Shannon Slater

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.

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

African American Women Who Receive Primary Anthracycline- and Taxane-Based Chemotherapy for Triple-Negative Breast Cancer Suffer Worse Outcomes Compared With White Women

TO THE EDITOR: We read with great interest the manuscript by Dawood et al from the M. D. Anderson Cancer Center (MDACC; Houston, TX) entitled, “Triple Receptor–Negative Breast Cancer: The Effect of Race on Response to Primary Systemic Treatment and Survival Outcomes.” We have also recently evaluated a data set of women with triple receptor–negative breast cancer who received primary systemic therapy (PST) at our institution and report our observations below. Breast cancer is a heterogeneous disease composed of a number of recognized biologic and pathologic subtypes. The so called “triple receptor–negative breast cancer,” used to describe all tumors that are estrogen receptor–, progesterone receptor–, and human epidermal growth factor receptor 2–negative, may include basal-like and non– basal-like tumors. Chemotherapy is the only systemic treatment available for women with primary triple receptor–negative breast cancer to improve long-term outcomes. Triple receptor–negative breast cancer is more prevalent among premenopausal African American women and is associated with a shorter disease-free interval and overall survival than white women. This disparity has been often attributed to lack of access to healthcare, poor follow-up, low socioeconomic status, body mass index, and possibly lower doses of adjuvant therapy. Dawood et al have previously reported that African American women with metastatic breast cancer are at greater risk of death compared with white women. African American women with triple receptor–negative breast cancer often present with a stage II or III breast cancer and may be recommended PST. Although PST does not improve disease-free survival or overall survival, it may enhance breast conservation and provide prognostic information to individual women. Women with poor response to PST and a large residual disease are more likely to suffer a recurrence and die of their disease compared with women with a pathological complete response (pCR). Based on available evidence and anecdotal observations, we hypothesized that African American women with triple receptor– negative breast cancers have worse outcomes in part because of resistance to standard chemotherapy. To test our hypothesis, we compared pCR rate, recurrence-free and overall survival, in African American versus white and other women with triple receptor–negative breast cancer who received preoperative anthracyclineand/or taxane-based therapy. In 2002, members of the multidisciplinary breast cancer program at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University Hospital (Baltimore, MD) developed an algorithm for PST management used by the clinicians from all specialties, which led to uniform identification, staging, and treatment criteria. Briefly, women with a clinical stage II or III breast cancer appropriate for PST based on International Consensus Recommendations had nodal evaluation by a fine-needle aspiration or sentinel node mapping before initiating chemotherapy. The breast cancer program screens and collects demographics and key medical characteristics on all new patients to the medical oncology clinic. All patients with a clinical stage II or III breast cancer at the time of initial consultation who received PST are included in the database. Approximately 2 to 4 weeks following PST, women undergo breast-conserving surgery or a mastectomy at the Johns Hopkins at the discretion of the treating surgeon and patient preference. Women with a positive axillary lymph node before chemotherapy undergo axillary lymph node dissection at time of definitive surgery. We reviewed the breast cancer PST database from May 2002 to December 2007. Eligible subjects for the investigation included women with histologically confirmed, estrogen receptor–, progesterone receptor–, and human epidermal growth factor receptor 2–negative, clinical stage II or III, invasive breast cancer who received chemotherapy with doxorubicin and cyclophosphamide, with or without a taxane before or after the doxorubicin and cyclophosphamide combination. The Johns Hopkins Medicine institutional review board provided an exemption for this retrospective review. We studied patient and tumor characteristics including age, race, family history, menopausal status, initial clinical stage, initial clinical tumor size and nodal status. Study end points included pCR (no residual invasive cancer in the breast and lymph nodes), recurrencefree survival, and overall survival in African American versus white/ other women. Differences in patient and tumor characteristics across race were compared with Fisher’s exact test, exact 2 test, or Wilcoxon rank sum test, where appropriate. Survival outcomes including recurrence-free survival and overall survival were analyzed using the Kaplan-Meier method and compared between race groups by the log-rank test. We identified 38 women that met the predefined criteria; 15 were African American, 23 were white or other race (Table 1). Consistent with the literature, African American women were more likely to be younger than 50 years of age and be preor perimenopausal at the time of initial diagnosis. There was no statistically significant difference between initial clinical stage and type of chemotherapy administered to the groups. Following therapy, 13% of the African American women had a pCR compared with 52% in white/other women (P .034). Fewer white/other women had a stage III residual pathological stage compared with African American women (12% and 53%, respectively). With a median follow-up of 2.1 years (range, 0.6 to 6.5 years), Kaplan-Meier curves indicate a trend for shorter recurrence-free survival (P .045) and overall survival (P .028) for African American women compared with white/other women (Fig 1). The trends remained similar after controlling for patient and tumor characteristics. JOURNAL OF CLINICAL ONCOLOGY C O R R E S P O N D E N C E VOLUME 27 NUMBER 22 AUGUST 1 2009

Individualized Molecular Analyses Guide Efforts (IMAGE): A Prospective Study of Molecular Profiling of Tissue and Blood in Metastatic Triple-Negative Breast Cancer

Purpose: The clinical utility of next-generation sequencing (NGS) in breast cancer has not been demonstrated. We hypothesized that we could perform NGS of a new biopsy from patients with metastatic triple-negative breast cancer (TNBC) in a clinically actionable timeframe. Experimental Design: We planned to enroll 40 patients onto a prospective study, Individualized Molecular Analyses Guide Efforts (IMAGE), to evaluate the feasibility of obtaining a new biopsy of a metastatic site, perform NGS (FoundationOne), and convene a molecular tumor board to formulate treatment recommendations within 28 days. We collected blood at baseline and at time of restaging to assess cell-free circulating plasma tumor DNA (ptDNA). Results: We enrolled 26 women with metastatic TNBC who had received ≥1 line of prior chemotherapy, and 20 (77%) underwent NGS of a metastatic site biopsy. Twelve (60%) evaluable patients received treatment recommendations within 28 days of consent. The study closed after 20 patients underwent NGS, based on protocol-specified interim futility analysis. Three patients went on to receive genomically directed therapies. Twenty-four of 26 patients had genetic alterations successfully detected in ptDNA. Among 5 patients, 4 mutations found in tumor tissues were not identified in blood, and 4 mutations found in blood were not found in corresponding tumors. In 9 patients, NGS of follow-up blood samples showed 100% concordance with baseline blood samples. Conclusions: This study demonstrates challenges of performing NGS on prospective tissue biopsies in patients with metastatic TNBC within 28 days, while also highlighting the potential use of blood as a more time-efficient and less invasive method of mutational assessment. Clin Cancer Res; 23(2); 379–86. ©2016 AACR.

Personalized Medicine in the Oncology Clinic: Implementation and Outcomes of the Johns Hopkins Molecular Tumor Board

Purpose Tumor genomic profiling for personalized oncology therapy is being widely applied in clinical practice even as it is being evaluated more formally in clinical trials. Given the complexities of genomic data and its application to clinical use, molecular tumor boards with diverse expertise can provide guidance to oncologists and patients seeking to implement personalized genetically targeted therapy in practice. Methods A multidisciplinary molecular tumor board reviewed tumor molecular profiling reports from consecutive referrals at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins over a 3-year period. The tumor board weighed evidence for actionability of genomic alterations identified by molecular profiling and provided recommendations including US Food and Drug Administration-approved drug therapy, clinical trials of matched targeted therapy, off-label use of such therapy, and additional tumor or germline genetic testing. Results One hundred fifty-five patients were reviewed. Actionable genomic alterations were identified in 132 patients (85%). Off-label therapies were recommended in 37 patients (24%). Eleven patients were treated off-label, and 13 patients were enrolled onto clinical trials of matched targeted therapies. Median progression-free survival of patients treated with matched therapies was 5 months (95% CI, 2.9 months to not reached), and the progression-free survival probability at 6 months was 43%(95% CI, 26% to 71%). Lack of locally available clinical trials was the major limitation on clinical actionability of tumor profiling reports. Conclusion The molecular tumor board recommended off-label targeted therapies for a quarter of all patients reviewed. Outcomes were heterogeneous, although 43% of patients receiving genomically matched therapy derived clinical benefit lasting at least 6 months. Until more data become available from precision oncology trials, molecular tumor boards can help guide appropriate use of tumor molecular testing to direct therapy.