Genomic instability is a hallmark of human cancers, including the 5% caused by human papillomavirus (HPV). Here we report a striking association between HPV integration and adjacent host genomic structural variation in human cancer cell lines and primary tumors. Whole-genome sequencing revealed HPV integrants flanking and bridging extensive host genomic amplifications and rearrangements, including deletions, inversions, and chromosomal translocations. We present a model of ''looping'' by which HPV integrant-mediated DNA replication and recombination may result in viral-host DNA concatemers, frequently disrupting genes involved in oncogenesis and amplifying HPV oncogenes E6 and E7. Our high-resolution results shed new light on a catastrophic process, distinct from chromothripsis and other mutational processes, by which HPV directly promotes genomic instability.

DNA double-strand breaks (DSBs) induce a signal transmitted by the ataxia-telangiectasia mutated (ATM) kinase, which suppresses illegitimate joining of DSBs and activates cell-cycle checkpoints. Here we show that a significant fraction of mature ATM-deficient lymphocytes contain telomere-deleted ends produced by failed end joining during V(D)J recombination. These RAG-1/2 endonuclease-dependent, terminally deleted chromosomes persist in peripheral lymphocytes for at least 2 weeks in vivo and are stable over several generations in vitro. Restoration of ATM kinase activity in mature lymphocytes that have transiently lost ATM function leads to loss of cells with terminally deleted chromosomes. Thus, maintenance of genomic stability in lymphocytes requires faithful end joining as well a checkpoint that prevents the long-term persistence and transmission of DSBs. Silencing this checkpoint permits DNA ends produced by V(D)J recombination in a lymphoid precursor to serve as substrates for translocations with chromosomes subsequently damaged by other means in mature cells.

To screen pancreatic carcinomas for chromosomal aberrations we have applied molecular cytogenetic techniques, including fluorescent in situ hybridization, comparative genomic hybridization, and spectral karyotyping to a series of nine established cell lines. Comparative genomic hybridization revealed recurring chromosomal gains on chromosome arms 3q, 5p, 7p, 8q, 12p, and 20q. Chromosome losses were mapped to chromosome arms 8p, 9p, 17p, 18q, 19p, and chromosome 21. The comparison with comparative genomic hybridization data from primary pancreatic tumors indicates that a specific pattern of chromosomal copy number changes is maintained in cell culture. Metaphase chromosomes from six cell lines were analyzed by spectral karyotyping, a technique that allows one to visualize all chromosomes simultaneously in different colors. Spectral karyotyping identified multiple chromosomal rearrangements, the majority of which were unbalanced. No recurring reciprocal translocation was detected. Cytogenetic aberrations were confirmed using fluorescent in situ hybridization with probes for the MDR gene and the tumor suppressor genes p16 and DCC. Copy number increases on chromosome 20q were validated with a probe specific for the nuclear receptor coactivator AIB1 that maps to chromosome 20q12. Amplification of this gene was identified in six of nine pancreatic cancer cell lines and correlated with increased expression.

A fundamental question in the biology of sex differences has eluded direct study in humans: How does sex-chromosome dosage (SCD) shape genome function? To address this, we developed a systematic map of SCD effects on gene function by analyzing genome-wide expression data in humans with diverse sex-chromosome aneuploidies (XO, XXX, XXY, XYY, and XXYY). For sex chromosomes, we demonstrate a pattern of obligate dosage sensitivity among evolutionarily preserved X-Y homologs and update prevailing theoretical models for SCD compensation by detecting X-linked genes that increase expression with decreasing X- and/or Y-chromosome dosage. We further show that SCD-sensitive sex-chromosome genes regulate specific coexpression networks of SCD-sensitive autosomal genes with critical cellular functions and a demonstrable potential to mediate previously documented SCD effects on disease. These gene coexpression results converge with analysis of transcription factor binding site enrichment and measures of gene expression in murine knockout models to spotlight the dosage-sensitive X-linked transcription factor ZFX as a key mediator of SCD effects on wider genome expression. Our findings characterize the effects of SCD broadly across the genome, with potential implications for human phenotypic variation.

To characterize patterns of global transcriptional deregulation in primary colon carcinomas, we did gene expression profiling of 73 tumors [Unio Internationale Contra Cancrum stage II (n = 33) and stage III (n = 40)] using oligonucleotide microarrays. For 30 of the tumors, expression profiles were compared with those from matched normal mucosa samples. We identified a set of 1,950 genes with highly significant deregulation between tumors and mucosa samples (P < 1eÀ7). A significant proportion of these genes mapped to chromosome 20 (P = 0.01). Seventeen genes had a >5-fold average expression difference between normal colon mucosa and carcinomas, including up-regulation of MYC and of HMGA1, a putative oncogene. Furthermore, we identified 68 genes that were significantly differentially expressed between lymph node-negative and lymph node-positive tumors (P < 0.001), the functional annotation of which revealed a preponderance of genes that play a role in cellular immune response and surveillance. The microarray-derived gene expression levels of 20 deregulated genes were validated using quantitative real-time reverse transcription-PCR in >40 tumor and normal mucosa samples with good concordance between the techniques. Finally, we established a relationship between specific genomic imbalances, which were mapped for 32 of the analyzed colon tumors by comparative genomic hybridization, and alterations of global transcriptional activity. Previously, we had conducted a similar analysis of primary rectal carcinomas. The systematic comparison of colon and rectal carcinomas revealed a significant overlap of genomic imbalances and transcriptional deregulation, including activation of the Wnt/B-catenin signaling cascade, suggesting similar pathogenic pathways.

To identify sequential alterations of the genome, transcriptome, and proteome during colorectal cancer progression, we have analyzed tissue samples from 36 patients, including the complete mucosa-adenoma-carcinoma sequence from 8 patients. Comparative genomic hybridization (CGH) revealed patterns of stage specific, recurrent genomic imbalances. Gene expression analysis on 9K cDNA arrays identified 58 genes differentially expressed between normal mucosa and adenoma, 116 genes between adenoma and carcinoma, and 158 genes between primary carcinoma and liver metastasis (P < 0.001). Parallel analysis of our samples by CGH and expression profiling revealed a direct correlation of chromosomal copy number changes with chromosome-specific average gene expression levels. Protein expression was analyzed by two-dimensional gel electrophoresis and subsequent mass spectrometry. Although there was no direct match of differentially expressed proteins and genes, the majority of them belonged to identical pathways or networks. In conclusion, increasing genomic instability and a recurrent pattern of chromosomal imbalances as well as specific gene and protein expression changes correlate with distinct stages of colorectal cancer progression. Chromosomal aneuploidies directly affect average resident gene expression levels, thereby contributing to a massive deregulation of the cellular transcriptome. The identification of novel genes and proteins might deliver molecular targets for diagnostic and therapeutic interventions.

To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations.

Our aim was to map and compare genomic imbalances in human papillomavirus (HPV)-positive and -negative squamous cell carcinomas of the tonsil. Twenty-five primary carcinomas were analyzed by comparative genomic hybridization. Fifteen (60%) were found to be HPV-positive by PCR, and the majority were HPV-16. There were statistically significant differences in the distribution of DNA gains and losses between the HPV-positive and -negative samples. Eleven of 15 HPV-positive samples (73%) showed gain on chromosome 3q24-qter, while only 4/10 (40%) HPV-negative samples had the same gain (p ‫؍‬ 0.049). Furthermore, 4/10 (40%) HPV-negative samples but no HPV-positive samples had gain on chromosome 7q11.2-q22 (p ‫؍‬ 0.017). As expected, and similar to previous studies, patients with an HPV-positive tumor had a statistically significantly better disease-specific survival than patients with an HPV-negative tumor (p ‫؍‬ 0.002). Head-and-neck cancer constitutes 3.4% of all cancer cases each year in Europe 1 and is the fifth most common cancer type in the United States. 2 In approximately 50 -60% of patients, the tumor has spread to regional lymph nodes by the time of diagnosis, and it is known that formation of metastases reduces the chance of survival by about 50%. 2 Treatment of head-and-neck cancer has not improved greatly over the last years, and the 5-year survival rate remains low. 3 The main reasons for the low survival rate are advanced tumor stage at detection, high prevalence of recurrence and multiple primary tumors. 3 The major risk factors of head-and-neck squamous cell carcinoma (HNSCC) in the Western world are smoking and alcohol consumption. However, during the past 2 decades the role of high-risk human papillomavirus (HPV) has been studied, and data supporting HPV as a causative agent in the development and progression of a subset of these cancers have accumulated. 4 -6 The overall frequency of HPV in HNSCC is around 25-30%, with considerable variability depending on the tumor location. 7 The highest frequency is reported from studies on tonsillar cancer, 7-9 where 35-70% of tumors are HPV-positive, most commonly with HPV-16 and/or HPV-33. Furthermore, we and others have shown that patients with HPV-positive tonsillar cancer have a statistically significant reduction in risk of death from cancer compared to patients with HPV-negative tumors and that this is independent of tumor stage. 4,8 In addition, in a relatively limited study, patients with high viral load tumors had a significantly better prognosis compared to patients with low viral load tumors. 10 The fact that HPV is a favorable predictive/prognostic factor in tonsillar cancer prompted us to analyze whether differences in the pattern of chromosomal gains and losses were correlated with the presence of HPV. We hypothesized that such differences could explain the variable clinical course of HPV-positive cancer.In high-risk HPV-positive cancer of the tonsil, the early proteins E6 and E7 are generally expressed, 11 and it is known that the presence of t...