Masafumi Inokuchi

Gene amplification of ESR1 in breast cancers-fact or fiction? A fluorescence in situ hybridization and multiplex ligation-dependent probe amplification study

Oestrogen receptor‐alpha (ERα), encoded by the ESR1 gene located on 6q25, is a nuclear transcription factor. Since it was reported in 2007 that more than 20% of breast cancers show ESR1 gene amplification, there has been considerable controversy about its frequency and clinical significance. We set out to assess the frequency and levels of ESR1 amplification in breast cancers. In a total of 106 breast needle biopsy specimens examined by immunohistochemistry, 78 tumours contained more than 10% ERα‐positive cancer cells. In fluorescence in situ hybridization (FISH) analysis with an ESR1‐specific probe, variously extended ESR1 signals were found in ERα‐expressing cells. Some of these were indistinguishable from large clustered signals generally accepted to mean high‐level gene amplification in homogeneously staining regions (HSRs), and could be considered to represent gene amplification. However, with RNase treatment, the ‘HSR‐like’ signals changed to small compact signals, and are thus thought to represent concentrated RNA. FISH using two differently labelled probes corresponding to the non‐overlapping 5′‐ and 3′‐end portions of the ESR1 gene on touch smears showed a preserved spatial relationship of the 3′ to 5′ sequence of ESR1, therefore strongly suggesting that the RNA consisted of primary transcripts. Using touch smears obtained from 51 fresh tumours, precise enumeration of ESR1 signals with a correction by the number of centromere 6 on FISH after RNase A treatment revealed that three tumours (5.9%) had tumour cells with one to three additional copies of ESR1 as predominant subpopulations. This infrequent and low level of gene amplification of ESR1 was also detected as a ‘gain’ of the gene by analysis with multiplex ligation‐dependent probe amplification (MLPA). The consistent results from immunohistochemistry, FISH, and MLPA in the present study settle the long‐standing debate concerning gene amplification of ESR1 in breast carcinoma. Copyright

Valproic acid inhibits proliferation of HER2-expressing breast cancer cells by inducing cell cycle arrest and apoptosis through Hsp70 acetylation

Breast cancer encompasses a heterogeneous group of diseases at the molecular level. It is known that chemo-sensitivity of breast cancer depends on its molecular subtype. We investigated the growth inhibitory effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, and the mechanism of this inhibition on four breast cancer cell lines with different molecular subtypes. The growth inhibitory effect of VPA in the four different breast cancer cell lines was investigated. The alteration of levels of p21 WAF1, cleaved caspase-3, acetylated Heat shock protein (Hsp) 90, acetylated Hsp70, and acetylated α-tubulin by VPA was examined in VPA-sensitive, human epidermal receptor 2 (HER2)-overexpressing SKBR3 cells. The cell growth inhibition of breast cancer cell lines was dependent on the dose and exposure time of VPA. The cell growth of HER2-overexpressing SKBR3 cell line was inhibited by VPA to a much greater degree than other cell lines studied. In SKBR3 cell line, VPA upregulated expression of p21 WAF1 and cleaved caspase-3 in the early phase. VPA markedly increased Hsp70 acetylation in a time-dependent manner but did not increase Hsp90 acetylation. Our data demonstrated that VPA inhibited cell proliferation and induced cell cycle arrest and apoptosis of HER2-overexpressing breast cancer cells. This anti-proliferation effect might be the direct function of VPA as an HDAC inhibitor. We propose an alternative mechanism whereby acetylation of Hsp70 disrupts the function of Hsp90 and leads to downregulation of its client proteins, including HER2 that might be the indirect function of VPA, in the sense that non-histone proteins are acetylated.

Increased E-selectin in hepatic ischemia-reperfusion injury mediates liver metastasis of pancreatic cancer

Several recent studies have reported that selectins are produced during ischemia-reperfusion injury, and that selectin ligands play an important role in cell binding to the endothelium and in liver metastasis. Portal clamping during pancreaticoduodenectomy with vessel resection for pancreatic head cancer causes hepatic ischemia-reperfusion injury, which might promote liver metastasis. We investigated the liver colonization of pancreatic cancer cells under hepatic ischemia-reperfusion and examined the involvement of E-selectin and its ligands. A human pancreatic cancer cell line (Capan-1) was injected into the spleen of mice after hepatic ischemia-reperfusion (I/R group). In addition, to investigate the effect of an anti-E-selectin antibody on liver colonization in the IR group, mice received an intraperitoneal injection of the anti-E-selectin antibody following hepatic ischemia-reperfusion and tumor inoculation (IR+Ab group). Four weeks later, mice were sacrificed and the number of tumor nodules on the liver was compared to mice without hepatic ischemia-reperfusion (control group). The incidence of liver metastasis in the I/R group was significantly higher (16 of 20, 80%) than that in the control group (6 of 20, 30%) (P<0.01). Moreover, mice in the I/R group had significantly more tumor nodules compared to those in the control group (median, 9.9 vs. 2.7 nodules) (P<0.01). In the I/R+Ab group, only 2 of 5 (40%) mice developed liver metastases. RT-PCR and southern blotting of the liver extracts showed that the expression of IL-1 and E-selectin mRNA after hepatic ischemia-reperfusion was significantly higher than the basal levels. Hepatic ischemia-reperfusion increases liver metastases and E-selectin expression in pancreatic cancer. These results suggest that E-selectin produced due to hepatic ischemia-reperfusion is involved in liver metastasis.

Two different types of ring-like enhancement on dynamic MR imaging in breast cancer: correlation with the histopathologic findings.

To describe two different types of “ring‐like enhancement” seen on dynamic magnetic resonance imaging (MRI) of breast cancer, and compare their histopathological features.

Extravasated platelet aggregation in liver zone 3 may correlate with the progression of sinusoidal obstruction syndrome following living donor liver transplantation: A case report

Sinusoidal obstruction syndrome (SOS), previously known as veno-occlusive disease, is relatively rare subsequent to liver transplantation (LT). SOS refractory to medical therapy, however, can result in centrilobular fibrosis, portal hypertension and liver failure. Although sinusoidal endothelial cell damage around central venules (zone 3) occurs early in the development of SOS, the detailed mechanism of SOS development and its association with thrombocytopenia are not yet completely understood. The present report describes a patient who experienced SOS with unexplained thrombocytopenia following living donor LT. The progression of SOS resulted in graft dysfunction and the patient succumbed. The presence of platelets in the liver allograft was assayed immunohistochemically using antibody to the platelet marker cluster of differentiation 42b (platelet glycoprotein Ib). Platelet aggregates were found attached to hepatocytes along the sinusoid and within the cytoplasm of hepatocytes, particularly in zone 3. By contrast, no staining was observed in zone 1. These findings suggested that extravasated platelet aggregation in the space of Disse and the phagocytosis of platelets by hepatocytes were initiated by sinusoidal endothelial cell damage due to the toxicity of the immunosuppressant tacrolimus or a corticosteroid pulse, and that platelet activation and degranulation may be at least partially involved in the mechanism responsible for SOS.

Low‑dose paclitaxel inhibits the induction of epidermal‑mesenchymal transition in the human cholangiocarcinoma CCKS‑1 cell line

Epidermal-mesenchymal transition (EMT) confers an advantage to cancer cells by improving their invasive capacity and metastatic potential. This phenomenon by which epidermal cells change into mesenchymal cells and therefore acquire a higher ability to automaticity, is considered a key process in cancer development. Transforming growth factor-β (TGF-β) is a significant factor for accelerating EMT through the activation of proteins, including members of the Smad pathway. Furthermore, previous studies have shown that low-dose paclitaxel (PTX) inhibits EMT in certain cell lines, including those of cancer cells. The present study determined whether low-dose PTX was able to inhibit EMT in a human cholangiocarcinoma CCKS-1 cell line that had been treated with TGF-β1. First, the cytotoxic concentration of PTX for the CCKS-1 cells was identified to be ~5 nM by MTT assay and dead cell staining. Therefore, the concentrations of PTX were set as 1 nM, 2.5 nM and 5 nM for the subsequent experiments. In the morphological investigation, the CCKS-1 cells changed into a spindle morphology and became separated by the administration of TGF-β1. However, low-dose PTX inhibited these changes and the morphology resembled the control cells in a dose-dependent manner. Similarly, immunofluorescence and immunoblotting investigations revealed that the CCKS-1 cells expressed mesenchymal markers following the administration of TGF-β1. However, low-dose PTX inhibited the expression of the mesenchymal markers and the CCKS-1 cells expressed the epithelial marker, E-cadherin. In particular, a concentration-dependent effect was observed in the immunoblotting experiments. These results show that PTX may be able to inhibit EMT in cancer cells, depending on the dose concentration.

Triple-negative Breast Cancer: Are the Imaging Findings Different Between Responders and Nonresponders to Neoadjuvant Chemotherapy?

RATIONALE AND OBJECTIVES The purpose of the present study was to evaluate the imaging findings of triple-negative breast cancer patients receiving neoadjuvant chemotherapy (NAC) and to investigate whether the findings are different between responders and nonresponders, enabling us to predict the final patient response. MATERIALS AND METHODS The subjects included 22 women ages 35-73 years (mean, 50.4 years) with 23 triple-negative breast cancers who underwent NAC. In all cases, a mammography, ultrasound, and magnetic resonance imaging (MRI) were performed a total of three times: before NAC, after the first half of NAC, and after NAC. The mass shape, mass margin, presence of clear intratumoral necrosis, and presence of intratumoral calcification were analyzed. The presence of clear intratumoral necrosis was evaluated on the MRI. If there was a very high signal intensity (similar to that of water) in the tumor on the fat-suppressed T2-weighted MRI scans, we judged it to be clear intratumoral necrosis. RESULTS An irregularly shaped mass (P = .018) and the presence of clear intratumoral necrosis (P = .044) were significantly associated with NAC nonresponse in triple-negative breast cancer patients. The mass margin and the presence of intratumoral calcification were not related to the effects after NAC. CONCLUSIONS In cases of triple-negative breast cancer involving clear intratumoral necrosis with an irregular mass shape, it is predicted that the effects of neoadjuvant chemotherapy will likely be poor, and therefore, the presence of such image findings may be useful for determining the optimal application of neoadjuvant chemotherapy.

Impact of histone deacetylase 1 and metastasis‑associated gene 1 expression in esophageal carcinogenesis

Animal models are important for the development of novel therapies for esophageal cancer. Histone deacetylase 1 (HDAC1)/metastasis-associated gene (MTA1) complexes inhibit p53 acetylation and thus, inhibit p53-induced apoptosis. The aim of the present study was to evaluate HDAC1 and MTA1 expression in esophageal carcinogenesis in rats. The rats underwent a total gastrectomy followed by esophagojejunostomy to induce chronic duodenal content reflux esophagitis. The rats were sacrificed sequentially at 20, 30, 40 and 50 weeks post-surgery and the esophagi were examined. Immunohistochemical analysis was conducted to assess the expression and localization of HDAC1 and MTA1. At 20 weeks post-surgery, squamous proliferative hyperplasia and Barrett’s metaplasia (BM) were observed. While, adenocarcinoma-associated BM and squamous cell carcinoma were observed at 30–50 weeks post-surgery. The nuclear expression of HDAC1 and MTA1 was observed in all of the stages of squamous carcinogenesis and adenocarcinogenesis, although not in the normal esophageal epithelium. The expression of HDAC1 and MTA1 may be involved in duodenoesophageal reflux-induced neoplastic transformation of the esophageal mucosa into cancer cells with squamous and adeno differentiation.

Peripheral hyperintense pattern on T2-weighted magnetic resonance imaging (MRI) in breast carcinoma: correlation with early peripheral enhancement on dynamic MRI and histopathologic findings.

To investigate the correlation between the peripheral hyperintense pattern of breast carcinoma on T2‐weighted images (T2WI) and the early peripheral enhancement (EPE) on dynamic magnetic resonance imaging (MRI) and to examine the histological characteristics involved in the causes thereof.

A phase I study of neoadjuvant chemotherapy with gemcitabine plus oral S-1 for resectable pancreatic cancer

The aim of this study was to determine the maximum-tolerated dose (MTD), the dose-limiting toxicity (DLT) and the recommended dose (RD) of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) plus oral S-1 in patients with resectable pancreatic cancer. Thirteen patients with radiologically proven resectable pancreatic cancer were included in this study. S-1 was administered orally for 14 consecutive days, and GEM was administered on days 8 and 15 for two pre-operative cycles. The dose of S-1 in this study was planned with fixed doses of GEM (1,000 mg/m2): 20, 30 and 40 mg/day for levels 0, 1 and 2, respectively. Treatment was initiated at level 1 in 3 patients, while adverse events occurred in 2 patients during the second course, leading to a dose reduction to level 0 for the 8 remaining patients. Two of the 10 patients enrolled at level 0 were excluded. Of the remaining 8 patients, GEM administration was terminated due to DLT on day 15, during the first course in 3 patients, while level 0 dosage reached MTD. Surgery was performed for the remaining 11 patients included in the study. Post-operative complications included pancreatic fistulas in 5 patients and Pseudomonas aeruginosa sepsis in 1 patient. Two of the 11 patients exhibited a partial response and 9 patients stable disease. Eight of the 11 tumor specimens showed histopathological evidence of tumor cell injury. In conclusion, NAC with GEM and S-1 was not well-tolerated in this study. However, pre-operative chemotherapy may be effective against pancreatic cancer. Therefore, it is necessary to reconsider NAC regimens for pancreatic cancer.