Array-CGH and multipoint FISH to decode complex chromosomal rearrangements

BACKGROUND: Recently, several high-resolution methods of chromosome analysis have been developed. It is important to compare these methods and to select reliable combinations of techniques to analyze complex chromosomal rearrangements in tumours. In this study we have compared array-CGH (comparative genomic hybridization) and multipoint FISH (mpFISH) for their ability to characterize complex rearrangements on human chromosome 3 (chr3) in tumour cell lines. We have used 179 BAC/PAC clones covering chr3 with an approximately 1 Mb resolution to analyze nine carcinoma lines. Chr3 was chosen for analysis, because of its frequent rearrangements in human solid tumours. RESULTS: The ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. Chr3 locus copy number was assessed by interphase and metaphase mpFISH. Totally 53 chr3 fragments were identified having copy numbers from 0 to 14. MpFISH results from the BAC/PAC clones and array-CGH gave mainly corresponding results. Each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpFISH. The analysis of the correlation between real copy number from mpFISH and the average normalized inter-locus fluorescence ratio (ANILFR) value detected by array-CGH demonstrated that copy number is a linear function of parameters that include the variable, ANILFR, and two constants, ploidy and background normalized fluorescence ratio. CONCLUSION: In most cases, the changes in copy number seen on array-CGH profiles reflected cumulative chromosome rearrangements. Most of them stemmed from unbalanced translocations. Although our chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels

Mandatory chromosomal segment balance in aneuploid tumor cells

Copyright: Copyright 2013 Elsevier B.V., All rights reserved.Background: Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost. Methods: Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids. Results: In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region. Conclusion: The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.publishersversionPeer reviewe

Fragile Genomic Sites Are Associated with Origins of Replication

Genome rearrangements are mediators of evolution and disease. Such rearrangements are frequently bounded by transfer RNAs (tRNAs), transposable elements, and other repeated elements, suggesting a functional role for these elements in creating or repairing breakpoints. Though not well explored, there is evidence that origins of replication also colocalize with breakpoints. To investigate a potential correlation between breakpoints and origins, we analyzed evolutionary breakpoints defined between Saccharomyces cerevisiae and Kluyveromyces waltii and S. cerevisiae and a hypothetical ancestor of both yeasts, as well as breakpoints reported in the experimental literature. We find that origins correlate strongly with both evolutionary breakpoints and those described in the literature. Specifically, we find that origins firing earlier in S phase are more strongly correlated with breakpoints than are later-firing origins. Despite origins being located in genomic regions also bearing tRNAs and Ty elements, the correlation we observe between origins and breakpoints appears to be independent of these genomic features. This study lays the groundwork for understanding the mechanisms by which origins of replication may impact genome architecture and disease

Segmental Duplications Arise from Pol32-Dependent Repair of Broken Forks through Two Alternative Replication-Based Mechanisms

The propensity of segmental duplications (SDs) to promote genomic instability is of increasing interest since their involvement in numerous human genomic diseases and cancers was revealed. However, the mechanism(s) responsible for their appearance remain mostly speculative. Here, we show that in budding yeast, replication accidents, which are most likely transformed into broken forks, play a causal role in the formation of SDs. The Pol32 subunit of the major replicative polymerase Polδ is required for all SD formation, demonstrating that SDs result from untimely DNA synthesis rather than from unequal crossing-over. Although Pol32 is known to be required for classical (Rad52-dependant) break-induced replication, only half of the SDs can be attributed to this mechanism. The remaining SDs are generated through a Rad52-independent mechanism of template switching between microsatellites or microhomologous sequences. This new mechanism, named microhomology/microsatellite-induced replication (MMIR), differs from all known DNA double-strand break repair pathways, as MMIR-mediated duplications still occur in the combined absence of homologous recombination, microhomology-mediated, and nonhomologous end joining machineries. The interplay between these two replication-based pathways explains important features of higher eukaryotic genomes, such as the strong, but not strict, association between SDs and transposable elements, as well as the frequent formation of oncogenic fusion genes generating protein innovations at SD junctions

Lineage-specific evolution of the vertebrate Otopetrin gene family revealed by comparative genomic analyses

Background: Mutations in the Otopetrin 1 gene (Otop1) in mice and fish produce an unusual bilateral vestibular pathology that involves the absence of otoconia without hearing impairment. The encoded protein, Otop1, is the only functionally characterized member of the Otopetrin Domain Protein (ODP) family; the extended sequence and structural preservation of ODP proteins in metazoans suggest a conserved functional role. Here, we use the tools of sequence-and cytogenetic-based comparative genomics to study the Otop1 and the Otop2-Otop3 genes and to establish their genomic context in 25 vertebrates. We extend our evolutionary study to include the gene mutated in Usher syndrome (USH) subtype 1G (Ush1g), both because of the head-to-tail clustering of Ush1g with Otop2 and because Otop1 and Ush1g mutations result in inner ear phenotypes. Results: We established that OTOP1 is the boundary gene of an inversion polymorphism on human chromosome 4p16 that originated in the common human-chimpanzee lineage more than 6 million years ago. Other lineage-specific evolutionary events included a three-fold expansion of the Otop genes in Xenopus tropicalis and of Ush1g in teleostei fish. The tight physical linkage between Otop2 and Ush1g is conserved in all vertebrates. To further understand the functional organization of the Ushg1-Otop2 locus, we deduced a putative map of binding sites for CCCTC-binding factor (CTCF), a mammalian insulator transcription factor, from genome-wide chromatin immunoprecipitation-sequencing (ChIP-seq) data in mouse and human embryonic stem (ES) cells combined with detection of CTCF-binding motifs. Conclusions: The results presented here clarify the evolutionary history of the vertebrate Otop and Ush1g families, and establish a framework for studying the possible interaction(s) of Ush1g and Otop in developmental pathways

Characterising chromosome rearrangements: recent technical advances in molecular cytogenetics

Genomic rearrangements can result in losses, amplifications, translocations and inversions of DNA fragments thereby modifying genome architecture, and potentially having clinical consequences. Many genomic disorders caused by structural variation have initially been uncovered by early cytogenetic methods. The last decade has seen significant progression in molecular cytogenetic techniques, allowing rapid and precise detection of structural rearrangements on a whole-genome scale. The high resolution attainable with these recently developed techniques has also uncovered the role of structural variants in normal genetic variation alongside single-nucleotide polymorphisms (SNPs). We describe how array-based comparative genomic hybridisation, SNP arrays, array painting and next-generation sequencing analytical methods (read depth, read pair and split read) allow the extensive characterisation of chromosome rearrangements in human genomes

Involvement of evolutionarily plastic regions in cancer associated CHR3 aberrations

A functional test to identify tumor antagonizing regions on chromosome 3 (chr3), called the Elimination Test, was developed in our group. It is based on microcell mediated transfer of human chr3 into mouse or human tumor cells and analysis of the monochromosomal hybrids after their growth in vivo. We identified three regions on 3p14-p22, which were frequently lost in derived tumors (common or frequently eliminated regions). In order to understand the role of these regions in tumor development, we continued the study following two leads: analysis of breakpoint clusters to identify and characterize possible instability features and search for tumor suppressor genes within the deleted regions. First, we identified and characterized a common eliminated region 1 (CER1) homologous sequence in mouse, where it was divided into two syntenic blocks on chromosome 9. In these blocks, the gene order and content was maintained with exception of two mouse gene duplications. Comparative analysis helped us to characterize five previously not identified mouse genes (Kiss et al. 2002). A more extensive comparative study of CER1 showed that its border regions are characterized by evolutionary plasticity: synteny breaks in several species, recent tandem gene duplications, retroposed pseudogene insertions, and horizontal evolution of the genes. Thus we showed that the cancer associated breakpoint regions have features of evolutionary plasticity. These results and other publications from our group suggested structural instability at the borders of eliminated regions identified by the Elimination Test (Darai et al. 2005) (Kost-Alimova et al. 2003; Kost-Alimova et al. 2004). As a next step, in order to analyze rearrangements of entire chr3 in human tumor cells, we developed and compared two high resolution methods, array-CGH and mpFISH. We proved that although our 1Mb chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels. In heterogeneous or normal cell contaminated samples the most precise analysis can be made by mpFISH due to its ability to give information at single cell level (Darai-Ramqvist et al. 2006). Using high resolution methods we analyzed ten carcinoma cell lines and identified two new hot spots of tumor breakpoints at 3p12-p13 and 3q21. These tumor breakpoint regions carried large segmental duplications, retrotransposable elements and satellite repeats, which participated in recent primate evolution and, as we suggest, are associated with structural chromosomal instability (CIN). CIN is an ongoing dynamic process. Therefore in order to prove that the instability at the breakpoint regions characterizes structural CIN phenotype and it is required for tumor development and progression, dynamic analysis of the tumors must be done. This may elucidate the mechanism of tumor development; and may help to develop CIN phenotype markers useful in choice of consequent treatment. Following the second lead of our study, we have analyzed a putative tumor suppressor gene LIMD1, which is located within the deleted central part of CER1. We found that it binds specifically to pRb and suppresses E2F driven transcription. A tumor suppressor effect of this gene was proven in in vitro and in vivo experiments, as well as in tumor biopsies (Sharp et al. 2004). In another part of the study we analyzed in details chr3 rearrangements in human renal cell carcinoma and nasopharyngeal carcinoma derived monochromosomal (chr3) hybrids and showed that aneuploid tumors maintain a mandatory chromosomal segment balance with stringency concerning no gain of 3p14-21 and no loss of 3q26-27. We concluded that the mechanism of tumor suppression by chr3 transfer is based on the alternative quantitative model. According to this model the tumor cell does not tolerate an increased dosage of the relevant gene or segment, and the lost part can be either of normal cell derived exogeneous or tumor derived endogenous origin (Kost-Alimova et al. 2007). First, we identified and characterized a common eliminated region 1 (CER1) homologous sequence in mouse, where it was divided into two syntenic blocks on chromosome 9. In these blocks, the gene order and content was maintained with exception of two mouse gene duplications. Comparative analysis helped us to characterize five previously not identified mouse genes (Kiss et al. 2002). A more extensive comparative study of CER1 showed that its border regions are characterized by evolutionary plasticity: synteny breaks in several species, recent tandem gene duplications, retroposed pseudogene insertions, and horizontal evolution of the genes. Thus we showed that the cancer associated breakpoint regions have features of evolutionary plasticity. These results and other publications from our group suggested structural instability at the borders of eliminated regions identified by the Elimination Test (Darai et al. 2005) (Kost-Alimova et al. 2003; Kost-Alimova et al. 2004). As a next step, in order to analyze rearrangements of entire chr3 in human tumor cells, we developed and compared two high resolution methods, array-CGH and mpFISH. We proved that although our 1Mb chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels. In heterogeneous or normal cell contaminated samples the most precise analysis can be made by mpFISH due to its ability to give information at single cell level (Darai-Ramqvist et al. 2006). Using high resolution methods we analyzed ten carcinoma cell lines and identified two new hot spots of tumor breakpoints at 3p12-p13 and 3q21. These tumor breakpoint regions carried large segmental duplications, retrotransposable elements and satellite repeats, which participated in recent primate evolution and, as we suggest, are associated with structural chromosomal instability (CIN). CIN is an ongoing dynamic process. Therefore in order to prove that the instability at the breakpoint regions characterizes structural CIN phenotype and it is required for tumor development and progression, dynamic analysis of the tumors must be done. This may elucidate the mechanism of tumor development; and may help to develop CIN phenotype markers useful in choice of consequent treatment. Following the second lead of our study, we have analyzed a putative tumor suppressor gene LIMD1, which is located within the deleted central part of CER1. We found that it binds specifically to pRb and suppresses E2F driven transcription. A tumor suppressor effect of this gene was proven in in vitro and in vivo experiments, as well as in tumor biopsies (Sharp et al. 2004). In another part of the study we analyzed in details chr3 rearrangements in human renal cell carcinoma and nasopharyngeal carcinoma derived monochromosomal (chr3) hybrids and showed that aneuploid tumors maintain a mandatory chromosomal segment balance with stringency concerning no gain of 3p14-21 and no loss of 3q26-27. We concluded that the mechanism of tumor suppression by chr3 transfer is based on the alternative quantitative model. According to this model the tumor cell does not tolerate an increased dosage of the relevant gene or segment, and the lost part can be either of normal cell derived exogeneous or tumor derived endogenous origin (Kost-Alimova et al. 2007)

Interventional and EBUS cytology in Sweden

Interventional cytology was first introduced in Sweden in the late 1940ies by Sixten Franzén at the Karolinska University Hospital in Solna, Stockholm. In the early 1950ies, Nils Söderström started using the technique at the University Hospital in Lund. Cytology was successively established as common practice at the pathology departments in Sweden, and e.g. Solna and Lund today have a high rate of cytological samples. Over the years new techniques, such as endobronchial ultrasound (EBUS)-guided fine-needle aspirations, and analyses have been introduced, contributing to the maintained value of cytology as a diagnostic method. In this article, we present a brief history and the current situation of cytology in Sweden with focus on interventional and EBUS cytology

Microenvironment-Dependent Phenotypic Changes in a SCID Mouse Model for Malignant Mesothelioma

Background and Aims: Malignant mesothelioma is an aggressive, therapy-resistant tumor. Mesothelioma cells may assume an epithelioid or a sarcomatoid phenotype, and presence of sarcomatoid cells predicts poor prognosis. In this study, we investigated differentiation of mesothelioma cells in a xenograft model, where mesothelioma cells of both phenotypes were induced to form tumors in severe combined immunodeficiency mice. Methods: Xenografts were established and thoroughly characterized using a comprehensive immunohistochemical panel, array comparative genomic hybridization (aCGH) of chromosome 3, fluorescent in situ hybridization, and electron microscopy. Results: Epithelioid and sarcomatoid cells gave rise to xenografts of similar epithelioid morphology. While sarcomatoid-derived xenografts had higher growth rates, the morphology and expression of differentiation-related markers was similar between xenografts derived from both phenotypes. aCGH showed a convergent genotype for both xenografts, resembling the original aggressive sarcomatoid cell sub-line. Conclusion: Human mesothelioma xenografts from sarcomatoid and epithelioid phenotypes converged to a similar differentiation state, and genetic analyses suggested that clonal selection in the mouse microenvironment was a major contributing factor. This thoroughly characterized animal model can be used for further studies of molecular events underlying tumor cell differentiation