Roger D. Palmer

Global microRNA profiles in cervical squamous cell carcinoma depend on Drosha expression levels

Gain of chromosome 5p is seen in over 50% of advanced cervical squamous cell carcinomas (SCCs), although the genes responsible for the selective advantage provided by this abnormality are poorly understood. In the W12 cervical carcinogenesis model, we observed that 5p gain was rapidly selected over ∼15 population doublings and was associated with the acquisition of a growth advantage and invasiveness. The most significantly upregulated transcript following 5p gain was the microRNA (miRNA) processor Drosha. In clinically progressed cervical SCC, Drosha copy‐number gain was seen in 21/36 clinical samples and 8/10 cell lines and there was a significant association between Drosha transcript levels and copy‐number gain. Other genes in the miRNA processing pathway, DGCR8, XPO5 and Dicer, showed infrequent copy‐number gain and over‐expression. Drosha copy‐number and expression were not elevated in pre‐malignant cervical squamous intraepithelial lesions. Importantly, global miRNA profiling showed that Drosha over‐expression in cervical SCC appears to be of functional significance. Unsupervised principal component analysis of a mixed panel of cervical SCC cell lines and clinical specimens showed clear separation according to Drosha over‐expression. miRNAs most significantly associated with Drosha over‐expression are implicated in carcinogenesis in other tissues, suggesting that they regulate fundamental processes in neoplastic progression. Our evidence suggests that copy‐number driven over‐expression of Drosha and consequent changes in miRNAs are likely to be important contributors to the selective advantage provided by 5p gain in cervical neoplastic progression. Copyright

Pediatric Malignant Germ Cell Tumors Show Characteristic Transcriptome Profiles

Malignant germ cell tumors (GCT) of childhood are rare and heterogeneous neoplasms thought to arise from primordial germ cells. They vary substantially in their natural history and show important clinical differences from their adult counterparts. To address the biological basis for these observations, we have undertaken a comprehensive analysis of global gene expression patterns in pediatric malignant GCTs and compared these findings with published data on adult testicular GCTs (TGCT). Our study included 27 primary tumors and assessed the principal malignant histologic types of pediatric GCT, yolk sac tumor (YST; n = 18), and seminoma (n = 9). Analysis of Affymetrix U133A GeneChip data was performed using the statistical software environment R, including gene set enrichment analysis, with cross-validation at the RNA and protein level. Unsupervised analysis showed complete separation of YSTs and seminomas by global gene expression profiles and identified a robust set of 657 discriminatory transcripts. There was no segregation of tumors of the same histology arising at different sites or at different ages within the pediatric range. In contrast, there was segregation of pediatric malignant GCTs and adult malignant TGCTs, most notably for the YSTs. The pediatric seminomas were significantly enriched for genes associated with the self-renewing pluripotent phenotype, whereas the pediatric YSTs were significantly enriched for genes associated with a differentiation and proliferation phenotype. We conclude that histologic type is the key discriminator in pediatric malignant GCTs and that the observed clinical differences between malignant GCTs of children and adults are mirrored by significant differences in global gene expression.

Functional evidence that Drosha overexpression in cervical squamous cell carcinoma affects cell phenotype and microRNA profiles.

Although gain of chromosome 5p is one of the most frequent DNA copy‐number imbalances in cervical squamous cell carcinoma (SCC), the genes that drive its selection remain poorly understood. In a previous cross‐sectional clinical study, we showed that the microRNA processor Drosha (located on chromosome 5p) demonstrates frequent copy‐number gain and overexpression in cervical SCC, associated with altered microRNA profiles. Here, we have conducted gene depletion/overexpression experiments to demonstrate the functional significance of up‐regulated Drosha in cervical SCC cells. Drosha depletion by RNA interference (RNAi) produced significant, specific reductions in cell motility/invasiveness in vitro, with a silent RNAi‐resistant Drosha mutation providing phenotype rescue. Unsupervised hierarchical clustering following global profiling of 319 microRNAs in 18 cervical SCC cell line specimens generated two groups according to Drosha expression levels. Altering Drosha levels in individual SCC lines changed the group into which the cells clustered, with gene depletion effects being rescued by the RNAi‐resistant mutation. Forty‐five microRNAs showed significant differential expression between the groups, including four of 14 that were differentially expressed in association with Drosha levels in clinical samples. miR‐31 up‐regulation in Drosha‐overexpressing samples/cell lines was the highest‐ranked change (by adjusted p value) in both analyses, an observation validated by northern blotting. These functional data support the role of Drosha as an oncogene in cervical SCC, by affecting expression of cancer‐associated microRNAs that have the potential to regulate numerous protein‐coding genes. Copyright

Malignant germ cell tumours of childhood: new associations of genomic imbalance

Malignant germ cell tumours (MGCTs) of childhood are a rare group of neoplasms that comprise many histological subtypes and arise at numerous different sites. Genomic imbalances have been described in these tumours but, largely because of the paucity of cases reported in the literature, it is unclear how they relate to abnormalities in adult MGCTs and impact on potential systems for classifying GCTs. We have used metaphase-based comparative genomic hybridisation to analyse the largest series of paediatric MGCTs reported to date, representing 34 primary tumours (22 yolk sac tumours (YSTs), 11 germinomatous tumours and one metastatic embryonal carcinoma) occurring in children from birth to age 16, including 17 ovarian MGCTs. The large dataset enabled us to undertake statistical analysis, with the aim of identifying associations worthy of further investigation between patterns of genomic imbalance and clinicopathological parameters. The YSTs showed an increased frequency of 1p- (P=0.003), 3p+ (P=0.02), 4q− (P=0.07) and 6q− (P=0.004) compared to germinomatous tumours. Gain of 12p, which is invariably seen in adult MGCTs, was present in 53% of primary MGCTs of children aged 5–16 and was also observed in four of 14 YSTs affecting children less than 5. Two of these cases (14% of MGCTs in children less than 5) showed gain of the 12p11 locus considered to be particularly relevant in adult MGCTs. Gain of 12p showed a significant association with gain of 12q. Conversely, MGCTs without 12p gain displayed a significantly increased frequency of loss on 16p (P=0.04), suggesting that this imbalance may contribute to tumour development in such cases. This data provides new insight into the biology of this under-investigated tumour group and will direct future studies on the significance of specific genetic abnormalities.

Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance

Background:Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes.Methods:A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays.Results:Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a ‘methylator’ phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype.Conclusion:Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

Variable methylation of the imprinted gene, SNRPN, supports a relationship between intracranial germ cell tumours and neural stem cells

Germ cell tumours (GCTs) are a diverse group of neoplasms all of which are generally believed to arise from germ cell progenitors (PGCs). Even those that form in the nervous system are likewise believed to be PGC-derived, despite being found a great distance from the normal location of germ cells. The primary evidence in favour of this model for the origins of intracranial GCTs is that they share molecular features with other GCTs. Those features include shared gene expression and a lack of methylation of imprinted genes, including SNRPN. Contrary to this model, we have proposed that endogenous neural stem cells of the brain are a more likely origin for these tumours. We show here that the lack of methylation of SNRPN that has previously been taken to indicate an origin for GCTs from PGCs is also seen in neural stem cells of mice and humans. We believe that, in the light of these and other recent observations, endogenous neural precursors of the brain are a more plausible origin for intracranial GCTs than are misplaced PGCs.

Analysis of the adenomatous polyposis coli (APC) gene in childhood and adolescent germ cell tumors

Aberrant Wnt signaling due to deregulation of Wnt regulators is implicated in the development and progression of numerous embryonal tumors. This study addresses the questions if activation of Wnt signaling in germ cell tumors (GCTs) arising during childhood and adolescence is associated with aberrations of the tumor suppressor adenomatous polyposis coli (APC), and whether APC aberrations might be responsible for progression from benign teratoma to malignant yolk sac tumor (YST).

Management of germ cell tumours in childhood

Abstract Germ cell tumours are a group of rare benign and malignant neoplasms of unknown cause, and may be gonadal or extragonadal (intracranial or extracranial). Teratomas are the most common and are usually benign. Malignant germ cell tumours often secrete the tumour markers alpha-fetoprotein and human chorionic gonadotrophin, which are useful in diagnosis and monitoring. The treatment is surgical for teratoma and non-metastatic gonadal tumours. Chemotherapy, reserved in the UK for stage 2–4 extracranial malignant germ cell tumours, achieves a 5-year survival of around 90%. Intracranial tumours are typically pineal or suprasellar. Intracranial germinomas are cured in over 90% of cases with radiotherapy or combined chemoradiotherapy, whereas non-germinomatous tumours fare less well despite combination chemotherapy and radiotherapy.

Abstract 3424: Malignant germ cell tumors display common microRNA profiles resulting in global changes in expression of mRNA targets

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background As malignant germ cell tumors (MGCTs) are thought to have a common origin from primordial germ cells, they may share fundamental biological abnormalities despite their clinical and histopathological heterogeneity. We tested the hypothesis that MGCTs are characterized by common patterns of microRNA (miRNA) expression that are functionally significant by affecting levels of mRNA targets. We further investigated relationships between miRNA profiles and transcription factor expression, in order to identify potential regulators of miRNA transcription in MGCTs. Methods We used the miRCURY microarray (Exiqon) to quantify global microRNA expression levels in 28 gonadal and extragonadal GCTs and 14 non-malignant samples (eight controls, six teratomas) from pediatric subjects. For 21 of these samples (17 MGCT, four non-malignant) global mRNA expression profiles had been obtained using the U133A GeneChip (Affymetrix). We compared our findings with a re-analysis of published qRT-PCR miRNA and U133A GeneChip mRNA profiling in adult gonadal MGCTs. Data were analysed using R and Bioconductor. We used the novel bioinformatic algorithm Sylamer to investigate enrichment and depletion of miRNA seed complementary regions (SCRs) in 3’UTRs of mRNAs ranked according to expression levels in MGCTs. For the pediatric MGCT samples we compared miRNA profiles with transcription factor expression using linear regression plots. Results miR-302 and miR-371∼373 clusters were universally over-expressed in MGCTs regardless of patient age (pediatric or adult), tumor histological type [yolk sac tumors (YSTs), seminomas or embryonal carcinomas] or anatomical site (gonadal or extragonadal). Sylamer demonstrated that the most significantly over-represented SCR in the 3’UTRs of down-regulated mRNAs was GCACTT, corresponding to the AAGUGC seed common to the six members of the miR-302a∼d and miR-372∼373 family. Gene Ontology (GO) analysis revealed that the down-regulated mRNAs containing this common SCR in pediatric MGCTs were members of major cancer-associated cellular pathways. The transcription factor SOX17 was highly significantly over-expressed in pediatric and adult MGCTs (adjusted p-value=3.0×10−4 and 1.4×10−11, respectively), and showed a strong positive correlation in matched pediatric samples with combined median expression values for miR-302a∼d and miR-372∼3 (p-value=4.9×10−4). Conclusion Our data suggests a fundamental role for miR-302 and miR-371∼373 clusters in the biology of MGCTs and identifies SOX17 as a candidate regulator of these clusters. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3424.