Diagnostic utility of trefoil factor families for the early detection of lung cancer and their correlation with tissue expression.

Trefoil factors (TFFs) are upregulated in numerous types of cancer, including those of the breast, the colon, the lung and the pancreas, suggesting their potential utility as biomarkers for screening. In the present study, the clinical relevance of serum or urinary TFFs as biomarkers were comprehensively evaluated and the correlation with TFF expression levels in lung cancer tissue was examined. Serum and urine were collected from 199 patients with lung cancer and 198 healthy individuals. Concentrations of serum and urinary TFF1, TFF2 and TFF3 were measured using ELISA and the potential of TFF levels to discriminate between cancer and non-cancer samples was evaluated. In 100 of the cancer cases, expression of TFF1-3 was analyzed using immunohistochemical staining of paraffin sections. Furthermore, the relationship between TFF levels and clinicopathological factors among these cancer cases was analyzed using immunohistochemistry of tissue specimens, quantified and statistically analyzed. While serum levels of all TFFs measured using ELISA were significantly higher in patients with lung cancer compared with those in healthy individuals, urinary TFFs were lower. Areas under the curve (AUC) of the receiver operating characteristic curves for serum/urinary TFF1, TFF2 and TFF3 were 0.709/0.594, 0.722/0.501 and 0.663/0.665, respectively. Furthermore, the combination of serum TFF1, TFF2, TFF3 and urinary TFF1 and TFF3 demonstrated the highest AUC (0.826). In the clinicopathological analysis, serum TFF1 was higher in the early pathological T-stage (pTis/1/2) compared with the later stage (pT3/4) and TFF2 was higher in the pN0/1 than the pN2 group. With regards to the histological types, urinary TFF1 was higher in squamous cell carcinoma than adenocarcinoma (AC), but TFF2 tended to be higher in AC. Using immunohistochemical analysis, although TFF1 and TFF3 expression showed positive correlation with serum concentrations, TFF2 was inversely correlated. In conclusion, serum and urinary TFF levels are promising predictive biomarkers, and their measurements provide a useful in vivo and non-invasive diagnostic screening tool. In particular, TFF1 and TFF3 could be surrogate markers of clinicopathological profiles of human lung cancer.

TFF-1 Functions to Suppress Multiple Phenotypes Associated with Lung Cancer Progression.

INTRODUCTION: Trefoil Factor (TFF) is a member of a protein family comprised of three isoforms, of which TFF-1 exhibits antithetical functions; promotion or suppression of cell proliferation, survival and invasion, depending on the cancer type. However, the pathobiological function of TFF-1 in lung carcinoma has been still unclear. METHODS: We examined the expression and secretion of TFF-1 using cultured human lung carcinoma cells by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay and quantitative real-time PCR analyses. The effects of TFF-1 on various phenotypes were analyzed in two cell lines, including those transfected with cDNA encoding TFF-1. Cell proliferation and death were examined by hemocytometer cell counting and by colorimetric viability/cytotoxicity assay. Cell cycle profile, migration and invasion were also examined by flow cytometry, wound healing assay and Matrigel Transwell assay, respectively. The effect of TFF-1 overexpression was confirmed by additional transfection of TFF-1-specific siRNA. RESULTS: Endogenous TFF-1 protein expression and secretion into the media were observed exclusively in adenocarcinoma-derived cell lines. Forced overexpression of TFF-1 drove cell cycle transition, while the proliferation decreased by 19% to 25% due to increased cell death. This cell death was predominantly caused by apoptosis, as assessed by the activation of caspase 3/7. Cell migration was also suppressed by 71% to 82% in TFF-1-transfected cells. The suppressive effect of TFF-1 on proliferation and migration was restored by transfection of TFF-1 siRNA. Moreover, invasion was also suppressed to 77% to 83% in TFF-1-transfected cells. CONCLUSION: These findings reveal that TFF-1 functions as a suppressor of cancer proliferation by induction of apoptosis, cell migration and invasion and thus may provide a synergistic target for potential treatment strategies for human lung carcinoma.

Neural EGFL like 2 expressed in myoepithelial cells and suppressed breast cancer cell migration.

Breast tissue has a branching structure that contains double-layered cells, consisting primarily of luminal epithelial cells inside and myoepithelial cells outside. Ductal carcinoma in situ (DCIS) still has myoepithelial cells surrounding the cancer cells. However, myoepithelial cells disappear in invasive ductal carcinoma. In this study, we detected expression of neural EGFL like (NELL) 2 and one of its receptors, roundabout guidance receptor (ROBO) 3, in myoepithelial and luminal epithelial cells (respectively) in normal breast tissue. NELL2 also was expressed in myoepithelial cells surrounding the non-cancerous intraductal proliferative lesions and DCIS. However, the expression level and proportion of NELL2-positive cells in DCIS were lower than those in normal and non-cancerous intraductal proliferative lesions. ROBO3 expression was decreased in invasive ductal carcinoma compared to that in normal and non-cancerous intraductal proliferative lesions. An evaluation of NELL2's function in breast cancer cell lines demonstrated that full-length NELL2 suppressed cell adhesion and migration in vitro. In contrast, the N-terminal domain of NELL2 increased cell adhesion in the early phase and migration in vitro in some breast cancer cells. These results suggested that full-length NELL2 protein, when expressed in myoepithelial cells, might serve as an inhibitor of breast cancer cell migration.

EphA2, a possible target of miR-200a, functions through the AKT2 pathway in human lung carcinoma.

We previously reported that miR-200a was highly up-regulated in lung carcinoma, exhibiting a copy number increase (CNI) of the AKT2 gene (AKT2+ group) in defined subsets, i.e., adenocarcinoma and early stages of carcinoma (pStage I/II). In this study, we searched possible targets of miR-200a in these subsets by IHC analyses focusing on the expression of known target proteins of miR-200a: beta-catenin, EphA2, ZEB1, PTEN, and YAP-1, as well as E-cadherin, the expression of which is suppressed by ZEB1. Among those 6 proteins, when all 38 cases of surgically resected specimens were analyzed as a whole, IHC score of ZEB1 was inversely (ρ=-.417) and E-cadherin was positively (ρ=.345) correlated with miR-200a expression. However, only EphA2 was inversely correlated with the expression of miR-200a in adenocarcinoma (ρ=-.496) and in pStage I/II group (ρ=-.547), while no correlation was seen in non-adenocarcinoma, squamous cell carcinoma, or pStage III carcinoma. Furthermore, by comparison of 3 groups categorized according to the AKT gene increase, only EphA2 was down-regulated to a statistically significant level in the AKT2+ group in both adenocarcinoma (p=.0447) and pStage I/II carcinoma (p=.0458). These results suggest that in lung carcinomas, higher Akt activation caused by increased AKT2 gene copy number leads to the upregulation of miR-200a, which exerts its function as a suppressor of EphA2 in adenocarcinoma and the early stages of carcinomas.

The relation between anti-TGBFR1 immunohistochemical reaction and low Ki67, small tumor size and high estrogen receptor expression in invasive breast cancer.

Most breast cancers are derived from the luminal epithelium, which composes the inside of the breast ductal structure. Ductal carcinoma in situ (DCIS) leads to invasive ductal carcinoma, but noncancerous intraductal proliferative lesions are also a risk factor for ductal carcinoma. The transforming growth factor beta (TGFB) signaling pathway behaves as a tumor suppressor in the early stage of cancer, and conversely as a tumor growth factor in invasive stages in several cancers. In this study, we performed immunohistochemistry with an antibody that detects the cytoplasmic region of TGFB receptor 1 (TGFBR1) and elucidated TGFBR1 protein expression in luminal epithelial cells of noncancerous breast ducts and in several cases of DCIS and invasive carcinoma. TGFBR1 expression was higher in noncancerous breast tissue than in cancerous tissue, and a difference in expression was also seen among histological subtypes. Comparing the expression level of TGFBR1 in cancer cells and clinico-pathological parameters, cases expressing low TGFBR1 tended to show low estrogen receptor expression, large tumor size (≥10 mm), and a high Ki67 labeling index. These data suggested that TGFBR1 protein expression may be related to the suppression of breast cancer cell growth.

Amplicons in breast cancers analyzed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

Gene amplification is a common event in breast cancer, and identifies actual and potential targets of molecular therapy. The aim of the present study was to determine the amplification status of 22 genes that are reportedly frequently amplified in breast cancers. An archive of 322 formalin-fixed and paraffin-embedded invasive breast cancer tissues were screened by multiple ligation-dependent probe amplification (MLPA) and a total of 906 gene loci judged as 'gain' or 'amplified' was further confirmed to have been amplified based on fluorescence in situ hybridization (FISH). The results showed that 109 of 322 tumors (34%) displayed gene amplification of at least one of the 22 genes. The frequencies of the amplification of four regions containing known driver oncogenes were as follows: 8p11 (ZNF703, FGFR1, ADAM9, IKBKB), 8q24 (MYC), 11q13 (CCND1, C11ORF30), and 17q11-21 (CPD, MED1, ERBB2, CDC6, TOP2A, MAPT) exhibited amplification in 9.6%, 9.6%, 12.4%, and 12.1% of the tumors, respectively. In addition to homogeneously staining region- or double-minute chromosome-type amplifications, a centromere-associated-type amplification was found in nine tumors. Co-localization of the amplicon on 8p11 and the amplicon on 11q13 in single cells was found in 10 tumors, and in six of those tumors the two amplicons constituted single amplification units. Similarly, an amplicon consisting of ERBB2 and its flanking genes on 17q12-21 co-localized with an amplicon on 8p11 in 10 tumors and with the amplicon on 11q13 in five tumors. Thus, precise and feasible analysis of gene amplification status can be obtained using a combination of MLPA and FISH.

Screening of the copy number increase of AKT in lung carcinoma by custom-designed MLPA.

Treatments for lung cancer include therapies targeting aberrant oncoproteins, but there remains a high medical need for novel therapies. Our previous studies showed that gene amplification/high-level polysomy of AKT1/2 occurs in more than 10% of lung carcinomas. Here, we describe multiplex ligation-dependent probe amplification analysis (MLPA) as a high-throughput method to evaluate copy number increases (CNIs) of AKT1/2 in lung carcinomas. The performance of MLPA using custom-made probes in formalin-fixed paraffin-embedded tissue was evaluated by comparing it to immunohistochemistry and fluorescence in situ hybridization analysis (FISH). By MLPA, we found 4 out of 30 samples harboring gene "gain" when the conventional cutoff value (> 1.3) was used. Two samples with gene amplification by FISH had MLPA values of 1.85 and 1.75, which were lower than the conventional cutoff for "amplification" (> 2.0). Moreover, samples with CNIs due to polysomy by FISH gave MLPA values between 1.13 and 1.47, so some samples had lower values than 1.3. The reasons appeared to be stromal contamination and the presence of carcinoma cells without CNIs. However, when we changed the cutoff for "gain" to the "average+2xstandard error", we detected CNIs in 10 samples, with only one each of false-positive and false-negative results. The sensitivity was 90% and the specificity was 98%. Consistently, all cases exhibiting CNI by this criteria revealed Akt activation. In conclusion, MLPA implemented with custom-made probes and an optimized cutoff value is a feasible screening method to semi-quantitatively detect oncogene aberrations, and may contribute to the design of individualized, molecularly targeted therapies against lung carcinoma.

Clinicopathological features of epiretinal membranes in eyes filled with silicone oil.

PURPOSE: The aim of this case series was to clarify the clinicopathological features of epiretinal membranes (ERMs) that developed in eyes after silicone oil (SO) tamponade to treat rhegmatog-enous retinal detachments (RRDs). PATIENTS AND METHODS: In the Department of Ophthalmology, Saitama Medical Center, Jichi Medical University, patients with idiopathic ERMs (23 eyes) and ERMs in eyes filled with SO (SO ERMs) after vitreous surgery to treat RRDs (nine eyes) were enrolled from July 2012 to March 2014. ERM tissues obtained intraoperatively were examined histopathologically. Besides the main outcome measure of the pathological findings of the ERM tissues, other outcome measures included the preoperative findings on optical coherence tomography (OCT) images and the surgical findings. RESULTS: Eight (89%) of nine eyes with SO ERMs had bilayered membranes composed of a firm layer on the retinal side with glial cells and extracellular matrix and a fragile sponge-like layer on the vitreous side. The sponge-like layer was composed of emulsified SO surrounded by macrophages. Quantitative analysis showed that the areas with cluster of differentiation 68 (CD68)-positive macrophages identified by immunohistochemistry in eyes with SO ERMs were significantly (P<0.001) larger than those in eyes with idiopathic ERMs. The findings on OCT images were consistent with the pathological features of the SO ERMs. Surgical removal of the SO ERMs was difficult because the sponge-like layer was fragile, and the underlying retina was also fragile due to inflammation. CONCLUSION: SO ERMs are bilayered membranes. Long-standing emulsified SO formed a sponge-like layer and SO (foreign body)-induced granulation and caused retinal inflammation in these eyes, making surgical removal difficult. A preoperative OCT examination is necessary to identify SO ERMs.

Regulation of p27 by ubiquitin ligases and its pathological significance in human lung carcinomas.

Down-regulation of cyclin-dependent kinase inhibitor protein p27, due to enhanced degradation, is frequently observed in various cancers. The ubiquitin ligases that mediate this degradation have been identified as S-phase kinase-associated protein-2 (Skp2), Kip1 ubiquitylation-promoting complex (KPC), and p53-inducible protein with RING-H2 domain (Pirh2) as well. We investigated the correlation among expression of these 3 ligases and p27 status in surgical specimens of human lung carcinomas by immunohistochemical analysis. Among 93 cases, expressions of p27, Skp2, KPC, and Pirh2 were found in 89.2%, 59.1%, 59.1%, and 67.7%, respectively. Down-regulation of p27 in cancer cells was frequently observed in adenocarcinoma (AC) and squamous cell carcinoma (SCC), but not in small cell carcinoma (SmCC). Overexpression of ubiquitin ligases was variously observed among histological types: Skp2 was more frequently observed in SCC and SmCC, KPC in SCC and Pirh2 in AC, followed by SCC. Several novel findings were obtained: (i) cytoplasmic p27 was observed in 8.6%, most frequently in SCC (13.3%), and correlated with nodal metastasis (P=.0044), (ii) significant inverse correlation between nuclear p27 and Pirh2 expression was observed by statistical analysis and at the cellular level, and (iii) cytoplasmic Pirh2 and total (cytoplasmic and/or nuclear) Pirh2 were significantly correlated with the nodal status (P=.0225, 0.0314), the pathological stage (P=.0213, 0.0475) and recurrence-free survival (P=.0194, 0.0482, respectively) in AC. Altogether, our data suggests that p27 and its cognate ubiquitin ligases are specifically involved in the clinical profiles, and thus, molecular targeting of these ubiquitin ligases, in particular, Pirh2, may have therapeutic value for human lung carcinomas.

Gene amplification of CCNE1, CCND1, and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

New and effective treatments for advanced gastric cancer are urgently needed. Cyclins E and D1 form a complex with cyclin-dependent kinase 2, 4, or 6 to regulate G1-S transition. The G1-S regulatory genes encoding cyclin E (CCNE1), cyclin D1 (CCND1), and CDK6 (CDK6) are frequently amplified in gastric cancer and may therefore influence molecularly targeted therapies against ERBB2 or EGFR when coamplified. A total of 179 formalin-fixed and paraffin-embedded gastric cancer specimens were examined for these gene amplifications by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization. Amplification of at least 1 G1-S regulatory gene was found in 35 tumors (CCNE1 amplification, 15% of samples; CCND1, 6%; CDK6, 1%). In 13 of the 35 tumors, dual-color fluorescence in situ hybridization identified coamplification of the G1-S regulatory genes with ERBB2, EGFR, and/or KRAS in single cancer nuclei. The observation that cells with G1-S regulatory gene amplification contained clonal subpopulations with coamplification of ERBB2, EGFR, or KRAS in 5 early and 3 advanced cancers suggests that amplification of the G1-S regulatory genes represents an early event, which precedes ERBB2, EGFR, or KRAS amplification. Amplified CCNE1, CCND1, and CDK6 in advanced gastric cancer may be potentially useful as direct targets for molecular therapy or for combination therapy with ERBB2 or EGFR inhibitors. Multiplex ligation-dependent probe amplification could be a useful tool for identification of patients who would benefit from such therapies.

Loss of YAP1 defines neuroendocrine differentiation of lung tumors.

YAP1, the main Hippo pathway effector, is a potent oncogene and is overexpressed in non-small-cell lung cancer (NSCLC); however, the YAP1 expression pattern in small-cell lung cancer (SCLC) has not yet been elucidated in detail. We report that the loss of YAP1 is a special feature of high-grade neuroendocrine lung tumors. A hierarchical cluster analysis of 15 high-grade neuroendocrine tumor cell lines containing 14 SCLC cell lines that depended on the genes of Hippo pathway molecules and neuroendocrine markers clearly classified these lines into two groups: the YAP1-negative and neuroendocrine marker-positive group (n = 11), and the YAP1-positive and neuroendocrine marker-negative group (n = 4). Among the 41 NSCLC cell lines examined, the loss of YAP1 was only observed in one cell line showing the strong expression of neuroendocrine markers. Immunostaining for YAP1, using the sections of 189 NSCLC, 41 SCLC, and 30 large cell neuroendocrine carcinoma (LCNEC) cases, revealed that the loss of YAP1 was common in SCLC (40/41, 98%) and LCNEC (18/30, 60%), but was rare in NSCLC (6/189, 3%). Among the SCLC and LCNEC cases tested, the loss of YAP1 correlated with the expression of neuroendocrine markers, and a survival analysis revealed that YAP1-negative cases were more chemosensitive than YAP1-positive cases. Chemosensitivity test for cisplatin using YAP1-positive/YAP1-negative SCLC cell lines also showed compatible results. YAP1-sh-mediated knockdown induced the neuroendocrine marker RAB3a, which suggested the possible involvement of YAP1 in the regulation of neuroendocrine differentiation. Thus, we showed that the loss of YAP1 has potential as a clinical marker for predicting neuroendocrine features and chemosensitivity.

MicroRNAs associated with increased AKT gene number in human lung carcinoma.

MicroRNA (miRNA) expression profiles were examined in 3 groups of lung carcinomas that had been stratified by increases in AKT1 or AKT2 gene number. Microarray analysis using 2000 probes revealed 87 miRNAs that were up-regulated and 32 down-regulated miRNAs in carcinomas harboring amplification or high-level polysomy of the AKT1 (AKT1+), as well as 123 up-regulated and 83 down-regulated miRNAs in those of the AKT2 genes (AKT2+), in comparison with carcinomas harboring disomy of both (AKTd/d). In total, 182 miRNAs were up-regulated in AKT1+ or AKT2+, compared with AKTd/d. Among these, 28 miRNAs were up-regulated in both the AKT1+ and AKT2+ groups, with a log2 ratio between 1.02 and 3.71 relative to AKTd/d group, including all miR-200 family members. Quantitative real-time polymerase chain reaction showed that carcinomas exhibiting lymph vessel invasion had significantly lower expression of miR-200a (P=.0230) and miR-200b (P=.0168), regardless of the status of the AKT genes. Moreover, a detailed statistical analysis revealed that, in adenocarcinoma and in the early stage of carcinomas (pathologic stage I/II), expression of miR-200a was higher in the AKT2+ group compared with the AKT1+ group, and these differences were statistically significant (P=.0334 and P=.0239, respectively). However, the expression of miR-200a was not significantly correlated with the expression of its target, the zinc finger E-box-binding homeobox 1 (ZEB1; P=.3801) or E-cadherin (P=.2840), a marker of the epithelial-mesenchymal transition. These results suggest that AKT2 can regulate miR-200a in a histology- or stage-specific manner and that this regulation is independent of subsequent involvement of miR-200a in epithelial-mesenchymal transition.

Association between AKT1 Gene Polymorphism rs2498794 and Smoking-Related Traits with reference to Cancer Susceptibility.

To clarify the potential role of variability within and around the AKT1 gene in smoking behaviors, we performed a single-nucleotide polymorphism (SNP) analysis of the AKT1 gene in an elderly Japanese cohort. Genotypes of the rs2498794 SNP, which is located in the fifth intron region of the AKT1 gene, were marginally but significantly associated with smoking duration in the total 999 samples of former and current smokers. Interestingly, this SNP had a marginally significant association with individual cancer history (past and current), especially in groups with a shorter smoking duration (<44 years) and fewer cigarettes per day (≤20). These data suggest that the rs2498794 polymorphism of the AKT1 gene is associated with a long smoking duration and may be involved in the predisposition to cancer when the smoking duration is short or the cigarettes per day is rate low.

Diverse involvement of isoforms and gene aberrations of Akt in human lung carcinomas.

Emerging evidence confirms a central role of Akt in cancer. To evaluate the relative contribution of deregulated Akt and their clinicopathological significance in lung carcinomas, overexpression, activation of Akt and AKT gene increases were investigated. Immunohistochemical staining for 108 cases revealed overexpression of total Akt, Akt1, Akt2 and Akt3 in 61.1, 47.2, 40.7 and 23.1%, respectively, and phosphorylated Akt in 42.6% of cases. Expression of total Akt, Akt2 and Akt3 were frequently observed in small cell carcinoma, but phosphorylated Akt and Akt1 were more frequently observed in squamous cell carcinoma. FISH analysis to evaluate gene increases of AKT1-3 revealed amplification of AKT1 in 4.2% and AKT1 increase by polysomy of chromosome 14 in 27.3% of cases. For AKT2, amplification was observed in 3.2% and polysomy of chromosome 19 in 26.3% of cases. AKT3 increase was observed in 40.0% of cases only by polysomy of chromosome 1. Although "FISH-positive" AKT1 and AKT2 gene increases (amplification/high-level polysomy) were found exclusively in the cases overexpressing total Akt, Akt1 or Akt2, respectively, AKT3 increase was irrelevant of Akt3 expression. Statistically, expressions of Akt2, p-Akt and cytoplasmic-p-Akt were correlated with lymph node metastasis (P = 0.0479, P = 0.0371 and P = 0.0310, respectively). Although AKT1 and AKT2 gene increase showed positive correlation with, or trend towards a positive correlation with tumor size (P = 0.0430, P = 0.0590, respectively), AKT3 did not. In conclusion, Akt isoforms are differentially involved in the pathological phenotype of lung carcinoma in a diverse manner. Because abnormality of Akt1/AKT1 and Akt2/AKT2 correlated with clinicopathological profiles, Akt1/2-specific targeting may open a novel therapeutic window for the group showing Akt deregulation.

Semi-comprehensive analysis of gene amplification in gastric cancers using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

The prognosis of patients with gastric carcinomas at an advanced stage still remains dismal, and therefore novel therapeutic modalities are urgently needed. Since the successful targeting of amplified ERBB2 with a humanized monoclonal antibody, the amplified genes of other receptor tyrosine kinases such as EGFR, FGFR2, and MET, as well as those of other cell regulator genes, are being considered as candidate targets of molecular therapy. The aim of the present study was to determine the amplification status of 26 genes, which are frequently amplified in solid cancers, in advanced gastric cancers. A total of 93 formalin-fixed and paraffin-embedded advanced gastric cancer tissues were examined by multiple ligation-dependent probe amplification, and 32 cases with 'gain' or 'amplified' status of 16 genes were further examined for the respective gene amplification by fluorescence in situ hybridization (FISH) and for the respective protein overexpression by immunohistochemistry. The frequencies of gene amplifications in advanced gastric cancers were as follows: ERBB2 (13 cases, 14%), FGFR2 (7 cases, 8%), MYC (7 cases, 8%), TOP2A (7 cases, 8%), MET (4 cases, 4%), MDM2 (4 cases, 4%), CCND1 (3 cases, 3%), FGF10 (2 cases, 3%), and EGFR (1 case, 1%). Amplification of the receptor tyrosine kinases genes occurred in a mutually exclusive manner except for one tumor in which ERBB2 and FGFR2 were both amplified but in different cancer cells. Co-amplification of ERBB2 and MYC, and EGFR and CCND1, in single nuclei but on different amplicons, was confirmed in one case each. Attempts at correlating the FISH status with the immunohistochemical staining pattern showed variable results from complete concordance to no correlation. In conclusion, combination of multiple ligation-dependent probe amplification and FISH analysis is a feasible approach for obtaining the semi-comprehensive genetic information that is necessary for personalized molecular targeted therapy.

Epidemiological and pathobiological profiles of Clostridium perfringens infections: review of consecutive series of 33 cases over a 13-year period.

BACKGROUND: Although Clostridium perfringens (C. perfringens) is well known as the causative agent of several forms of enteric disease, precise epidemiological and pathobiological aspects are still unknown. METHODS: We retrospectively reviewed the culture results of samples collected in our hospital from 2001 through 2013. In addition, for the detection and toxinogenic typing of C. perfringens, polymerase-chain-reaction amplification (PCR)-based rapid analysis was performed in 6 cases using DNA extracted from paraffin-embedded tissues. RESULTS: A total of 35 samples from 33 cases were positive for C. perfringens, representing an incidence of 0.017% (35/205, 114). Among 33 patients, 21 patients manifested sepsis and 7 patients had bacteremia. One of the septic cases was complicated by fatal intravascular hemolysis and thus, the prevalence was estimated at 3.0% among C. perfringens infections (1/33). The direct causative disease or state for C. perfringens infection was identified in 18 patients: surgery or intervention for cancers, 8 patients; chemotherapy for cancer, 2 patients; surgery or intervention for non-neoplastic disease, 6 patients; liver cirrhosis, 3 patients, etc. PCR-based toxinogenic typing of C. perfringens detected the alpha-toxin gene only in tissue from a patient who died of massive hemolysis; none of the toxin genes could be amplified in the other 5 cases examined. CONCLUSIONS: The prevalence of overt C. perfringens infection is low, but upon detection, infected patients should be carefully monitored for fatal acute hemolysis caused by type A C. perfringens. Furthermore, PCR-based rapid detection of C. perfringens and toxinogenic typing by archival pathological material is applicable as a diagnostic tool.

Clonal profiling of mixed lobular and ductal carcinoma revealed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

A needle biopsy of a mass in the right breast of a 36-year-old woman revealed invasive ductal carcinoma (IDC), and approximately 20% of cancer cells showed unequivocal membranous staining with the HercepTest. After systemic therapy with trastuzumab and paclitaxel followed by FEC (fluorouracil + epirubicin + cyclophosphamide), a right mastectomy was performed. By histological and immunohistochemical examinations, the resected tumor consisted mainly of E-cadherin-negative invasive lobular carcinoma (ILC), and the rest was ERBB2-positive IDC; thus, the diagnosis was mixed ductal and lobular carcinoma. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization (FISH) analyses revealed that ILC and IDC shared high-level amplification of CCND1 in homogeneously staining regions (HSR) and that IDC had an additional HSR-type amplicon of ERBB2. These findings strongly indicate that IDC and ILC had a common precursor cell with CCND1 amplification. Review of the biopsy specimen with FISH showed IDC with gene amplifications of CCND1 and ERBB2 as a minor component, IDC without amplification of CCND1 or ERBB2 as a major component, and a minute portion of ILC with CCND1 amplification. We speculate that chemotherapy and trastuzumab caused a marked reduction in IDC; however, ILC with CCND1 amplification was resistant to chemotherapy and grew.

Intratumoral heterogeneous amplification of ERBB2 and subclonal genetic diversity in gastric cancers revealed by multiple ligation-dependent probe amplification and fluorescence in situ hybridization.

A humanized monoclonal antibody against ERBB2 is used in neoadjuvant therapy for patients with gastric cancer. A critical factor in determining patient eligibility and predicting outcomes of this therapy is the intratumoral heterogeneity of ERBB2 amplification in gastric adenocarcinomas. The aims of this study are to assess the underlying mechanisms of intratumoral heterogeneity of ERBB2 amplification; to characterize the diversity of coamplified oncogenes such as EGFR, FGFR2, MET, MYC, CCND1, and MDM2; and to examine the usefulness of multiple ligation-dependent probe amplification (MLPA) in the semicomprehensive detection of these gene amplifications. A combined analysis of immunohistochemistry and fluorescence in situ hybridization revealed ERBB2-amplified cancer cells in 51 of 475 formalin-fixed, paraffin-embedded gastric adenocarcinomas. The fraction of amplification-positive cells in each tumor ranged from less than 10% to almost 100%. Intratumoral heterogeneity of ERBB2 amplification, defined as less than 50% of cancer cells positive for ERBB2 amplification, was found in 41% (21/51) of ERBB2-amplified tumors. The combined analysis of MLPA and fluorescence in situ hybridization revealed that ERBB2 was coamplified with EGFR in 7 tumors, FGFR2 in 1 tumor, and FGFR2 and MET in 1 tumor; however, the respective genes were amplified in mutually exclusive cells. Coamplified ERBB2 and MYC coexisted within single nuclei in 4 tumors, and one of these cases had suspected coamplification in the same amplicon of ERBB2 with MYC. In conclusion, the amplification status of ERBB2 and other genes can be obtained semicomprehensively by MLPA and could be useful to plan individualized molecularly targeted therapy against gastric cancers.

Significance of Akt activation and AKT gene increases in soft tissue tumors.

To clarify the aberrations of AKT genes, their protein products and clinicopathologic significance in bone and soft tissue tumors, expression profiles of total Akt, its isoforms and activated Akt, and increases in copy number of AKT1/AKT2 genes were examined. Immunohistochemical analysis in 77 cases revealed overexpression of total Akt, Akt1, Akt2, and phosphorylated Akt in 84.4%, 67.5%, 72.7%, and 71.4%, respectively. Positive results were also observed in benign lesions but at a lower frequency. Overexpression of Akt1 was more frequent than that of Akt2 in well-differentiated liposarcoma (6/7 versus 3/7 cases) and schwannoma (4/4 versus 1/4 cases), whereas Akt2 overexpression and Akt activation were more frequent than Akt1 overexpression in malignant nerve sheath (3/4 and 4/4, respectively, versus 2/4 cases) and muscular tumors (8/9 and 8/9 versus 4/9 cases). By fluorescence in situ hybridization analysis, increase of gene copy number was observed in 13.3% for AKT1 and in 25.0% for AKT2 due to polysomy of chromosome 14 or 19, respectively, but not gene amplification. One case of schwannoma exhibited polysomy of both chromosomes 14 and 19. Akt activation was correlated with total Akt cytoplasmic localization (P = .0031) and subsequent metastasis (P = .0454). Moreover, AKT2 gene increase correlated with tumor size (P = .0352) and metastasis (P = .0344). In conclusion, in a defined subset of bone and soft tissue tumors, including benign tumors, Akt was frequently overexpressed and activated, and AKT1/2 copy number was increased. Because abnormality of Akt/AKT correlated with clinicopathologic profiles, novel therapies targeting isoform-specific Akts may be useful for these particular types of tumors.