassiadou PP, Veneti SZ, Kyrkou KA, et al: Presence of epidermal growth factor receptor in breast smears of cyst fluids: Relationship to electrolyte ratios and pH concentration. Cancer Detect Prev 16:113-118, 1992 7. Ayer DE, Laherty CD, Lawrence QA, Armstrong AP, et al: Mad proteins contain a dominant transcription repression domain. Mol Cell Biol 16:5772-5581, 1996 8. Bacus JW, Bacus JV Quality control in image cytometry: DNA ploidy. J Cell Biochem 19 (suppl G):153-164, 1994 9. Bacus JW, Bacus J V A method of correcting DNA ploidy measurements in tissue sections. Mod Pathol 7652-664, 1994 10. Bargou RC, Daniel PT, Mapara MY, et al: Expression of the bcl-2 gene family in normal and malignant breast tissue: Low bax-alpha expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer 60854-859, 1995 11. Baylin SB, Herman JG, Graff JR, et al: Alterations in DNA methylation: A fundamental aspect of neoplasia. Adv Cancer Res 72141-196, 1998 12. Bertin N, Clezardin P, Kubiak R, et a1 Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues: Correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia. Cancer Res 57396-399, 1997 13. Binder C, Marx D, Binder L, et al: Expression of bux in relation to bcl-2 and other predictive parameters in breast cancer. AM Oncol 7:129-133, 1996

MODELS FOR EARLY CHEMOPREVENTION TRIALS IN BREAST CANCER

1011

14. Boiko IV,Mitchell MF, Pandey DK, et a1 DNA image cytometric measurement as a surrogate end point biomarker in a Phase I trial of a0difluoromethylomithine for cervical intraepithelial neoplasia. Cancer Epidemiol Biomarkers Prev 6:849-855, 1997 Bacus JV,et al: Properties of intraepithelial neoplasia relevant 15. Boone CW, Bacus JW, to cancer chemoprevention and to the development of surrogate end points for clinical trials. Proc SOCExp Biol Med 216:151-165, 1997 16. Brown LF, Berse B, Jackman RW, et al: Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer. Hum Pathol 26%-91, 1995 17. Bruning PF, Van Doom J, Bonfrer JM, et al: Insulin-like growth-factor-binding protein 3 is decreased in early-stage operable pre-menopausal breast cancer. Int J Cancer 62266-270, 1995 18. Byrne C: Studying mammographic density: Implications for understanding breast cancer. J Natl Cancer Inst 89531-533, 1997 19. Cassano E, Coopmans deYoldi G, et al: Mammographic patterns in breast cancer chemoprevention with fenretinide (4-HPR). Eur J Cancer 29A:2161-2163, 1993 20. Clarke RB, Howell A, Anderson E: Type I insulin-like growth factor receptor gene expression in normal human breast tissue treated with oestrogen and progesterone. Br J Cancer 75:251-257, 1997 21. Clarke RB, Laidlaw IJ, Jones LJ, et al: Effect of tamoxifen on Ki67 labelling index in human breast tumours and its relationship to oestrogen and progesterone receptor status. Br J Cancer 67606-611, 1993 22. Clezardin P, Frappart L, Clerget M, et al: Expression of thrombospondin (TSP1) and its receptors (CD36 and CD.51) in normal, hyperplastic, and neoplastic human breast. Cancer Res 53:1421-1430, 1993 23. Costa A, Decensi A, DePalo A, et al: Breast cancer chemoprevention with retinoids and tamoxifen. Proc Am Assoc Cancer Res 37655, 1996 24. Crissman JD, Visscher DW, Kubus J: Image cytophotometric DNA analysis of atypical hyperplasias and intraductal carcinomas of the breast. Arch Pathol Lab Med 114124912.53, 1990 25. Davidoff AM, Kerns BJ, Iglehart JD, et a1 Maintenance of p53 alterations throughout breast cancer progession. Cancer Res 51:2605-2610, 1991 26. Dickson RB, Lippman ME: Oncogenes and suppressor genes. In Harris JR, Lippman ME, Morrow M, et a1 (eds): Diseases of the Breast. Philadelphia, JB Lippincott, 1996, pp 221-229 27. Dupont WD, Page D L Risk factors for breast cancer in women with proliferative breast disease. N Engl J Med 312145-151, 198.5 28. Ernster VL, Barclay J, Kerlikowske K, et al: Incidence of a treatment for ductal carcinoma in situ of the breast. JAMA 275913-918, 1996 29. Fabian CJ, Kamel S, Zalles C, et al: Biomarkers predictive of short interval cancer development in high risk women. The Breast Journal 3 (suppl):4248, 1997 30. Fabian CJ, Zalles C, Kamel S, et a1 Prevalence of aneuploidy, overexpressed ER and EGFR in random breast aspirates of women at high and low risk for breast cancer. Breast Cancer Res Treat 30263-274, 1994 31. Fabian CJ, Zalles C, Kamel S, et al: Breast cytology and biomarkers obtained by random fine needle aspiration: Use in risk assessment and early chemoprevention trials. J Cell Biochem Suppl 28101-110, 1997 32. Fabian CJ, Kamel S, Zalles CM, et al: Identification of a chemoprevention cohort from a population of women at high risk for breast cancer. J Cell Biochem 25S112-122,1996 33. Fabian CJ, Kamel S, Kimler BF, et al: Potential use of biomarkers in breast cancer risk assessment and chemoprevention trials. The Breast Journal 1236-242, 1995 34. Fabian C, Zalles C, Kamel S, et al: Biomarker and cytologic abnormalities in women at high and low risk for breast cancer. J Cell Biochem 17 (suppl G):153-160, 1993 35. Fisher B, Costantino J, Redmond C, Fisher E, et al: Lumpectomy compared with lumpectomy and radiation therapy for the treatment of intraductal breast cancer. N Engl J Med 328:1581-1586, 1993 36. Dunn BK, Kramer BS, Ford L: Phase 111, large-scale chemoprevention trials. Hematol Oncol Clin North Am 12:1019, 1998

1012

FABIAN et a1

37. Formelli F, Cleris L, Cavadini E, et al: Analysis of fenretinide (4-HPR) effects on plasma IGF-1 levels in relation with age and chemopreventive efficacy in breast cancer patients. Proc Am Assoc Cancer Res 37185, 1996 38. Freedman LS, Schatxkin A, Shiffman MH: Statistical validation of intermediate markers of precancer for use as endpoints in chemoprevention trials. J Cell Biochem 16 (Suppl G):27-32, 1992 39. Friedrichs K, Franke F, Gjorn-Wieland L, et al: CD44 isoforms correlate with cellular differentiation but not with prognosis in human breast cancer. Cancer Res 55:54245433, 1995 40. Gann P, Chatterton R, Vogelsong K, et al: Mitogenic growth factors in breast fluid obtained from healthy women: Evaluation of biological and extraneous sources of variability. Cancer Epidemiol Biomarkers Prev 6:421428, 1997 41. Gasparini G, Toi M, Gion M, et al: Prognostic significance of vascular endothelial growth factor protein in node-negative breast carcinoma. J Natl Cancer Inst 89:139147, 1997 42. Goodman GE: The clinical evaluation of cancer chemoprevention agents: Defining and contrasting phase I, 11, and I11 objectives. Cancer Res 52:2752~-2757s,1992 43. Graff JR, Herman JG, Lapidus RG, et al: E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res 55:51955199, 1995 44. Grizzle WE, Myers RB, Manne U, et al: Factors affecting immunohistochemical evaluation of biomarker expression in neoplasia. In Hanasek M, Walaszek 2 (eds): Methods in Molecular Biology, vol XX, Tumor Marker Protocols. Humana Press, Totowa, NJ, 1998 45. Grizzle WE, Meyers RB, Oelschlager DK: Prognostic biomarkers in breast cancer: Factors affecting immunohistochemical evaluation. The Breast Journal 1:243-250, 1995 46. Guidi AJ, Schnitt SJ, Fischer L, et al: Vascular permeability factor (vascular endothelial growth factor) expression and angiogenesis in patients with ductal carcinoma in situ of the breast. Cancer 80:1945-1953, 1997 47. Haagensen CD: Lobular neoplasia (lobular carcinoma in situ). In Haagensen CD (ed): Diseases of the Breast, ed 3. Philadelphia, WB Saunders, 1986, pp 192-241 48. Hankinson SE, Willet WC, Colditz GA, et al: A prospective assessment of plasma insulin-like growth factor levels in breast cancer risk. Proc SOCEpidemiol Res, 1997, abstract 286 49. Henricksson M, Luscher 8: Proteins of the Myc network Essential regulators of cell growth and differentiation. Adv Cancer Res 68:109-182, 1996 50. Hu W, Mitchell MF, Boiko I, et al: Decreased PCNA expression in cervical premalignant lesions after chemoprevention by aGdifluoro-methylornithine (DFMO).Proc Am Assoc Cancer Res 37:185, 1996 51. Hurlin PJ, Foley KP, Ayer DE, et al: Regulation of Myc and Mad during epidermal differentiation and HPV-associated tumorigenesis. Oncogene 11:2487-2501, 1995 52. Jacquemier JD, Rolland PH, Vague D, et al: Relationships between steroid receptor and epithelial cell proliferation in benign fibrocystic disease of the breast. Cancer 49:2534-2536, 1992 53. Jernstrom HC, Olsson H, Borg A: Reduced testosterone, 17 beta-oestradiol and sexual hormone binding globulin, and increased insulin-like growth factor-1 concentrations, in healthy nulligravid women aged 19-25 years who were first and/or second degree relatives to breast cancer patients. Eur J Cancer Prev 6:330-340, 1997 54. Juu A, Bang P, Hertel N, et al: Serum insulin-like growth factor I in 1030 healthy children, adolescents, and adults: Relation to age, sex, stage of puberty, testicular size, and body mass index. J Clin Endocrinol Metab 78:744-752, 1995 55. Kalkhoven E, Roelen BA, de Winter JP, et al: Resistance to transforming growth factor beta and activin due to reduced receptor expression in human breast tumor cell lines. Cell Growth Differ 6:1151-1161, 1995 56. Kamd A, Giruis NA, Weaver-Feldhaus J, et al: A cell cycle regulator potentially involved in genesis of many tumor types. Science 264:436440, 1994 57. Kelloff GJ, Boone CW, Crowell JA, et al: Development of breast cancer chemopreventive drugs. The Breast Journal 1:271-283, 1995

MODELS FOR EARLY CHEMOPREVENTION TRIALS IN BREAST CANCER

1013

58. Kelloff GJ, Boone CW, Crowell JA, et al: Risk biomarkers and current strategies for cancer chemoprevention. J Cell Biochem 25:l-14, 1996 59. Kelloff GJ, Boone CW, Steele VE, et al: Progress in cancer chemoprevention: Perspectives on agent selection and short-term clinical intervention trials. Cancer Res 54(suppl):2015!3-2024S, 1994 60. Kelloff GJ, Boone CW, Steele VE, et al: Mechanistic considerations in chemopreventive drug development. J Cell Biochem 20 (suppl G):l-24,1994 61. Kelloff GJ, Crowell JA, Boone CW, et al: Strategy and planning for chemopreventive drug development: Clinical development plans. J Cell Biochem 20:55-62,1994 62. Keyomarsi K, OLeary N, Molnar G, et al: Cyclin-E, a potential prognostic marker for breast cancer. Cancer Res 54:380-385, 1994 63. Khan SA, Rogers AM, Obanda JA, et a1 Estrogen receptor expression of benign breast epithelium and its association with breast cancer. Cancer Res 5499S997, 1994 64. Khan SA: Estrogen and progesterone receptors in benign breast epithelium. The Breast Journal 1:251-261, 1995 65. Khan SA, Rogers MA, Khurana KK, et al: Estrogen receptor expression in benign breast epithelium and breast cancer risk. J Natl Cancer Inst 90:3742, 1998 66. King EB, Chew KL, Duarte L, et a1 Image cytometric classification of premalignant breast disease in fine needle aspirates. Cancer 62:114-124, 1988 67. Knight JA, Boyd NF, Greenberg C, et a1 Nutrients associated with reduction of mammographic breast density in premenopausal women in a randomized controlled dietary intervention breast cancer prevention trial. Proc Am Assoc Cancer Res 39:89, 1998 68. Lagios MD, Westdahl PR, Margolin FR, et al: Duct carcinoma in situ. Relationship of extent of noninvasive disease to the frequency of occult invasion, multicentricity, lymph node metastases, and short-term treatment failures. Cancer 50:1309-1314, 1982 69. Lagios M D Heterogeneity of duct carcinoma in situ (DCIS): Relationship of grade and subtype analysis to local recurrence and risk of invasive transformation. Cancer Lett 9097-102, 1995 70. Lagios MD: Classification of duct carcinoma in situ (DCIS) with a characterization of high grade lesions: Defining cohorts for chemoprevention trials. J Cell Biochem 25S108-111, 1996 71. Lambird PA, Shelley W M The spatial distribution of lobular in situ mammary carcinoma. JAMA 210-689, 1969 72. Leek RD, Kaklamanis L, Pezzella F, et al: Bcl-2 in normal human breast and carcinoma, association with oestrogen receptor-positive, epidermal growth factor receptor-negative tumours and in situ cancer. Br J Cancer 69:135-139, 1994 73. LeRoith D Insulin-like growth factors and cancer. Ann Intern Med 12254-59, 1995 74. Manne U, Myers RB, Moron C, et al: Prognostic significance of Bcl-2 expression and p53 nuclear accumulation in colorectal adenocarcinomas. Int J Cancer 74:346-358,1997 75. Markopoulos C, Berger U, Wilson P, et a1 Oestrogen receptor content of normal breast cells and breast carcinomas throughout the menstrual cycle. Br Med J 296:13491351, 1988 76. Marshall CJ, Schumann GB, Ward JH, et al: Cytologic identification of clinically occult proliferative breast disease in women with a family history of breast cancer. Am J Clin Pathol 95:157-165, 1991 77. Marshall LM, Hunter DJ, Connolly JL, et al: Risk of breast cancer associated with atypical hyperplasia of lobular and ductal types. Cancer Epidemiol Biomarkers Prev 6:297-301, 1997 78. Masood S Cytomorphology of fibrocystic change, high risk, premalignant breast lesions. The Breast Journal 1:210-221, 1995 79. Masood S, Frykberg ER, McLellan GL, et al: Prospective evaluation of radiologically directed fine-needle aspiration biopsy of nonpalpable breast lesions. Cancer 66:14801487, 1990 80. Miller BA, Ries LAG, Hankey BF, et al: Breast cancer trends. Incidence, mortality and survival. SEER Cancer Statistics Review: 1973-1990. Bethesda, MD, National Cancer Institute, 1993, NIH Pub1 No. 93-2789

1014

FABIAN et a1

81. Millikan R, Hulka B, Thor A, et al: p53 mutations in benign breast tissue. J Clin Oncol 13~2293-2300,1995 82. Modan B, Lubin F, Alfandary E, et a1 Breast cancer following benign breast disease-a nationwide study. Breast Cancer Res Treat 46:45, 1997 83. Moll UM, Riou G, Levine AJ: Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc Natl Acad Sci USA 89:7262-7266, 1992 84. Monaghan P, Perusinghe NP, Nicholson RI,et al: Growth factor stimulation of proliferating cell nuclear antigen (PCNA) in human breast epithelium in organ culture. Cell Biol Int Rep 15:561-570, 1991 85. Moses HL, Serra R Regulation of differentiation by TGF-beta. Curr Opin Genet Dev 6581-586, 1996 86. Nass SJ, Rosfjord EC, Dickson R B Regulation of cell-cycle progression and cell death in breast cancer. Breast J 315-25, 1997 87. Plenary Session. Presented at the American Society of Clinical Oncology 34th Annual Meeting. May 1619,1998, Los Angeles 88. Olsen H, Jernstrom H, Alm P, et a1 Proliferation of the breast epithelium in relation to menstrual-cycle phase, hormonal use and reproductive factors. Breast Cancer Res Treat 40187-196, 1996 89. Ottesen GL, Graversen HP, Blichert-Toft M, et al: Lobular carcinoma in situ of the female breast Short-term results of a prospective nationwide study. Am J Surg Pathol 1714-21, 1993 90. Page DL, Dupont W D Anatomic markers of human premalignancy and risk of breast cancer. Cancer 663326-1335, 1990 91. Page DL, Dupont WD, Rogers LW, Rados MS: Atypical hyperplastic lesions of the female breast. A long-term follow-up study. Cancer 55:2698-2708, 1985 92. Page DL, Kidd TE Jr, Dupont WD, et al: Lobular neoplasia of the breast: Higher risk for subsequent invasive cancer by more extensive disease. Hum Pathol 22:12321239,1991 93. Peterson OW, Hoyer PE, Van Deurs B: Frequency and distribution of estrogen receptor-positive cells in normal non-lactating human breast tissue. Cancer Res 4757485751, 1987 94. Petrakis N L Nipple aspirate fluid in epidemiologic studies of breast disease. Epidemiol Rev 15:188-195, 1993 95. Pezzino V, Papa V, Milazzo G, et al: Insulin-like growth factor I (IGF-I) receptors in breast cancer. Ann NY Acad Sci 78439-201, 1996 96. Pike MC, Spicer DV, Dahmoush L, et a1 Estrogens, progestins, normal breast cell proliferation, and breast cancer risk. Epidemiol Rev 1517-35, 1993 97. Pollak M, Costantino J, Polychronakos C, et al: Effect of tamoxifen on serum insulinlike growth factor I levels in stage I breast cancer patients. J Natl Cancer Inst 821693-1697, 1990 98. Pollak J: Effects of adjuvant tamoxifen therapy on growth hormone and insulin-like growth factor I (IGF-I) physiology. In Salmon SE (ed): Adjuvant Therapy of Cancer, VII. Philadelphia, JB Lippincott, 1993, pp 43-55 99. Poller DN, Roberts EC, Bell JA, et al: p53 protein expression in mammary ductal carcinoma in situ: Relationship to immunohistochemical expression of estrogen receptor and c-erbB-2 protein. Hum Pathol 24:463468, 1993 100. Porter PL, Malone KE, Heagerty PJ, et al: Expression of cell cycle regulators p27Kipl and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med 3:222-225, 1997 101. Posner MC, Wolmark N Non-invasive breast carcinoma. Breast Cancer Res Treat 21~155-164, 1992 102. Potten CS, Watson RJ, Williams GT, et al: The effect of age and menstrual cycle upon proliferative activity of the normal human breast. Br J Cancer 58:163-170, 1988 103. Resnik JL, Reichart DB, Huey K, et al: Elevated insulin-like growth factor I receptor autophosphorylation and kinase activity in human breast cancer. Cancer Res 58:11591164, 1998 104. Roberts JM, Porter PL, Daling JR, et al: Expression of cell-cycle regulatory proteins in

MODELS FOR EARLY CHEMOPREVENTION TRIALS IN BREAST CANCER

1015

normal and neoplastic cells: Prognostic implications of cyclin E and p27 protein levels in young women with breast cancer. Proc Am SOCClin Oncol35:41, 1997 105. Rosai J: Borderline epithelial lesions of the breast. Am J Surg Pathol 15:209-221, 1991 106. Sabourin JC, Martin A, Baruch J, et al: bcl-2 expression in normal breast tissue during menstrual cycle. Int J Cancer 59:1-6, 1994 107. Saftlas AF, Hoover RN, Brinton LA, et al: Mammographic densities and risk of breast cancer. Cancer 672833-2838, 1991 108. Said T, Medina D: Cell cyclins and cyclin-dependent kinase activities in mouse mammary tumor development. Carcinogenesis 165323-830, 1995 109. Sauter ER, Daly M, Linahan K, et al: Prostate specific antigen levels in nipple aspirate fluid correlate with breast cancer risk. Cancer Epidemiol Biomarkers Prev 5:967-970, 1996 110. Sauter ER, Ross E, Daly M, et al: Nipple aspirate fluid: A promising non-invasive method to identify cellular markers of breast cancer risk. Br J Cancer, 76:494-501, 1997 111. Schatzkin A, Freedman LS, Dorgan J, et a1 Surrogate end points in cancer research: A critique. Cancer Epidemiol Biomarkers Prev 5:947-953, 1996 112. Schmitt FC, Leal C, Lopes C: p53 protein expression and nuclear DNA content in breast intraductal proliferations. J Pathol 176:233-241, 1995 113. Schwartz G F Sub-clinical ductal carcinoma in situ of the breast: Selection for treatment by local excision alone. The Breast Journal 24144, 1996 114. Schwartz GF, Patchefsky AS, Finklestein SD, et al: Nonpalpable in situ ductal carcinoma of the breast. Predictors of multicentricity and microinvasion and implications for treatment. Arch Surg 124:29-32, 1989 115. Serre C-M, Clezardin P, Frappart L, et al: Distribution of thrombospondin and integrin a, in DCIS, invasive ductal and lobular human breast carcinomas: Virchows Arch 427365-372, 1995 116. Shaulsky G, Goldfinger N, Peled A, et al: Involvement of wild-type p53 protein in the cell cycle requires nuclear localization. Cell Growth Differ 2:661467, 1991 117. Siitonen SM, Isola JJ, Rantala IS, et al: Intratumor variation in cell proliferation in breast carcinoma as determined by antiproliferating cell nuclear antigen monoclonal antibody automated image analysis. Am J Clin Pathol 99:2&231, 1993 118. Silva JS, Georgiade GS, Dilley WG, et al: Menstrual cycle-dependent variations of breast cyst fluid proteins and sex steroid receptors in the normal human breast. Cancer 51:1297-1302, 1983 119. Silverstein MJ, Lagios MD, Craig PH, et a1 A prognostic index for ductal carcinoma in situ of the breast. Cancer 772267-2274, 1996 120. Simpson J, Quan D, Clark P: Amplification of cyclin D1 in noninvasive breast cancer. Proc Am Assoc Cancer Res 37209, 1996 121. Skolnick MH, Cannon-Albright LA, Goldgar DE, et al: Inheritance of proliferative breast disease in breast cancer kindreds. Science 250:1715-1700, 1990 122. Soderqvist G, von Schoultz 8, Tani E, et al: Estrogen and progesterone receptor content in breast epithelial cells from healthy women during the menstrual cycle. Am J Obstet Gynecol 168:874-879, 1993 123. Spicer DV, Ursin G, Parisky YR, et al: Changes in mammographic densities induced by a hormonal contraceptive designed to reduce breast cancer risk. J Natl Cancer Inst 86:431436, 1994 124. Stanley MW, Henry-Stanley MJ, Zera R Atypia in breast fine-needle aspiration smears correlates poorly with the presence of a prognostically significant proliferative lesion of ductal epithelium. Hum Pathol24:630-635, 1993 125. Stenmark-Askmalm M, Stal 0, Sullivan S, et a1 Cellular accumulation of p53 protein: An independent prognostic factor in stage I1 breast cancer. Eur J Cancer 30A175180, 1994 126. Stoll BA: Breast cancer: Further metabolic-endocrine risk markers? Br J Cancer 76:1652-1654, 1997 127. Stomper PC, Saneli PC, VanSlyke MA, et al: Does tamoxifen cause mammographic changes? A longitudinal study of postmenopausal women. Breast Dis 7191-195,1994

1016

FABIAN et al

128. Tan P, Cady B, Wanner M, et al: The cell cycle inhibitor p27 is an independent prognostic marker in small (Tla.Jinvasive breast carcinomas. Cancer Res 57:12591263, 1997 129. Tavassoli FA: Mammary intraepithelial neoplasia: A translational classification system for the intraductal epithelial proliferations. The Breast Journal 3:48-58, 1997 130. Tavassoli FA, Man Y Morphofunctional features of intraductal hyperplasia, atypical intraductal hyperplasia, and various grades of intraductal carcinoma. The Breast Journal 1:155-162, 1995 131. Tavassoli FA, Man Y Expression of ER, PR, p53, KI-67 in proliferative hyperplastic and malignant apocrine lesions of the breast. Mod Pathol 8:26A, 1994 132. Tavassoli FA, Norris HJ: A comparison of the results of long-term followup for atypical intraductal hyperplasia and intraductal hyperplasia of the breast. Cancer 65~518-529,1990 133. Torrisi R, Parodi S, Pensa F, et al: Effect of fenretinide on the insulin-like growth factor (IGF) system in early breast cancer patients. Proc Am Assoc Cancer Res 37185, 1996 134. Tsuda H, Fukotomi T, Hirohashi S Pattern of genetic alteration in intraductal breast neoplasm associated with histological type and grade. Clin Cancer Res 1:261-267, 1995 135. Tsutsumi Y, Naber SP, DeLellis RA, et al: newoncogene protein and epidermal growth factor receptor are independently expressed in benign and malignant breast tissues. Hum Pathol 21:750-758, 1990 136. Tuccari G, Rizzo A, Muscara M, et al: PCNA/cyclin expression in breast carcinomas: Its relationships with Ki-67, ER, PgR immunostaining and clinico-pathologic aspects. Pathologica 85(1095):47-55, 1993 137. The uniform approach to breast fine-needle aspiration biopsy. Diagn Cytopathol 16295-311, 1997 138. Van de Vijver MJ, Peterse JL, Mooi WJ, et al: Neu-protein overexpression in breast cancer: Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage I1 breast cancer. N Engl J Med 319:1239-1245, 1988 139. Van Den Berg CL, Yee D Insulin-like growth factor I (IGF-I) enhances MCF-7 cell growth in vitro even in the presence of Adriamycin and taxol. Proc Am Assoc Cancer Res 37220, 1996 140. van Dierdendonck, Wijsman JH, Keijzer A, et al: Cell-cycle related staining patterns of anti-proliferative cell nuclear antigen monoclonal antibodies. Comparison with BrdUrd labeling and Ki-67 staining. Am J Pathol 138:1165-1172, 1991 141. Van Diest PJ, Baak JPA: Diagnostic and prognostic implications of morphometry and DNA cytometry in breast cytology. In Mouriquand J (ed): Diagnosis of Non-palpable Breast Lesions: Ultrasonography Controlled Fine Needle Aspiration. New York, Karger, 1993, pp 43-53 142. Vatten L, Kvikstad A, Nymoen E: Incidence and mortality of breast cancer related to body height and living conditions during childhood and adolescence. Eur T Cancer 28:128-1$, 1992 143. Voluert OV, Stellmach V, Bouch N The modulation of thrombosuondin and other nat;rally occurring inhibitors of angiogenesis during tumor progre'ssion. Breast Cancer Res Treat 36:119-126, 1995 144. Walker RA, Dearing SJ, Lane DP, et al: Expression of p53 protein in infiltrating and in situ breast carcinoma. J Pathol 165:203-211, 1991 145. Wang TC, Cardiff RD, Zukerberg L, et al: Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature 369:669-671, 1994 146. Ward JH, Marshall CJ, Schumann GB, et al: Detection of proliferative breast disease using four quadrant fine needle aspiration. J Natl Cancer Inst 82964-966, 1990 147. Watkins LF: Decreasing expression of insulin-like growth factor binding protein-3 with progression of preneoplastic breast epithelial cell lines. Proc Am Assoc Cancer Res 37218, 1996 148. Wrensch M, Petrakis NL, King EB, et al: Breast cancer risk associated with abnormal cytology in nipple aspirates of breast fluid and prior history of breast biopsy. Am J Epidemiol 137829-833, 1993

MODELS FOR EARLY CHEMOPREVENTION TRIALS IN BREAST CANCER

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149. Xu XC, Sneige N, Liu X, et al: Progressive decrease in nuclear retinoic acid receptor p messenger RNA level during breast carcinogenesis. Cancer Res 574992-4996, 1997 150. Zalles C, Kimler BF, Kame1 S, et al: Cytologic patterns in random aspirates from women at high and low risk for breast cancer. The Breast Journal 1:343-349, 1995 Address reprint requests to Carol J. Fabian, MD University of Kansas Cancer Center University of Kansas Medical Center 3901 Rainbow Boulevard Kansas City, KS 66160-7820