Akihiko Osaki

Prognostic significance of co-expression of c-erbB-2 oncoprotein and epidermal growth factor receptor in breast cancer patients

The expression of c-erbB-2 oncoprotein and epidermal growth factor receptor (EGFR) was examined by immunocytochemical and radioreceptor assays in 115 patients with primary breast cancer. In 48 of 115 patients (42%), the assays were found to be positive for the expression of c-erbB-2 oncoprotein, and, in 44 of 115 (35%) patients, the assays were positive for the expression of EGFR. There was no correlation between the expression of c-erbB-2 oncoprotein and EGFR. Clinical survey demonstrated that both c-erbB-2 oncoprotein expression and EGFR expression have independent prognostic values. Furthermore, when patients were divided into three groups on the basis of the expression of both c-erbB-2 oncoprotein and EGFR, those who were found to be positive for the expression of both c-erbB-2 oncoprotein and EGFR showed a worse prognosis than other groups. These results suggest that the combination of the expression of both c-erbB-2 oncoprotein and EGFR may be important in selecting patients who have a poor prognosis.

Estrogen-related receptor γ modulates cell proliferation and estrogen signaling in breast cancer

Breast cancer is primarily a hormone-dependent tumor that can be regulated by status of steroid hormones including estrogen and progesterone. Estrogen-related receptors (ERRs) are orphan nuclear receptors most closely related to estrogen receptor (ER) and much attention has been recently paid to the functions of ERRs in breast cancer in terms of the interactions with ER. In the present study, we investigated the expression of ERRγ in human invasive breast cancers by immunohistochemical analysis (n=110) obtained by radical mastectomy. Nuclear immunoreactivity of ERRγ was detected in 87 cases (79%) and tended to correlate with the lymph node status. No significant associations were observed with other clinicopathological characteristics, including the expression levels of both estrogen and progesterone receptors. In MCF-7 breast cancer cells, we demonstrated that ERRγ mRNA was up-regulated dose-dependently by estrogen, and that this up-regulation of ERRγ mRNA by estrogen was abolished by ICI 182,780 treatment. We also demonstrated that exogenously transfected ERRγ increased MCF-7 cell proliferation. Furthermore, ERRγ enhanced estrogen response element (ERE)-driven transcription in MCF-7 cells. In 293T cells, ERRγ could also stimulate ERE-mediated transcription with or without ERα. These results suggest that ERRγ plays an important role as a modulator of estrogen signaling in breast cancer cells.

FOXP1, an estrogen-inducible transcription factor, modulates cell proliferation in breast cancer cells and 5-year recurrence-free survival of patients with tamoxifen-treated breast cancer.

Breast cancer is primarily a hormone-dependent tumor that can be regulated by the status of steroid hormones, including estrogen and progesterone. Forkhead box P1 (FOXP1) is a member of the forkhead box transcription factor family and has been reported to be associated with various types of tumors. In the present study, we investigated the expression of FOXP1 in 133 human invasive breast cancers, obtained by core biopsy, by immunohistochemical analysis. Nuclear immunoreactivity of FOXP1 was detected in 89 cases (67%) and correlated positively with tumor grade and hormone receptor status, including estrogen receptor alpha (ERα) and progesterone receptor, and negatively with pathological tumor size. In ERα-positive MCF-7 breast cancer cells, we demonstrated that FOXP1 mRNA was upregulated by estrogen and increased ERα recruitment to ER binding sites identified by ChIP-on-chip analysis within the FOXP1 gene region. We also demonstrated that proliferation of MCF-7 cells was increased by exogenously transfected FOXP1 and decreased by FOXP1-specific siRNA. Furthermore, FOXP1 enhanced estrogen response element-driven transcription in MCF-7 cells. Finally, FOXP1 immunoreactivity was significantly elevated in relapse-free breast cancer patients treated with tamoxifen. These results suggest that FOXP1 plays an important role in proliferation of breast cancer cells by modulating estrogen signaling and that FOXP1 immunoreactivity could be associated with the estrogen dependency of clinical breast cancers, which may predict favorable prognosis in the patients treated with tamoxifen.

Optical imaging of tumor vascularity associated with proliferation and glucose metabolism in early breast cancer: clinical application of total hemoglobin measurements in the breast.

BackgroundNear-infrared optical imaging targeting the intrinsic contrast of tissue hemoglobin has emerged as a promising approach for visualization of vascularity in cancer research. We evaluated the usefulness of diffuse optical spectroscopy using time-resolved spectroscopic (TRS) measurements for functional imaging of primary breast cancer.MethodsFifty-five consecutive TNM stageI/II patients with histologically proven invasive ductal carcinoma and operable breast tumors (<5 cm) who underwent TRS measurements were enrolled. Thirty (54.5%) patients underwent 18F-fluoro-deoxy-glucose (FDG) positron emission tomography with measurement of maximum tumor uptake. TRS was used to obtain oxyhemoglobin, deoxyhemoglobin, and total hemoglobin (tHb) levels from the lesions, surrounding normal tissue, and contralateral normal tissue. Lesions with tHb levels 20% higher than those present in normal tissue were defined as “hotspots,” while others were considered “uniform.” The findings in either tumor type were compared with clinicopathological factors.Results“Hotspot” tumors were significantly larger (P = 0.002) and exhibited significantly more advanced TNM stage (P = 0.01), higher mitotic counts (P = 0.01) and higher levels of FDG uptake (P = 0.0004) compared with “uniform” tumors; however, other pathological variables were not significantly different between the two groups.ConclusionsOptical imaging for determination of tHb levels allowed for measurement of tumor vascularity as a function of proliferation and glucose metabolism, which may be useful for prediction of patient prognosis and potential response to treatment.

Successful treatment of leptomeningeal metastases from breast cancer using the combination of trastuzumab and capecitabine: a case report

We report a case of metastatic breast cancer with leptomeninges and multiple bone metastases that showed an excellent response to the combination of trastuzumab and capecitabine; therapeutic effect was evaluated by MRI at follow-up. A 44-year-old woman underwent modified radical mastectomy in February 1997. In April 2003, a tumor at the right basis cerebri and multiple bone metastases were noted, and in October 2003, she underwent enucleation of the tumor. Histopathologically, the tumor was consistent with a basal skull metastasis from breast cancer. In March 2004, the patient began to experience pain, weakness, and paresthesia of both legs. She was diagnosed, with leptomeningeal metastasis (LM) from breast cancer using MRI. In December 2005, the combination of trastuzumab and capecitabine administered as sixth-line treatment was very effective for LM. Although it is generally very difficult to diagnose LM and assess the therapeutic effect with MRI, in this case, it was possible. To our knowledge, there has been no report in the literature describing the combination of trastuzumab and capecitabine for LM from breast cancer. Although the mechanism underlying the efficacy of this combination is still unknown, the treatment would be worth trying because of its few side effects in extensively treated patients with LM from breast cancer. To confirm the antitumor efficacy of trastuzumab and capecitabine, however, further investigations are required.

In vivo imaging of eribulin-induced reoxygenation in advanced breast cancer patients: a comparison to bevacizumab

Background:Eribulin mesylate (eribulin) is a first-in-class halichondrin B-based microtubule dynamics inhibitor. To compare the anti-angiogenic activity of eribulin to that of bevacizumab, we compared tumour vessel remodelling and reoxygenation between the two agents.Methods:Patients with advanced breast cancer with stage III/IV were eligible for the study. Patients were assigned to receive either eribulin or single-agent bevacizumab. Tissue concentrations of oxyhaemoglobin (O2Hb) and deoxyhaemoglobin (HHb), and oxygen saturation (SO2) of breast tumours before and day 7 after the first infusion were repeatedly measured using diffuse optical spectroscopic imaging (DOSI). A pair of blood samples was collected for multiplex biomarker studies.Results:Baseline DOSI measurement of all 29 patients (eribulin, n=14 and bevacizumab, n=15) revealed significantly higher tumour concentrations of O2Hb and HHb than that in the normal breast tissue. After eribulin treatment, DOSI revealed a significant decrease in HHb concentration and increased SO2 during the observation period. This trend was not observed for bevacizumab. Instead, bevacizumab significantly decreased the concentration of O2Hb. The multiplex biomarker study revealed that both eribulin and bevacizumab decreased plasma concentrations of VEGF and bFGF, but only eribulin treatment suppressed the plasma concentration of TGF-β1.Conclusions:Eribulin, but not bevacizumab, treatment increased tumour SO2. Suppression of TGF-β1 by eribulin could have a favourable anti-angiogenic effect. Our results suggest that differences in vascular remodelling between these two agents may account for their different effects on tumour reoxygenation.

Early Reduction in Standardized Uptake Value After One Cycle of Neoadjuvant Chemotherapy Measured by Sequential FDG PET/CT is an Independent Predictor of Pathological Response of Primary Breast Cancer

To the Editor: F-fluorodeoxyglucose (FDG) levels on positron emission tomography (PET) reflect glucose metabolism in cancer cells (1). Currently, FDG PET combined with computed tomography (FDG PET ⁄ CT) has become employed as a non-invasive method for imaging glucose metabolism in tumors (2,3). The aim of the present study was to evaluate whether early metabolic changes after one cycle of neoadjuvant chemotherapy in FDG uptake evaluated by maximal standardized uptake value (SUVmax) could predict a pathological response of primary breast cancers. Thirty-two tumors in 30 patients having primary invasive breast cancer were investigated. All patients had received a standard neoadjuvant chemotherapy regimen comprising four cycles of epirubicine (90 mg ⁄ m) and cyclophosphamide (600 mg ⁄ m) on a triweekly basis and sequential use of 12 cycles of weekly paclitaxel (80 mg ⁄ m) (25 patients) or four cycles of triweekly docetaxel (60 mg ⁄ m) (five patients). The procedure of FDG PET ⁄ CT has been described (4). Sequential FDG PET ⁄ CT (Biograph LSO Emotion; 3D model, Siemens, Germany) was performed at the baseline (baseline PET ⁄ CT), after one cycle of chemotherapy (PET ⁄ CT2), after four cycles of chemotherapy (PET ⁄ CT3), and prior to surgery (PET ⁄ CT4). Tumors showing a 40% reduction or more in SUVmax on PET ⁄ CT2, when compared with the baseline PET ⁄ CT, were defined as metabolic responders and those showing a change of less than 40% in SUVmax were considered to be metabolic nonresponders (Fig. 1). Baseline characteristics of patients and tumors, determined by conventional modalities, are shown in Table 1. Baseline SUVmax was significantly higher in metabolic responders [10.2 ± 6.4 SD] than nonresponders (6.7 ± 3.1 SD) (p = 0.05). The percentage of tumors with nuclear grade 3 was significantly higher among the metabolic responders (71%) than among the nonresponders (16%) (p = 0.03). There were no significant differences between metabolic responders and nonresponders with regard to patient age, T-stage, nodal status, hormone receptor status, or HER2 status. The average degree of decrease in SUVmax at PET ⁄ CT2 in comparison with the baseline SUVmax was 57.9% (±11.7 SD) in metabolic responders and 10.3% (±15.7 SD) in metabolic nonresponders (p < 0.0001). Clinical response after completion of chemotherapy was measured using ultrasound or CT combined with FDG PET, and evaluated based on RECIST. On the basis of clinical response, five (71%) and two (29%) of the seven tumors with a metabolic response exhibited partial response (PR) and complete response (CR), respectively, while 18 (72%) of the 25 nonresponding tumors had PR. No nonresponding tumors showed CR. Metabolic responders showed a significantly excellent clinical response rate (100%) in terms of PR or CR in comparison with that (72%) for non-responding tumors (p = 0.001) (Table 2a). Histological criteria for assessment of therapeutic response were based on General Rules for Clinical and Pathological Recording of Breast Cancer 2008 (5). Among a total of 32 tumors, six (19%) and two (6%) were found to have grade 3, or pathological complete response (pCR), and to have grade 2b, or near pCR, where only a few residual invasive cancer cells were seen, respectively. Among the seven tumors with a metabolic response, three (43%) and two (29%) showed pCR and near pCR, respectively. Among the 25 tumors without metabolic response, Address corresopondence and reprint requests to: Hitoshi Tsuda, MD, PhD, Department of Basic Pathology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan, or e-mail: hstsuda@ gmail.com.

Axillary ultrasound examination is useful for selecting patients optimally suited for sentinel lymph node biopsy after primary systemic chemotherapy

BACKGROUND Controversy surrounds the reliability of sentinel lymph node biopsy after primary systemic chemotherapy. In this study, we assessed axillary ultrasound for selecting patients most likely to optimally benefit from biopsy. METHODS The study included 87 patients who received primary systemic chemotherapy and underwent a sentinel lymph node biopsy followed by axillary lymph node dissection. Lymph nodes >10 mm in diameter, irregularly swollen, round, and homogeneously hypoechoic without an echo-rich center were considered axillary ultrasound positive. RESULTS In axillary ultrasound-negative patients before and after primary systemic chemotherapy, identification, sensitivity, and false-negative rates were 81%, 100%, and 0%, respectively. However, in patients whose lymph nodes converted from positive to negative after primary systemic chemotherapy, these values were 83%, 70.8%, and 29.2%, respectively. CONCLUSIONS Axillary ultrasound-negative patients before and after primary systemic chemotherapy were suitable for sentinel lymph node biopsy. Axillary ultrasound should be used during primary systemic chemotherapy and before surgery.

miR-378a-3p modulates tamoxifen sensitivity in breast cancer MCF-7 cells through targeting GOLT1A.

Breast cancer is a hormone-dependent cancer and usually treated with endocrine therapy using aromatase inhibitors or anti-estrogens such as tamoxifen. A majority of breast cancer, however, will often fail to respond to endocrine therapy. In the present study, we explored miRNAs associated with endocrine therapy resistance in breast cancer. High-throughput miRNA sequencing was performed using RNAs prepared from breast cancer MCF-7 cells and their derivative clones as endocrine therapy resistant cell models, including tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF-7 cells. Notably, miR-21 was the most abundantly expressed miRNA in MCF-7 cells and overexpressed in TamR and LTED cells. We found that miR-378a-3p expression was downregulated in TamR and LTED cells as well as in clinical breast cancer tissues. Additionally, lower expression levels of miR-378a-3p were associated with poor prognosis for tamoxifen-treated patients with breast cancer. GOLT1A was selected as one of the miR-378a-3p candidate target genes by in silico analysis. GOLT1A was overexpressed in breast cancer specimens and GOLT1A-specific siRNAs inhibited the growth of TamR cells. Low GOLT1A levels were correlated with better survival in patients with breast cancer. These results suggest that miR-378a-3p-dependent GOLT1A expression contributes to the mechanisms underlying breast cancer endocrine resistance.

Immunohistochemical detection of breast cancer stem cells in hormone receptor-positive breast cancer and their role in response to endocrine therapy and clinical outcome.

Background: Breast cancer stem cells are rich in triple negative or human epidermal growth factor receptor-2-positive breast cancers. The role of these stem cells in hormone receptor-positive breast cancers is unknown. Therefore, we launched this retrospective biomarker analysis to clarify the role of stem cells in relation with endocrine therapy in hormone receptor-positive breast cancer. Methods: Formalin-fixed paraffin-embedded tissue samples from primary stage IV, hormone receptor-positive breast cancers prior to endocrine therapy were obtained from 4 cancer centers in Japan between 1999 and 2008. We examined the expression of ALDH1 and CD44/CD24 in breast tissue and correlated the results with response to endocrine therapy and patient prognosis. Results: ALDH1-positive and CD44+CD24– cancer cells were found in 16% of 92 samples and 27% of 77 samples, respectively. Response to endocrine therapy was similar in the ALDH1-positive and ALDH1-negative tumor groups, and between CD44+CD24– and other tumor groups. After a median follow-up period of 793 days, neither ALDH1 positivity nor CD44+CD24– status of tumor cells was related to progression-free survival (ALDH1 positive vs. negative, 378 vs. 292 days, p = 0.53; CD44+CD24– vs. others, 224 vs. 269 days, p = 0.52) or overall survival (ALDH1 positive vs. negative, 1,348 vs. 1,479 days, p = 0.17; CD44+CD24– vs. others, 1,071 vs. 1,462 days, p = 0.54). Conclusions: There is no correlation between biomarker expression and outcome in hormone receptor-positive breast cancer.