High-resolution mapping identifies a commonly amplified 11q13.3 region containing multiple genes flanked by segmental duplications

DNA amplification of the 11q13 region is observed frequently in many carcinomas. Within the amplified region several candidate oncogenes have been mapped, including cyclin D1, TAOS1 and cortactin. Yet, it is unknown which gene(s) is/are responsible for the selective pressure enabling amplicon formation. This is probably due to the use of low-resolution detection methods. Furthermore, the size and structure of the amplified 11q13 region is complex and consists of multiple amplicon cores that differ between different tumor types. We set out to test whether the borders of the 11q13 amplicon are restricted to regions that enable DNA breakage and subsequent amplification. A high-resolution array of the 11q13 region was generated to study the structure of the 11q13 amplicon and analyzed 29 laryngeal and pharyngeal carcinomas and nine cell lines with 11q13 amplification. We found that boundaries of the commonly amplified region were restricted to four segments. Three boundaries coincided with a syntenic breakpoint. Such regions have been suggested to be putatively fragile. Sequence comparisons revealed that the amplicon was flanked by two large low copy repeats known as segmental duplications. These segmental duplications might be responsible for the typical structure and size of the 11q13 amplicon. We hypothesize that the selection for genes through amplification of the 11q13.3 region is determined by the ability to form DNA breaks within specific regions and, consequently, results in large amplicons containing multiple genes. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Detailed analysis of an amplified region at chromosome 11q13 in malignant tumors.

We have examined the amplification unit at chromosome band 11q13 in 12 primary tumors and one cancer cell line (A431) with 15 DNA markers. The amplified region and size varied from one tumor to another; the smallest amplicon was estimated to be 700 kb long and the largest was 4.5 Mb long at maximum, on the basis of a physical map. Furthermore, the DNA amplified in tumors was not always continuous, because in three cases one locus within the amplicon was not amplified. The amplified region common to all 13 cancers consisted of 500 kb of DNA which incorporated eight defined loci (BCL-1, cCI11-524, cCI11-283, cCI11-234, HBI-1, cCI11-454, HSTF1, and INT2). As two of them, cCI11-524 and cCI11-454, were found to contain DNA sequences conserved in other species, one or both of these loci might encode the gene(s) that may be associated with progression of these tumors.     

Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.     

Integrated cytogenetic and high-resolution array CGH analysis of genomic alterations associated with MYCN amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.     

From amplification to gene in thyroid cancer: a high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization.

Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. We now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. We used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3-6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKCepsilon), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKCepsilon as a previously unmapped candidate gene involved in thyroid tumorigenesis.     

Megabase-scale analysis of the origin of N-myc amplicons in human neuroblastomas.

In order to elucidate the initiation of the N-myc gene amplification, we have analyzed the original structures of the N-myc amplicons among 38 human neuroblastomas. Nineteen DNAs isolated from the N-myc amplicons recognized a continuous stretch totally encompassing a 5.5 megabase region spanning the normal N-myc gene. The co-amplification profiles with these DNAs showed that two of them, which mapped into a 300 kb region flanking the N-myc gene, were commonly amplified in most specimens, while others were differentially amplified among various subsets. These profiles enabled us to divide the N-myc amplicons into several groups and outline their original domains as a continuous stretches, pointing to the existence of 'consensus sites' for the ends of the initial domains in the original region. In one cell line, the domain was found to be several times larger than that of the derivative amplicon; and the rearranged sites identified within the amplicons, which showed no site specificity, were consistent with those deduced from the domain structure. These results lead to a model in which N-myc gene amplification is initiated at some consensus sites by a preferential mechanism and followed by a random loss of the domain structures during subsequent stages.     

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.     

Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization

We reinvestigated rearrangements occurring in region q13 of chromosome 11 aiming to: (i) describe heterogeneity of the observed structural alterations, (ii) estimate amplicon size and (iii) identify of oncogenes involved in laryngeal cancer progression as potential targets for therapy. The study included 17 cell lines derived from laryngeal cancers and 34 specimens from primary laryngeal tumors. The region 11q13 was analyzed by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and gene expression microarray. Next, quantitative real time PCR was used for chosen genes to confirm results from aCGH and gene expression microarray. The observed pattern of aberrations allows to distinguish three ways, in which gain and amplification involving 11q13 region may occur: formation of a homogeneously staining region; breakpoints in/near 11q13, which lead to the three to sevenfold increase of the copy number of 11q13 region; the presence of additional copies of the whole chromosome 11. The minimal altered region of gain and/or amplification was limited to ~1.8 Mb (chr.11:69,395,184–71,209,568) and comprised mostly 11q13.3 band which contain 12 genes. Five, out of these genes (CCND1, ORAOV1, FADD, PPFIA1, CTTN) had higher expression levels in comparison to healthy controls. Apart from CCND1 gene, which has an established role in pathogenesis of head and neck cancers, CTTN, ORAOV1 and FADD genes appear to be oncogene-candidates in laryngeal cancers, while a function of PPFIA1 requires further studies.     

Pattern of evolution of amplicons during the development of multiple drug resistance in Djungarian hamster cells

Six cloned DNA fragments representing different portions of the genomic region amplified in multidrug resistant Djungarian hamster cells were used to study amplicon variations in a large number of the resistant cell lines. Expressed correlation exists between the degree of 3 cloned sequences amplification and the level of multidrug resistance. Three other cloned regions amplify coordinately with the latter ones at the initial steps of selection. Later their amplification halts and they mao even eliminate from amplicons of highly resistant cells. The rates and order of elimination of these sequences vary among different independently derived series of multidrug resistant cell lines.     

Molecular cytogenetic characterization of pancreas cancer cell lines reveals high complexity chromosomal alterations

Karyotype analysis can provide clues to significant genes involved in the genesis and growth of pancreas cancer. The genome of pancreas cancer is complex, and G-band analysis cannot resolve many of the karyotypic abnormalities seen. We studied the karyotypes of 15 recently established cell lines using molecular cytogenetic tools. Comparative genomic hybridization (CGH) analysis of all 15 lines identified genomic gains of 3q, 8q, 11q, 17q, and chromosome 20 in nine or more cell lines. CGH confirmed frequent loss of chromosome 18, 17p, 6q, and 8p. 14/15 cell lines demonstrated loss of chromosome 18q, either by loss of a copy of chromosome 18 (n = 5), all of 18q (n = 7) or portions of 18q (n = 2). Multicolor FISH (Spectral Karyotyping, or SKY) of 11 lines identified many complex structural chromosomal aberrations. 93 structurally abnormal chromosomes were evaluated, for which SKY added new information to 67. Several potentially site-specific recurrent rearrangements were observed. Chromosome region 18q11.2 was recurrently involved in nine cell lines, including formation of derivative chromosomes 18 from a t(18;22) (three cell lines), t(17;18) (two cell lines), and t(12;18), t(15;18), t(18;20), and ins(6;18) (one cell line each). To further define the breakpoints involved on chromosome 18, YACs from the 18q11.2 region, spanning approximately 8 Mb, were used to perform targeted FISH analyses of these lines. We found significant heterogeneity in the breakpoints despite their G-band similarity, including multiple independent regions of loss proximal to the already identified loss of DPC4 at 18q21.     

Bacterial artificial chromosome library for genome‐wide analysis of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene‐amplified CHO DR1000L‐4N cell line for genome‐wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC‐FISH). Thirteen BAC‐FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR‐deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165‐kb DNA region containing exogenous Dhfr was cloned from the BAC library using high‐density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome. Biotechnol. Bioeng. 2009; 104: 986–994. © 2009 Wiley Periodicals, Inc.     

Construction and application of a full-coverage, high-resolution, human chromosome 8q genomic microarray for comparative genomic hybridization.

BACKGROUND: Array-based comparative genomic hybridization (aCGH) enables genome-wide quantitative delineation of genomic imbalances. A high-resolution contig array was developed specifically for chromosome 8q because this chromosome arm is frequently altered in many human cancers. METHODS: A minimal tiling path contig of 702 8q-specific bacterial artificial chromosome (BAC) clones was generated with a novel computational tool (BAC Contig Assembler). BAC clones were amplified by degenerative oligonucleotide primer (DOP) polymerase chain reaction and subsequently printed onto glass slides. For validation of the array DNA samples of gastroesophageal and prostate cancer cell lines, and chronic myeloid leukemia specimens were used, which were previously characterized by multicolor fluorescence in situ hybridization and conventional CGH. RESULTS: Single and double copy gains were confidently demonstrated with the 8q array. Single copy loss and high-level amplifications were accurately detected and confirmed by bicolor fluorescence in situ hybridization experiments. The 8q array was further tested with paraffin-embedded prostate cancer specimens. In these archival specimens, the copy number changes were confirmed. In fresh and archival samples, additional alterations were disclosed. In comparison with conventional CGH, the resolution of the detected changes was much improved, which was demonstrated by an amplicon of 0.7 Mb and a deletion of 0.6 Mb, both spanned by only six BAC clones. CONCLUSIONS: A comprehensive array is presented, which provides a high-resolution method for mapping copy number alterations on chromosome 8q.     

High Resolution Genome-Wide Analysis of Chromosomal Alterations in Burkitt's Lymphoma

Additional chromosomal abnormalities are currently detected in Burkitt''s lymphoma. They play major roles in the progression of BL and in prognosis. The genes involved remain elusive. A whole-genome oligonucleotide array CGH analysis correlated with karyotype and FISH was performed in a set of 27 Burkitt''s lymphoma-derived cell lines and primary tumors. More than half of the 145 CNAs<2 Mb were mapped to Mendelian CNVs, including GSTT1, glutathione s-transferase and BIRC6, an anti-apoptotic protein, possibly predisposing to some cancers. Somatic cell line-specific CNVs localized to the IG locus were consistently observed with the 244 K aCGH platform. Among 136 CNAs >2 Mb, gains were found in 1q (12/27), 13q (7/27), 7q (6/27), 8q(4/27), 2p (3/27), 11q (2/27) and 15q (2/27). Losses were found in 3p (5/27), 4p (4/27), 4q (4/27), 9p (4/27), 13q (4/27), 6p (3/27), 17p (3/27), 6q (2/27),11pterp13 (2/27) and 14q12q21.3 (2/27). Twenty one minimal critical regions (MCR), (range 0.04–71.36 Mb), were delineated in tumors and cell lines. Three MCRs were localized to 1q. The proximal one was mapped to 1q21.1q25.2 with a 6.3 Mb amplicon (1q21.1q21.3) harboring BCA2 and PIAS3. In the other 2 MCRs, 1q32.1 and 1q44, MDM4 and AKT3 appeared as possible drivers of these gains respectively. The 13q31.3q32.1 <89.58–96.81> MCR contained an amplicon and ABCC4 might be the driver of this amplicon. The 40 Kb 2p16.1 <60.96–61> MCR was the smallest gained MCR and specifically encompassed the REL oncogene which is already implicated in B cell lymphomas. The most frequently deleted MCR was 3p14.1 <60.43–60.53> that removed the fifth exon of FHIT. Further investigations which combined gene expression and functional studies are essential to understand the lymphomagenesis mechanism and for the development of more effective, targeted therapeutic strategies.     

Fine mapping of the constitutional translocation t(11;22)(q23;q11)

Translocation t(11;22)(q23;q11) is the most common constitutional reciprocal translocation in man. Balanced carriers are phenotypically normal, except for decreased fertility, an increased spontaneous abortion rate and a possible predisposition to breast cancer in some families. Here, we report the high resolution mapping of the t(11;22)(q23;q11) breakpoint. We have localised the breakpoint, by using fluorescence in situ hybidisation (FISH) walking, to a region between D11S1340 and WI-8564 on chromosome 11, and D22S134 and D22S264 on chromosome 22. We report the isolation of a bacterial artificial chromosome (BAC) clone spanning the breakpoint in 11q23. We have narrowed down the breakpoint to an 80-kb sequenced region on chromosome 11 and FISH analysis has revealed a variation of the breakpoint position between patients. In 22q11, we have sequenced two BACs (BAC2280L11 and BAC41C4) apparently mapping to the region; these contain low copy repeats (LCRs). Southern blot analysis with probes from BAC2280L11 has revealed different patterns between normal controls and translocation carriers, indicating that sequences similar/identical to these probes flank the translocation breakpoint. The occurrence of LCRs has previously been associated with genomic instability and "unclonable" regions. Hence, the presence of such repeats renders standard translocation breakpoint cloning techniques ineffective. Thus, we have used high resolution fiber-FISH to study this region in normal and translocation cases by using probes from 22q11, LCRs and 11q23. We demonstrate that the LCR containing the gap in 22q11 is probably substantially larger than the previous estimates of 100 kb. Using fiber-FISH, we have localised the breakpoint in 22q11 to approximately 20-40 kb from the centromeric border of the LCR (i.e. the telomeric end of AC006547) and have confirmed the breakpoint position on 11q23.     

Enhanced Expression of ANO1 in Head and Neck Squamous Cell Carcinoma Causes Cell Migration and Correlates with Poor Prognosis

Head and neck squamous cell carcinoma (HNSCC) has the potential for early metastasis and is associated with poor survival. Ano1 (Dog1) is an established and sensitive marker for the diagnosis of gastrointestinal stromal tumors (GIST) and has recently been identified as a Ca(2+) activated Cl(-) channel. Although the ANO1 gene is located on the 11q13 locus, a region which is known to be amplified in different types of human carcinomas, a detailed analysis of Ano1 amplification and expression in HNSCC has not been performed. It is thus still unclear how Ano1 contributes to malignancy in HNSCC. We analyzed genomic amplification of the 11q13 locus and Ano1 together with Ano1-protein expression in a large collection of HNSCC samples. We detected a highly significant correlation between amplification and expression of Ano1 and showed that HNSCC patients with Ano1 protein expression have a poor overall survival. We further analyzed the expression of the Ano1 protein in more than 4'000 human samples from 80 different tumor types and 76 normal tissue types and detected that besides HNSCC and GISTs, Ano1 was rarely expressed in other tumor samples or healthy human tissues. In HNSCC cell lines, expression of Ano1 caused Ca(2+) activated Cl(-) currents, which induced cell motility and cell migration in wound healing and in real time migration assays, respectively. In contrast, knockdown of Ano1 did not affect intracellular Ca(2+) signaling and surprisingly did not reduce cell proliferation in BHY cells. Further, expression and activity of Ano1 strongly correlated with the ability of HNSCC cells to regulate their volume. Thus, poor survival in HNSCC patients is correlated with the presence of Ano1. Our results further suggest that Ano1 facilitates regulation of the cell volume and causes cell migration, which both can contribute to metastatic progression in HNSCC.     

SHANK2 is a frequently amplified oncogene with evolutionarily conserved roles in regulating Hippo signaling

Dysfunction of the Hippo pathway enables cells to evade contact inhibition and provides advantages for cancerous overgrowth.However,for a significant portion of human cancer,how Hippo signaling is perturbed remains unknown.To answer this question,we performed a genome-wide screening for genes that affect the Hippo pathway in Drosophila and cross-referenced the hit genes with human cancer genome.In our screen,Prosap was identified as a novel regulator of the Hippo pathway that potently affects tissue growth.Interestingly,a mammalian homolog of Prosap,SHANK2,is the most frequently amplified gene on 11 q13,a major tumor amplicon in human cancer.Gene amplification profile in this 11q13 amplicon clearly indicates selective pressure for SHANK2 amplification.More importantly,across the human cancer genome,SHANK2 is the most frequently amplified gene that is not located within the Myc amplicon.Further studies in multiple human cell lines confirmed that SHANK2 overexpression causes deregulation of Hippo signaling through competitive binding for a LATS1 activator,and as a potential oncogene,SHANK2 promotes cellular transformation and tumor formation in vivo.In cancer cell lines with deregulated Hippo pathway,depletion of SHANK2 restores Hippo signaling and ceases cellular proliferation.Taken together,these results suggest that SHANK2 is an evolutionarily conserved Hippo pathway regulator,commonly amplified in human cancer and potently promotes cancer.Our study for the first time illustrated oncogenic function of SHANK2,one of the most frequently amplified gene in human cancer.Furthermore,given that in normal adult tissues,SHANK2 s expression is largely restricted to the nervous system,SHANK2 may represent an interesting target for anticancer therapy.     

Detection of novel amplicons in prostate cancer by comprehensive genomic profiling of prostate cancer cell lines using oligonucleotide-based arrayCGH

BACKGROUND: The purpose of this study was to prove the feasibility of a longmer oligonucleotide microarray platform to profile gene copy number alterations in prostate cancer cell lines and to quickly indicate novel candidate genes, which may play a role in carcinogenesis. METHODS/RESULTS AND FINDINGS: Genome-wide screening for regions of genetic gains and losses on nine prostate cancer cell lines (PC3, DU145, LNCaP, CWR22, and derived sublines) was carried out using comparative genomic hybridization on a 35,000 feature oligonucleotide microarray (arrayCGH). Compared to conventional chromosomal CGH, more deletions and small regions of gains, particularly in pericentromeric regions and regions next to the telomeres, were detected. As validation of the high-resolution of arrayCGH we further analyzed a small amplicon of 1.7 MB at 9p13.3, which was found in CWR22 and CWR22-Rv1. Increased copy number was confirmed by fluorescence in situ hybridization using the BAC clone RP11-165H19 from the amplified region comprising the two genes interleukin 11 receptor alpha (IL11-RA) and dynactin 3 (DCTN3). Using quantitative real time PCR (qPCR) we could demonstrate that IL11-RA is the gene with the highest copy number gain in the cell lines compared to DCTN3 suggesting IL11-RA to be the amplification target. Screening of 20 primary prostate carcinomas by qPCR revealed an IL11-RA copy number gain in 75% of the tumors analyzed. Gain of DCTN3 was only found in two cases together with a gain of IL11-RA. CONCLUSIONS/SIGNIFICANCE: ArrayCGH using longmer oligonucleotide microarrays is feasible for high-resolution analysis of chomosomal imbalances. Characterization of a small gained region at 9p13.3 in prostate cancer cell lines and primary prostate cancer samples by fluorescence in situ hybridization and quantitative PCR has revealed interleukin 11 receptor alpha gene as a candidate target of amplification with an amplification frequency of 75% in prostate carcinomas. Frequent amplification of IL11-RA in prostate cancer is a potential mechanism of IL11-RA overexpression in this tumor type.     

利用染色体区段混合BAC探针鉴定食管癌细胞中的染色体畸变

染色体畸变是恶性肿瘤细胞的重要遗传学特征, 文章旨在应用BAC DNA克隆鉴定食管癌细胞中的染色体臂和染色体区段的畸变。针对染色体各区段选取5~10个1 Mb BAC DNA, 分别混合制备成特定染色体区段的BAC DNA混合克隆, 然后将染色体臂上覆盖所有区段的上述混合克隆进一步混合制备成特定染色体臂BAC DNA混合克隆。利用简并寡核苷酸引物聚合酶链反应(Degenerate oligonucleotide primed PCR, DOP-PCR)标记染色体臂探针, 利用切口平移法(Nick translation)标记染色体区段探针, 并对食管癌细胞中期染色体进行荧光原位杂交(Fluorescence in situ hybridization, FISH)分析。正常人外周血淋巴细胞中期染色体FISH结果显示, 上述方法标记的探针具有较高的特异性。进一步利用染色体臂混合探针, 确定了多个食管癌细胞中的染色体重排所涉及的特定染色体臂; 利用染色体区段混合探针, 鉴定出KYSE140的t(1q;7q)衍生染色体中1q上的断点范围位于1q32-q41。文章成功建立了1 Mb BAC DNA混合克隆探针标记技术, 并鉴定出多个食管癌细胞中的染色体臂和染色体区段畸变, 不仅为利用M-FISH技术鉴定肿瘤细胞中的染色体畸变提供了更为准确的方法, 而且还可能进一步将该法推广应用于恶性血液病的核型分析以及产前诊断。