Common Region Of Amplification Research Articles

Abstract 4357: Comparing amplification of 12q13-q14 and 12q15 chromosomal regions across cancer types through genomic and transcriptome analysis

Abstract Gene amplification, or an increase in copy number of a confined region on the chromosome arm, has been identified as a critical genetic event that contributes to the development of various cancers. There is increased expression of certain genes within the amplified regions, which alters normal cell growth and survival pathways, and contributes to tumorigenesis. Although previous studies show that some regions are amplified in more than one cancer type, direct analyses comparing the size and gene composition of these amplified regions across tumor types have not been performed. In the current study, we used copy number and RNA sequencing data from our published data and The Cancer Genome Atlas (TCGA) to characterize the commonly affected 12q13-q14 and 12q15 chromosomal regions in rhabdomyosarcoma, glioblastoma multiforme, lung adenocarcinoma, and liposarcoma. Based on our analysis of copy number data from high-density single-nucleotide polymorphism arrays, we observed a 0.08 Mb common region of overlap of the 12q13-q14 amplicons and a 0.20 Mb common region of overlap of the 12q15 amplicons across these tumor types. Differential gene expression analysis between amplified and nonamplified samples showed that OS9, TSPAN31, CDK4, CYP27B1, METTL1, EEF1AKMT3, and TSFM were overexpressed by the 12q13-q14 amplicons. Similarly, MDM2 and CPM were overexpressed by the 12q15 chromosomal amplicons. In addition to the common region of overlap, regions of tumor-type specific amplification were also found in our analysis. The 12q13-q14 amplicon in fusion-positive rhabdomyosarcoma extended 0.48 Mb toward the centromere, while the 12q13-q14 amplicons in dedifferentiated liposarcoma and lung adenocarcinoma extended 0.56 Mb and 0.96 Mb, respectively, toward the telomere. For the 12q15 region, the amplicon in lung adenocarcinoma extended 1.3 Mb toward the centromere, while the amplicons in dedifferentiated liposarcoma and fusion-negative rhabdomyosarcoma extended 1.4 Mb and 1.6 Mb, respectively, toward the telomere. Gene expression analyses showed that some genes from these tumor-specific regions of amplification were preferentially overexpressed in the corresponding tumor types. For example, four genes from the fusion-positive rhabdomyosarcoma-specific 12q13-q14 amplicon (NEMP1, NAB2, SHMT2, and R3HDM2) and two genes from the lung adenocarcinoma-specific 12q15 amplicon (MDM1 and RAP1B) were specifically overexpressed in these two tumor types. Our findings indicate that, in addition to common regions of amplification across multiple tumor types, there are tumor-specific amplified regions and overexpressed genes that indicate the presence of unique features within each cancer category affecting these amplification events. Citation Format: Prasantha L. Vemu, Gregory E. Hoy, Wenyue Sun, Jack Shern, Javed Khan, Frederic G. Barr. Comparing amplification of 12q13-q14 and 12q15 chromosomal regions across cancer types through genomic and transcriptome analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4357.

Application of high-resolution genomic profiling in the differential diagnosis of liposarcoma

BackgroundRarity and heterogeneity of liposarcomas (LPS) make their diagnosis difficult even for sarcoma-experts pathologists. The molecular mechanism underlying the development and progression of liposarcomas (LPS) remains only partially known. In order to identify and compare the genomic profiles, we analyzed array-based comparative genomic hybridization (array-CGH) profiles of 66 liposarcomas, including well-differentiated (WDLPS), dedifferentiated (DDLPS) and myxoid (MLPS) subtypes.ResultsCopy number aberrations (CNAs) were identified in 98% of WDLPS and DDLPS and in 95% of MLPS cases. The minimal common region of amplification at 12q14.1q21.1 was observed in 96% of WDLPS and DDLPS cases. Four regions of CNAs, including losses of chromosome 6, 11 and 13 and gains of chromosome 14 were classified as recurrent in DDLPS; at least one was identified in 74% of DDLPS tumors. The DDLPS-associated losses were much more common in tumors with increased genomic complexity. In MLPS, the most frequent CNAs were losses of chromosome 6 (40%) and gains of chromosome 1 (30%), with the minimal overlapping regions 6q14.1q22.31 and 1q25.1q32.2, respectively.ConclusionsOur findings show that the application of array-CGH allows to delineate clearly the genomic profiles of WDLPS, DDLPS and MLPS that reflect biological differences between these tumors. Although CNAs varied widely, the subtypes of tumors have characteristic genomic profiles that could facilitate the differential diagnosis of LPS subtypes, especially between WDLPS and DDLPS.

Evaluation of multiplex ligation dependent probe amplification (MLPA) for identification of acute lymphoblastic leukemia with an intrachromosomal amplification of chromosome 21 (iAMP21) in a Brazilian population.

BackgroundAn intrachromosomal amplification of chromosome 21 (iAMP21) defines a unique subgroup of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The finding of three or more extra copies of the RUNX1 gene by fluorescence in situ hybridization (FISH) is internationally used to define an iAMP21. Genomic profiling of chromosome 21 has been suggested for assisting diagnostic case identification. Due to limitations of comparative genomic hybridization, in terms of a routine application as first line-screening tests we evaluated the multiplex ligation-dependent probe amplification (MLPA) SALSA P327_A1 and P327_B1 probe sets for detecting chromosome 21 copy number alterations in Brazilian childhood BCP-ALL.ResultsIn 74 out of 368 patients gain of genetic material was detected. For data confirmation RUNX1 directed FISH was performed. Cells with ≥5 RUNX1 signals (n = 9) were considered as “true iAMP21” while <5 RUNX1 signals (n = 41) were counted as evidence for additional copies of intact chromosomes 21. All patients with an iAMP21 had high MLPA peak ratios (≥1.8), while the majority of patients with <5 RUNX1 presented low MLPA peak ratios (<1.8). Observed differences gained statistical strength by comparing probes located within the common region of amplification. Next, a principal component analysis was performed in order to illustrate distribution of cases according to their MLPA peak profile in two dimensions. Cases with an iAMP21 mostly clustered together, however additional cases with <5 RUNX1 signals or no available FISH data located in proximity.ConclusionsMLPA qualified as a high throughput technique that could be employed in future studies for a critical comparison with data obtained by FISH, especially in cases where metaphase nuclei are not available. Taking submicroscopic aberrations into account examined by MLPA, cases exhibiting an “iAMP21 like” peak ratio profile but <5 RUNX1 signals should be considered as candidates for this chromosomal abnormality.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-015-0147-2) contains supplementary material, which is available to authorized users.

Clonal Progression Including Evolution Of An iAMP21 Chromosome In Xenograft Models Of BCP-ALL

Insight into the role of recurrent genomic changes arising in leukemia has depended largely on the functional analysis of cell lines and transgenic animals. However not all acquired abnormalities are represented in available cell lines and a few are too complex to be faithfully engineered in animal models. One such abnormality is intrachromosomal amplification of chromosome 21 (iAMP21), a heterogeneous cytogenetic rearrangement, with a distinct clinical profile, occurring in 2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The most highly amplified segment of iAMP21 varies in size and copy number but has always included a 5.1 Mb common region of amplification (CRA). Regions flanking the most highly amplified segment have profiles that may be step-like, or more complex combinations of lower level amplification, normal copy number and deletion. Abnormalities at other chromosomal locations such as deletions of IKZF1, CDKN2A/B or RB1 and rearrangements activating CRLF2 co-occur with iAMP21 but have never been shown to precede its formation.To investigate clonal evolution and to provide a resource for future functional studies, we established xenografts from three cases of iAMP21 BCP-ALL in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Signs of disease were seen between 6 and 8 months after intrafemoral injection and autopsy revealed marked splenomegaly in all cases. Cells isolated from the bone marrow and spleen, were positive for human B cell markers CD19 and CD10. Disease onset was more rapid (3-6 months) when primografts were transplanted and cells from these secondary mice were used to establish leukemia in a third generation. Whole body imaging of mice transplanted with xenograft cells, transduced with a lentivirus expressing luciferase, revealed the rapid spread of disease from the injection site to the contralateral femur, sternum, pelvis, ribs, spine and skull, with the spleen becoming the major site of engraftment at late stage disease.Comparison of SNP 6.0 array profiles of presentation and xenografts samples revealed several examples of post-transplant genomic progression which, with the exception of a biallelic deletion of the CDKN2A/B locus, had not previously been reported in iAMP21 patients. Additional copies of chromosomes 9 and 12 in patient samples were lost in xenografts resulting in copy number neutral loss of heterozygosity (CNN-LOH) for chromosome 12 but not 9. Analysis of further primary iAMP21 BCP-ALL cases identified two with CNN-LOH for part of the long arm of chromosome 12, suggesting involvement of an imprinted locus in this region.iAMP21 chromosomes were retained in all xenografts, supporting evidence that it is a primary abnormality. However, as these rearrangements have been considered to be stable once formed, we were surprised to observe acquisition of a non–contiguous deletion of chromosome 21 in one secondary mouse and all of its tertiary recipients. The deletion was proximal to the CRA and resulted in extension of pre-existing haploinsufficient regions by 6.6 Mb. Evolution of this iAMP21 chromosome was confirmed by fluorescence in-situ hybridisation (FISH) using probes targeting the deletion, with loss of signal observed in all cells from mice carrying the deletion but in no cells from the presentation sample or unaffected mice. Although we cannot rule out that the deletion arose in response to adaptive pressure specific to the xeno-environment, opportunities to observe similar progression in patients have been limited because FISH analysis has generally targeted only the CRA and few paired presentation/relapse samples have been analysed by genomic array. While it remains likely that overrepresentation of oncogenes within the CRA acts as a primary driver of iAMP21 BCP-ALL, the additional deletion may highlight the position of tumour suppressor genes involved in leukemia progression. As these patients are treated as a single clinical entity, an important implication of these data is that the position and extent of deletions in iAMP21 rearrangements may influence response to therapy.In conclusion, xenografts of iAMP21 BCP-ALL closely resemble human primary disease and will make valuable pre-clinical models for basic and translational research. As models for the study of clonal evolution they support previous evidence that iAMP21 is a primary abnormality and have revealed novel forms of clonal progression. Disclosures:No relevant conflicts of interest to declare.

Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia

AbstractIntrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that has a dismal outcome when treated with standard therapy. For improved diagnosis and risk stratification, the initiating genetic events need to be elucidated. To investigate the genetic basis of BCP-ALL, genomes of 94 iAMP21 patients were interrogated by arrays, FISH, and multiplex ligation-dependent probe amplification. Most copy number alterations targeted chromosome 21, reinforcing the complexity of this chromosome. The common region of amplification on chromosome 21 was refined to a 5.1-mb region that included RUNX1, miR-802, and genes mapping to the Down syndrome critical region. Recurrent abnormalities affecting genes in key pathways were identified: IKZF1 (22%), CDKN2A/B (17%), PAX5 (8%), ETV6 (19%), and RB1 (37%). Investigation of clonal architecture provided evidence that these abnormalities, and P2RY8-CRLF2, were secondary to chromosome 21 rearrangements. Patient outcome was uniformly poor with standard therapy irrespective of the presence or absence of these changes. This study has provided evidence that chromosome 21 instability is the only anomaly among those so far investigated that is common to all iAMP21 patients, and therefore the initiating event is likely to be found among the complex structural rearrangements of this abnormal chromosome.

Genomic characterization of asymptomatic CT-detected lung cancers

Computed tomography (CT) screening of lung cancer allows the detection of early tumors. The objective of our study was to verify whether initial asymptomatic lung cancers, identified by high-resolution low-dose CT (LD-CT) on a high-risk population, show genetic abnormalities that could be indicative of the early events of lung carcinogenesis. We analyzed 78 tumor samples: 21 (pilot population) from heavy smokers with asymptomatic non-screening detected early-stage lung cancers and 57 from 5203 asymptomatic heavy smoker volunteers, who underwent a LD-CT screening study. During surgical resection of the detected tumors, tissue samples were collected and short-term cultures were started for karyotype evaluation. Samples were classified according to the normal (NK) or aneuploid (AK) karyotype. The NK samples were further analyzed by the Affymetrix single-nucleotide polymorphisms (SNPs) technology. Metaphase spreads were obtained in 73.0% of the selected samples: 80.7% showed an AK. A statistically significant correlation was found between presence of vascular invasion and abnormal karyotype. A total of 10 NK samples were suitable for SNPs analysis. Subtle genomic alterations were found in eight tumors, the remaining two showing no evidence to date of chromosomal aberrations anywhere in the genome. Two common regions of amplification were identified at 5p and 8p11. Mutation analysis by direct sequencing was conducted for the K-RAS, TP53 and EGFR genes, confirming data already described for heavy smokers. We show that: (i) the majority of screening-detected tumors are aneuploid; (ii) early-stage tumors tend to harbor a less abnormal karyotype; (iii) whole genome analysis of NK tumors allows for the detection of common regions of copy number variation (such as amplifications at 5p and 8p11), highlighting genes that might be considered candidate markers of early events in lung carcinogenesis.

What Is the Initiating Mechanism of iAMP21 in Childhood B Cell Precursor ALL?.

Abstract 581Patients with iAMP21 (intrachromosomal amplification of chromosome 21) represent a distinct cytogenetic subgroup of childhood B cell precursor ALL (BCP-ALL; incidence ∼2%) with a poor prognosis on standard therapy (29% EFS at 5 years). From cytogenetic studies the amplification of chromosome 21 appears to be the primary genetic change, however, whether this or associated abnormalities provide the initiating event of leukaemogenesis remains to be elucidated. Currently fluorescence in situ hybridisation (FISH) with probes directed to the RUNX1 gene provides the only reliable detection method. Knowledge of the underlying genetic mechanism will lead to improved diagnosis and determine those patients at high risk of relapse who require more intensive therapy. Our previous study using genomic arrays (1Mb aCGH, n=10; NimbleGen chromosome 21 custom oligonucleotide array, n=5) identified a common region of amplification (CRA) on 21q of 6.6 Mb and a common region of deletion (CRD) of 3.3 Mb within the telomeric region in 100% and 70% of patients, respectively. However, expression profiling (Affymetrix U133A, n=8) indicated that no important genes within the CRA or CRD were differentially expressed. We now have clinical, cytogenetic and FISH data on 94 iAMP21 patients (44 F, 50 M; median age 9.5y, range 2-30); the largest iAMP21 cohort investigated to date. Higher resolution analysis of 17/94 diagnostic samples by aCGH (Agilent 185K platform) have refined these regions to 5.1 Mb and 2.5 Mb in 100% and 88% of patients, respectively. Recent genome mapping has highlighted the presence of the microRNA miR-802 within the CRA and shown this region overlaps with the Down Syndrome Critical Region at 21q22.3 encompassing the genes DSCR1, DSCR3 and DYRK1. Detailed analysis of chromosome 21 identified recurrent breakpoints centromeric of the CRD within three genes: PDE9A (n=2), COL6A2 (n=2) and DSCAM (n=2). The role of these genes in the initiation of the chromosomal instability associated with iAMP21 is currently under investigation. Global analysis of the iAMP21 genome identified an average of 13 copy number alterations (CNA) per case with recurrent deletions of the leukaemia associated genes PAX5 (n=2), IKZF1 (n=4) and CDKN2A (n=4). To determine the incidences of PAX5, IKZF1 and CDKN2A deletions among the cohort of 94 iAMP21 patients, FISH using probes targeting these genes was carried out on availabel samples. IKZF1 (exons 3-6) was deleted at the highest incidence of 21% (13/62), while CDKN2A and PAX5 were deleted at a lower incidence of 17% (12/71) and 8% (4/48), respectively. The high incidence of IKZF1 deletions found in this patient group correlates with their association with high risk ALL. We have recently reported a deletion within the PAR1 region of the sex chromosomes, centromeric of the CRLF2 gene involving CSF2RA and IL3RA, which leads to deregulated expression of CRLF2. This deletion occurred at an incidence of 23% (17/73), as determined by FISH. Among pairs of matched diagnostic and relapse samples; one had this deletion at both diagnosis and relapse while the second had a deletion in the diagnostic sample only. Gain of an additional × chromosome was identified in 28% (18/65). Of note is that the 3 aberrations: abnormalities of chromosome 21, gain of the × chromosome and high incidence of deletions centromeric of CRLF2 in this patient group mirror the findings in Down Syndrome (DS) ALL, although we have not identified any mutations of the JAK2 kinase domain among this cohort of iAMP21 patients to date. This genome-wide study provides further support for the proposal that amplification of genes within the CRA on chromosome 21 is the primary event driving leukaemogenesis in iAMP21 patients. It has also highlighted that the 3 abnormalities in common with DS ALL and/or deletions of IKZF1, which all occur at a high incidence, contribute towards the progression of this high risk subtype of childhood BCP-ALL. Disclosures:No relevant conflicts of interest to declare.

Genomic characterization of early-stage asymptomatic lung cancers

11106 Background: CT screening of lung cancer allows the detection of very small tumors. The objective of our study was to verify whether a selected population of initial asymptomatic lung cancers show genetic abnormalities able to mark the early events of lung carcinogenesis. Methods: A pilot study was performed for the standardization of the technique, enrolling heavy smokers with asymptomatic early stage lung cancers non-screening detected. Since 2004, a low dose CT screening study enrolled 5201 asymptomatic heavy smokers volunteers. Tissue samples were collected from patients of these two groups who underwent surgical resection in our institute and short-term cultures were started from tumoral tissues. Cultures were suitably treated for the analysis of metaphase spreads. Samples were classified according to the normal (N) or aneuploid (A) karyotype. N samples were further analyzed by the Affymetrix HuSNP 50K technology. Results: 101 tumor samples were analyzed (33 collected during a pilot project and 78 as part of the screening program). Metaphase spreads were obtained in 70% (71/101) of the selected samples: 77% (55/71) showed an aneuploid karyotype, while 20% (14/71) resulted as normal, 3% (2/71) were uncharacterized. The subgroup of tumors with p-stage IA showed aneuploidy in 70% (24/34) of cases with a mean size of 15 mm. A correlation between tumor size and chromosome number was shown, having small size tumors a higher probability to show a normal karyotype. 8/14 of the N samples were found suitable for SNPs analysis. Subtle genomic alterations were found in 4 of the analyzed patients, the remaining 4 lacking to date of an evidence for the presence of chromosomal aberrations throughout the genome. A common region of amplification was identified at 8p11 in 2 samples and is currently under investigation. Conclusions: i) The majority of screening detected tumors presented aneuploidy despite very initial stage. ii) A correlation between tumor size and ploidy status exists in early lung cancers. ii) Genomic scanning is able to detect subtle region of chromosomal alterations in early lung cancer patients. No significant financial relationships to disclose.

Identification of New Genomic Lesions in Childhood ALL without Known Genetic Aberrations: A Microarray Study of Gene Expression and Genotype Data.

Despite the risk stratification based on the prognostic relevant translocations, approximately 20–25% of childhood ALL patients cannot be classified according to genetic hallmarks. Recent studies based on single nucleotide polymorphism (SNP) analyses showed that Loss of Heterozygosity (LOH), with or without loss of genetic material, is frequently related to childhood ALL and AML. Moreover, there is an increasing interest in integrating the gene expression profiles with chromosomal localizations to identify new genetic subgroups. We applied an integrated approach composed of DNA index estimation, PCR (and/or RT-PCR) and cytogenetics, to exclude patients with known molecular and cytogenetics aberrations. Aim of the study was to identify criptic abnormalities in childhood ALL patients by performing an integrative analysis of gene expression and copy number changes (CNC) data. The 30 patients included in our study met the following inclusion criteria: a) B-cell precursor childhood ALL b) DNA index 1; c) negativity at t(4;11), t(12;21), t(9;22), t(1;19) RT-PCR screening; d) cytogenetics revealing normal karyotype or not technically feasible. Patients genomic DNA has been analyzed by the Affymetrix GeneChip Mapping 100K SNP arrays to identify genomic regions of LOH and CNC. In parallel, biotin-labeled cRNA has been synthesized and hybridized on HG-U133Plus2 Probe Arrays in accordance with the MILE (mircroarray innovation in leukemia) protocols; all samples were part of the B-ALL class without known genetic aberrations.The presence of del(9)(p21) was found in 9/30 (30%) patients, with an homozygous commonly deleted region involving CDKN2A and CDKN2B genes in 7/30. Hemizygous losses on 9p13 were found in two cases. Four patients (13,3%) showed the hemizygous deletion of chromosome 12p13.2, with a commonly deleted region involving the ETV6 gene. In one ETV6 deleted patient FISH analysis identified a translocation involving the first ETV6 exon and an unknown partner gene. Four patients, including three ETV6 deleted, showed the presence of the intrachromosomal amplification of chromosome 21 (iAMP21) with a common region of amplification between 31.3–43.5 Mb and a common region of deletion in 2/4 patients between 43.5–47 Mb. Four patients showed large regions of LOH not associated with CNC. Three patients did not show CNC and random microdeletions were found in single cases. Remission samples were analyzed for 5 patients carrying different aberrations, showing small regions of LOH in both the diagnosis and remission samples in 4 cases. No CNC was found in the remission samples. We found a statistically significant reduction for the expression of the CDKN2A transcript located within the (9)(p21) region. We are applying a locally adaptive statistical procedure to identify genes whose expression signals are specifically modulated by CNC. The integration of genomic variation and gene expression profiling might may be useful to identify new hidden genetic lesions, and to learn how critical regulators of tumor are linked to genomic alterations in cancer cells.

Overlapping 3q28 amplifications in the COMA cell line and undifferentiated primary sarcoma

Historically, amplicon mapping and characterization of double minute (dmin) chromosomes content have been the ways to pinpoint important oncogenes. The COMA cell line established from a sarcoma contains DMs, some of them composed of material of the long arm of chromosome 3. To identify putative oncogenes on 3q that may be included in these dmins, we have analyzed the COMA cell line by microarray-based comparative genomic hybridization (array-CGH). We have detected the amplification of 1-Mb segment at 3q28, which contains the genes LPP, FLJ42393, and hsa-mir-28. Fluorescence in situ hybridization experiments confirmed the presence of numerous copies of 3q28 segment included in dmins. Further screening of eight undifferentiated primary sarcomas with 3q gains previously detected by chromosome CGH disclosed, in two cases, amplifications at 3q28 overlapping the 1-Mb segment amplified in COMA. To isolate target genes upregulated by gene dosage effect, we measured the transcription levels of every gene (in the RefSeq collection) located in the common region of amplification, selected expressed sequence tags (ESTs) and the micro-RNA hsa-mir-28 in the COMA cell line compared to one MFH cell line without alteration at 3q28. Expression levels of all transcripts were almost similar in both cell lines, except for two ESTs (AI338598 and BX118304) showing a 20-fold increase. These two transcripts are poorly characterized and their contribution to MFH carcinogenesis is difficult to evaluate.

Complex genomic alterations and gene expression in acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21

We have previously identified a unique subtype of acute lymphoblastic leukemia (ALL) associated with a poor outcome and characterized by intrachromosomal amplification of chromosome 21 including the RUNX1 gene (iAMP21). In this study, array-based comparative genomic hybridization (aCGH) (n = 10) detected a common region of amplification (CRA) between 33.192 and 39.796 Mb and a common region of deletion (CRD) between 43.7 and 47 Mb in 100% and 70% of iAMP21 patients, respectively. High-resolution genotypic analysis (n = 3) identified allelic imbalances in the CRA. Supervised gene expression analysis showed a distinct signature for eight patients with iAMP21, with 10% of overexpressed genes located within the CRA. The mean expression of these genes was significantly higher in iAMP21 when compared to other ALL samples (n = 45). Although genomic copy number correlated with overall gene expression levels within areas of loss or gain, there was considerable individual variation. A unique subset of differentially expressed genes, outside the CRA and CRD, were identified when gene expression signatures of iAMP21 were compared to ALL samples with ETV6-RUNX1 fusion (n = 21) or high hyperdiploidy with additional chromosomes 21 (n = 23). From this analysis, LGMN was shown to be overexpressed in patients with iAMP21 (P = 0.0012). Genomic and expression data has further characterized this ALL subtype, demonstrating high levels of 21q instability in these patients leading to proposals for mechanisms underlying this clinical phenotype and plausible alternative treatments.

A Breakage-Fusion-Bridge Cycle Generated by Telomeric Loss May Underlie the dup(21q) of Acute Lymphoblastic Leukemia.

A highly complex, rearranged chromosome 21, arising from duplication of 21q and associated with amplification of RUNX1 [dup(21q)], is linked to a poor prognosis in acute lymphoblastic leukemia (ALL). Using BAC array-based CGH (Spectral Genomics 1Mb, USA) (aCGH), we originally identified a characteristic pattern of imbalance, with common regions of amplification (CRA) and deletion (CRD) along 21q, in a series of 10 patients. The extent of these regions was refined to 6.6Mb (between 33.192 and 39.796Mb) and 3.3Mb (between 43.7 and 47Mb, which included sub-telomeric sequences), respectively, by tiling-path oligonucleotide-based aCGH (NimbleGen Inc., USA) (n=15). Six BAC clones (including a sub-telomeric one) from the 1Mb arrays, corresponding to the variable regions of amplification along 21q were used as probes for interphase FISH (iFISH) in 48 patients. The same CRA was confirmed in all, while the CRD was observed in 34/46 (77%) of them. iFISH showed that the degree of amplification corresponding to each chromosomal region differed between patients. Although these techniques, which measure genomic copy number changes, showed consistent patterns of amplification and deletion along 21q, it was intriguing as to why, at the cytogenetic level, the abnormal chromosome 21 had many different forms. The same locus-specific probes, applied to metaphases from 10 patients, revealed complex signal patterns, unique to each patient, in which the signals from each probe were distributed along 21q in unexpected positions relative to each other. Often signals from the same probe were located to more than one region of the abnormal chromosome 21. These findings were indicative of duplications of the chromosomal regions to which the probes were located, in combination with intricate intrachromosomal rearrangements, including inversions. We were able to illustrate these unique rearrangements by multicolor FISH banding (XCyte21, Metasystems, Germany) in two cases. Supervised gene expression analysis (HG-U133A arrays, Affymetrix, USA) showed a distinct signature for eight patients with dup(21q). Genomic copy number correlated with overall gene expression levels within areas of gain or loss. However, there was considerable inter-genic differences and variation between individuals. Of the 40 genes contained within the amplicon, no promising targets were upregulated when compared to patients with high hyperdiploidy, comprising at least one additional copy of chromosome 21. Collectively, our results have provided evidence for extensive intrachromosomal rearrangements and instabilityn of 21q in these patients. Although there was no evidence of other established chromosomal changes, the clonal heterogeneity we observed in the karyotypes was indicative of genome-wide chromosomal instability. These types of genomic alterations, arising from a series of chromatid breaks and reunions, which lead to intrachromosomal amplifications and deletions, are the hallmark of the breakage-fusion-bridge (BFB) cycle in solid tumors. This is the first time that cytogenetic features linked to the BFB mechanism have been described in ALL. We hypothesize that the loss or abnormal functioning of telomeric sequences may be the causal event behind this poor-risk 21q abnormality.

Expression and copy number analysis of TRPS1, EIF3S3 and MYC genes in breast and prostate cancer.

The long arm of chromosome 8 is one of the most common regions of amplification in cancers of several organs, especially carcinomas of the breast and prostate. TRPS1, MYC and EIF3S3 genes are located in one of the minimal regions of amplification, 8q23–q24, and have been suggested to be the target genes of the amplification. Here, our goal was to study copy number and expression of the three genes in order to investigate the significance of the genes in breast and prostate cancer. By using fluorescence in situ hybridisation (FISH), we first found that TRPS1 and EIF3S3 were amplified together in about one-third of hormone-refractory prostate carcinomas. Next, we analysed the mRNA expression of the three genes by real-time quantitative RT–PCR and the gene copy number by FISH in six breast and five prostate cancer cell lines. Breast cancer cell line, SK-Br-3, which contained the highest copy number of all three genes, showed overexpression of only EIF3S3. Finally, the expression levels of TRPS1, EIF3S3 and MYC were measured in freshly frozen clinical samples of benign prostate hyperplasia (BPH), as well as untreated and hormone-refractory prostate carcinoma. The TRPS1 and MYC expression levels were similar in all prostate tumour groups, whereas EIF3S3 expression was higher (P=0.029) in prostate carcinomas compared to BPH. The data suggest that the expression of EIF3S3 is increased in prostate cancer, and that one of the mechanisms underlying the overexpression is the amplification of the gene.

Identification of novel regions of amplification and deletion within mantle cell lymphoma DNA by comparative genomic hybridization.

We have carried out comparative genomic hybridization (CGH) analysis on archival biopsy material from a series of 30 UK mantle cell lymphomas. The most frequent aberrations were gains of 3q (21 cases), 6p (19 cases), 7q (8 cases), 12p (8 cases), 12q (9 cases) and 17q11q21 (8 cases), and losses of 1p13p32 (10 cases), 5p13p15.3 (9 cases), 6q14q27 (11 cases), 8p (7 cases), 11q13q23 (8 cases) and 13q (18 cases). Nineteen cases (63%) had a common region of amplification at 3q28q29, which was highly amplified in three cases, suggesting the presence of a mantle cell lymphoma (MCL)-related oncogene in this region. There was a minimal common region of deletion at 6q25q26 in nine cases (30%). No MCL-specific locus has previously been identified on chromosome 6 and this region may contain a tumour suppressor gene specifically implicated in the development of this subtype of lymphoma. An increased number of chromosome aberrations, gain of Xq and loss of 17p were all significantly associated with a worse prognosis. A greater understanding of the genetics of mantle cell lymphoma may allow the identification of prognostic factors which will aid the identification of appropriate treatment regimens.

Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer.

The biological significance of DNA amplification in cancer is thought to be due to the selection of increased expression of a single or few important genes. However, systematic surveys of the copy number and expression of all genes within an amplified region of the genome have not been performed. Here we have used a combination of molecular, genomic, and microarray technologies to identify target genes for 17q23, a common region of amplification in breast cancers with poor prognosis. Construction of a 4-Mb genomic contig made it possible to define two common regions of amplification in breast cancer cell lines. Analysis of 184 primary breast tumors by fluorescence in situ hybridization on tissue microarrays validated these results with the highest amplification frequency (12.5%) observed for the distal region. Based on GeneMap'99 information, 17 known genes and 26 expressed sequence tags were localized to the contig. Analysis of genomic sequence identified 77 additional transcripts. A comprehensive analysis of expression levels of these transcripts in six breast cancer cell lines was carried out by using complementary DNA microarrays. The expression patterns varied from one cell line to another, and several overexpressed genes were identified. Of these, RPS6KB1, MUL, APPBP2, and TRAP240 as well as one uncharacterized expressed sequence tag were located in the two common amplified regions. In summary, comprehensive analysis of the 17q23 amplicon revealed a limited number of highly expressed genes that may contribute to the more aggressive clinical course observed in breast cancer patients with 17q23-amplified tumors.

Novel amplification unit at chromosome 3q25-q27 in human prostate cancer.

In prostate carcinoma, amplification of the genes c-MYC, Her2/NEU, and the androgen receptor gene has been documented, with gene amplification being related to progressive tumor growth. Recently, using comparative genomic hybridization (CGH), we provided evidence for DNA copy number gains at chromosome 3q25-q26 in prostate cancer [Sattler et al.: Prostate 39:79-86, 1999]. In this study, additional prostatic tumors were evaluated by CGH to determine the frequency of DNA overrepresentation at 3q. Comparative PCR and Southern blot analyses were applied to determine whether known genes are involved in DNA copy number gains. By CGH, DNA copy number gains, all of which involved chromosome region 3q25-q26, were disclosed in 50% of the prostate tumors analyzed. There was no evidence for high-level amplification. The analysis of 12 genes from 3q25-q27 by comparative PCR revealed amplification in 6 (35.3%) of 17 tumors tested. Amplification was detected for the genes IL12A, MDS1, SLC2A2, and SOX2, with coamplification of three genes in two tumors. IL12A was amplified as single gene in three tumors and in a subline of the DU145 cell line, SLC2A2 in one tumor. Our studies revealed a novel amplification unit at 3q25-q27 in prostate carcinoma, with the genes IL12A, MDS1, SLC2A2, and SOX2 being located within the amplification unit. A common region of amplification was evident spanning the IL12A gene locus at 3q25-q26.2. Possibly, IL12A indicates an adjacent, till now unidentified gene which is important in the development of prostate cancer.