The Evolution of Single Cell-derived Colorectal Cancer Cell Lines is Dominated by the Continued Selection of Tumor Specific Genomic Imbalances, Despite Random Chromosomal Instability

Intratumor heterogeneity is a major challenge in cancer treatment. To decipher patterns of chromosomal heterogeneity, we analyzed six colorectal cancer cell lines by multiplex interphase FISH (miFISH). The mismatch repair deficient cell lines DLD-1 and HCT116 had the most stable copy numbers, whereas aneuploid cell lines (HT-29, SW480, SW620 and H508) displayed a higher degree of instability. We subsequently assessed the clonal evolution of single cells in two CRC cell lines, SW480 and HT-29, which both have aneuploid karyotypes but different degrees of chromosomal instability. The clonal compositions of the single cell-derived daughter lines, as assessed by miFISH, differed for HT-29 and SW480. Daughters of HT-29 were stable, clonal, with little heterogeneity. Daughters of SW480 were more heterogeneous, with the single cell-derived daughter lines separating into two distinct populations with different ploidy (hyper-diploid and near-triploid), morphology, gene expression and tumorigenicity. To better understand the evolutionary trajectory for the two SW480 populations, we constructed phylogenetic trees which showed ongoing instability in the daughter lines. When analyzing the evolutionary development over time, most single cell-derived daughter lines maintained their major clonal pattern, with the exception of one daughter line that showed a switch involving a loss of APC. Our meticulous analysis of the clonal evolution and composition of these colorectal cancer models shows that all chromosomes are subject to segregation errors, however, specific net genomic imbalances are maintained. Karyotype evolution is driven by the necessity to arrive at and maintain a specific plateau of chromosomal copy numbers as the drivers of carcinogenesis

Single-Cell Genetic Analysis Reveals Insights into Clonal Development of Prostate Cancers and Indicates Loss of PTEN as a Marker of Poor Prognosis

Gauging the risk of developing progressive disease is a major challenge in prostate cancer patient management. We used genetic markers to understand genomic alteration dynamics during disease progression. By using a novel, advanced, multicolor fluorescence in situ hybridization approach, we enumerated copy numbers of six genes previously identified by array comparative genomic hybridization to be involved in aggressive prostate cancer [TBL1XR1, CTTNBP2, MYC (alias c-myc), PTEN, MEN1, and PDGFB] in six nonrecurrent and seven recurrent radical prostatectomy cases. An ERG break-apart probe to detect TMPRSS2-ERG fusions was included. Subsequent hybridization of probe panels and cell relocation resulted in signal counts for all probes in each individual cell analyzed. Differences in the degree of chromosomal and genomic instability (ie, tumor heterogeneity) or the percentage of cells with TMPRSS2-ERG fusion between samples with or without progression were not observed. Tumors from patients that progressed had more chromosomal gains and losses, and showed a higher degree of selection for a predominant clonal pattern. PTEN loss was the most frequent aberration in progressers (57%), followed by TBL1XR1 gain (29%). MYC gain was observed in one progresser, which was the only lesion with an ERG gain, but no TMPRSS2-ERG fusion. According to our results, a probe set consisting of PTEN, MYC, and TBL1XR1 would detect progressers with 86% sensitivity and 100% specificity. This will be evaluated further in larger studies

Genomic changes defining the progression of human colorectal and cervical tumors

Defining changes during the carcinogenesis and progression of tumors is a major way to obtain a better understanding of the mechanisms of cancer development. We therefore investigated the cacinogenesis process in the colon-rectum and in the uterine cervix by different cellchemical, immunohistochemical and cytogenetic methods. Cell proliferation, assessed by immunohistochemical detection of the Ki-67 antigen (MIB 1 antibody), DNA ploify, determined by image cytometry, expression of the tumor suppressor gene p53 (antibody DO1) and the cycline- dependent kinase inhibitor p21/WAF1 (antibody EA10), detected immunohistochemically, and chromosomal aberrations, determined by comparative genomic hybridization (CGH), were monitored in a series of normal mucosa, low and high grade ademonas and adenocarcinomas of the colon-rectum (mainly pT3-tumors)and in a series of normal epithelium, mild, moderate and severe dysplasias/CIS and early stage squamous cell carcinomas (stage I) of the uterin cervix. The cervix material was also tested for human papillomavirus (HPW) infection. To investigate the behaviour of this panel of markers during tumor progression we additionally examined a material of advanced cervical carcinomas (stage II-IV). A comparison of the two carcinogenesis models showed that in both systems proliferation increases rapidly with increasing grade of atypia of the lesions. 60-90% of the tumor cells of the high grade adenomas, the severe dysplasias/CIS and the invasive carcinomas stained positively for MIB1. DNA ploidy measurements showed that aneuoploidy in colorectal carcinogenesis often developed via triploid DNA distribution patterns while in cervical lesions a tetraploidisation of the cells preceded crude aneuploidy. Carcinomas of both the colon and the uterine cervix showed high percentages of aneuploidy (80-95%) Thirty-eight percent (6/16) of the colon carcinomas exhibited stron expression of p53 protein but were negative for p21/WAF1 expression which could possibly be interpreted as being due to a mutation of the p53 gene. Almost all cervical carcinomas showed a weak p53 expression combined with a relatively strong p21-WAF1 expression. p53 PCR-SSCP did not reveale any mutations in 10 stage I cervix carcinomas (SSCP data not published) indicating that the immunohistochemically detected p53 expression is due to wild type p53. All of the severe cervical dysplasias, 90% of the stage I cervix carcinomas and 83% of the advanced cervix carcinomas (stage IIb-IV) were positive for high risk HPW infection. High risk HPW E6 region is known to be able to degrade p53 wild type protein which could explain why there is no need to block the p53-p21/WAF1 cell cycle control pathway by mutations of the p53 gene. On the other hand it is not clear whether the strong expression of p21/WAF1 indicates that the inactivation of p53 via the E6-proteins is incomplete, or whether p21/WAF1 expression is up-regulated via alternative regulatory pathways. The screening for chromosomal aberrations showed that the striking major CGH event in cervical cancer was a gain of the long arm of chromosome 3 which was detected in 1 of 13 severe dysplasias, 9 of 10 stage I carcinomas and 23 of 30 advanced cervical carcinomas, wild colorectal carcinomas exhibited a large number of different changes with gains on 20q (12/16 cases), 13q (8/16 cases), 8q (8/16) and 7p (8/16) and losses on 8p (7/16) and 18q (6/16) as the most frequent ones. In advanced cervical carcinomas acquisition of genetic material also occurred frequently on chromosomes 1q (14/30) and 5p (9/30) and recurrent losses were mapped to chromosomes 3p (15/30), 2q (10/30), 6q (8/30) and 13q (8/30). The average number of chromosomal aberrations (ANCA) was 3,8 for the stage I cervical carcinomas, 5.6 for the colorectal adenocarcinomas (mainly pT3-tumors) and 8.2 for the advanced cervical carcinomas stage IIb-IV indicating that ANCA is tumor stage-correlated. The investigation of anal carcinomas as an example of another HPV-related tumor revealed a very similar picture to the results obtained for the uterine cervix concerning proliferation, DNA ploidy and p21/WAF1 expression. One exceptional case showed a strong p53 expression, p21/WAF1 negativity and a high level copy number increase on 2p23-24. 56% of the cases which could be evaluated by HPV-analysis tested positively. Non- random copy number increases of chromosomes 17 (9/23) and 19 (14/23) and chromome arm 3q (7/23) and consistent losses on 4p (7/23), 11q (9/23), 13q (6/23) and 18q (8/23) could be observed. The average number of chromosomal aberrations (ANCA) was 4.8. Major differences in the carcinogenesis of colorectal cancer and the HPV-related cancers of the uterine cervix and the anus concerning DNA ploidy, p53-p21/WAF1 expression pattern and chromosomal aberrations can be observed. A recurrent pattern of chromosomal aberrations defines each of the carcinoma types. The most common chromosomal aberration in cervical carcinogenesis is a gain on chromosome 3q which becomes visible at the transition from severe dysplasia/CIS to early stage carcinomas. 3q gain also plays a major role in HPV-infected anal carcinomas. Progression of cervical carcinomas is correlated to an increased frequency of specific chromosomal aberrations with 3q gain still playing the predominant role. Key words: colorectal, cervical, anal neoplasias; proliferation, DNA ploidy, p53 and p21/WAF1 expression, HPV, CGH ISBN 91-628-2251-

Automated Analysis of Protein Expression and Gene Amplification within the Same Cells of Paraffin-Embedded Tumour Tissue

Background: The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH) negatively influence each other which often results in suboptimal staining. Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell

Algorithms to model single gene, single chromosome, and whole genome copy number changes jointly in tumor phylogenetics.

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

FISHtrees 3.0: Tumor Phylogenetics Using a Ploidy Probe.

Advances in fluorescence in situ hybridization (FISH) make it feasible to detect multiple copy-number changes in hundreds of cells of solid tumors. Studies using FISH, sequencing, and other technologies have revealed substantial intra-tumor heterogeneity. The evolution of subclones in tumors may be modeled by phylogenies. Tumors often harbor aneuploid or polyploid cell populations. Using a FISH probe to estimate changes in ploidy can guide the creation of trees that model changes in ploidy and individual gene copy-number variations. We present FISHtrees 3.0, which implements a ploidy-based tree building method based on mixed integer linear programming (MILP). The ploidy-based modeling in FISHtrees includes a new formulation of the problem of merging trees for changes of a single gene into trees modeling changes in multiple genes and the ploidy. When multiple samples are collected from each patient, varying over time or tumor regions, it is useful to evaluate similarities in tumor progression among the samples. Therefore, we further implemented in FISHtrees 3.0 a new method to build consensus graphs for multiple samples. We validate FISHtrees 3.0 on a simulated data and on FISH data from paired cases of cervical primary and metastatic tumors and on paired breast ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Tests on simulated data show improved accuracy of the ploidy-based approach relative to prior ploidyless methods. Tests on real data further demonstrate novel insights these methods offer into tumor progression processes. Trees for DCIS samples are significantly less complex than trees for paired IDC samples. Consensus graphs show substantial divergence among most paired samples from both sets. Low consensus between DCIS and IDC trees may help explain the difficulty in finding biomarkers that predict which DCIS cases are at most risk to progress to IDC. The FISHtrees software is available at ftp://ftp.ncbi.nih.gov/pub/FISHtrees

Detection of Genomic Amplification of the Human Telomerase Gene TERC, a Potential Marker for Triage of Women with HPV-Positive, Abnormal Pap Smears

The vast majority of invasive cervical carcinomas harbor additional copies of the chromosome arm 3q, resulting in genomic amplification of the human telomerase gene TERC. Here, we evaluated TERC amplification in routinely collected liquid based cytology (LBC) samples with histologically confirmed diagnoses. A set of 78 LBC samples from a Swedish patient cohort were analyzed with a four-color fluorescence in situ hybridization probe panel that included TERC. Clinical follow-up included additional histological evaluation and Pap smears. Human papillomavirus status was available for all cases. The correlation of cytology, TERC amplification, human papillomavirus typing, and histological diagnosis showed that infection with high-risk human papillomavirus was detected in 64% of the LBC samples with normal histopathology, in 65% of the cervical intraepithelial neoplasia (CIN)1, 95% of the CIN2, 96% of the CIN3 lesions, and all carcinomas. Seven percent of the lesions with normal histopathology were positive for TERC amplification, 24% of the CIN1, 64% of the CIN2, 91% of the CIN3 lesions, and 100% of invasive carcinomas. This demonstrates that detection of genomic amplification of TERC in LBC samples can identify patients with histopathologically confirmed CIN3 or cancer. Indeed, the proportion of TERC-positive cases increases with the severity of dysplasia. Among the markers tested, detection of TERC amplification in cytological samples has the highest combined sensitivity and specificity for discernment of low-grade from high-grade dysplasia and cancer