Fit-for-Purpose Ki-67 Immunohistochemistry Assays for Breast Cancer.

New therapies are being developed for breast cancer, and in this process, some "old" biomarkers are reutilized and given a new purpose. It is not always recognized that by changing a biomarker's intended use, a new biomarker assay is created. The Ki-67 biomarker is typically assessed by immunohistochemistry (IHC) to provide a proliferative index in breast cancer. Canadian laboratories assessed the analytical performance and diagnostic accuracy of their Ki-67 IHC laboratory-developed tests (LDTs) of relevance for the LDTs' clinical utility. Canadian clinical IHC laboratories enrolled in the Canadian Biomarker Quality Assurance Pilot Run for Ki-67 in breast cancer by invitation. The Dako Ki-67 IHC pharmDx assay was employed as a study reference assay. The Dako central laboratory was the reference laboratory. Participants received unstained slides of breast cancer tissue microarrays with 32 cases and performed their in-house Ki-67 assays. The results were assessed using QuPath, an open-source software application for bioimage analysis. Positive percent agreement (PPA, sensitivity) and negative percent agreement (NPA, specificity) were calculated against the Dako Ki-67 IHC pharmDx assay for 5%, 10%, 20%, and 30% cutoffs. Overall, PPA and NPA varied depending on the selected cutoff; participants were more successful with 5% and 10%, than with 20% and 30% cutoffs. Only 4 of 16 laboratories had robust IHC protocols with acceptable PPA for all cutoffs. The lowest PPA for the 5% cutoff was 85%, for 10% was 63%, for 20% was 14%, and for 30% was 13%. The lowest NPA for the 5% cutoff was 50%, for 10% was 33%, for 20% was 50%, and for 30% was 57%. Despite many years of international efforts to standardize IHC testing for Ki-67 in breast cancer, our results indicate that Canadian clinical LDTs have a wide analytical sensitivity range and poor agreement for 20% and 30% cutoffs. The poor agreement was not due to the readout but rather due to IHC protocol conditions. International Ki-67 in Breast Cancer Working Group (IKWG) recommendations related to Ki-67 IHC standardization cannot take full effect without reliable fit-for-purpose reference materials that are required for the initial assay calibration, assay performance monitoring, and proficiency testing.

Chitinase 3-like 1 promotes macrophage recruitment and angiogenesis in colorectal cancer.

Chitinase 3-like 1 (CHI3L1), one of the mammalian members of the chitinase family, is expressed in several types of human cancer, and elevated serum level of CHI3L1 is suggested to be a biomarker of poor prognosis in advanced cancer patients. However, the overall biological function of CHI3L1 in human cancers still remains unknown. Studies were performed to characterize the role of CHI3L1 in cancer pathophysiology utilizing human colorectal cancer samples and human cell lines. Plasma protein and tissue mRNA expression levels of CHI3L1 in colorectal cancer were strongly upregulated. Immunohistochemical analysis showed that CHI3L1 was expressed in cancer cells, and CHI3L1 expression had a significant association with the number of infiltrated macrophages and microvessel density (MVD). By utilizing transwell migration and tube-formation assays, overexpression of CHI3L1 in SW480 cells (human colon cancer cells) enhanced the migration of THP-1 cells (human macrophage cells) and HUVECs (human endothelial cells), and the tube formation of HUVECs. The knockdown of CHI3L1 by RNA interference or the neutralization of CHI3L1 by anti-CHI3L1 antibody displayed strong suppression of CHI3L1-induced migration and tube formation. Cell proliferation assay showed that CHI3L1 overexpression significantly enhanced the proliferation of SW480 cells. Enzyme-linked immunosorbent assay (ELISA) analysis showed that CHI3L1 increased the secretion of inflammatory chemokines, IL-8 and monocyte chemoattractant protein-1 (MCP-1), from SW480 cells through mitogen-activated protein kinase (MAPK) signaling pathway. Both neutralization of IL-8 or MCP-1 and inhibition or knockdown of MAPK in SW480 cells significantly inhibited CHI3L1-induced migration and tube formation. In a xenograft mouse model, overexpression of CHI3L1 in HCT116 cells (human colon cancer cells) enhanced the tumor growth as well as macrophage infiltration and MVD. In conclusion, CHI3L1 expressed in colon cancer cells promotes cancer cell proliferation, macrophage recruitment and angiogenesis. Thus, the inhibition of CHI3L1 activity may be a novel therapeutic strategy for human colorectal cancer.

Inflammation-induced repression of tumor suppressor miR-7 in gastric tumor cells.

Inflammation has an important role in cancer development through various mechanisms. It has been shown that dysregulation of microRNAs (miRNAs) that function as oncogenes or tumor suppressors contributes to tumorigenesis. However, the relationship between inflammation and cancer-related miRNA expression in tumorigenesis has not yet been fully understood. Using K19-C2mE and Gan mouse models that develop gastritis and gastritis-associated tumors, respectively, we found that 21 miRNAs were upregulated, and that 29 miRNAs were downregulated in gastric tumors in an inflammation-dependent manner. Among these miRNAs, the expression of miR-7, a possible tumor suppressor, significantly decreased in both gastritis and gastric tumors. Moreover, the expression of miR-7 in human gastric cancer was inversely correlated with the levels of interleukin-1ß and tumor necrosis factor-α, suggesting that miR-7 downregulation is related to the severity of inflammatory responses. In the normal mouse stomach, miR-7 expression was at a basal level in undifferentiated gastric epithelial cells, and was induced during differentiation. Moreover, transfection of a miR-7 precursor into gastric cancer cells suppressed cell proliferation and soft agar colony formation. These results suggest that suppression of miR-7 expression is important for maintaining the undifferentiated status of gastric epithelial cells, and thus contributes to gastric tumorigenesis. Although epigenetic changes were not found in the CpG islands around miR-7-1 of gastritis and gastric tumor cells, we found that activated macrophage-derived small molecule(s) (<3 kDa) are responsible for miR-7 repression in gastric cancer cells. Furthermore, the miR-7 expression level significantly decreased in the inflamed gastric mucosa of Helicobacter-infected mice, whereas it increased in the stomach of germfree K19-C2mE and Gan mice wherein inflammatory responses were suppressed. Taken together, these results indicate that downregulation of tumor suppressor miR-7 is a novel mechanism by which the inflammatory response promotes gastric tumorigenesis.

Chromosomal instability by beta-catenin/TCF transcription in APC or beta-catenin mutant cells.

Adenomatous polyposis coli (APC/Apc) gene encodes a key tumor suppressor whose mutations activate beta-catenin/T-cell factor (TCF)-mediated transcription (canonical Wnt signaling). Here, we show that Wnt signaling can cause chromosomal instability (CIN). As an indicator of CIN, we scored anaphase bridge index (ABI) in mouse polyps and ES cells where Wnt signaling was activated by Apc or beta-catenin mutations. We found three to nine times higher ABI than in wild-type controls. Furthermore, karyotype analysis confirmed that the Wnt signal-activated ES cells produced new chromosomal aberrations at higher rates; hence CIN. Consistently, expression of dominant-negative TCFs in these cells reduced their ABI. We also found that Wnt signal activation increased phosphorylation of Cdc2 (Cdk1) that inhibited its activity, and suppressed apoptosis upon exposure of the cells to nocodazole or colcemid. The data suggest that Wnt signaling stimulates the cells to escape from mitotic arrest and apoptosis, resulting in CIN. In human gastric cancer tissues with nuclear beta-catenin, ABI was significantly higher than in those without. These results collectively indicate that beta-catenin/TCF-mediated transcription itself increases CIN through dysregulation of G2/M progression.

Hypoxia induced by benign intestinal epithelial cells is associated with cyclooxygenase-2 expression in stromal cells through AP-1-dependent pathway.

Cyclooxygenase-2 (COX-2) plays important roles in tumor development. Especially in the early-stage colorectal tumors, COX-2 expression is often observed in the tumor stroma. However, the mechanism regulating such stromal expression of COX-2 remains unknown. In the present study, we simulated the indirect interaction between epithelial cells and stromal cells in the process of colorectal tumor development using an in vitro co-culture model in which NIH3T3 fibroblasts were co-cultured with 'sparsely' or 'densely' populated intestinal epithelial cells, Intestine-407 as a model of premalignant or benign intestinal epithelial cells, and DLD-1 and Caco-2 as models of malignant epithelial cells. COX-2 expression in NIH3T3 fibroblasts was upregulated when co-cultured with the 'dense' epithelial cells regardless of their character. Interestingly, there was pericellular hypoxia in the vicinity of NIH3T3 fibroblasts when co-cultured with 'dense' epithelial cells, and the recovery of the partial pressure of oxygen level resulted in the reduction of enhanced COX-2 expression only in NIH3T3 fibroblasts co-cultured with 'dense' Intestine-407 cells. Furthermore, COX-2 expression was also reduced by the inhibition of transcription factor AP-1. Thus, pericellular hypoxia of the stromal cells caused by densely populated epithelial cells may be one of the potent COX-2 enhancers before completion of malignant transformation during intestinal tumor development.

Group size determined by fusion and fission. A mathematical modelling with inclusive fitness.

We consider a mathematical model for the group size determination by the intra-reactions, self-growth, ostracism and fission within a group, and by the inter-reactions, immigration and fusion between two groups. In some group reactions, a conflict between two groups occurs about the reaction to change the group size. We construct a mathematical model to consider such conflict, taking into account the inclusive fitness of members in each group. In the conflict about the fusion between two groups, our analysis shows that the smaller group wants to fuse, while the larger does not. Also the criterion to resolve the conflict is discussed, and some numerical examples are given, too. It is concluded that, depending on the deviation in the total cost paid for the conflict by counterparts, the group reactions could result in a terminal group size different from that reached only by a sequence of outsider's immigrations into a group.

Usefulness of famotidine in functional dyspepsia patient treatment: comparison among prokinetic, acid suppression and antianxiety therapies.

BACKGROUND: The treatment of functional dyspepsia is controversial. AIM: The purpose of this paper is to clarify the initial effect of prokinetic, acid suppression and antianxiety treatment for functional dyspepsia patients. PATIENTS AND METHODS: Sixty-four functional dyspepsia patients without Helicobacter pylori infection were randomly assigned to 15 mg/day of mosapride, 40 mg/day of famotidine, or 30 mg/day of tandospirone during an 8-week treatment. Individual functional dyspepsia symptoms were evaluated with 4 cm visual analogue scale before and at 2, 4 and 8 weeks after treatment. RESULTS: Among 64 enrolled patients, 62 completed the study. Within 2 weeks, visual analogue scale score in the mosapride-treated group decreased from 2.29 +/-0.14 to 1.57 +/- 0.20; in the famotidine from 2.04 +/- 0.16 to 1.09 +/- 0.12 (mean +/- S.E.). Therefore, there were significant improvements of functional dyspepsia symptoms in mosapride- and famotidine-treated patients (P <0.01). Furthermore, famotidine was significantly more effective than mosapride (P < 0.05). On the contrary, visual analogue scale score in the tandospirone therapy was 2.23 +/- 0.20 and 2.13 +/- 0.22 before and at 2 weeks, respectively, without any significant improvement. CONCLUSIONS: A treatment regimen of famotidine at 40 mg/day had a significant favourable effect on the clinical outcome in functional dyspepsia patients.

Effect of CYP2C19 polymorphism on the safety and efficacy of omeprazole in Japanese patients with recurrent reflux oesophagitis.

BACKGROUND: The polymorphic enzyme cytochrome P450 2C19 affects omeprazole metabolism. This influence on metabolism might affect serum gastrin levels, and safety, during long-term treatment of reflux oesophagitis. AIM: To examine the relationship between cytochrome P450 2C19 genotype and the safety profile of long-term omeprazole treatment. METHODS: A total of 119 Japanese patients with recurrent reflux oesophagitis underwent cytochrome P450 2C19 genotyping prior to receiving daily omeprazole 10 mg or 20 mg for 6-12 months, during which adverse event frequency, serum gastrin levels and endoscopic findings were monitored. RESULTS: The incidences of adverse events, serious adverse events and adverse events leading to withdrawal did not differ between homozygous extensive metabolizer (n = 46), heterozygous extensive metabolizer (n = 53) or poor metabolizer (n = 20) groups. In all genotype groups, serum gastrin increased during the first 3 months of dosing but stabilized thereafter. No significant differences were seen either in the rate of reflux oesophagitis healing or symptom improvement among genotype groups. CONCLUSIONS: Long-term treatment with omeprazole was well-tolerated in Japanese patients, irrespective of their cytochrome P450 2C19 metabolic genotype, indicating that dose adjustment depending on metabolic genotype is not required during treatment with omeprazole.

Possible role of REG Ialpha protein in ulcerative colitis and colitic cancer.

BACKGROUND AND AIMS: Although regenerating gene (REG) Ialpha protein may be involved in the inflammation and carcinogenesis in the gastrointestinal tract, its pathophysiological role in ulcerative colitis (UC) and the resulting colitic cancer remains unclear. We investigated expression of the REG Ialpha gene and its protein in UC and colitic cancer tissues. We examined whether cytokines are responsible for REG Ialpha gene expression and whether REG Ialpha protein has a trophic and/or an antiapoptotic effect on colon cancer cells. METHODS: Expression of REG Ialpha mRNA and its gene product in UC tissues was analysed by real time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. The effects of cytokines on REG Ialpha promoter activity were examined in LoVo cells by luciferase reporter assay. The effects of REG Ialpha protein on growth and H(2)O(2) induced apoptosis were examined in LoVo cells by MTT and TUNEL assays, respectively. RESULTS: REG Ialpha protein was strongly expressed in inflamed epithelium and in dysplasias and cancerous lesions in UC tissues. The level of REG Ialpha mRNA expression in UC tissues correlated significantly with severity of inflammation and disease duration. REG Ialpha promoter activity was enhanced by stimulation with interferon gamma or interleukin 6. REG Ialpha protein promoted cell growth and conferred resistance to H(2)O(2) induced apoptosis in LoVo cells. REG Ialpha protein promoted Akt phosphorylation and enhanced Bcl-xL and Bcl-2 expression in LoVo cells. CONCLUSIONS: The REG Ialpha gene is inducible by cytokines and its gene product may function as a mitogenic and/or an antiapoptotic factor in the UC-colitic cancer sequence.

Jaw-opening dystonia (Brueghel's syndrome) associated with cavum septi pellucidi and Verga's ventricle - a case report.

Jaw-opening dystonia (oromandibular dystonia with jaw-opening; Brueghel's syndrome) is a rare condition, and only a limited number of cases have been reported in the literature. However, many patients may remain undiscovered or misdiagnosed, like a patient described previously. A case (40-year-old man) of jaw-opening dystonia (oromandibular dystonia with jaw-opening; Brueghel's syndrome) is reported. In this case, brain anomalies, cavum septi pellucidi and Verga's ventricle, were observed on magnetic resonance imaging of the brain. This case and a review of the literature indicate the presence of organic factors in the etiology of Brueghel's syndrome. The etiological relationship of brain anomalies in Bruegel's syndrome is discussed.

Regression of fundic gland polyps following acquisition of Helicobacter pylori.

The prevalence of Helicobacter pylori infection is very low in patients with fundic gland polyps (FGPs) of the stomach. We report here two cases with multiple FGPs that regressed following new H pylori acquisition. Patient Nos I and II had multiple FGPs in normal fundic mucosa without inflammatory changes or atrophy. Both were not infected with H pylori. Following acquisition of H pylori infection however, all FGPs in both patients completely disappeared except for one FGP in patient No I. Although the size of the remaining polyp in patient No I was greatly reduced after H pylori acquisition, it became enlarged again after eradication. Interestingly, in the remaining polyp, we found an activating beta-catenin gene mutation whereas no such mutations were detected in FGPs of patient No II. Thus H pylori infection may have an inhibitory effect on the development of FGPs.