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A R Glover, J T Zhao, J C Ip, J C Lee, B G Robinson, A J Gill, P S H Soon, and S B Sidhu

Adrenocortical carcinoma (ACC) is an aggressive malignancy with high rates of recurrence following surgical resection. Long noncoding RNAs (lncRNAs) play an important role in cancer development. Pathogenesis of adrenal tumours have been characterised by mRNA, microRNA and methylation expression signatures, but it is unknown if this extends to lncRNAs. This study describes lncRNA expression signatures in ACC, adrenal cortical adenoma (ACA) and normal adrenal cortex (NAC) and presents lncRNAs associated with ACC recurrence to identify novel prognostic and therapeutic targets. RNA was extracted from freshly frozen tissue with confirmation of diagnosis by histopathology. Focused lncRNA and mRNA transcriptome analysis was performed using the ArrayStar Human LncRNA V3.0 microarray. Differentially expressed lncRNAs were validated using quantitative reverse transcriptase-PCR and correlated with clinical outcomes. Microarray of 21 samples (ten ACCs, five ACAs and six NACs) showed distinct patterns of lncRNA expression between each group. A total of 956 lncRNAs were differentially expressed between ACC and NAC, including known carcinogenesis-related lncRNAs such as H19, GAS5, MALAT1 and PRINS (P≤0.05); 85 lncRNAs were differentially expressed between ACC and ACA (P≤0.05). Hierarchical clustering and heat mapping showed ACC samples correctly grouped compared with NAC and ACA. Sixty-six differentially expressed lncRNAs were found to be associated with ACC recurrence (P≤0.05), one of which, PRINS, was validated in a group of 20 ACCs and also found to be associated with metastatic disease on presentation. The pathogenesis of adrenal tumours extends to lncRNA dysregulation and low expression of the lncRNA PRINS is associated with ACC recurrence.

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P S H Soon, A J Gill, D E Benn, A Clarkson, B G Robinson, K L McDonald, and S B Sidhu

The management of adrenocortical tumors (ACTs) is complex. The Weiss score is the present most widely used system for ACT diagnosis. An ACT is scored from 0 to 9, with a higher score correlating with increased malignancy. However, ACTs with a score of 3 can be phenotypically benign or malignant. Our objective is to use microarray profiling of a cohort of adrenocortical carcinomas (ACCs) and adrenocortical adenomas (ACAs) to identify discriminatory genes that could be used as an adjunct to the Weiss score. A cohort of Weiss score defined ACCs and ACAs were profiled using Affymetrix HGU133plus2.0 genechips. Genes with high-discriminatory power were identified by univariate and multivariate analyses and confirmed by quantitative real-time reverse transcription PCR and immunohistochemistry (IHC). The expression of IGF2, MAD2L1, and CCNB1 were significantly higher in ACCs compared with ACAs while ABLIM1, NAV3, SEPT4, and RPRM were significantly lower. Several proteins, including IGF2, MAD2L1, CCNB1, and Ki-67 had high-diagnostic accuracy in differentiating ACCs from ACAs. The best results, however, were obtained with a combination of IGF2 and Ki-67, with 96% sensitivity and 100% specificity in diagnosing ACCs. Microarray gene expression profiling accurately differentiates ACCs from ACAs. The combination of IGF2 and Ki-67 IHC is also highly accurate in distinguishing between the two groups and is particularly helpful in ACTs with Weiss score of 3.

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M Bullock, C O'Neill, A Chou, A Clarkson, T Dodds, C Toon, M Sywak, S B Sidhu, L W Delbridge, B G Robinson, D L Learoyd, D Capper, A von Deimling, R J Clifton-Bligh, and A J Gill

Identification of BRAFV600E in thyroid neoplasia may be useful because it is specific for malignancy, connotes a worse prognosis, and is the target of novel therapies currently under investigation. Sanger sequencing is the ‘gold standard’ for mutation detection but is subject to sampling error and requires resources beyond many diagnostic pathology laboratories. In this study, we compared immunohistochemistry (IHC) using a BRAF V600E mutation-specific MAB to Sanger sequencing on DNA from formalin-fixed paraffin-embedded tissue, in a well-characterized cohort of 101 papillary thyroid carcinoma (PTC) patients. For all cases, an IHC result was available; however, five cases failed Sanger sequencing. Of the 96 cases with molecular data, 68 (71%) were BRAF V600E positive by IHC and 59 (61%) were BRAF V600E positive by sequencing. Eleven cases were discordant. One case was negative by IHC and initially positive by sequencing. Repeat sequencing of that sample and sequencing of a macrodissected sample were negative for BRAF V600E. Of ten cases positive by IHC but negative by sequencing on whole sections, repeat sequencing on macrodissected tissue confirmed the IHC result in seven cases (suggesting that these were false negatives of sequencing on whole sections). In three cases, repeat sequencing on recut tissue remained negative (including using massive parallel sequencing), but these cases demonstrated relatively low neoplastic cellularity. We conclude that IHC for BRAF V600E is more sensitive and specific than Sanger sequencing in the routine diagnostic setting and may represent the new gold standard for detection of BRAF V600E mutation in PTC.

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V H M Tsang, T Dwight, D E Benn, G Y Meyer-Rochow, A J Gill, M Sywak, S Sidhu, D Veivers, C M Sue, B G Robinson, R J Clifton-Bligh, and N R Parker

miR-210 is a key regulator of response to hypoxia. Pheochromocytomas (PCs) and paragangliomas (PGLs) with germline SDHx or VHL mutations have pseudohypoxic gene expression signatures. We hypothesised that PC/PGLs containing SDHx or VHL mutations, and succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumours (GISTs), would overexpress miR-210 relative to non-SDH or -VHL-mutated counterparts. miR-210 was analysed by quantitative PCR in i) 39 PC/PGLs, according to genotype (one SDHA, five SDHB, seven VHL, three NF1, seven RET, 15 sporadic, one unknown) and pathology (18 benign, eight atypical, 11 malignant, two unknown); ii) 18 GISTs, according to SDHB immunoreactivity (nine SDH-deficient and nine SDH-proficient) and iii) two novel SDHB-mutant neurosphere cell lines. miR-210 was higher in SDHx- or VHL-mutated PC/PGLs (7.6-fold) compared with tumours without SDHx or VHL mutations (P=0.0016). miR-210 was higher in malignant than in unequivocally benign PC/PGLs (P=0.05), but significance was lost when benign and atypical tumours were combined (P=0.08). In multivariate analysis, elevated miR-210 was significantly associated with SDHx or VHL mutation, but not with malignancy. In GISTs, miR-210 was higher in SDH-deficient (median 2.58) compared with SDH-proficient tumours (median 0.60; P=0.0078). miR-210 was higher in patient-derived neurosphere cell lines containing SDHB mutations (6.5-fold increase) compared with normal controls, in normoxic conditions (P<0.01). Furthermore, siRNA-knockdown of SDHB in HEK293 cells increased miR-210 by 2.7-fold (P=0.001) under normoxia. Overall, our results suggest that SDH deficiency in PC, PGL and GISTs induces miR-210 expression and substantiates the role of aberrant hypoxic-type cellular responses in the development of these tumours.

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Michael A Hahn, Viive M Howell, Anthony J Gill, Adele Clarkson, Graham Weaire-Buchanan, Bruce G Robinson, Leigh Delbridge, Oliver Gimm, Wolfgang D Schmitt, Bin T Teh, and Deborah J Marsh

The tumor suppressor HRPT2/CDC73 is mutated in constitutive DNA from patients with the familial disorder hyperparathyroidism–jaw tumor syndrome and in ∼70% of all parathyroid carcinomas. In a number of HRPT2 mutant tumors however, expression of the encoded protein parafibromin is lost in the absence of a clear second event such as HRPT2 allelic loss or the presence of a second mutation in this tumor suppressor gene. We sought to determine whether hypermethylation of a 713 bp CpG island extending 648 nucleotides upstream of the HRPT2 translational start site and 65 nucleotides into exon 1 might be a mechanism contributing to the loss of expression of parafibromin in parathyroid tumors. Furthermore, we asked whether mutations might be present in the 5′-untranslated region (5′-UTR) of HRPT2. We investigated a pool of tissue from 3 normal parathyroid glands, as well as 15 individual parathyroid tumor samples including 6 tumors with known HRPT2 mutations, for hypermethylation of the HRPT2 CpG island. Methylation was not identified in any specimens despite complete loss of parafibromin expression in two parathyroid carcinomas with a single detectable HRPT2 mutation and retention of the wild-type HRPT2 allele. Furthermore, no mutations of a likely pathogenic nature were identified in the 5′-UTR of HRPT2. These data strongly suggest that alternative mechanisms such as mutation in HRPT2 intronic regions, additional epigenetic regulation such as histone modifications, or other regulatory inactivation mechanisms such as targeting by microRNAs may play a role in the loss of parafibromin expression.