Prognostic value of Ki67 in localized prostate carcinoma: a multi-institutional study of >1000 prostatectomies.

BACKGROUND: Expanding interest in and use of active surveillance for early state prostate cancer (PC) has increased need for prognostic biomarkers. Using a multi-institutional tissue microarray resource including over 1000 radical prostatectomy samples, we sought to correlate Ki67 expression captured by an automated image analysis system with clinicopathological features and validate its utility as a clinical grade test in predicting cancer-specific outcomes. METHODS: After immunostaining, the Ki67 proliferation index (PI) of tumor areas of each core (three cancer cores/case) was analyzed using a nuclear quantification algorithm (Aperio). We assessed whether Ki67 PI was associated with clinicopathological factors and recurrence-free survival (RFS) including biochemical recurrence, metastasis or PC death (7-year median follow-up). RESULTS: In 1004 PCs (∼4000 tissue cores) Ki67 PI showed significantly higher inter-tumor (0.68) than intra-tumor variation (0.39). Ki67 PI was associated with stage (P<0.0001), seminal vesicle invasion (SVI, P=0.02), extracapsular extension (ECE, P<0.0001) and Gleason score (GS, P<0.0001). Ki67 PI as a continuous variable significantly correlated with recurrence-free, overall and disease-specific survival by multivariable Cox proportional hazard model (hazards ratio (HR)=1.04-1.1, P=0.02-0.0008). High Ki67 score (defined as ⩾5%) was significantly associated with worse RFS (HR=1.47, P=0.0007) and worse overall survival (HR=2.03, P=0.03). CONCLUSIONS: In localized PC treated by radical prostatectomy, higher Ki67 PI assessed using a clinical grade automated algorithm is strongly associated with a higher GS, stage, SVI and ECE and greater probability of recurrence.

Axl receptor tyrosine kinase is a potential therapeutic target in renal cell carcinoma.

BACKGROUND: Axl plays multiple roles in tumourigenesis in several cancers. Here we evaluated the expression and biological function of Axl in renal cell carcinoma (RCC). METHODS: Axl expression was analysed in a tissue microarray of 174 RCC samples by immunostaining and a panel of 11 normal tumour pairs of human RCC tissues by western blot, as well as in RCC cell lines by both western blot and quantitative PCR. The effects of Axl knockdown in RCC cells on cell growth and signalling were investigated. The efficacy of a humanised Axl targeting monoclonal antibody hMAb173 was tested in histoculture and tumour xenograft. RESULTS: We have determined by immunohistochemistry (IHC) that Axl is expressed in 59% of RCC array samples with moderate to high in 20% but not expressed in normal kidney tissue. Western blot analysis of 11 pairs of tumour and adjacent normal tissue show high Axl expression in 73% of the tumours but not normal tissue. Axl is also expressed in RCC cell lines in which Axl knockdown reduces cell viability and PI3K/Akt signalling. The Axl antibody hMAb173 significantly induced RCC cell apoptosis in histoculture and inhibited the growth of RCC tumour in vivo by 78%. The hMAb173-treated tumours also had significantly reduced Axl protein levels, inhibited PI3K signalling, decreased proliferation, and induced apoptosis. CONCLUSIONS: Axl is highly expressed in RCC and critical for RCC cell survival. Targeting Axl is a potential approach for RCC treatment.

Transgenic expression of the forkhead box M1 transcription factor induces formation of lung tumors.

The forkhead box m1 (Foxm1 or Foxm1b) protein (previously called HFH-11B, Trident, Win or MPP2) is abundantly expressed in human non-small cell lung cancers where it transcriptionally induces expression of genes essential for proliferation of tumor cells. In this study, we used Rosa26-Foxm1 transgenic mice, in which the Rosa26 promoter drives ubiquitous expression of Foxm1 transgene, to identify new signaling pathways regulated by Foxm1. Lung tumors were induced in Rosa26-Foxm1 mice using the 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT) lung tumor initiation/promotion protocol. Tumors from MCA/BHT-treated Rosa26-Foxm1 mice displayed a significant increase in the number, size and DNA replication compared to wild-type mice. Elevated tumor formation in Rosa26-Foxm1 transgenic lungs was associated with persistent pulmonary inflammation, macrophage infiltration and increased expression of cyclooxygenase-2 (Cox-2), Cdc25C phosphatase, cyclin E2, chemokine ligands CXCL5, CXCL1 and CCL3, cathepsins and matrix metalloprotease-12. Cell culture experiments with A549 human lung adenocarcinoma cells demonstrated that depletion of Foxm1 by either short interfering RNA transfection or treatment with Foxm1-inhibiting ARF 26-44 peptide significantly reduced Cox-2 expression. In co-transfection experiments, Foxm1 protein-induced Cox-2 promoter activity and directly bound to the -2566/-2580 bp region of human Cox-2 promoter.

Downstream signalling and specific inhibition of c-MET/HGF pathway in small cell lung cancer: implications for tumour invasion.

The c-MET receptor can be overexpressed, amplified, or mutated in solid tumours including small cell lung cancer (SCLC). In c-MET-overexpressing SCLC cell line NCI-H69, hepatocyte growth factor (HGF) dramatically induced c-MET phosphorylation at phosphoepitopes pY1230/1234/1235 (catalytic tyrosine kinase), pY1003 (juxtamembrane), and also of paxillin at pY31 (CRKL-binding site). We utilised a global proteomics phosphoantibody array approach to identify further c-MET/HGF signal transduction intermediates in SCLC. Strong HGF induction of specific phosphorylation sites in phosphoproteins involved in c-MET/HGF signal transduction was detected, namely adducin-alpha [S724], adducin-gamma [S662], CREB [S133], ERK1 [T185/Y187], ERK1/2 [T202/Y204], ERK2 [T185/Y187], MAPKK (MEK) 1/2 [S221/S225], MAPKK (MEK) 3/6 [S189/S207], RB [S612], RB1 [S780], JNK [T183/Y185], STAT3 [S727], focal adhesion kinase (FAK) [Y576/S722/S910], p38alpha-MAPK [T180/Y182], and AKT1[S473] and [T308]. Conversely, inhibition of phosphorylation by HGF in protein kinase C (PKC), protein kinase R (PKR), and also CDK1 was identified. Phosphoantibody-based immunohistochemical analysis of SCLC tumour tissue and microarray established the role of c-MET in SCLC biology. This supports a role of c-MET activation in tumour invasive front in the tumour progression and invasion involving FAK and AKT downstream. The c-MET serves as an attractive therapeutic target in SCLC, as shown through small interfering RNA (siRNA) and selective prototype c-MET inhibitor SU11274, inhibiting the phosphorylation of c-MET itself and its downstream molecules such as AKT, S6 kinase, and ERK1/2. Investigation of mechanisms of invasion and, ultimately, metastasis in SCLC would be very useful with these signal transduction molecules.

Topoisomerase IIalpha in Wilms' tumour: gene alterations and immunoexpression.

BACKGROUND: Topoisomerase IIalpha (topoIIalpha) is an essential enzyme gene in regulating DNA structure and cell proliferation and is encoded by the TOP2A. Using cDNA microarray analysis, TOP2A has been reported to be one of the top genes overexpressed in Wilms' tumour. AIM: To evaluate the role of TopoIIalpha in Wilms' tumorigenesis and its prognostic value. METHODS: TOP2A gene copy numbers were determined using the fluorescence in situ hybridisation technique, and protein expression levels of TopoIIalpha by immunostaining in 39 samples of primary and 18 samples of metastatic Wilms' tumour. RESULTS: TOP2A gene amplification was detected only in anaplastic Wilms' tumours, and none of the Wilms' tumours showed deletion of the TOP2A gene. TopoIIalpha protein overexpression was detected in 97% of Wilms' tumours, and correlated strongly with proliferation, as measured by Ki-67 (r = 0.85). The high TopoIIalpha expression was associated with the presence of vascular invasion, prominent apoptosis, metastases and adverse clinical outcomes (p<0.05). CONCLUSIONS: Our findings suggest that TopoIIalpha overexpression in Wilms' tumours is caused by a change at the transcription level, except for anaplastic Wilms' tumours, in which gene amplification was present. High levels of TopoIIalpha protein are correlated with tumour aggressiveness. The assessment of TopoIIalpha expression in Wilms' tumour may have prognostic value.

Alpha-methylacyl-CoA racemase: a multi-institutional study of a new prostate cancer marker.

AIM: To test whether alpha-methylacyl-CoA racemase (AMACR) is a sensitive and specific marker of prostate cancer. METHODS AND RESULTS: The expression levels of AMACR mRNA were measured by real-time polymerase chain reaction. A total of 807 prostatic specimens were further examined by immunohistochemistry specific for AMACR. Quantitative immunostaining analyses were carried out by using the ChromaVision Automated Cellular Imaging System and the Ariol SL-50 Imaging System, respectively. AMACR mRNA levels measured in prostatic adenocarcinoma were 55 times higher than those in benign prostate tissue. Of 454 cases of prostatic adenocarcinoma, 441 were positive for AMACR, while 254 of 277 cases of benign prostate were negative for AMACR. The sensitivity and specificity of AMACR immunodetection of prostatic adenocarcinomas were 97% and 92%, respectively. Both positive and negative predictive values were 95%. By automatic imaging analyses, the AMACR immunostaining intensity and percentage in prostatic adenocarcinomas were also significantly higher than those in benign prostatic tissue (105.9 versus 16.1 for intensity, 45.7% versus 0.02% and 35.03% versus 4.64% for percentage, respectively). CONCLUSIONS: We have demonstrated the promising features of AMACR as a biomarker for prostate cancer in this large series and the potential to develop automated quantitative diagnostic tests.

Expression of NOD2 in Paneth cells: a possible link to Crohn's ileitis.

BACKGROUND AND AIMS: Genetic variation in NOD2 has been associated with susceptibility to Crohn's disease (CD) and specifically with ileal involvement. The reason for the unique association of NOD2 mutations with ileal disease is unclear. To identify a possible link, we tested expression of NOD2 in intestinal tissue of CD patients and controls. PATIENTS AND METHODS: Fifty five specimens of ileum or colon from 21 CD patients, seven ulcerative colitis (UC) patients, and five controls with pathology other than CD or UC were stained for NOD2 using an immunoperoxidase method. RESULTS: Using a monoclonal antibody against NOD2 developed in our laboratory, we detected uniform expression of NOD2 in terminal ileum Paneth cells from controls and patients as well as in metaplastic Paneth cells in the colon. Mechanical purification showed enriched expression of NOD2 mRNA in ileal crypts. In Paneth cells, NOD2 was located in the cytosol in close proximity to the granules that contain antimicrobial peptides. We detected minimal NOD2 in the villous epithelium of the ileum or in the colonic epithelium from both CD patients and controls. CONCLUSIONS: These results suggest a role for NOD2 in the regulation of Paneth cell mediated responses against intestinal bacteria and a plausible mechanism to explain the selective association of NOD2 mutations with ileal disease. The impaired capacity of CD associated mutations to sense luminal bacteria may result in increased susceptibility to certain gut microbes.

Gastrin-releasing peptide is a mitogen and a morphogen in murine colon cancer.

Little is known about the factors involved in regulating the appearance, or differentiation, of solid tumors including those arising from the colon. We herein demonstrate that the mitogen gastrin-releasing peptide (GRP) is a morphogen, critically important in regulating the differentiation of murine colon cancer. Although epithelial cells lining the mouse colon do not normally express GRP and its receptor (GRP-R), both are aberrantly expressed by all better differentiated cancers in wild-type C57BL/6J mice treated with the carcinogen azoxymethane. Whereas small tumors in both wild-type and GRP-R-deficient (i.e., GRP-R-/-) mice are histologically similar, larger tumors become better differentiated in the former but degenerate into more poorly differentiated mucinous adenocarcinomas in the latter. This alteration in phenotype is attributable to GRP increasing focal adhesion kinase expression in GRP-R-expressing tumors. Consistent with GRP acting as a mitogen, GRP/GRP-R coexpressing tumors in wild-type animals also contain more proliferating cells than those occurring in GRP-R-/- mice. Yet tumors are similarly sized in animals of either genotype receiving azoxymethane for identical times, a finding attributable to the significantly higher number of apoptotic cells detected in GRP/GRP-R coexpressing cancers. Thus, these findings indicate that although GRP is a mitogen, aberrant expression does not result in increased tumor growth. Rather, the mitogenic properties of GRP are subordinate to it acting as a morphogen, where it and its receptor are critically involved in regulating colon cancer histological progression by promoting a well-differentiated phenotype.

Proliferative activity of oxyphilic (Hurthle) cells in reactive and neoplastic thyroid lersions.

The proliferative potential of oxyphilic (Hurthle) cells (HCs) present in neoplastic and non-neoplastic thyroid lesions is uncertain. To estimate the HCs ability to proliferate and to determine whether their proliferative activity correlates with the biologic behavior of different thyroid oxyphilic lesions, we selected 31 cases of chronic lymphocytic (Hashimoto's) thyroiditis and 28 oxyphilic (Hurthle cell) thyroid tumors, including 12 adenomas and 16 carcinomas. Seven histologically normal thyroid specimens from euthyroid patients served as control tissue. The proliferative activity of HCs was evaluated by means of a double immunostaining for Ki67 and a mitochondrial antigen (which specifically recognizes oxyphilic cells). Oxyphilic cells in thyroiditis had a low proliferative activity (PA: 0.55%), although higher than that of normal thyroid parenchyma (PA: 0.06%). Neoplastic HC lesions had a mean proliferative activity of 1.56% and 6.26% in adenomas and carcinomas, respectively. A statistically significant difference was observed between proliferative activity of non-neoplastic and neoplastic lesions (p<0.01), but not within the tumor group, between adenomas and carcinomas. In addition, HC carcinomas had a statistically significant positive correlation between proliferative activity and tumor size (p<0.01) and the presence of necrosis (p<0.001).