Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex genetic alterations in cervical cancer

BackgroundCervical carcinoma develops as a result of multiple genetic alterations. Different studies investigated genomic alterations in cervical cancer mainly by means of metaphase comparative genomic hybridization (mCGH) and microsatellite marker analysis for the detection of loss of heterozygosity (LOH). Currently, high throughput methods such as array comparative genomic hybridization (array CGH), single nucleotide polymorphism array (SNP array) and gene expression arrays are available to study genome-wide alterations. Integration of these 3 platforms allows detection of genomic alterations at high resolution and investigation of an association between copy number changes and expression.ResultsGenome-wide copy number and genotype analysis of 10 cervical cancer cell lines by array CGH and SNP array showed highly complex large-scale alterations. A comparison between array CGH and SNP array revealed that the overall concordance in detection of the same areas with copy number alterations (CNA) was above 90%. The use of SNP arrays demonstrated that about 75% of LOH events would not have been found by methods which screen for copy number changes, such as array CGH, since these were LOH events without CNA. Regions frequently targeted by CNA, as determined by array CGH, such as amplification of 5p and 20q, and loss of 8p were confirmed by fluorescent in situ hybridization (FISH). Genome-wide, we did not find a correlation between copy-number and gene expression. At chromosome arm 5p however, 22% of the genes were significantly upregulated in cell lines with amplifications as compared to cell lines without amplifications, as measured by gene expression arrays. For 3 genes, SKP2, ANKH and TRIO, expression differences were confirmed by quantitative real-time PCR (qRT-PCR).ConclusionThis study showed that copy number data retrieved from either array CGH or SNP array are comparable and that the integration of genome-wide LOH, copy number and gene expression is useful for the identification of gene specific targets that could be relevant for the development and progression in cervical cancer.

[1]  Benjamin M. Bolstad,et al.  affy - analysis of Affymetrix GeneChip data at the probe level , 2004, Bioinform..

[2]  R. Bookstein,et al.  High‐density screen of human tumor cell lines for homozygous deletions of loci on chromosome arm 8p , 1999, Genes, chromosomes & cancer.

[3]  P. Eilers,et al.  Substantial changes in gene expression of Wnt, MAPK and TNFalpha pathways induced by TGF-beta1 in cervical cancer cell lines. , 2005, Carcinogenesis.

[4]  M. Sehested,et al.  Up-regulation of ALG-2 in hepatomas and lung cancer tissue. , 2003, The American journal of pathology.

[5]  P. Lichter,et al.  Comparative genomic hybridization: uses and limitations. , 2000, Seminars in hematology.

[6]  F. Speleman,et al.  Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes , 2002, Genome Biology.

[7]  P. Bates,et al.  OPCML at 11q25 is epigenetically inactivated and has tumor-suppressor function in epithelial ovarian cancer , 2003, Nature Genetics.

[8]  J. McDougall,et al.  Genomic Changes and HPV Type in Cervical Carcinoma (44497) , 2000, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[9]  M. Nishimura,et al.  Microsatellite instability is a late event in the carcinogenesis of uterine cervical cancer , 2000, Gynecologic oncology.

[10]  A. Zetterberg,et al.  Amplification of the telomerase reverse transcriptase (hTERT) gene in cervical carcinomas , 2002, Genes, chromosomes & cancer.

[11]  H. Ngan,et al.  Semi-quantitative fluorescent PCR analysis identifies PRKAA1 on chromosome 5 as a potential candidate cancer gene of cervical cancer. , 2006, Gynecologic oncology.

[12]  H. Chung,et al.  Gene copy number change events at chromosome 20 and their association with recurrence in gastric cancer patients. , 2005, Clinical cancer research : an official journal of the American Association for Cancer Research.

[13]  R. Hruban,et al.  Copy‐number methods dramatically underestimate loss of heterozygosity in cancer , 2006, Genes, chromosomes & cancer.

[14]  B. Ylstra,et al.  BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH) , 2006, Nucleic acids research.

[15]  Xueli Fan,et al.  Regulation of cell cycle progression and apoptosis by the papillomavirus E6 oncogene. , 2004, Critical reviews in eukaryotic gene expression.

[16]  R. Clarke,et al.  AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines , 2006, BMC Cancer.

[17]  H. Klinger,et al.  Genomic alterations in cervical carcinoma: losses of chromosome heterozygosity and human papilloma virus tumor status. , 1996, Cancer research.

[18]  H. Ngan,et al.  Genetic abnormalities and HPV status in cervical and vulvar squamous cell carcinomas. , 2005, Cancer genetics and cytogenetics.

[19]  G. Bastert,et al.  Prospective clinical study comparing DNA flow cytometry and HPV typing as predictive tests for persistence and progression of CIN I/II. , 2001, Cytometry.

[20]  L. Harris,et al.  Induction of topoisomerase II activity after ErbB2 activation is associated with a differential response to breast cancer chemotherapy. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[21]  Keith W. Jones,et al.  Whole genome DNA copy number changes identified by high density oligonucleotide arrays , 2004, Human Genomics.

[22]  Bhuvanesh Singh,et al.  Comprehensive molecular cytogenetic characterization of cervical cancer cell lines , 2003, Genes, chromosomes & cancer.

[23]  M. Trková,et al.  The application of comparative genomic hybridization to previously karyotyped cervical cancer cell lines. , 2000, Cancer genetics and cytogenetics.

[24]  A. Schauer,et al.  Genomic alterations of the c-myc protooncogene in relation to the overexpression of c-erbB2 and Ki-67 in human breast and cervix carcinomas , 2005, Journal of Cancer Research and Clinical Oncology.

[25]  D. Kingsley,et al.  Role of the mouse ank gene in control of tissue calcification and arthritis. , 2000, Science.

[26]  Rafael A Irizarry,et al.  Exploration, normalization, and summaries of high density oligonucleotide array probe level data. , 2003, Biostatistics.

[27]  H. Tanke,et al.  Insights from genomic microarrays into structural chromosome rearrangements , 2005, American journal of medical genetics. Part A.

[28]  M. J. van de Vijver,et al.  Loss of heterozygosity for defined regions on chromosomes 3, 11 and 17 in carcinomas of the uterine cervix. , 1998, British Journal of Cancer.

[29]  M. Sakamoto,et al.  Comparative genomic hybridization detects genetic alterations during early stages of cervical cancer progression , 2002, Genes, chromosomes & cancer.

[30]  B. Ylstra,et al.  Microarray‐based comparative genomic hybridization and its applications in human genetics , 2004, Clinical genetics.

[31]  Shirley A. Miller,et al.  A simple salting out procedure for extracting DNA from human nucleated cells. , 1988, Nucleic acids research.

[32]  C. Lundsteen,et al.  Comparative genomic hybridization reveals a recurrent pattern of chromosomal aberrations in severe dysplasia/carcinoma in situ of the cervix and in advanced‐stage cervical carcinoma , 1999, Genes, chromosomes & cancer.

[33]  H. Tanke,et al.  Genomic array and expression analysis of frequent high-level amplifications in adenocarcinomas of the gastro-esophageal junction. , 2006, Cancer genetics and cytogenetics.

[34]  P. Heinrich,et al.  Oncostatin M-induced activation of stress-activated MAP kinases depends on tyrosine 861 in the OSM receptor and requires Jak1 but not Src kinases. , 2006, Cellular signalling.

[35]  T. Ried,et al.  In situ hybridization with fluoresceinated DNA. , 1991, Nucleic acids research.

[36]  M. J. van de Vijver,et al.  Genetic alterations during the progression of squamous cell carcinomas of the uterine cervix , 1999, Genes, chromosomes & cancer.

[37]  H. Höfler,et al.  Distinct cytogenetic alterations in squamous intraepithelial lesions of the cervix revealed by laser‐assisted microdissection and comparative genomic hybridization , 1998, Cancer.

[38]  M. Stanley,et al.  Amplification of chromosome 5p correlates with increased expression of Skp2 in HPV-immortalized keratinocytes , 2003, Oncogene.

[39]  P. Eilers,et al.  Substantial changes in gene expression of Wnt, MAPK and TNFα pathways induced by TGF-β1 in cervical cancer cell lines , 2005 .

[40]  M. McAsey,et al.  Endoglin Expression as a Measure of Microvessel Density in Cervical Cancer , 2000, Obstetrics and gynecology.

[41]  A. Monroy,et al.  Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines , 2003, BMC Cancer.

[42]  Bradley P. Coe,et al.  High‐resolution chromosome arm 5p array CGH analysis of small cell lung carcinoma cell lines , 2005, Genes, chromosomes & cancer.

[43]  G. Fleuren,et al.  Differential expression of the calcium sensing receptor and combined loss of chromosomes 1q and 11q in parathyroid carcinoma , 2004, The Journal of pathology.

[44]  G. Fleuren,et al.  Allelic loss and prognosis in carcinoma of the uterine cervix , 1998, International journal of cancer.

[45]  F. Jiang,et al.  RNA silencing of S-phase kinase-interacting protein 2 inhibits proliferation and centrosome amplification in lung cancer cells , 2005, Oncogene.

[46]  P. Meltzer,et al.  Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[47]  Luc Girard,et al.  An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays. , 2004, Cancer research.

[48]  G. Sauter,et al.  TRIO amplification and abundant mRNA expression is associated with invasive tumor growth and rapid tumor cell proliferation in urinary bladder cancer. , 2004, The American journal of pathology.

[49]  J. Inazawa,et al.  Amplification and overexpression of SKP2 are associated with metastasis of non-small-cell lung cancers to lymph nodes. , 2004, The American journal of pathology.

[50]  W. Loging,et al.  Elevated expression of ribosomal protein genes L37, RPP-1, and S2 in the presence of mutant p53. , 1999, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[51]  Paul H. C. Eilers,et al.  Quantile smoothing of array CGH data , 2005, Bioinform..

[52]  G. Fleuren,et al.  Cytokine profile of cervical cancer cells. , 2001, Gynecologic oncology.

[53]  Z. Szallasi,et al.  A signature of chromosomal instability inferred from gene expression profiles predicts clinical outcome in multiple human cancers , 2006, Nature Genetics.

[54]  G. Fleuren,et al.  Recurrent integration of human papillomaviruses 16, 45, and 67 near translocation breakpoints in new cervical cancer cell lines. , 1999, Cancer research.

[55]  P. Rao,et al.  Chromosomal amplifications, 3q gain and deletions of 2q33-q37 are the frequent genetic changes in cervical carcinoma , 2004, BMC Cancer.

[56]  R. Chaganti,et al.  Allelotype analysis of cervical carcinoma. , 1994, Cancer research.

[57]  Stanley Letovsky,et al.  GDB: the Human Genome Database , 1998, Nucleic Acids Res..

[58]  M. Mansukhani,et al.  Genetic analysis identifies putative tumor suppressor sites at 2q35–q36.1 and 2q36.3–q37.1 involved in cervical cancer progression , 2003, Oncogene.

[59]  N. Tchurikov,et al.  Molecular Mechanisms of Epigenetics , 2005, Biochemistry (Moscow).

[60]  E. Schröck,et al.  Advanced‐stage cervical carcinomas are defined by a recurrent pattern of chromosomal aberrations revealing high genetic instability and a consistent gain of chromosome arm 3q , 1997, Genes, chromosomes & cancer.

[61]  Eytan Domany,et al.  Relationship of gene expression and chromosomal abnormalities in colorectal cancer. , 2006, Cancer research.

[62]  J. Sisken,et al.  MORPHOLOGICAL AND KINETIC ASPECTS OF MITOTIC ARREST BY AND RECOVERY FROM COLCEMID , 1966, The Journal of cell biology.

[63]  E. Jordanova,et al.  High‐resolution multi‐parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin‐embedded tissues , 2005, The Journal of pathology.

[64]  P. Gariglio,et al.  High correlation between molecular alterations of the c-myc oncogene and carcinoma of the uterine cervix. , 1987, Cancer research.

[65]  G. Thomas,et al.  Fine deletion mapping of chromosome 8p in non‐small‐cell lung carcinoma , 1999, International journal of cancer.

[66]  C J L M Meijer,et al.  Increased gene copy numbers at chromosome 20q are frequent in both squamous cell carcinomas and adenocarcinomas of the cervix , 2006, The Journal of pathology.