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Simple Summary: Breast cancer tumors are considered to have intratumoral, as well as human epidermal growth factor receptor 2 (HER2), heterogeneity. Tumors with high intratumoral heterogeneity (ITH) have demonstrated therapeutic resistance. However, studies on cancer heterogeneity as a prognostic factor in breast cancer have been limited. Therefore, we evaluated HER2 ITH, which was manifested by the shape of HER2 fluorescence in situ hybridization amplification distributed histograms (HER2 FISH distributions) with the HER2 gene copy number within a tumor sample. We aimed to determine whether high HER2 heterogeneity is clinically significant for poor prognosis due to resistance to postoperative adjuvant therapy with HER2-targeted agents in primary breast cancer. Indeed, we were able to show herein that high HER2 heterogeneity is significantly associated with poorer prognosis in patients with HER2-positive breast cancer. These results indicated that high HER2 heterogeneity is a factor predicting poor prognosis in patients with HER2-positive breast cancer. Our present observation seemed to be clinically important, because it is expected that our HER2 FISH distribution analysis of heterogeneity might be a convenient and clinically useful method for the prognosis prediction of patients after HER2 adjuvant therapy. Purpose: Breast cancer tumors frequently have intratumoral heterogeneity (ITH). Tumors with high ITH cause therapeutic resistance and have human epidermal growth factor receptor 2 (HER2) heterogeneity in response to HER2-targeted therapies. This study aimed to investigate whether high HER2 heterogeneity levels were clinically related to a poor prognosis for HER2-targeted adjuvant therapy resistance in primary breast cancers. Methods: This study included patients with primary breast cancer (n = 251) treated with adjuvant HER2-targeted therapies. HER2 heterogeneity was manifested by the shape of HER2 fluorescence in situ hybridization amplification (FISH) distributed histograms with the HER2 gene copy number within a tumor sample. Each tumor was classified into a biphasic grade graph (high heterogeneity [HH]) group or a monophasic grade graph (low heterogeneity [LH]) group based on heterogeneity. Both groups were evaluated for disease-free survival (DFS) and overall survival (OS) for a median of ten years of annual follow-up. Results: Of 251 patients with HER2-positive breast cancer, 46 (18.3%) and 205 (81.7%) were classified into the HH and LH groups, respectively. The HH group had more distant metastases and a poorer prognosis than the LH group (DFS: p < 0.001 (HH:63% vs. LH:91% at 10 years) and for the OS: p = 0.012 (HH:78% vs. LH:95% at 10 years). Conclusions: High HER2 heterogeneity is a poor prognostic factor in patients with HER2-positive breast cancer. A novel approach to heterogeneity, which is manifested by the shape of HER2 FISH distributions, might be clinically useful in the prognosis prediction of patients after HER2 adjuvant therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: ESR1 mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors (AI). These mutations are common in metastatic breast cancer (MBC). In past reports, mutations in primary tumors were so rare that they were thought to occur de novo with AI therapy. Conversely, previous studies using droplet digital polymerase chain reaction (ddPCR) have suggested the existence of ESR1-mutant minor clones with low allele frequencies; however, no large-scale studies have been conducted. In this study, we attempted to detect ultra-rare ESR1 mutations in primary breast cancer tumors using locked nucleic acid (LNA)-clamp ddPCR, and 28 ESR1 mutations were found in 27 patients. Most of these mutations were minor clones with a variant allele frequency of <0.1% which may have been overlooked by conventional methods. LNA-clamp ddPCR can also be applied to detect other gene mutations, which would be very useful. ESR1 mutations in breast cancer are one of the mechanisms of resistance to aromatase inhibitors. These mutations are common in metastatic breast cancer; however, these are rare in primary breast cancer. However, these data have been analyzed mainly in formalin-fixed, paraffin-embedded tissue; thus, rare mutations that may be present in primary breast cancer may be overlooked. In this study, we developed a highly sensitive mutation detection method called locked nucleic acid (LNA)-clamp droplet digital PCR (ddPCR) and validated it. The mutation detection sensitivity was substantiated to 0.003%. Then, we used this method to analyze ESR1 mutations in fresh-frozen (FF) tissues of primary breast cancer. cDNA extracted from the FF tissues of 212 patients with primary breast cancers were measured. Twenty-eight ESR1 mutations were found in twenty-seven (12.7%) patients. Sixteen (7.5%) patients had Y537S mutations and twelve (5.7%) had D538G mutations. Two mutations with a variant allele frequency (VAF) of ≥0.1% and twenty-six mutations with a VAF of <0.1% were found. By using this LNA-clamp ddPCR, this study demonstrated the presence of minor clones with a VAF of <0.1% in primary breast cancer. [ABSTRACT FROM AUTHOR]

Bone metastasis significantly affects the quality of life of patients with metastatic breast cancer, and can shorten overall survival. Identifying patients with early-stage breast cancer at high risk for bone metastasis and preventing bone metastasis may lead to a better quality of life and prolonged survival. The present study investigated whether serum tartrate-resistant acid phosphatase-5b (TRACP-5b), a bone turnover marker, can be a prognostic factor for bone metastasis. Female patients who underwent resectable breast surgery between May 2002 and August 2006 were consecutively investigated. A total of 304 patients with a median follow-up of 3,722 days were retrospectively analyzed. TRACP-5b levels in sera prepared from patients' blood drawn preoperatively without any presurgical treatments were measured using an enzyme-linked immunosorbent assay. The cutoff of TRACP-5b levels, in order to separate patients into high and low TRACP-5b groups, was set at median (347 mU/dl). The associations of clinicopathological factors, including TRACP-5b, with bone metastasis-free interval (BMFI), which was defined as the duration between surgery and the diagnosis of bone metastasis at any time point, were examined. Multivariate analysis of various clinicopathological features revealed that lymph node metastasis and histological grade were independent factors associated with BMFI (P=0.017 and 0.030, respectively). In patients with node-positive breast cancer (n=114), a high TRACP-5b level and a high grade were significantly and independently associated with worse BMFI (log-rank P=0.041 and 0.011, respectively). In conclusion, these findings indicated that TRACP-5b may predict bone metastasis in patients with node-positive breast cancer. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to elucidate the clinicopathological characteristics of breast ductal carcinomas in situ (DCIS) with aldehyde dehydrogenase 1 (ALDH1)-positive cancer stem cells (CSCs). Methods: DCIS (n = 194) were subjected to immunohistochemical staining and results were examined for associations with various clinicopathological parameters. The ALDEFLUOR assay for breast cancer cell lines was also performed to determine the proportion of CSCs according to intrinsic subtype. Results: DCIS with ALDH1-positive CSCs were significantly (p < 0.05) more likely to be estrogen receptor-α (ER)-negative, progesterone receptor (PgR)-negative, Ki67-positive and HER2-positive tumors. Luminal A subtype (8.6%) showed a significantly (p < 0.001) lower ALDH1 positivity than the other subtypes [luminal B (50.0%), luminal HER2 (36.8%), HER2 (35.3%) and triple-negative subtype (26.7%)]. Double immunostaining revealed that ALDH1-positive CSCs did not overlap with ER-, PgR- or Ki67-positive tumor cells but did overlap with HER2-positive tumor cells. The percentage of ALDEFLUOR-positive CSCs was lower in the luminal A cell lines (0.02% for T-47D and 0.4% for MCF7) than in the luminal HER2 (9.1% for BT-474 and 9.5% for MDA-MB-361), HER2 (13.9% for AU565 and 33.2% for SK-BR-3) and triple-negative cell lines (28.4% for MDA-MB-231 and 30.7% for MDA-MB-468). Conclusions: Our results suggest that most CSCs in DCIS are in the G0 phase (Ki67 negative) and do not express ER or PgR, but they do express HER2 in HER2-positive tumors. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]