<p>Protontherapy is a rapidly expanding radiotherapy modality where accelerated proton beams are used to precisely deliver the dose to the tumor target but is generally considered ineffective against radioresistant tumors. Proton-Boron Capture Therapy (PBCT) is a novel approach aimed at enhancing proton biological effectiveness. PBCT exploits a nuclear fusion reaction between low-energy protons and B atoms, i.e. p+ B→ 3α (p-B), which is supposed to produce highly-DNA damaging α-particles exclusively across the tumor-conformed Spread-Out Bragg Peak (SOBP), without harming healthy tissues in the beam entrance channel. To confirm previous work on PBCT, here we report new in-vitro data obtained at the 62-MeV ocular melanoma-dedicated proton beamline of the INFN-Laboratori Nazionali del Sud (LNS), Catania, Italy. For the first time, we also tested PBCT at the 250-MeV proton beamline used for deep-seated cancers at the Centro Nazionale di Adroterapia Oncologica (CNAO), Pavia, Italy. We used Sodium Mercaptododecaborate (BSH) as B carrier, DU145 prostate cancer cells to assess cell killing and non-cancer epithelial breast MCF-10A cells for quantifying chromosome aberrations (CAs) by FISH painting and DNA repair pathway protein expression by western blotting. Cells were exposed at various depths along the two clinical SOBPs. Compared to exposure in the absence of boron, proton irradiation in the presence of BSH significantly reduced DU145 clonogenic survival and increased both frequency and complexity of CAs in MCF-10A cells at the mid- and distal SOBP positions, but not at the beam entrance. BSH-mediated enhancement of DNA damage response was also found at mid-SOBP. These results corroborate PBCT as a strategy to render protontherapy amenable towards radiotherapy-resilient tumor. If coupled with emerging proton FLASH radiotherapy modalities, PBCT could thus widen the protontherapy therapeutic index.</p>

Digital object identifier (DOI): 10.3389/fonc.2021.682647

Mitochondria play a vital role in proliferation and differentiation and their remodeling in the course of differentiation is related to the variable energy and metabolic needs of the cell. In this work, we show a distinctive mitochondrial remodeling in human induced pluripotent stem cells differentiated into neural or mesenchymal progenitors. While leading to upregulation of the citrate synthase-α-ketoglutarate dehydrogenase segment of the Krebs cycle and increased respiratory chain activities and respiration in the mesenchymal stem cells, the remodeling in the neural stem cells resulted in downregulation of α-ketoglutarate dehydrogenase, upregulation of isocitrate dehydrogenase 2 and the accumulation of α-ketoglutarate. The distinct, lineage-specific changes indicate an involvement of these Krebs cycle enzymes in cell differentiation.

Digital object identifier (DOI): 10.1016/j.bbrc.2019.02.033

Use of ionizing radiation (IR) in various industrial, medical and other applications can potentially increase the risk of medical, occupational or accidental human exposure. Additionally, in the event of a radiological or nuclear (R/N) incident, several tens of hundreds and thousands of people are likely to be exposed to IR. IR causes serious health effects including mortality from acute radiation syndrome and therefore it is imperative to determine the absorbed radiation dose, which will enable physicians in making an appropriate clinical 'life-saving' decision. The 'Dicentric Chromosome Assay (DCA)' is the gold standard for estimating the absorbed radiation dose but its performance is time consuming and laborious. Further, timely evaluation of dicentric chromosomes (DCs) for dose estimation in a large number of samples provides a bottleneck because of a limited number of trained personnel and a prolonged time for manual analysis. To circumvent some of these technical issues, we developed and optimized a miniaturized high throughput version of DCA (mini-DCA) in a 96-microtube matrix with bar-coded 1.4 ml tubes to enable the processing of a large number of samples. To increase the speed of DC analysis for radiation dose estimation, a semi-automated scoring was optimized using the Metafer DCScore algorithm. The accuracy of mini-DCA in dose estimation was verified and validated though comparison with conventional DCA performed in 15 ml conical tubes. The mini-DCA considerably reduced the sample processing time by a factor of 4 when compared to the conventional DCA. Further, the radiation doses estimated by mini-DCA using the triage mode of scoring (50 cells or 30 DCs) were similar to that of conventional DCA using 300-500 cells. The mini-DCA coupled with semi-automated DC scoring not only reduced the sample processing and analysis times by a factor of 4 but also enabled the processing of a large number of samples at once. Our mini-DCA method, once automated for high throughput robotic platforms, will be an effective radiological triage tool for mass casualty incidents.

Digital object identifier (DOI): 10.1093/rpd/ncy127

<p>To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30-/CD15- cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (-/-)(NSG) mice. Using cell sorting, we demonstrate that CD30-/CD15- subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30-/CD15- cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Our HL animal model requires only 10³ cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30-/CD15- cells exhibiting high telomerase activity and telomere dysfunction.</p>

Digital object identifier (DOI): 10.3390/cancers10110414

Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

Digital object identifier (DOI): 10.3390/cancers10070233

<p>Mutations in the gene encoding Gasdermin A3 (Gsdma3) have been described to cause severe skin phenotypes, including loss of sebaceous glands and alopecia, in mice. We discovered a novel C-terminal mutation in Gsdma3 in a new mouse line and characterized a less frequently reported corneal phenotype, likely caused by degeneration of Meibomian glands of the inner eyelid. We used histologic methods to evaluate the effects of the C+/H- mutation on sebaceous gland and skin morphology as well as Meibomian glands of the inner eyelid and corneal tissue. Chromosomal aberrations were excluded by karyogram analyses. The mutation was identified by Sanger sequencing of candidate genes. Analyses of skin samples from affected mice confirmed the frequently reported phenotypes associated with mutations in Gsdma3: Degeneration of sebaceous glands and complete loss of pelage. Immunologic staining of corneal samples suggested an inflammatory response with signs of neovascularization in half of the affected older mice. While the corneal phenotype was observed at irregular time points, mainly after 6 months, its appearance coincided with a degeneration of Meibomian glands in the eyelids of affected animals. The mutation described herein is associated with inflammation and neovascularization of corneal tissue. Simultaneous degeneration of Meibomian glands in affected animals suggested a change in tear-film composition as the underlying cause for the corneal phenotype. Our data further support that different pathogenic mechanisms underlie some of the reported mutations in Gsdma3.</p>

Digital object identifier (DOI): 10.1167/iovs.17-22658

Foulds defined, "Tumor progression (as a) permanent, irreversible qualitative change in one or more of its characters" (Cancer Res. 1954). Accordingly progressions, such as metastases and acquired drug-resistance, were since found to be subspecies of cancers with conserved and numerous new chromosomes. Here we ask whether cancers acquire numerous new chromosomes gradually or simultaneously in progressions. The currently prevailing theory of Nowell (Science, 1976) holds that unexplained "genetic instability" generates "variant sublines (with) changes in chromosome number" and that "clonal" progressions arise by "stepwise selection of more aggressive sublines". The literature, however, contains many examples of "immediate" selections of progressions with numerous new chromosomes - notably experimentally initiated fusions between cancers and heterologous cells. Furthermore, the stepwise progression theory predicts intermediate sublines of cancers with multiple non-clonal additions of new chromosomes. However, the literature does not describe such intermediates. In view of these inconsistencies with stepwise progression we test here a saltational theory, in which the inherent variability of cancer-specific aneuploidy generates "immediate" progressions with individual clonal karyotypes, transcriptomes and phenotypes in single steps. Using cell fusion as an established controllable model of "immediate" progression, we generated seven immortal murine hybridomas by fusing immortal murine myeloma cells and normal antibody-producing B-cells with polyethylene glycol within a few minutes. These immortal hybridomas contained individual sets of 71 to 105 clonal chromosomes, compared to the 52 chromosomes of the parental myeloma. Thus the myeloma had gained 19 to 53 new clonal chromosomes in seven individual hybridomas in a single step. Furthermore, no stable intermediates were found, as would be predicted by a saltational process. We conclude that random fusions between myelomas and normal B-cells generate clonal hybridomas with multiple, individual chromosomes in single steps. Similar single-step mechanisms may also generate the "late" clonal progressions of cancers with gains of numerous new chromosomes and thus explain the absence of intermediates. Latency would reflect the low probability of rare stochastic progressions. In conclusion, the karyotypic clonality of hybridomas and spontaneous progressions suggests karyotypic alterations as proximate causes of neoplastic progressions. Since cancer-specific aneuploidy catalyzes karyotypic variation, the degree of aneuploidy predicts the clinical risk of neoplastic progression onfirming classical predictions based on DNA content

Digital object identifier (DOI): 10.1186/s13039-017-0350-4

Protontherapy is hadrontherapy’s fastest-growing modality and a pillar in the battle against cancer. Hadrontherapy’s superiority lies in its inverted depth-dose profile, hence tumour-confined irradiation. Protons, however, lack distinct radiobiological advantages over photons or electrons. Higher LET (Linear Energy Transfer) 12C-ions can overcome cancer radioresistance: DNA lesion complexity increases with LET, resulting in efficient cell killing, i.e. higher Relative Biological Effectiveness (RBE). However, economic and radiobiological issues hamper 12C-ion clinical amenability. Thus, enhancing proton RBE is desirable. To this end, we exploited the p + 11B → 3α reaction to generate high-LET alpha particles with a clinical proton beam. To maximize the reaction rate, we used sodium borocaptate (BSH) with natural boron content. Boron-Neutron Capture Therapy (BNCT) uses 10B-enriched BSH for neutron irradiation-triggered alpha particles. We recorded significantly increased cellular lethality and chromosome aberration complexity. A strategy combining protontherapy’s ballistic precision with the higher RBE promised by BNCT and 12C-ion therapy is thus demonstrated.

The mechanisms behind the transmission of chromosomal aberrations (CA) remain unclear, despite a large body of work and major technological advances in chromosome identification. We reevaluated the transmission of CA to second- and third-division cells by telomere and centromere (TC) staining followed by M-FISH. We scored CA in lymphocytes of healthy donors after in vitro irradiation and those of cancer patients treated by radiation therapy more than 12 years before. Our data demonstrate, for the first time, that dicentric chromosomes (DCs) decreased by approximately 50% per division. DCs with two centromeres in close proximity were more efficiently transmitted, representing 70% of persistent DCs in ≥M3 cells. Only 1/3 of acentric chromosomes (ACs), ACs with four telomeres, and interstitial ACs, were paired in M2 cells and associated with specific DCs configurations. In lymphocytes of cancer patients, 82% of detected DCs were characterized by these specific configurations. Our findings demonstrate the high stability of DCs with two centromeres in close proximity during cell division. The frequency of telomere deletion increased during cell cycle progression playing an important role in chromosomal instability. These findings could be exploited in the follow-up of exposed populations.

Digital object identifier (DOI): 10.1038/s41598-017-03198-7

<p>The ability of parabens to promote the appearance of multiple cancer hallmarks in breast epithelium cells provides grounds for regulatory review of the implication of the presence of parabens in human breast tissue. It is well documented that telomere dysfunction plays a significant role in the initiation of genomic instability during carcinogenesis in human breast cancer. In the present study, we evaluated the genotoxic effect of ethyl 4-hydroxybenzoate (ethyl-paraben), with and without metabolic activation (S9), in studies following OECD guidelines. We observed a significant increase in genotoxic damage using the Mouse Lymphoma Assay and in vitro micronucleus (MN) tests in the L5178Y cell line in the presence of S9 only after a short exposure. A high frequency of MN was observed in the TK6 cells after a short exposure (3 h) in the presence of S9 and a long exposure (26 h) without S9. We found significant increases in the MN frequency and induced chromosomal aberrations in the lymphocytes of only one donor after ethyl-paraben exposure in the presence of S9 after a short exposure. Cytogenetic characterization of the paraben-treated cells demonstrated telomere shortening associated with telomere loss and telomere deletions in L5178Y and TK6 cells and lymphocytes of the paraben sensitive-donor. In a control cohort of 68 human lymphocytes, telomere length and telomere aberrations were age-dependent and showed high inter-individual variation. This study is the first to link telomere shortening and the genotoxic effect of ethyl paraben in the presence of S9 and raises the possibility that telomere shortening may be a proxy for underlying inter-individual sensitivity to ethyl-paraben. Copyright © 2016 John Wiley &amp; Sons, Ltd.</p>

Digital object identifier (DOI): 10.1002/jat.3425

Evidence supports a role of 17&-estradiol (E2) in carcinogenesis and the large majority of breast carcinomas are dependent on estrogen. The anti-estrogen tamoxifen (TAM) is widely used for both treatment and prevention of breast cancer; however, it is also carcinogenic in human uterus and rat liver, highlighting the profound complexity of its actions. The nature of E2- or TAM-induced chromosomal damage has been explored using relatively high concentrations of these agents, and only some numerical aberrations and chromosomal breaks have been analyzed. This study aimed to determine the effects of low doses of E2 and TAM (10(&8 )mol L(&1) and 10(&6 )mol L(&1) respectively) on karyotypes of MCF7, T47D, BT474, and SKBR3 breast cancer cells by comparing the results of conventional karyotyping and multi-FISH painting with cell proliferation. Estrogen receptor (ER)-positive (+) cells showed an increase in cell proliferation after E2 treatment (MCF7, T47D, and BT474) and a decrease after TAM treatment (MCF7 and T47D), whereas in ER& cells (SKBR3), no alterations in cell proliferation were observed, except for a small increase at 96 h. Karyotypes of both ER+ and ER& breast cancer cells increased in complexity after treatments with E2 and TAM leading to specific chromosomal abnormalities, some of which were consistent throughout the treatment duration. This genotoxic effect was higher in HER2+ cells. The ER&/HER2+ SKBR3 cells were found to be sensitive to TAM, exhibiting an increase in chromosomal aberrations. These in vitro results provide insights into the potential role of low doses of E2 and TAM in inducing chromosomal rearrangements in breast cancer cells.

Digital object identifier (DOI): 10.1530/ERC-16-0078

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

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

Telomeres are specific structures that protect chromosome ends and act as a biological clock, preventing normal cells from replicating indefinitely. Mammalian telomeres are replicated throughout S-phase in a predetermined order. However, the mechanism of this regulation is still unknown. We wished to investigate this phenomenon under physiological conditions in a changing environment, such as the immortalization process to better understand the mechanism for its control. We thus examined the timing of human telomere replication in normal and SV40 immortalized cells, which are cytogenetically very similar to cancer cells. We found that the timing of telomere replication was globally conserved under different conditions during the immortalization process. The timing of telomere replication was conserved despite changes in telomere length due to endogenous telomerase reactivation, in duplicated homologous chromosomes, and in rearranged chromosomes. Importantly, translocated telomeres, possessing their initial subtelomere, retained the replication timing of their homolog, independently of the proportion of the translocated arm, even when the remaining flanking DNA is restricted to its subtelomere, the closest chromosome-specific sequences (inferior to 500 kb). Our observations support the notion that subtelomere regions strongly influence the replication timing of the associated telomere.

Digital object identifier (DOI): 10.1038/srep32510

<p>The genetic basis of metastasis is still unclear because metastases carry individual karyotypes and phenotypes, rather than consistent mutations, and are rare compared to conventional mutation. There is however correlative evidence that metastasis depends on cancer-specific aneuploidy, and that metastases are karyotypically related to parental cancers. Accordingly we propose that metastasis is a speciation event. This theory holds that cancer-specific aneuploidy varies the clonal karyotypes of cancers automatically by unbalancing thousands of genes, and that rare variants form new autonomous subspecies with metastatic or other non-parental phenotypes like drug-resistance - similar to conventional subspeciation. To test this theory, we analyzed the karyotypic and morphological relationships between seven cancers and corresponding metastases. We found (1) that the cellular phenotypes of metastases were closely related to those of parental cancers, (2) that metastases shared 29 to 96% of their clonal karyotypic elements or aneusomies with the clonal karyotypes of parental cancers and (3) that, unexpectedly, the karyotypic complexity of metastases was very similar to that of the parental cancer. This suggests that metastases derive cancer-specific autonomy by conserving the overall complexity of the parental karyotype. We deduced from these results that cancers cause metastases by karyotypic variations and selection for rare metastatic subspecies. Further we asked whether metastases with multiple metastasis-specific aneusomies are assembled in one or multiple, sequential steps. Since (1) no stable karyotypic intermediates of metastases were observed in cancers here and previously by others, and (2) the karyotypic complexities of cancers are conserved in metastases, we concluded that metastases are generated from cancers in one step - like subspecies in conventional speciation. We conclude that the risk of cancers to metastasize is proportional to the degree of cancer-specific aneuploidy, because aneuploidy catalyzes the generation of subspecies, including metastases, at aneuploidy-dependent rates. Since speciation by random chromosomal rearrangements and selection is unpredictable, the theory that metastases are karyotypic subspecies of cancers also explains Foulds' rules, which hold that the origins of metastases are "abrupt" and that their phenotypes are "unpredictable."</p>

Digital object identifier (DOI): 10.1186/s13039-016-0297-x

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

Fluorescence in situ hybridisation assays designed for del(7q) detection uncover more complex rearrangements in myeloid leukaemia cell lines ABSTRACT Chromosome 7 abnormalities are associated with poor prognosis in myeloid leukaemia. The pathogenetic mechanisms chromosome 7 rearrangements and lead to malignancy are still poorly understood. The use of leukaemia- derived cell lines might be a useful tool to shed some light on these mechanisms. The cytogenetic characterisation of these cell lines is therefore important for the understanding of the genetic alterations leading to the disease. We carried out fluorescence in situ hybridisation (FISH) on three different myeloid leukaemia-derived cell lines (GDM-1, GF-D8 and K562). These were selected on the basis of harbouring rearrangements of chromosome 7. The probes used in these experiments were whole and partial chromosome paints, Multiplex-fluorescence in situ hybridisation (M-FISH) probes as well as locus specific probes for the 7q22, 7q31 and 7q36 regions. Our study confirmed the chromosome 7 abnormalities previously reported in the cell lines GDM-1 and GF-D8. We refined one of the rearrangements of chromosome 7 in the K562 cell line and reported some discrepancies with the data published in earlier reports. With this study, we confirm the importance of using a series of FISH that arise from probes to characterise chromosomal abnormalities in detail, as some rearrangements might go under-detected or mis-interpreted. Moreover, we highlight the importance of monitoring cell lines broadly used in research, as these can lose or acquire characteristics as they evolve in time in different laboratories.

In the WHO classification, double or triple-hit lymphoma depicts rare and aggressive lymphomas displaying BCL2 and/or MYC and/or BCL6 gene rearrangements that are categorized as B-cell lymphomas unclassified, with features intermediate between diffuse B-cell lymphoma and Burkitt lymphoma. Bacher et al.2 described an interesting series of 10 cases of such neoplasms. In addition, they reported the two first cases displaying four different lymphoma-specific events (quadruple hit) involving the genes MYC, BCL2, BCL6 and CCND1. We describe here a third case occurring in a 79-year-old male patient suffering from paraesthesias for 4 months who was referred for polyneuritis in a context of poor general condition. Clinical examination showed the presence of numerous axillary, supraclavicular, mediastinal and inguinal lymphadenopathies, neuro-meningeal invasion and skin infiltration. The biopsy of a left arm skin nodule revealed large proliferating cells (Ki-67 80%) stained by anti-CD20, BCL2 and BCL6 antibodies, CD10 and CD23 remaining negative, consistent with the diagnosis of diffuse large B-cell lymphoma (DLBCL), not otherwise specified. Blood cell counts showed 8.1 × 109/l leukocytes, 13.2 g/dl hemoglobin, 166 × 109/l platelets. LDH and β-2 microglobulin were elevated (989 U/I and 9.14 mg/l, respectively). Blood cell film examination showed the presence of 28% abnormal lymphocytes (medium sized, with intense basophilia, irregular nuclear contours, slightly clumped chromatin and frequent prominent nucleoli) suggestive of a high grade lymphoma. Flow cytometry revealed a lambda immunoglobulin light chain restriction. These cells expressed pan-B markers such as CD19, CD20, FMC7, CD22, with weak CD5 and CD43 positivity. CD10 and 23 were negative. Both the morphology and immunophenotype of the blood cells favored a pleomorphic mantle cell lymphoma (MCL) aggressive variant diagnosis. Cytogenetic study performed in the WBCs found a complex hyperdiploid karyotype (47 chromosomes, Figure 1) with a t(3;22) translocation involving the BCL6 and IGL genes, a structural abnormality of chromosome 8 resulting in juxtaposition of 5′ MYC and BCL2 in fluorescence in situ hybridization (with break of the MYC probe), a derivative chromosome 18 from a t(14;18) translocation with fusion of 5′IGH and BCL2, and a t(11;14) complex translocation involving IGH and CCND1 (Figure 2). Other numeral (trisomy 12) and structural abnormalities (involving the 1, 7, 14 and 21 chromosomes) were also detected (Figure 1). Overexpression of cyclin D1 was detected in the WBCs by real-time quantitative PCR, as well as in the skin lesion using immunochemistry. Anti-SOX11 antibody staining was found to be negative. Chemotherapy combining rituximab, ifosfamide, cytosine arabinoside and intrathecal methotrexate was initiated, but the patient died 4 months after the diagnosis. This third case of quadruple-hit lymphoma underlines the complexity of the classification of such aggressive malignancies. Initial rearrangement of the CCND1 gene characterizes MCL that may harbor in very rare cases additional rearrangements of MYC or BCL6, but histological transformation to typical large cell lymphoma is not retained in the WHO classification. In addition, cyclin D1 overexpression is considered to be a rare feature in DLBCL. Recently, Ok et al.3 proposed to reclassify DLBCL with expression of cyclin D1, CCND1 chromosomal rearrangement and CD5 positivity as an aggressive pleomorphic MCL variant. However, no observation of multiple lymphoma-specific gene rearrangements was described in that study. Juskevicius et al.4 suggest the existence of a ‘gray zone’ in which morphologic, clinical and genetic features are insufficient to segregate lymphomas with overexpression of cyclin D1/translocations involving CCND1 between blastoid MCL and cyclin D1-positive DLBCL. Regarding the immunophenotyping and molecular data, our case is possibly a genetically unstable aggressive pleomorphic MCL variant, which acquired three additional genetic hits.

Digital object identifier (DOI): 10.1038/bcj.2015.99

<p>Despite over 50 years of research, it remains unclear how the DNA tumor viruses SV40 and Polyoma cause cancers. Prevailing theories hold that virus-coded Tumor (T)-antigens cause cancer by inactivating cellular tumor suppressor genes. But these theories don't explain four characteristics of viral carcinogenesis: (1) less than one in 10,000 infected cells become cancer cells, (2) cancers have complex individual phenotypes and transcriptomes, (3) recurrent tumors without viral DNA and proteins, (4) preneoplastic aneuploidies and immortal neoplastic clones with individual karyotypes. As an alternative theory we propose that viral carcinogenesis is a form of speciation, initiated by virus-induced aneuploidy. Since aneuploidy destabilizes the karyotype by unbalancing thousands of genes it catalyzes chain reactions of karyotypic and transcriptomic evolutions. Eventually rare karyotypes evolve that encode cancer-specific autonomy of growth. The low probability of forming new autonomous cancer-species by random karyotypic and transcriptomic variations predicts individual and clonal cancers. Although cancer karyotypes are congenitally aneuploid and thus variable, they are stabilized or immortalized by selections for variants with cancer-specific autonomy. Owing to these inherent variations cancer karyotypes are heterogeneous within clonal margins. To test this theory we analyzed karyotypes and phenotypes of SV40-infected human, rat and mouse cells developing into neoplastic clones. In all three systems we found (1) preneoplastic aneuploidies, (2) neoplastic clones with individual clonal but flexible karyotypes and phenotypes, which arose from less than one in 10,000 infected cells, survived over 200 generations, but were either T-antigen positive or negative, (3) spontaneous and drug-induced variations of neoplastic phenotypes correlating 1-to-1 with karyotypic variations. Since all 14 virus-induced neoplastic clones tested contained individual clonal karyotypes and phenotypes, we conclude that these karyotypes have generated and since maintained these neoplastic clones. Thus SV40 causes cancer indirectly, like carcinogens, by inducing aneuploidy from which new cancer-specific karyotypes evolve automatically at low rates. This theory explains the (1) low probability of carcinogenesis per virus-infected cell, (2) the individuality and clonal flexibility of cancer karyotypes, (3) recurrence of neoplasias without viral T-antigens, and (4) the individual clonal karyotypes, transcriptomes and immortality of virus-induced neoplasias - all unexplained by current viral theories.</p>

Digital object identifier (DOI): 10.1186/s13039-015-0183-y

A century of research has established that cancers arise from tissues exposed to carcinogens only after long latencies of years to decades and have individual clonal karyotypes. Since speciation from known precursors also depends on long latencies and new species also have individual karyotypes, we and others have recently proposed that carcinogenesis is a form of speciation. According to this theory karyotypic evolutions generate new cancer species from normal cells as follows: Carcinogens induce aneuploidy (Figure 1). By unbalancing thousands of genes aneuploidy automatically destabilizes the karyotype and thus catalyzes random karyotypic variations. Selections of variants with proliferative phenotypes form non-clonal hyperplasias with persistently varying karyotypes. Very rare karyotypic variations form new cancer species with individual clonal karyotypes. Despite destabilization by the resulting congenital aneuploidies, cancer karyotypes are stabilized within narrow margins of variation by clonal selections for cancer-specific autonomy. Because all non-cancerous aneuploidies are unstable, all aneusomies of prospective cancers are joined in single-steps, rather than gradually. Since this mechanism is very inefficient, it predicts long latent periods from carcinogens to cancers and individual clonal cancer karyotypes. Here we have tested the predicted roles of karyotypic evolutions during the time course of carcinogenesis in an established experimental system. In this system injection of nitrosourea induces in female rats non-invasive mammary hyperplasias ("tumors") after two or more months, and invasive carcinomas after six or more months. Accordingly four specific predictions were tested: (1) Invasive cancers are late and carry individual clonal karyotypes and phenotypes, (2) Persistent hyperplasias carry non-clonal karyotypes, (3) Non-clonal hyperplasias generate clonal cancers spontaneously but rarely, (4) Cancer-karyotypes arise with all individual clonal aneusomies in single-steps. All four predictions were experimentally confirmed. Our results along with the literature reveal a coherent karyotypic mechanism of carcinogenesis: Carcinogens induce aneuploidy. The inherent instability of aneuploidy automatically catalyzes new karyotypic variations. Aneuploid karyotypes with proliferative phenotypes form varying non-clonal hyperplasias. Rare variations form cancer species with individual clonal karyotypes, which are stabilized by clonal selection for autonomy. The low odds of this mechanism explain the long latencies of carcinogenesis, the individuality and karyotypic clonality of cancers.

Digital object identifier (DOI): 10.1186/s13039-014-0071-x