11q13 Region Research Articles

Karyotypic evolution in squamate reptiles: comparative gene mapping revealed highly conserved linkage homology between the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Lacertilia) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae, Serpentes)

The butterfly lizard (Leiolepis reevesii rubritaeniata) has the diploid chromosome number of 2n = 36, comprising two distinctive components, macrochromosomes and microchromosomes. To clarify the conserved linkage homology between lizard and snake chromosomes and to delineate the process of karyotypic evolution in Squamata, we constructed a cytogenetic map of L. reevesii rubritaeniata with 54 functional genes and compared it with that of the Japanese four-striped rat snake (E. quadrivirgata, 2n = 36). Six pairs of the lizard macrochromosomes were homologous to eight pairs of the snake macrochromosomes. The lizard chromosomes 1, 2, 4, and 6 corresponded to the snake chromosomes 1, 2, 3, and Z, respectively. LRE3p and LRE3q showed the homology with EQU5 and EQU4, respectively, and LRE5p and LRE5q corresponded to EQU7 and EQU6, respectively. These results suggest that the genetic linkages have been highly conserved between the two species and that their karyotypic difference might be caused by the telomere-to-telomere fusion events followed by inactivation of one of two centromeres on the derived dicentric chromosomes in the lineage of L. reevesii rubritaeniata or the centric fission events of the bi-armed macrochromosomes and subsequent centromere repositioning in the lineage of E. quadrivirgata. The homology with L. reevesii rubritaeniata microchromosomes were also identified in the distal regions of EQU1p and 1q, indicating the occurrence of telomere-to-telomere fusions of microchromosomes to the p and q arms of EQU1.

Newly acquired chromosome Abnormalities During Course of CLL: a Retrospective Collection Data From 2 Spanish Centers.

Abstract 4384 BackgroundDifferent recurrent cytogenetic abnormalities have shown clear prognostic value in patients with Chronic Lymphocytic Leukemia (CLL). Acquisition of cytogenetic aberrations during evolution of the disease (i.e. clonal evolution) may have clinical implications. Objective of the studyTo analyze the frequency of clonal evolution and its potential clinical consequences. Methods and MaterialsWe retrospectively collect data from 80 patients diagnosed of CLL, who had been made at least two Fluorescence In Situ Hybridation (FISH) studies, with probes for gene ATM (11q22), gene p53 (17p13), 13q14 region and centromeric probe for chromosome 12. Clonal evolution was found in 16 out of 80 cases (20%), 8 male and 8 women, median age 70 years (41-80). Initial FISH was normal in 9 cases, deletion (del) of 13q14 region was observed in 5, and trisomy of chromosome 12 (+12) in 2 cases. Median of previous lines of treatment were 1 (0-6): Chlorambucil 7, Fludarabine (F) 1, Cyclophosphamide (C) 1, FC combination 3, FC plus Rituximab (FCR) 2, Alemtuzumab 3, CVP 2, CHOP 1, autologous stem cell transplantation (SCT) 1, Bendamustine 4. By flow cytometry, 5/9 analyzed cases were CD38-positive and 3/6 were ZAP70-positive. Out of 13 treated patients, complete response (CR) criteria (by 2008 iwCLL guidelines) were reached in 2, and PR in 8. Median duration of response (DR) were 14 months (0-42), and median time to retreatment (TTR) were 18 months (1-60). ResultsAs clonal evolution, FISH abnormalities detected were: del (p53) in 8 cases, del (ATM) in 7 cases (2 associated with p53 deletion); del(13q) in 3 (homozigous in 2 patients). Treatment were given to 12 patients (75%), indicated in 10 for progressive adenopathies (6/7 cases with del(ATM) y 7/8 with del(p53)), 5 for anemia and 4 for thrombocytopenia (all of them had del(p53)), 3 for short LDT and 3 for B symptoms. Rescue regimens were: FC (4), FR (1), FCR (1), Pentostatin+R (1), Alemtuzumab (1), Cyclophosfamide (2), CHOP+R (1), allogeneic SCT (1). 7/12 responded, including 1 case that reached CR. Median DR was 5 months (0-29), with TTR 9 months (1-41). As following lines they had: Alemtuzumab +/- Methylprednisolone (3), Chlorambucil (1), CVP (1), CHOP (1), Rituximab (1), FR (1), Bendamustine (1). After a median follow-up of 9 months (3-60) from clonal evolution, 5/16 had died, after 3-32 months (median 9). 4 of them died because of disease progression and/or infectious complications. 1 patient had a second malignancy, dying from metastatic prostate cancer. ConclusionsThis retrospective series shows the relevance of clonal evolution in CLL. Selection of clones with deletion of ATM or p53 genes frequently associates with adenopathy progression, while p53 deletion seems to correlate more specifically with cytopenias. Treatment-free and overall survival seem to get compromised. Usefulness of performing FISH at regular intervals might warrant further studies. Disclosures:No relevant conflicts of interest to declare.

High-Resolution SNP 6.0 Array Profiling Discloses Novel Genomic Aberrations in Polycythemia Vera and Essential Thrombocythemia.

Abstract 1898Poster Board I-921 Introduction:JAK2V617F mutation is detected in more than 90% of cases of polycythemia vera (PV) and in about 50% of cases of essential thrombocythemia (ET). Recently, JAK2-exon 12 and MPL mutations have been reported in myeloproliferative neoplasms (MPN). All these three mutations have a disease causing potential. There are still 50% of ET and PMF patients negative for JAK2V617F mutation. Thus, further genetic analysis to identify novel disease-related aberrations in MPN is required. Methods:We performed whole genome analysis on granulocytic DNA of 45 MPN patients (19 PV and 26 ET) using the single nucleotide polymorphism (SNP) Array 6.0 platform [Affymetrix 6.0]. Genotypes were analyzed using Genotyping Console 3.0.2. Data were normalized against a commercial and an own set of reference samples. All patients had JAK2V617F screening performed. Clinical and analytical data and results of cytogenetics study performed at diagnosis of MPN were colected from clinical reports. Results:From 45 MPN patients, 41 had normal cytogenetics at diagnosis; there were no data concerning cytogenetics study from the rest 4 patients. Using SNP-A 6.0 platform, we detected aberrations (gain or loss of the molecular material) in the following regions: 1q12, 9p1, 17q21, 4q, 3q26 and 8p12. Aberrations in a region 1q12 (n=14) were presented in 8 of PV (gain in 3 and loss in 5) and in 6 of ET (gain in 2 and loss in 4) patients. Aberrations in a region 9p1 (n=27) were observed in 11 of PV (gain in 6 and loss in 5) and in 16 of ET (gain in 10 and loss in 6) patients. Aberrations in a region 17q21 (n=26) were presented in 11 of PV (gain in 3 and loss in 8) and in 15 of ET (gain in 9 and loss in 6) patients. Aberrations in the region 4q (n=20) were detected in 8 of PV (gain in 5 and loss in 3) and in 12 of ET (gain in 9 and loss in 3) patients. Interestingly, only the gain of the molecular material was detected in a region 3q26 (n=12; 5 PV and 7 ET patients). In case of aberration in a region 8p12 (n=20), all 12 ET patients presented gain of the molecular material, whereas PV patients had gain (n=3) or loss (n=3) of the molecular material. There were no relation between the presence of these aberrations and the status of the JAK2V617F mutation, analytical data or clinical outcome of the patients. Conclusions:1. As we know, patients with ET have low frequency of cytogenetics aberrations. Nevertheless, using SNP-A 6.0 platform [Affymetrix 6.0], it is possible to detect new genomic aberrations in these group of patients. 2. According to our results, gain in 8p12 region is especially related to ET patients. Recently has been reported that, in a 8p12 region there is localized gen INDOL1 that, may be involved in the inhibition of immune response to tumours. 3. Gain in a 3q26 region can be related with both PV and ET. In this region, there are localized two microRNA: hsa-mir-1263 and has-mir-720. 4. SNP-A 6.0 technology should not replace conventional cytogenetics in study of MPN patients. However, SNP-A 6.0 platform, as a high resolution assay, can be useful in identification of new genomic abnormalities that may be relevant for pathogenesis of MPN. Disclosures:No relevant conflicts of interest to declare.

A High Resolution Comparative Genomic Hybridization Array of Adult T-Cell Leukemia-Lymphoma in Individuals of African Descent.

Abstract 4241Adult T-cell leukaemia/lymphoma (ATLL) is a highly aggressive malignancy with a poor prognosis caused by the human T-cell lymphotropic virus type-1 (HTLV-I). While no specific chromosome or genetic abnormalities have been clearly proven to contribute to the pathogenesis of ATLL, recent comparative genomic hybridization (CGH) studies have demonstrated frequent genetic lesions (gains and losses) involving specific chromosomal regions containing a number of genes of potential cancer relevance. In this study, using a high resolution Agilent platform, we performed a comprehensive CGH analysis of 60 ATLL DNA specimens obtained from individuals of African descent from the United States, the Caribbean, and Brazil. Our preliminary results demonstrate the presence of some alterations already reported by others, such as gains in chromosomes 1q22, 4p16, 7p22, 8q24, 9q33-34, 14q31-q32, 16q16, 18q23, 19p13 and deletions at 6q14, 9p21 and q21 and 14q11. Additionally, we have observed several genetic alterations not previously reported in a significant percentage of our ATLL tumors, such as specific gains in regions of chromosomes 1q, 2q, 5p, and 17p and q, and deletions in 13q and 15q. Most of these regions harbor potential cancer related genes but some contain genes of unknown function. Our preliminary results confirm there are common genetic alterations between ATLL patients of Japanese and African origin, but also distinct genetic lesions between both ethnic groups perhaps contributing to the pathogenesis of ATLL. A more comprehensive analysis including validated findings will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

Gene Dosage Effects in B-Chronic Lymphocytic Leukemia with Del 13q14.

Abstract 2640Poster Board II-616 Introduction:Deletion of 13q14 (del13q14) is the most common abnormality of B-chronic lymphocytic leukemia (CLL). However, for a long time investigations of this region did not detect the relevant pathogenetic mechanism. Micro-RNA genes MIRN15a and MIRN16-1 located on 13q14.3 were postulated to close this gap. Down-regulated expression of these miRNAs was shown to increase the anti apoptotic B cell lymphoma 2 (Bcl2) proteins. However, relevance and frequency of deregulated micro-RNA genes was differentially described. To understand the influence of deletion of chromosomal region 13q14 on gene expression, chromosomal abnormalities detected by single nucleotide polymorphism (SNP) chips were compared with gene expression analyzed by gene expression profiling in CLL. Furthermore, impact of deletion 13q14 on MIRN15A and MIRN16-1 expression and correlation to BCL2 protein expression were investigated. Methods:15 B-CLL cases harboring del 13q14 were investigated for the extend of deletion with the 50k Xbal SNP array. Gene expression of the genes located in the aberrant region evaluated by Affymetrix U133A gene chip of 13 of these cases was compared between cases with and without this aberration. FISH analysis was done to validate SNP-data. Expression of MIRN15a and MIRN16-1 was evaluated by real-time PCR from further 20 B-CLL with TaqMan MicroRNA assays. From the 25 cases Western blot analysis for Bcl2 was performed. Results:SNP-chip and FISH analysis with probes for regions RB1, D13S25 and D13S319 gave consistent results. The minimal deleted region of del13q14 reaches from physical position 49543165 to 50272626 and mostly includes the location of MIRN15a and 16-1. 10 gene probes were located in the aberrant chromosomal 13q14 region and entered statistical analysis. In 4 of 5 genes with monoallelic deletions gene expression was significantly reduced. In regions harbouring mono- and biallelic deletions in 4 of 5 genes the monoallelic deleted cases showed decreased expression compared to the normal cases. Evaluation of MIRN15a and MIRN16-1 revealed strong down-regulation of expression in CLL harbouring biallelic del13q14 compared to other CLL and healthy B-cells (p=0.002; p=0.002), whereas there were no statistically significant differences between CLL with or without monoallelic deletion or healthy B-cells (p=0.534; p=0.665). Bcl2 Western blot analysis revealed stronger Bcl2 expression in CLL harbouring del13q14 in contrast to CLL without this abnormality (p=0.002). However, no difference between cases with mono- or biallelic del13q14 was detected (p=0.400). Conclusion:SNP chip analysis is a helpful tool to detect chromosomal abnormalities on a high resolution. Although detailed genetic analyses failed to demonstrate the consistent involvement of any of the genes located in the deleted region of B-CLL harboring del13q14, reduced expression occurred in most of these genes indicating gene dosage effects. MIRN15a and MIRN16-1 expression are frequently reduced in B-CLL with biallelic but not monoallelic deletion of 13q14 without an influence on Bcl2 protein levels. Disclosures:Off Label Use: intrapleural and intraperitoneal use of rituximab.

13q Deletion Size Predicts Disease Progression and Response to Treatment in Patients with Chronic Lymphocytic Leukaemia.

Abstract 671Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease characterised by a variable clinical course. Chromosomal abnormalities involving 13q14 are the most common genetic alterations in CLL, occurring in ∼60% of cases and when identified as a sole abnormality, are associated with a favourable clinical outcome. A minimally deleted region (MDR), found in most cases, encompasses DLEU2, DLEU1, miR15a and miR16-1. However, recent SNP profiling data showed that 13q deletions extends beyond the MDR and exhibit considerable heterogeneity both in size and location (Ouillette et al, Cancer Res 2008 68(4);1012). Furthermore, this study showed that larger genomic deletions were associated with a higher Rai stage and were enriched in treated patients. To explore this association further, we performed an initial analysis on samples taken at diagnosis from 100 Stage A patients, of whom 50% had stable disease for over 10 years, and on pre-treatment samples from 100 patients enrolled on the LRF CLL4 trial who had either a complete or partial response to fludarabine and cyclophosphamide [n=200] using the Affymetrix SNP6.0 microarray platform. Deletions of 13q were identified in 99/200 patients (49.5%), the majority (94%) of which included a mono- or biallelic deletion of the MDR. The smallest deletion in this series was 130Kb, while the largest was an 82Mb terminal deletion (mean deletion size 7.25Mb). Furthermore, we identified 12 cases with acquired copy number neutral LOH affecting 13q, exclusive in the presence of a bi-allelic deletion of the MDR. In agreement with the previous study we demonstrated that the size and genomic location of the deletions were highly heterogeneous. Common deletion breakpoints within a 1.9Mb region on 13q between genomic locations 48.7 and 50.6Mb defined 42 cases with small deletions. The remaining 57 cases (58%) showed deletions extending further towards the telomere or centromere and defined those cases with a large deletion. The presence of a large 13q deletion at diagnosis was associated with disease progression (p<0.03) and in cases with unmutated VH genes, a median treatment free survival of 3 months compared to 18 months for cases with small deletions (p<0.003). In the CLL4 trial samples, large deletions were associated with partial rather than complete response to treatment (p<0.04). Interestingly, the deletion sub-types defined by Ouillette et al were not associated with disease progression or response to treatment in our series. To extend this observation we designed a series of custom 13q oligonucleotides for multiplex ligation-dependant probe amplification (MLPA) and are in the process of screening an unselected cohort of 500 CLL patients. Of the 50 patients currently screened with MLPA, five cases analyzed with both the SNP6.0 platform and MLPA showed 100% concordance. The remaining 45 cases had a 13q deletion identified by FISH and confirmed the association between 13q deletion size and clinical outcome (Figure, p<0.03). Furthermore, this analysis shows that this association is independent of VH gene mutational status and the presence of an 11q or 17p deletion. These findings suggest that whilst 13q abnormalities are generally associated with a favourable outcome, aberrant expression of gene(s) on 13q outside of the MDR adversely affect outcome. We are currently refining the 13q region associated with adverse outcome to identify causative gene loci. In conclusion, 13q deletion size represents a new biomarker for predicting outcome of CLL, whose target gene(s) could provide new therapeutic strategies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Identification of susceptibility loci at 7q31 and 9p13 for bipolar disorder in an isolated population

We performed a linkage analysis on 23 Finnish families with bipolar disorder and originating from the North-Eastern region of Finland, using the Illumina Linkage Panel IV (6K) Array with an average intermarker spacing of 0.65 cM across the genome. We detected genome-wide significant evidence for linkage of mood disorder (bipolar disorder type I, II, or not otherwise specified, manic type of schizoaffective psychosis, cyclothymia, or recurrent depression) to chromosomes 7q31 (LOD = 3.20) and 9p13.1 (LOD = 4.02). Analyzing the best markers on the complete set of 179 Finnish bipolar families supported the findings on chromosome 9p13 (maximum LOD score of 3.02 at position 383 Mb, immediately upstream of the centromere). This region harbors several interesting candidate genes, including contactin associated protein-like 3 (CNTNAP3) and aldehyde dehydrogenase 1 (ALDH1B1). For the 7q31 locus, only one extended pedigree and ten families originating from the same late settlement region in North-Eastern Finland provided evidence for linkage, suggesting that a gene predisposing to bipolar disorder is enriched in that region. Candidate genes of interest in this locus include potassium-voltage-gated channel, member 2 (KCND2) and calcium-dependent activator protein for secretion 2 (CADPS2). The loci on the centromeric region of 9p13 and the telomeric region of 7q31 may represent susceptibility loci for mood disorder in the Finnish population.

Detection of chromosomal abnormalities associated with chronic lymphocytic leukemia: what is the best method?

B-cell chronic lymphocytic leukemia (CLL) follows a heterogeneous clinical course, for which several biological markers may predict clinical outcome. Cytogenetic aberrations are considered major prognostic indicators for predicting the survival of CLL patients. Given the difficulties in obtaining abnormal metaphases in CLL, fluorescent in situ hybridization (FISH) with specific probes is generally used to detect the most frequent abnormalities. To determine the best strategy for identifying cytogenetic abnormalities, we compared results obtained by FISH analysis on peripheral blood mononuclear cells with those obtained by FISH after culture with mitogens. We studied 46 CLL patients selected from two different institutions. The most frequent structural aberrations leading to loss of genetic material were loss of the 13q14 region, in 32 cases (70%), and loss of TP53 in 11 cases (24%). Of the 46 cases patients, 10 patients (21.7%) had deletion of the ATM locus at 11q22 and 8 patients (17%) had trisomy 12. Results could be interpreted as discordant in four cases in which the abnormal clone was small and both values were around the threshold. FISH performed on stimulated cells detected the same frequency of abnormalities as FISH performed without culture. This equivalence between the two techniques allows performing both conventional cytogenetic and FISH analyses on the same sample.

Association and interaction analyses of eight genes under asthma linkage peaks

Linkage studies have implicated the 2q33, 9p21, 11q13 and 20q13 regions in the regulation of allergic disease. The aim of this study was to test genetic variants in candidate genes from these regions for association with specific asthma traits. Ninety-five single nucleotide polymorphisms (SNP) located in eight genes (CD28, CTLA4, ICOS, ADAM23, ADAMTSL1, MS4A2, CDH26 and HRH3) were genotyped in >5000 individuals from Australian (n = 1162), Dutch (n = 99) and Danish (n = 303) families. Traits tested included doctor-diagnosed asthma, atopy, airway obstruction, total serum immunoglobulin (Ig) E levels and eosinophilia. Association was tested using both multivariate and univariate methods, with gene-wide thresholds for significance determined through simulation. Gene-by-gene and gene-by-environment analyses were also performed. There was no overall evidence for association with seven of the eight genes tested when considering all genetic variation assayed in each gene. The exception was MS4A2 on chromosome 11q13, which showed weak evidence for association with IgE (gene-wide P < 0.05, rs502581). There were no significant gene-by-gene or gene-by-environment interaction effects after accounting for the number of tests performed. The individual variants genotyped in the 2q33, 9p21 and 20q13 regions do not explain a large fraction of the variation in the quantitative traits tested or have a major impact on asthma or atopy risk. Our results are consistent with a weak effect of MS4A2 polymorphisms on the variation of total IgE levels.

Characterization of candidate gene copy number alterations in the 11q13 region along with BRAF and NRAS mutations in human melanoma

Amplification of the 11q13 chromosomal region is a common event in primary melanomas. Several candidate genes are localized at this sequence; however, their role in melanoma has not been clearly defined. The aim of this study was to develop an accurate method for determining the amplification pattern of six candidate genes that map to this amplicon core and to elucidate the possible relationship between BRAF, NRAS mutations and CCND1 copy number alterations, all of which are key components of the MAP kinase pathway. Characterization of gene copy numbers was performed by quantitative PCR and, as an alternative method, fluorescence in situ hybridization was used to define the CCND1 amplification pattern at the single cell level. Samples with amplified CCND1 (32%) were further analyzed for copy number alterations for the TAOS1, FGF3, FGF19, FGF4 and EMS1 genes. Coamplification of the CCND1 and TAOS1 was present in 15% of tumors and was more frequent in ulcerated lesions (P=0.017). Furthermore, 56% of primary melanomas had either BRAF or NRAS mutations, but these two mutations were not present in any of the lesions analyzed. Of these cases, 34% also had CCND1 amplification. There was a significant relationship between NRAS activating mutations and UV exposure (P=0.005). We did not find correlations between CCND1 gene amplification status and any of the patients' clinicopathological parameters. However, CCND1 amplification simultaneously with either BRAF or NRAS activation mutations was observed mainly in primary tumors with ulcerated surfaces (P=0.028). We assume that coamplification of these candidate genes in the 11q13 region or CCND1 gene alterations along with either BRAF or NRAS mutations might be more important for prognosis than the presence of these alterations alone.

Microsatellite Instability Is a Common Finding in Multiple Myeloma

PurposeMicrosatellite instability (MSI) occurs as a result of sliding in the DNA sequences from shortening or elongation of the repeat zones of DNA during replication. Such abnormalities can normally be corrected by the enzymes coded by the DNA mismatch repair (MMR) genes. Therefore, detection of MSI is considered to be a sign of disorder of the MMR genes and is interpreted as a replication error phenotype. Patients and MethodsWe evaluated the MSI in 5 different loci in the 14q32 region of immunoglobulin heavy chain IgH gene in 26 newly diagnosed patients with multiple myeloma (MM). ResultsFifty-four percent of the patients disclosed MSI and at least 1 locus but no significant association of MSI was found between different clinical stages and the MM subtype. MSI was not found in 5 light-chain myeloma patients. ConclusionAlthough our case number is small, probably the genomic instability in heavy-chain MM may be a common finding and probably plays a critical role in the MM pathogenesis.

Correlation between losses ofIGHor its segments and deletions of 13q14 in t(11;14) (q13;q32) multiple myeloma

Multiple myeloma (MM) is a malignancy of the plasma cells (PCs) characterized by a wide variety of genetic and chromosomal abnormalities. In recent years, major attention was drawn to the significance of chromosomal aberrations involving chromosome arm 13q and the IGH region on chromosome band 14q32 as a prognostic indicator in MM. In this study we applied a combined cell morphology and FISH method for the analysis of coexistence of t(11;14)(q13;q32) with deletions of the long arm of chromosome 13 (Delta13) in PCs from 51 MM patients using several probes for the 13q14, 11q13, and IGH regions. We found 15 different variants of the t(11;14) that are the consequence of different 11q13 breakpoints and various deletions of Variable (del IGH Var) or Constant (del IGH Const) IGH segments and also duplications and losses of the IGH gene on the normal nontranslocated chromosome 14 as well as IGH/Cyclin D1 (CCND1) fusion on der(14) and CCND1/IGH fusions on der(11). A strong association between Delta13 and specific variants of t(11;14) was found: variants with deletion of the IGH gene or its segments were found only in MM cases with deleted chromosome 13, while the common translocation t(11;14) was found only in the MM cases with normal chromosome arm 13q. In contrast, we did not find any association between Delta13 and deletions of the IGH gene or its segments in the MM patients with t(4;14)(p16;q32).

Common genetic changes in leiomyosarcoma and gastrointestinal stromal tumour: implication for ataxia telangiectasia mutated involvement

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract. Formerly GISTs were commonly classified histologically as leiomyosarcomas; however, they are now known to arise from the interstitial cells of Cajal. Majority of GISTs overexpress KIT and have characteristic mutations within the gene, which are the targets of drug treatment with tyrosine kinase inhibitors. Leiomyosarcoma is a malignant tumour of smooth muscle differentiation and falls into a group of sarcomas that show complex karyotypic changes with no consistent recurrent genetic abnormality. We have used comparative genomic hybridization in combination with fluorescence in situ hybridization to determine genetic differences between the tumour types. We found leiomyosarcomas and GISTs share common regions of chromosomal 13q and 11q imbalance, in addition to more specific 1p and 8p losses in leiomyosarcoma and 15q and 22q losses in GISTs. More importantly, we have shown for the first time a deletion in the ataxia telangiectasia mutated (ATM) gene locus with decreased/absent expression of ATM protein, and amplification in the region 13q21-q32 in both tumour types, suggesting both regions may play a role in leiomyosarcoma and GIST biology.

9300 Expression alterations of genes located on the 7q31 region in human malignant melanomas

9300 Expression alterations of genes located on the 7q31 region in human malignant melanomas

Searching for Genes for Cleft Lip and/or Palate Based on Breakpoint Analysis of a Balanced Translocation t(9;17)(q32;q12)

Identification of the breakpoints of disease-associated chromosome rearrangements can provide informative clues to a positional cloning approach for genes responsible for inherited diseases. Recently, we found a three-generation Japanese family segregating balanced chromosome translocation t(9;17)(q32;q12). One of the subjects had cleft lip and palate. We examined whether regions near the breakpoint could be associated with cleft lip and/or palate. We determined the breakpoints involved in the translocation by fluorescence in situ hybridization analysis and subsequent long-range polymerase chain reaction. In order to study the role of these disrupted regions in nonsyndromic cleft lip and/or palate, we performed mutation analysis and a haplotype-based transmission disequilibrium test using tagging single-nucleotide polymorphisms in the flanking regions of the breakpoints in white and Filipino nonsyndromic cleft lip and/or palate populations. Sequence analysis demonstrated that two genes, SLC31A1 (solute carrier family 31 member 1) on chromosome 9 and CCL2 (chemokine ligand 2) on chromosome 17, were rearranged with the breaks occurring within their introns. It is interesting that SLC31A1 lies closed to BSPRY (B-box and SPRY domain), which is a candidate for involvement with cleft lip and/or palate. Some of the variants in BSPRY and CCL2 showed significant p values in the cleft lip and/or palate population compared with the control population. There was also statistically significant evidence of transmission distortion for haplotypes on both chromosomes 9 and 17. The data support previous reports that genes on chromosomal regions of 9q and 17q play an important role in facial development.

Application of fluorescence in-situ hybridization technique in multiple myeloma

To investigate the common chromosome abnormalities of the patients with multiple myeloma in China and the relationships of cytogenetic abnormalities and clinical features. In interphase fluorescence in-situ hybridization (FISH) analysis, a panel of probes including D13S319 (13q14.3), RB1(RB1 gene), IgH (14q32), P53(17p13), 1q21(1q21 gene) was used to study the cytogenetic abnormalities of 31 patients with multiple myeloma; and the clinical implications of cytogenetic abnormalities were investigated. The frequencies of the partial deletion of chromosome 13, translocation involving the 14q32 region, abnormalities in 1q21 and deletion of 17p13 were 45%, 68%, 50%, and 35% in the study, respectively. The abnormalities of both the partial deletion of chromosome 13 and translocation involving the 14q32 region were found in 35% of the patients. 79% of the patients with del (13q) had 14q32 translocations simultaneously. All the patients with positive detection of probe D13S319 were found to have translocation of 14q32 at the same time. There were correlations between the partial deletion of chromosome 13 and translocation involving the 14q32 region. The overall response rate of induction treatment was 67.7%. No significant difference was found in patients with positive or negative cytogenetic abnormalities of del(13q), 14q32 translocation, del(17p13), and 1q21 abnormalities. 13q deletion, IgH rearrangement, chromosome 1 abnormality and 17p13 deletion are the common cytogenetic abnormalities of MM patients in China. There is a significant correlation between the presence of 14q32 translocations and chromosome 13 deletion in MM patients.

Predictive value of gain of 3q for cervical dysplasia and potential utility in the clinical management of LSIL

5583 Background: Physicians need better tools for determining whether an LSIL will progress to HSIL or regress. Research demonstrates that the chromosome 3q26 region is found to be amplified in patients with cervical cancer and that the frequency of this 3q gain increases with dysplasia severity. The objective of this study was to demonstrate that 3q can be used as a risk stratification tool to identify patients likely to regress/progress. Methods: Slide-based, archival thin-layer specimens and longitudinal follow-up data for 52 patients (47 usable cases) originally diagnosed as LSIL were obtained from independent cytopathology laboratories. Slides were destained, then hybridized with a single-copy probe for the chromosome 3q26 region and a probe for the centromeric alpha-repeat sequence of chromosome 7, using standard FISH methods. Results: Of the 47 cases analyzed, 30 showed a negative FISH test result for a sensitivity of 92% (95% CI of 64, 100), specificity of 85% (95% CI of 69, 95) and a negative predictive value of 97% (95% CI of 83, 100). In a subset of cases a biopsy was obtained. Of the 32 cases with tissue diagnosis, 11 women had progressed to CIN2+, of which 10 were FISH positive; and 18 of 21 women with tissue diagnosis of ≤CIN1 were FISH negative, for a sensitivity of 91% (95% CI of 59, 100), specificity of 86% (95% CI of 64, 97) and a negative predictive value of 95% (95% CI of 74, 100). If the analysis is confined to the 36 cases for which follow-up of at least 12 months after the index LSIL was available, 26 showed a negative FISH test result for a sensitivity of 100% (95% CI of 48, 100), specificity of 84% (95% CI of 66, 95) and a negative predictive value of 100% (95% CI of 87, 100). Of the 22 cases with tissue diagnosis, 4 women had progressed to CIN2+, all FISH positive; and 16 of 18 women with ≤CIN1 were FISH negative, for a sensitivity of 100% (95% CI of 40, 100), specificity of 89% (95% CI of 65, 99) and a negative predictive value of 100% (95% CI of 79, 100). Conclusions: Analysis of 47 specimens demonstrated the ability of 3q gain to predict disease progression/regression in cytology samples. 3q gain can serve as a risk stratification tool for LSIL cases. [Table: see text]

SNP genotyping of a sclerosing rhabdomyosarcoma: reveals highly aneuploid profile and a specific MDM2/ HMGA2 amplification

Since the first description of sclerosing rhabdomyosarcoma in 2000, 19 pediatric cases have been reported in the literature. However, it is debated whether sclerosing rhabdomyosarcoma represents a specific rhabdomyosarcoma entity or a variant of embryonal or alveolar rhabdomyosarcoma. To date, 6 sclerosing rhabdomyosarcoma karyotypes and 1 sclerosing rhabdomyosarcoma comparative genomic hybridization profile have been reported. We present the first whole-genome tumoral genotyping of a sclerosing rhabdomyosarcoma by high-density single nucleotide polymorphism array. The single nucleotide polymorphism genotyping revealed a complex pattern including gains and losses of whole chromosomes and an amplification of the 12q13-15 region. Amplification of the 12q13-q15 region containing SAS, GLI, CDK4, and MDM2 has been observed in rhabdomyosarcoma. In the present case, the 2 amplified target genes were MDM2 and HMGA2, excluding CDK4. The identification of a specific MDM2-HGMA2 amplicon excluding CDK4 has only been described so far in well-differentiated and dedifferentiated liposarcoma. Further studies are needed to assess if this anomaly is a specific marker of sclerosing rhabdomyosarcoma.

The ring 14 syndrome: Clinical and molecular definition

The ring 14 (r14) syndrome is a rare condition, whose precise clinical and genetic characterization is still lacking. We analyzed a total of 20 patients with r14 and another 9 patients with a linear 14q deletion. The ring was complete, with no apparent loss of chromosome material, in 6 cases; a terminal 14q deletion, varying in size from 0.65 to 5 Mb, was detected in the remaining 14 cases. Deleted ring chromosomes were 70% paternal and 30% maternal. UPD (14) was never detected. With respect to the linear 14q deletions, three were proximal, varying in size from 4 to 7.2 Mb, and six distal, varying in size from 4.8 to 20 Mb. The majority of the linear deletions were also of paternal origin, and UPD (14) was excluded in all cases. Clinically, the r14 syndrome was characterized by a recognizable phenotype, consisting of shortness of stature, a distinctive facial appearance, microcephaly, scoliosis, and ocular abnormalities, which included abnormal retinal pigmentation, strabismus, glaucoma, and abnormal macula. All patients except one had mental retardation. Drug-resistant epilepsy was another highly consistent finding. Aggressive and hyperactive behavior was noted in about half of the patients. Based on genotype-phenotype correlations, we could deduce that retinal abnormalities, epilepsy, microcephaly, and mental retardation map within the proximal 14q11.2-q12 region. Likewise, behavior disorders and scoliosis could be assigned to the 14q32 region.

Similar gene expression profiles of sporadic, PGL2-, and SDHD-linked paragangliomas suggest a common pathway to tumorigenesis

BackgroundParagangliomas of the head and neck are highly vascular and usually clinically benign tumors arising in the paraganglia of the autonomic nervous system. A significant number of cases (10–50%) are proven to be familial. Multiple genes encoding subunits of the mitochondrial succinate-dehydrogenase (SDH) complex are associated with hereditary paraganglioma: SDHB, SDHC and SDHD. Furthermore, a hereditary paraganglioma family has been identified with linkage to the PGL2 locus on 11q13. No SDH genes are known to be located in the 11q13 region, and the exact gene defect has not yet been identified in this family.MethodsWe have performed a RNA expression microarray study in sporadic, SDHD- and PGL2-linked head and neck paragangliomas in order to identify potential differences in gene expression leading to tumorigenesis in these genetically defined paraganglioma subgroups. We have focused our analysis on pathways and functional gene-groups that are known to be associated with SDH function and paraganglioma tumorigenesis, i.e. metabolism, hypoxia, and angiogenesis related pathways. We also evaluated gene clusters of interest on chromosome 11 (i.e. the PGL2 locus on 11q13 and the imprinted region 11p15).ResultsWe found remarkable similarity in overall gene expression profiles of SDHD -linked, PGL2-linked and sporadic paraganglioma. The supervised analysis on pathways implicated in PGL tumor formation also did not reveal significant differences in gene expression between these paraganglioma subgroups. Moreover, we were not able to detect differences in gene-expression of chromosome 11 regions of interest (i.e. 11q23, 11q13, 11p15).ConclusionThe similarity in gene-expression profiles suggests that PGL2, like SDHD, is involved in the functionality of the SDH complex, and that tumor formation in these subgroups involves the same pathways as in SDH linked paragangliomas. We were not able to clarify the exact identity of PGL2 on 11q13. The lack of differential gene-expression of chromosome 11 genes might indicate that chromosome 11 loss, as demonstrated in SDHD-linked paragangliomas, is an important feature in the formation of paragangliomas regardless of their genetic background.