Penny Powers

Detection of Tumor PIK3CA Status in Metastatic Breast Cancer Using Peripheral Blood

Purpose: We sought to evaluate the feasibility of detecting PIK3CA mutations in circulating tumor DNA (ctDNA) from plasma of patients with metastatic breast cancer using a novel technique called BEAMing. Experimental Design: In a retrospective analysis, 49 tumor and temporally matched plasma samples from patients with breast cancer were screened for PIK3CA mutations by BEAMing. We then prospectively screened the ctDNA of 60 patients with metastatic breast cancer for PIK3CA mutations by BEAMing and compared the findings with results obtained by screening corresponding archival tumor tissue DNA using both sequencing and BEAMing. Results: The overall frequency of PIK3CA mutations by BEAMing was similar in both patient cohorts (29% and 28.3%, respectively). In the retrospective cohort, the concordance of PIK3CA mutation status by BEAMing between formalin-fixed, paraffin-embedded (FFPE) samples and ctDNA from temporally matched plasma was 100% (34 of 34). In the prospective cohort, the concordance rate among 51 evaluable cases was 72.5% between BEAMing of ctDNA and sequencing of archival tumor tissue DNA. When the same archival tissue DNA was screened by both sequencing and BEAMing for PIK3CA mutations (n = 41 tissue samples), there was 100% concordance in the obtained results. Conclusions: Analysis of plasma-derived ctDNA for the detection of PIK3CA mutations in patients with metastatic breast cancer is feasible. Our results suggest that PIK3CA mutational status can change upon disease recurrence, emphasizing the importance of reassessing PIK3CA status on contemporary (not archival) biospecimens. These results have implications for the development of predictive biomarkers of response to targeted therapies. Clin Cancer Res; 18(12); 3462–9. ©2012 AACR.

Preclinical and Clinical Evaluation of Intraductally Administered Agents in Early Breast Cancer

Intraductal administration of chemotherapeutic agents may reduce new breast cancer formation and offer a less toxic treatment regimen than intravenous therapy. Repairing the Ductwork Breast cancer is typically treated intravenously with chemotherapeutic drugs, poisons that permeate the entire body and cause toxic side effects, such as hair loss, pain, and nausea. Because most breast tumors originate in the cellular lining of the breast ducts, Stearns and colleagues designed a gentler, more local treatment regimen that gets right to the source: intraductal drug injection. The authors first tested intraductal treatment with five different chemotherapeutic agents, including paclitaxel and doxorubicin, on rats with mammary tumors, at doses comparable to what might be used in the clinic in actual patients. Compared to saline-treated or untreated control animals, the rats treated intraductally had fewer tumors in the mammary glands, with minimal side effects. Stearns et al. then enrolled 17 women in a phase 1 clinical trial to examine intraductal treatment using one chemical agent, pegylated liposomal doxorubicin. Their localized delivery to the breast ducts resulted in considerably lower systemic concentrations of the drug compared to intravenous administration, suggesting that the intraductal approach is a less toxic alternative to standard chemotherapy. This clinical trial also indicates that approved agents can be delivered to the breast ducts in an outpatient setting. Longer-term studies in more women will be necessary to determine the efficacy of intraductal chemotherapy. Intraductal treatment could be especially useful for women with premalignant lesions or those at high risk of developing breast cancer, thus drastically improving upon their other, less attractive options of breast-removal surgery or surveillance (termed “watch and wait”). It’s not yet routine practice, but direct treatment of the breast ductwork with cancer-fighting drugs promises to be a safer, less painful method for controlling cancer. Most breast cancers originate in the epithelial cells lining the breast ducts. Intraductal administration of cancer therapeutics would lead to high drug exposure to ductal cells and eliminate preinvasive neoplasms while limiting systemic exposure. We performed preclinical studies in N-methyl-N′-nitrosourea–treated rats to compare the effects of 5-fluorouracil, carboplatin, nanoparticle albumin-bound paclitaxel, and methotrexate to the previously reported efficacy of pegylated liposomal doxorubicin (PLD) on treatment of early and established mammary tumors. Protection from tumor growth was observed with all five agents, with extensive epithelial destruction present only in PLD-treated rats. Concurrently, we initiated a clinical trial to establish the feasibility, safety, and maximum tolerated dose of intraductal PLD. In each eligible woman awaiting mastectomy, we visualized one ductal system and administered dextrose or PLD using a dose-escalation schema (2 to 10 mg). Intraductal administration was successful in 15 of 17 women with no serious adverse events. Our preclinical studies suggest that several agents are candidates for intraductal therapy. Our clinical trial supports the feasibility of intraductal administration of agents in the outpatient setting. If successful, administration of agents directly into the ductal system may allow for “breast-sparing mastectomy” in select women.

TBCRC 008: Early Change in 18F-FDG Uptake on PET Predicts Response to Preoperative Systemic Therapy in Human Epidermal Growth Factor Receptor 2–Negative Primary Operable Breast Cancer

Epigenetic modifiers, including the histone deacetylase inhibitor vorinostat, may sensitize tumors to chemotherapy and enhance outcomes. We conducted a multicenter randomized phase II neoadjuvant trial of carboplatin and nanoparticle albumin-bound paclitaxel (CP) with vorinostat or placebo in women with stage II/III, human epidermal growth factor receptor 2 (HER2)–negative breast cancer, in which we also examined whether change in maximum standardized uptake values corrected for lean body mass (SULmax) on 18F-FDG PET predicted pathologic complete response (pCR) in breast and axillary lymph nodes. Methods: Participants were randomly assigned to 12 wk of preoperative carboplatin (area under the curve of 2, weekly) and nab-paclitaxel (100 mg/m2 weekly) with vorinostat (400 mg orally daily, days 1–3 of every 7-d period) or placebo. All patients underwent 18F-FDG PET and research biopsy at baseline and on cycle 1 day 15. The primary endpoint was the pCR rate. Secondary objectives included correlation of change in tumor SULmax on 18F-FDG PET by cycle 1 day 15 with pCR and correlation of baseline and change in Ki-67 with pCR. Results: In an intent-to-treat analysis (n = 62), overall pCR was 27.4% (vorinostat, 25.8%; placebo, 29.0%). In a pooled analysis (n = 59), we observed a significant difference in median change in SULmax 15 d after initiating preoperative therapy between those achieving pCR versus not (percentage reduction, 63.0% vs. 32.9%; P = 0.003). Patients with 50% or greater reduction in SULmax were more likely to achieve pCR, which remained statistically significant in multivariable analysis including estrogen receptor status (odds ratio, 5.1; 95% confidence interval, 1.3–22.7; P = 0.023). Differences in baseline and change in Ki-67 were not significantly different between those achieving pCR versus not. Conclusion: Preoperative CP with vorinostat or placebo is associated with similar pCR rates. Early change in SULmax on 18F-FDG PET 15 d after the initiation of preoperative therapy has potential in predicting pCR in patients with HER2-negative breast cancer. Future studies will further test 18F-FDG PET as a potential treatment-selection biomarker.

Biomarker Modulation following Short-Term Vorinostat in Women with Newly Diagnosed Primary Breast Cancer

Purpose: Agents that target the epigenome show activity in breast cancer models. In preclinical studies, the histone deacetylase inhibitor vorinostat induces cell-cycle arrest, apoptosis, and differentiation. We evaluated biomarker modulation in breast cancer tissues obtained from women with newly diagnosed invasive disease who received vorinostat and those who did not. Experimental Design: Tumor specimens were collected from 25 women who received up to 6 doses of oral vorinostat 300 mg twice daily and from 25 untreated controls in a nonrandomized study. Candidate gene expression was analyzed by reverse transcription PCR (RT-PCR) using the Oncotype DX 21-gene assay, and by immunohistochemistry for Ki-67 and cleaved caspase-3. Matched samples from treated women were analyzed for gene methylation by quantitative multiplex methylation-specific PCR (QM-MSP). Wilcoxon nonparametric tests were used to compare changes in quantitative gene expression levels pre- and post-vorinostat with changes in expression in untreated controls, and changes in gene methylation between pre- and post-vorinostat samples. Results: Vorinostat was well tolerated and there were no study-related delays in treatment. Compared with untreated controls, there were statistically significant decreases in the expression of proliferation-associated genes Ki-67 (P = 0.003), STK15 (P = 0.005), and Cyclin B1 (P = 0.03) following vorinostat, but not in other genes by the Oncotype DX assay, or in expression of Ki-67 or cleaved caspase-3 by immunohistochemistry. Changes in methylation were not observed. Conclusions: Short-term vorinostat administration is associated with a significant decrease in expression of proliferation-associated genes in untreated breast cancers. This demonstration of biologic activity supports investigation of vorinostat in combination with other agents for the management of breast cancer. Clin Cancer Res; 19(14); 4008–16. ©2013 AACR.

Combination Epigenetic Therapy in Advanced Breast Cancer with 5-Azacitidine and Entinostat: A Phase II National Cancer Institute/Stand Up to Cancer Study.

Purpose: In breast cancer models, combination epigenetic therapy with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor led to reexpression of genes encoding important therapeutic targets, including the estrogen receptor (ER). We conducted a multicenter phase II study of 5-azacitidine and entinostat in women with advanced hormone-resistant or triple-negative breast cancer (TNBC). Experimental Design: Patients received 5-azacitidine 40 mg/m2 (days 1–5, 8–10) and entinostat 7 mg (days 3, 10) on a 28-day cycle. Continuation of epigenetic therapy was offered with the addition of endocrine therapy at the time of progression [optional continuation (OC) phase]. Primary endpoint was objective response rate (ORR) in each cohort. We hypothesized that ORR would be ≥20% against null of 5% using Simon two-stage design. At least one response was required in 1 of 13 patients per cohort to continue accrual to 27 per cohort (type I error, 4%; power, 90%). Results: There was one partial response among 27 women with hormone-resistant disease (ORR = 4%; 95% CI, 0–19), and none in 13 women with TNBC. One additional partial response was observed in the OC phase in the hormone-resistant cohort (n = 12). Mandatory tumor samples were obtained pre- and posttreatment (58% paired) with either up- or downregulation of ER observed in approximately 50% of posttreatment biopsies in the hormone-resistant, but not TNBC cohort. Conclusions: Combination epigenetic therapy was well tolerated, but our primary endpoint was not met. OC phase results suggest that some women benefit from epigenetic therapy and/or reintroduction of endocrine therapy beyond progression, but further study is needed. Clin Cancer Res; 23(11); 2691–701. ©2016 AACR.

Abstract 4666: A phase 2 study investigating the safety, efficacy and surrogate biomarkers of response of 5-azacitidine (5-AZA) andentinostat (MS-275) in patients with triple-negative advanced breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: In preclinical breast cancer models, combination epigenetic therapy with a DNA methyltransferase inhibitor (DNMTI) and a histone deacetylase inhibitor (HDACI) yield superior estrogen receptor (ER) re-expression and greater restoration of tamoxifen responsiveness than either agent alone. We conducted a multicenter phase II clinical trial to evaluate the DNMTI 5-azacitidine (5-AZA) and the HDACI entinostat in women with advanced breast cancer. Methods: Women with advanced HER2-negative, either triple-negative (TN; ER/progesterone receptor [PR]/HER2-negative) or hormone-resistant breast cancer received 5-AZA 40 mg/m2 (SQ, days 1-5, 8-10) and entinostat 7 mg (PO, days 3,10) every 28 days. Primary endpoint: objective response rate (ORR) in each group. Secondary endpoints: safety, tolerability, survival, clinical benefit rate. Exploratory endpoints: pharmacokinetics, pharmacogenetics, change in candidate gene re-expression/methylation in circulating DNA and mandatory tumor samples. Patients are offered ongoing study therapy at progression with addition of hormonal therapy (optional continuation phase). Sample size: Simon two-stage design with interim analysis after 13 patients per cohort (1st stage). If ≥1 response, accrual will continue for total of 27 per cohort (2nd stage). Null hypothesis: ORR at most 5% against alternative hypothesis that is at least 20% with type I error 4% and power 90%. Preclinical TN/ ER-positive xenograft studies assessing 5-AZA impact were also performed. Results: Thirteen evaluable patients were enrolled in 1st stage of TN cohort. Median age was 47 (31-67), median prior chemotherapies 3 (1-5), 77% white/33% black, 77% visceral disease. Median cycles received 2 (1-4). Therapy was well tolerated, most common grade 3/4 treatment related adverse events leucopenia and neutropenia (23% each). No responses observed following 1st stage and this cohort was closed. Median 1.5 additional cycles (optional continuation phase) received by 4 patients with no responses to date. Exposure to 5-AZA (Cmax=1134±1670ng/mL; AUCINF=939±724 ng*h/mL) was slightly higher than previous studies, entinostat (Cmin=0.78±0.65ng/mL) was similar. Hormone-resistant cohort proceeded to 2nd stage as 1 partial response observed. Final results will be reported once accrual complete. Ongoing preclinical studies suggest that ER-positive is more sensitive than TN breast cancer to 5-AZA. Conclusion: Combination epigenetic therapy with agents, dose and schedule described was well tolerated but not associated with clinical activity in advanced TN breast cancer. Correlative analyses will be presented at meeting. Promising preclinical findings suggest epigenetic therapy may be efficacious in ER-positive breast cancer. Citation Format: Roisin M. Connolly, Rachel C. Jankowitz, Cynthia A. Zahnow, Zhe Zhang, Michelle A. Rudek, Stacie C. Jeter, Shannon Slater, Penny Powers, Antonio C. Wolff, John Fetting, Adam M. Brufsky, Richard Piekarz, Nita Ahuja, George Somlo, Augustin Garcia, Steven Baylin, Nancy E. Davidson, Vered Stearns. A phase 2 study investigating the safety, efficacy and surrogate biomarkers of response of 5-azacitidine (5-AZA) andentinostat (MS-275) in patients with triple-negative advanced breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4666. doi:10.1158/1538-7445.AM2013-4666

A phase 1 study assessing the feasibility and safety of intraductal pegylated liposomal doxorubicin (PLD) in women awaiting mastectomy

Methods Women 18 or older with a known breast cancer awaiting a mastectomy were eligible. Neoadjuvant chemotherapy was allowed. Women with T4 features, prior breast irradiation, or procedures that in the opinion of the investigator may have altered the breast ductal system were excluded. Participants underwent nipple aspiration and ductal cannulation using a dose escalation schema. The first 3 women received 5 mL intraductal dextrose only. We determined serial doxorubicin and doxorubicinol concentrations in plasma and nipple aspirate fluid using LC/MS/ MS. We injected blue dye into the treated duct just prior to mastectomy and obtained tissue for pharmacokinetic and biomarker analysis.