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

Vasomotor symptoms have a significant impact on the quality of life of breast cancer patients. Clinical trials to determine the safest and the most effective ways to relieve these symptoms are needed.

BACKGROUND Obesity has been associated with inferior outcomes in operable breast cancer, but the relation between body mass index (BMI) and outcomes by breast cancer subtype has not been previously evaluated. METHODS The authors evaluated the relation between BMI and outcomes in 3 adjuvant trials coordinated by the Eastern Cooperative Oncology Group that included chemotherapy regimens with doxorubicin and cyclophosphamide, including E1199, E5188, and E3189. Results are expressed as hazard ratios (HRs) from Cox proportional hazards models (HR >1 indicates a worse outcome). All P values are 2-sided. RESULTS When evaluated as a continuous variable in trial E1199, increasing BMI within the obese (BMI, ≥30 kg/m2) and overweight (BMI, 25-29.9 kg/m2) ranges was associated with inferior outcomes in hormone receptor-positive, human epidermal growth receptor 2 (HER-2)/neu-negative disease for disease-free survival (DFS; P = .0006) and overall survival (OS; P = .0007), but not in HER-2/neu–overexpressing or triple-negative disease. When evaluated as a categorical variable, obesity was associated with inferior DFS (HR, 1.24; 95% confidence interval [CI], 1.06-1.46; P = .0008) and OS (HR, 1.37; 95% CI, 1.13-1.67; P = .002) in hormone receptor-positive disease, but not other subtypes. In a model including obesity, disease subtype, and their interaction, the interaction term was significant for OS (P = .02) and showed a strong trend for DFS (P = .07). Similar results were found in 2 other trials (E5188, E3189). CONCLUSIONS In a clinical trial population that excluded patients with significant comorbidities, obesity was associated with inferior outcomes specifically in patients with hormone receptor-positive operable breast cancer treated with standard chemohormonal therapy.

Summary GATA3 plays an integral role in breast luminal cell differentiation and is implicated in breast cancer progression. GATA3 immunohistochemistry is a useful marker of breast cancer; however, its use in specific subtypes is unclear. Here, we evaluate GATA3 expression in 86 invasive ductal carcinomas including triple-negative, Her-2, and luminal subtypes, in addition to 13 metaplastic carcinomas and in 34 fibroepithelial neoplasms. In addition, we report GATA3 expression in matched primary and metastatic breast carcinomas in 30 patients with known estrogen receptor (ER), progesterone receptor (PR), and Her-2 status, including 5 with ER and/or PR loss from primary to metastasis. Tissue microarrays containing 5 to 10 cores per tumor were stained for GATA3, scored as follows: 0 (0–5%), 1+ (6%–25%), 2+ (26%–50%), 3+ (51%–75%), and 4+ (>75%). GATA3 labeling was seen in 67% (66/99) of primary ductal carcinomas including 43% of triple-negative and 54% of metaplastic carcinomas. In contrast, stromal GATA3 labeling was seen in only 1 fibroepithelial neoplasm. GATA3 labeling was seen in 90% (27/30) of primary breast carcinomas in the paired cohort, including 67% of triple-negative carcinomas. GATA3 labeling was overwhelmingly maintained in paired metastases. Notably, GATA3 was maintained in all “luminal loss” metastases, which showed ER and/or PR loss. In conclusion, GATA3 expression is maintained between matched primary and metastatic carcinomas including ER-negative cases. GATA3 can be particularly useful as a marker for metastatic breast carcinoma, especially triple-negative and metaplastic carcinomas, which lack specific markers of mammary origin. Finally, GATA3 labeling may help distinguish metaplastic carcinoma from malignant phyllodes tumors.

A B S T R A C T PurposeGranulocyte-macrophage colony-stimulating factor (GM-CSF) -secreting tumor vaccines have demonstrated bioactivity but may be limited by disease burdens and immune tolerance. We tested the hypothesis that cyclophosphamide (CY) and doxorubicin (DOX) can enhance vaccine-induced immunity in patients with breast cancer. Patients and MethodsWe conducted a 3 ϫ 3 factorial (response surface) dose-ranging study of CY, DOX, and an HER2-positive, allogeneic, GM-CSF-secreting tumor vaccine in 28 patients with metastatic breast cancer. Patients received three monthly immunizations, with a boost 6 to 8 months from study entry. Primary objectives included safety and determination of the chemotherapy doses that maximize HER2-specific immunity. Results Twenty ConclusionFirst, immunotherapy with an allogeneic, HER2-positive, GM-CSF-secreting breast tumor vaccine alone or with CY and DOX is safe and induces HER2-specific immunity in patients with metastatic breast cancer. Second, the immunomodulatory activity of low-dose CY has a narrow therapeutic window, with an optimal dose not exceeding 200 mg/m 2 . Third, factorial designs provide an opportunity to identify the most active combination of interacting drugs in patients. Further investigation of the impact of chemotherapy on vaccine-induced immunity is warranted.

Purpose Population-based studies have shown improved survival for patients diagnosed with metastatic breast cancer (MBC) over time, presumably due to the availability of new and more effective therapies. The objective of this analysis was to determine if survival improved for patients who developed distant recurrence of breast cancer after receiving adjuvant therapy. Methods Adjuvant chemotherapy trials coordinated by the Eastern Cooperative Oncology Group (ECOG) that accrued patients between 1978–2002 were reviewed. Survival following distant recurrence was estimated for progressive time periods, and adjusted for baseline covariates in a Cox proportional hazards model. Results Of the 13,785 patients who received adjuvant chemotherapy in 11 trials, 3447 (24.4%) developed distant recurrence; median survival following recurrence was 20 months (95% confidence intervals: 19, 21). Factors associated with inferior survival included shorter distant recurrence free interval (DRFI), ER- and PR-negative disease number of positive axillary nodes at diagnosis and black race (p<0.0001 for all). When time-period of recurrence was added to the model, it was not significantly associated with survival for the general population with recurrence. Survival improved over time only in hormone-receptor negative patients with a DRFI ≤ 3 years, both among the 5 recent and entire trial datasets (p=0.01 and p=0.05 respectively). Conclusions In contrast to reports from population-based studies, we did not observe general improvement in survival over the last three decades for patients who developed distant recurrence after adjuvant chemotherapy after adjusting for DRFI. Improved survival for hormone-receptor negative patients with a short DRFI suggests benefit from trastuzumab.

Purpose: A comprehensive comparison of biomarker expression between patients' primary breast carcinoma (PBC) and their metastatic breast carcinomas (MBC) has not been done. Experimental Design: We did rapid autopsies (postmortem intervals,1-4 hours) on10 consenting patients who died of MBC.We constructed single-patient tissue microarrays from the patients' archived PBC and multiple different MBCs harvested at autopsy, which were immunohistochemically labeled for multiple biomarkers. Methylation of multiple gene promoters was assessed quantitatively on dissected PBC and MBC samples. Results: Extensive heterogeneity was observed between PBC and their paired MBC, as well as among multiple MBC from the same patient. Estrogen and progesterone receptors tended to be uniformly down-regulated in metastases. E-cadherin was down-regulated in a subset of the MBC of one case.Variable overexpression in MBC compared with the PBC was observed for cyclooxygenase-2 (five cases), epidermal growth factor receptor (EGFR; four cases), MET (four cases), and mesothelin (four cases). No case strongly overexpressed HER-2/neu by immunohistochemistry, but eight cases showed variable protein expression ranging from negative to equivocal (2+) in different MBC. In one case, variable low-level HER-2/neu gene amplification was found. EGFR and METoverexpression were restricted to the four basal-type cancers. EGFR protein overexpression did not correlate with EGFR gene amplification. Multigene promoter hypermethylation of RASSF1a, HIN1, cyclin D2,Twist, estrogen receptor a, APC1, and RARb was overall very similar in the PBC and all MBCs in all cases. Conclusions: Therapeutic targets identified in the PBC or even some MBC may not reflect targets present in all metastatic sites.

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