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Distinct patterns of DNA methylation characterize the epigenetic landscape of promyelocytic leukemia/retinoic acid receptor-α (PML-RARα)-associated acute promyelocytic leukemia (APL). We previously reported that the microRNAs (miRNAs) clustered on chromosome 14q32 are overexpressed only in APL. Here, using high-throughput bisulfite sequencing, we identified an APL-associated hypermethylation at the upstream differentially methylated region (DMR), which also included the site motifs for the enhancer blocking protein CCCTC-binding factor (CTCF). Comparing the profiles of diagnostic/remission paired patient samples, we show that hypermethylation was acquired in APL in a monoallelic manner. The cytosine guanine dinucleotide status of the DMR correlated with expression of the miRNAs following a characteristic position-dependent pattern. Moreover, a signature of hypermethylation was also detected in leukemic cells from an established transgenic PML-RARA APL mouse model at the orthologous region on chromosome 12, including the CTCF binding site located upstream from the mouse miRNA cluster. These results, together with the demonstration that the region does not show DNA methylation changes during myeloid differentiation, provide evidence that 14q32 hypermethylation is implicated in the pathogenesis of APL. We propose a model in which loss of imprinting at the 14q32 domain leads to overexpression of the miRNAs in APL.

Since the introduction of cisplatin into the clinic, the treatment of patients with a variety of solid tumors including testicular germ cell tumors, ovarian and lung cancers, has dramatically improved. One of the main causes for therapeutic failure in these malignancies is the development of drug resistance. Testicular germ cell tumors (TGCTs), the most common malignancy in young men, exhibit extreme sensitivity to cisplatin-based chemotherapy, making them an ideal model for investigating the mechanisms of cisplatin chemo-sensitivity and resistance. TGCT development and pathogenesis have been well studied but little is known about the genetic background in chemo-resistant cases. We investigated genomic differences between three TGCT parental cell lines and their cisplatin resistant derivatives. Using 10K single nucleotide polymorphism (SNP) microarray analysis, we identified two small chromosomal regions with consistent copy number changes across all three pairs of resistant cell lines. These were an 8.7 Mb region at 6q26-27, which displayed consistent copy number gain and a 0.3 Mb deletion involving 4 SNPs at 10p14. Both the chromosomal gain and loss were confirmed by fluorescence in situ hybridization. The significance of these regions should be further investigated as they may contain key genes involved in the development of chemo- resistance to cisplatin-based treatment in TGCTs and other cancers. © 2008 Wiley-Liss, Inc.

BACKGROUND: The incidence of human papillomavirus-associated vulval neoplasia is increasing worldwide; yet the associated genetic changes remain poorly understood. METHODS: We have used single-nucleotide polymorphism microarray analysis to perform the first high-resolution investigation of genome-wide allelic imbalance in vulval neoplasia. Our sample series comprised 21 high-grade vulval intraepithelial neoplasia and 6 vulval squamous cell carcinomas, with paired non-lesional samples used to adjust for normal copy number variation. RESULTS: Overall the most common recurrent aberrations were gains at 1p and 20, with the most frequent deletions observed at 2q, 3p and 10. Copy-neutral loss of heterozygosity at 6p was a recurrent event in vulval intraepithelial neoplasia. The pattern of genetic alterations differed from the characteristic changes we previously identified in cutaneous squamous cell carcinomas. Vulval neoplasia samples did not exhibit gain at 5p, a frequent recurrent aberration in a series of cervical tumours analysed elsewhere using an identical protocol. CONCLUSION: This series of 27 vulval samples comprises the largest systematic genome-wide analysis of vulval neoplasia performed to date. Despite shared papillomavirus status and regional proximity, our data suggest that the frequency of certain genetic alterations may differ in vulval and cervical tumours.

Historically, genes targeted by recurrent chromosomal deletions have been identified within the smallest genomic region shared in all patients, the minimally deleted region (MDR). However, deletions this small do not occur in all patients and are a simplification of the impact larger heterogeneous deletions have during carcinogenesis. We use the example of 13q14 deletions in chronic lymphocytic leukemia to show that genes outside MDRs are associated with disease progression. Genomic profiling of 224 patients identified 205 copy number alterations on chromosome 13 in 132 cases. Deletions including DLEU2 were heterogeneous (845 Kb-96.2 Mb) and identified two breakpoint cluster regions within short interspersed nuclear elements proximal to DLEU2 and within long interspersed nuclear elements/L1 repeats distal to GUCY1B2. After defining a deletion class on the basis of size and location, we show that (a) at diagnosis, larger deletions (class II) were associated with a significantly increased risk of disease progression (odds ratio=12.3; P=0.005), (b) in progressive patients, class II deletions were enriched (P=0.02) and (c) this association was independent of IgVH mutational status, ZAP70 expression and ATM/TP53 deletion. Deletion of a 1 Mb gene cluster (48.2-49.2 Mb), including SETDB2, PHF11 and RCBTB1, was significantly associated (P

Early genetic events in the development of high-grade serous ovarian cancer (HGSOC) may define the molecular basis of the profound structural and numerical instability of chromosomes in this disease. To discover candidate genetic changes we sequentially passaged cells from a karyotypically normal hTERT immortalised human ovarian surface epithelial line (IOSE25) resulting in the spontaneous formation of colonies in soft agar. Cell lines transformed ovarian surface epithelium 1 and 4 (TOSE 1 and 4) established from these colonies had an abnormal karyotype and altered morphology, but were not tumourigenic in immunodeficient mice. TOSE cells showed loss of heterozygosity (LOH) at TP53, increased nuclear p53 immunoreactivity and altered expression profile of p53 target genes. The parental IOSE25 cells contained a missense, heterozygous R175H mutation in TP53, whereas TOSE cells had LOH at the TP53 locus with a new R273H mutation at the previous wild-type TP53 allele. Cytogenetic and array CGH analysis of TOSE cells also revealed a focal genomic amplification of CXCR4, a chemokine receptor commonly expressed by HGSOC cells. TOSE cells had increased functional CXCR4 protein and its abrogation reduced epidermal growth factor receptor (EGFR) expression, as well as colony size and number. The CXCR4 ligand, CXCL12, was epigenetically silenced in TOSE cells and its forced expression increased TOSE colony size. TOSE cells had other cytogenetic changes typical of those seen in HGSOC ovarian cancer cell lines and biopsies. In addition, enrichment of CXCR4 pathway in expression profiles from HGSOC correlated with enrichment of a mutated TP53 gene expression signature and of EGFR pathway genes. Our data suggest that mutations in TP53 and amplification of the CXCR4 gene locus may be early events in the development of HGSOC, and associated with chromosomal instability.

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. Cancer Res; 70(13); 5207–12. ©2010 AACR.

A novel exon in AML1-ETO negatively influences the clonogenic potential of the t(8;21) in acute myeloid leukemia

The acquisition of uniparental disomy (aUPD) in acute myeloid leukemia (AML) results in homozygosity for known gene mutations. Uncovering novel regions of aUPD has the potential to identify previously unknown mutational targets. We therefore aimed to develop a map of the regions of aUPD in AML. Here, we have analyzed a large set of diagnostic AML samples (n = 454) from young adults (age: 15-55 years) using genotype arrays. Acquired UPD was found in 17% of the samples with a nonrandom distribution particularly affecting chromosome arms 13q, 11p, and 11q. Novel recurrent regions of aUPD were uncovered at 2p, 17p, 2q, 17q, 1p, and Xq. Overall, aUPDs were observed across all cytogenetic risk groups, although samples with aUPD13q (5.4% of samples) belonged exclusively to the intermediate-risk group as defined by cytogenetics. All cases with a high FLT3-ITD level, measured previously, had aUPD13q covering the FLT3 gene. Significantly, none of the samples with FLT3-ITD(-)/FLT3-TKD(+) mutation exhibited aUPD13q. Of the 119 aUPDs observed, the majority (87%) were due to mitotic recombination while only 13% were due to nondisjunction. This study demonstrates aUPD is a frequent and significant finding in AML and pinpoints regions that may contain novel mutational targets.

BACKGROUND: Oral submucous fibrosis (OSF) is a high-risk pre-cancerous condition where 7‐13% of these patients develop head and neck squamous cell carcinoma (HNSCC). To date there is no cancer predictive markers for OSF patients. Genomic instability hallmarks early genetic events during malignant transformation causing loss of heterozygosity (LOH) and chromosomal copy number abnormality. However, to date there is no study on genomic instability in OSF. Although this condition is known as a high-risk pre-cancerous condition, there is no data regarding the genomic status of this disease in terms of genetic susceptibility to malignant transformation. METHODS: In this study, we investigated the existence of genetic signatures for carcinogenesis in OSF. We employed the high-resolution genome-wide Affymetrix Mapping single nucleotide polymorphism microarray

In a small fraction ( approximately 2%) of cases of childhood acute lymphoblastic leukemia (ALL) clinical presentation of leukemia is preceded, some 2-9 months earlier, by a transient, remitting phase of nonclassical aplastic anemia, usually in connection with infection. The potential "preleukemic" nature of this prodromal phase has not been fully explored. We have retrospectively analyzed the blood and bone marrow of a child who presented with aplastic anemia 9 months before the development of ETV6-RUNX1 fusion gene positive ALL. High resolution SNP genotyping arrays identified 11 regions of loss of heterozygosity, with and without concurrent copy number changes, at the presentation of ALL. In all cases of copy number change, the deletion or gain identified by single nucleotide polymorphism (SNP) analysis was confirmed in the ALL blasts by FISH. Retrospective analysis of aplastic phase bone marrow showed that the ETV6-RUNX1 fusion was present along with all of the additional genetic changes assessed, albeit subclonal to ETV6-RUNX1. These data identify for the first time the leukemic genotype of an aplasia preceding clinical ALL and indicate that multiple secondary genetic abnormalities can contribute to a dominant subclone several months before a diagnosis of ALL. These data have implications for the biology of ALL and for management of similar patients.

International audience; 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 approximately 116 000 single nucleotide polymorphism (SNP) loci. We detected frequent gains of 1q, 2, 3, 5, 7p, 8q, 11, 14q and 17q (> or =78% of cases), and losses of 1p, 3p, 6, 9p, 13q, 14q, 17p and 18q (> or =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

Many techniques have been developed in recent years for genome-wide analysis of genetic alterations, but no current approach is capable of rapidly identifying all chromosome rearrangements with precise definition of breakpoints. Combining multiple color fluorescent in situ hybridization and high-density single nucleotide polymorphism array analyses, we present here an approach for high resolution karyotyping and fast identification of chromosome breakpoints. We characterized all of the chromosome amplifications and deletions, and most of the chromosome translocation breakpoints of three prostate cancer cell lines at a resolution which can be further analyzed by sequence-based techniques. Genes at the breakpoints were readily determined and potentially fused genes identified. Using high-density exon arrays we simultaneously confirmed altered exon expression patterns in many of these breakpoint genes. © 2007 Wiley-Liss, Inc.

Cancers with chromosomal instability (CIN) are held to be aneuploid/polyploid with multiple large-scale gains/deletions, but the processes underlying CIN are unclear and different types of CIN might exist. We investigated colorectal cancer cell lines using array-comparative genomic hybridization (CGH) for copy number changes and single-copy number polymorphism (SNP) microarrays for allelic loss (LOH). Many array-based CGH changes were not found by LOH because they did not cause true reduction-to-homozygosity. Conversely, many regions of SNP-LOH occurred in the absence of copy number change, comprising an average per cell line of 2 chromosomes with complete LOH; 1-2 terminal regions of LOH (mitotic recombination); and 1 interstitial region of LOH. SNP-LOH detected many novel changes, representing possible locations of uncharacterized tumor suppressor loci. Microsatellite unstable (MSI+) lines infrequently showed gains/deletions or whole-chromosome LOH, but their near-diploid karyotypes concealed mitotic recombination frequencies similar to those of MSI− lines. We analyzed p53 and chromosome 18q (SMAD4) in detail, including mutation screening. Almost all MSI− lines showed LOH and/or deletion of p53 and 18q; some near-triploid lines had acquired three independent changes at these loci. We found consistent results in primary colorectal cancers. Overall, the distributions of mitotic recombination and whole-chromosome LOH in the MSI− cell lines differed significantly from random, with some lines having much higher than expected levels of these changes. Moreover, lines with more LOH changes had significantly fewer copy number changes. These data suggest that CIN is not synonymous with copy number change and some cancers have a specific tendency to whole-chromosome deletion and regain or to mitotic recombination. (Cancer Res 2006; 66(7): 3471-9)

The genotype of a tumor determines its biology and clinical behavior. The genetic alterations associated with the unique embryonal morphology of nonseminomatous subtypes of testicular germ cell tumors remain to be established. Using single nucleotide polymorphism microarray analysis, we found in all of the 15 nonseminomas analyzed, large-scale chromosomal homozygosities, most of which were not associated with relative chromosome loss. This unusual genotype, distinguishing nonseminoma from seminomas and other human tumors, may be associated with the special embryonal development morphologic transition of this malignancy. Based on these genetic data, we hypothesized a new potential origin of nonseminomas through sperm fusion. Nonrandom involvement of certain chromosomes also suggests that genes on these chromosome regions may play an important role in nonseminoma development.

Genome-wide analysis of single nucleotide polymorphisms in 64 acute myeloid leukemias has revealed that ∼20% exhibited large regions of homozygosity that could not be accounted for by visible chromosomal abnormalities in the karyotype. Further analysis confirmed that these patterns were due to partial uniparental disomy (UPD). Remission bone marrow was available from five patients showing UPD in their leukemias, and in all cases the homozygosity was found to be restricted to the leukemic clone. Two examples of UPD11p were shown to be of different parental origin as indicated by the methylation pattern of the H19 gene. Furthermore, a previously identified homozygous mutation in the CEBPA gene coincided with a large-scale UPD on chromosome 19. These cryptic chromosomal abnormalities, which seem to be nonrandom, have the characteristics of somatic recombination events and may define an important new subclass of leukemia.

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.

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.

This is an open-access paper.-- et al., ATM mutation and BIRC3 deletion and/or mutation have independently been shown to have prognostic significance in chronic lymphocytic leukemia. However, the relative clinical importance of these abnormalities in patients with a deletion of 11q encompassing the ATM gene has not been established. We screened a cohort of 166 patients enriched for 11q-deletions for ATM mutations and BIRC3 deletion and mutation and determined the overall and progression-free survival among the 133 of these cases treated within the UK LRF CLL4 trial. SNP6.0 profiling demonstrated that BIRC3 deletion occurred in 83% of 11q-deleted cases and always co-existed with ATM deletion. For the first time we have demonstrated that 40% of BIRC3-deleted cases have concomitant deletion and mutation of ATM. While BIRC3 mutations were rare, they exclusively occurred with BIRC3 deletion and a wildtype residual ATM allele. In 11q-deleted cases, we confirmed that ATM mutation was associated with a reduced overall and progression-free survival comparable to that seen with TP53 abnormalities, whereas BIRC3 deletion and/or mutation had no impact on overall and progression-free survival. In conclusion, in 11q-deleted patients treated with first-line chemotherapy, ATM mutation rather than BIRC3 deletion and/or mutation identifies a subgroup with a poorer outcome., The authors would like to thank Leukaemia and Lymphoma Research, the Kay Kendall Leukaemia Fund and Wessex Medical Research for funding this study. The UK LRF CLL4 trial was funded by a core grant from Leukaemia and Lymphoma Research, with associated research work supported by the MRC (G8223452) and Cancer Research UK, and laboratory studies by the Arbib Foundation, Schering Healthcare UK, Schering AG, Germany and Leukaemia and Lymphoma Research.

The interstitial insertion of genetic material from one chromosome into another can achieve the type of gene-gene fusions more usually associated with chromosome translocations. An example of such an interstitial insertion, which has created an MLL-AF10 fusion in an acute myeloid leukaemia, has been analysed at the genomic level. The genomic fusion, which resulted in the juxtaposition of 3' AF10 sequence to 5' MLL sequence, was identified within MLL and AF10 intronic sequences. It was further established that the remaining 3' MLL sequence, from exon 6 onwards, was fused to novel sequence of unknown origin (named FM3 for fused to MLL 3'). The points of fusion of these 5' and 3' portions of MLL matched to adjacent nucleotides and lay between exons 5 and 6. The FM3 sequence was shown to be from chromosome arm 10p and located close to AF10 in a proximal position. It was subsequently demonstrated that in the leukaemia a third fusion existed between 5' AF10 and the FM3 sequence at a point immediately downstream from its fusion to MLL. It was therefore concluded that the MLL-AF10 gene fusion is the result of a simultaneous transposition of genetic material into the MLL gene and the joining of the remaining free ends on chromosome 10. This kind of event, characterised completely here for the first time, is a means to achieve a fusion when the genes involved lie in opposite orientations and results in three genomic junctions.

Many human cancers present as multifocal lesions. Understanding the clonal origin of multifocal cancers is of both etiological and clinical importance. The molecular basis of multifocal prostate cancer has previously been explored using a limited number of isolated markers and, although independent origin is widely believed, the clonal origin of multifocal prostate cancer is still debatable. We attempted to address clonal origin using a genome-wide copy-number analysis of individual cancer and high-grade prostatic intraepithelial neoplasia (HGPIN) lesions. Using Affymetrix array 6.0 copy-number analysis, we compared the genomic changes detected in 48 individual cancer and HGPIN lesions, isolated from 18 clinically localized prostate cancer cases. Identical genomic copy-number changes, shared by all same-case cancer foci, were detected in all 13 informative cases displaying multiple tumor foci. In addition, individual HGPIN lesions in the two multifocal-HGPIN cases available shared identical genomic changes. Commonly known genomic alterations, including losses at 6q15, 8p21.3-8p21.2, 10q23.2-10q23.31, 16q22.3, 16q23.2-16q23.3 and 21q22.2-21q22.3 regions and gain of 8q24.3 were the most frequently detected changes in this study and each was detected in all same-case foci in at least one case. Microarray data were confirmed by fluorescence in situ hybridization in selected foci. Our high-resolution genome-wide copy-number data suggest that many multifocal cases derive from a single prostate cancer precursor clone and that this precursor may give rise to separate HGPIN foci and may further progress to multifocal invasive prostate cancer. These findings, which demonstrate the monoclonal origin of multifocal prostate cancer, should significantly enhance our understanding of prostate carcinogenesis. © 2012 Wiley Periodicals, Inc.

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% of patients still relapse. It is important to identify these patients already at diagnosis to ensure proper risk stratification. We have investigated 11 paired diagnostic and relapse samples with single nucleotide polymorphism array and mutation analyses of FLT3, KRAS, NRAS and PTPN11 in order to identify changes associated with relapse and to ascertain the genetic evolution patterns. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P

Three overlapping yeast artificial chromosomes (YACs) spanning a 780 kb region of DNA around the CD3 locus on chromosome 11 have been isolated and characterised. The individual cloned regions have been mapped by in situ hybridisation to chromosome band 11 q23, and a restriction enzyme map of this region has been constructed. The positions of these clones in relation to a series of leukaemia-associated chromosomal translocations has also been determined. It was concluded that, although two clones lay entirely proximal to the breakpoints examined, the third clone (13HH4) encompassed the breakpoints for the translocations t(4;11), t(6;11), and t(9;11). The t(9;11) was observed in an acute myeloid leukaemia in a patient previously treated for an unrelated malignancy. It would thus appear that the breakpoints at chromosome band 11q23 occurring in therapy-related leukaemias are in the same region as those found in adult and childhood acute leukaemias and may result from a common underlying mechanism.

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.

To investigate if genomic instability exists in tumors with cytogenetically normal karyotypes, we analyzed four diploid cancer cell lines A204, CAL51, CH1 and SK-UT-1B. We detected subtle genomic changes in all four cell lines. More of these alterations were found in A204 and CH1 than in the microsatellite unstable lines CAL51 and SK-UT-1B. The number of de novo, non-clonal chromosome rearrangements was also significantly higher in CH1 than CAL51 and SK-UT-1B (p = 0.001). This study reveals multiple genomic abnormalities in tumors with near normal karyotypes and suggests that genomic instability may be essential in cancer development.

Cutaneous squamous cell carcinomas (SCC) are the second most commonly diagnosed cancers in fair-skinned people; yet the genetic mechanisms involved in SCC tumorigenesis remain poorly understood. We have used single nucleotide polymorphism (SNP) microarray analysis to examine genome-wide allelic imbalance in 16 primary and 2 lymph node metastatic SCC using paired non-tumour samples to counteract normal copy number variation. The most common genetic change was loss of heterozygosity (LOH) on 9p, observed in 13 of 16 primary SCC. Other recurrent events included LOH on 3p (9 tumors), 2q, 8p, and 13 (each in 8 SCC) and allelic gain on 3q and 8q (each in 6 tumors). Copy number-neutral LOH was observed in a proportion of samples, implying that somatic recombination had led to acquired uniparental disomy, an event not previously demonstrated in SCC. As well as recurrent patterns of gross chromosomal changes, SNP microarray analysis revealed, in 2 primary SCC, a homozygous microdeletion on 9p23 within the protein tyrosine phosphatase receptor type D (PTPRD) locus, an emerging frequent target of homozygous deletion in lung cancer and neuroblastoma. A third sample was heterozygously deleted within this locus and PTPRD expression was aberrant. Two of the 3 primary SCC with PTPRD deletion had demonstrated metastatic potential. Our data identify PTPRD as a candidate tumor suppressor gene in cutaneous SCC with a possible association with metastasis.

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.

Basal cell carcinoma is the most common human cancer with increasing incidence reported worldwide. Despite the aberrant signaling role of the Hedgehog pathway, little is known about the genetic mechanisms underlying basal cell carcinomas. Towards a better understanding of global genetic events, we have employed the Affymetrix Mapping 10K single nucleotide polymorphism (SNP) microarray technique for “fingerprinting” genomewide allelic imbalance in 14 basal cell carcinoma–blood pair samples. This rapid high-resolution SNP genotyping technique has revealed a somatic recombination event–uniparental disomy, leading to a loss of heterozygosity (LOH), as a key alternative genetic mechanism to allelic imbalances in basal cell carcinomas. A highly conserved LOH region at 9q21-q31 was found in 13 of 14 (93%) basal cell carcinomas. Further statistical and fluorescence in situ hybridization analyses confirmed that the 9q LOH was a result of uniparental disomy in 5 of 13 (38%) basal cell carcinomas. De novo mutations in the Patched 1 gene (PTCH) were found in 9 of 13 (69%) basal cell carcinomas with 9q LOH. A second important locus, containing LOH at 6q23-q27 was found in 5 of 14 (36%) basal cell carcinomas, suggesting that the presence of an additional putative tumor suppressor gene may be contributing to basal cell carcinoma development. This study shows that the rate of 9q LOH in basal cell carcinomas has been previously underestimated. Furthermore, we provide the first evidence that uniparental disomy due to somatic recombination constitutes one of the mechanisms of LOH in basal cell carcinoma tumorigenesis.

Δ9-Tetrahydrocannabinol (THC) is the active metabolite of cannabis. THC causes cell death in vitro through the activation of complex signal transduction pathways. However, the role that the cannabinoid 1 and 2 receptors (CB1-R and CB2-R) play in this process is less clear. We therefore investigated the role of the CB-Rs in mediating apoptosis in 3 leukemic cell lines and performed microarray and immunoblot analyses to establish further the mechanism of cell death. We developed a novel flow cytometric technique of measuring the expression of functional receptors and used combinations of selective CB1-R and CB2-R antagonists and agonists to determine their individual roles in this process. We have shown that THC is a potent inducer of apoptosis, even at 1 × IC50 (inhibitory concentration 50%) concentrations and as early as 6 hours after exposure to the drug. These effects were seen in leukemic cell lines (CEM, HEL-92, and HL60) as well as in peripheral blood mononuclear cells. Additionally, THC did not appear to act synergistically with cytotoxic agents such as cisplatin. One of the most intriguing findings was that THC-induced cell death was preceded by significant changes in the expression of genes involved in the mitogen-activated protein kinase (MAPK) signal transduction pathways. Both apoptosis and gene expression changes were altered independent of p53 and the CB-Rs.

Chromosome 6 has been shown to contain at band q27 a minimal region of deletion associated with epithelial ovarian cancers and AF6, a gene disrupted in acute myeloid leukemia with t(6;11)(q27;q23). Using rapid amplification of cDNA ends by polymerase chain reaction, the breakpoint in AF6 was confirmed and a cDNA clone identified. This clone was used as a probe to screen a chromosome 6 cosmid library, and a single cosmid C-109F0645 was isolated. By fluorescence in situ hybridization, C-109F0465 was found to map distal to the critically deleted region associated with ovarian malignancies. AF6 is therefore distinct from and lies telomeric to this region.

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) functions are associated with various cancers, and the inhibition of HDAC has been found to arrest disease progression. Here, we have investigated the gene expression profiles of leukaemic cells in response to the HDAC inhibitor trichostatin A (TSA) using oligonucleotide microarrays. Nucleosomal histone acetylation was monitored in parallel and the expression profiles of selected genes were confirmed by quantitative polymerase chain reaction (PCR). A large number of genes (9% of the genome) were found to be similarly regulated in CCRF-CEM and HL-60 cells in response to TSA, and genes showing primary and secondary responses could be distinguished by temporal analysis of gene expression. A small fraction of genes were highly sensitive to histone hyper-acetylation, including XRCC1, HOXB6, CDK10, MYC, MYB, NMI and CBFA2T3 and many were trans-acting factors relevant to cancer. The most rapidly repressed gene was MKRN3, an imprinted gene involved in the Prader-Willi syndrome.

The t(10;11)(p13;q14-21) is a non-random translocation described in acute lymphoblastic and myeloid leukaemias. It results in the fusion of the gene CALM, which encodes a clathrin assembly protein, on 11q14 to the gene AF10, a putative transcription factor on 10p13. Here we describe for the first time, the occurrence of a CALM-AF10 fusion in a case of acute megakaryoblastic leukaemia. Fluorescence in situ hybridisation and reverse transcriptase polymerase chain reaction were used to confirm the presence of a CALM-AF10 fusion. A novel splice variant of CALM missing nt 1927-2091 was also detected. Though CALM is a cytoplasmic protein, the chimaeric fusion product is able to localise to both the nucleus and cytoplasm. Analysis of the fusion variants suggests, however, that the critical fusion product is likely to be cytoplasmic and contain the interactive leucine zipper of AF10.

Chronic lymphocytic leukemia (CLL) is associated with profound defects in immune function, resulting in failure of anti-tumor immunity and increased susceptibility to infection. We have previously demonstrated alterations in gene expression profiles of T cells from CLL patients, which translate into functional defects in T-cell immune synapse formation, motility and cytotoxicity (Gorgun et al. JCI 2005; Ramsay et al. JCI 2008, Blood 2013). However a comparison of the transcriptome of natural killer (NK) cells from CLL patients and controls has not been investigated. NK cells were isolated from the peripheral blood of patients with CLL and healthy donors, followed by gene expression profiling using the Affymetrix U133Plus2.0 platform. 117 probes showed a >2-fold decrease in expression while only 18 probes showed a >2-fold increase in expression (adjusted p-value < 0.05) in CLL NK cells compared to healthy donor NK cells. Strikingly, 52 out of the 117 significantly down-regulated probes (44.4%) were for interferon-inducible genes including STAT1 (Signal Transducers and Activator 1), SOCS1 (Suppressor of cytokine signaling 1), interferon regulatory factor genes IRF7 and IRF9, and oligoadenylate synthetase genes OAS1, OAS2, and OAS3. The majority of these genes were inducible by both type 1 and type 2 interferons. Many of these genes have been implicated in host immunity to viral infections, and so it is possible that decreased NK-cell responsiveness to interferon contributes to the increased susceptibility of CLL patients to viruses. Notably, there was also altered expression of signaling pathways in common with T cells from CLL patients, with dysregulation of the cytoskeleton genes RAB3GAP1, RAB38, and EPHA1 and down-regulation of JUN mirroring the dysregulated JNK-signaling and the altered actin cytoskeleton pathways we have found in T cells from CLL patients. These changes were not due to differences in the relative frequencies of CD56DIM and CD56BRIGHTNK cells. Lenalidomide has significant clinical activity in CLL. It is not directly toxic to tumor cells in vitro, but instead is thought to activate anti-tumor immunity and block pro-tumor micro-environmental factors. NK-cell proliferation has been shown to correlate with clinical response to lenalidomide in CLL (Chanan Khan et al. BJ Haem 2011). Therefore, we investigated the effect of lenalidomide treatment on the gene expression profiles of NK cells from CLL patients in comparison to healthy controls. PBMCs from CLL patients or healthy controls were cultured in the presence of 1μM lenalidomide or vehicle control for 48 hours, followed by NK-cell isolation, RNA extraction and gene expression profiling. There were striking differences in the effect of lenalidomide on NK cells from CLL patients compared with healthy NK cells. In CLL NK cells, lenalidomide repaired the down-regulation of interferon-inducible genes, by increasing the expression of genes such as OAS3, IFIT1, IFI44L, IFIT3, OAS1, PDK4, and ACTN1. Pathway analysis highlighted the effect of lenalidomide on inducing interferon signaling, showing significant activation of interferon α, γ, and λ as upstream regulators. While many of the interferon-inducible genes were up-regulated >3-fold in CLL NK cells, only OAS3 was significantly up-regulated in healthy NK cells with lenalidomide. Furthermore, the gene for IFNγ, IFNG, was actually significantly down-regulated in healthy NK cells by this agent. Lenalidomide also significantly down-regulated the expression of 5 genes encoding killer-cell immunoglobulin-like receptors (KIRs): KIR2DL1, KIR2DL2, KIR2DS3, KIR2DS4, and KIR3DL2, in healthy NK cells, but did not significantly down-regulate KIR genes in the CLL NK-cell dataset. Lenalidomide treatment did have some overlapping effects on CLL and healthy NK cells, including up-regulation of genes ARL11, CYFIP, and CORO1B that regulate the actin cytoskeleton pathway. In conclusion, NK cells from CLL patients have down-regulation of interferon response genes and pathways known to regulate normal immune function in response to bacteria and viruses. Lenalidomide has a differential effect on CLL and healthy NK cells: in CLL NK cells it repairs defective interferon responses, whereas in healthy NK cells it down-regulates inhibitory pathways. Disclosures: Riches: Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Abstract 790 Several investigators have defined gene expression profiling (GEP) signatures in Diffuse Large B-cell Lymphoma (DLBCL) enriched for macrophage and stromal genes, suggesting an active innate immune response against lymphoma. We hypothesize that the malignant B-cells drive tumor-associated macrophage (TAM) dysfunction in a subset of patients with DLBCL which is relevant for their biology and prognosis. We performed GEP on TAM from diagnostic DLBCL in order to recognise key genes and pathways open to functional validation. Single cell suspensions from 8 DLBCL and 8 reactive lymph nodes were used in this study. TAMs were flow sorted using CD36 expression. cDNA synthesis and amplification was performed using the Nugen Ovation Pico WTA system and the Affymetrix GeneChip human gene 1.0 ST platform was used. We used bioinformatics analysis of GEP data from DLBCL whole tumors, DLBCL purified B-cells, in vitro manipulated macrophages and other immune cells in order to define macrophage-enriched genes as well as specific M1/M2 signatures. We identified a 221 gene signature that significantly distinguished DLBCL TAMs from control macrophages, with 165 genes upregulated and 56 genes downregulated. Moreover, a comparative transcriptome analysis of 22 diverse immune cell phenotypes/activation states (IRIS: GSE22886) revealed that 26% of these genes are highly macrophage-enriched. Gene Ontology analysis revealed an over-representation for transcripts involved in inflammatory response (p 6.8×10−23), wound healing (p 1.9×10−20), chemotaxis (p 3.2 ×10−9), and cell motility (p 1.7×10−7). Upregulated genes in TAMs included well known M1 (complement components, CXCL9 or CXCL10) as well as M2 genes (MSR, CD163 or MARCO) (Table 1). In our signature, there was enrichment for M1 compared to M2 genes as defined by bioinformatics analysis. TAMs showed overexpression of the CSF1R gene as well as the chemokines CCL2 and CCL5, suggesting an autocrine feed-back loop of macrophage chemotaxis and survival in DLBCL. Moreover, TAMs showed upregulation of the lymphocyte attractants CCL20, CXCL9 and CXCL10, together with T-cell immunosupressants indoleamine 2,3-dioxygenase 1 and PD-L1, which would support a role for macrophages in T-cell recruitment and dysfunction in DLBCL. We also saw strong upregulation of 7 metallothionein isoforms in TAMs. These are proteins known to be expressed in macrophages and linked to response to oxidative damage, modulation of inflammation and cell proliferation. However their role in cancer microenvironment is unclear. We describe for the first time the GEP from DLBCL TAMs. The TAM transcriptome has partial overlapping genes with both M1 and M2 gene signatures, but also has a characteristic GEP potentially driven by their presence in the DLBCL microenvironment. Although further molecular and functional validation is required, this data provides a platform of genes which serve as excellent candidates for future exploration to understand DLBCL pathogenesis and to define new therapeutic targets. Table 1: Genes of particular interest represented in our TAM signature. Probe collapse was done using the highest expression. The Benjamini-Hochberg multiple hypothesis test was used to determine significance (FDR 0.05). Gene IDs Gene description Log2 Fold Change Adjusted p value MT1E-H, L, M, X, MT2A metallothionein 1E-H, 1M, 1X, 2A 2.3 – 4.4 0.00145 - 0.00017 C1QA, B and C complement component 1, q subcomponent, A, B and C chains 3.1 – 3.6 0.00136 - 0.00255 C2 complement component 2 3.2 0.00136 CCL2, 4, 5, 8, 20 chemokine (C-C motif) ligand 2, 4, 5, 8 and 20 2.4 - 3.7 0.046 - 0.00130 CD163 CD163 molecule 4.6 0.00025 CD14 CD14 molecule 3.4 0.00091 CD274 CD274 molecule 3.7 0.00447 CSF1R colony stimulating factor 1 receptor 2.1 0.00886 CXCL9-11 chemokine (C-X-C motif) ligand 9, 10 and 11 4.2 – 4.4 0.012 - 0.00158 FCGR1B Fc fragment of IgG, high affinity Ib, receptor (CD64) 4.9 0.00091 FCGR2A Fc fragment of IgG, low affinity IIa, receptor (CD32) 3.3 0.00139 FCGR3A Fc fragment of IgG, low affinity IIIa, receptor (CD16a) 5.2 0.00011 IDO1 indoleamine 2,3-dioxygenase 1 4.8 0.00232 MARCO macrophage receptor with collagenous structure 2.6 0.02029 MSR1 macrophage scavenger receptor 1 3.7 0.01467 Disclosures: Gribben: Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Tumor suppressor genes (TSGs) play critical roles in preventing tumorigenesis and they are frequently inactivated in tumours. Recently developed high-density microarrays can detect subchromosomal deletions, recurrence of which usually indicates the location of TSGs within the deleted region. We analyzed testicular germ cell tumour (TGCT) clinical samples using SNP arrays and found a frequent small deletion on the region 6q25.3 containing only one known gene, ZDHHC14. While its cellular function is unknown, ZDHHC14 belongs to the recently discovered DHHC family, which are predicted to be involved in protein palmitoylation, a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Consistently, we found a dramatic under-expression of ZDHHC14 mRNA and protein in TGCTs, and this associated with chemoresistance. Oncomine database mining showed that ZDHHC14 is also under-expressed in lymphoma, liposarcoma, brain, kidney, lung and colorectal cancers. Thus, it appears that ZDHHC14 downregulation may be involved in other cancers. We studied ZDHHC14 expression in prostate cancer (PCa), detecting a decrease at mRNA and protein level. We also detected that ZDHHC14 mRNA was downregulated in a pilot study on breast cancer samples. As genomic loss of the ZDHHC14 region was only detected in a small number of PCa samples, we checked whether promoter hypermethylation was the cause for ZDHHC14 downregulation. However, no changes in methylation status were found. We then sequenced the whole genomic region surrounding ZDHHC14 by next generation sequencing in TGCTs and PCa and found several mutations in the promoter, the coding region, as well as in intronic regions. Finally, we tested the function of ZDHHC14 in cell-based studies. We generated a 293 T-REx tetracycline inducible ZDHHC14 overexpressing stable cell line, which showed that ZDHHC14 overexpression decreased cell viability. The induction of apoptosis by ZDHHC14 overexpression was detected both by FACS and caspase 7 and PARP cleavage analyses. This was confirmed by transient ZDHHC14 overexpression in the PCa cell line 22RV1. In vivo we xenografted mice using both tetracycline inducible ZDHHC14 overexpressing 293 T-REx cells and control cells transfected with the empty vector. ZDHHC14 expression was induced by tetracycline at the beginning of inoculation and we detected that ZDHHC14 overexpression blocked tumour initiation completely. In conclusion, these results implicate ZDHHC14 as a tumour suppressor gene commonly inactivated in human cancers, indicating that it might exert its tumor suppressor role through the induction of programmed cell death. This is the first study showing the involvement of ZDHHC14 in a specific pathway, the classic caspase-dependent apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4858. doi:1538-7445.AM2012-4858

Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Prostate cancer is the most common male cancer in Western countries, but much less frequent in Asian countries. We systematically investigated genomic changes in prostate cancers from UK and China to determine genetic similarity and difference and potential alternative mechanisms of prostate carcinogenesis in these high and low cancer incidence populations. Using Affymetrix array 6.0 microarrays, we analyzed genome-wide genomic alterations in 32 UK and 39 Chinese prostate cancer samples. Distinct difference in genomic alterations between Western and Chinese prostate cancers were found, including loss of 21q22 and PTEN deletion, which are the most common genomic changes in Western prostate cancer but rarely detected in the Chinese samples. To further evaluate the difference between Western and Chinese samples, FISH analysis for PTEN deletion and ERG rearrangements and immunohistochemistry analysis for PTEN and ERG expressions were applied to UK (n=160) and Chinese (n=143) prostate cancer tissue microarrays (TMAs). PTEN deletion and ERG rearrangements were found at a significantly higher frequency in samples from UK (42.3% and 37.4%) than China (14.3% and 7.5% respectively) (P In conclusion, we compared genetic alterations in prostate cancer samples from UK and China and found there are significant differences between the two groups, commencing at pre-invasive, HGPIN, stage. Our findings suggest that tumours arise in Western and Chinese populations by alternative pathogenetic mechanisms. These alterations, differentially presented in these two populations may be key genetic changes underlying the regional/ethnic difference in clinical incidence and may be induced by specific environmental and/or genetic risk factors that Western men are exposed to. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3878. doi:10.1158/1538-7445.AM2011-3878

Prostate cancer is commonly detected as multiple foci in a single prostate gland and it is of etiological and clinical importance to identify the origin of these multifocal cancer lesions. Due to the limited number of markers analyzed, previous studies to address this issue are inconclusive. A high-resolution genome-wide study should address this issue better. Therefore, we have performed a genome-wide analysis using Affymetrix Array 6.0 of individually laser-capture microdissected foci to compare the genetic similarities and/or differences of these foci within the same prostate gland. Fluorescence in situ hybridization was used to confirm SNP array results. A total of 43 cancer and high-grade prostatic intraepithelial neoplasia (PIN) foci from 16 prostate cancer cases were analyzed for genomic copy number changes. Copy number changes from same-case foci were compared to determine the genetic relationship between the foci. In many cases, we found extensive genetic differences between different foci isolated from the same case. However, omniclonal copy number gains and losses were detected in 11/11 informative case-matched multifocal tumor cases and 8/8 informative case-matched PIN and tumor cases, suggesting that multiple cancer foci arise from a common prostate cancer precursor clone. In addition, a higher degree of similarity was observed between PIN and adjacent cancer foci, as compared to distant lesions. The strong genetic similarities between PIN and prostate cancer foci from the same region suggest clonal expansion of separate PIN lesions to invasive cancer. Based on the monoclonal model, we examined the step-wise accumulation of these genomic copy number changes. Loss of 10q23.2-10q23.31 and 16q were commonly shared in same-case tumor foci and, in many cases, also matched PIN foci, suggesting that they are early events in prostate carcinogenesis. Other common genomic changes, such as loss of 6q, 8p and 21q22.2-21q22.3, were found less frequently in all same-case foci and rarely in PIN lesions, suggesting these genetic events occur at a relatively later stage. In conclusion, using a high-density SNP array, we reveal a monoclonal origin for the majority of multifocal prostate cancer samples analyzed. From these SNP array data, we have also begun to elucidate the step-wise genetic events in prostate carcinogenesis, including early events such as loss of 10q23.2-10q23.31 and 16q Taken together, these findings should significantly enhance our understanding of prostate carcinogenesis and potentially affect the clinical management of this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4831. doi:10.1158/1538-7445.AM2011-4831

An example of a homogeneously staining region (hsr), occurring in an acute myeloid leukaemia (M5) on chromosome 11 in the region of bands q23–q24, has been analysed. In situ hybridisation using yeast artificial chromosome (YAC) DNA demonstrated that the amplification did not include the CD3 gene cluster and did not affect the human trithorax gene known to be disrupted by translocations at 11q23. In contrast, the amplification was shown to include the sequence D11S543 which has been previously mapped to chromosome band 11q24. High resolution analysis using confocal microscopy allowed the individual amplicons to be visualised, and it was shown that the hsr consisted of an 8-fold amplification of the region surrounding the probe D11S543. From previous estimates of human chromosome size it was possible to calculate that the hsr was composed of amplicons approximately 10 megabases in length. It was concluded that the region amplified did not extend as far as the translocation breakpoints occurring at 11q23 in acute leukaemias. © 1993 Wiley-Liss, Inc.

Prostate cancer is a highly heterogeneous disease both in terms of its pathology and clinical presentation. Pathologically, multiple cancer foci are commonly detected in a single prostate gland. Previously, microsatellite analysis has been used to analyse the origin of multifocal prostate cancer cases. However, it is still debatable whether these separate cancer foci originate from a single clone, or arise independently. To date no extensive high-resolution genome-wide study has been performed to address this. For this reason, we have performed a genome-wide analysis using Affymetrix Array 6.0 with 18 million gene probes to compare the genetic similarities and/or differences of multiple cancer foci within the same prostate gland. We considered two lesions to be separate if they were located O1 cm apart throughout the depth of the tissue. Cancer foci, high-grade prostatic intraepithelial neoplasia (PIN), and matched normal prostate tissue were identified and microdissected separately from H+E stained fresh-frozen tissue sections. SNP 6.0 array data were analysed to determine the genetic relationship between the cancer foci. In many cases we found extensive genetic differences between different cancer foci isolated from the same case. However, all same-case cancer foci and/or PIN shared at least one common genetic alteration, suggesting that multiple cancer foci arise from a common prostate cancer precursor clone at stage of PIN or earlier. In addition, a higher degree of similarity was observed between PIN and adjacent cancer foci, as compared to distant lesions. The strong genetic similarities between PIN and prostate cancer foci from the same region suggest that clonal expansion of separate PIN lesions may account for the multifocal nature of prostate cancer. BREAKPOINT PATTERN ANALYSIS IN SOLID TUMORS

Background The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation. Results The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP) mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH) and copy number variations (CNV). FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3) and segmental LOH (6q25.1-6q25.3). Conclusion We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant upregulation of FOXM1 serves as a 'first hit' where cells acquire genomic instability which in turn predisposes cells to a 'second hit' whereby DNA-damage checkpoint response (eg. p53 or p16) is abolished to allow damaged cells to proliferate and accumulate genetic aberrations/mutations required for cancer initiation.

166 Recurrence of acute myeloid leukemia AML has a poor prognosis with only 20% of adults surviving to 5 years. Therefore it is of importance to identify molecular changes that explain the pathogenesis of relapsed AML. Previous studies had not identified consistently acquired cytogenetic changes at relapse. Recently, acquired uniparental disomy due to mitotic recombination was described in 40% of relapsed AML (Raghavan et al 2008). Most of the events lead to homozygosity for FLT3 mutations. This study aimed to discover if there are further genetic abnormalities acquired at disease recurrence that cannot be identified by conventional cytogenetics, i.e. microdeletions or gains. Twenty-one presentation and relapse paired AML patient blood and marrow samples were stored with consent at St Bartholomew's Hospital, London. Eleven patient samples had a normal karyotype at diagnosis, two had favourable prognosis cytogenetics (inv(16) and t(8;21)) and others had varying numerical cytogenetic abnormalities and rearrangements associated with an intermediate prognosis. DNA from the samples was analysed by array based high-resolution single nucleotide polymorphism (SNP) genotyping (Affymetrix Human SNP array 6.0). Data was analysed using Partek Genome Browser (Partek, MO). In all cases, the leukemia infiltrate of the marrow or blood was greater than 60% and most cases were greater than 90% allowing accurate identification of DNA copy number changes. Abnormalities of a size that would be identified by cytogenetics were disregarded. Using segmentation analysis using a p-value less than 0.001, over 400 microdeletions and gains were detected that were acquired at relapse in the 21 pairs. Each of the copy number changes was less than 2 megabases in size. One AML sample with a normal karyotype at diagnosis and trisomy 8 and add(9)(q34) at relapse had not acquired any microdeletions or gains. In contrast, in other samples as many as 69 microdeletions/gains were detected. There was no correlation between increased complexity of the karyotype of the leukemia and the number of microdeletions/gains. Several of the acquired microdeletions/gains were in regions containing genes known to be involved in AML, including a deletion of 234Kb at 13q12.2 involving FLT3 and CDX2 , and an acquired deletion at 21p11.2 of 150Kb involving exons encoding the runt domain of RUNX1 . Another copy number gain was detected at the MLL locus, suggestive of partial tandem duplication. Other detected locations are in [Table 1][1]. | Location by cytoband | Copy number change | Size / Kb | P value | Gene | |:--------------------:| ------------------ | --------- | ------- | ---------- | | 13q12.2 | Deletion | 234 | 10−33 | FLT3, CDX2 | | 21q22.12 | Deletion | 150 | 10−13 | RUNX1 | | 11q23.3 | Gain | 5.1 | 0.0099 | MLL | | 11p15.4 | Gain | 83 | 0.00001 | NUP98 | | 17q21.31 | Deletion | 8.0 | 0.0007 | BRCA1 | * The results indicate that recurrent AML may be associated with the deletion or gain of several genes involved in leukaemogenesis. Many other locations are involved throughout the genome, suggesting at least some of these are also involved in the clonal evolution of the leukaemia at recurrence. Further studies should identify novel genes from these regions involved in the pathogenesis of AML. Table 1 Disclosures: No relevant conflicts of interest to declare. [1]: #T1

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.