Chromothripsis is the massive but highly localized chromosomal rearrangement in response to a one-step catastrophic event, rather than an accumulation of a series of subsequent and random alterations. Chromothripsis occurs commonly in various human cancers and is thought to be associated with increased malignancy and carcinogenesis. However, the causes and consequences of chromothripsis remain unclear. Therefore, to identify the mechanism underlying the generation of chromothripsis, we investigated whether chromothripsis could be artificially induced by ionizing radiation. We first elicited DNA double-strand breaks in an oral squamous cell carcinoma cell line HOC313-P and its highly metastatic subline HOC313-LM, using Single Particle Irradiation system to Cell (SPICE), a focused vertical microbeam system designed to irradiate a spot within the nuclei of adhesive cells, and then established irradiated monoclonal sublines from them, respectively. SNP array analysis detected a number of chromosomal copy number alterations (CNAs) in these sublines, and one HOC313-LM-derived monoclonal subline irradiated with 200 protons by the microbeam displayed multiple CNAs involved locally in chromosome 7. Multi-color FISH showed a complex translocation of chromosome 7 involving chromosomes 11 and 12. Furthermore, whole genome sequencing analysis revealed multiple de novo complex chromosomal rearrangements localized in chromosomes 2, 5, 7, and 20, resembling chromothripsis. These findings suggested that localized ionizing irradiation within the nucleus may induce chromothripsis-like complex chromosomal alterations via local DNA damage in the nucleus.

Digital object identifier (DOI): 10.18632/oncotarget.7186

Biliary tract carcinoma is a rare malignancy with multiple causes, which underlie the different genetic and molecular profiles. Cancer cell lines are affordable models, reflecting the characteristics of the tumor of origin. They represent useful tools to identify molecular targets for treatment. Here, we established and characterized from biological, molecular, and genetic point of view, an Italian intrahepatic cholangiocarcinoma cell line (ICC), the MT-CHC01. MT-CHC01 cells were isolated from a tumor-derived xenograft. Immunophenotypical characterization was evaluated both at early and after stabilization passages. In vitro biological, genetic, and molecular features were also investigated. In vivo tumorigenicity was assessed in NOD/SCID mice. MT-CHC01cells retain epithelial cell markers, EPCAM, CK7, and CK19, and some stemness and pluripotency markers, i.e., SOX2, Nanog, CD49f/integrin-α6, CD24, PDX1, FOXA2, and CD133. They grow as a monolayer, with a population double time of about 40 h; they show a low migration and invasion potential. In low attachment conditions, they are able to form spheres and to growth in anchorage-independent manner. After subcutaneous injection, they retain in vivo tumorigenicity; the expression of biliary markers as CA19-9 and CEA were maintained from primary tumor. The karyotype is highly complex, with a hypotriploid to hypertriploid modal number (3n+/-) (52 to 77 chromosomes); low level of HER2 gene amplification, TP53 deletion, gain of AURKA were identified; K-RAS G12D mutation were maintained from primary tumor to MT-CHC01 cells. We established the first ICC cell line derived from an Italian patient. It will help to study either the biology of this tumor or to test drugs both in vitro and in vivo.

Digital object identifier (DOI): 10.1007/s13277-015-4215-3

All colorectal cancer cell lines except RKO displayed active β-catenin/TCF regulated transcription. This feature of RKO was noted in familial colon cancers; hence our aim was to dissect its carcinogenic mechanism. MFISH and CGH revealed distinct instability of chromosome structure in RKO. Gene expression microarray of RKO versus 7 colon cancer lines (with active Wnt signaling) and 3 normal specimens revealed 611 differentially expressed genes. The majority of the tested gene loci were susceptible to LOH in primary tumors with various β-catenin localizations as a surrogate marker for β-catenin activation. The immunohistochemistry of selected genes (IFI16, RGS4, MCTP1, DGKI, OBCAM/OPCML, and GLIPR1) confirmed that they were differentially expressed in clinical specimens. Since epigenetic mechanisms can contribute to expression changes, selected target genes were evaluated for promoter methylation in patient specimens from sporadic and hereditary colorectal cancers. CMTM3, DGKI, and OPCML were frequently hypermethylated in both groups, whereas KLK10, EPCAM, and DLC1 displayed subgroup specificity. The overall fraction of hypermethylated genes was higher in tumors with membranous β-catenin. We identified novel genes in colorectal carcinogenesis that might be useful in personalized tumor profiling. Tumors with inactive Wnt signaling are a heterogeneous group displaying interaction of chromosomal instability, Wnt signaling, and epigenetics.

Digital object identifier (DOI): 10.1155/2016/6089658

Human tumors using the alternative lengthening of telomeres (ALT) exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction-driven chromosomal instability in neoplasia (CIN) in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines. We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted. We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs) were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth.

A variant of del(20q), an isochromosome of the long arm with the loss of an interstitial part of 20q, ider(20q), has been reported in patients with myeloid diseases (Li et al, 2004). About 40 cases with this rearrangement have been reported up to 2012 (reviewed by Mullier et al, 2012). Molecular cytogenetic and array techniques have been used for mapping of the deleted region on 20q (Douet-Guilbert et al, 2009). The proximal breakpoints are consistently located in the 20q11.21 band, and the distal breakpoints span from band 20q13.13 to band 20q13.33.

<p>We report the case of an 84 years old prostate cancer patient with severe side effects after radiotherapy in 2006. He was cytogenetically analysed in 2009 and in 2012 in a comparative study for individual radiosensitivity of prostate cancer patients. No other patient had clonal aberrations, but this patient showed ring chromosomes in the range of 21-25% of lymphocytes. He received 5 cycles of 5-fluorouracil/folic acid for chemotherapy of sigmoid colon carcinoma in 2003, three years before radiotherapy of prostate cancer. Blood samples were irradiated ex vivo with Cs-137 γ-rays (0.7Gy/min) in the G0-phase of the cell cycle. 100 FISH painted metaphases were analysed for the control and the irradiated samples each. Multicolour in situ hybridisation techniques like mFISH and mBand as well as MYC locus, telomere and centromere painting probes were used to characterise ring metaphases. Metaphase search and autocapture was performed with a Zeiss Axioplan 2 imaging microscope followed by scoring and image analysis using Metafer 4/ISIS software (MetaSystems). In 2009 chromosome 8 rings were found in about 25% of lymphocytes. Rings were stable over time and increased to about 30% until 2012. The ring chromosome 8 always lacked telomere signals and a small amount of rings displayed up to four centromere signals. In aberrant metaphases 8pter and 8qter were either translocated or deleted. Further analyses revealed that the breakpoint at the p arm is localised at 8p21.2-22. The breakpoint at the q arm turned out to be distal from the MYC locus at 8q23-24. We hypothesise that the ring chromosome 8 has been developed during the 5 FU/folic acid treatments in 2003. The long term persistence might be due to clonal expansion of a damaged but viable hematopoietic stem cell giving rise to cycling progenitor cells that permit cell survival and proliferation.</p>

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines.

<p>Myelodysplastic syndrome (MDS) represents a group of clonal hematological disorders characterized by progressive cytopenia, and reflects to defects in erythroid, myeloid and megakaryocytic maturation. MDS is more frequently observed in older aged patients with cytogenetic abnormalities like monosomy of chromosome(s) 5 and/or 7. In 50% of de novo MDS cases, chromosomal aberrations are found and rearrangements involving the retinoblastoma (RB1) gene in 13q14 are found. Here, we are presenting a case report of a rare biclonal MDS with a karyotype of 45, XY,-4, der(6)t(4;6)(p15.1;p21.3), der(8)t(4;8)(q31.2;q22), t(13;16)(q21.3;p11.2)11/45, XY, der(7)t(7;13)(p22.2~22.3;q21.3),-13 9. The patient was diagnosed according to WHO classification as refractory anemia with excess of blasts (RAEB-II).Immunophenotyping was positive for CD11b, CD11c, CD10, CD13, CD15, CD16 and CD33. We report, a novel and cytogenetically rare case of a biclonal MDS with complex chromosomal aberrations and deletion of RB1-gene in both clones. These findings are associated with a poor prognosis as the patient died 3 months after diagnosis.</p>

ABSTRACT: The chromosomal translocation (11;14)(q13;q32) rearranging the locus for cyclin D1 (CCND1) to that of the immunoglobulin heavy chain (IGH) can be found in virtually all cases of mantle cell lymphoma (MCL), while other CCND1 translocations are extremely rare. As CCND1 overexpression and activation is a hallmark of MCL it is regarded as a central biological mechanism in the development and maintenance of this disease.Here we present a patient initially diagnosed with chronic lymphocytic leukemia (CLL) where chromosome banding analysis revealed, among other aberrations, a translocation (11;22)(q13;q11.2). We show by fluorescence in situ hybridization (FISH) analysis that on chromosome 22 the immunoglobulin light chain lambda (IGL) is involved in this cytogenetic aberration. Additionally, we demonstrate the resulting overexpression of CCND1 on the RNA and protein level, thereby consolidating the new diagnosis of a MCL-like B-cell neoplasia. Summing up, we described a rare case of t(11;22)(q13;q11.2) in a MCL-like neoplasia and showed that this aberration leads to an overexpression of CCND1 which is regarded as a key biological feature in MCL. This case underlines the importance of cytogenetic analyses especially in atypical cases of B cell lymphomas.

Multicolour fluorescence in situ hybridisation (M-FISH) and multicolour banding (M-BAND) are advanced chromosome painting techniques combining multiple chromosome- or region-specific paints in one step. M-FISH identifies all chromosomes or chromosome arms at once, whereas M-BAND identifies the different regions of a single chromosome. The use of either or both can improve the accuracy of karyotyping and help identify cryptic chromosome rearrangements. These probes are prepared by pooling multiple chromosome- or chromosome region-specific DNA libraries, each labelled with a unique combination of fluorochromes. Commercial probes are available, avoiding the need for probe preparation. In the protocol described here, a commercial probe is used. Well-spread metaphases are prepared according to standard techniques, followed by alkaline denaturation and application of the denatured probe. After an incubation period, the slides are washed. A fluorescence microscope with filter sets specific to the fluorescent labels is used for analysis, together with specialised image analysis software. The software interprets the combination of fluorochromes to identify each chromosome and produce a false colour image specific for each chromosome or region. The single colour galleries - which show the hybridisation patterns of the individual fluorochromes - are useful to help interpret and confirm the false colour images produced by the software, including ambiguous signals.

Chromosome aberrations are important prognostic markers in multiple myeloma (MM), but their identification may be hampered by complexity of the karyotypes. Using multicolor fluorescence in situ hybridization (mFISH), we found cryptic aberrations in 7 of 10 patients with a complex karyotype. Moreover, in addition to typical aberrations involving 1q, 13q, 14q and 17p and structural aberrations in chromosomes 1, 6, 9 and 19, (iso)dicentric chromosomes and whole-arm translocations were detected. These chromosome aberrations were generated by breaks in heterochromatic regions indicating an increased breakage of these regions, which may predispose to the generation of chromosome aberrations in multiple myeloma.

<p>The purpose of the present study was to investigate as to what extent differences in the linear energy transfer (LET) are reflected at the chromosomal level. For this study human lymphocytes were exposed to 9.5 MeV/u C-ions (1 or 2 Gy, LET=175 keV/microm) or X-rays (1-6 Gy), harvested at 48, 72 or 96 h post-irradiation and aberrations were scored in first cycle metaphases using 24 color fluorescence in situ hybridization (mFISH). Additionally, in selected samples aberrations were measured in prematurely condensed G2-phase cells. Analysis of the time-course of aberrations in first cycle metaphases showed a stable yield of simple and complex exchanges after X-ray irradiation. In contrast, after C-ion exposure the yields profoundly increased with harvesting time complicating the estimation of the frequency of aberrations produced by high LET particles within the entire cell population. This is especially true for the yield of complex exchanges. Complex aberrations dominate the aberration spectrum produced by C-ions. Their fraction was about 50\% for the two measured doses. In contrast, isodoses of X-rays induced smaller proportions of complex aberrations (i.e. 5% and 15%, respectively). For both radiation qualities the fraction of complexes did not change with harvesting time. As expected from the different dose deposition of high and low LET radiation, complex exchanges produced by high LET C-ions involved more breaks and more chromosomes than those induced by isodoses of X-rays. Noteworthy, C-ions but not X-rays induced a small number of complex chromatid-isochromatid exchanges that are not expected for cells exposed in the G0-phase. The results obtained so far for cells arrested in G2-phase confirm these patterns. Altogether our data show that the increased effectiveness of C-ions for the induction of aberrations in first cycle cells is determined by complex exchanges, whereas for simple exchanges the relative biological effectiveness is about one.</p>

Interphase chromosomes are divided into discrete domains, with limited overlapping and movement. We explored the role of nuclear topology in the formation of chromosome aberrations by irradiating normal human fibroblasts with high-energy heavy ions from different directions. Cells with elliptical nuclei were grown in an aligned manner onto micrometer grooved culturing substrates to have a predetermined orientation with respect to the accelerated iron ions. Particles were directed either perpendicular to the cell layer or along the major or minor axis of the nucleus. Analysis of chromosome aberrations by mFISH showed that, at the same radiation dose, the yield of chromosomal damage and its complexity are largely modified by the irradiation geometry. The results demonstrate that the architecture of the cell nucleus determines the formation of chromosomal rearrangements.

Papillary thyroid carcinoma (PTC) is a known radiation-induced tumor. Rearrangements in human chromosome 10 and in particular intrachromosomal exchanges are often associated with PTC formation. In this study we measured intrachromosomal exchanges in human thyroid follicular cells exposed to sparsely or densely ionizing radiation. Assuming that inversions in chromosome 10 are a biomarker of PTC risk, we estimated the relative biological effectiveness (RBE) of heavy ions using a molecular marker in vitro. The analysis of chromosomal aberrations was performed with the mBAND technique, which allows detection of both inter- and intrachromosomal exchanges. Our results do not show any significant increase in the yield of intrachanges in samples exposed to heavy ions compared to X rays. Within the constraints imposed by the experimental model we used, we conclude that heavy ions would not necessarily be more effective than X rays in the induction of thyroid cancer.

Cancers are clones of autonomous cells defined by individual karyotypes, much like species. Despite such karyotypic evidence for causality, three to six synergistic mutations, termed oncogenes, are generally thought to cause cancer. To test single oncogenes, they are artificially activated with heterologous promoters and spliced into the germ line of mice to initiate cancers with collaborating spontaneous oncogenes. Because such cancers are studied as models for the treatment of natural cancers with related oncogenes, the following must be answered: 1) which oncogenes collaborate with the transgenes in cancers; 2) how do single transgenic oncogenes induce diverse cancers and hyperplasias; 3) what maintains cancers that lose initiating transgenes; 4) why are cancers aneuploid, over- and underexpressing thousands of normal genes? Here we try to answer these questions with the theory that carcinogenesis is a form of speciation. We postulate that transgenic oncogenes initiate carcinogenesis by inducing aneuploidy. Aneuploidy destabilizes the karyotype by unbalancing teams of mitosis genes. This instability thus catalyzes the evolution of new cancer species with individual karyotypes. Depending on their degree of aneuploidy, these cancers then evolve new subspecies. To test this theory, we have analyzed the karyotypes and phenotypes of mammary carcinomas of mice with transgenic SV40 tumor virus- and hepatitis B virus-derived oncogenes. We found that (1) a given transgene induced diverse carcinomas with individual karyotypes and phenotypes; (2) these karyotypes coevolved with newly acquired phenotypes such as drug resistance; (3) 8 of 12 carcinomas were transgene negative. Having found one-to-one correlations between individual karyotypes and phenotypes and consistent coevolutions of karyotypes and phenotypes, we conclude that carcinogenesis is a form of speciation and that individual karyotypes maintain cancers as they maintain species. Because activated oncogenes destabilize karyotypes and are dispensable in cancers, we conclude that they function indirectly, like carcinogens. Such oncogenes would thus not be valid models for the treatment of cancers.

BACKGROUND AND PURPOSE: To investigate the cytogenetic damage in blood lymphocytes of patients treated for prostate cancer with different radiation qualities and target volumes. MATERIALS AND METHODS: Twenty patients receiving carbon-ion boost irradiation followed by IMRT or IMRT alone for the treatment of prostate cancer entered the study. Cytogenetic damage induced in peripheral blood lymphocytes of these patients was investigated at different times during the radiotherapy course using Giemsa staining and mFISH. A blood sample from each patient was taken before initiation of radiation therapy and irradiated in vitro to test for individual radiosensitivity. In addition, in vitro dose-effect curves for the induction of chromosomal exchanges by X-rays and carbon ions of different energies were measured. RESULTS: The yield of chromosome aberrations increased during the therapy course, and the frequency was lower in patients irradiated with carbon ions as compared to patients treated with IMRT with similar target volumes. A higher frequency of aberrations was measured by increasing the target volume. In vitro, high-LET carbon ions were more effective than X-rays in inducing aberrations and yielded a higher fraction of complex exchanges. The yield of complex aberrations observed in vivo was very low. CONCLUSION: The investigation showed no higher aberration yield induced by treatment with a carbon-ion boost. In contrast, the reduced integral dose to the normal tissue is reflected in a lower chromosomal aberration yield when a carbon-ion boost is used instead of IMRT alone. No cytogenetic #signature# of exposure to densely ionizing carbon ions could be detected in vivo.

To characterise the radiation response of human hematopoietic stem and progenitor cells (HSPC) with respect to X and carbon ion irradiation.HSPC from peripheral blood of healthy donors treated with granulocyte-colony stimulating factor (G-CSF) were enriched for the transmembrane glycoprotein CD34 (cluster of differentiation) and irradiated with X rays or carbon ions (29 keV/microm monoenergetic beam and 60-85 keV/microm spread-out Bragg peak), mimicking radiotherapy conditions. Apoptotic cell death, cell cycle progression and the frequency of chromosomal aberrations were determined.After radiation exposure no inhibition in the progression of the cell cycle was detected. However, an enhanced frequency of apoptotic cells and an increase in aberrant cells were observed, both effects being more pronounced for carbon ions than X rays, resulting in a relative biological effectiveness (RBE) of 1.4-1.7. The fraction of complex-type aberrations was higher following carbon ion exposure.RBE values of carbon ions are low, as expected for radiosensitive cells. The observed frequencies of apoptotic cells and chromosome aberrations in HSPC are similar to those reported for human peripheral blood lymphocytes suggesting that at least with respect to apoptosis and chromosomal aberrations mature lymphocytes reflect the respective radiation responses of their proliferating progenitors.

We measured residual cytogenetic damage in the progeny of human peripheral blood lymphocytes exposed to 1 GeV/ nucleon iron ions or gamma rays. Arm-specific DNA probes for chromosome 1 were used to detect aberrations as a function of dose in cells harvested 144 h after exposure. In addition, arm-specific mFISH was applied to samples exposed to a single dose of 2 Gy. These methods allowed the detection of interarm intrachanges (pericentric inversions) in addition to interchanges. The ratio of these types of aberrations (F ratio) has been proposed as a fingerprint of exposure to densely ionizing radiation. The fractions of aberrant cells in the progeny of cells exposed to iron ions were similar to those in the population exposed to gamma rays, possibly because many rearrangements induced by heavy ions ultimately lead to cell death. Simple inter- and intrachanges were also similar, but more complex rearrangements were found in cells that survived after exposure to iron ions. We did not find a significant difference in the ratio of simple interchanges to simple intrachanges for the two radiation types. However, iron ions induced a much higher frequency of events involving both inter- and intrachanges. We conclude that these complex rearrangements represent a hallmark of exposure to heavy ions and may be responsible of the decrease of the F ratio with increasing LET reported in the literature in some in vitro and in vivo experiments.

In 1957/58 the British Government conducted a series of nuclear tests in the mid-Pacific codenamed Operation Grapple, which involved several naval vessels from Britain and New Zealand. Two New Zealand frigates with 551 personnel onboard were stationed at various distances between 20 and 150 nautical miles from ground zero. In the present study we applied the cytomolecular technique mFISH (multicolour fluorescent in situ hybridisation) to investigate a potential link between chromosome abnormalities and possible past radiation exposure in New Zealand nuclear test veterans who participated in Operation Grapple. Compared to age matched controls, the veterans showed significantly higher (P < 0.0001) frequencies of chromosomal abnormalities (275 translocations and 12 dicentrics in 9,360 cells vs. 96 translocations and 1 dicentric in 9,548 cells in the controls), in addition to a significant excess of CCRs (complex chromosomal rearrangements) in the veterans. A Kolmogorov-Smirnoff test showed that the distributions of translocations for the two groups were significantly different.

<p>BACKGROUND: The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods. RESULTS: Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15%-36%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total. CONCLUSION: These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.</p>