Prognostic value of serum tartrate­resistant acid phosphatase­5b for bone metastasis in patients with resectable breast cancer.

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.

High HER2 Intratumoral Heterogeneity Is a Predictive Factor for Poor Prognosis in Early-Stage and Locally Advanced HER2-Positive Breast Cancer.

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.

Evaluation of RASSF1A methylation in the lysate of sentinel lymph nodes for detecting breast cancer metastasis: A diagnostic accuracy study.

The restriction enzyme-based digital methylation-specific polymerase chain reaction (RE-dMSP) assay is useful for diagnosing sentinel lymph node (SN) metastasis in patients with breast cancer, by detecting tumor-derived methylated Ras association domain-containing protein 1 (RASSF1A). In addition, this assay has high concordance (95.0%) with one-step nucleic acid amplification (OSNA). The present study aimed to perform RE-dMSP using OSNA lysate from more patients and to re-evaluate its clinical usage. Overall, 418 SNs from 347 patients were evaluated using both OSNA and RE-dMSP. The concordance rate was 83.3% (348/418). RASSF1A methylation of the primary tumors was negative in 36 patients. When these patients were excluded, the concordance rate improved to 88.2% (330/374). Of the 79 OSNA-negative cases, 19 were RE-dMSP-positive, although all were positive for cytokeratin 19 expression in the primary tumor, suggesting that RE-dMSP can detect tumor-derived DNA with a higher sensitivity. The percent of methylated reference of the breast tumors showed a wide variety in the 16 OSNA-positive/RE-dMSP-negative cases, and such variability of methylation could have affected the results in these patients. In conclusion, although RE-dMSP can diagnose SN metastasis with high sensitivity and accuracy, and can be a supplementary tool to OSNA in breast cancer, RE-dMSP showed certain discordance with OSNA and critically depended on the absence or heterogeneity of DNA methylation in breast tumors. Further research is expected to develop an assay targeting other DNA alterations, such as mutations.

Detection of Ultra-Rare ESR1 Mutations in Primary Breast Cancer Using LNA-Clamp ddPCR.

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.

Hypercalcemia affected in metastatic breast cancer patients without bone metastasis: report of three cases.

BACKGROUND: Since humoral hypercalcemia of malignancy (HHM) in breast cancer patients without bone metastasis is rare, the clinical features of this condition are not fully understood. CASE PRESENTATION: During the recent 12 years, 3602 patients were diagnosed with breast cancer in our institution, and only three patients developed HHM without bone metastasis. They were all recurrent breast cancer patients with visceral metastases including the lung and the liver. It took no more than 2 months since symptomatic onset to hospitalization because of hypercalcemia. The maximum serum calcium concentrations were 15.0 mg/dL or higher. All patients had symptoms related to hypercalcemia. Treatment of hypercalcemia including hydration, calcitonin, bisphosphonate, and diuretics was initially effective in the three patients. However, two of three cases were eventually fatal because of unsuccessful treatment of breast cancer. CONCLUSIONS: The common features of HHM without bone metastasis in breast cancer patients include acute onset, severe symptomatic hypercalcemia, and presence of visceral metastasis. Treatment of hypercalcemia decreased serum calcium level in a short period, while successful treatment of breast cancer was essential for a long-term management of HHM. This report provides a consideration to help elucidate the pathophysiology and medical care of breast cancer patients with HHM without bone metastasis.

Molecular Barcode Sequencing of the Whole Ligand Binding Domain of the ESR1 Gene in Cell-Free DNA from Patients with Metastatic Breast Cancer.

ESR1 mutations in breast cancer are known as one of the mechanisms of resistance to aromatase inhibitors. These mutations often occur in the hotspot regions in the ligand binding domain (LBD), but comprehensive mutational analysis has shown that mutations are observed throughout the whole LBD. We previously developed a molecular barcode sequencing (MB-NGS) technique to detect ESR1 hotspot mutations in plasma with high sensitivity. In this study, we have developed a multiplex MB-NGS assay that covers the whole LBD of ESR1. The assay demonstrated that the background errors in the plasma DNA of 10 healthy controls were below 0.1%; thus, the limit of detection was set at 0.1%. We analyzed the plasma DNA of 54 patients with estrogen receptor-positive metastatic breast cancer. Seventeen mutations were detected in 13 patients (24%), with variant allele frequencies ranging from 0.13% to 10.67%, including six rare mutations with a variant allele frequency <1.0% and a novel nonhotspot mutation (A312V). Three patients had double mutations located in the same amplicons, and it was revealed that the double mutations were located in different alleles. ESR1 hotspot mutations were associated with a longer duration of aromatase inhibitor treatment under metastatic conditions and to liver metastasis. The multiplex MB-NGS assay is useful for the sensitive and comprehensive detection of mutations throughout the whole LBD of ESR1. Our assay can be applied to any specific target region of interest using tailor-made primers and can result in minimized sequencing volume and cost.

Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

PURPOSE: We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). METHODS: The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). RESULTS: MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. CONCLUSION: In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.