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Background Human epidermal growth factor 2 (HER2) is an effective therapeutic target in breast cancer; however, resistance to anti-HER2 agents such as trastuzumab and lapatinib develops. In a preclinical model, an HDAC inhibitor epigenetically reversed the resistance of cancer cells to trastuzumab and showed synergistic efficacy with lapatinib in inhibiting growth of trastuzumab-resistant HER2-positive (HER2+) breast cancer. Methods A phase 1b, dose escalation study was performed to assess maximum tolerated dose, safety/toxicity, clinical efficacy and explored pharmacodynamic biomarkers of response to entinostat combined with lapatinib with or without trastuzumab. Results The combination was safe. The MTD was lapatinib, 1000 mg daily; entinostat, 12 mg every other week; trastuzumab, 8 mg/kg followed by 6 mg/kg every 3 weeks. Adverse events included diarrhoea (89%), neutropenia (31%), and thrombocytopenia (23%). Neutropenia, thrombocytopenia and hypokalaemia were noted. Pharmacodynamic assessment did not yield conclusive results. Among 35 patients with evaluable response, PR was observed in 3 patients and CR in 3 patients, 1 maintained SD for over 6 months. Discussion This study identified the MTD of the entinostat, lapatinib, and trastuzumab combination that provided acceptable tolerability and anti-tumour activity in heavily pre-treated patients with HER2+ metastatic breast cancer, supporting a confirmatory trial.

There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2−) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SETER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors (ESR1 and PGR) and robust to preanalytical and analytical influences. We tested SETER/PR index in biopsies of metastastic HR+/HER2− breast cancer against the treatment outcomes in 140 patients. Then we customized the SETER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SETER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors (PFS: HR 0.534, 95% CI 0.299 to 0.955, p = 0.035; OS: HR 0.315, 95% CI 0.157 to 0.631, p = 0.001). Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1−98% of ESR1 transcripts (all had high SETER/PR index). A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SETER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2− breast cancer, especially in the absence of mutated ESR1 transcript., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: Neoadjuvant chemotherapy (NACT) is an integral component for locally advanced and large operable breast cancer. The sequence of taxanes followed by anthracyclines has been the standard of care for almost 20 years. Eribulin (E) is a synthetic analogue of halichondrin B with distinct mechanism of action as microtubule dynamics inhibitor. The FDA approved E in 11/2010 for the treatment of patients (pts) with metastatic breast cancer who have previously received at least two chemotherapeutic regimens for the treatment of metastatic disease. Research Hypothesis: Sequential administration of eribulin followed by FAC/FEC-regimen, would have greater pathologic complete response (pCR) rate than sequential administration of paclitaxel followed by FAC/FEC-regimen as primary systemic therapy for woman with operable breast cancer. Methods: This is a phase II, randomized, single institution, open label study. Pts were randomized 1:1 to receive E (1.4 mg/m2 d1 and d8 q 21 days x 4) or paclitaxel (P) (80 mg/m2 weekly x12). Both arms received FAC/FEC regimen x 4 doses followed by surgery. Eligible pts were women age 18 or older, Karnosfky PS 80 – 100, histologically confirmed invasive breast cancer, clinical T2-T3, N0-3, M0, HER2-negative. Baseline LVEF of > 50% and normal hematology, liver and kidney laboratory function tests. Primary endpoint was pathologic complete response (pCR/RCB-0) assessed by residual cancer burden (RCB). [Symmans F, 2007]. This protocol (2012-0167) IRB of The University of Texas, MD Anderson Cancer Center. Results: A preplanned interim analysis aimed to validate trial assumption was conducted after treatment of 54 randomized pts. Between 8/2012 to 7/2014, 54 pts were randomized and 49 were evaluable for pCR(27 P arm and 22 E arm). Tumor response by RCB is shown in the table. pCR rates were 30% and 4.5% in the P and E arm, respectively. Table 1.ResponsePaclitaxel - FAC/FEC Arm (N=27)Eribulin - FAC/FEC Arm (N=22)RCB 0 (pCR)8 (30%)1 (4.5%)RCB I6 (22.2%)1 (4.5%)RCB II9 (33%)10 (45%)RCB III4 (14.8%)10 (45%) 53 pts were evaluable for toxicity. The combination was safe with mostly grade 1 and 2 toxicities in both arms. In the P arm grade 3 peripheral neuropathy and neutropenia was seen in 3% and 7%, respectively. In the E arm one patient died due to multiorgan failure during cycle 1. There was no other grade 3-5 toxicity. Biomarker analysis using CTCs by AdnaTest Breast were evaluated in 39 pts at baseline. 5/39 pts were positive for CTCs. 3 pts had transcripts for EpCAM, 1 for Muc-1 and another had both. 30 pts had an additional sample post therapy. 2 pts were positive for CTC at baseline as well as at follow up (FU) visit at 180 days. None of the samples showed CTC-EMT at baseline or at FU visits. Conclusions: The interim analysis demonstrated that E arm lead to significantly lower pCR/RCB1 rate compared to P arm. Ongoing biomarker analyses include TIL, hot spot mutation analysis (HSMA) and molecular inversion probes (MIP) will be presented at the time of the meeting. Clinical trial information: NCT01593020. Citation Format: Alvarez RH, Koenig KB, Ensor JE, Ibrahim NK, Chavez-MacGregor M, Litton JK, Schwartz Gomez JK, Cyriac A, Krishnamurty S, Caudle AS, Shaitelman SF, Whitman GJ, Booser DJ, Reuben JM, Valero V. A randomized phase II neoadjuvant (NACT) study of sequential eribulin followed by FAC/FEC-regimen compared to sequential paclitaxel followed by FAC/FEC-regimen in patients (pts) with operable breast cancer not overexpressing HER-2. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-14-04.

Background: Our in vitro and in vivo preclinical data showed that entinostat enhances the efficacy of lapatinib in HER2 positive (HER2+) breast cancer cells via FOXO3-mediated Bim1 expression, which resulted in enhanced apoptosis in HER2 targeted therapy (lapatinib and trastuzumab)-resistant breast cancer (IBC and non-IBC) cells [Lee et al.]. Based on these findings, we conducted a phase 1b trial of entinostat to determine the maximal tolerated dose (MTD) in combination with lapatinib alone and in combination with lapatinib and trastuzumab for metastatic HER2+ breast cancer patients (pts), who progressed on trastuzumab. Method: This was a single-center, open-label phase 1b study to evaluate the dose limiting toxicity (DLT) and determine MTD. 3+3 dose escalation schedule was used for Cohorts 1 and 2. Pts received lapatinib and entinostat (Cohort 1) or entinostat, lapatinib, and trastuzumab (Cohort 2). Initial dose of lapatinib 1250mg in Cohort 1 and 1000mg for Cohort 2 to match standard dose in combination with trastuzumab dose. In Cohort 1, entinostat was given PO on day 1 and 15 every 28 days cycle at dose levels 10 mg (level 0), 12 mg (level 1), or 15 mg (level 2). The dose levels for Cohort 2 were 12 mg (co-level 0) or 15 mg (co-level 1) on day 1 and 15 every 28 days cycle. While lapatinib and entinostat were given 28 days cycle due to entinostat dosing, the dosing of trastuzumab followed approved schedule every 21 days starting at 8mg/kg loading followed by 6mg/kg q 3 wks in Cohort 2 and 3. After the MTD of entinostat in cohort 2 was determined at 12mg, an expansion cohort of 10 pts (cohort 3) was conducted. Results: Median age was 52 (26-69 yrs). Median number of prior trastuzumab-based regimens was 2 (1-6), 8 pts had lapatinib containing treatment prior to the trial, including 5 pts who had clinical benefit. 16 had ER+ and 13 ER negative, and 9 had IBC. Clinical efficacy and toxicity of treatment is summarized in table 1. Out of 14 pts who had clinical benefit (CR, PR, SD), 6 had IBC. Three pts are still on therapy (1CR, 1PR, 1SD). Table 1. Clinical Efficacy, Toxicity of combination Receptor StatusResponseGrade 3 toxicityGrade 4 toxicityCohort 1HER2+/ER- (N=8) HER2+/ER+ (N=7)CR (N=1; 8M), SD (N=4;1,2,4M)Lapatinib dose reduction: 3 pts Rash (2) Abdominal pain + dyspnea (1)Entinostat dose reduction: 2pts Neutropenia (1 at 12mg, 1 at 15mg)Cohort 2/3HER2+/ER- (N=8) HER2+/ER+ (N=6)CR (N=2; 3,6M), PR (N=2;4,5M) SD (N=5;1,2,4,6M)Lapatinib dose reduction: 2 pts Diarrhea (N=1 at 12mg N=1 at 10mg) Entinostat dose reduction: 5 pts Neutropenia (N=2 at 12 mg) Leukopenia (N=1 at 12mg) Anemia (N=1 at 12mg)Entinostat dose reduction: 2pts Hypokalemia (N=1 at 12mg) Thrombocytopenia (N=1 at 15mg)CR: complete response, PR: partial response, SD: stable disease, N=number of pts, M=months Conclusion: MTD was reached at 12mg q 2wkly entinostat, lapatinib 1000 mg daily and trastuzumab 8 mg/kg followed by 6mg/kg q 3 wks. This combination was safe and had promising clinical efficacy in patients with trastuzumab-resistant metastatic HER2+ breast cancer including IBC, warranting further study. Citation Format: Lim B, Jackson S, Alvarez RH, Ibrahim NK, Willey JS, Murthy RK, Booser DJ, Giordano SH, Barcenas CH, Brewster A, Walters RS, Brown PH, Tripathy D, Valero V, Ueno NT. A single-center, open-label phase 1b study of entinostat, and lapatinib alone, and in combination with and trastuzumab in patients with HER2+ metastatic breast cancer after progression on trastuzumab. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-22.

Background: Approximately 30% of TNBCs are characterized by microarray as claudin-low, mesenchymal or mesenchymal stem cell-like and, unlike basal TNBCs, these tumors frequently harbor aberrations in the PI3K/AKT/mTOR axis, raising the possibility of targeting this axis to enhance chemotherapy response. Assays to clinically identify mesenchymal TNBCs are under development, but published results confirm that up to 30% are metaplastic breast cancers (MpBCs), a chemo-refractory group of tumors that contain a mixture of epithelial and mesenchymal components, making them identifiable by microscopy. As such, MpBCs serve as surrogates of response for potential regimens to treat mesenchymal TNBC. Methods: Patients (pts) with advanced TNBC (N=64) were treated with liposomal doxorubicin (D), bevacizumab (A) and the mTOR inhibitors temsirolimus (T) or everolimus (E). D and A were administered IV on day 1 with T (IV on days 1, 8 and 15) or E (continuous daily oral administration) using 21 day cycles. Response was assessed every 6 weeks using RECIST. When available, archived tissue was evaluated for aberrations in the PI3K pathway using standard assays. Results: Fifty-two MpBC pts were treated with DAT (N=39) or DAE (N=13). Median age was 58 (range 37-79); median # of prior regimens for metastatic disease was 1 (range 0-5). The objective response rate (ORR) was 21% [complete response (CR)=4 (8%); partial response (PR)=7 (13%)] and 10 (19%) pts had stable disease (SD)≥6 months for a clinical benefit rate (CBR) of 40%. Tissue was available for testing in 43 pts and 32 (74%) had a PI3K pathway activating aberration (Table 1). Response According to PI3K Pathway AberrationPI3K Pathway AberrationN (%)CRPRSD≥6monthsCBRORRAny PI3K Pathway Aberration*32 (74)46444%31%PIK3CA Mutation19 (59)23447%26%p.H1047R12 (38)21350%25%p.E545K6 (19)02150%33%p.G1007R1 (3)010100%100%p.E545A1 (3)0000%0%p.H1047Y1 (3)0000%0%p.K111E1 (3)0000%0%p.E542K1 (3)0000%0%PIK3CA Amplification1 (3)010100%100%PTEN Mutation5 (16)0000%0%PTEN Loss5 (16)02040%40%AKT1 p.E17K Mutation2 (6)0000%0%AKT2 Amplification1 (3)100100%100%PIK3R1 Mutation2 (6)01050%50%NF2 Mutation1 (3)100100%100%No PI3K Pathway Aberration11 (26)00545%0%*Some tumors had >1 aberration detected PI3K pathway activation was associated with a significant improvement in ORR (31 vs 0%; P=0.043) but not CBR (44 vs 45%; P=1.000) or progression-free survival (median 5.1 vs 2.9 months; P=0.352). A pt with 5 year+ durable CR (on maintenance everolimus) had a mutation in NF2. To emphasize the importance of pt selection, it is notable that 12 pts with non-metaplastic TNBC were also treated with DAT, and only 1 pt had a response (CR/PR=1; SD≥6 months=0), for a CBR that was significantly worse than pts with MpBC (8 vs 40%; P=0.045). Conclusions: Using MpBC as a surrogate of response, DAT/DAE has significantly better activity in mesenchymal compared to non-selected TNBC. Response is enhanced in pts with PI3K pathway activation. DAT/DAE should be tested in non-metaplastic, mesenchymal TNBC once a diagnostic assay is available. Citation Format: Basho RK, Gilcrease M, Murthy RK, Helgason T, Booser DJ, Karp DD, Meric-Bernstam F, Wheler JJ, Valero V, Albarracin C, Litton J, Chavez-MacGregor M, Ibrahim NK, Murray JL, Koenig KB, Hong D, Subbiah V, Kurzrock R, Janku F, Moulder S. Targeting the PI3K/AKT/mTOR pathway for the treatment of mesenchymal triple-negative breast cancer (TNBC): Evidence of efficacy and proof of concept from a phase I trial with dose expansion of mTOR inhibition in combination with liposomal doxorubicin and bevacizumab. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-02.

Background: Our preclinical data show that entinostat enhances the efficacy of lapatinib in HER2 positive (HER2+) breast cancer cell lines via FOX-03-mediated Bim1 expression. In-vitro and in- vivo, the combination of entinostat and lapatinib enhanced apoptosis in lapatinib-resistant cells. We conducted a phase I study with the primary objective to determine the RP2D of entinostat plus lapatinib in patients with HER2+ metastatic breast cancer with progressive disease (PD) after trastuzumab treatment. Methods: This was a single-center, open-label study to evaluate the dose limiting toxicity (DLT) and determine the MTD of every-other-week entinostat plus daily lapatinib in 28-day cycles. Patients with locally recurrent or metastatic breast cancer in whom trastuzumab had failed were enrolled. DLT was defined as: any toxicity resulting in 14 or more days of treatment delay, febrile neutropenia (NTP), grade 4 NTP for over 7 days, any grade 3 or higher non-hematologic toxicity, grade 3 nausea, vomiting, diarrhea or electrolyte imbalance lasting over 48 hours despite adequate supportive care, or a platelet count less than 10,000. Grading was assigned according to common toxicity criteria (CTC 4). A standard 3+3 dose escalation design was used. Entinostat was given at 10 mg (level 0), 12 mg (level 1), or 15 mg (level 2) by mouth every 14 days. Lapatinib was given at 1250 mg by mouth daily. Toxicity was evaluated on day 15 and day 28 for C1 and C2, and at the end of each cycle thereafter. Results: Fifteen patients with HER2+ metastatic breast cancer were enrolled. Median age was 52 years (range 26-69), median of all prior systemic treatment was 4 (range 1- 12), and median prior trastuzumab-based regimens was 2 (range 1-6). Eight patients had prior lapatinib exposure. Seven patients had ER+/HER2+ tumors. Median number of cycles completed was 2 (range 1-13). DLT was observed in 0 of 3 patients at level 0, 1 of 6 at level 1, and 1 of 6 at level 2. 3 patients at level 1 had lapatinib dose reductions because of grade 3 rash (n=2) or grade 3 dyspnea (n=1) or grade 3 abdominal pain (n=1). 2 patients at level 2 had entinostat dose reductions because of grade 4 NTP in cycle 1 (n=1) or grade 3 NTP in cycle 5 and grade 4 neutropenia in cycle 6 (n=1). Five patients had SD (defined per 2009 RECIST guidelines): two at dose level 1 for 6 months and 13 months, three at level 2 for 3, 4, and 8 months. Median time to progression was 2 months (range 1-13). The most common treatment related adverse events were fatigue (n=15), myalgia (n=14), nausea (n=14), diarrhea (n=13), anemia (n=11), and rash (n=11). Conclusion: MTD was not reached. Cumulative toxicity of entinostat plus lapatinib was fairly well tolerated. The combination therapy suggests there was clinical activity in at-least 5 patients who had SD in this cohort of heavily pretreated trastuzumab-resistant MBC. Data from the EG104900 clinical trial showed that lapatinib plus trastuzumab significantly improved median overall survival compared with lapatinib alone. Therefore, this study was modified after 15 patients to add trastuzumab to the combination. We are currently conducting a phase 1b trial to determine the MTD of entinostat, trastuzumab and lapatinib. Citation Format: Jie Willey, Ricardo H Alvarez, Daniel J Booser, Sharon H Giordano, Ana M Gonzalez-Angulo, James L Murray, Vincente Valero, Jangsoon Lee, Naoto T Ueno, Summer A Jackson, Fahad A Faruqi. A phase I clinical trial of entinostat and lapatinib in patients with trastuzumab refractory HER2-positive metastatic breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-19-22.

Bone is the most common site of metastasis of breast cancer, affecting most women with advanced disease. Procollagen type I N-terminal propeptide (P1NP), osteocalcin, CTX, and IL-6 are markers of bone turnover. Our objective was to determine whether serum levels of these proteins have clinical utility as predictors of breast cancer metastasis to bone. Blood was collected before treatment from 164 patients with stage I-III breast cancer from September 2001 to December 2008. Serum levels of P1NP, CTX, IL-6, and OC were measured using an automated immunoassay system. Correlations of the levels of these markers with time to bone metastasis development and with overall survival (OS) rate were assessed using Cox proportional hazards regression analysis and the Kaplan-Meier method. Fifty-five patients with stage I-III disease at the time of blood sample collection subsequently experienced metastasis to bone. A baseline P1NP level of at least 75 ng/mL predicted increased risk of bone metastasis (hazard ratio, 2.7 [95 % confidence interval, 1.2-6.0]; P = 0.031) and a poor OS rate (P = 0.031). Serum P1NP levels at or above 75 ng/mL correlate with a short time to development of bone metastasis and low overall survival in patients with stage I-III breast cancer.

Purpose: Our goal was to examine whether borderline estrogen receptor (ER)-positive cancers, defined as 1–10% positivity by immunohistochemistry (IHC), show the same global gene expression pattern and high ESR1 mRNA expression as ER-positive cancers or are more similar to ER-negative cancers. Patients and methods: ER status was determined by IHC in 465 primary breast cancers and with Affymetrix U133A gene chip (ESR1 mRNA gene expression: Probe set = 205225_at). We compared expressions of ESR1 mRNA and a 106-probe set ER-associated gene signature score between ER-negative (n=183), 1–9% (n=25), exactly 10% (n=6), and > 10% ER-positive (n=251) cancers. We also assessed the molecular class of the borderline ER-positive cases using the PAM-50 classifier. Results: Among the 1–9%, 10% and > 10% IHC positive cases, 24%, 67% and 92% were also ER-positive by ESR1 mRNA expression. The average ESR1 expression was significantly higher in the > 10% IHC-positive cohorts compared to the 1–9% or completely negative cases but in these latter two cohorts ER expression was similarly low. The average ER gene signature scores were similar for the ER-negative and 1–9% IHC-positive cases, but significantly lower than in > 10% ER-positive cases. None of the 1–9% ER-positive cases were classified as Luminal A, 2 were Luminal B and 12 were Basal-like. Among the 10% ER-positive cases, 2 were Luminal A and 1 was Luminal B. Conclusion: Overall, 24% of the 1–9% and 67% of the 10% ER-positive cancers show ESR1 mRNA levels and gene signatures that are consistent with ER-positive, potentially endocrine sensitive tumors. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-09.

BACKGROUND: Inhibition of mammalian target of rapamycin with everolimus may improve the efficacy of taxanes. Everolimus and docetaxel are both metabolized by CYP3A4, which could result in a pharmacokinetic (PK) interaction. METHODS: Fifteen patients with metastatic breast cancer were treated with docetaxel (doses of 40-75 mg/m2 intravenously on day 1 of a 21-day cycle) in combination with everolimus (doses ranging from 20 to 50 mg orally on days 1 and 8 of a 21-day cycle) in a phase 1 trial using the continuous reassessment method to determine maximum tolerated dose. The first 2 patients developed a dose-limiting toxicity (neutropenic infection), prompting a mandatory dose reduction and PK evaluation of both everolimus and docetaxel for patients enrolled in subsequent dosing cohorts. RESULTS: Fifteen patients were treated. Dose-limiting toxicity included grade 3 mucositis (n = 1), prolonged grade 4 neutropenia (n = 1), and grade 3 infection/febrile neutropenia (n = 3). Day 8 of everolimus was commonly held for neutropenia despite a dose reduction in docetaxel to 40 mg/m2. Eleven patients underwent complete PK evaluation for everolimus, and 9 patients underwent complete PK evaluation for both everolimus and docetaxel. Widely variable changes in clearance were seen for both drugs, and the study was terminated because of lack of efficacy and concerns regarding toxicity seen with the combination. CONCLUSIONS: Weekly everolimus in combination with docetaxel every 3 weeks was associated with excessive neutropenia and variable clearance of both drugs, making combination therapy unpredictable, even at low doses of both drugs. Cancer 2012. © 2011 American Cancer Society.

Several oncologic and cardiovascular organizations have recommended routine left ventricular ejection fraction (LVEF) evaluation for all patients prior to anthracycline chemotherapy for breast cancer. Nevertheless, there is no clinical or cost-effective evidence in support of such practice. Clinical

There is limited prognostic information to identify breast cancer patients who are at risk for late recurrences after adjuvant or neoadjuvant systemic therapy (AST). We evaluated the residual risk of recurrence and prognostic factors of 2838 patients with stage I–III breast cancer who were treated with AST between January 1, 1985, and November 1, 2001, and remained disease free for 5 years. Residual recurrence-free survival was estimated from the landmark of 5 years after AST to date of first recurrence or last follow-up using the Kaplan–Meier method. The log-rank test (two-sided) was used to compare groups. Residual recurrence-free survival rates at 5 and 10 years were 89% and 80%, respectively, and 216 patients developed a recurrence event. The 5-year residual risks of recurrence for patients with stage I, II, and III cancers were 7% (95% confidence interval [CI] = 3% to 15%), 11% (95% CI = 9% to 13%), and 13% (95% CI = 10% to 17%), respectively (P = .02). In multivariable analysis, stage, grade, hormone receptor status, and endocrine therapy were associated with late recurrences. Breast cancer patients have a substantial residual risk of recurrence, and selected tumor characteristics are associated with late recurrences.

Background Mesenchymal/metaplastic breast cancers (MpBCs) are often triple-negative (TNBC), and chemo-refractory, and can harbor phosphoinositide 3-kinase (PI3kinase) alterations; thus, therapy with mTor inhibitors may demonstrate activity. Patients and methods Patients with mesenchymal/MpBC treated with temsirolimus-based regimens were evaluated. Mutational analyses [polymerase chain reaction (PCR)-based DNA sequencing method, mass spectrometric detection (Sequenom MassARRAY), or next-generation sequencing] as well as loss of phosphatase and tensin homolog (PTEN) (immunohistochemistry) were performed (archived tissue when available). Results Twenty-three patients (one of whom was on two separate trials) were treated using temsirolimus-containing regimens: temsirolimus alone (n = 1 patient) or combined with the following: liposomal doxorubicin and bevacizumab (DAT, n = 18); liposomal doxorubicin (DT, n = 1); paclitaxel and bevacizumab (TAT, n = 2); paclitaxel (TT, n = 1); carboplatin and bevacizumab (CAT, n = 1). Response rate [complete response (CR) + partial response (PR)] was 25% across all regimens; 32% in the anthracycline-based regimens [DAT and DT (CR = 2, PR = 4; N = 19)]. An additional two patients achieved stable disease (SD) ≥6 months [total SD ≥6 months/CR/PR = 8 (33%)]. Molecular aberrations in the PI3K pathway were common: PIK3CA mutation = 6/15 (40%), PTEN mutation = 3/11 (27%), and PTEN loss = 2/11 (18%). A point mutation in the NF2 gene (K159fs*16; NF2 alterations can activate mTor) was found in one patient who attained CR (3+ years). Of the eight patients who achieved SD ≥6 months/CR/PR, all 4 patients with available tissue had a molecular aberration that activate the PIK3CA/Akt/mTOR axis: PIK3CA mutation = 2; PTEN loss = 1; NF2 aberration = 1. Conclusions DAT has activity in MpBCs including complete CRs. Molecular aberrations that can activate the PI3 K/Akt/mTOR axis are common in MpBC.

2 Hematopathology, 3 Pathology, 5 Background: Bortezomib (VELCADE � ) is a potent inhibitor of the 26S proteasome with broad antitumor activity. We performed a phase II study of bortezomib to evaluate its clinical effects in patients with metastatic breast cancer. Patients and methods: Twelve patients with metastatic breast cancer were treated with bortezomib (VELCADE � ) at a dosage of 1.5 mg/m 2 administered biweekly for 2 weeks with 1 week of rest in a 21-day cycle. The primary objective was clinical response rate. Toxicity and pharmacodynamics data were also obtained. Results: No objective responses were observed. One patient had stable disease, and 11 others experienced disease progression. The median survival time was 4.3 months (range, 0.9-37 months). The most common grade 3 or 4 toxicities included fatigue (58%; n= 7) and skin rash (33%; n= 4). The mean inhibition of specific chymotryptic activity was 53.1% (± 13.33%). A statistically significant reduction in the plasma interleukin-6 level was seen (P = 0.0354). Conclusion: Bortezomib was well tolerated but showed limited clinical activity against metastatic breast cancer when used as a single agent. The future development of this agent for the treatment of breast cancer should be guided

Background: We developed SET4 as a targeted, droplet-based, next-generation RNA sequencing assay to measure both the SETER/PR index of gene expression and the percent of estrogen receptor (ER) transcripts with point mutation in the ligand-binding domain (LBD) in metastatic biopsies of Stage IV breast cancer. The SETER/PR index (31 genes) is a cumulative measure of gene expression for transcripts associated with ER and progesterone receptor (PR), excluding those with a direct role in proliferation. High SETER/PR could indicate increased sensitivity to endocrine therapy, whereas LBD mutations indicate resistance but might also induce high SETER/PR. Methods: Targeted needle biopsies from a metastatic site were prospectively obtained from 82 patients with HR+/HER2- breast cancer at time of any progression event, and preserved in RNAlater. Samples were prepared for targeted sequencing on a MiSeq by combining purified total RNA with SET4 primers and RT-PCR master mix into single molecule-formatted picodroplets using a RainDrop Source instrument, followed by thermal cycling and sample indexing. Calculated SETER/PR index and percent ER transcripts with LBD mutations were evaluated as continuous variables and compared to progression-free and overall survival using Cox regression analysis with log-rank test, if the next treatment after biopsy included endocrine therapy. Results: The average read depth for the LBD of the ER transcript was 33,475X (range: 1230-180,889X), confidently detecting mutation at 1% frequency. LBD mutations were identified in 17% (14/82) of metastases (range of mutated transcripts 1%-98%). LBD mutations (>10% of transcripts) were only observed in metastases with higher SETER/PR (above the median). In patients who next received endocrine therapy (n=58), higher SETER/PR predicted longer progression-free (PFS) (HR=0.37, p=0.0004, Δ median PFS 9 months) and overall survival (OS) (HR=0.49, p=0.03). The predictions were more pronounced in patients without LBD mutation (PFS HR=0.32, p=0.0001, Δ median PFS 13 months; OS HR=0.42, p=0.01). Currently, there are insufficient cases with LBD mutation for reliable survival analysis. Conclusion: The SET4 assay measured the percent of ER transcripts with activating LBD mutation (≥1% prevalence) and also downstream ER/PR-related transcription. High SETER/PR predicted longer PFS and OS with endocrine therapy. While activating LBD mutations may be associated with endocrine resistance of Stage IV cancer, they were associated with high SETER/PR index. Consequently, metastatic cancers with high SETER/PR index and no LBD mutation in ER transcripts were particularly sensitive to endocrine therapy. This single assay unraveled a possible interaction between genotype, phenotype, and treatment outcome; and is currently being evaluated in a larger cohort of patients. Citation Format: Rosanna Lau, Lily Fu, Michael Samuels, Rashmi K. Murthy, Bruno Sinn, Jane Yu, Rebekah Gould, Jennifer Litton, Alda Tam, Stacy Moulder, Daniel Booser, Debu Tripathy, Vicente Valero, Fraser Symmans. A targeted RNA-seq assay to measure activating ER mutations and ER/PR-associated gene expression predicts sensitivity to endocrine therapy for metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1796. doi:10.1158/1538-7445.AM2017-1796

BACKGROUND The purpose of the current study was to evaluate the objective response rate and possibility of breast-conserving surgery using neoadjuvant tamoxifen in the multimodality treatment, including surgery and radiotherapy, of elderly or frail patients with locally advanced breast carcinoma. METHODS Forty-seven patients age > 75 years or age < 75 years with comorbid conditions and locally advanced breast carcinoma were treated with neoadjuvant tamoxifen (20 mg/day) for 3–6 months. This was followed by surgery and radiotherapy when feasible and adjuvant tamoxifen for 5 years or until disease recurrence. RESULTS The median age of the patients was 72 years (range, 48–86 years). Approximately 22% had T3 lesions, 57% had T4 lesions, 22% were Stage II (AJCC Manual for Staging Cancer, 3rd edition), and 78% were Stage III. Eighty percent were estrogen receptor positive. After 6 months of treatment with neoadjuvant tamoxifen, a response rate of 47% was observed, including a complete response rate of 6%. Twenty-nine patients (62%) were rendered free of disease by surgery, including 5 with breast-conserving procedures. After a median follow-up of 40 months, 23 patients (49%) remained disease free. The median survival time had not been reached at the time of last follow-up. No major toxicity was observed, with the exception of one patient who developed a possible tamoxifen-related Stage I endometrial carcinoma. The estimated 2-year and 5-year progression free and overall survival rates were 50% and 41%, and 83% and 59%, respectively. CONCLUSIONS The results of the current study show that neoadjuvant tamoxifen was effective in the treatment of elderly or frail patients with locally advanced breast carcinoma with estrogen receptor positive tumors, and resulted in a reasonable response rate, including complete responses and good overall survival. Cancer 2000;88:2054–60. © 2000 American Cancer Society.

Background: Uncontrolled studies have reported encouraging outcomes for patients with high-risk primary breast cancer treated with high-dose chemotherapy and autologous hematopoietic stem cell support. We conducted a prospective randomized trial to compare standard-dose chemotherapy with the same therapy followed by high-dose chemotherapy. Patients and Methods: Patients with 10 or more positive axillary lymph nodes after primary breast surgery or patients with four or more positive lymph nodes after four cycles of primary (neoadjuvant) chemotherapy were eligible. All patients were to receive eight cycles of 5-fluorouracil, doxorubicin (Adriamycin), and cyclophosphamide (FAC). Patients were stratified by stage and randomly assigned to receive two cycles of high-dose cyclophosphamide, etoposide, and cisplatin with autologous hematopoietic stem cell support or no additional chemotherapy. Tamoxifen was planned for postmenopausal patients with estrogen receptor-positive tumors and chest wall radiotherapy was planned for all. All P values are from two-sided tests. Results: Seventy-eight patients (48 after primary surgery and 30 after primary chemotherapy) were registered. Thirty-nine patients were randomly assigned to FAC and 39 to FAC followed by high-dose chemotherapy. After a median follow-up of 6.5 years, there have been 41 relapses. In intention-to-treat analyses, estimated 3-year relapse-free survival rates were 62% and 48% for FAC and FAC/high-dose chemotherapy, respectively (P = .35), and 3-year survival rates were 77% and 58%, respectively (P = .23). Overall, there was greater and more frequent morbidity associated with high-dose chemotherapy than with FAC; there was one septic death associated with highdose chemotherapy. Conclusions: No relapse-free or overall survival advantage was associated with the use of high-dose chemotherapy, and morbidity was increased with its use. Thus, high-dose chemotherapy is not indicated outside a clinical trial. [J Natl Cancer Inst 2000;92:225‐33] Patients with stage III breast cancer or patients with stage II breast cancer and multiple positive axillary lymph nodes have an approximately 80% relapse rate at 5 years if treated only with locoregional therapy (1‐3). With optimal combined modality therapies, approximately 30% of the patients with stage IIIB breast cancer, 50% of those with stage IIIA breast cancer, and

The aim of this study was to compare the time-to progression and overall survival (OS) in patients with metastatic breast cancer (MBC) with and without deleterious BRCA1/2 mutations. 195 women with MBC who were referred for BRCA genetic testing between 1997 and 2011 were included in the study. Logistic regression models and Cox proportional hazards models were fit to determine the associations between clinical variables and outcomes. Of 195 women with MBC, 21 % (n = 41) were positive for BRCA1/2 mutations. The number of metastatic sites at the time of metastatic disease was not different between BRCA1 versus BRCA2 carriers versus non-carriers (P = 0.77). The site of first metastasis was visceral-only in 70 % of BRCA1 carriers compared to 9 % in BRCA2 carriers and 37 % in non-carriers (P = 0.001). Median follow-up was 2.8 years. BRCA non-carriers and BRCA2 carriers had a longer time-to progression and OS compared to BRCA1 carriers (median time-to progression = 1.3 vs. 0.9 vs. 0.7 years; P = 0.31, and median OS = 4.88 vs. 4.94 vs. 1.34 years; P = 0.0065). In a multivariate model, no association was identified between BRCA positivity and time-to-event outcomes (P > 0.28). In addition, patients with triple-negative MBC carried a poorer prognosis irrespective of their BRCA status (P = 0.058 and P = 0.15 for the interaction term of BRCA status and triple-negative for time-to progression and OS, respectively). Our data indicate that BRCA1 carriers diagnosed with MBC have worse outcomes compared to BRCA2 carriers and non-carriers. However, the differences in outcome did not reach statistical significance likely due to small sample sizes.

Background: Metaplastic breast cancers (MpBCs) are a chemo-refractory group of tumors that contain a component of squamous and/or mesenchymal differentiation identifiable by light microscopy. MpBCs contain a high frequency of aberrations in the PI3K/AKT/mTOR pathway, making this pathway a potential target for therapy. Methods: Patients with advanced MpBC (N = 52) were treated with liposomal doxorubicin (D), bevacizumab (A) and the mTOR inhibitors temsirolimus (T) or everolimus (E). D and A were administered IV on day 1 with T (IV on days 1, 8 and 15) or E (continuous daily oral administration) using 21 day cycles. All tumors were evaluated to assess histology of metaplasia (spindle, mixed spindle vs non-spindle cell). Response was assessed every 6 weeks using RECIST. When available, archived tissue was evaluated for aberrations in the PI3K pathway using standard assays. Results: Fifty-two MpBC patients were treated with DAT (N = 39) or DAE (N = 13). Median age was 58 (range 37-79); median number of prior regimens for metastatic disease was 1 (range 0-5). The objective response rate (ORR) was 21% [complete response (CR) = 4 (8%); partial response (PR) = 7 (13%)] and 10 (19%) pts had stable disease (SD)≥6 months for a clinical benefit rate (CBR) of 40%. Tissue was available in 43 pts and 32 (74%) had a PI3K pathway activating aberrations. PI3K pathway aberration was associated with a significant improvement in ORR (31 vs 0%; P = 0.04) but not CBR (44 vs 45%; P = 1.00) or progression-free survival (median 5 vs 3 months; P = 0.35). The most frequent PI3K pathway aberration was mutation in PIK3CA, occurring in 19 patients. Outcomes were similar if mutations of PIK3CA were located in the helical or kinase domain (ORR 25% vs 27%; P = 1.00 and CBR 38% vs 47%; P = 1.00, respectively). Spindle cell was the most frequent metaplastic histology seen, occurring in 18 tumors and mixed with other metaplastic histologies including squamous, chondroid and osseous in 14 additional tumors, while 20 tumors had non-spindle cell morphologies. The incidence of PI3K pathway aberration was similar across histologies (61% in spindle vs 67% in mixed spindle vs 60% in non-spindle cell). Tumors with mixed histology had lower ORR, CBR and PFS, but this was not statistically significant, likely due to small numbers in each cohort: ORR 22% in spindle vs 7% in mixed spindle vs 30% in non-spindle cell, P = 0.27; CBR 50% in spindle vs 21% in mixed spindle vs 40% in non-spindle cell, P = 0.25; and PFS median 4 months in spindle vs 2 months in mixed spindle vs 5 months in non-spindle cell, P = 0.68. Conclusions: Response to mTOR inhibition is enhanced in MpBCs with PI3K pathway aberrations. However, specific aberrations in PIK3CA do not lead to differential response to mTOR inhibition. PI3K pathway aberrations and response to mTOR inhibition are seen across all histologies of MpBC, and the response is not enhanced in particular histologies. Citation Format: Reva K. Basho, Michael Gilcrease, Rashmi K. Murthy, Thorunn Helgason, Daniel J. Booser, Daniel D. Karp, Funda Meric-Bernstam, Kenneth R. Hess, Shelley M. Herbrich, Vicente Valero, Constance Albarracin, Jennifer Litton, Mariana Chavez-MacGregor, Nuhad K. Ibrahim, James L. Murray, Kimberly B. Koenig, David Hong, Vivek Subbiah, Razelle Kurzrock, Filip Janku, Stacy Moulder. Targeting the PI3K/AKT/mTOR pathway for the treatment of metaplastic breast cancer: Does location of PIK3CA mutation or histology affect response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2273.

We hypothesized that distinct biological processes might be associated with prognosis and chemotherapy sensitivity in the different types of breast cancers.We performed gene set analyses with BRB-ArrayTools statistical software including 2331 functionally annotated gene sets (ie, lists of genes that correspond to a particular biological pathway or biochemical function) assembled from Ingenuity Pathway Analysis and Gene Ontology databases corresponding to almost all known biological processes. Gene set analysis was performed on gene expression data from three cohorts of 234, 170, and 175 patients with HER2-normal lymph node-negative breast cancer who received no systemic adjuvant therapy to identify gene sets associated prognosis and three additional cohorts of 198, 85, and 62 patients with HER2-normal stage I-III breast cancer who received preoperative chemotherapy to identify gene sets associated with pathological complete response to therapy. These analyses were performed separately for estrogen receptor (ER)-positive and ER-negative breast cancers. Interaction between gene sets and survival and treatment response by breast cancer subtype was assessed in individual datasets and also in pooled datasets. Statistical significance was estimated with permutation test. All statistical tests were two-sided.For ER-positive cancers, from 370 to 434 gene sets were associated with prognosis (P ≤ .05) and from 209 to 267 gene sets were associated with chemotherapy response in analysis by individual dataset. For ER-positive cancers, 131 gene sets were associated with prognosis and 69 were associated with pathological complete response (P ≤.001) in pooled analysis. Increased expression of cell cycle-related gene sets was associated with poor prognosis, and B-cell immunity-related gene sets were associated with good prognosis. For ER-negative cancers, from 175 to 288 gene sets were associated with prognosis and from 212 to 285 gene sets were associated with chemotherapy response. In pooled analyses of ER-negative cancers, 14 gene sets were associated with prognosis and 23 were associated with response. Gene sets involved in sphingolipid and glycolipid metabolism were associated with better prognosis and those involved in base excision repair, cell aging, and spindle microtubule regulation were associated with chemotherapy response.Different biological processes were associated with prognosis and chemotherapy response in ER-positive and ER-negative breast cancers.

A combination of uracil and ftorafur (UFT) was developed to combine the cytotoxic effects of 5-fluorouracil (5-FU) with convenient oral dosing. Leucovorin was combined with UFT to further potentiate the effect of 5-FU on tumor cells. Orally administered UFT and leucovorin provided higher peak plasma concentrations of 5-FU and prolonged therapeutic 5-FU concentrations compared with continuous infusion of 5-FU.Ninety-one patients with metastatic breast cancer who had been previously treated with anthracyclines and/or taxanes were treated with UFT and leucovorin, given orally, for the first 28 days of a 35-day cycle. The total daily dose of UFT was 300 mg/m(2), administered in 3 doses of 100 mg/m(2) each every 8 hours. The primary endpoint was time to disease progression (TTP). Secondary objectives included overall tumor response rate (overall response equals complete response plus partial response) and overall survival.Of the 91 patients enrolled, 70 were evaluable for efficacy. Although no complete responses were observed, 7 patients had partial responses, for an overall response rate of 10% in the evaluable population. The median TTP for the evaluable population was 10 weeks, and the proportion of patients who were free of disease progression at 6 months was 23%. The median overall survival was 59.4 weeks for all patients enrolled. Common, drug-relatedor = grade 3 adverse events (graded according to National Cancer Institute Common Toxicity Criteria, version 2) included diarrhea, vomiting, abdominal pain, and nausea.The combination of UFT and leucovorin administered orally in a 3-times-daily regimen was found to have modest activity. Grade 3 toxicities were manageable with appropriate dose adjustments in patients with metastatic breast cancer previously treated with anthracyclines and/or taxanes.

Introduction: As part of the MicroArray Quality Control (MAQC)-II project, this analysis examines how the choice of univariate feature-selection methods and classification algorithms may influence the performance of genomic predictors under varying degrees of prediction difficulty represented by three clinically relevant endpoints. Methods: We used gene-expression data from 230 breast cancers (grouped into training and independent validation sets), and we examined 40 predictors (five univariate feature-selection methods combined with eight different classifiers) for each of the three endpoints. Their classification performance was estimated on the training set by using two different resampling methods and compared with the accuracy observed in the independent validation set. Results: A ranking of the three classification problems was obtained, and the performance of 120 models was estimated and assessed on an independent validation set. The bootstrapping estimates were closer to the validation performance than were the cross-validation estimates. The required sample size for each endpoint was estimated, and both gene-level and pathway-level analyses were performed on the obtained models. Conclusions: We showed that genomic predictor accuracy is determined largely by an interplay between sample size and classification difficulty. Variations on univariate feature-selection methods and choice of classification algorithm have only a modest impact on predictor performance, and several statistically equally good predictors can be developed for any given classification problem.

Purpose: Preclinical data showed enhancement of breast cancer cell death when G3139 was combined with anthracyclines and taxanes. We evaluated the efficacy and safety of a Bcl-2 antisense oligonucleotide, G3139, in combination with doxorubicin (A) and docetaxel (T) in patients with locally advanced breast cancer (LABC). Experimental Design: Following a brief phase I to determine the phase II dose, patients with locally advanced breast cancer received G3139 administered by continuous i.v. infusion for 5 to 7 days with bolus A (50 mg/m2) and T (75 mg/m2) administered on either day 3 or 6 of therapy with G3139. Cycles were repeated every 21 days × 6 in the neoadjuvant setting. Serial plasma samples were obtained for pharmacokinetic analysis. Tissue samples were obtained before and after therapy for pharmacodynamic analysis of Bcl-2 expression. Results: Thirty patients (median age, 49 years; range, 24-71 years) received 160 cycles. During the phase I portion of the trial, the dose of G3139 was escalated from 3 to 7 mg/kg/d (i.v. for 5 days) in combination with AT. During the phase II portion of the trial, several doses and schedules of G3139 were evaluated. There were no pathologic complete responses. Pharmacodynamic studies showed limited Bcl-2 down-regulation in the primary tumors. Conclusions: G3139 in combination with doxorubicin and docetaxel is well tolerated. No pathologic complete response was seen and pharmacodynamic studies showed very little down-regulation of Bcl-2 in primary tumors, perhaps related to issues with insufficient drug delivery to the intact tumor.

Background: Novel molecular therapies for metastatic breast cancer (MBC) are necessary to improve the dismal prognosis of this condition. Imatinib mesylate (Gleevec®) inhibits several protein tyrosine kinases, including platelet-derived growth factor receptor (PDGFR) and c-kit, which are preferentially expressed in tumor cells. We tested the activity of imatinib mesylate in MBC with overexpression of PDGFR or c-kit. Additionally, we sought to determine the biological correlates and immunomodulatory effects. Patients and methods: Thirteen patients were treated with Imatinib administered orally at 400 mg p.o. b.i.d. (800 mg/day), until disease progression. All patients demonstrated PDGFR-β overexpression and none showed c-kit expression. Results: No objective responses were observed among the 13 patients treated in an intention-to-treat analysis. All patients experienced disease progression, with a median time to progression of 1.2 months. Twelve patients have died, and the median overall survival was 7.7 months. No patient had a serious adverse event. Imatinib therapy had no effect on the plasma levels of the angiogenesis-related cytokines, vascular endothelial growth factor, PDGF, b-fibroblast growth factor, and E-selectin. Immune studies showed imatinib inhibits interferon-γ production by TCR-activated CD4+ T cells. Conclusion: Imatinib as a single agent has no clinical activity in PDGFR-overexpressing MBC and has potential immunosuppressive effects.

Primary systemic therapy (PST) is the standard approach to the management of patients with locally advanced breast cancer (LABC). The authors hypothesized that the intratumoral administration of a nonreplicating adenoviral vector (Ad5) that contains the human wild-type p53, AdCMV-p53, combined with chemotherapy, could increase the efficacy of PST as measured by pathologic complete response.: In a prospective, open-label, Phase II trial, 13 patients with LABC were treated with 6 3-week cycles of PST, which consisted of intratumoral injections of Ad5CMV-p53 for 2 consecutive days plus docetaxel and doxorubicin followed by surgery. p53 status was determined at baseline and was assessed immediately after the first injection (up to 48 hours). Clinical response was assessed by clinical and radiologic methods.: The trial was terminated early, because none of the patients achieved a pathologic complete response. The median age was 56 years (range, 39-71 years), and the median tumor size was 8 cm (range, 5-11 cm). Eight patients (73%) had a p53 mutation. Serial biopsies showed an increase in p53 messenger RNA (mRNA) and p21(WAF1/Cip1) mRNA. All 12 evaluable patients achieved an objective clinical response. The surgical specimens revealed scattered tumor cells with extensive tumor-infiltrate leukocytes (predominantly T-lymphocytes). At a median follow-up of 37 months (range, 30-41 months), 4 patients (30%) developed systemic recurrence, and 2 patients died. The estimate breast cancer-specific survival rate at 3 years was 84% (95% confidence interval, 65.7-100%). There was no increase in systemic toxicity.: Ad5CMV-p53 combined with PST is safe, active, and associated with local immunomodulatory effects. The promising clinical activity of this combination deserves further investigation in randomized studies.

BACKGROUND Accurate assessment of HER-2 status is necessary prior to anti-HER-2 antibody (trastuzumab) therapy for metastatic breast carcinoma. However, controversy exists regarding whether to assess HER-2 status in the primary tumor or in metastatic lesions. It is also unclear whether HER-2 status can change during disease progression or after chemotherapy. METHODS Breast carcinoma samples from 60 women with known HER-2 status in both primary tumors and paired metastases (locoregional disease, n = 43 patients; distant disease, n = 17 patients) were reviewed retrospectively. Thirty-two patients underwent chemotherapy before their metastatic lesions were sampled, including 18 patients who received neoadjuvant chemotherapy and 14 patients who received adjuvant chemotherapy. The HER-2 gene was examined by fluorescence in situ hybridization either in paraffin-embedded tissue samples (48 primary tumors and 9 metastatic tumors) or in fine-needle aspirates (12 primary tumors and 51 metastatic tumors). HER-2 gene amplification was defined as a HER-2:chromosome 17 signal ratio ≥ 2.0. RESULTS The HER-2 status of primary and metastatic tumors agreed in 58 of 60 patients (97%), including 18 (30%) amplified tumors and 40 (67%) nonamplified tumors. A discrepancy in HER-2 status was observed in specimens from two patients in which HER-2 amplification was detected in the primary tumor but not the metastatic tumors. In one patient, three foci of tumor nodules were found in the same breast; the HER-2 status was assessed in only one of them, which showed amplification; however, HER-2 amplification was not detected in the axillary lymph node metastasis. In another patient, the HER-2 gene was amplified in the primary tumor but not in the liver metastasis. No metastases showed HER-2 amplification without amplification in the primary tumor. Locoregional and distant metastases demonstrated similar concordance rates with their corresponding primary tumors (98% and 94%, respectively). Complete concordance of HER-2 status was found between primary tumors prior to chemotherapy and metastases that were sampled after chemotherapy. CONCLUSIONS The HER-2 status in breast carcinoma generally was stable during metastasis, whether to locoregional or distant sites. Chemotherapy did not modify the HER-2 status in metastatic lesions. Therefore, HER-2 amplification can be evaluated reliably in material from either primary or metastatic tumors in most patients. Further study with larger series is warranted to elucidate the significance of discordant results. Cancer 2005. © 2005 American Cancer Society.

Purpose: TAS-108 is a novel steroidal anti-estrogen compound that has a strong binding affinity to the estrogen receptor and, in preclinical studies, has antitumor activity against tamoxifen-resistant breast cancer cell lines. The objective of this study was to investigate the safety and the pharmacokinetics in patients with previously treated advanced breast cancer. Experimental Design: TAS-108 was administered orally once daily starting at 40 mg/day, with dose escalations of 60, 80, 120, and 160 mg/day. A minimum of three patients were enrolled in each dose level, and, if no drug-related grade 3 or higher adverse events were seen in the first 14 days, the next cohort of patients was treated at the next level. Pharmacokinetic data were obtained on day 1, 2, 15, and 28 of the first course. Results: A total of 16 patients were enrolled, and most had received six to seven prior therapies. Clinical toxicities included nausea, vomiting, hot flashes, headache, weakness and fatigue; all were grade 1–2. TAS-108 had no effect on endometrial thickness based on trans-vaginal ultrasound evaluation. The average duration of therapy was 17.4 weeks (range, 4–60 weeks). The mean terminal half-life ranged from 8.0 to 10.7 hour in the interval of 12 to 24 hours postdose. The mean Cmax ranged from 2.8 to 21.0 ng/mL and AUC0-t from 15.1 to 148.7 ng·h/mL, this showed a linear correlation with the dose. Conclusions: TAS-108 was well tolerated in the doses studied with no maximum tolerated dose. The drug has linear pharmacokinetics, and in this heavily treated patient population, there was evidence of biological antitumor activity. A multi-institutional phase II study is planned.

We evaluated the feasibility and efficacy of a reduced-intensity conditioning (RIC) regimen of fludarabine and melphalan to achieve rapid complete donor chimerism after allogeneic stem cell transplantation (SCT) in patients with metastatic solid tumors. Between January 1999 and January 2003, 8 patients with metastatic breast cancer (BC) and 15 with metastatic renal cell carcinoma (RCC) underwent allogeneic SCT after an RIC regimen of 5 days of fludarabine and 2 days of melphalan. Filgrastim-mobilized stem cells from HLA-identical related or unrelated donors were infused. Prophylaxis for graft-versus-host disease (GVHD) consisted of tacrolimus and methotrexate. All 22 evaluable patients had 100% donor chimerism at day 30 and at all measurement times thereafter. One patient died 19 days after SCT. Nine patients (39%) had grades II to IV acute GVHD and 10 patients (43%) had chronic GVHD. Five patients (22%) died of nonrelapse treatment-related complications. Treatment-related disease response was seen in 10 patients (45%), with 3 complete responses, 2 partial responses, and 5 minor responses. Fludarabine-melphalan is a feasible and effective RIC regimen for allogeneic SCT in metastatic BC and RCC. It induces rapid complete donor chimerism without the need for donor lymphocyte infusion. Tumor regression associated with GVHD is consistent with graft-versus-tumor effect. (Blood. 2003;102:3829-3836)

In Phase I, the purpose was to determine the maximum tolerated dose and pharmacokinetics of docetaxel plus cyclophosphamide (DC) with and without granulocyte colony-stimulating factor in the treatment of patients with solid tumors. For Phase II, the purpose was to determine the safety and efficacy of this combination as first-line treatment in patients with metastatic breast cancer (MBC).In Phase I (45 patients), docetaxel was escalated from 60 mg/m(2) to 85 mg/m(2), and cyclophosphamide from 600 mg/m(2) to 800 mg/m(2). Pharmacokinetic evaluation of docetaxel was performed in 19 patients with MBC. In Phase II (34 patients), patients received cyclophosphamide (600 mg/m(2)) followed by docetaxel (75 mg/m(2)), i.v.In Phase I, the dose-limiting toxicity was neutropenia-related events. The maximum tolerated dose for DC was 75 mg/m(2)/700 mg/m(2) in solid tumor patients treated previously and 75 mg/m(2)/800 mg/m(2) for patients not treated previously for MBC. Dose escalation of docetaxel75 mg/m(2) was not tolerated, despite prophylactic granulocyte colony-stimulating factor treatment. In Phase II, 71% of patients received prior anthracycline therapy. Neutropenic fever requiring i.v. antibiotics occurred in 6 patients (19%). One patient had grade 3 neuropathy. There was no cardiotoxicity. The overall Phase II intent-to-treat objective response rate was 65% (complete responses, 12%). The median overall survival was 22 months, and the median time to progression was 6 months.DC combination therapy is an active regimen with acceptable toxicity and is appropriate regardless of prior anthracycline therapy. In view of the high activity and lack of cardiotoxicity, this combination warrants additional investigation in early stage breast cancer and in combination with trastuzumab.

To determine the toxicity and immunogenicity of the HER-2/neu, HLA-A2-restricted peptide E75 in patients with metastatic breast and ovarian cancer, 14 patients were vaccinated with escalating amounts of E75 (100, 500, and 1000 microg) mixed with 250 microg granulocyte macrophage colony-stimulating factor as adjuvant. Each vaccine dose was administered in a total volume of 1.5 ml divided into four intradermal injections and administered weekly for 4 weeks, followed by monthly boosts for a total of 10 injections. Vaccinations were well tolerated without significant toxicity. Blood was drawn before, at 8 weeks, and up to 13-16 months after vaccination for measurement of cellular immunity. Seven of 8 patients tested had significant delayed type hypersensitivity to E75 defined as5 mm induration. Peripheral blood mononuclear cells from 5 of 9 patients tested proliferated to E75 with a stimulation index ofor = 2.0. Of 8 vaccinated patients tested for induction of a CTL response, 4 responded to stimulation by autologous dendritic cells plus cytokines by eliciting E75-specific lytic activity consistent with the presence of activated/memory cells, 2 others after in vitro stimulation with E75 + interleukin-12 +/- anti-CD152(33KD), whereas 2 others did not respond. Four patients with E75-specific CTLs present specifically recognized E75 on indicator tumors as demonstrated by cold-target inhibition of tumor lysis. These 4 patients showed E75-specific IFN-gamma production. peripheral blood mononuclear cell from 3 of these patients proliferated to E75, but stimulation indices were higher in the prevaccine samples. All 4 of the patients showed DTH responses to E75. These results demonstrate that vaccination with E75+ granulocyte macrophage colony-stimulating factor can induce both peptide-specific IFN-gamma and epitope specific CTLs, which lyse HER-2/neu+ tumors in stage IV patients.

This Phase II study was designed to determine the efficacy and toxicity of combination doxorubicin and paclitaxel as front-line treatment for metastatic breast cancer.Eligible patients had no prior anthracycline or taxane therapy and normal cardiac function. They were treated with bolus doxorubicin 60 mg/m2, followed by paclitaxel 200 mg/m2, as either 1- or 3-h infusions for six to seven cycles. Single-agent paclitaxel was continued thereafter. Serial multiple gated acquisition scans were performed, and endomyocardial biopsies were performed for consenting patients.Eighty-two patients were enrolled with a median age of 53 years (range, 32-78 years). Of 79 evaluable patients, 58.2% had an objective response (3.8% complete response + 54.4% partial response), 34.2% had stable disease, and 7.6% had progressive disease. With median follow-up of 37.5 months, median time to progression was 7 months; median survival was 31 months. Multiple gated acquisition scans were performed in 82 of 82 patients at baseline, 75 of 82 patients at a total doxorubicin dose of 60-180 mg/m2, 62 of 68 patients at 200-300 mg/m2, 18 of 52 patients at 310-360 mg/m2, and 4 of 8 patients at 420 mg/m2. Median ejection fractions were 62.5, 60, 57.5, 52.5, and 32%, respectively. Fifteen of 82 (18.3%) patients had a decrease in ejection fractionor = 15% to an absolute ejection fractionor = 50%. Eight of these 15 patients (53%) developed clinical congestive heart failure: 4 of 8 (50%) who received a total doxorubicin dose of 420 mg/m2 versus 4 of 74 (5.4%) who received a doseor = 360 mg/m2 (P = 0.002).When the doxorubicin dose exceeds 360 mg/m2, the combination of bolus doxorubicin and paclitaxel presents unacceptable cardiac risk.

Paclitaxel has significant antitumor activity in patients with metastaticbreast cancer who have been previously treated with or exposed to anthracycline-containing chemotherapy. In this prospective randomized trial, the role of paclitaxel was evaluated in an adjuvant setting to determine its impact on reducing the risk of recurrence in patients with operable breast cancer.Five hundred twenty-four patients were randomized to be treated either with 4 cycles of paclitaxel followed by 4 cycles of combination therapy with 5-fluorouracil, Adriamycin, and cyclophosphamide (Pac/FAC) or with 8 cycles of FAC alone. Patients with intact primary breast cancer received the initial 4 cycles of paclitaxel or 4 cycles of FAC in a neoadjuvant setting. Planned duration of therapy was the same in all patients. After completion of 8 cycles of chemotherapy, those patients who wereor =50 years and whose tumors were positive for estrogen receptors received tamoxifen for 5 years.Ninety-two patients have had a recurrence after a median follow-up of 60 months with a range of 5-89 months. Estimated disease-free survival at 48 months was 0.83 for FAC and 0.86 for Pac/FAC group. The difference between the two groups was not statistically significant (P = 0.09). The overall estimated hazard ratio for Pac/FAC compared with FAC derived by fitting the Cox regression model and incorporating terms for prognostic factors was 0.66.Preliminary results suggest that the addition of paclitaxel to a FAC regimen of adjuvant or neoadjuvant therapy may further reduce the risk of disease recurrence; however, differences were not statistically significant. At the time of this analysis, there have been 47 deaths. The survival data are too preliminary to permit meaningful evaluation of the impact of paclitaxel on mortality.

The skeleton is the most frequent site of metastatic disease in breast cancer, involving cortical and trabecular bone with or without extension into adjacent bone marrow. A subset of breast cancer patients develop bone dominant disease without metastases to other organs over a prolonged period of time. An attractive strategy is to utilize bone targeted radiotherapy to address this site of disease with minimal systemic toxicity. This study was designed to determine the safety and preliminary efficacy of 166Ho-DOTMP, which localizes to bone providing radiation to adjacent marrow and malignant cells. The limiting toxicity is myelosuppression, which can be overcome by autologous stem cell transplantation. We studied this strategy in subjects with breast cancer metastatic to confined to the bone. Treatment occurred between 1997 and 1999. Subjects were eligible if they were If uptake was adequate, a therapeutic dose was to be administered in one of 2 dose levels, receiving 22 Gy (n=3) or 28 Gy (n=3). Treatment was followed by autologous stem cell transplantation when the ongoing radiation dose to marrow was 3 acute toxicity aside from the expected profound myelosuppression. All subjects treated in the trial had prompt trilineage hematologic recovery post transplant. Two patients developed hemorrhagic cystitis at approximately two years following therapy which resolved in both patients. One of these patients also had GI bleeding and pseudomembranous colitis unrelated to the 166Ho DOTMP treatment. One subject developed MDS, but was found to have a pre-existing trisomy 8 by FISH analysis from the peripheral blood stem cell product collected prior to treatment on this study. Two patients achieved complete remission and remain progression-free over 5 years following study entry. Four relapsed and died of progressive disease; all recurred in extraosseous sites with two also having disease in the bone marrow. Median time to progression was 10 months. Conclusions: Treatment with 166Ho DOTMP delivers targeted radiotherapy to the skeleton with an acceptable toxicity profile. Two of 6 patients with bone-only metastases achieved sustained complete responses. Further studies are warranted to evaluate this strategy for treatment of bone metastases.

Background Radiation is a standard component of treatment for patients with locoregional recurrence (LRR) of breast cancer following mastectomy. The current study reports the results of a 10% radiation dose escalation in these patients. Methods 159 patients treated at MD Anderson Cancer Center between 1994-2006 with isolated LRR after mastectomy alone were reviewed. Patients in the standard treatment group (65 pts, 40.9%) were treated to 50 Gy comprehensively plus a boost of 10 Gy. The dose escalated group (94 pts, 59.1%) was treated to 54 Gy comprehensively and a minimum 12 Gy boost. Median dose in the standard dose and dose escalated group was 60 Gy (±1 Gy, 95% CI) and 66 Gy (±0.5 Gy, 95% CI) respectively. Median follow up for living patients was 94 months from time of recurrence. Results The actuarial five year locoregional control (LRC) rate was 77% for the entire study population. The five year overall survival and disease-free survival was 55% and 41%, respectively. On multivariate analysis, initial tumor size (p = 0.03), time to initial LRR (p = 0.03), absence of gross tumor at the time of radiation (p = 0.001) and Her2 status (p = 0.03) were associated with improved LRC. Five year LRC rates were similar in patients with a complete response to chemotherapy without surgery and patients with a complete surgical excision (77% vs 83%, p = NS), compared to a 63% LRC rate in patients with gross disease at the time of radiation (p = 0.024). LRC rates were 80% in the standard dose group and 75% in the dose escalated group (p = NS). Conclusions While LRR following mastectomy is potentially curable, distant metastasis and local control rates remain suboptimal. Radiation dose escalation did not appear to improve LRC. Given significant local failure rates, these patients are good candidates for additional strategies to improve their outcomes.

Background: We performed next generation targeted sequencing of all known human protein kinases (n=530 genes) and 56 additional cancer related genes (BRCA1,2, p53, PTEN, etc..) in 112 samples to assess the kinase mutation landscape of breast cancer. Gene expression profiling was also performed on RNA from each specimen and DNA copy number variations were assessed in a subset of 59 cases using array CGH. The three types of genomic data were mapped to canonical biological pathways to identify frequently genomically disturbed pathways in these cancers. Methods: DNA and RNA extracted from fine needle biopsies of 92 breast cancers were analyzed, 20 samples were sequenced in duplicates or triplicates to assess technical variation of the results. Targeted sequencing was performed with Agilent SureSelect Human Kinome kit and the SOLiD sequencing platform. Gene expression profiling and array CGH were performed with Affymetrix U133A chips and Agilent 244K CGH array. Sequence data was mapped to hg19, functional impact scores were calculated with SWIFT, canonical pathways were obtained from the Broad Institute. Results: 0.1% of the entire genome was sequenced and >80% of target base pairs had > 20-fold coverage. The mean number of single nucleic acid variants (SNV) and indels per sample were 1043 (range: 493–1583, about 60% homozygous) and 159 (range: 75–269) respectively, 97% of SNVs and 78% of indels were already represented in dbSNP or COSMIC data bases. About 20% of SNVs were predicted to alter kinase or other biological function. The mean number of functionally high impact SNVs was 28 per sample (range 11–47). In addition to known p53 and PI3K mutations we detected frequent mutations in BRCA1 (20%) and observed several predicted high impact SNVs in HER2 (20%) as well as in many MAPK family enzymes. Not all SNVs were distributed equally across disease subsets, SNVs in ULK4, BMP2K, PALB2, ALPK3 were more frequent in triple negative cancers (TNBC) whereas SNVs in EPHA2 was more common in ER+ cancers. Among TNBC, those with residual cancer after neo-adjuvant chemotherapy (n=22) had significantly higher rates of SNVs in HUNK, TRPM7, NEK1 and HER3 compared to cases with pathologic complete response (n=25). When high impact SNVs, DNA copy number alterations and gene over-expression (relative to normal breast n=45) observed in individual cases were mapped to biological pathways a complex network of anomalies emerged for each case. Conclusion: We observed several known mutations in cancer genes and also detected many SNVs in important regulatory genes that were previously described as functional, germ-line variants with experimentally validated or suspected impact on protein function. Individual cancers have unique combinations of these events. This suggest that cancers arise in the context of complex genomic “germ line noise” which may determine which types of somatic events can or cannot “drive” individual cancers. The data also suggest therapeutic hypotheses about what biological pathways should be targeted in individuals and in subsets of cancers. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S6-4.

Prediction of high probability of survival from standard cancer treatments is fundamental for individualized cancer treatment strategies.To develop a predictor of response and survival from chemotherapy for newly diagnosed invasive breast cancer.Prospective multicenter study conducted from June 2000 to March 2010 at the M. D. Anderson Cancer Center to develop and test genomic predictors for neoadjuvant chemotherapy. Patients were those with newly diagnosed ERBB2 (HER2 or HER2/neu)-negative breast cancer treated with chemotherapy containing sequential taxane and anthracycline-based regimens (then endocrine therapy if estrogen receptor [ER]-positive). Different predictive signatures for resistance and response to preoperative (neoadjuvant) chemotherapy (stratified according to ER status) were developed from gene expression microarrays of newly diagnosed breast cancer (310 patients). Breast cancer treatment sensitivity was then predicted using the combination of signatures for (1) sensitivity to endocrine therapy, (2) chemoresistance, and (3) chemosensitivity, with independent validation (198 patients) and comparison with other reported genomic predictors of chemotherapy response.Distant relapse-free survival (DRFS) if predicted treatment sensitive and absolute risk reduction ([ARR], difference in DRFS between 2 predicted groups) at median follow-up (3 years).Patients in the independent validation cohort (99% clinical stage II-III) who were predicted to be treatment sensitive (28%) had 56% (95% CI, 31%-78%) probability of excellent pathologic response and DRFS of 92% (95% CI, 85%-100%), with an ARR of 18% (95% CI, 6%-28%). Survival was predicted in ER-positive (30% predicted sensitive; DRFS, 97% [95% CI, 91%-100%]; ARR, 11% [95% CI, 0.1%-21%]) and ER-negative (26% predicted sensitive; DRFS, 83% [95% CI, 68%-100%]; ARR, 26% [95% CI, 4%-48%]) subsets and was significant in multivariate analysis. Other genomic predictors showed paradoxically worse survival for patients predicted to be responsive to chemotherapy.A genomic predictor combining ER status, predicted chemoresistance, predicted chemosensitivity, and predicted endocrine sensitivity identified patients with high probability of survival following taxane and anthracycline chemotherapy.

A prospective randomized study was done to determine the effect of different doxorubicin (Adriamycin [ADR], Adria Laboratories, Columbus, OH) administration (schedules every week versus every 3 weeks) on the productivity of a cyclophosphamide, ADR, cisplatin (CAP) chemotherapy regimen for patients with non-small cell lung cancer (NSCLC). Electrocardiograms, multigated cardiac scans, echocardiograms, and endomyocardial biopsies were done serially for cardiac monitoring. Of 102 patients, 47 ahd inoperable limited disease (LD), 47 had extensive disease (ED), and eight had no evidence of disease. In the last group chemotherapy was given adjuvantly. Fifty-one patients were entered into each treatment arm. The groups were formed according to extent of disease and were comparable in terms of patient characteristics. In these groups, the overall response rates using both schedules in LD patients were similar: in patients without chest irradiation previously, there was a response of 35% with ADR weekly, and 31% with ADR triweekly; in LD patients with chest irradiation previously, the response was 20% with ADR weekly, and 25% with ADR triweekly; and in ED patients, 16% with ADR weekly, and 11% with ADR triweekly. There was no significant difference in survival between the two treatment groups. However, results for all responders suggested a longer duration of response with weekly than with triweekly ADR (complete plus partial response: 35.8 versus 11.4 weeks, P = 0.06; minor response: 34 versus 11.5 weeks, P = 0.003, respectively). Results also suggested that weekly ADR was less cardiotoxic than triweekly ADR: 29% of patients in the former group had no changes or only minor changes in endomyocardial biopsy results, whereas all patients in the latter group had at least grade 0.5 changes at a similar dosage. The median doses of weekly ADR were higher at the same endomyocardial biopsy-defined toxicity levels. No correlation was found between toxic effects defined by endomyocardial biopsy results and those defined by noninvasive monitoring techniques, although the number of patients assessed was small. Weekly ADR produced less granulocytopenia and a lower incidence of fever (6% versus 16%, P less than 0.001) than did triweekly ADR. Alopecia, nausea, vomiting, and diarrhea were significantly less for weekly ADR than triweekly Adr (P less than 0.0005, less than 0.0005, and less than 0.005, respectively). These data suggest that weekly ADR can achieve the same therapeutic results as the standard triweekly regimen with less cardiotoxicity, myelotoxicity, alopecia, diarrhea, nausea, and vomiting in patients with NSCLC.