IntroductionThe status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8+ TILs and FOXP3+ TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC).MethodsOne hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8+ TIL and FOXP3+ TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8+ TIL and FOXP3+ TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated.ResultsTNBC patients with high CD8+ TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8+ TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537–6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029–4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8+ TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499–9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8+ TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively).ConclusionsThis is the first study to demonstrate that high CD8+ TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0632-x) contains supplementary material, which is available to authorized users.

Differences in the pathogenesis of microsatellite stable (MSS) sporadic colorectal cancers (CRCs) between left‐sided CRC (LC) and right‐sided CRC (RC) have not been clarified. To identify pathogenesis‐related genomic differences between MSS CRCs within the two locations, we performed a comprehensive molecular analysis using crypt isolation with samples from 92 sporadic CRCs. Microsatellite instability (MSI; high and low/negative) and DNA methylation status (low methylation epigenome; intermediate methylation epigenome [IME] or high methylation epigenome [HME]) were determined using polymerase chain reaction (PCR) microsatellite analysis and PCR‐bisulfite pyrosequencing, respectively. Additionally, mutations in the TP53, KRAS, BRAF and PIK3CA genes were examined using PCR‐bisulfite pyrosequencing (for KRAS and BRAF mutations) or PCR‐single conformation polymorphism (for TP53 and PIK3CA mutations), followed by sequencing of aberrant bands. Finally, a genome‐wide study using a copy number alteration (CNA)‐targeted single nucleotide polymorphism array was performed. Ninety‐two CRCs were classified into 71 MSS and 21 MSI phenotypes. We examined 71 CRCs with the MSS phenotype (LC, 56; RC, 15). Mutations in KRAS were associated with RC with the MSS phenotype, whereas mutations in TP53 were more frequently found in LC with the MSS phenotype. There were significant differences in the frequencies of KRAS and TP53 mutations in the IME between LC and RC with the MSS phenotype. Although CNA gains were associated with LC with the MSS phenotype, CNA losses were not major alterations associated with the MSS phenotype. These findings suggested that the molecular pathogenesis of the MSS phenotype in LC was different from that in RC.

(1) The mechanisms of tumor suppression are exerted by down-regulation or ablation of a variety of oncogene products including transactivaters such as nuclear factor-kappa B (NF-κB), growth signal transducers such as the Wnt signal molecule, β-catenin, and several growth factors and their receptors, and so on.(2) Curcumin also has anti-invasion, antimetastasis, and antiangiogenesis potential. (3,4) This potential is desirable for new cancer therapeutic agents. β-Catenin is a key molecule of APC (adenomatous polyposis coli)-derived colorectal carcinogenesis (5) that can be phosphorylated as a result of its association with APC, glycogen synthase kinase (GSK)-3β, and then degraded in the ubiquitination pathway. Accumulated β-catenin in the cytoplasm is transferred into the nucleus and acts together with lymphoid enhancer binding factor-1/T-cell-factor family proteins as a transactivator of other oncogenes, including c-myc and cyclin D1.(7-9) The low toxicity of curcumin is also advantageous for clinical use; however, its hydrophobicity, low absorption, and easy degradation are disadvantageous.(1) Recent clinical and preclinical studies of colorectal cancer patients and animals have suggested that curcumin bioavailability is limited. (10)(11)(12) To improve this low bioavailability, we synthesized a series of curcumin analogs and successfully screened an analog named bearing a 30-to 50-fold enhanced tumor suppressive potential against various types of cancers in vitro.(13) The molecular mechanisms of the tumor suppressive effects of GO-Y030 were similar to those of curcumin, including cell cycle arrest, down-regulation of oncogene activities such as NF-κB and Wnt signal transactivation, and induction of caspase 3 activity. The familial adenomatous polyposis (FAP) mouse is a very good model, because it develops adenomas through the same molecular mechanism as in human colorectal carcinogenesis, including the APC tumor suppressor gene. The chemopreventive ability of curcumin was assessed in an FAP model mouse, Min, harboring a truncation mutation at codon 850 with the potential to prevent adenoma formation. (12,14) To examine the bioavailability of synthesized analog and its toxicity, the FAP mouse model was applied. In this study, oral administration of GO-Y030 improved chemopreventive ability in FAP mouse without apparent toxicities in vivo. Materials and MethodsAnimal experiment. Genotyping of Apc 580D/+ mice were carried out by the polymerase chain reaction method as described previously.(15) Apc 580D/+ mice were fed either the basal diet (HFD32; CLEA Japan, Tokyo, Japan) basal diet containing 0.1% (weight/weight) curcumin, or basal diet containing 0.1% or 0.5% GO-Y030. All animal experiments were conducted in accordance with the guidelines set by Tohoku University.Compound. GO-Y030 was synthesized as previously described.Immunohistochemistry. The deparaffinized 4-μm specimens were heated in 1 mM ethylenediaminetetraacetic acid (EDTA)/ 10 mM Tris buffer (pH = 9.0) at 95°C for 40 min. After washing the specimens ...

Vasohibin-1 is a recently identified negative feedback regulator of angiogenesis induced by VEGF-A and bFGF. In this study, we first evaluated mRNA expression of vasohibin-1 and CD31 in 39 Japanese female breast carcinoma specimens including 22 invasive ductal carcinoma (IDC) and 17 ductal carcinoma in situ (DCIS) using a real-time quantitative RT-PCR (QRT-PCR) with LightCycler system. In addition, we also immunolocalized vasohibin-1 and CD31 and compared their immunoreactivity to nuclear grades and histological grades of 100 carcinoma cases (50 IDC and 50 DCIS). There were no statistically significant differences of CD31 mRNA expression and the number of CD31 positive vessels between DCIS and IDC (P = 0.250 and P = 0.191, respectively), whereas there was a statistically significant difference in vasohibin-1 mRNA expression and the number of vasohibin-1 positive vessels in DCIS and IDC (P = 0.022 and P £ 0.001, respectively). There was a significant positive correlation between vasohibin-1 mRNA level and Ki-67 labeling index in DCIS (r 2 = 0.293, P £ 0.001). In addition, vasohibin-1 mRNA expression was correlated with high nuclear and histological grades in DCIS cases and a significant positive correlation was detected between the number of vasohibin-1 positive vessels and Ki-67 labeling index or nuclear grade or Van Nuys classification of carcinoma cells (P £ 0.001, respectively). These results all indicate the possible correlation between aggressive biological features in DCIS including increased tumor cell proliferation and the status of neovascularization determined by vasohibin-1 immunoreactivity. (Cancer Sci 2010; 101: 1051-1058 B reast cancer is one of the most common malignancies in woman worldwide and its morbidity has recently increased.(1) Numerous factors have been reported to be associated with development of breast cancer including angiogenesis. Angiogenesis or the formation of new blood vessel networks not only plays a pivotal role in human normal development, but also in pathological conditions such as inflammatory diseases and neoplasms.(2) A switch to the actively angiogenic phenotype is in general considered to be dependent upon an in situ balance between stimulatory and inhibitory factors of angiogenesis. (2,3) Therefore, numerous studies have been reported on the mechanisms of control or regulation of angiogenesis since the discovery of endothelium-specific proangiogenic factors, namely vascular endothelial growth factor (VEGF) and angiopoietin family proteins.(2) In addition, other molecules involved in this process of angiogenesis, including pigment epithelium derived factor (PEDF), platelet factor 4, angiostantin and endostatin, have been proposed as angiogenesis inhibitors. (2,4) Vasohibin-1 has been very recently identified as one of the first established negative feedback regulators of angiogenesis, from an extensive microarray analysis originally designed to identify genes up-regulated by VEGF in cultured vascular endothelial cells. (2,(5)(6)(7)(8) Vasohibin-1 was subsequently demonst...

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BackgroundLittle is known about the roles of Notch signaling in cholangiocarcinoma (CC). The expression of hairy and enhancer of split 1 (Hes-1) has not been investigated yet in resected specimens of CC. Notch signaling has been reported to be related to cancer stem cell (CSC) like properties in some malignancies. Our aim is to investigate the participation of Notch signaling in resected specimens of extrahepatic CC (EHCC) and to evaluate the efficacy of CC cells with CSC-like properties by Notch signaling blockade.MethodsFirst, the expression of Notch1, 2, 3, 4 and Hes-1 was examined by immunohistochemistry in 132 resected EHCC specimens. The clinicopathological characteristics in the expression of Notch receptors and Hes-1 were investigated. Second, GSI IX, which is a γ-secretase-inhibitor, was used for Notch signaling blockade in the following experiment. Alterations of the subpopulation of CD24+CD44+ cells, which are surface markers of CSCs in EHCC, after exposure with GSI IX, gemcitabine (GEM), and the combination of GSI IX plus GEM were assessed by flow cytometry using the human CC cell lines, RBE, HuCCT1 and TFK-1. Also, anchorage-independent growth and mice tumorigenicity in the cells recovered by regular culture media after GSI IX exposure were assessed.ResultsNotch1, 2, 3, 4 and Hes-1 in the resected EHCC specimens were expressed in 50.0, 56.1, 42.4, 6.1, and 81.8 % of the total cohort, respectively. Notch1 and 3 expressions were associated with poorer histological differentiation (P = 0.008 and 0.053). The patients with the expression of at least any one of Notch1-3 receptors, who were in 80.3 % of the total, exhibited poorer survival (P = 0.050). Similarly, the expression of Hes-1 tended to show poor survival (P = 0.093). In all of the examined CC cell lines, GSI IX treatment significantly diminished the subpopulation of CD24+CD44+ cells. Although GEM monotherapy relatively increased the subpopulation of CD24+CD44+ cells in all lines, GSI IX plus GEM attenuated it. Anchorage-independent growth and mice tumorigenicity were inhibited in GSI IX-pretreated cells in RBE and TFK-1 (P < 0.05).ConclusionAberrant Notch signaling is involved with EHCC. Inhibition of Notch signaling is a novel therapeutic strategy for targeting cells with CSC-like properties.

To investigate epithelial cell proliferation and oncoprotein expression of the serrated adenoma, a term that has been used synonymously with mixed hyperplastic and adenomatous polyp, immunohistochemical staining using polyclonal antibodies against Ki-67 and p53, and a Bcl-2 monoclonal antibody, was performed and the results compared with those in hyperplastic polyps and tubular adenomas. A total of 20 serrated adenomas all characterized by a serrated glandular pattern, contained immature goblet cells, upper crypt zone mitotic figures, and a few nucleoli within the epithelial cells. Twenty hyperplastic polyps and 20 tubular adenomas (all with low-grade dysplasia) were examined, and lesions that contained separate areas of hyperplastic and adenomatous glands were excluded. The Ki-67-positive rate in the middle zone of the crypts in serrated adenomas was significantly higher than in hyperplastic polyps but lower than in tubular adenomas; a similar tendency was also noted for the upper zone. Both serrated adenomas and hyperplastic polyps demonstrated Bcl-2-positive reactivity that was essentially limited to the lower crypt zone, while in contrast, involvement in tubular adenomas often extended to the middle zone. No p53 overexpression was found in any category. These results suggest that serrated adenomas may be committed to independent growth.

Matricellular proteins such as osteopontin (OPN), galectin-9 (Gal-9), and tenascin-C (TN-C) are expressed not only under normal physiological conditions, but also during infection, inflammation and tumorigenesis. Plasma concentrations of matricellular proteins were studied to determine their diagnostic value as potential markers of tuberculosis (TB) activity. It was found that concentrations of OPN and TN-C were higher in patients with active TB than in healthy controls and individuals with latent infection. Moreover, LTBI patients had higher concentrations of OPN than did healthy controls. Gal-9 concentrations did not differ significantly between groups. Concentrations of matricellular proteins were higher in pleural fluid than in the plasma of patients with TB. Expression of matricellular proteins was also investigated in TB granulomas and other granulomatous diseases. Positive OPN and Gal-9 staining was observed in TB and sarcoidosis granulomas, but not in Crohn disease granulomas. The fibrotic ring around granulomas stained positive for TN-C in TB and sarcoidosis, but not in Crohn disease. Of the three matricellular proteins studied, OPN and TN-C may serve as reliable plasma markers for monitoring TB activity, whereas Gal-9 seems to be expressed more at the site of infection than in the systemic circulation.