Multiplex ligation-dependent probe amplification: a diagnostic tool for simultaneous identification of different genetic markers in glial tumors

Genetic aberrations in tumors are predictive for chemosensitivity and survival. A test is needed that allows simultaneous detection of multiple changes and that is widely applicable in a routine diagnostic setting. Multiplex ligation-dependent probe amplification (MLPA) allows detection of DNA copy number changes of up to 45 loci in one relatively simple, semiquantitative polymerase chain reaction-based assay. To assess the applicability of MLPA, we performed MLPA analysis to detect relevant genetic markers in a spectrum of 88 gliomas. The vast majority of these tumors (n = 79) were previously characterized by comparative genomic hybridization. With MLPA kit P088 (78 cases), complete and partial loss of 1p and 19q were reliably identified, even in samples containing only 50% tumor DNA. Distinct 1p deletions exist with different clinically prognostic consequences, and in contrast to the commonly used diagnostic strategies (loss of heterozygosity or fluorescent in situ hybridization 1p36), P088 allows detection of such distinct 1p losses. Combining P088 with P105 will further increase the accurate prediction of clinical behavior because this kit identified markers (EGFR, PTEN, and CDKN2A) of high-grade malignancy in 41 cases analyzed. We conclude that MLPA is a reliable diagnostic tool for simultaneous identification of different region-specific genetic aberrations of tumors.     

Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.     

Adrenocortical carcinoma survival rates correlated to genomic copy number variants

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy accounting for between 0.02% and 0.2% of all cancer deaths. Surgical removal offers the only current potential for cure. Unfortunately, ACC has undergone metastatic spread in 40% to 70% of patients at the time of diagnosis. Standard chemotherapy with mitotane is often ineffective with intolerable side effects. The modern molecular technology of comparative genomic hybridization allows the examination of DNA for chromosomal alterations, which can lend biological insight into cancer processes. Genomes of 25 ACC clinical samples were queried on the Agilent 44K Human Genome comparative genomic hybridization array detecting regions of chromosomal gain and loss within the tumor population. Commonly shared amplifications appearing in > or =50% of tumors at P < or = 10(-4) include regions within chromosomes 5, 7, 12, 16q, and 20. Deleted genomic regions within ACC include portions of chromosomes 1, 3p, 10q, 11, 14q, 15q, 17, and 22q. Genomic aberrations in regions associated with differential survival (P < or = 0.05) and presence in > or =20% of tumors include amplifications of 6q, 7q, 12q, and 19p. Deletions within stratified survival groups include localized regions within 3, 8, 10p, 16q, 17q, and 19q. Statistical analysis of this genetic landscape reveals a set of chromosomal aberrations strongly associated with survival in an accumulation-dependent fashion. These regions may hold prognostic indicators and offer therapeutic targets that can be used to develop novel treatments for aggressive tumors.     

Analysis of the relationship between sex and chromosomal aberrations in colorectal cancer by comparative genomic hybridization

Colorectal cancer is thought to be more common in men than in women. The chromosomal locations of DNA gains and losses in surgical specimens of colorectal tumours were detected by comparative genomic hybridization and were compared by gender. Five chromosomal regions, 7p, 8p, 8q, Xp and Xq, contained multiple gains that were significantly more common in males than in females, and within these regions, the differences were significant for Xp21, Xp11.3, Xp11.4 and Xq26. Regions 1p, 3q, 11q, 12p, 12q and 15q contained multiple sites of gain that were significantly more common in females than in males. Tumours from male and female patients showed significantly more losses at 11p and 15q, and at 4q and Xq, respectively. The fact that gains in X-chromosomal regions were detected with a significantly higher frequency in tumours from male patients suggests that the difference between the genders might be explained by X-chromosomal inactivation.     

Multicolor fluorescence in situ hybridization and comparative genomic hybridization reveal molecular events in lung adenocarcinomas and squamous cell lung carcinomas

We have used the molecular cytogenetic techniques of multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH) to analyze two established lung cancer cell lines (A549, H520), 80 primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples in order to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q and 22q to be commonly over-represented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be under-represented. In lung adenocarcinomas the most common gains were found in 16p13 (50%); while in squamous cell lung carcinomas the common gains were found in 17q21 (45%) and these alterations were observed to be associated with their specific pathological subtype. In conclusion, the present study contributes to the molecular biological characterization in lung adenocarcinomas and squamous cell lung carcinomas and through evaluation of molecular events to the recently emergent focus on novel markers for lung cancer treatment.     

Genomic characterization of chromosome 8 pericentric trisomy

We present a patient with trisomy 8p11.21q11.21 associated with language, gross motor, fine motor, and cognitive delay. Furthermore, using array‐based comparative genomic hybridization, we identify the specific genes duplicated in our patient.     

应用原位杂交技术比较肺腺癌和肺鳞癌的内在分子机制

目的分析肺腺癌及肺鳞癌肿瘤中染色体异常变化和基因组的改变。探讨肺腺癌及肺鳞癌细胞遗传物质的改变,揭示这两种NSCLC主要亚型发生发展的内在本质及其与临床特征之间的关系。方法应用M-FISH技术,检测肺腺癌及肺鳞癌细胞株各1株,观察其染色体数目及结构畸变情况,应用CGH技术对80例肺腺癌组织80例肺鳞癌组织中所提取的全基因组DNA进行检测,比较这两种类型的NSCLC全基因组的变化。结果M-FISH结果显示肺腺癌及肺鳞癌细胞中存在许多复杂的染色体重排,其中5、6、11、12、17号染色体最频繁参于染色体间的易位。CGH发现,在160例肺癌标本基因组中,最常见的扩增区域足1 q,2 p,3 q,5 p,5 q,7 p,8 q,11 q,12 q,14 q,16 p,17 p.19 p,20 q,21 q,22 q。最常见的缺失区域是2 q.3 p,4 p,5 q,7 q,8 p,9 p.13 q,14 q,17 p。在肺腺癌中最常见的扩增位点足16p13,阳性率达50%.而肺鳞癌中最常见的扩增位点是17q21(45%),并且这两个位点的变异在肺腺癌和肺鳞癌之间有着显著性的差异(P<0.05)。结论M- FISH和CGH技术是研究肺癌基因组变化的强有力的工具,本实验中发现的基因改变町能代表了与肺腺癌及肺鳞癌特有的病理,诊断相关和候选基因。     

Detection of MYCN amplification and chromosome 1p36 loss in neuroblastoma by cDNA microarray comparative genomic hybridization.

BACKGROUND: In the last decade, microarray technology has been extensively used to evaluate gene expression profiles and genome imbalances. We have developed a microarray-based comparative genomic hybridization (CGH) approach to identify MYCN gene amplification and 1p36 chromosome loss, two markers of tumor aggressiveness in neuroblastoma. AIM: The aim was to use microarray CGH technology to detect the two major prognostic markers for neuroblastoma, MYCN amplification and 1p36 chromosome deletion, in neuroblastoma patients and, therefore, confirm the usefulness of this approach in this cancer. METHODS: DNA was purified from 16 tumors containing at least 90% malignant neuroblasts and collected at the onset of disease. Pooled fluorescent-labeled reference and neuroblastoma tumor genomic DNA was hybridized to epoxide-coated glass slides on laboratory-made complementary DNA microarray. The microarray contained cDNA mapped at the 1p36.33-36.1 chromosomal region and MYCN gene. cDNA from the 2q33-q34 and 12p13 chromosomes was used as a control and Arabidopsis thaliana DNA was spotted to control unspecific hybridization. Fluorescence in situ hybridization analysis was also performed to validate results from the microarray CGH. RESULTS: Both MYCN amplification and 1p36 chromosome deletion were detected by microarray CGH. The sensitivity and specificity for 1p36 loss detection were 66.7% and 90.0%, respectively. The method had a sensitivity of 66.7% and specificity of 90.9% to detect MYCN amplification. DISCUSSION: Our results demonstrated that the microarray CGH can be efficiently applied to study DNA gain and loss of specific chromosome regions.     

Genomic Variation by Whole-Genome SNP Mapping Arrays Predicts Time-to-Event Outcome in Patients with Chronic Lymphocytic Leukemia: A Comparison of CLL and HapMap Genotypes

Genomic abnormalities, such as deletions in 11q22 or 17p13, are associated with poorer prognosis in patients with chronic lymphocytic leukemia (CLL). We hypothesized that unknown regions of copy number variation (CNV) affect clinical outcome and can be detected by array-based single-nucleotide polymorphism (SNP) genotyping. We compared SNP genotypes from 168 untreated patients with CLL with genotypes from 73 white HapMap controls. We identified 322 regions of recurrent CNV, 82 of which occurred significantly more often in CLL than in HapMap (CLL-specific CNV), including regions typically aberrant in CLL: deletions in 6q21, 11q22, 13q14, and 17p13 and trisomy 12. In univariate analyses, 35 of total and 11 of CLL-specific CNVs were associated with unfavorable time-to-event outcomes, including gains or losses in chromosomes 2p, 4p, 4q, 6p, 6q, 7q, 11p, 11q, and 17p. In multivariate analyses, six CNVs (ie, CLL-specific variations in 11p15.1-15.4 or 6q27) predicted time-to-treatment or overall survival independently of established markers of prognosis. Moreover, genotypic complexity (ie, the number of independent CNVs per patient) significantly predicted prognosis, with a median time-to-treatment of 64 months versus 23 months in patients with zero to one versus two or more CNVs, respectively (P = 3.3 × 10⁻⁸). In summary, a comparison of SNP genotypes from patients with CLL with HapMap controls allowed us to identify known and unknown recurrent CNVs and to determine regions and rates of CNV that predict poorer prognosis in patients with CLL.Several recurrent genomic abnormalities have been identified in chronic lymphocytic leukemia (CLL) using traditional cytogenic methods and had prognostic importance.^1,2 For example, deletions in chromosome 11q22 (10% to 20% of CLL cases), which includes the ataxia-telangiectasia mutated (ATM) gene, or 17p13 (3% to 5% of CLL cases), which includes the tumor protein p53 (TP53) gene, have been associated with rapid disease progression, treatment resistance, and inferior survival in patients.^1–5 Thus, fluorescence in situ hybridization analysis of interphase nuclei using a panel of probes is commonly part of the clinical workup of CLL cases at diagnosis of CLL.^1,6 In addition to the common recurrent chromosomal gains and losses, the presence of balanced or unbalanced translocations and cytogenetic complexity (three or more abnormalities) have been associated with shorter treatment-free and overall survival (OS).^2,7–9 Furthermore, the acquisition of new and high-risk abnormalities during the clinical course of disease can render CLL B cells resistant to therapy.^10,11 These data indicate that genomic instability is common in CLL, and that genetic abnormalities evolve over time, with a subset of changes that correlate with therapeutic resistance and/or prognosis.Modern array-based genomic technologies, such as array comparative genomic hybridization and single-nucleotide polymorphism (SNP) genotyping, allow rapid and sensitive identification of small DNA copy number changes that are beyond the resolution of conventional cytogenetic techniques. For example, we and other researchers have demonstrated that bacterial artificial chromosome and oligonucleotide-based array comparative genomic hybridization allow the identification of novel recurrent genomic imbalances in CLL involving chromosomes 5q, 2p, 8, 19, and 22.^12,13 Although the average resolution of bacterial artificial chromosome arrays is approximately 1 Mb, commercially available SNP arrays offer the possibility of genome-wide DNA genotyping at a mean resolution of <1000 bp by assessing thousands or millions of SNP loci per patient. Several groups have evaluated such SNP arrays as a screening tool for copy number abnormalities in various types of cancers. SNP arrays applied to CLL have identified acquired chromosomal imbalances in 66% to 82% of investigated cases.^14–18 However, the interpretation of results from high-resolution DNA microarrays, especially without comparison of tumor to matched nontumor DNA, can be challenging. In particular, SNP arrays detect constitutional copy number variants and allelic polymorphisms, which tend to be smaller (median, approximately 150 kb) than acquired genomic aberrations in tumors.¹⁹ It is conceivable that both acquired and constitutional copy number changes, both included in the term copy number variation (CNV) herein, provide a genetic advantage or disadvantage to leukemic cells, and contribute to disease stage and clinical outcome. Considerable evidence from family and case-control studies suggests a nonexclusive, but presumably polygenic, predisposition for CLL by the presence of DNA variants in distinct susceptibility loci, such as 2q37.3, 8q24.21, 15q21.3, and 16q24.1.^20–22In the current study, we assessed total CNV by genome-wide SNP genotyping in 168 previously untreated patients with CLL using the Illumina610Quadv1 BeadChips (Illumina Inc., San Diego, CA). We developed analytical methods that allow us to determine acquired and constitutional CNVs associated with CLL, and to use the genome-wide copy number data for time-to-event outcome prognostication, without the availability of matched nontumor DNA. We hypothesized that CNVs acquired by CLL cells or inherently involved in CLL biological characteristics, are detected more frequently in SNP genotypes from patients with CLL than in SNP genotypes from healthy HapMap individuals who had been assessed on the same array platform. Furthermore, we hypothesized that any CNVs of biological interest in CLL should be associated with clinical time-to-event outcomes. We calculated CLL-specific CNVs as segments that were identified with statistical significance more frequently in patients with CLL than in HapMap controls. These segments included known recurrent abnormalities in chromosomes 2, 6, 11, 12, 13, and 17, as well as previously unknown CNVs in CLL. By using either all or CLL-specific CNV, we designed multivariate models that determined individual CNVs and increasing levels of genotypic complexity to predict time-to-event outcomes independent of established markers of prognosis.     

Genomic changes in gliomas detected using single nucleotide polymorphism array in formalin-fixed, paraffin-embedded tissue: superior results compared with microsatellite analysis

Deletion or loss of heterozygosity (LOH) in chromosomes 1p and 19q in oligodendrogliomas (ODGs) have diagnostic, prognostic, and therapeutic implications. Current clinical assays are limited because the probes or primers interrogate only limited genomic segments. We investigated the use of single nucleotide polymorphism (SNP) arrays for identifying genomic changes in gliomas from FFPE tissues. DNA was extracted from FFPE tissues of 30 brain tumor cases (15 ODGs and 15 non-ODGs) and assayed on the Illumina array with 300,000 markers. SNP results were compared with standard short tandem repeat (STR) assays of chromosomes 1p and 19q. Fifteen ODGs had LOH by STR and deletion by array on both 1p and 19q. Ten non-ODGs had no evidence of LOH on 1p and 19q by STR, seven of which had no abnormalities for these chromosomes; three had partial deletions by SNP array. Five non-ODG cases had partial LOH or deletion by both assays. No major discordance was found between SNP array and STR results. Advantages of SNP arrays include no need for an accompanying normal sample, the ability to find small segmental deletions, the potential to distinguish between deletions and copy neutral LOH, and whole-genome screening to allow discovery of new, significant loci. Assessment of genomic changes in routine glioma specimens using SNP arrays is feasible and has great potential as an accurate clinical diagnostic test.     

Molecular cytogenetic characterization of esophageal cancer detected by comparative genomic hybridization

Aim: Detection of cytogenetic alterations in esophageal cancer (EC). A total of 40 cases of primary EC and their paired nearby nontumor tissues were collected. The comparative genomic hybridization (CGH) is the technique that brings out the gains and losses of chromosome fragments and was applied to determine the aberrations from the tissue DNA. In noncancer tissues, the gains were at 19p (5/40, 13%), 20q (5/40, 13%), and losses at 9p (13/40, 33%), 2q (10/40, 25%), 12q (10/40, 25%), 13q (10/40, 25%), 5q (9/40, 23%), 6q (9/40, 23%), 7q (9/40, 23%), and 8p (9/40, 23%). Two cases in nontumor tissues showed no CGH change. In the 40 cases of primary EC, the gains were at 8q (10/40, 25%), 3q (9/40, 23%), 2q (7/40, 18%), and 13q (7/40, 18%), and the losses were at 1q (8/40, 20%), 4q (8/40, 20%), 3p (7/40, 18%), 5q (7/40, 18%), and 18q (7/40, 18%) in comparison with paired nearby noncancerous tissues. We found that the loss aberrations were on 1q, 2p, 3p, 5q, 6q, 9p, 11p, 15q, 16q, 18q, 21q and gains on 20p in both tumor and nontumor tissues; nevertheless, ?4p, ?7q, ?8p, ?10q, ?12q, ?13q, ?14q and +17p, +19q, +22q were only found in nontumor tissues and +1q, +2pq, +3q, ?4q, +4q, +5q, 7p, +8q, +10q, +12q, +13q, +14q ?17p, ?19pq, ?22q in EC. From these results, we suggest that most of the tissues near the cancer parts of EC may be considered as a precancerous region. The alteration between cancer and noncancer tissues may play a role in the development of EC. J. Clin. Lab. Anal. 24:167–174, 2010.© 2010 Wiley‐Liss, Inc.     

脑星形细胞肿瘤的MR灌注成像和病理分级对照研究

目的研究MR灌注成像(PWI)在脑星形细胞肿瘤术前分级中的应用价值.方法对28例脑星形细胞肿瘤病人行MR PWI,低级别星形细胞瘤9例,间变型星形细胞瘤12例,胶质母细胞瘤7例.依次行常规MRI及MR PWI,首先得到信号强度-时间曲线,并合成相对脑血流容积(rCBV)图,计算出最大rCBV比率.结果三组最大rCBV比率分别为(3.23±0.97), (5.23±0.33),(6.09±0.70),三组之间比较均有显著性差异.结论 MR PWI能有效地在术前评价星形细胞肿瘤的病理级别.     

淋巴瘤比较基因组杂交研究

目的评价比较基因组杂交(CGH)技术在淋巴瘤研究中的应用价值。方法采用CGH技术检测20例淋巴瘤患者核型异常,部分病例与核型分析比较。结果20例淋巴瘤患者CGH核型异常检出率为60%。8例进行常规核型分析的患者,CGH检测出4例常规核型分析未发现的异常。8例弥漫大B细胞淋巴瘤5例发现染色体拷贝数改变,其中3例发现13号染色体长臂扩增。5例滤泡性淋巴瘤中3例分别检测出7q11-21和15q11-14扩增和18号三体。4例小淋巴细胞淋巴瘤中1例6q16-25缺失,1例发现复杂核型异常:1p21-23扩增伴有2p12-ter,9p13-ter,10p13-ter缺失。结论淋巴瘤患者存在多种染色体异常,CGH是一有用的分析淋巴瘤核型异常的分子细胞遗传学工具。     

Glioma test array for use with formalin-fixed, paraffin-embedded tissue: array comparative genomic hybridization correlates with loss of heterozygosity and fluorescence in situ hybridization

Array-based comparative genomic hybridization (aCGH) is a powerful, high-throughput tool for whole genome analysis. Until recently, aCGH could only be reproducibly performed on frozen tissue samples and with significant tissue amounts. For brain tumors however, paraffin-embedded tissue blocks from small stereotactic biopsies may be the only tissue routinely available. The development of methods to analyze formalin-fixed, paraffin-embedded (FFPE) material therefore has the potential to impact molecular diagnosis in a significant way. To this end, we constructed a BAC array representing chromosomes 1, 7, 19, and X because 1p/19q deletion and EGFR gene amplification provide clinically relevant information for glioma diagnosis. We also optimized a two-step labeling procedure using an amine-modified nucleotide for generating aCGH probes. Using this approach, we analyzed a series of 28 FFPE oligodendroglial tumors for alterations of chromosomes 1, 7, and 19. We also independently assayed these tumors for 1p/19q deletion by fluorescence in situ hybridization and by loss of heterozygosity analyses. The concordance between aCGH, standard loss of heterozygosity and fluorescence in situ hybridization was nearly 100% for the chromosomes analyzed. These results suggest that aCGH could offer an improved molecular diagnostic approach for gliomas because of its ability to detect clinically relevant molecular alterations in small FFPE specimens.     

FHACT: the FISH-based HPV-associated cancer test that detects nonrandom gain at four genomic loci as biomarkers of disease progression

Despite implementation of screening programs for human papillomavirus (HPV)-associated cancers, in particular cervical, scientific studies continue to uncover viral and host biomarkers that could serve to further optimize the detection of individuals with underlying or at risk for developing precancer or cancer. Nonrandom host somatic chromosomal alterations are frequently shared across HPV-associated cancers, but with varying frequencies, potentially with functional roles. At least for 3q26 gain, there is firm preliminary evidence to support that this genomic alteration can serve as a biomarker of disease progression of cervical cancer. In the current review, the FISH-based HPV-associated cancer test is described that enables detection of genomic imbalance at four loci (3q26, 5p15, 20q13, centromere 7) in a single hybridization on a cell-by-cell basis in cytology specimens. When implemented as a secondary screening assay, the FISH-based HPV-associated cancer test could assist in the detection of clinically relevant HPV-associated disease and help guide patient management decisions.     

Copy number gains in 11q13 and 8q24 [corrected] are highly linked to prognosis in cutaneous malignant melanoma

Relating specific genetic alterations to prognosis may help improve prognostication in melanoma, may identify key oncogenic drivers in cancer, and may assist in developing targeted therapies. Characteristic genetic alterations in melanoma include chromosomal copy number aberrations. We evaluated 97 melanomas (55 metastasizing and 42 nonmetastasizing) after a minimum 5-year follow-up in a case-control study using fluorescence in situ hybridization, targeting commonly altered chromosomal loci in melanoma. Eight probes arranged in two panels were used, and 11 parameters were evaluated. Parameters showing a statistically significant difference between the metastasizing and nonmetastasizing groups were evaluated with multivariate logistic regression analysis to compare their prognostic potential with other traditional prognostic markers used by the American Joint Committee on Cancer. Four of 11 parameters evaluated, including CCND1 (alias Bcl-1) gain, CCND1 r-gain, MYC (alias c-myc) gain, and MYC r-gain, had a statistically significant difference in the metastasizing versus nonmetastasizing group. All four parameters maintained statistical significance when evaluated in separate multivariate logistic regression analyses that included the seven currently used American Joint Commission on Cancer prognosticators in melanoma. In multivariate analyses, these four parameters were second only to ulceration in their prognostic potential. Copy number changes at 11q13 and 8q24 [corrected] harboring CCND1 and MYC, respectively, are highly associated with prognosis. Fluorescence in situ hybridization targeting these loci may be a useful standardized prognostic marker in melanoma skin cancer.     

Comparative genomic hybridization of hepatocellular carcinoma: correlation with fluorescence in situ hybridization in paraffin-embedded tissue.

BACKGROUND: Proto-oncogene MYC, mapped to chromosomal band 8q24 and the genes for hepatocyte growth factor (HGF at 7q21) and its receptor, MET, at chromosomal band 7q31, have an important role in the biology and growth of normal and neoplastic liver. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies have reported frequent abnormalities of chromosomes 1 and 8 in hepatocellular carcinomas (HCCs) of various clinical and pathological stages. Chromosome 7 involvement is reported to be less frequent. MATERIALS AND METHODS: Frozen tissue from 17 HCCs was used for CGH analysis and sections of corresponding formalin-fixed, paraffin-embedded HCC tissue were used for dual-color FISH with locus-specific (LSI-cMYC for chromosome 8q24 and LSI D7S486 for chromosome 7q31) and centromeric probes, CEP8 (8p11.1-q11.2) and CEP7 (7p11.1-q11.2) (Vysis, Inc, Downers Grove, IL). This study intended to determine the pattern of chromosomal aberrations in early-stage (incidental) HCC and large surgically resected HCC, and also compared the efficiency and usefulness of the two cytogenetic methods. RESULTS: CGH showed abnormalities on chromosomes 1q, 5q, 7q, 8q, 9, 10, 13q, 15, 16, 17p, 18q, 19, 20, 21, 22, and X. Gains of 8q were noted in 50% of the HCCs, including five cases of incidental HCCs by CGH. Increase in copy numbers of MYC detected by FISH was noted in 25% of tumors that had shown 8q gains by CGH and in five cases with no chromosome abnormalities noted by CGH. Three cases with 7q31 copy number abnormalities were found by FISH in addition to those detected by CGH. CONCLUSION: Combined use of CGH and FISH may provide important information about early and/or primary genetic changes in the development of HCC.     

Amplification and overexpression of the EMS 1 oncogene,a possible prognostic marker,in human hepatocellular carcinoma

DNA amplification in cancer cells frequently involves oncogenes whose increased expression confers a selective advantage on tumor cell growth. In an attempt to identify novel oncogenes involved in hepatocarcinogenesis, representational difference analysis (RDA) was performed using DNA from a primary human hepatocellular carcinoma (HCC) that showed high-level DNA amplifications on chromosomes 1p32 and 11q13 by comparative genomic hybridization. Ten amplification fragments were isolated by RDA, and when used to probe Southern blots of tumor DNA, there was a 5- to 50-fold increase in hybridization intensity relative to normal DNA. The sequence of one amplification product matched that of the EMS1 oncogene, which is located on chromosome 11q13 and is amplified in other cancers. We detected EMS1 amplification in 3 of 17 primary HCC. Overexpression of EMS1 mRNA was observed in 12 of 14 HCC cell lines in the absence of gene amplification or an increased copy-number of the gene. The EMS1 gene encodes cortactin, a cortical actin-associated protein that is a substrate for Src kinase and is involved in cytoskeleton organization. Alterations of the EMS1 gene that lead to overexpression of cortactin may be associated with tumor development in HCC. EMS1 amplification and overexpresion is indicative of unfavorable prognosis in several cancers and may have similar prognostic implications in liver cancer.     

Duplication of 19q (13.2-13.31) associated with comitant esotropia: A case report

BACKGROUNDComitant esotropia is the most common form of strabismus. It is caused by heterogeneous environmental and genetic risk factors. The pure duplication of the long arm of chromosome 19 is a rare abnormality. Only 8 patients with partial trisomy of the long arm of chromosome 19q have been reported to date. Here, we describe a girl with pure duplication of 19q, who was diagnosed with congenital esotropia, microcephaly, and gallbladder agenesis. CASE SUMMARYThe patient was diagnosed with esotropia when she was 1-year-old. The Krimsky method showed +50 prism diopters in the primary gaze position. No additional abnormal findings were observed following slit lamp and fundus examination, but the features of the full-field electroretinogram showed a decreased amplitude and increased implicit times. Magnetic resonance imaging showed ventriculomegaly with thinning of the corpus callosum and splenium in her brain. A 4.42 Mb mosaic duplication within 19q13.2-q13.31 region (chr19:39,343,725 to 43,762,586) was detected by microarray comparative genomic hybridization. CONCLUSIONStrabismus is reported in many live borns with pure duplication of 19q. This important clinical characteristic indicates that the candidate genes fundamental for this phenotype may be narrowed to genes within the 19q13.3-q13.31 region. There were two candidate genes observed that may contribute to the comitant esotropia phenotype, namely XRCC1 (19:43,543,311) and SMG9 (19:43,727,991).