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Purpose: The US Food and Drug Administration approved fam-trastuzumab deruxtecan-nxki (DS-8201a, T-DXd) for the treatment of adult patients with unresectable or metastatic human epidermal growth factor receptor 2 (HER2)-low (immunohistochemistry 1 + or immunohistochemistry 2+/in situ hybridization-) breast cancer who have received a prior chemotherapy in the metastatic setting or developed disease recurrence during or within 6 months of completing adjuvant chemotherapy., Patients and Methods: Approval was based on DESTINY-Breast04, a phase III, randomized, open-label, multicenter trial in patients with unresectable or metastatic HER2-low breast cancer, determined at a central laboratory. A total of 557 patients were randomly assigned (2:1) to receive either T-DXd 5.4 mg/kg intravenously once every 3 weeks (n = 373) or physicians' choice of chemotherapy (n = 184)., Results: The study met its primary efficacy end point of progression-free survival (PFS) by blinded independent central review assessment in the hormone receptor-positive (HR+) cohort (N = 494) with an estimated hazard ratio (HR) of 0.51(95% CI, 0.40 to 0.64; P < .0001). Key secondary end points were also met, including PFS in the intent-to-treat population with an HR of 0.50 (95% CI, 0.40 to 0.63; P < .0001), overall survival (OS) in the HR+ cohort with an HR of 0.64 (95% CI, 0.48 to 0.86; P = .0028) and OS in the intent-to-treat with an HR of 0.64 (95% CI, 0.49 to 0.84; P = .0010). The safety profile of T-DXd was consistent with previously approved indications, and no new safety signals were observed in this study population., Conclusion: The approval of T-DXd in HER2-low metastatic breast cancer was based on statistically significant and clinically meaningful PFS and OS improvements observed in the DESTINY-Breast04 trial and represents the first approved therapy specifically for the treatment of HER2-low metastatic breast cancer.

Post-translational modification of arginine to citrulline is catalyzed by members of the peptidylarginine deiminase (PAD) family. Dysregulation of this catalysis is a significant driver of the pathogenesis of numerous inflammatory diseases, including cancer. However, dysregulation of PAD activity has not been examined in breast cancer with respect to hormone receptor status. In this study, we measured PAD enzyme levels using Western blotting and investigated protein citrullination using a mass spectrometry-based proteomics approach in primary estrogen receptor negative (ER-) or positive (ER+) breast tumor and matched adjacent normal tissue. Our findings reveal 72 and 41 citrullinated proteins in ER- tumor and adjacent healthy tissue, respectively, where 20 of these proteins are common between the two groups. We detected 64 and 49 citrullinated proteins in ER+ tumor and adjacent healthy tissue, respectively, where 32 proteins are common. Interestingly, upon comparison of ER- and ER+ tumor tissue, only 32 citrullinated proteins are shared between the two and the rest are unique to the tumor's receptor status. Using the STRING database for protein-protein interaction network analysis, these proteins are involved in protein-folding events (i.e., heat shock proteins) in ER- samples and blood-clotting events (i.e., fibulin) in ER+ samples. Constituents of the extracellular matrix structure (i.e., collagen and fibrinogen) were found in both. Herein, we establish evidence that supports the role of this unique post-translational modification in breast cancer biology. Finally, to aid drug discovery against citrullination, we developed a liquid chromatography-ultraviolet method to measure PAD enzymatic activity and optimized glucagon-like peptide II to quantitatively measure the ability of PADs to citrullinate its substrate.

On December 16, 2020, the FDA granted regular approval to margetuximab-cmkb (MARGENZA), in combination with chemotherapy, for the treatment of adult patients with HER2-positive (HER2+) metastatic breast cancer who have received two or more prior anti-HER2 regimens, at least one of which was for metastatic disease. Approval was based on data from SOPHIA, a multicenter, randomized, open-label, active controlled study comparing margetuximab with trastuzumab, in combination with chemotherapy. The primary efficacy endpoint was progression-free survival (PFS) by blinded independent central review. SOPHIA demonstrated a 0.9-month difference in median PFS between the two treatment arms [5.8 vs. 4.9 months, respectively; stratified HR, 0.76 (95% confidence interval: 0.59-0.98; P = 0.0334)]. Overall survival (OS) was immature at the data cut-off date of September 10, 2019. Infusion-related reactions (IRR) are an important safety signal associated with margetuximab plus chemotherapy. In SOPHIA, 13% of patients treated with margetuximab plus chemotherapy reported IRRs, of which 1.5% were grade 3. The most commonly reported adverse drug reactions (>10%) with margetuximab in combination with chemotherapy were fatigue/asthenia, nausea, diarrhea, vomiting, constipation, headache, pyrexia, alopecia, abdominal pain, peripheral neuropathy, arthralgia/myalgia, cough, decreased appetite, dyspnea, IRR, palmar-plantar erythrodysesthesia, and extremity pain. Overall, the favorable risk-benefit profile for margetuximab when added to chemotherapy supported its approval for the intended indication., (©2021 American Association for Cancer Research.)

Purpose: The US Food and Drug Administration approved abemaciclib in combination with endocrine therapy (ET) for the adjuvant treatment of adult patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative, node-positive, early breast cancer (EBC) at high risk of recurrence and a Ki-67 score ≥ 20%., Patients and Methods: The approval was based on monarchE, a phase III, open-label, 2-cohort, multicenter trial of patients with EBC randomly assigned to receive abemaciclib plus ET (n = 2,808) or ET alone (n = 2,829). Abemaciclib was given at 150 mg orally twice daily for 2 years., Results: Invasive disease-free survival (IDFS) in the intent-to-treat population was statistically significant at the second IDFS interim analysis (IA; March 2020; hazard ratio [HR; 95% CI], 0.747 [0.598 to 0.932]; P = .0096); however, only 12.5% of patients had completed adjuvant therapy, and the HR for overall survival (OS) was > 1. A prespecified, controlled analysis of IDFS in patients with Ki-67 ≥ 20% in cohort 1 was statistically significant at the final IDFS analysis (July 2020; HR [95% CI], 0.643 [0.475 to 0.872]; P = .0042). At the first OS IA (April 2021), the majority of patients had completed adjuvant therapy, IDFS remained consistent, and potential detriment in OS was not observed for this subgroup (HR [95% CI], 0.767 [0.511 to 1.152]). The HR for OS in the intent-to-treat population at OS IA remained > 1 (HR [95% CI], 1.091 [0.818 to 1.455]). More patients in the abemaciclib plus ET arm experienced treatment emergent adverse events (all grades 98.4% v 88.8%, grade 3 ≥ 49.7% v 16.3%)., Conclusion: The approval of abemaciclib in adjuvant EBC was limited to patients with high risk of recurrence and Ki-67 ≥ 20%, given their favorable benefit:risk with a statistically significant IDFS advantage and no observed detriment on survival., Competing Interests: Flora MulkeyEmployment: BeiGene (I)No other potential conflicts of interest were reported.

Objectives: Many trials conclude "no clinically meaningful detriment" to health-related quality of life (HRQL) or function between arms, even when notable differential toxicity is observed. Mean change from baseline analyses of function or HRQL can possibly obscure important change in subgroups experiencing symptomatic toxicity. We evaluate the impact of diarrhea, a key treatment arm toxicity, on patient-reported HRQL and functioning in clinical trials submitted to US Food and Drug Administration., Methods: This study used 4 randomized, breast cancer trials (adjuvant to late-line metastatic) as case examples. Diarrhea, physical functioning (PF), and global health status and quality of life (GHS/QoL) from the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-C30 were analyzed at baseline and approximately 3 and 6 months., Results: Generally, patients reporting very much diarrhea at months 3 and 6 had worse PF (9-19 points lower) and GHS/QoL (16-19 points lower) than patients reporting no diarrhea regardless of treatment arm. In the change from baseline analysis, patients reporting very much diarrhea also experienced a greater decrease in PF (6-13 points) and GHS/QoL (6-16 points) versus patients reporting no diarrhea in both arms., Conclusions: In trials with moderate to large differences in symptomatic toxicity by arm, reporting "no meaningful difference in functioning and HRQL between arms" based on mean change from baseline analysis is insufficient and may obscure important impacts on subgroups experiencing symptomatic adverse events. Additional exploratory analyses with simple data visualizations evaluating functioning or HRQL in patient subgroups experiencing expected symptomatic toxicities can further inform the safety and tolerability of an investigational agent., (Copyright © 2021 International Society for Pharmacoeconomics and Outcomes Research, Inc. Published by Elsevier Inc. All rights reserved.)

Over the last decade, the treatment of patients with breast cancer has been greatly impacted by the approval of multiple drugs and indications. This summary describes 30 FDA approvals of treatments for breast cancer from 2010 to 2020. The trial design endpoints, results, and regulatory considerations are described for each approved indication. Of the 30 indications, 23 (76.6%) received regular and 7 (23.3%) received accelerated approval. Twenty-six approvals were granted in metastatic breast cancer (MBC) and four in early breast cancer. Approval decisions for the 26 MBC indications were initially supported by progression-free survival (PFS) in 21 (80.8%), overall survival (OS) or a combination of OS and PFS in two (7.7%), and objective response rate (ORR) in three (11.5%). The four approvals in early breast cancer utilized pathologic complete response (pCR) in one (25%) and invasive disease-free survival (iDFS) in three (75%) trials. Among the 30 indications, 22 received priority review, seven were granted Breakthrough Therapy Designation, and 10 applications participated in one or more pilot Oncology Center of Excellence regulatory review initiatives, including Real Time Oncology Review, Assessment Aid, and Project Orbis. FDA initiatives to advance breast cancer drug development are also described., (©2021 American Association for Cancer Research.)

Background: The European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire Core 30 Physical Functioning subscale is a widely used patient-reported outcome measure that quantifies cancer patients' physical functioning. Strong floor/ceiling effects can affect a scale's sensitivity to change. The aim of this study was to characterize floor/ceiling effects of the physical functioning domain in patients with advanced/metastatic breast cancer enrolled in commercial clinical trials and a community-based trial., Methods: The clinical trial cohort comprised patients from 5 registrational trials submitted to the Food and Drug Administration for review (2010-2017). The community cohort comprised a subgroup of patients from the Alliance Patient Reported Outcomes to Enhance Cancer Treatment (PRO-TECT) trial. The distribution of patient responses to Physical Functioning items and the summed score were assessed at the baseline and 3-month follow-up for both cohorts. Descriptive statistics were used to determine floor/ceiling effects at the item and scale levels., Results: The clinical trial cohort and the community cohort consisted of 2407 and 178 patients, respectively. Twenty-four percent or more of the respondents reported "not at all" for having trouble/needing help with each Physical Functioning item across both cohorts and measurement time points. Fourteen to twenty percent of the patients scored perfectly (100 of 100) on the Physical Functioning subscale summary measure (where higher scores indicated better physical functioning) across both cohorts and time points., Conclusions: Minor floor effects and notable ceiling effects were found at the item and scale levels of the Physical Functioning subscale, regardless of cohort, and this creates some uncertainty about its ability to detect changes in physical functioning among high-functioning patients. Investigators may consider adding additional high-functioning items from the EORTC's item library to more accurately describe the impact of anticancer treatment on patients' physical functioning., (© 2021 American Cancer Society.)

Autophagy drives drug resistance and drug-induced cancer cell cytotoxicity. Targeting the autophagy process could greatly improve chemotherapy outcomes. The discovery of specific inhibitors or activators has been hindered by challenges with reliably measuring autophagy levels in a clinical setting. We investigated drug-induced autophagy in breast cancer cell lines with differing ER/PR/Her2 receptor status by exposing them to known but divergent autophagy inducers each with a unique molecular target, tamoxifen, trastuzumab, bortezomib or rapamycin. Differential gene expression analysis from total RNA extracted during the earliest sign of autophagy flux showed both cell- and drug-specific changes. We analyzed the list of differentially expressed genes to find a common, cell- and drug-agnostic autophagy signature. Twelve mRNAs were significantly modulated by all the drugs and 11 were orthogonally verified with Q-RT-PCR (Klhl24, Hbp1, Crebrf, Ypel2, Fbxo32, Gdf15, Cdc25a, Ddit4, Psat1, Cd22, Ypel3). The drug agnostic mRNA signature was similarly induced by a mitochondrially targeted agent, MitoQ. In-silico analysis on the KM-plotter cancer database showed that the levels of these mRNAs are detectable in human samples and associated with breast cancer prognosis outcomes of Relapse-Free Survival in all patients (RSF), Overall Survival in all patients (OS), and Relapse-Free Survival in ER+ Patients (RSF ER+). High levels of Klhl24, Hbp1, Crebrf, Ypel2, CD22 and Ypel3 were correlated with better outcomes, whereas lower levels of Gdf15, Cdc25a, Ddit4 and Psat1 were associated with better prognosis in breast cancer patients. This gene signature uncovers candidate autophagy biomarkers that could be tested during preclinical and clinical studies to monitor the autophagy process., Competing Interests: The authors have declared that no competing interests exist.

Background: Cyclin-dependent kinase 4/6 inhibitors (CDKIs) are oral targeted agents approved for use in combination with endocrine therapy as first-line or second-line treatment of patients with hormone receptor-positive, HER2-negative, advanced or metastatic breast cancer. We previously reported the pooled analyses of progression-free survival in patients in specific clinicopathological subgroups, all of whom received consistent benefit from the addition of a CDKI to hormonal therapy. Here, we report the pooled overall survival results in patients treated with a CDKI and fulvestrant., Methods: In this exploratory analysis, we pooled individual patient data from three phase 3 randomised trials of CDKI or placebo in combination with fulvestrant in patients with breast cancer submitted to the US Food and Drug Administration and approved before Aug 1, 2020, in support of marketing applications. All analysed patients were aged at least 18 years, had an Eastern Cooperative Oncology Group performance status of 0-1, had hormone receptor-positive, HER2-negative advanced or metastatic breast cancer, and received at least one dose of CDKI or placebo in combination with fulvestrant. The median overall survival was estimated using Kaplan-Meier methods, and hazard ratios (HRs) with corresponding 95% CIs were estimated using Cox regression models. Patients were analysed collectively, by number of previous lines of systemic endocrine therapy in any disease setting (first-line or endocrine naive vs second-line and later), and in various clinicopathological subgroups of interest. The estimated median overall survival was not reported by group when the pooled population included patients treated across lines of therapy because of potential patient heterogeneity. All results presented are considered exploratory and hypothesis generating., Findings: Across the three pooled trials, 1960 patients were randomly assigned between Oct 7, 2013, and June 10, 2016 (12 patients were not treated and 1296 [66%] patients were randomly assigned to CDKI and 652 [33%] to placebo). In all treated patients (n=1948), the estimated HR for overall survival was 0·77 (95% CI 0·68-0·88), with a median follow-up of 43·7 months (IQR 37·8-47·7) and deaths in 935 (48%) of the 1948 patients. The difference in estimated median overall survival was 7·1 months, favouring CDKIs. In patients who received CDKIs or placebo in combination with fulvestrant as first-line systemic endocrine therapy (two trials; n=396), the estimated HR for overall survival was 0·74 (95% CI 0·52-1·07), with a median follow-up of 39·4 months (IQR 37·0-42·2). 123 (31%) of these patients died. The difference in estimated median overall survival could not be calculated because median overall survival was not estimable (95% CI 50·9-not estimable) in the CDKI group and was 45·7 months (95% CI 41·7-not estimable) in the placebo group. In patients who received CDKIs or placebo in combination with fulvestrant as second-line or later systemic endocrine therapy (three trials; n=1552), the estimated HR for overall survival was 0·77 (95% CI 0·67-0·89), with a median follow-up of 45·1 months (95% CI 39·2-48·5). 812 (52%) of these patients died. The difference in estimated median overall survival was 7·0 months, favouring CDKIs., Interpretation: The addition of CDKIs to fulvestrant resulted in a consistent overall survival benefit in all pooled patients and within most clinicopathological subgroups of interest. These findings support the existing standard of care of CDKIs plus fulvestrant for the treatment of patients with hormone receptor-positive, HER2-negative, advanced breast cancer., Funding: None., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

On December 20, 2019, the FDA granted accelerated approval to fam-trastuzumab deruxtecan-nxki [DS-8201a; T-DXd; tradename ENHERTU (Daiichi Sankyo)] for the treatment of adult patients with unresectable or metastatic HER2-positive breast cancer who have received two or more prior anti-HER2-based regimens in the metastatic setting. Approval was based on data from study DS8201-A-U201 (DESTINY-Breast01) with supportive safety data from study DS8201-A-J101. The primary efficacy endpoint in DESTINY-Breast01 was overall response rate (ORR) based on confirmed responses by blinded independent central review (ICR) using RECIST v1.1 in all participants who were assigned to receive the recommended dose of 5.4 mg/kg while secondary endpoints included duration of response (DoR). The confirmed ORR based on ICR in these 184 patients was 60.3% [95% confidence interval (CI): 52.9-67.4] and the median DoR was 14.8 months (95% CI: 13.8-16.9). Interstitial lung disease, including pneumonitis, was experienced in patients treated with T-DXd and can be severe, life threatening, or fatal. In addition, neutropenia and left ventricular dysfunction were included as Warnings and Precautions in labeling. Other important common adverse reactions were nausea, fatigue, vomiting, alopecia, constipation, decreased appetite, anemia, diarrhea, and thrombocytopenia. Overall, the totality of efficacy and safety data supported the accelerated approval of T-DXd for the intended indication., (©2021 American Association for Cancer Research.)

Purpose: Three cyclin-dependent kinase 4/6 inhibitors (CDKIs) are approved by the US Food and Drug Administration for the treatment of patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer in combination with hormonal therapy (HT). We hypothesized that on an individual basis, efficacy outcomes and adverse event (AE) development can be predicted using baseline patient and tumor characteristics., Methods: Individual-level data from seven randomized controlled trials submitted to the US Food and Drug Administration for new or supplemental marketing applications of CDKIs were pooled. Progression-free survival (PFS), overall survival (OS), and AE prediction models were developed for specific treatment regimens (HT v HT plus CDKI). An individual's characteristics were used in all models simultaneously to create a group of predicted outcomes that are comparable across treatment settings., Results: Accuracy of the PFS and OS prediction models for HT were 66% and 64%, respectively, with the strongest predictors being menopausal status and therapy line. The corresponding AE prediction models resulted in an average area under the curve of 0.613. Accuracy of the PFS and OS prediction models for HT plus CDKI were 62% and 63%, respectively, with the strongest predictors being histologic grade for both. The corresponding AE prediction models resulted in an average area under the curve of 0.639., Conclusion: This exploratory analysis demonstrated that models of efficacy outcomes and AE development can be developed using baseline patient and tumor characteristics. Comparison of paired models can inform treatment selection for individuals on the basis of the patient's personalized goals and concerns. Although use of CDKIs is standard of care in the first- or second-line setting, this model provides prognostic information that may inform individual treatment decisions., Competing Interests: Yutao GongEmployment: BeiGeneStock and Other Ownership Interests: BeiGene Jennifer J. GaoEmployment: WorldCare Clinical LLC (part time) Gideon M. BlumenthalEmployment: MerckStock and Other Ownership Interests: MerckNo other potential conflicts of interest were reported.

On June 29, 2020, the FDA approved pertuzumab, trastuzumab, and hyaluronidase-zzxf subcutaneous injection (Phesgo) for the treatment of patients with HER2-positive early-stage and metastatic breast cancer. Patients should be selected for therapy based on an FDA-approved companion diagnostic test. Approval was primarily based on the FeDeriCa trial, a randomized, open-label, multicenter comparability study of pertuzumab, trastuzumab, and hyaluronidase-zzxf subcutaneous injection compared with intravenous pertuzumab and intravenous trastuzumab administered in the neoadjuvant and adjuvant settings with chemotherapy for the treatment of patients with early breast cancer. The pharmacokinetic endpoints were, first, to demonstrate that the exposure of subcutaneous pertuzumab was not inferior to that of intravenous pertuzumab, and then to demonstrate that the exposure of subcutaneous trastuzumab was not inferior to that of intravenous trastuzumab. The primary endpoints were met with the observed lower limit of the two-sided 90% confidence intervals above the prespecified noninferiority margins. The most common adverse reactions were alopecia, nausea, diarrhea, anemia, and asthenia. The totality of the evidence demonstrated comparability of the subcutaneous product to intravenous, allowing for extrapolation and approval of all breast cancer indications for which intravenous trastuzumab and pertuzumab are approved., (©2020 American Association for Cancer Research.)

On May 24, 2019, the FDA granted regular approval to alpelisib in combination with fulvestrant for postmenopausal women, and men, with hormone receptor (HR)-positive, HER2-negative, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated, advanced or metastatic breast cancer as detected by an FDA-approved test following progression on or after an endocrine-based regimen. Approval was based on the SOLAR-1 study, a randomized, double-blind, placebo-controlled trial of alpelisib plus fulvestrant versus placebo plus fulvestrant. The primary endpoint was investigator-assessed progression-free survival (PFS) per RECIST v1.1 in the cohort of trial participants whose tumors had a PIK3CA mutation. The estimated median PFS by investigator assessment in the alpelisib plus fulvestrant arm was 11 months [95% confidence interval (CI), 7.5-14.5] compared with 5.7 months (95% CI, 3.7-7.4) in the placebo plus fulvestrant arm (HR, 0.65; 95% CI, 0.50-0.85; two-sided P = 0.001). The median overall survival was not yet reached for the alpelisib plus fulvestrant arm (95% CI, 28.1-NE) and was 26.9 months (95% CI, 21.9-NE) for the fulvestrant control arm. No PFS benefit was observed in trial participants whose tumors did not have a PIK3CA mutation (HR, 0.85; 95% CI, 0.58-1.25). The most common adverse reactions, including laboratory abnormalities, on the alpelisib plus fulvestrant arm were increased glucose, increased creatinine, diarrhea, rash, decreased lymphocyte count, increased gamma glutamyl transferase, nausea, increased alanine aminotransferase, fatigue, decreased hemoglobin, increased lipase, decreased appetite, stomatitis, vomiting, decreased weight, decreased calcium, decreased glucose, prolonged activated partial thromboplastin time, and alopecia., (©2020 American Association for Cancer Research.)

On April 17, 2020, the FDA approved tucatinib in combination with trastuzumab and capecitabine for the treatment of patients with advanced unresectable or metastatic HER2-positive breast cancer, including patients with brain metastases, who have received one or more prior anti-HER2-based regimens in the metastatic setting. This was the first new molecular entity evaluated under Project Orbis, an FDA Oncology Center of Excellence initiative, which supports concurrent review of oncology drugs by multiple global health authorities. Approval was based on the HER2CLIMB trial, which randomized patients to receive tucatinib or placebo with trastuzumab and capecitabine. Tucatinib demonstrated efficacy compared with placebo in progression-free survival [PFS; HR: 0.54; 95% confidence interval (CI): 0.42-0.71; P < 0.00001] and overall survival (OS; HR: 0.66; 95% CI, 0.50-0.87; P = 0.00480). Patients with either treated and stable or active brain metastases made up 48% of the study population. PFS in patients with brain metastases confirmed benefit (HR: 0.48; 95% CI, 0.34-0.69; P < 0.00001). The benefit in patients with brain metastases allowed for inclusion of this specific population in the indication. Important safety signals included diarrhea and hepatotoxicity which are listed under Warnings and Precautions. This article summarizes the FDA thought process and data supporting the favorable benefit-risk profile and approval of tucatinib., (©2020 American Association for Cancer Research.)

Background: Although the Adjuvant Paclitaxel and Trastuzumab (APT) trial has been adopted clinically, single-arm trials have limitations, and interest remains whether these patients with small node-negative human epidermal growth factor receptor 2 (HER2)-positive early breast cancer (EBC) would benefit from more intensive chemotherapy. This analysis explored whether external controls can contextualize single-arm studies to add to clinical decision making in the use of de-escalated therapy in patients with low-risk HER2-positive EBC., Patients and Methods: Patient-level data from five randomized trials supporting drug approval in adjuvant HER2-positive EBC were pooled, and patients with low-risk EBC were selected (n = 1770). Patients treated concurrently with trastuzumab and either anthracycline/cyclophosphamide/taxane/trastuzumab (ACTH) or taxane/carboplatin/trastuzumab (TCH; n = 1366) were matched (1:1) to patients treated with paclitaxel and trastuzumab (TH) in the APT trial (n = 406) using propensity scores. Patients treated with anthracycline/cyclophosphamide/taxane (ACT; n = 374) were also matched (1:1) to those treated with TH. Propensity scores were estimated using covariates of age, tumor stage, estrogen receptor status, progesterone receptor status, and histological grade., Results: After matching, the estimated probabilities of invasive disease-free survival (iDFS) at 3 and 5 years were 98.6% and 96.5% in the TH arm, and 96.6% and 92.9% in the ACTH/TCH arm, respectively. The estimated probabilities of overall survival (OS) at 3 and 5 years were 99.7% and 99.3% in the TH arm, and 99.0% and 97.4% in the ACTH/TCH arm, respectively. Comparing the TH arm with the ACT arm in the matched sample, the estimated difference in iDFS was 7.5% (TH 98.8% and ACT 91.3%) at 3 years and 12.6% (TH 96.1% and ACT 83.5%) at 5 years. The estimated difference in OS was 2.6% (TH 100% and ACT 97.4%) at 3 years, and 5.3% (TH 99.3% and ACT 94.0%) at 5 years., Conclusions: Our analyses suggest that patients' outcomes in both arms were in general similar, thus providing additional reassurance regarding de-escalation of therapy., Competing Interests: Disclosure The authors have declared no conflicts of interest., (Published by Elsevier Ltd.)

On May 3, 2019, the FDA granted regular approval to ado-trastuzumab emtansine (KADCYLA), for the adjuvant treatment of patients with HER2-positive early-breast cancer (EBC) who have residual invasive disease after neoadjuvant taxane-based chemotherapy and trastuzumab-based treatment. Approval was based on data from the KATHERINE trial, which randomized patients to receive ado-trastuzumab emtansine or trastuzumab. At 3 years, the event-free rate for invasive disease-free survival in the ado-trastuzumab emtansine arm was 88.3% [95% confidence interval (CI), 85.8-90.7] compared with 77.0% (95% CI, 73.8-80.7) in the trastuzumab arm, (HR, 0.50; 95% CI, 0.39-0.64; P < 0.0001). Results from secondary endpoints, subgroup analyses, and sensitivity analyses generally supported the primary efficacy endpoint results. Common adverse reactions (>25% and higher incidence in ado-trastuzumab emtansine arm) with ado-trastuzumab emtansine were fatigue, nausea, increased transaminases, musculoskeletal pain, hemorrhage, thrombocytopenia, headache, peripheral neuropathy, and arthralgia. Ado-trastuzumab emtansine is the first drug approved for the treatment of patients with residual disease after neoadjuvant treatment and surgery. This article summarizes the FDA review and the data supporting the approval of ado-trastuzumab emtansine as a component of treatment for patients with HER2-positive EBC with residual disease., (©2020 American Association for Cancer Research.)

The evolution of thought in assessing benefit in clinical trials of systemic therapy for metastatic breast cancer (MBC) is well documented, with most agents garnering regulatory approval based either on an advantage in overall survival (OS), time to progression (TTP), or progression-free survival (PFS) over an existing standard of care or objective response rate (ORR). Previous guidance for industry on clinical trial endpoints for the approval of cancer drugs and biologics was provided by the U.S. Food and Drug Administration (FDA) in 2007 and recently updated in 2018. The more recent FDA guidance recognizes that advances in science are facilitating the development of oncology products, which "may also result in the identification of additional endpoints that may be used to support approval of oncology products." This article critically addressed the evolution of thought on the advancement of clinical trials in MBC, from various stakeholder perspectives. Despite the term "stakeholder," the objective of all co-authors and parties concerned is to promote and inform the optimal design, conduct, and reporting of clinical trials for women with advanced breast cancer toward improving and extending lives. This article provides an overview of the evolving perspectives on this issue from the physician, regulatory agency, and patient and/or advocate points of view.

Background: Cyclin-dependent kinase 4/6 inhibitors (CDKIs) are indicated with endocrine therapy as first-line or second-line treatment for hormone receptor-positive, HER2-negative, advanced or metastatic breast cancer. We aimed to investigate the benefit of adding CDKIs to endocrine therapy in patients whose tumours might have differing degrees of endocrine sensitivity., Methods: We pooled individual patient data from all phase 3 randomised breast cancer trials of CDKIs plus endocrine therapy submitted to the US Food and Drug Administration before Jan 1, 2019, in support of marketing applications. Our pooled analysis included all randomly assigned patients in these trials who received at least one dose of CDKI or placebo with endocrine therapy (an aromatase inhibitor [letrozole or anastrazole] or fulvestrant). We did prespecified subgroup analyses in patients with progesterone receptor-negative disease; patients with a disease-free interval of 12 months or less; patients with de-novo metastases, lobular histology, and bone-only disease; patients with visceral metastases; and patients aged up to 40 years. Patients who were not treated, who received tamoxifen as endocrine therapy, or who were treated with an aromatase inhibitor but who had received previous chemotherapy in the metastatic setting (not first-line) were excluded from our pooled analyses. All studies had a primary endpoint of investigator-assessed progression-free survival, defined as time from date of randomisation to the initial date of documented cancer progression or death, whichever occurred first. Median progression-free survival was estimated with Kaplan-Meier methods. Hazard ratios (HR) with 95% CIs for progression-free survival were estimated by means of Cox regression models., Findings: The seven studies meeting this study's inclusion criteria were done between Feb 22, 2013, and Nov 3, 2017, with a median duration of follow-up of 19·7 months (IQR 15·9-25·9). 4200 patients were included in the pooled analysis, of whom 1320 received an aromatase inhibitor plus a CDKI, 932 received placebo plus an aromatase inhibitor, 1296 received fulvestrant plus a CDKI, and 652 received fulvestrant plus placebo. Across all seven pooled trials, the difference in estimated median progression-free survival was 8·8 months in favour of CDKI plus endocrine therapy over placebo plus endocrine therapy (range across the trials 6·8-13·3 months; HR 0·59, 95% CI 0·54-0·64). Progression-free survival results favoured the CDKI group in all prespecified clinicopathological subgroups analysed, with similar HRs to that for the broader intended-use population. In first-line aromatase inhibitor-treated patients (n=2252), the median progression-free survival in the CDKI plus aromatase inhibitor group was 28·0 months (95% CI 25·3-29·1) versus 14·9 months (14·0-16·7) in the placebo plus aromatase inhibitor group (difference 13·1 months; range across the trials 13·0-13·3 months; HR 0·55, 95% CI 0·49-0·62). In first-line fulvestrant-treated patients (n=396), the median progression-free survival was 18·6 months (95% CI 14·8-23·5) in the placebo plus fulvestrant group and not estimable (22·4 to not estimable) in the CDKI plus fulvestrant group (difference not estimable; HR 0·58, 95% CI 0·42-0·80). In the patients treated with fulvestrant in the second-line setting and beyond (n=1552), the difference in estimated median progression-free survival between the CDKI plus fulvestrant group and the placebo plus fulvestrant group was 6·9 months in favour of the CDKI group (range across the trials 5·5-7·3 months; HR 0·56, 95% CI 0·49-0·64)., Interpretation: Since the addition of CDKI to endocrine therapy seemed to benefit all clinicopathological subgroups of interest in this pooled analysis, further research is needed to identify patient subgroups for whom endocrine therapy alone might be appropriate for first-line or second-line treatment of hormone receptor-positive, HER2-negative metastatic breast cancer., Funding: None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Many older women will be treated with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor and an aromatase inhibitor (AI), given US Food and Drug Administration approval of three agents in this class. The current pooled analysis examines the efficacy and safety of this combination in older women., Patients and Methods: We pooled data from three randomized controlled studies (N = 1,827) of different CDK4/6 inhibitors in combination with an AI for initial treatment of postmenopausal women with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer. The effect of age on progression-free survival was evaluated using Kaplan-Meier estimates and a Cox proportional hazards regression model., Results: For patients age 75 years or older (n = 198) who were treated with a CDK4/6 inhibitor and an AI, hazard ratio was 0.49 (95% CI, 0.31 to 0.76) with an estimated median progression-free survival of 31.1 months (95% CI, 20.2 months to not reached) versus 13.7 months (95% CI, 10.9 months to 24.9 months) for those treated with an AI. Incidence of grade 3 to 4 adverse events was 88.8% in patients age 75 years and older and 73.4% in patients younger than age 75 years. Patients age 75 years or older reported a decline in quality-of-life measures using the EQ-5D regardless of treatment with AI alone or with the addition of a CDK4/6 inhibitor., Conclusion: There was similar efficacy with a CDK4/6 inhibitor in combination with an AI compared with AI alone for first-line treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer in older women compared with younger patients. Patients older than age 75 years experienced higher rates of toxicity, dose modifications, and a decrease from baseline in quality-of-life measures.

On December 20, 2017, the FDA granted regular approval to pertuzumab in combination with trastuzumab and chemotherapy for the adjuvant treatment of patients with HER2-positive early breast cancer (EBC) at high risk of recurrence. Approval was based on data from the APHINITY trial, which randomized patients to receive pertuzumab or placebo in combination with trastuzumab and chemotherapy. After 45.4-month median follow-up, the proportion of invasive disease-free survival (IDFS) events in the intent-to-treat population was 7.1% ( n = 171) in the pertuzumab arm and 8.7% ( n = 210) for placebo [hazard ratio (HR), 0.82; 95% confidence interval (CI), 0.67-1.00; P = 0.047]. The proportion of IDFS events in patients with hormone receptor-negative disease was 8.2% ( n = 71) and 10.6% ( n = 91) in the pertuzumab and placebo arms, respectively (HR, 0.76; 95% CI, 0.56-1.04). The proportion of IDFS events for patients with node-positive disease was 9.2% ( n = 139) and 12.1% ( n = 181) in the pertuzumab and placebo arms, respectively (HR, 0.77; 95% CI, 0.62-0.96). Adverse reactions in ≥30% of patients receiving pertuzumab were diarrhea, nausea, alopecia, fatigue, peripheral neuropathy, and vomiting. From a regulatory standpoint, the benefits of the addition of pertuzumab to adjuvant treatment outweighed the risks for patients with EBC at high risk of recurrence., (©2018 American Association for Cancer Research.)

On July 17, 2017, the FDA approved neratinib (NERLYNX; Puma Biotechnology, Inc.) for the extended adjuvant treatment of adult patients with early-stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy. Approval was based on data from ExteNET, a randomized, double-blind, placebo-controlled multicenter trial. Women with early-stage HER2-positive breast cancer and within 2 years of completing adjuvant trastuzumab were randomized to neratinib ( n = 1,420) or placebo ( n = 1,420) for 1 year. The primary endpoint was invasive disease-free survival (iDFS), defined as the time between randomization date to first occurrence of invasive recurrence (local/regional, ipsilateral, or contralateral breast cancer), distant recurrence, or death from any cause, with 2 years and 28 days of follow-up. The trial showed a statistically significant treatment effect favoring neratinib with a stratified HR of 0.66 [95% confidence interval (CI), 0.49-0.90, P = 0.008]. The estimated iDFS rate at 2 years was 94.2% (95% CI, 92.6%-95.4%) in patients treated with neratinib versus 91.9% (95% CI, 90.2%-93.2%) in those receiving placebo. Diarrhea was the most common adverse event (AE), with a 40% incidence of grade 3 or 4 diarrhea, and represents the most common AE leading to treatment discontinuation. Other frequent AEs (>10% incidence) were nausea, abdominal pain, fatigue, vomiting, rash, stomatitis, decreased appetite, and muscle spasms. Other than diarrhea, neratinib is associated with a low incidence of severe AEs; toxicities are generally reversible and manageable with dose interruptions, dose reductions, and/or standard medical care. This article summarizes FDA decision-making and data supporting the neratinib approval. Clin Cancer Res; 24(15); 3486-91. ©2018 AACR See related commentary by Unni et al., p. 3483 ., (©2018 American Association for Cancer Research.)

On March 13, 2017, the FDA approved ribociclib (KISQALI; Novartis Pharmaceuticals Corp.), a cyclin-dependent kinase 4/6 inhibitor, in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with hormone receptor (HR)-positive, HER2-negative advanced or metastatic breast cancer. The approval was based on a randomized, double-blind, placebo-controlled, international clinical trial (MONALEESA-2). A total of 668 patients were randomized to receive either ribociclib plus letrozole ( n = 334) or placebo plus letrozole ( n = 334). An improvement in progression-free survival (PFS) was observed in patients receiving ribociclib plus letrozole compared with patients receiving placebo plus letrozole [HR = 0.556; 95% confidence interval (CI), 0.429-0.720]. Overall response rate (ORR) in patients with measurable disease was 52.7% (95% CI, 46.6-58.9) in the ribociclib plus letrozole arm and 37.1% (95% CI, 31.1-43.2) in the placebo plus letrozole arm. Overall survival data were immature. The most common adverse reactions observed in 20% or more of patients taking ribociclib were neutropenia, nausea, fatigue, diarrhea, leukopenia, alopecia, vomiting, constipation, headache, and back pain. This article summarizes FDA decision-making and data supporting the approval of ribociclib. Clin Cancer Res; 24(13); 2999-3004. ©2018 AACR See related commentary by Spring and Bardia, p. 2981 ., (©2018 American Association for Cancer Research.)

Purpose The outcome and proportion of patients with bone-only (BO) metastatic breast cancer (MBC) has not been well described. We sought to describe the differential outcomes of patients with BO MBC in clinical trials and explore whether there was a discrepancy in radiographic reads between investigator and blinded independent central review. Methods We pooled and analyzed data on 10,521 patients from 13 prospective trials submitted for MBC treatment in initial or supplemental New Drug or Biologics License Applications from 2005. Three subsets were evaluated: BO, bone with other metastases (BWO), and no bone metastases (NBM). Early discordance rate and late discordance rate were calculated from 3,733 and 2,813 patients subject to a blinded independent central review, respectively. Results Bone metastases were identified in 49% (range: 42% to 73%) of patients across trials. BO disease was present in 12.5% (range: 4% to 26%), dependent on subtype. Investigator-assessed progression-free survival (PFS) and overall survival (OS) for the pooled trials demonstrated improved outcomes for the BO subgroup compared with other subgroups (BO v BWO PFS hazard ratio [HR], 0.64; 95% CI, 0.591 to 0.696; BO v NBM PFS HR, 0.70; 95% CI, 0.65 to 0.76; BO v BWO OS HR, 0.56; 95% CI, 0.50 to 0.61; BO v NBM OS HR, 0.68; 95% CI, 0.61 to 0.76). The BO subgroup has a higher early discordance rate and lower late discordance rate than the BWO and NBM subgroups. Conclusion To our knowledge, this review is the largest analysis to date of the BO subgroup of MBC and suggests this subgroup may have a distinct natural history. There also seems to be a difference in how the local investigators assessed progression events in the BO subgroup when compared with the other two groups.

Ki-67 expression is correlated with cell proliferation and is a prognostic marker for various cancers; however, its function is unknown. Here we demonstrate that genetic disruption of Ki-67 in human epithelial breast and colon cancer cells depletes the cancer stem cell niche. Ki-67 null cells had a proliferative disadvantage compared to wildtype controls in colony formation assays and displayed increased sensitivity to various chemotherapies. Ki-67 null cancer cells showed decreased and delayed tumor formation in xenograft assays, which was associated with a reduction in cancer stem cell markers. Immunohistochemical analyses of human breast cancers revealed that Ki-67 expression is maintained at equivalent or greater levels in metastatic sites of disease compared to matched primary tumors, suggesting that maintenance of Ki-67 expression is associated with metastatic/clonogenic potential. These results elucidate Ki-67's role in maintaining the cancer stem cell niche, which has potential diagnostic and therapeutic implications for human malignancies.

Circulating cell-free DNA (ccfDNA)--first identified in 1947--is "naked" DNA that is free-floating in the blood, and derived from both normal and diseased cells. In the 1970s, scientists observed that patients with cancer had elevated levels of ccfDNA as compared to their healthy, cancer-free counterparts. The maternal fetal medicine community first developed techniques to identify the small fraction of fetal-derived ccfDNA for diagnostic purposes. Similarly, due to the presence of tumor-specific (somatic) variations in all cancers, the fraction of circulating cell-free plasma tumor DNA (ptDNA) in the larger pool of ccfDNA derived from normal cells can serve as extremely specific blood-based biomarkers for a patient's cancer. In theory this "liquid biopsy" can provide a real-time assessment of molecular tumor genotype (qualitative) and existing tumor burden (quantitative). Historically, the major limitation for ptDNA as a biomarker has been related to a low detection rate; however, current and developing techniques have improved sensitivity dramatically. In this chapter, we discuss these methods, including digital polymerase chain reaction and various approaches to tagged next-generation sequencing.

On February 3, 2015, the FDA granted accelerated approval to palbociclib (IBRANCE, Pfizer Inc.), an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), for use in combination with letrozole for the treatment of postmenopausal women with estrogen receptor (ER)-positive, HER2-negative advanced breast cancer as initial endocrine-based therapy for their metastatic disease. The approval is based on a randomized, multicenter, open-label phase I/II trial (PALOMA-1) in 165 patients randomized to palbociclib (125 mg orally daily for 21 consecutive days, followed by 7 days off treatment) plus letrozole (2.5 mg orally daily) or letrozole alone. The phase II portion of the trial was divided into two cohorts: cohort 1 enrolled 66 biomarker-unselected patients and cohort 2 enrolled 99 biomarker-positive patients. The major efficacy outcome measure was investigator-assessed progression-free survival (PFS). A large magnitude of improvement in PFS was observed in patients receiving palbociclib plus letrozole compared with patients receiving letrozole alone (HR, 0.488; 95% confidence interval, 0.319-0.748). Multiple sensitivity analyses were supportive of clinical benefit. The most common adverse reaction in patients receiving palbociclib plus letrozole was neutropenia. This article summarizes the FDA thought process and data supporting accelerated approval based on PALOMA-1 that may be contingent upon verification and description of clinical benefit in the ongoing and fully accrued confirmatory trial PALOMA-2., (©2015 American Association for Cancer Research.)

Objectives: Circulating plasma DNA is being increasingly used for biomedical and clinical research as a substrate for genetic testing. However, cell lysis can occur hours after venipuncture when using standard tubes for blood collection, leading to an increase in contaminating cellular DNA that may hinder analysis of circulating plasma DNA. Cell stabilization agents can prevent cellular lysis for several days, reducing the need for immediate plasma preparation after venipuncture, thereby facilitating the ease of blood collection and sample preparation for clinical research. However, the majority of cell stabilizing reagents have not been formally tested for their ability to preserve circulating plasma tumor DNA., Design & Methods: In this study, we compared the properties of two cell stabilizing reagents, the cell-free DNA BCT tube and the PAXgene tube, by collecting blood samples from metastatic breast cancer patients and measuring genome equivalents of plasma DNA by droplet digital PCR. We compared wild type PIK3CA genome equivalents and also assayed for two PIK3CA hotspot mutations, E545K and H1047R., Results: Our results demonstrate that blood stored for 7 days in BCT tubes did not show evidence of cell lysis, whereas PAXgene tubes showed an order of magnitude increase in genome equivalents, indicative of considerable cellular lysis., Conclusions: We conclude that BCT tubes can prevent lysis and cellular release of genomic DNA of blood samples from cancer patients when stored at room temperature, and could therefore be of benefit for blood specimen collections in clinical trials., (Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Clinical genetic testing of BRCA1 and BRCA2 is commonly performed to identify specific individuals at risk for breast and ovarian cancers who may benefit from prophylactic therapeutic interventions. Unfortunately, it is evident that deleterious BRCA1 alleles demonstrate variable penetrance and that many BRCA1 variants of unknown significance (VUS) exist. In order to further refine hereditary risks that may be associated with specific BRCA1 alleles, we performed gene targeting to establish an isogenic panel of immortalized human breast epithelial cells harboring eight clinically relevant BRCA1 alleles. Interestingly, BRCA1 mutations and VUS had distinct, quantifiable phenotypes relative to isogenic parental BRCA1 wild type cells and controls. Heterozygous cells with known deleterious BRCA1 mutations (185delAG, C61G and R71G) demonstrated consistent phenotypes in radiation sensitivity and genomic instability assays, but showed variability in other assays. Heterozygous BRCA1 VUS cells also demonstrated assay variability, with some VUS demonstrating phenotypes more consistent with deleterious alleles. Taken together, our data suggest that BRCA1 deleterious mutations and VUS can differ in their range of tested phenotypes, suggesting they might impart varying degrees of risk. These results demonstrate that functional isogenic modeling of BRCA1 alleles could aid in classifying BRCA1 mutations and VUS, and determining BRCA allele cancer risk.

The PIK3CA gene encodes for the p110 alpha isoform of PI3 kinase and is one of the most frequently mutated oncogenes in human cancers. However, the mechanisms by which PIK3CA mutations activate cell signaling are not fully understood. Here we used a phosphoproteomic approach to compare differential phosphorylation patterns between human breast epithelial cells and two isogenic somatic cell knock in derivatives, each harboring a distinct PIK3CA mutation. We demonstrated differential phosphorylation patterns between isogenic cell lines containing a PIK3CA helical domain mutation (E545K) compared to cells with a PIK3CA kinase domain mutation (H1047R). In particular, the receptor tyrosine kinase, HER3, showed increased phosphorylation at tyrosine 1328 in H1047R cells versus E545K cells. Genetic studies using shRNA demonstrated that H1047R cells have a profound decrease in growth factor independent proliferation upon HER3 knock down, but this effect was attenuated in E545K cells. In addition, HER3 knock down led to reductions in both PI3 kinase and MAP kinase pathway activation in H1047R cells, but in E545K cells only PI3 kinase pathway diminution was observed. These studies demonstrate the power of using paired isogenic cell lines for proteomic analysis to gain new insights into oncogenic signal transduction pathways., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.

Loss-of-heterozygosity (LOH) analysis of archival tumor tissue can aid in determining the clinical significance of BRCA variants. Here we describe an approach for assessing LOH in formalin-fixed, paraffin-embedded (FFPE) tissues using variant-specific probes and droplet digital polymerase chain reaction (ddPCR). We evaluated LOH in 2 related breast cancer patients harboring a rare missense BRCA2 variant of unknown clinical significance (c.6966G>T; M2322I). Conventional PCR followed by Sanger sequencing suggested a change in allelic abundance in the FFPE specimens. However, we found no evidence of LOH as determined by the allelic ratio (wild type-variant) for BRCA2 in both patients' archival tumor specimens and adjacent normal control tissues using ddPCR. In summary, these experiments demonstrate the utility of ddPCR to quickly and accurately assess LOH in archival FFPE tumor tissue., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection., Experimental Design: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR., Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery., Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients., (©2014 American Association for Cancer Research.)

Digital polymerase chain reaction is a new technology that enables detection and quantification of cancer DNA molecules from peripheral blood. Using this technique, we identified mutant PIK3CA DNA in circulating ptDNA (plasma tumor DNA) from a patient with concurrent early stage breast cancer and non-small cell lung cancer. The patient underwent successful resection of both her breast and lung cancers, and using standard Sanger sequencing the breast cancer was shown to harbor the identical PIK3CA mutation identified in peripheral blood. This case report highlights potential applications and concerns that can arise with the use of ptDNA in clinical oncology practice., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown., Experimental Design: We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to "correct" PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background., Results: Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206., Conclusions: AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies., (©2013 AACR.)

Significant advances over the past decade have enabled scientists to obtain increasingly detailed molecular profiles of breast cancer. The recent analysis by The Cancer Genome Atlas published in the September 2012 issue of Nature is the most comprehensive description of breast cancer 'omics' to date. This study is impressive in its scope and scale, with the findings reconfirming the heterogeneity of breast cancer and highlighting the future challenges in translating these findings for clinical benefit.

The combination of the mTOR inhibitor everolimus with the aromatase inhibitor exemestane was evaluated in the randomized Phase III BOLERO-2 trial. Research has indicated that aberrant signaling through the mTOR pathway is associated with resistance to endocrine therapies. The BOLERO-2 trial examined the effects on progression-free survival of the addition of everolimus to exemestane in a patient population of postmenopausal, hormone receptor-positive, advanced breast cancer. At the interim analysis, the median progression-free survival assessed by local investigators was 6.9 months for everolimus plus exemestane versus 2.8 months for placebo plus exemestane (hazard ratio: 0.43; p < 0.001), and by central assessment was 10.6 versus 4.1 months, respectively (hazard ratio: 0.36; p < 0.001). The everolimus plus exemestane arm showed greater number of grade 3 and 4 adverse events. This study suggests that the addition of everolimus to exemestane is a potential viable treatment option for this patient population.