The transition of normal epithelium to invasive carcinoma occurs sequentially. In colorectal and cervical carcinogenesis, this transition is reflected by histomorphologically defined grades of increasing dysplasia that untreated may progress to invasive disease. In an attempt to understand the role of chromosomal aberrations during tumorigenesis we have applied comparative genomic hybridization using DNA extracted from defined stages of colorectal and cervical tumors, from low‐ and high‐grade astrocytic tumors and from diploid and aneuploid breast carcinomas. Genetic instability, as measured by the number of chromosomal copy alterations per case, increases significantly at the transition from precursor lesions to invasive carcinomas and continues to increase with tumor stage. Aggressive tumors have a higher number of copy alterations per case. High‐level copy number changes (amplifications) become more prevalent in advanced‐stage disease. Subtractive karyograms of chromosomal gains and losses were used to map tumor stage‐specific chromosomal aberrations and clearly showed that nonrandom chromosomal aberrations occur during disease progression. In colorectal and cervical tumors, chromosomal copy number changes were correlated with nuclear DNA content, proliferative activity, expression levels of the tumor suppressor gene TP53, and the cyclin‐dependent kinase inhibitor p21/WAF1, as well as the presence of viral genomes. Here we summarize and review the results of this comprehensive phenotype/genotype correlation and discuss the relevance of stage‐specific chromosomal aberrations with respect to diagnostic applications. Genes Chromosomes Cancer 25:195–204, 1999. Published 1999 Wiley‐Liss, Inc.
Ductal carcinoma in situ (DCIS) is a precursor lesion of invasive ductal carcinoma (IDC) of the breast. To understand the dynamics of genomic alterations in this progression, we used four multicolor fluorescence in situ hybridization probe panels consisting of the oncogenes COX2, MYC, HER2, CCND1, and ZNF217 and the tumor suppressor genes DBC2, CDH1, and TP53 to visualize copy number changes in 13 cases of synchronous DCIS and IDC based on single-cell analyses. The DCIS had a lower degree of chromosomal instability than the IDC. Despite enormous intercellular heterogeneity in DCIS and IDC, we observed signal patterns consistent with a nonrandom distribution of genomic imbalances. CDH1 was most commonly lost, and gain of MYC emerged during progression from DCIS to IDC. Four of 13 DCISs showed identical clonal imbalances in the IDCs. Six cases revealed a switch, and in four of those, the IDC had acquired a gain of MYC. In one case, the major clone in the IDC was one of several clones in the DCIS, and in another case, the major clone in the DCIS became one of the two major clones in the IDC. Despite considerable chromosomal instability, in most cases the evolution from DCIS to IDC is determined by recurrent patterns of genomic imbalances, consistent with a biological continuum.
Invasive cervical carcinomas frequently reveal additional copies of the long arm of chromosome 3. The detection of this genetic aberration in diagnostic samples could therefore complement the morphological interpretation. We have developed a triple-color DNA probe set for the visualization of chromosomal copy number changes directly in thin-layer cervical cytology slides by fluorescence in situ hybridization. The probe set consists of a BAC contig that contains sequences for the RNA component of the human telomerase gene (TERC) on chromosome band 3q26, and repeat sequences specific for the centromeres of chromosomes 3 and 7 as controls. In a blinded study, we analyzed 57 thin-layer slides that had been rigorously screened and classified as normal (n ؍ 13), atypical squamous cells (ASC, n ؍ 5), low-grade squamous intraepithelial lesions (LSIL, n ؍ 14), and highgrade squamous intraepithelial lesions ( Cytologic screening 1 has greatly reduced incidence and mortality of cervical cancer in industrialized nations.2 In developing countries, however, cervical cancer remains a health problem of tremendous proportions. If detected in a timely manner, cervical cancer precursors, especially high-grade squamous intraepithelial lesions (HSILs) can be effectively treated, sparing patients the morbidity and mortality resulting from invasive cancer. Despite its success as a public health measure, a single cytologic examination is relatively insensitive, poorly reproducible and frequently yields equivocal results. Inadequate sampling, the scarcity of aberrant cells in some samples and the subjectivity of morphological interpretation are recognized limitations of cytology. 2,3 In addition, equivocal and mild cytologic abnormalities are extremely common in young women, but most of these lesions regress spontaneously, even when caused by oncogenic types of human papillomaviruses, 4,5 which play a crucial role in the pathogenesis of cervical cancer. 6,7 This has prompted efforts to discover other biomarkers and other screening techniques with the potential to supplement cytologic screening. 8 -13
Invasive cervical carcinomas almost invariably carry extra copies of chromosome arm 3q, resulting in a gain of the human telomerase gene (TERC). This provided the rationale for the development of a multicolor fluorescence in situ hybridization (FISH) probe set as a diagnostic tool for the direct detection of TERC gains in Pap smears. We previously used this probe set to show that cervical intraepithelial neoplasia (CIN) 2 and CIN3 lesions could be distinguished from normal samples, atypical squamous cell of undetermined significance (ASCUS) and CIN1, with a sensitivity and specificity exceeding 90%, independent of the cytomorphological assessment. In the current study, we explored whether gain of 3q and amplification of TERC could predict progression from CIN1/CIN2 to CIN3 and invasive carcinoma. We applied our probe set to a series of 59 previously stained Pap smears for which repeat Pap smears and clinical follow-up were available. The samples included CIN1/ CIN2 lesions that progressed to CIN3 (progressors), CIN1/CIN2 lesions that regressed spontaneously (regressors), and normal Pap smears from women who subsequently developed CIN3 or cervical cancer. Here, we show that progressors displayed a gain of 3q whereas none of the regressors showed this genetic aberration. These data suggest that 3q gain is required for the transition from CIN1/CIN2 to CIN3 and that it predicts progression. Of note, 3q gain was found in 33% of cytologically normal Pap smears from women who were diagnosed with CIN3 or invasive cervical carcinoma after a short latency. The sensitivity of our test for predicting progression from CIN1/CIN2 to CIN3 was 100% and the specificity, ie, the prediction of regression, was 70%. We conclude that the detection of 3q gain and amplification of TERC in routinely collected Pap smears can assist in identifying lowgrade lesions with a high progression risk and in decreasing false-negative cytological screenings.
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...
We present methods to construct phylogenetic models of tumor progression at the cellular level that include copy number changes at the scale of single genes, entire chromosomes, and the whole genome. The methods are designed for data collected by fluorescence in situ hybridization (FISH), an experimental technique especially well suited to characterizing intratumor heterogeneity using counts of probes to genetic regions frequently gained or lost in tumor development. Here, we develop new provably optimal methods for computing an edit distance between the copy number states of two cells given evolution by copy number changes of single probes, all probes on a chromosome, or all probes in the genome. We then apply this theory to develop a practical heuristic algorithm, implemented in publicly available software, for inferring tumor phylogenies on data from potentially hundreds of single cells by this evolutionary model. We demonstrate and validate the methods on simulated data and published FISH data from cervical cancers and breast cancers. Our computational experiments show that the new model and algorithm lead to more parsimonious trees than prior methods for single-tumor phylogenetics and to improved performance on various classification tasks, such as distinguishing primary tumors from metastases obtained from the same patient population.
Motivation: Development and progression of solid tumors can be attributed to a process of mutations, which typically includes changes in the number of copies of genes or genomic regions. Although comparisons of cells within single tumors show extensive heterogeneity, recurring features of their evolutionary process may be discerned by comparing multiple regions or cells of a tumor. A useful source of data for studying likely progression of individual tumors is fluorescence in situ hybridization (FISH), which allows one to count copy numbers of several genes in hundreds of single cells. Novel algorithms for interpreting such data phylogenetically are needed, however, to reconstruct likely evolutionary trajectories from states of single cells and facilitate analysis of tumor evolution.Results: In this article, we develop phylogenetic methods to infer likely models of tumor progression using FISH copy number data and apply them to a study of FISH data from two cancer types. Statistical analyses of topological characteristics of the tree-based model provide insights into likely tumor progression pathways consistent with the prior literature. Furthermore, tree statistics from the resulting phylogenies can be used as features for prediction methods. This results in improved accuracy, relative to unstructured gene copy number data, at predicting tumor state and future metastasis.Availability: Source code for software that does FISH tree building (FISHtrees) and the data on cervical and breast cancer examined here are available at ftp://ftp.ncbi.nlm.nih.gov/pub/FISHtrees.Contact: sachowdh@andrew.cmu.eduSupplementary information: Supplementary data are available at Bioinformatics online.
Cell fusion in vitro has been used to study cancer, gene mapping and regulation, and the production of antibodies via hybridomas. However, in-vivo heterosynkaryon formation by cell-cell fusion has received less attention. This investigation describes the spontaneous fusion of a human glioblastoma with normal hamster cells after xenogeneic transplantation, resulting in malignant cells that express both human and hamster genes and gene products, and retention of glioblastoma traits with an enhanced ability to metastasize. Three of 7 human genes found showed translation of their proteins during serial propagation in vivo or in vitro for years; namely, CD74, CXCR4, and PLAGL2, each implicated with malignancy or glioblastoma. This supports the thesis that genetic hybridization of cancer and normal cells can transmit malignancy and also, as first described herein, regulatory genes involved in the tumor’s organotypic morphology. Evidence also is increasing that even cell-free human cancer DNA can induce malignancy and transfer genetic information to normal cells. Hence, we posit that the transfer of genetic information between tumor and stromal cells, whether by cell-cell fusion or other mechanisms, is implicated in the progression of malignancy, and may further define the crosstalk between cancer cells and their stromal neighbors.
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