Identification of prognostic biomarkers for glioblastomas using protein expression profiling

A set of proteins reflecting the prognosis of patients have clinical significance since they could be utilized as predictive biomarkers and/or potential therapeutic targets. With the aim of finding novel diagnostic and prognostic markers for glioblastoma (GBM), a tissue microarray (TMA) library consisting of 62 GBMs and 28 GBM-associated normal spots was constructed. Immunohistochemistry against 78 GBM-associated proteins was performed. Expression levels of each protein for each patient were analyzed using an image analysis program and converted to H-score [summation of the intensity grade of staining (0-3) multiplied by the percentage of positive cells corresponding to each grade]. Based on H-score and hierarchical clustering methods, we divided the GBMs into two groups (n=19 and 37) that had significantly different survival lengths (p<0.05). In the two groups, expression of nine proteins (survivin, cyclin E, DCC, TGF-β, CDC25B, histone H1, p-EGFR, p-VEGFR2/3, p16) was significantly changed (q<0.05). Prognosis-predicting potential of these proteins were validated with another independent library of 82 GBM TMAs and a public GBM DNA microarray dataset. In addition, we determined 32 aberrant or mislocalized subcellular protein expression patterns in GBMs compared with relatively normal brain tissues, which could be useful for diagnostic biomarkers of GBM. We therefore suggest that these proteins can be used as predictive biomarkers and/or potential therapeutic targets for GBM.     

Predictive and prognostic markers in human glioblastomas

Opinion statement Glioblastomas (GBMs) are among the most aggressive of all known human tumors. The median survival times remain in the 12- to 15-month range despite aggressive surgery, radiation, and chemotherapy. Through molecular and genetic profiling efforts, underlying mechanisms of resistance to these therapies are becoming better understood. The present standard of care has been shaped by the recently reported phase III study by the European Organisation for Research and Treatment of Cancer and the National Cancer Institute of Canada, which found that the addition of temozolomide (TMZ) to radiation therapy significantly improved outcome compared with radiation alone. However, careful examination of these data reveals that not all GBM patients benefited from the addition of TMZ to radiation therapy. A companion correlative study found that GBM patients with tumors with MGMT promoter methylation appeared to derive the greatest benefit from the addition of TMZ to radiation therapy. Although this finding is provocative, it should be kept in mind that this study was performed retrospectively and that prospective validation is required before MGMT methylation can be used for clinical stratification purposes. However, this study does show promise for the tailoring of future treatments according to the molecular and genetic profiles of an individual's tumor rather than using the “one-glove-fits-all≓ approach that is currently being followed. As more effective “smart drugs≓ are developed, molecular and genetic profiling will assume even greater importance in this regard.     

Pathological findings and prognostic factors in recurrent glioblastomas

Glioblastomas, which are the most common primary intracranial tumor, are associated with the poorest survival time, which is typically 1–2?years. Age at initial diagnosis, Karnofsky performance score, and O⁶-methylguanine DNA-methyltransferase (MGMT) promoter methylation status are the most well-documented predictors of survival in patients with newly diagnosed glioblastoma. Few studies have examined prognostic factors in patients with recurrent glioblastomas. At relapse, the pathological features of glioblastomas are affected by tumor regrowth and the influence of chemoradiotherapy during the initial treatment. Morphological transformations at recurrence include quantitative changes in tumor cells, such as the presence of giant cells and gemistocytic cell formation, radiation necrosis, and vascular structural changes. Therefore, we should carefully examine pathological findings at recurrence. In this report, we analyzed MGMT promoter status, the MIB-1 index, and the pathology of tumor samples at the first (primary tumor) and second (recurrent tumor) surgeries and clarified prognostic factors in patients with recurrent cases. In the multivariate analysis, we showed that MIB-1 indexes at the time of the second surgery (p?=?0.004) persisted as a significant independent prognostic factor in survival of patients with recurrent glioblastoma.     

Correlation between the prognostic value and the expression of the stem cell marker CD133 and isocitrate dehydrogenase1 in glioblastomas

Cancer stem cells are thought to be responsible for tumor recurrence and resistance in glioblastomas. An isocitrate dehydrogenase1 (IDH1) mutation, affecting codon132 of the isocitrate dehydrogenase1 gene, has prognostic significance in glioblastomas. We investigated whether stem cell marker expression [CD133, CD34, and vascular endothelial growth factor (VEGF)] and IDH1 mutation correlate with clinical factors and prognosis in glioblastoma. CD133, CD34, and VEGF expression was evaluated by immunohistochemistry in 67 cases of glioblastoma identified between 2005 and 2012. IDH1 mutation was assessed by immunohistochemistry, peptide-nucleic-acid mediated PCR clamping, and direct gene sequencing. Diffuse CD133 expression was detected in 12 (17.9 %) cases and was associated with poor overall survival (OS) (P = 0.010) and progression-free survival (P = 0.017). CD34 and VEGF expression were not associated with prognosis in these samples. IDH1 mutation was detected in ten (14.9 %) cases. Eight were clinically secondary tumors and two were primary tumors (P < 0.001); the mean age of the secondary tumor patients was significantly younger (P = 0.001, 41.20 vs. 59.14). IDH1-positive patients had longer OS than IDH1-negative patients (25.78 vs. 22.95 months), but this difference was not significant. In addition, IDH1 and CD34 expression showed a negative correlation (P = 0.024). Multivariate analysis showed that age, extent of surgery, and diffuse CD133 expression correlated with OS. CD133 may be a survival marker for glioblastoma. Further characterization of CD133, IDH1, and vascular markers in glioblastoma may help identify new therapeutic targets.     

Prognostic significance of c-Met expression in glioblastomas

BACKGROUD:

The authors investigated whether expression of c‐Met protein in glioblastomas is associated with overall survival and biologic features representing tumor invasiveness in patients with glioblastomas.

METHODS:

Paraffin‐embedded specimens of glioblastomas from 62 patients treated in a single institution were assessed by immunohistochemical (IHC) analysis of c‐Met expression. On the basis of the clinical data for these patients, the association between c‐Met expression and clinicobiologic features representing tumor invasiveness was analyzed.

RESULTS:

c‐Met overexpression was detected in 29.0% (18 of 62) of glioblastomas. In patients with c‐Met overexpression, the median survival was 11.7 months (95% confidence interval [95% CI], 9.9 months‐13.5 months), compared with a median survival of 14.3 months (95% CI, 7.6 months‐21.0 months) for patients whose tumors had no or little expression of c‐Met (P = .031). On the radiographic analysis, 9 of 18 patients (50%) with tumors overexpressing c‐Met demonstrated invasive and multifocal lesions on the initial magnetic resonance images, whereas only 9 of 44 patients (20.5%) with tumors that expressed no or little c‐Met demonstrated these features (P = .030). Using immunohistochemistry, we also found a significant association between c‐Met expression and matrix metalloproteinase‐2,‐9 (P = .020 and P = .013). Furthermore, Myc overexpression was found to be closely correlated with c‐Met overexpression on IHC analysis (P = .004).

CONCLUSIONS:

The authors suggest that c‐Met overexpression is associated with shorter survival time and poor treatment response in glioblastomas, the mechanism for which is elevated tumor invasiveness on the molecular and clinical phenotypes. This implies that more effective therapeutic strategies targeting c‐Met receptors may have important clinical implication. Cancer 2009. © 2008 American Cancer Society.     

原发性胶质母细胞瘤和继发性胶质母细胞瘤中MMP-2蛋白及MDM2蛋白的表达和意义

目的:探讨MMP-2蛋白及MDM2蛋白在原发性胶质母细胞瘤和继发性胶质母细胞瘤中的表达差异性及其意义。方法:免疫组化SP二步法检测72例胶质母细胞瘤（36例原发性胶质母细胞瘤和36例继发性胶质母细胞瘤）石蜡包埋标本及10例正常脑组织石蜡包埋标本MMP-2蛋白和MDM2蛋白的表达情况,分析上述蛋白在两种病理类型的胶质母细胞瘤中的表达差异情况。结果:在原发性和继发性胶质瘤标本中,MMP-2蛋白的阳性表达率分别为63.9%和86.1%;MDM2蛋白的阳性表达率分别为61.1%和80.6%。MMP-2蛋白及MDM2蛋白在原发性和继发性胶质母细胞瘤中的表达差异存在统计学意义（P＜0.05）。结论:MMP-2蛋白及MDM2蛋白在原发性、继发性胶质母细胞瘤中的表达差异可反映它们的不同分子遗传学特征。     

COX-2在胶质母细胞瘤中的表达及与肿瘤增殖和凋亡的关系

目的: 探讨环氧化酶-2(COX-2)在胶质母细胞瘤中的表达,及其与肿瘤凋亡和增殖的关系.方法: 采用免疫组化S-P法检测34例星形细胞瘤患者和10例正常脑组织COX-2和Bcl-2、Ki-67的表达,用TUNEL法检测肿瘤细胞凋亡,并计算其凋亡指数(AI).结果: 胶质母细胞瘤组织中COX-2的阳性表达率明显高于对照组;Bcl-2和Ki-67在肿瘤组织中的表达均高于对照组,且和COX-2表达呈正相关性.结论: 胶质母细胞瘤中COX-2的过表达可抑制肿瘤细胞的凋亡,促进细胞增殖,其抑制凋亡的作用可能是通过提高Bcl-2的阳性表达实现的,在胶质母细胞瘤的发生和发展中起重要作用.     

Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration

Background:

Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with distinct molecular profiles. Gene expression profiling previously identified sonic hedgehog (SHH) as part of a gene signature that is differentially regulated in IBC patients.

Methods:

The effects of reducing GLI1 levels on protein expression, cell proliferation, apoptosis and migration were determined by immunoblots, MTT assay, Annexin-V/PI assay and conventional and automated cell migration assays.

Results:

Evaluation of a panel of breast cancer cell lines revealed elevated GLI1 expression, typically a marker for hedgehog-pathway activation, in a triple-negative, highly invasive IBC cell line, SUM149 and its isogenic-derived counterpart rSUM149 that has acquired resistance to ErbB1/2 targeting strategies. Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis. Further, GLI1 suppression in these cell lines significantly inhibited cell migration as assessed by a wound-healing assay compared with MCF-7, a non-invasive cell line with low GLI1 expression. A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement.

Conclusion:

Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.     

IL-6 gene amplification and expression in human glioblastomas

The aggressiveness of human gliomas appears to be correlated with the upregulation of interleukin 6 (IL-6) gene. Using quantitative PCR methods, we detected amplification and expression of the IL-6 gene in 5 of 5 primary glioblastoma samples and in 4 of 5 glioblastoma cell lines. This finding suggests that the amplification of IL-6 gene may be a common feature in glioblastomas and may contribute to the IL-6 over-expression.     

Proliferating cell nuclear antigen and p53 expression as prognostic factors in T1-2MO prostatic adenocarcinoma

The expression of proliferating cell nuclear antigen and p53 protein was analysed by immunocytochemical methods (PC10, CMI antisera) in 139 patients with T1-2MO prostatic adenocarcinomas followed-up for > 12 years. p53 protein was expressed in 21 (15%) tumours (15%), the fraction of positive nuclei being very low (mean SE, 1% ± 0.7%). Accumulation of p53 protein in epithelial cells was independent of tumour stage and Gleason score, and had no effect on prognosis. In 4 cases, p53 protein was expressed only in stromal cells. The fraction of PCNA-positive nuclei (evaluable in 116 cases) was higher in T2 than in T1 tumours (p < 0.001); furthermore, high Gleason score was positively correlated with PCNA positivity (p < 0.001). A finding of over 5% of PCNA-positive nuclei predicted progression in T and M categories and were a sign of poor outcome. The fraction of PCNA-positive stromal-cell nuclei was related to T-category with a borderline significance (p = 0.06). In a multi-variate analysis of the prognostic factors, independent predictors of survival included Gleason score (p < 0.001), fraction of PCNA-positive nuclei (p = 0.013), observation before therapy (p = 0.05), and T-category (p = 0.07) in that order of significance. The results suggest that overexpression of p53 protein is of marginal prognostic value in local prostatic adenocarcinomas, whereas direct measurement of cell proliferation by PCNA immunolabelling provides important prognostic information in T1-2MO tumours, in addition to the Gleason score.     

The prognostic relevance of molecular alterations in glioblastomas for patients age < 50 years

BACKGROUND: In patients with glioblastoma, age < 50 years was identified as a consistent prognostic variable. In addition, the prognosis for these patients may be determined by a complex interaction between age and genetic alterations. The objective of the current study was the molecular analysis of glioblastomas from adult patients age < 50 years ("young adults"). METHODS: The authors analyzed a set of 189 glioblastoma specimens. Fluorescence in situ hybridization was performed with a set of 10 chromosome probes (1p36, 1q25, centomere probe 7 [CEP7], 7p12/epidermal growth factor receptor gene (EGFR), CEP9, 9p21/p16, CEP10, 10q23/phosphatase and tesnin homolog gene (PTEN), 19p13, and 19q13). RESULTS: Patient age < 40 years was associated strongly with a favorable prognosis. Patients age > or = 40 years frequently showed EGFR amplification, loss of 9p, loss of 10q, and gain of chromosome 19. The patients with - 19q were age < 40 years. The survival was shorter for patients with EGFR amplification, gain of chromosome 7, loss of 9p, loss of 10q, and gain of chromosome 19. In contrast, the patients who had tumors with gain of chromosome 9 or loss of 19q had more favorable outcomes. In a multivariate analysis, gain of chromosome 9 (P = 0.026) and loss of 10q23 (P = 0.007) reached the level of independent prognostic value. In addition, the prognostic value of molecular alterations in patients age < 40 years and patients age > 40 years were examined separately. Consequently, EGFR amplification, - 9p, and + 9 were significant for both age groups, whereas gain of chromosome 7 and loss of 10q showed clinical importance only among patients age > 40 years. CONCLUSIONS: Adult patients age < 50 years with glioblastoma had molecularly distinct disease, and the age-dependent heterogeneity seen on the chromosomal level also applied at the clinical level.     

Negative prognostic impact of low absolute CD4+ T cell counts in peripheral blood in mantle cell lymphoma

Tumor microenvironment and host immunity are closely related to outcome in patients with mantle cell lymphoma (MCL). However, few researchers have focused on the prognostic value of peripheral blood lymphocyte subsets counts. The purpose of this study was to investigate the prognostic value of lymphocyte subsets and absolute monocyte counts. Sixty‐eight patients were analyzed retrospectively. Absolute CD4⁺ T cell counts (ACD4C), CD8⁺ T cell counts, nature killer cell counts, and CD4/CD8 ratios were assessed by peripheral blood flow cytometry and correlated with clinical parameters and long‐term outcomes. The median follow‐up for all patients was 21 months and the median survival time was 44 months. The overall survival (OS) rate at 1, 3, and 5 years was 80%, 51%, and 41%, respectively. In our cohort, high absolute monocyte count, and low ACD4C and CD4/CD8 ratio were associated with unfavorable OS (P = 0.029, P = 0.027, and P = 0.045, respectively) by univariate analysis. Multivariate analysis indicated that low ACD4C was a significant predictor of unfavorable OS (P = 0.004) independent of the simplified MCL International Prognostic Index (P = 0.048) in patients treated with or without rituximab (P = 0.011). Low CD4⁺ T cell counts proved to be a significant predictor of unfavorable OS in patients with MCL.