Tracy Chaplin

MicroRNA miR-181a correlates with morphological sub-class of acute myeloid leukaemia and the expression of its target genes in global genome-wide analysis.

MicroRNAs (miRNAs) are short single-stranded RNAs that have a potentially important role in gene regulation. Using a quantitative real-time polymerase chain reaction assay specific to the mature miRNA, the expression level of a selected group of haematopoietic tissue-specific miRNAs was measured across a set of 30 primary adult acute myeloid leukaemia (AML) with a normal karyotype. The expression levels of each miRNA were correlated with the genome-wide mRNA expression profiles in the same leukaemias. This revealed that miR-181a correlated strongly with the AML morphological sub-type and with the expression of genes previously identified through sequence analysis as potential interaction targets. Three other miRNAs, miR-10a, miR-10b and miR-196a-1, showed a clear correlation with HOX gene expression.

Distinctive Patterns of MicroRNA Expression Associated with Karyotype in Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML) is the most common acute leukaemia in adults; however, the genetic aetiology of the disease is not yet fully understood. A quantitative expression profile analysis of 157 mature miRNAs was performed on 100 AML patients representing the spectrum of known karyotypes common in AML. The principle observation reported here is that AMLs bearing a t(15;17) translocation had a distinctive signature throughout the whole set of genes, including the up regulation of a subset of miRNAs located in the human 14q32 imprinted domain. The set included miR-127, miR-154, miR-154*, miR-299, miR-323, miR-368, and miR-370. Furthermore, specific subsets of miRNAs were identified that provided molecular signatures characteristic of the major translocation-mediated gene fusion events in AML. Analysis of variance showed the significant deregulation of 33 miRNAs across the leukaemic set with respect to bone marrow from healthy donors. Fluorescent in situ hybridisation analysis using miRNA-specific locked nucleic acid (LNA) probes on cryopreserved patient cells confirmed the results obtained by real-time PCR. This study, conducted on about a fifth of the miRNAs currently reported in the Sanger database (microrna.sanger.ac.uk), demonstrates the potential for using miRNA expression to sub-classify cancer and suggests a role in the aetiology of leukaemia.

Activation of the ERK/MAPK pathway: a signature genetic defect in posterior fossa pilocytic astrocytomas

We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549–BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto‐inhibitory domains of BRAF and RAF1, which are replaced in‐frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low‐grade gliomas. Copyright

Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.

Genome-wide analysis of acute myeloid leukemia with normal karyotype reveals a unique pattern of homeobox gene expression distinct from those with translocation-mediated fusion events.

Gene expression profiles were determined from presentation peripheral blood and bone marrow samples of 28 patients with acute myeloid leukemia (AML). Hierarchical clustering sorted the profiles into separate groups, each representing one of the major cytogenetic classes in AML [i.e., t(8;21), t(15;17), inv(16), 11q23, and normal karyotype]. Statistical group comparison identified genes whose expression was strongly correlated with these chromosomal classes. Moreover, the normal karyotype AMLs were characterized by distinctive up‐regulation of certain members of the class I homeobox A and B gene families, implying a common underlying genetic lesion. These data reveal novel diagnostic and therapeutic targets and demonstrate the potential of microarray‐based dissection of AML.

FOXM1 Upregulation Is an Early Event in Human Squamous Cell Carcinoma and it Is Enhanced by Nicotine during Malignant Transformation

Background Cancer associated with smoking and drinking remains a serious health problem worldwide. The survival of patients is very poor due to the lack of effective early biomarkers. FOXM1 overexpression is linked to the majority of human cancers but its mechanism remains unclear in head and neck squamous cell carcinoma (HNSCC). Methodology/Principal Findings FOXM1 mRNA and protein expressions were investigated in four independent cohorts (total 75 patients) consisting of normal, premalignant and HNSCC tissues and cells using quantitative PCR (qPCR), expression microarray, immunohistochemistry and immunocytochemistry. Effect of putative oral carcinogens on FOXM1 transcriptional activity was dose-dependently assayed and confirmed using a FOXM1-specific luciferase reporter system, qPCR, immunoblotting and short-hairpin RNA interference. Genome-wide single nucleotide polymorphism (SNP) array was used to ‘trace’ the genomic instability signature pattern in 8 clonal lines of FOXM1-induced malignant human oral keratinocytes. Furthermore, acute FOXM1 upregulation in primary oral keratinocytes directly induced genomic instability. We have shown for the first time that overexpression of FOXM1 precedes HNSCC malignancy. Screening putative carcinogens in human oral keratinocytes surprisingly showed that nicotine, which is not perceived to be a human carcinogen, directly induced FOXM1 mRNA, protein stabilisation and transcriptional activity at concentrations relevant to tobacco chewers. Importantly, nicotine also augmented FOXM1-induced transformation of human oral keratinocytes. A centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, both located within a consensus loci (10q23), were found to be novel targets of FOXM1 and their expression correlated tightly with HNSCC progression. Conclusions/Significance This study cautions the potential co-carcinogenic effect of nicotine in tobacco replacement therapies. We hypothesise that aberrant upregulation of FOXM1 may be inducing genomic instability through a program of malignant transformation involving the activation of CEP55 and HELLS which may facilitate aberrant mitosis and epigenetic modifications. Our finding that FOXM1 is upregulated early during oral cancer progression renders FOXM1 an attractive diagnostic biomarker for early cancer detection and its candidate mechanistic targets, CEP55 and HELLS, as indicators of malignant conversion and progression.

Distinct Genomic Alterations in Prostate Cancers in Chinese and Western Populations Suggest Alternative Pathways of Prostate Carcinogenesis

Prostate cancer is significantly more common in Western men than in Asian men, but the basis for this difference remains unknown. Because genomic studies of Asian prostate cancer are very limited, we used a genome-wide approach to reveal the genomic alterations in Chinese prostate cancers. We found a significant reduction in the frequency of certain somatic genomic changes that are commonly found in Western prostate cancers, including the 21q22.2-22.3 deletion, which involves the TMPRSS2:ERG fusion gene, and 10q deletion, which causes PTEN inactivation. Array results were confirmed by PCR-based molecular copy-number counting in selected samples. The different frequencies of these genomic changes were further evaluated by fluorescent in situ hybridization and immunohistochemistry analyses of tissue microarray samples. These alterations might be key genetic changes underlying the regional/ethnic difference in clinical incidence and might be induced by specific environmental and/or genetic risk factors that Western men are exposed to. Our findings suggest that tumors arise in Western and Chinese populations by alternative pathogenetic mechanisms.

Genome-wide DNA copy number analysis in pancreatic cancer using high-density single nucleotide polymorphism arrays.

To identify genomic abnormalities characteristic of pancreatic ductal adenocarcinoma (PDAC) in vivo, a panel of 27 microdissected PDAC specimens were analysed using high-density microarrays representing ∼116 000 single nucleotide polymorphism (SNP) loci. We detected frequent gains of 1q, 2, 3, 5, 7p, 8q, 11, 14q and 17q (⩾78% of cases), and losses of 1p, 3p, 6, 9p, 13q, 14q, 17p and 18q (⩾44%). Although the results were comparable with those from array CGH, regions of those genetic changes were defined more accurately by SNP arrays. Integrating the Ensembl public data, we have generated ‘gene’ copy number indices that facilitate the search for novel candidates involved in pancreatic carcinogenesis. Copy numbers in a subset of the genes were validated using quantitative real-time PCR. The SKAP2/SCAP2 gene (7p15.2), which belongs to the src family kinases, was most frequently (63%) amplified in our sample set and its recurrent overexpression (67%) was confirmed by reverse transcription–PCR. Furthermore, fluorescence in situ hybridization and in situ RNA hybridization analyses for this gene have demonstrated a significant correlation between DNA copy number and mRNA expression level in an independent sample set (P<0.001). These findings indicate that the dysregulation of SKAP2/SCAP2, which is mostly caused by its increased gene copy number, is likely to be associated with the development of PDAC.

Microdeletions are a general feature of adult and adolescent acute lymphoblastic leukemia: Unexpected similarities with pediatric disease

We present here a genome-wide map of abnormalities found in diagnostic samples from 45 adults and adolescents with acute lymphoblastic leukemia (ALL). A 500K SNP array analysis uncovered frequent genetic abnormalities, with cryptic deletions constituting half of the detected changes, implying that microdeletions are a characteristic feature of this malignancy. Importantly, the pattern of deletions resembled that recently reported in pediatric ALL, suggesting that adult, adolescent, and childhood cases may be more similar on the genetic level than previously thought. Thus, 70% of the cases displayed deletion of one or more of the CDKN2A, PAX5, IKZF1, ETV6, RB1, and EBF1 genes. Furthermore, several genes not previously implicated in the pathogenesis of ALL were identified as possible recurrent targets of deletion. In total, the SNP array analysis identified 367 genetic abnormalities not corresponding to known copy number polymorphisms, with all but two cases (96%) displaying at least one cryptic change. The resolution level of this SNP array study is the highest used to date to investigate a malignant hematologic disorder. Our findings provide insights into the leukemogenic process and may be clinically important in adult and adolescent ALL. Most importantly, we report that microdeletions of key genes appear to be a common, characteristic feature of ALL that is shared among different clinical, morphological, and cytogenetic subgroups.

Segmental uniparental disomy is a commonly acquired genetic event in relapsed acute myeloid leukemia

Despite advances in the curative treatment of acute myeloid leukemia (AML), recurrence will occur in the majority of cases. At diagnosis, acquisition of segmental uniparental disomy (UPD) by mitotic recombination has been reported in 15% to 20% of AML cases, associated with homozygous mutations in the region of loss of heterozygosity. This study aimed to discover if clonal evolution from heterozygous to homozygous mutations by mitotic recombination provides a mechanism for relapse. DNA from 27 paired diagnostic and relapsed AML samples were analyzed using genotyping arrays. Newly acquired segmental UPDs were observed at relapse in 11 AML samples (40%). Six were segmental UPDs of chromosome 13q, which were shown to lead to a change from heterozygosity to homozygosity for internal tandem duplication mutation of FLT3 (FLT3 ITD). Three further AML samples had evidence of acquired segmental UPD of 13q in a subclone of the relapsed leukemia. One patient acquired segmental UPD of 19q that led to homozygosity for a CEBPA mutation 207C>T. Finally, a single patient with AML acquired segmental UPD of chromosome 4q, for which the candidate gene is unknown. We conclude that acquisition of segmental UPD and the resulting homozygous mutation is a common event associated with relapse of AML.