Alterations in tumour suppressor gene p53 in human gliomas from Indian patients

Alterations in the tumour suppressor p53 gene are among the most common defects seen in a variety of human cancers. In order to study the significance of the p53 gene in the genesis and development of human glioma from Indian patients, we checked 44 untreated primary gliomas for mutations in exons 5-9 of the p53 gene by PCR-SSCP and DNA sequencing. Sequencing analysis revealed six missense mutations. The incidence of p53 mutations was 13.6% (6 of 44). All the six mutations were found to be located in the central core domain of p53, which carries the sequence-specific DNA-binding domain. These results suggest a rather low incidence but a definite involvement of p53 mutations in the gliomas of Indian patients

Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM

Identification of potential serum biomarkers of glioblastoma: serum osteopontin levels correlate with poor prognosis

Background: The aim of this study is to identify serum biomarkers with classification and prognosis utility for astrocytoma, in particular glioblastoma (GBM). Methods: Our previous glioma microarray database was mined to identify genes that encode secreted or membrane-localized proteins. Subsequent analysis was done using significant analysis of microarrays, followed by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemical validation in tumor tissues, ELISA and Western blot validation in sera, and correlation with survival of GBM patients. Results: Significant analysis of microarrays identified 31 upregulated and 3 downregulated genes specifically in GBMs. RT-qPCR validation on an independent set of samples confirmed the GBM-specific differential expression of several genes, including three upregulated (CALU, CXCL9, and TIMP1) and two downregulated (GPX3 and TIMP3) novel genes. With respect to osteopontin (OPN), we show the GBM-specific upregulation by RT-qPCR and immunohistochemical staining of tumor tissues. Elevated serum OPN levels in GBM patients were also shown by ELISA and Western blot. GBM patients with high serum OPN levels had poorer survival than those with low serum OPN levels (median survival 9 versus 22 months respectively; P = 0.0001). Further, we also show high serum TIMP1 levels in GBM patients compared with grade II/III patients by ELISA and downregulation of serum GPX3 and TIMP3 proteins in GBMs compared with normal control by Western blot analysis. Conclusions: Several novel potential serum biomarkers of GBM are identified and validated. High serum OPN level is found as a poor prognostic indicator in GBMs. Impact: Identified serum biomarkers may have potential utility in astrocytoma classification and GBM prognosis

Novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis

Purpose: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. Experimental Design: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II - diffuse astrocytoma, grade III - anaplastic astrocytoma, and grade IV - glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). Results: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage-inducible α (GADD45α) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45α and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45α conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. Conclusions: Our study reveals that GADD45α and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45α overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients

Shift in AP-2α localization characterizes astrocytoma progression

Activator protein 2α (AP-2α) has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of AP-2α in astrocytoma samples of different grades both at the RNA level, by real-time qPCR and at the protein level, by immunohistochemistry, and to examine its correlation, if any, with patient outcome. Five Grade I, 14 Grade II, 18 Grade III, 72 Grade IV samples and 13 normal brain controls were included. We did not find any clear pattern of regulation at the RNA level with tumor grade. The RNA expression levels however, correlated to a large extent with the nuclear AP-2α staining in these samples (72.09%; 31/43). Further, we did not find a complete loss of nuclear AP-2α expression in the higher grades, in contrast to previous reports. Interestingly, we found cytoplasmic AP-2α expression in a majority of higher grade astrocytomas (Grade IV-85%; 33/39 and Grade III-74%; 14/19) in comparison to lower grades (Grade I-0%; 0/5 and Grade II-37.5%; 3/8) suggesting that the translocation of this protein from the nucleus to the cytoplasm may be responsible for the increased malignancy. The nuclear expression in these grades was found to be concomitantly reduced. Within GBMs, we found that decreased nuclear expression was indicative of a better prognosis. The striking observation was the shift in localization of this protein from the nucleus to the cytoplasm with increasing tumor grade, pointing to a crucial role for this transcription factor in the progression of astrocytomas

Alterations in tumour suppressor gene p53 in human gliomas from Indian patients

Alterations in the tumour suppressor p53 gene are among the most common defects seen in a variety of human cancers. In order to study the significance of the p53 gene in the genesis and development of human glioma from Indian patients, we checked 44 untreated primary gliomas for mutations in exons 5-9 of the p53 gene by PCR-SSCP and DNA sequencing. Sequencing analysis revealed six missense mutations. The incidence of p53 mutations was 13.6% (6 of 44). All the six mutations were found to be located in the central core domain of p53, which carries the sequence-specific DNA-binding domain. These results suggest a rather low incidence but a definite involvement of p53 mutations in the gliomas of Indian patients

Shift in AP−2αAP-2\alpha Localization Characterizes Astrocytoma Progression

Activator protein $2\alpha$ $(AP‑2\alpha)$ has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of $(AP‑2\alpha)$ in astrocytoma samples of different grades both at the RNA level, by real‑time qPCR and at the protein level, by immunohistochemistry, and to examine its correlation, if any, with patient outcome. Five Grade I, 14 Grade II, 18 Grade III, 72 Grade IV samples and 13 normal brain controls were included. We did not find any clear pattern of regulation at the RNA level with tumor grade. The RNA expression levels however, correlated to a large extent with the nuclear $(AP‑2\alpha)$ staining in these samples (72.09%; 31/43). Further, we did not find a complete loss of nuclear $(AP‑2\alpha)$ expression in the higher grades, in contrast to previous reports. Interestingly, we found cytoplasmic $(AP‑2\alpha)$ expression in a majority of higher grade astrocytomas (Grade IV—85%; 33/39 and Grade III—74%; 14/19) in comparison to lower grades (Grade I—0%; 0/5 and Grade II—37.5%; 3/8) suggesting that the translocation of this protein from the nucleus to the cytoplasm may be responsible for the increased malignancy. The nuclear expression in these grades was found to be concomitantly reduced. Within GBMs, we found that decreased nuclear expression was indicative of a better prognosis. The striking observation was the shift in localization of this protein from the nucleus to the cytoplasm with increasing tumor grade, pointing to a crucial role for this transcription factor in the progression of astrocytomas

Differential Expression Of Igfbp Isoforms In Astrocytoma: Prognostic Value Of Igfbp-2,-3, And-5 In Glioblastoma Patients

Insulin-like growth factor binding proteins (IGFBP) are known to modulate the actions of insulin-like growth factors (IGF) mainly by controlling the amount of free IGF that can bind to its receptors. IGFBP are also known to regulate cell survival in an IGF-independent pathway. However, they can have a complex bidirectional effect on tumorigenesis. Among the various IGFBP, the expression and role of IGFBP2 in gliomas has been extensively studied. In an earlier gene-expression profiling study on the various grades of astrocytoma, we identified the upregulation of IGFBP-2, - 5, and -7 in glioblastoma tumors. In order to establish a role for all the IGFBP isoforms in the progression and prognosis of astrocytoma, we analyzed the gene expressions of IGFBP-1 to –5, -7 to -10, and -L1 by real-time quantitative polymerase chain reaction (RT-PCR) and IGFBP-2, -3, -4, -5, and -7 by immunohistochemistry (IHC) in a cohort of 50 diffusely infiltrating astrocytoma tissues. There were greater than two-fold upregulations in the transcript levels of IGFBP-2, -3, and -4 and in the majority of glioblastomas (GBM) relative to anaplastic astrocytomas (AA). On the other hand, IGFBP-5 and -7 were upregulated in the majority of malignant astrocytomas (AA and GBM). In contrast to these observations, IGFBP-9 transcript levels were found to be downregulated in both AA and GBM tumors. Overexpression of IGFBP -2, -3, -4, –5, and -7 was observed by IHC maximally in the tumor cells of GBM. Notably, we found that in an independent set of GBM patients, expression of IGFBP-2 alone and coexpression of IGFBP-2 and -5 in the tumor were indicative of shorter survival. In addition, when IGFBP-3 coexpressed with epidermal growth factor receptor, it indicated shorter patient survival. Our study suggested associations between IGFBP-2, -3, -4, -5, and -7 and malignant transformation in diffusely infiltrating astrocytomas, with maximal expression in GBM. We also showed that overexpression of IGFBP-2, -3, and -5 isoforms portended poor prognosis in GBM patients

Cockayne syndrome - A Clinical, Radiological, Audiological And Chromosomal Study

We report two brothers of Cockayne syndrome (CS) with progressive growth retardation, microcephaly, bird headed facies with sunken eyes, cutaneous photosensitivity, retinits pigmentosa, sensorineural deafness, spasticity ataxia, neuropathy and intracranial calcifiactions. These clinical with radiological features of cortical and cerebellar atrophy with basal ganglionic calcification and presence of consanguinity in parents and chromosome studies showing sister chromatid exchange in less than 6% strongly supported the diagnosis of Cockyne syndrome and differentiated it from Bloom′s syndrome and xeroderma pigmentosa. Without genetic analysis or tests for defective DNA repair, the diagnosis is mostly clinical