Abstract ED01-02: Use of presurgical models to screen active agents for breast intraepithelial neoplasia.
Authors: De Censi Andrea , Lazzeroni Matteo , Pruneri Giancarlo 
Published: June 2012

Abstract
In order to increase the efficiency of drug development in breast cancer treatment and prevention, window-of-opportunity (WOP), pre-surgical models are being used to screen the activity of the candidate agents and to characterize their mechanism of action. Ki-67 labeling index (LI) is one of the most studied biomarkers of cell proliferation. Several groups including ours are increasingly being using the pre-versus post-treatment change in Ki-67 malignant cells as a surrogate biomarker to screen drugs' therapeutic and preventive activity in as much as the decrease in this proliferation-related antigen after short term presurgical treatment has prognostic value and may predict the efficacy of anti cancer drugs1. In the present presentation we will show the feasibility of the WOP presurgical model to assess also the breast cancer preventive potential of the candidate agent by assessing the effect on Ki-67 LI on adjacent ductal intraepithelial neoplasia and distant ductal hyperplasia in a cost-effective manner. In other words, the window-of opportunity trial model can exploit the existence of the field cancerization, which is well documented in breast cancer, to get important clues on the preventive effect of the investigational agent. Moreover, recent studies have shown that Ki-67 LI is positively associated with carcinogenesis progression and its level of staining in atypical lesions in core biopsies of non-malignant lesions may predict subsequent breast cancer risk, going from a 10-year cumulative incidence of breast cancer of 3% for lesions with Ki-67 LI &lt;2% to 14% in those with Ki-67 LI ≥2%.
Understanding potential survival mechanisms, such as autophagy, that might be functioning in DCIS lesions provides strategies for arresting invasion at the pre-malignant stage.
The first WOP trial performed by our group in collaboration with Royal Marsden NHS Trust evaluated the effect of two doses of raloxifene in 143 postmenopausal women (50–80 years of age), with stage I or II primary breast cancer, randomly assigned to 14 days of placebo, 60 mg/day raloxifene, or 300 mg twice daily (600 mg/day) of raloxifene before surgery. In this exploratory trial, 60 mg/day raloxifene showed a significant antiproliferative effect in ER-positive breast cancer, as demonstrated by the decrease in Ki67, with no effect in ER-negative cancer. Interestingly, there was no dose-response relationship between 60 and 600 mg/day, with the lower dose being selected for phase III trials.
We further expanded the concept of the WOP model by designing a pharmacodynamic trial to select the best biological dose of tamoxifen. The effects of tamoxifen at 1 mg/day or 5 mg/day were compared with the standard dose of 20 mg/day on breast cancer Ki-67 LI and blood biomarkers associated with the risk of breast cancer, cardiovascular disease, and bone fracture. We randomly assigned 120 women with estrogen receptor (ER)-positive breast cancer to tamoxifen at 1, 5, or 20 mg/day for 4 weeks before surgery. Labeling of the tumor proliferation marker Ki-67 was determined before (baseline) and after treatment. Labeling of Ki-67 decreased in all three tamoxifen groups, with no difference in the magnitude of reduction among groups (P =.81). Relative to baseline, Ki-67 expression decreased by a median of 15.0% (95% confidence interval = 0.0% to 24.1%) among the tamoxifen groups but increased by 12.8% (95% confidence interval = 0.0% to 19.6%) among the nonrandomized control groups. We observed dose-concentration relationships for measurements of tamoxifen and its metabolites in serum and cancer tissue. The high tamoxifen concentration in breast tissue confirmed its preferential accumulation there, with the drug and metabolite concentrations being 2–15 times those of the corresponding serum level. Importantly, with a tamoxifen dose of 1 mg/day, levels of 4-hydroxytamoxifen in tissue were approximately 10–20 times greater than the 50% inhibitory concentration range observed in different MCF-7 breast cancer cell clones after 48–72 hours, namely, 0.5–5 nM or 0.2–2 ng/mL.
We have recently conducted a randomized, double blind, placebo controlled, WOP trial with lapatinib2, a reversible tyrosine kinase inhibitor which targets both HER-2 and EGFR tyrosine kinases, in 60 women with treatment-naïve T1–2, N0–1, HER-2 positive breast cancer. Lapatinib (or matching placebo) was administered for 3 weeks prior to surgery at 1500 mg once daily. The primary endpoint was to assess lapatinib biomarker (including the primary endpoint, Ki-67 LI) and clinical activity in invasive breast cancer, adjacent ductal intraepithelial neoplasia (DIN, which comprises ductal carcinoma in situ and atypical ductal hyperplasia), and distant ductal hyperplasia without atypia (DH). Ki-67 LI increased progressively in association with disease stage, increasing in the placebo arm, for example, by medians of 3% in DH to 20% in DIN to 30% in invasive cancer. Ki-67 LI in cancer tissue decreased by a mean (±SD) of 9.3% (±34.2) in the lapatinib arm and increased by 15.1% (±30.9) in the placebo arm (P = 0.008). Compared with placebo, lapatinib reduced Ki-67 significantly more in ER-negative tumors (by 34.8%; P = 0.01) but not significantly more in ER-positive tumors (by 12.3%; P = 0.2) and reduced Ki-67 more (non significantly) in cytosol PTEN-overexpressing tumors (P = 0.057). The prevalence of DIN in post-treatment surgical specimens of both arms was similar (70%-76%), with a median Ki-67 of 15% (range, 5–35%) on lapatinib versus 20% (5–60%) on placebo (P = 0.067). The prevalence of DH also was similar in both arms (&gt;90%), with a median Ki-67 of 1% (1–7%) on lapatinib versus 3% (1–5%) on placebo (P = 0.006). Other results of lapatinib versus placebo, respectively, were as follows: Median tumor diameter at surgery of 18mm(11 mm-57 mm) versus 24mm(10 mm-37 mm; P = 0.009); partial response of 13.6% versus 3.7%, stable disease of 59.1% versus 40.7%, and progression of 27.3% versus 55.6% (P-trend = 0.035). Our findings indicated that short-term lapatinib decreased cell proliferation in DIN, DH, and invasive HER-2-positive (especially ER-negative) breast cancer, thus providing the rationale for further clinical development of lapatinib for breast cancer prevention in high-risk patients, including those with HER-2-positive DIN.
With a similar study design, we have recently completed the accrual of 200 operable breast cancer patients, randomized to metformin or placebo for 4 weeks before surgery. The rationale is based on the evidence that hyperinsulinemia and insulin resistance worsen breast cancer prognosis, increase breast cancer risk, and may partly explain the obesity-breast cancer risk association. Insulin may promote tumorigenesis via a direct effect on epithelial tissues, or indirectly by affecting other modulators, such as insulin-like growth factors, sex hormones, and adipokines. Metformin, an oral biguanide which sensitizes insulin action at peripheral level and inhibits hepatic gluconeogenesis, is the first-line treatment of type-2 diabetes, can substantially reduce diabetes incidence in obese women with glucose intolerance, and has a good tolerability profile and very low cost. Epidemiological studies showed a significant risk reduction in cancer incidence and mortality in diabetic patients on metformin relative to other antidiabetic drugs, including positive results specifically in breast cancer3. Preclinical studies suggested that metformin may have different mechanisms of tumor inhibition via insulin dependent and independent pathways, including activation of AMPK through LKB1 and ATM, with inhibition of cancer proliferation and induction of apoptosis in breast cancer cell lines. These findings have prompted interest in metformin as an anticancer agent, including an ongoing adjuvant trial in women with breast cancer after primary therapy. We conducted a randomized, phase-II, double-blind, placebo-controlled trial in women with stage I-IIa breast cancer candidate to elective surgery who received either metformin or placebo (1:1 ratio) for 4 weeks prior to surgery. Patients were randomly assigned to either metformin, 850 mg tablets or placebo once daily from day 1 to 3 to adapt to gastrointestinal symptoms, followed by two 850 mg tablets after dinner from days 4 to 28. The primary study endpoint is Ki-67 in breast cancer surgical specimen and in adjacent DIN and distant ductal hyperplasia overall and by insulin resistance and BMI. Preliminary results on the first 100 patients enrolled: adverse events were in line with the known toxicity profile of metformin, with a higher incidence of gastrointestinal symptoms, mostly low grade diarrhea and nausea of transient duration. Notably, compliance was extremely high and there were no cases of hypoglycemia in this non-diabetic population. Interestingly, the prevalence of ductal intraepithelial neoplasia was 91% (median Ki-67 LI, 10%) in samples both adjacent and distant from the tumor, whereas the prevalence of ductal hyperplasia was 77% (median Ki-67LI, 2%) in samples distant from the tumor. Updated results will be presented at the conference
In conclusion, our data support the hypothesis that the pre-surgical model is suitable to unveil the field cancerization effect present in breast surgical specimens of women with breast cancer. This model may provide insight into the drug's preventive potential against tumor adjacent dysplastic (or intraepithelial neoplastic) cells and distant ductal hyperplastic cells. Although the issue as to whether cancer adjacent DCIS or DIN is a different entity from de novo DCIS/DIN remains unsolved, our model provides a practical and feasible tool to screen active agents in a cost-effective manner.
References:
1. Decensi A et al. Ann Oncol 2010;22:582.
2. Decensi A et al. Cancer Prev Res 2011;4(8):1181
3. Decensi A et al., Cancer Prev Res 2010;3:1451.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr ED01-02.