Background
The aim of our study is to find microRNAs (miRNAs) associated with sentinel lymph node metastasis (SLNM) and to develop a prediction model for SLNM in ER-positive and HER2-negative ...(ER+/HER2−) breast cancer.
Patients and Methods
In the present study, only ER+/HER2− primary breast cancer was considered. The discovery set for SLNM-associated miRNAs included 10 tumors with and 10 tumors without SLNM. The training and validation sets both included 100 tumors. miRNA expression in tumors was examined comprehensively by miRNA microarray in the discovery set and by droplet digital PCR in the training and validation sets.
Results
In the discovery set, miR-98, miR-22, and miR-223 were found to be significantly (
P
< 0.001, fold-change > 2.5) associated with SLNM. In the training set, we constructed the prediction model for SLNM using miR-98, tumor size, and lymphovascular invasion (LVI) with high accuracy (AUC, 0.877). The accuracy of this prediction model was confirmed in the validation set (AUC, 0.883), and it outperformed the conventional Memorial Sloan Kettering Cancer Center nomogram. In situ hybridization revealed the localization of miR-98 expression in tumor cells.
Conclusions
We developed a prediction model consisting of miR-98, tumor size, and LVI for SLNM with high accuracy in ER+/HER2− breast cancer. This model might help decide the indication for SLN biopsy in this subtype.
Zinc is an essential element, necessary for sustaining all life. Zinc deficiency causes taste impairments, immune deficiency, skin problems, and growth and mental retardation. Recent reports suggest ...that zinc is associated with an increased risk of cancer, although it is still unclear whether zinc or its transporters are involved in cancer progression. Here we show that zinc and its transporter ZIP10 are involved in the invasive behavior of breast cancer cells. The screening of clinical samples for ZIP10 mRNA expression suggested that ZIP10 was significantly associated with the metastasis of breast cancer to the lymph node. In addition, the expression of ZIP10 mRNA was higher in the invasive and metastatic breast cancer cell lines MDA‐MB‐231 and MDA‐MB‐435S than in less metastatic breast cancer cell lines, such as MCF7, T47D, ZR75‐1 and ZR75‐30. In in vitro cell migration assays, the depletion of zinc transporter ZIP10 and intracellular zinc inhibited the migratory activity of MDA‐MB‐231 and MDA‐MB‐435S cells. These results showed that zinc and ZIP10 play an essential role in the migratory activity of highly metastatic breast cancer cells, and suggest ZIP10 as a possible marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies. (Cancer Sci 2007; 98: 692–697)
Trastuzumab is a drug that targets the receptor tyrosine kinase HER2 and is essential for the treatment of HER2-positive breast cancer. Resistance to the drug leads to severe consequences, including ...disease recurrence, tumor enlargement, and metastasis. We hypothesized that trastuzumab treatment might be associated with phenotypic switching in HER2-positive breast cancer cells (BCCs), enabling them to escape and survive the effect of trastuzumab.
We conducted comprehensive immunophenotyping to detect phenotypic changes in HER2-positive BCCs treated with trastuzumab, based on criteria determined a priori. Based on immunophenotyping results, we characterized the vascular phenotypes of HER2-positive BCCs by western blotting, real-time RT-PCR, and tube formation assay. The vascular phenotype of tumor cells from clinical samples was evaluated by staining with periodic acid-Schiff and an anti-CD31 antibody. We explored small molecule inhibitors that suppress tube formation and determined the inhibitory mechanism.
Out of 242 cell surface antigens, 9 antigens were significantly upregulated and 3 were significantly downregulated by trastuzumab treatment. All upregulated antigens were related to endothelial and stem cell phenotypes, suggesting that trastuzumab treatment might be correlated to switching to a vascular phenotype, namely, vasculogenic mimicry (VM). Several VM markers were upregulated in trastuzumab-treated cells, but these cells did not form tubes on Matrigel, a functional hallmark of VM. Upon analysis of three trastuzumab-resistant HER2-positive cell lines, we found that all three cell lines showed tube formation on Matrigel in the presence of angiogenic growth factors including EGF, FGF2, IGF1, or VEGF. Clinically, VM channels significantly increased in surviving cancer cell clusters of surgically removed tumors pretreated with trastuzumab and chemotherapy compared to both surgically removed tumors without prior systemic treatment and tumors biopsied before presurgical treatment with trastuzumab. Finally, we found that salinomycin completely suppressed VM in all three trastuzumab-resistant cell lines through disruption of actin cytoskeletal integrity.
VM promotes metastasis and worsens patient outcomes. The present study indicates that HER2-positive BCCs can exhibit VM in an angiogenic microenvironment after eventually acquiring trastuzumab resistance. The clinical finding supports this in vitro observation. Thus, targeting VM might provide a therapeutic benefit to patients with HER2-positive breast cancer.
We attempted to develop a highly sensitive and specific method for the detection of circulating tumor DNA (ctDNA) using a digital PCR (dPCR) assay for
PIK3CA
mutations (i.e., H1047R, E545K, and ...E542K) in primary breast cancer patients. The sensitivity of the dPCR assay for the mutant alleles was examined using cell lines with
PIK3CA
mutations and proved to be 0.01 %. Serum samples were collected pre-operatively from 313 stage I–III breast cancer patients, of whom 110 were found to have
PIK3CA
mutant tumors. The serum samples from these patients with
PIK3CA
mutant tumors were subjected to the dPCR assay, and 25 (22.7 %) were found to be positive. No
PIK3CA
mutant ctDNA was detected in the serum samples of 50 healthy women and 30 breast cancer patients with
PIK3CA
non-mutant tumors. The patients with
PIK3CA
mutant ctDNA were dichotomized into mutant ctDNA-high (ctDNA
high
) and ctDNA-low (ctDNA
low
) groups based on the median. The ctDNA
high
patients exhibited significantly shorter recurrence-free survival (RFS;
P
= 0.0002) and overall survival rates (OS;
P
= 0.0048) compared to those exhibited by the combined ctDNA
low
patient and ctDNA-free patient group. Multivariate analysis revealed that ctDNA
high
status significantly predicted poor RFS and OS and did so independently of conventional histological parameters. These results suggest that dPCR is a highly sensitive and specific method for the detection of
PIK3CA
mutant ctDNA and that ctDNA
high
but not ctDNA
low
status is a significant and independent prognostic factor for primary breast cancer patients.
Purpose
Resistance against anti-HER2 drugs in HER2-positive breast cancer is a major obstacle to the improving prognosis. Transforming growth factor β (TGFβ) is a cytokine involved in the acquisition ...of more malignant phenotypes through epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. The aim of this study was to investigate the effects of TGFβ and its downstream SMAD pathway on resistance to anti-HER2 drugs.
Methods
HER2-positive breast cancer cell lines were stimulated with TGFβ for 14 days. Then, the sensitivity to trastuzumab and lapatinib and the expression levels of various EMT and CSC markers were examined. The correlation of nuclear SMAD3 expression in untreated breast tumor tissues with trastuzumab efficacy in neoadjuvant settings was examined. The effect of a small-molecule inhibitor of SMAD3 (SIS3) on resistance to anti-HER2 drugs was explored.
Results
We found that continuous activation of the TGFβ-SMAD3 pathway induced resistance to anti-HER2 drugs and CSC traits in HER2-positive breast cancer cells. The induction of drug resistance by TGFβ required strong activation of SMAD3. In fact, activated SMAD3 regulated multiple genes that harbor SMAD-binding elements and are involved in trastuzumab resistance. Nuclear SMAD3 expression in tumor tissue was inversely correlated with sensitivity to neoadjuvant treatment with trastuzumab. SIS3 not only prevented the acquisition of resistance to anti-HER2 drugs but also restored trastuzumab sensitivity in trastuzumab-resistant cells.
Conclusions
This study indicates that the TGFβ-SMAD3 pathway plays an important role in the induction and maintenance of resistance to anti-HER2 drugs. Thus, SMAD3 is a potential therapeutic target that can inhibit resistance and restore sensitivity to anti-HER2 drugs.
Anti-HER2-autoantibodies (HER2-AAbs) are found in breast cancer patients as well as healthy individuals. However, the clinical relevance of the antibodies is unknown. We established an enzyme-linked ...immunosorbent assay with high sensitivity and quantified serum HER2-AAbs in 100 healthy women, 100 untreated patients with ductal carcinoma in situ (DCIS), and 500 untreated patients with invasive breast carcinoma (IBC). The associations between the levels of HER2-AAbs and breast cancer risk, and recurrence-free survival, were examined. High levels of HER2-AAbs were significantly associated with a reduced risk of DCIS (odds ratio OR 0.19,
P
= 4.6 × 10
−7
) or IBC (OR 0.31,
P
= 3.7 × 10
−7
). Subgroup analysis of IBC revealed a stronger association of HER2-AAbs with a reduced risk of the hormone receptor (HR)
−
/HER2
+
subtype (OR 0.12) than the other subtypes (HR
+
/HER2
−
OR = 0.32, HR
+
/HER2
+
OR 0.38, and HR
−
/HER2
−
OR 0.29). When we set the cutoff of HER2-AAbs at 20 ng/mL, recurrence-free survival of HER2-AAb-positive patients (
N
= 74) was significantly better than that of HER2-AAb-negative patients (
N
= 426) (
P
= 0.015). Univariate and multivariate analyses demonstrated that HER2-AAbs, as well as histological grade, were independently and significantly (
P
= 0.0065 and 0.049, respectively) associated with recurrence-free survival. Our exploratory study suggests a protective effect of naturally occurring HER2-AAbs on the development of primary and recurrent breast cancer. Further studies on HER2-AAbs are warranted.
Background
Everolimus is a mechanistic-target-of-rapamycin (mTOR) inhibitor bearing a potent antitumor effect against hormone receptor-positive breast cancer. Here, we report the case of a patient ...with recurrent breast cancer who developed osteomyelitis during the treatment with everolimus plus exemestane.
Case presentation
A 56-year-old woman with early-stage breast cancer underwent right mastectomy and axillary lymph node dissection at the age of 45. Four years after the surgery, she experienced relapse at the chest wall. Radiotherapy was performed on the chest wall, including the sternum, and denosumab was administered. After several regimens of hormonal therapies, everolimus in combination with exemestane was administered. Three months later, the patient visited our clinic because of continuous fever. A computed tomography scan showed an osteolytic change in the sternal bone with pneumomediastinum, which indicated sternal osteomyelitis. Extensive debridement followed by secondary reconstruction of the chest wall was successfully performed.
Conclusions
Everolimus may cause osteomyelitis of the affected bone as a result of tumor necrosis. Everolimus-induced osteomyelitis may be manageable by extensive debridement performed without delay.
Expression of specific breast cancer stem cells (BCSCs) is seen in aggressive tumors, but their regulation is unclear. Epigenetic changes influence gene expression and are implicated in breast cancer ...progression. We hypothesized that promoter methylation regulates specific BCSC-related genes CD44 , CD133 , CD24 , MSH1 (alias, Musashi-1 ), and ALDH1 and that this epigenetic profile can identify aggressive subtypes, such as triple-negative breast cancer (TNBC). Methylation analysis was performed using MassARRAY EpiTYPER sequencing; CpG-rich sites were identified in the promoter regions of BCSC genes, except ALDH1 . These sites were screened by treatment with 5-aza-2′-deoxycytidine in four TN and five non-TNBC cell lines. The specific regulatory CpG site demonstrating the most significant inverse correlation between CpG site methylation and mRNA expression was identified for CD44 , CD133 , and Musashi-1 , but not for CD24 . Methylation of CD44 , CD133 , and Musashi-1 was evaluated in 91 American Joint Committee on Cancer stage I to III primary breast cancer tumors, and these sites were significantly hypomethylated in TNBC versus non-TNBC. The IHC staining of primary tumors with the highest and lowest methylation levels revealed the strongest staining in hypomethylated specimens, suggesting that hypomethylation leads to gene activation. We demonstrate that methylation is a significant mechanism regulating CD44 , CD133 , and Musashi-1 , and that gene hypomethylation correlates with TNBC. Assessment of epigenetic changes in BCSC genes may provide a more accurate classification of TNBC and could be developed as potential therapeutic targets.
Purpose
Liquid biopsy using digital PCR (dPCR) has been widely used for the screening of
ESR1
mutations, since they are frequently identified in the hotspot. However, dPCR is limited to the known ...mutations. Therefore, we aimed to analyze the utility of next-generation sequencing (NGS) to discover novel
ESR1
mutations.
Methods
Whole exon sequencing of the
ESR1
gene using NGS was performed in 16 primary and 47 recurrent tumor samples and 38 plasma samples from hormone receptor-positive metastatic breast cancer patients. Functional analyses were then performed for the novel mutations we detected.
Results
We identified no mutations in primary tumors and six mutations in five recurrent tumors, including three types of known mutations (Y537C, Y537N, and D538G) and two novel mutations (E279V and G557R). We also identified seven mutations in five plasma samples, including three types of known mutations (S463P, Y537S, and D538G) and one mutation not reported in COSMIC database (L536H). All nine patients with
ESR1
mutations were treated with aromatase inhibitors (AIs) prior to sampling, and the mutations were frequently detected in patients who received AI treatments in the metastatic setting. Among the three novel mutations (E279V, L536H, and G557R), L536H, but not E279V and G557R, showed ligand-independent activity. All three mutant proteins showed nuclear localization and had no relation with non-genomic ER pathways.
Conclusions
Although the molecular mechanisms of the E279V and G557R mutations remain unclear, our data suggest the utility of NGS as a liquid biopsy for metastatic breast cancer patients and the potential to identify novel
ESR1
mutations.
Purpose
Prediction models for late (> 5 years) recurrence in ER-positive breast cancer need to be developed for the accurate selection of patients for extended hormonal therapy. We attempted to ...develop such a prediction model focusing on the differences in gene expression between breast cancers with early and late recurrence.
Methods
For the training set, 779 ER-positive breast cancers treated with tamoxifen alone for 5 years were selected from the databases (GSE6532, GSE12093, GSE17705, and GSE26971). For the validation set, 221 ER-positive breast cancers treated with adjuvant hormonal therapy for 5 years with or without chemotherapy at our hospital were included. Gene expression was assayed by DNA microarray analysis (Affymetrix U133 plus 2.0).
Results
With the 42 genes differentially expressed in early and late recurrence breast cancers in the training set, a prediction model (42GC) for late recurrence was constructed. The patients classified by 42GC into the late recurrence-like group showed a significantly (
P
= 0.006) higher late recurrence rate as expected but a significantly (
P
= 1.62 × E−13) lower rate for early recurrence than non-late recurrence-like group. These observations were confirmed for the validation set, i.e.,
P
= 0.020 for late recurrence and
P
= 5.70 × E−5 for early recurrence.
Conclusion
We developed a unique prediction model (42GC) for late recurrence by focusing on the biological differences between breast cancers with early and late recurrence. Interestingly, patients in the late recurrence-like group by 42GC were at low risk for early recurrence.
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