Mitsuhiro Hayashi

NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

Clinical significance of Ki-67 in neoadjuvant chemotherapy for primary breast cancer as a predictor for chemosensitivity and for prognosis

BackgroundNeoadjuvant chemotherapy (NAC) is one of the main strategies for patients with locally advanced breast cancer. In recent years several biological markers such as estrogen receptor (ER), progesterone receptor (PgR), and HER2 were discovered to be predictive factors for the effectiveness of NAC to help individualize treatment. In this retrospective study, we focused on Ki-67 as a biological marker and examined the correlation between Ki-67 and chemosensitivity, and the prognosis after the start of treatment.Patients and methodsBetween July 1996 and March 2008, 148 patients with tumors ≥3xa0cm in diameter or lymph node metastases received NAC and surgery. The items investigated were ER/PgR and Ki-67 from core needle biopsy. The treatment regimens were EC in 36 cases, ET in 51 cases, and FEC-DOC in 61 cases. The patients with FEC-DOC regimen had smaller tumors and higher Ki-67 values than the others.ResultsClinical response (cCRxa0+xa0cPR) was 79.7%, and the pathological complete response (pCR) was 14.2%. Multivariate analysis revealed that Ki-67 was significantly related to pCR. Moreover, there was no pathological responder in cases with Ki-67xa0<xa025%. The Ki-67 values significantly decreased after NAC (median from 45.0 to 17.5%). Patients with cCR had significantly lower Ki-67 values after NAC than those with cPR, cSD, and cPD. There was a significant difference in the Ki-67 value in terms of the presence and the absence of recurrence (median 26.0% with recurrence vs. 12% without recurrence). The disease-free survival (DFS) rate after the start of treatment was significantly higher in the patients with Ki-67xa0<xa012% after NAC than those with Ki-67xa0≥xa012%.ConclusionThe Ki-67 value before NAC was a significant predictive factor for the effectiveness of NAC. The Ki-67 values after NAC significantly decreased and correlated with clinical response and DFS. Therefore, higher Ki-67 values (≥25%) before NAC as well as lower values (<12%) after NAC might be clinically significant for treating patients.

PET/CT supports breast cancer diagnosis and treatment

The application of positron emission tomography with 18F fluoro-2-deoxy-d-glucose (FDG-PET) has remarkably improved the management of cancer patients. However, some caution is necessary in the interpretation of FDG-PET images. Because of its low spatial resolution, it is difficult to identify the anatomical location of radiotracer uptake and to distinguish between normal physiological accumulation and pathological uptake. A novel combined PET/CT system has been developed that improves the capacity to correctly localize and interpret FDG uptake. Although only a few studies have been conducted on the potential role of PET/CT in the management of breast cancer patients, the advantage of this modality compared with PET alone should be relevant for application in the field of breast cancer. In this review, we describe the clinical impact of PET/CT on breast cancer diagnosis compared with PET alone with respect to disease restaging, treatment monitoring, preoperative staging and primary diagnosis. In addition, the possible role of PET/CT with iodine contrast is noted for evaluation of intra-ductal spreading.

A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.

Utility of FDG-PET/CT in the Evaluation of the Response of Locally Advanced Breast Cancer to Neoadjuvant Chemotherapy

Neoadjuvant chemotherapy (NAC) is effective in down-staging a primary tumor before surgery, and quick differentiation between responders to NAC and nonresponders is needed. We investigated the utility of [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT) in evaluating the therapeutic effectiveness of NAC. We investigated 25 patients who underwent NAC for stage II and III noninflammatory breast cancer. FDG-PET/CT was undertaken before and after NAC to determine the maximum standardized uptake value (SUVmax) reduction rate. Findings were compared with postoperative histopathologic evaluation of therapeutic response. It was not possible to accurately assess tumor response to NAC using CT. However, using the SUVmax reduction rate, we noted a significant difference (P=0.0420) between patients who were responsive and nonresponsive to NAC. The sensitivity and specificity were as high as 83.3% and 78.9%, respectively. This study demonstrated that FDG-PET/CT can differentiate responders from nonresponders. This improves patient management by avoiding unnecessary chemotherapy.

Evaluation of the heterogeneous tissue distribution of erlotinib in lung cancer using matrix-assisted laser desorption ionization mass spectrometry imaging

Although drug distribution in tumor tissues has a significant impact on efficacy, conventional pharmacokinetic analysis has some limitations with regard to its ability to provide a comprehensive assessment of drug tissue distribution. Erlotinib is a tyrosine kinase inhibitor that acts on the epidermal growth factor receptor; however, it is unclear how this drug is histologically distributed in lung cancer. We used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze erlotinib distribution in the tumor and normal lung tissues of a mouse xenograft model and patient with non-small cell lung cancer. LC-MS/MS showed that the erlotinib tissue concentration in the xenograft tumor tissue was clearly lower than that in the normal tissue at the time of maximum blood concentration. MALDI-MSI showed the heterogeneous distribution of erlotinib at various levels in the murine tissues; interestingly, erlotinib was predominantly localized in the area of viable tumor compared to the necrotic area. In the patient-derived tissue, MALDI-MSI showed that there were different concentrations of erlotinib distributed within the same tissue. For drug development and translational research, the imaging pharmacokinetic study used the combination of MALDI-MSI and LC-MS/MS analyses may be useful in tissues with heterogeneous drug distribution.

MALDI mass spectrometry imaging of erlotinib administered in combination with bevacizumab in xenograft mice bearing B901L, EGFR-mutated NSCLC cells

Combination therapy of erlotinib plus bevacizumab improves progression-free survival of patients with epidermal growth factor receptor–mutated (EGFR-mutated) advanced non–small-cell lung cancer (NSCLC) compared with erlotinib alone. Although improved delivery and distribution of erlotinib to tumours as a result of the normalization of microvessels by bevacizumab is thought to be one of the underlying mechanisms, there is insufficient supporting evidence. B901L cells derived from EGFR-mutated NSCLC were subcutaneously implanted into mice, and mice were treated with bevacizumab or human IgG followed by treatment with erlotinib. The distribution of erlotinib in their tumours at different times after erlotinib administration was analysed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). We also analysed the distribution of erlotinib metabolites and the distribution of erlotinib in tumours refractory to erlotinib, which were established by long-term treatment with erlotinib. We found that erlotinib was broadly diffused in the tumours from B901L-implanted xenograft mice, independently of bevacizumab treatment. We also found that erlotinib metabolites were co-localized with erlotinib and that erlotinib in erlotinib-refractory tumours was broadly distributed throughout the tumour tissue. Multivariate imaging approaches using MALDI MSI as applied in this study are of great value for pharmacokinetic studies in drug development.

Mucocele-like tumor associated with ductal carcinoma in situ diagnosed as mucinous carcinoma by fine-needle aspiration cytology: report of a case.

Mucocele-like tumors (MLTs) of the breast are rare, with only 11 cases reported from Japan and 35 cases from other countries. MLTs of the breast were first described by Rosen in 1986. They are believed to be related to atypical ductal hyperplasia, ductal carcinoma, or mucinous carcinoma. It is difficult to diagnose this tumor preoperatively, and especially difficult to differentiate between benign and malignant forms. We report a case of MLT associated with ductal carcinoma in situ, which was initially diagnosed as fibroadenoma by mammography and ultrasonography, and as mucinous carcinoma by fine-needle aspiration cytology. We discuss the characteristic findings of imaging and the appropriate clinical treatment of this tumor. The characteristic image first signals the possibility of this tumor, following which the diagnosis can be confirmed by pathological examination of a fully excised tumor specimen. Breast-conserving surgery is recommended because of the low risk of high-grade malignancy, even when malignancy is confirmed, and lymph node dissection may be avoided.

Distribution of erlotinib in rash and normal skin in cancer patients receiving erlotinib visualized by matrix assisted laser desorption/ionization mass spectrometry imaging

Background The development of skin rashes is the most common adverse event observed in cancer patients treated with epidermal growth factor receptor-tyrosine kinase inhibitors such as erlotinib. However, the pharmacological evidence has not been fully revealed. Results Erlotinib distribution in the rashes was more heterogeneous than that in the normal skin, and the rashes contained statistically higher concentrations of erlotinib than adjacent normal skin in the superficial skin layer (229 ± 192 vs. 120 ± 103 ions/mm2; P = 0.009 in paired t-test). LC-MS/MS confirmed that the concentration of erlotinib in the skin rashes was higher than that in normal skin in the superficial skin layer (1946 ± 1258 vs. 1174 ± 662 ng/cm3; P = 0.028 in paired t-test). The results of MALDI-MSI and LC-MS/MS were well correlated (coefficient of correlation 0.879, P < 0.0001). Conclusions Focal distribution of erlotinib in the skin tissue was visualized using non-labeled MALDI-MSI. Erlotinib concentration in the superficial layer of the skin rashes was higher than that in the adjacent normal skin. Methods We examined patients with advanced pancreatic cancer who developed skin rashes after treatment with erlotinib and gemcitabine. We biopsied both the rash and adjacent normal skin tissues, and visualized and compared the distribution of erlotinib within the skin using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The tissue concentration of erlotinib was also measured by liquid chromatography-tandem mass spectrometry (LC–MS/MS) with laser microdissection.

Heterogeneous distribution of alectinib in neuroblastoma xenografts revealed by matrix-assisted laser desorption ionization mass spectrometry imaging: a pilot study

The penetration of the anaplastic lymphoma kinase (ALK) inhibitor alectinib in neuroblastomas and the relationship between alectinib and ALK expression are unknown. The aim of this study was to perform a quantitative investigation of the inter‐ and intra‐tumoural distribution of alectinib in different neuroblastoma xenograft models using matrix‐assisted laser desorption ionization MS imaging (MALDI‐MSI).