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Journal of cell science, 132
March, 2019

Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML deficiency.

Han, Mingqi, Napier, Christine E, Frölich, Sonja, Teber, Erdahl, Wong, Ted, Noble, Jane R, Choi, Eugene H Y, Everett, Roger D, Cesare, Anthony J, Reddel, Roger R

<p>Cancers that utilize the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of promyelocytic leukemia nuclear bodies (PML NBs) that are required for intrinsic immunity to various viruses. Here, we asked whether ATRX deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in infecting ATRX-deficient cells than wild-type ATRX-expressing cells. Infection of co-cultured primary and ATRX-deficient cancer cells revealed that mutant HSV-1 selectively killed ATRX-deficient cells. Sensitivity to mutant HSV-1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.</p>

Digital object identifier (DOI): 10.1242/jcs.222349

Cell death & disease, 10, 186
February, 2019

Type 3 inositol 1,4,5-trisphosphate receptor has antiapoptotic and proliferative role in cancer cells.

Rezuchova, Ingeborg, Hudecova, Sona, Soltysova, Andrea, Matuskova, Miroslava, Durinikova, Erika, Chovancova, Barbora, Zuzcak, Michal, Cihova, Marina, Burikova, Monika, Penesova, Adela, Lencesova, Lubomira, Breza, Jan, Krizanova, Olga

Although the involvement of type 1 (IP R1) and type 2 (IP R2) inositol 1,4,5-trisphosphate receptors in apoptosis induction has been well documented in different cancer cells and tissues, the function of type 3 IP R (IP R3) is still elusive. Therefore, in this work we focused on the role of IP R3 in tumor cells in vitro and in vivo. We determined increased expression of this receptor in clear cell renal cell carcinoma compared to matched unaffected part of the kidney from the same patient. Thus, we hypothesized about different functions of IP R3 compared to IP R1 and IP R2 in tumor cells. Silencing of IP R1 prevented apoptosis induction in colorectal cancer DLD1 cells, ovarian cancer A2780 cells, and clear cell renal cell carcinoma RCC4 cells, compared to apoptosis in cells treated with scrambled siRNA. As expected, silencing of IP R3 and subsequent apoptosis induction resulted in increased levels of apoptosis in all these cells. Further, we prepared a DLD1/IP R3_del cell line using CRISPR/Cas9 gene editing method. These cells were injected into nude mice and tumor's volume was compared with tumors induced by DLD1 cells. Lower volume of tumors originated from DLD1/IP R3_del cells was observed after 12 days, compared to wild type DLD1 cells. Also, the migration of these cells was lesser compared to wild type DLD1 cells. Apoptosis under hypoxic conditions was more pronounced in DLD1/IP R3_del cells than in DLD1 cells. These results clearly show that IP R3 has proliferative and anti-apoptotic effect in tumor cells, on contrary to the pro-apoptotic effect of IP R1.

Digital object identifier (DOI): 10.1038/s41419-019-1433-4

Oncogenesis, 7, 62
August, 2018

Chromosomal instability-induced senescence potentiates cell non-autonomous tumourigenic effects.

He, Qianqian, Au, Bijin, Kulkarni, Madhura, Shen, Yang, Lim, Kah J, Maimaiti, Jiamila, Wong, Cheng Kit, Luijten, Monique N H, Chong, Han C, Lim, Elaine H, Rancati, Giulia, Sinha, Indrajit, Fu, Zhiyan, Wang, Xiaomeng, Connolly, John E, Crasta, Karen C

Chromosomal instability (CIN), a high rate of chromosome loss or gain, is often associated with poor prognosis and drug resistance in cancers. Aneuploid, including near-polyploid, cells contain an abnormal number of chromosomes and exhibit CIN. The post-mitotic cell fates following generation of different degrees of chromosome mis-segregation and aneuploidy are unclear. Here we used aneuploidy inducers, nocodazole and reversine, to create different levels of aneuploidy. A higher extent of aneuploid and near-polyploid cells in a given population led to senescence. This was in contrast to cells with relatively lower levels of abnormal ploidy that continued to proliferate. Our findings revealed that senescence was accompanied by DNA damage and robust p53 activation. These senescent cells acquired the senescence-associated secretory phenotype (SASP). Depletion of p53 reduced the number of senescent cells with concomitant increase in cells undergoing DNA replication. Characterisation of these SASP factors demonstrated that they conferred paracrine pro-tumourigenic effects such as invasion, migration and angiogenesis both in vitro and in vivo. Finally, a correlation between increased aneuploidy and senescence was observed at the invasive front in breast carcinomas. Our findings demonstrate functional non-equivalence of discernable aneuploidies on tumourigenesis and suggest a cell non-autonomous mechanism by which aneuploidy-induced senescent cells and SASP can affect the tumour microenvironment to promote tumour progression.

Digital object identifier (DOI): 10.1038/s41389-018-0072-4

Annals of diagnostic pathology, 34, 1–12
June, 2018

Review with novel markers facilitates precise categorization of 41 cases of diagnostically challenging, "undifferentiated small round cell tumors". A clinicopathologic, immunophenotypic and molecular analysis.

Machado, Isidro, Yoshida, Akihiko, Morales, María Gema Nieto, Abrahão-Machado, Lucas Faria, Navarro, Samuel, Cruz, Julia, Lavernia, Javier, Parafioriti, Antonina, Picci, Piero, Llombart-Bosch, Antonio

<p>Despite extensive immunohistochemical (IHC) and molecular studies combined with morphologic findings, a group of round/ovoid cell tumors histologically similar to Ewing sarcomas (ES) but lacking EWSR1-rearrangements may remain unclassifiable. We retrospectively analyzed 41 Ewing-like tumors (formalin-fixed, paraffin-embedded) previously determined as negative or non-informative for EWSR1-rearrangements by FISH and/or RT-PCR. A new histopathology revision and additional IHC and molecular analyses were carried out in order to investigate whether additional IHC and/or molecular testing in combination with the morphological findings may help in reaching a definitive diagnosis. Almost all the tumors (n=40) involved soft tissue and/or bone and half the patients died of disease. In the archival cases all diagnoses were Ewing sarcoma (ES), Ewing-like sarcoma (ELS), myoepithelial tumor and undifferentiated sarcoma (US). In the new review all the tumors were re-classified as, ES (n=16), Ewing-like tumor with EWSR1 rearrangement and amplification and possible EWSR1-NFATC2 gene fusion (n=1), CIC-rearranged sarcomas or undifferentiated sarcoma, most consistent with CIC-rearranged sarcoma (n=7), sarcoma with BCOR-alteration or undifferentiated sarcoma, consistent with BCOR-associated sarcoma (n=3), neuroblastoma (n=2), unclassifiable neoplasm with neuroblastic differentiation (n=1), malignant rhabdoid tumor (n=2), lymphoblastic lymphoma (n=1), clear cell sarcoma of the gastrointestinal tract (n=1), small cell carcinoma (n=1), sclerosing rhabdomyosarcoma (n=1), desmoplastic small round cell tumor (n=1), malignant peripheral sheath nerve tumor (n=1), poorly-differentiated synovial sarcoma (n=1), Possible gastrointestinal stromal tumor/GIST with predominant round cells (n=1) and possible SMARCA4-deficient-sarcoma (n=1). NKX2.2, ETV4 and BCOR immunoreactivity was observed in all ES, CIC-rearranged sarcomas and sarcomas with BCOR alteration, respectively. CIC-rearrangement by FISH was observed in many of the CIC-rearranged sarcomas. Our analysis of 41 Ewing-like tumors confirms that there may be a significant pathological and IHC overlap among Ewing-like tumors, with prognostic and therapeutic impacts. Additional IHC (NKX2.2, ETV4 and BCOR) and molecular studies including FUS, CIC or BCOR analysis may support the final diagnosis when FISH or RT-PCR fail to detect EWSR1-rearrangements. Any molecular findings should always be interpreted in relation to the specific clinical and pathological context.</p>

Digital object identifier (DOI): 10.1016/j.anndiagpath.2017.11.011

PloS one, 13, e0193213
June, 2018

Impact of 9p deletion and p16, Cyclin D1, and Myc hyperexpression on the outcome of anaplastic oligodendrogliomas.

Michaud, Karine, de Tayrac, Marie, D'Astous, Myreille, Paquet, Claudie, Gould, Peter Vincent, Saikali, Stéphan

<p>To study the presence of 9p deletion and p16, cyclin D1 and Myc expression and their respective diagnostic and prognostic interest in oligodendrogliomas. We analyzed a retrospective series of 40 consecutive anaplastic oligodendrogliomas (OIII) from a single institution and compared them to a control series of 10 low grade oligodendrogliomas (OII). Automated FISH analysis of chromosome 9p status and immunohistochemistry for p16, cyclin D1 and Myc was performed for all cases and correlated with clinical and histological data, event free survival (EFS) and overall survival (OS). Chromosome 9p deletion was observed in 55% of OIII (22/40) but not in OII. Deletion was highly correlated to EFS (median = 29 versus 53 months, p&lt;0.0001) and OS (median = 48 versus 83 months, p&lt;0.0001) in both the total cohort and the OIII population. In 9p non-deleted oligodendrogliomas, p16 hyperexpression correlated with a shorter OS (p = 0.02 in OII and p = 0.0001 in OIII) whereas lack of p16 expression was correlated to a shorter EFS and OS in 9p deleted OIII (p = 0.001 and p = 0.0002 respectively). Expression of Cyclin D1 was significantly higher in OIII (median expression 45% versus 14% for OII, p = 0.0006) and was correlated with MIB-1 expression (p&lt;0.0001), vascular proliferation (p = 0.002), tumor necrosis (p = 0.04) and a shorter EFS in the total cohort (p = 0.05). Hyperexpression of Myc was correlated to grade (median expression 27% in OII versus 35% in OIII, p = 0.03), and to a shorter EFS in 9p non-deleted OIII (p = 0.01). Chromosome 9p deletion identifies a subset of OIII with significantly worse prognosis. The combination of 9p status and p16 expression level identifies two distinct OIII populations with divergent prognosis. Hyperexpression of Bcl1 and Myc appears highly linked to anaplasia but the prognostic value is unclear and should be investigated further.</p>

Digital object identifier (DOI): 10.1371/journal.pone.0193213

eLife, 7, 3122
May, 2018

Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations.

Sun, Jiying, Shi, Lin, Kinomura, Aiko, Fukuto, Atsuhiko, Horikoshi, Yasunori, Oma, Yukako, Harata, Masahiko, Ikura, Masae, Ikura, Tsuyoshi, Kanaar, Roland, Tashiro, Satoshi

Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities.

Digital object identifier (DOI): 10.7554/eLife.32222

Cancers, 10, e0193213
May, 2018

The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes.

M'kacher, Radhia, Cuceu, Corina, Al Jawhari, Mustafa, Morat, Luc, Frenzel, Monika, Shim, Grace, Lenain, Aude, Hempel, William M, Junker, Steffen, Girinsky, Theodore, Colicchio, Bruno, Dieterlen, Alain, Heidingsfelder, Leonhard, Borie, Claire, Oudrhiri, Noufissa, Bennaceur-Griscelli, Annelise, Moralès, Olivier, Renaud, Sarah, Van de Wyngaert, Zoé, Jeandidier, Eric, Delhem, Nadira, Carde, Patrice

<p>We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30-/CD15- cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival ( &lt; 10 ), event-free survival ( &lt; 10 ), and freedom from progression ( &lt; 10 ) and the presence of an ALT profile in lymph nodes of EBV+ patients. The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients.</p>

Digital object identifier (DOI): 10.3390/cancers10060169

Nanoscale, 10, 4320–4331
March, 2018

Super-resolution localization microscopy of radiation-induced histone H2AX-phosphorylation in relation to H3K9-trimethylation in HeLa cells.

Hausmann, Michael, Wagner, Emma, Lee, Jin-Ho, Schrock, Gerrit, Schaufler, Wladimir, Krufczik, Matthias, Papenfuß, Franziska, Port, Matthias, Bestvater, Felix, Scherthan, Harry

<p>Ionizing radiation (IR)-induced damage confers functional and conformational changes to nuclear chromatin associated with DNA single and double strand breaks. This leads to the activation of complex DNA repair machineries that aim to preserve the integrity of the DNA molecule. Since hetero- and euchromatin are differentially accessible to DNA repair pathways, local chromatin re-arrangements and structural changes are among the consequences of an activated DNA damage response. Using super-resolution localization microscopy (SRLM), we investigated the X-ray-induced repositioning of γ-H2AX and histone H3K9me3 heterochromatin marks in the nuclei of HeLa cells. Aliquots of cells exposed to different IR doses (0.5, 1 and 2 Gy) were fixed at certain repair times for SRLM imaging. The number and size of nano-scale γ-H2AX molecule signal clusters detected increased with rising irradiation doses, with the number and size being the highest 0.5 h after irradiation. With growing repair time both the number and size of γ-H2AX nano-clusters decreased. Eight hours after irradiation, the number of clusters reached control levels, in agreement with the disappearance of most IR-induced foci seen by conventional microscopy. SRLM investigation of heterochromatin marks in spatial relation to γ-H2AX clusters showed that on average the heterochromatin density was high in the vicinity of γ-H2AX, which is in agreement with the observation that DSBs seem to relocate to the surface of heterochromatin clusters for DNA repair. The data demonstrate the potential of pointillist images obtained by SRLM for quantitative investigations of chromatin conformation changes and repair-protein recruitment on the nanoscale as measures for a radiation response.</p>

Digital object identifier (DOI): 10.1039/c7nr08145f

Nature communications, 9, 1048
March, 2018

Integrative genomic profiling of large-cell neuroendocrine carcinomas reveals distinct subtypes of high-grade neuroendocrine lung tumors.

George, Julie, Walter, Vonn, Peifer, Martin, Alexandrov, Ludmil B, Seidel, Danila, Leenders, Frauke, Maas, Lukas, Müller, Christian, Dahmen, Ilona, Delhomme, Tiffany M, Ardin, Maude, Leblay, Noemie, Byrnes, Graham, Sun, Ruping, De Reynies, Aurélien, McLeer-Florin, Anne, Bosco, Graziella, Malchers, Florian, Menon, Roopika, Altmüller, Janine, Becker, Christian, Nürnberg, Peter, Achter, Viktor, Lang, Ulrich, Schneider, Peter M, Bogus, Magdalena, Soloway, Matthew G, Wilkerson, Matthew D, Cun, Yupeng, McKay, James D, Moro-Sibilot, Denis, Brambilla, Christian G, Lantuejoul, Sylvie, Lemaitre, Nicolas, Soltermann, Alex, Weder, Walter, Tischler, Verena, Brustugun, Odd Terje, Lund-Iversen, Marius, Helland, Åslaug, Solberg, Steinar, Ansén, Sascha, Wright, Gavin, Solomon, Benjamin, Roz, Luca, Pastorino, Ugo, Petersen, Iver, Clement, Joachim H, Sänger, Jörg, Wolf, Jürgen, Vingron, Martin, Zander, Thomas, Perner, Sven, Travis, William D, Haas, Stefan A, Olivier, Magali, Foll, Matthieu, Büttner, Reinhard, Hayes, David Neil, Brambilla, Elisabeth, Fernandez-Cuesta, Lynnette, Thomas, Roman K

<p>Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (<em>n</em> = 60) and transcriptomic (<em>n</em> = 69) analysis of 75 LCNECs and identify two molecular subgroups: "type I LCNECs" with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and "type II LCNECs" enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1 /DLL3 /NOTCH , type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1 /DLL3 /NOTCH , and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors.</p>

Digital object identifier (DOI): 10.1038/s41467-018-03099-x

Scientific reports, 8, 1032
January, 2018

The ten-year evolutionary trajectory of a highly recurrent paediatric high grade neuroepithelial tumour with MN1:BEND2 fusion.

Burford, Anna, Mackay, Alan, Popov, Sergey, Vinci, Maria, Carvalho, Diana, Clarke, Matthew, Izquierdo, Elisa, Avery, Aimee, Jacques, Thomas S, Ingram, Wendy J, Moore, Andrew S, Frawley, Kieran, Hassall, Timothy E, Robertson, Thomas, Jones, Chris

<p>Astroblastomas are rare brain tumours which predominate in children and young adults, and have a controversial claim as a distinct entity, with no established WHO grade. Reports suggest a better outcome than high grade gliomas, though they frequently recur. Recently, they have been described to overlap with a newly-discovered group of tumours described as'high grade neuroepithelial tumour with MN1 alteration' (CNS HGNET-MN1), defined by global methylation patterns and strongly associated with gene fusions targeting MN1. We have studied a unique case of astroblastoma arising in a 6 year-old girl, with multiple recurrences over a period of 10 years, with the pathognomonic MN1:BEND2 fusion. Exome sequencing allowed for a phylogenetic reconstruction of tumour evolution, which when integrated with clinical, pathological and radiological data provide for a detailed understanding of disease progression, with initial treatment driving tumour dissemination along four distinct trajectories. Infiltration of distant sites was associated with a later genome doubling, whilst there was evidence of convergent evolution of different lesions acquiring distinct alterations targeting NF-κB. These data represent an unusual opportunity to understand the evolutionary history of a highly recurrent childhood brain tumour, and provide novel therapeutic targets for astroblastoma/CNS HGNET-MN1.</p>

Digital object identifier (DOI): 10.1038/s41598-018-19389-9

British journal of cancer
2018

Heterogeneous MYCN amplification in neuroblastoma: a SIOP Europe Neuroblastoma Study.

Berbegall, Ana P, Bogen, Dominik, Pötschger, Ulrike, Beiske, Klaus, Bown, Nick, Combaret, Valérie, Defferrari, Raffaella, Jeison, Marta, Mazzocco, Katia, Varesio, Luigi, Vicha, Ales, Ash, Shifra, Castel, Victoria, Coze, Carole, Ladenstein, Ruth, Owens, Cormac, Papadakis, Vassilios, Ruud, Ellen, Amann, Gabriele, Sementa, Angela R, Navarro, Samuel, Ambros, Peter F, Noguera, Rosa, Ambros, Inge M

<p>In neuroblastoma (NB), the most powerful prognostic marker, the MYCN amplification (MNA), occasionally shows intratumoural heterogeneity (ITH), i.e. coexistence of MYCN-amplified and non-MYCN-amplified tumour cell clones, called heterogeneous MNA (hetMNA). Prognostication and therapy allocation are still unsolved issues. The SIOPEN Biology group analysed 99 hetMNA NBs focussing on the prognostic significance of MYCN ITH. Patients &lt;18 months (18 m) showed a better outcome in all stages as compared to older patients (5-year OS in localised stages: &lt;18 m: 0.95 ± 0.04, &gt;18 m: 0.67 ± 0.14, <em>p</em> = 0.011; metastatic: &lt;18 m: 0.76 ± 0.15, &gt;18 m: 0.28 ± 0.09, <em>p</em> = 0.084). The genomic 'background', but not MNA clone sizes, correlated significantly with relapse frequency and OS. No relapses occurred in cases of only numerical chromosomal aberrations. Infiltrated bone marrows and relapse tumour cells mostly displayed no MNA. However, one stage 4s tumour with segmental chromosomal aberrations showed a homogeneous MNA in the relapse. This study provides a rationale for the necessary distinction between heterogeneous and homogeneous MNA. HetMNA tumours have to be evaluated individually, taking age, stage and, most importantly, genomic background into account to avoid unnecessary upgrading of risk/overtreatment, especially in infants, as well as in order to identify tumours prone to developing homogeneous MNA.</p>

Digital object identifier (DOI): 10.1038/s41416-018-0098-6

Journal of clinical pathology
2018

KRAS fluorescence in situ hybridisation testing for the detection and diagnosis of pancreatic adenocarcinoma.

Shiroma, Noriyuki, Arihiro, Koji, Oda, Miyo, Orita, Makoto

The aim of our study was to analyse correlations between mutation status, chromosomal changes that affect status in cells from pancreatic tumours. We collected 69 cases of surgically resected pancreatic ductal adenocarcinoma (PDA) and seven cases of chronic pancreatitis (CP). Chromosomal abnormalities of and CEP12 were detected using fluorescence in situ hybridisation (FISH). The number of CEP12 signals per cell ranged from 1.78 to 2.04 and 1.46 to 4.88 in CP and PDA samples, respectively, while the number of signals per cell ranged from 1.94 to 2.06 and 1.88 to 8.18 in CP and PDA samples, respectively. The 'chromosomal instability index', which was defined as the percentage of cells with any chromosomal abnormality, was over 5.7 times greater in PDA than in CP. We performed mutation analysis by direct sequencing and found that tumours with mutations have a significantly higher mean signal per cell from PDA samples compared with tumours with wild-type amplification was noted in 10% of cases. Although we found that lymph node metastasis and distal metastasis of PDA were more frequent in cases with amplification, this was not correlated with overall survival. Using a threshold of 40%, we found that the chromosomal instability index robustly discriminated PDA cells from CP cells. Based on these findings, we concluded that FISH testing of using cytology samples may represent an accurate approach for the diagnosis of PDA.

Digital object identifier (DOI): 10.1136/jclinpath-2018-205002

Basic and clinical andrology, 28, 5
2018

Is sperm FISH analysis still useful for Robertsonian translocations? Meiotic analysis for 23 patients and review of the literature.

Lamotte, Anna, Martinez, Guillaume, Devillard, Françoise, Hograindleur, Jean-Pascal, Satre, Véronique, Coutton, Charles, Harbuz, Radu, Amblard, Florence, Lespinasse, James, Benchaib, Mehdi, Bessonnat, Julien, Brouillet, Sophie, Hennebicq, Sylviane

<p>Robertsonian translocations (RobT) are common structural chromosome rearrangements where carriers display a majority of chromosomally balanced spermatozoa from alternate segregation mode. According to some monotony observed in the rates of balanced segregation, is sperm FISH analysis obsolete for RobT carriers? Retrospective cohort research study on 23 patients analyzed in our center from 2003 to 2017 and compared to the data of 187 patients in literature from 1983 to 2017.Robertsonian translocation carriers were divided in six groups according to the chromosomes involved in the translocation: 9 patients from our center and 107 from literature carrying 45,XY,der(13;14) karyotype, 3 and 35 patients respectively with 45,XY,der(14;21), 5 and 11 patients respectively with 45,XY,der(13;15), 4 and 7 patients respectively with 45,XY,der(14;15), 1 and 4 patients respectively with 45,XY,der(13;22),and 1 and 10 patients respectively with 45,XY,der(14;22). Alternate segregation mode is predominant in our group of Robertsonian translocation carriers with 73.45% ± 8.05 of balanced spermatozoa (min 50.92%; max 89.99%). These results are compliant with the data from literature for all translocations types (<em>p</em> &gt; 0.05) and are consistent among the different types of Robertsonian translocations (<em>p</em> &gt; 0.05) except for der(13;15) that exhibit lower balanced spermatozoa rates (<em>p</em> &gt; 0.05 versus der(13;14), der(14;21), (13;21) and der(15;22)). Normozoospermic patients also display a significantly (<em>p</em> &lt; 0.01) higher rate of balanced sperm cells than patients with abnormal seminograms whatever the defect implied. According to the discrepancies observed between der(13;15) and all the other Rob T carriers, the differences observed among patients presenting normal and abnormal sperm parameters and the input in genetical counselling, sperm FISH does not seem obsolete for these patients. Moreover, it seems important to collect more data for rare RobT.</p>

Digital object identifier (DOI): 10.1186/s12610-018-0069-z

International journal of radiation biology, 93, 58–64
January, 2017

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB).

Moquet, Jayne, Barnard, Stephen, Staynova, Albena, Lindholm, Carita, Monteiro Gil, Octávia, Martins, Vanda, Rößler, Ute, Vral, Anne, Vandevoorde, Charlot, Wojewódzka, Maria, Rothkamm, Kai

<p>Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma-H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Whole blood and separated lymphocyte samples were homogenously irradiated with (60)Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.</p>

Digital object identifier (DOI): 10.1080/09553002.2016.1207822

Radiation research, 185, 658–667
June, 2016

Telomere Length in Aged Mayak PA Nuclear Workers Chronically Exposed to Internal Alpha and External Gamma Radiation.

Scherthan, Harry, Sotnik, Natalia, Peper, Michel, Schrock, Gerrit, Azizova, Tamara, Abend, Michael

<p>Telomeres consist of GC-rich DNA repeats and the "shelterin" protein complex that together protect chromosome ends from fusion and degradation. Telomeres shorten with age due to incomplete end replication and upon exposure to environmental and intrinsic stressors. Exposure to ionizing radiation is known to modulate telomere length. However, the response of telomere length in humans chronically exposed to radiation is poorly understood. Here, we studied relative telomere length (RTL) by IQ-FISH to leukocyte nuclei in a group of 100 workers from the plutonium production facility at the Mayak Production Association (PA) who were chronically exposed to alpha-emitting ((239)Pu) radiation and/or gamma (photon) radiation, and 51 local residents serving as controls, with a similar mean age of about 80 years. We applied generalized linear statistical models adjusted for age at biosampling and the second exposure type on a linear scale and observed an age-dependent telomere length reduction. In those individuals with the lowest exposure, a significant reduction of about 20% RTL was observed, both for external gamma radiation (≤1 Gy) and internal alpha radiation (≤0.05-0.1 Gy to the red bone marrow). In highly exposed individuals (&gt;0.1 Gy alpha, 1-1.5 Gy gamma), the RTL was similar to control. Stratification by gender revealed a significant (∼30%) telomere reduction in low-dose-exposed males, which was absent in females. While the gender differences in RTL may reflect different working conditions, lifestyle and/or telomere biology, absence of a dose response in the highly exposed individuals may reflect selection against cells with short telomeres or induction of telomere-protective effects. Our observations suggest that chronic systemic exposure to radiation leads to variable dose-dependent effects on telomere length.</p>

Digital object identifier (DOI): 10.1667/RR14271.1

Front Neurol, 7, 23
2016

Lesion Size Is Exacerbated in Hypoxic Rats Whereas Hypoxia-Inducible Factor-1 Alpha and Vascular Endothelial Growth Factor Increase in Injured Normoxic Rats: A Prospective Cohort Study of Secondary Hypoxia in Focal Traumatic Brain Injury.

Thelin, Eric Peter, Frostell, Arvid, Mulder, Jan, Mitsios, Nicholas, Damberg, Peter, Aski, Sahar Nikkhou, Risling, M\aarten, Svensson, Mikael, Morganti-Kossmann, Maria Cristina, Bellander, Bo-Michael

Hypoxia following traumatic brain injury (TBI) is a severe insult shown to exacerbate the pathophysiology, resulting in worse outcome. The aim of this study was to investigate the effects of a hypoxic insult in a focal TBI model by monitoring brain edema, lesion volume, serum biomarker levels, immune cell infiltration, as well as the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF).Female Sprague-Dawley rats (n = 73, including sham and naive) were used. The rats were intubated and mechanically ventilated. A controlled cortical impact device created a 3-mm deep lesion in the right parietal hemisphere. Post-injury, rats inhaled either normoxic (22\% O2) or hypoxic (11\% O2) mixtures for 30 min. The rats were sacrificed at 1, 3, 7, 14, and 28 days post-injury. Serum was collected for S100B measurements using ELISA. Ex vivo magnetic resonance imaging (MRI) was performed to determine lesion size and edema volume. Immunofluorescence was employed to analyze neuronal death, changes in cerebral macrophage- and neutrophil infiltration, microglia proliferation, apoptosis, complement activation (C5b9), IgG extravasation, HIF-1α, and VEGF.The hypoxic group had significantly increased blood levels of lactate and decreased pO2 (p < 0.0001). On MRI post-traumatic hypoxia resulted in larger lesion areas (p = 0.0173), and NeuN staining revealed greater neuronal loss (p = 0.0253). HIF-1α and VEGF expression was significantly increased in normoxic but not in hypoxic animals (p < 0.05). A trend was seen for serum levels of S100B to be higher in the hypoxic group at 1 day after trauma (p = 0.0868). No differences were observed between the groups in cytotoxic and vascular edema, IgG extravasation, neutrophils and macrophage aggregation, microglia proliferation, or C5b-9 expression.Hypoxia following focal TBI exacerbated the lesion size and neuronal loss. Moreover, there was a tendency to higher levels of S100B in the hypoxic group early after injury, indicating a potential validity as a biomarker of injury severity. In the normoxic group, the expression of HIF-1α and VEGF was found elevated, possibly indicative of neuro-protective responses occurring in this less severely injured group. Further studies are warranted to better define the pathophysiology of post-TBI hypoxia.

Digital object identifier (DOI): 10.3389/fneur.2016.00023

Nat Commun, 7, 10529
2016

RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

Lescale, Chloé, Abramowski, Vincent, Bedora-Faure, Marie, Murigneux, Valentine, Vera, Gabriella, Roth, David B., Revy, Patrick, de Villartay, Jean-Pierre, Deriano, Ludovic

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly.

Digital object identifier (DOI): 10.1038/ncomms10529

Dental materials : official publication of the Academy of Dental Materials, 31, 1335–1344
November, 2015

Dental composite components induce DNA-damage and altered nuclear morphology in gingiva fibroblasts.

Styllou, Marianthi, Reichl, Franz-Xaver, Styllou, Panorea, Urcan, Ebru, Rothmund, Lena, Hickel, Reinhard, Högg, Christof, Scherthan, Harry

<p>Released dental composite components can damage human gingival fibroblasts (HGFs) and their DNA. The cytotoxicity, chromatin condensation and the induction of DNA double strand breaks (DSBs) by different compounds of dental composites was investigated using an improved γ-H2AX focus assay. HGFs were incubated with the monomers: bisphenol-A-ethoxylate-dimethacrylate (Bis-DMA), bisphenol-A-glycerolate-dimethacrylate (BisGMA), ethyltriethylen glycol methacrylate (ETEGMA), glycidyl methacrylate (GMA), 1,6-hexandiol-dimethycrylate (HDDMA), trimethylolpropane ethoxylate triacrylate (TMPTA), and acrylamide (ACR). DSBs were determined by enumerating γ-H2AX and 53BP1 foci colocalized at DSBs. A concentration-dependent induction of DSBs was found in the order: GMA&gt;BisGMA&gt;ACR&gt;Bis-DMA&gt;HDDMA&gt;TMPTA&gt;ETEGMA. HGFs exposure to GMA (0.3mM) and to BisGMA (0.09mM) induced the highest rate of DSB foci, i.e. 12-fold and 8-fold, respectively, relative to control (0.33 DSB foci/cell). At the highest concentrations (EC50) prominent changes in the chromatin morphology of HGF cell nuclei, i.e. compaction of nuclear chromatin and reduction of the area covered by the ovoid fibroblast nuclei, were observed. Nuclear condensation was significantly induced by GMA (1.7-fold at 0.3mM) and BisGMA (1.6-fold at 0.09mM), which correlated with the highest numbers of induced DSB foci (GMA, BisGMA, 3.9 and 2.6 foci/cell, respectively). The improved γ-H2AX/53BP1 focus assay revealed a concentration-dependent increase in DSBs for all tested substances. Furthermore, concentration-dependent changes in HGF cell nucleus morphology was noted, demonstrating genotoxic effects of the substances tested.</p>

Digital object identifier (DOI): 10.1016/j.dental.2015.08.156

Cancers, 281-295
2015

A Novel Three-Colour Fluorescence in Situ Hybridization Approach for the Detection of t(7;12)(q36;p13) in Acute Myeloid Leukaemia Reveals New Cryptic Three Way Translocation t(7;12;16)

Abdulbasit Naiel, Michael Vetter, Olga Plekhanova, Elena Fleischman, Olga Sokova, Grigory Tsaur, Jochen Harbott, Sabrina Tosi

The t(7;12)(q36;p13) translocation is a recurrent chromosome abnormality that involves the ETV6 gene on chromosome 12 and has been identified in 20-30% of infant patients with acute myeloid leukaemia (AML). The detection of t(7;12) rearrangements relies on the use of fluorescence in situ hybridization (FISH) because this translocation is hardly visible by chromosome banding methods. Furthermore, a fusion transcript HLXB9-ETV6 is found in approximately 50% of t(7;12) cases, making the reverse transcription PCR approach not an ideal screening method. Considering the report of few cases of variant translocations harbouring a cryptic t(7;12) rearrangement, we believe that the actual incidence of this abnormality is higher than reported to date. The clinical outcome of t(7;12) patients is believed to be poor, therefore an early and accurate diagnosis is important in the clinical management and treatment. In this study, we have designed and tested a novel three-colour FISH approach that enabled us not only to confirm the presence of the t(7;12) in a number of patients studied previously, but also to identify a cryptic t(7;12) as part of a complex rearrangement. This new approach has proven to be an efficient and reliable method to be used in the diagnostic setting.

Modern Pathology, 402-411
2014

Multiple genetic alterations in primary cutaneous large B-cell lymphoma, leg type support a common lymphomagenesis with activated B-cell-like diffuse large B-cell lymphoma

Anne Pham-Ledard, Martina Prochazkova-Carlotti, Laetitia Andrique, David Cappellen, Béatrice Vergier, Fabian Martinez, Florent Grange, Tony Petrella, Marie Beylot-Barry, Jean-Philippe Merlio

<p>Primary cutaneous large B-cell lymphoma, leg type has been individualized from nodal diffuse large B-cell lymphoma. The objective of this study was to screen primary cutaneous large B-cell lymphoma, leg type for genetic alterations recently described in nodal diffuse large B-cell lymphoma. Skin biopsies from 23 patients were analyzed for IRF4, BCL2, BCL6, and MYC expression. FISH testing was performed for BCL2, BCL6, MYC with separation probes and for CDKN2A and PRDM1/BLIMP1 deletion. Multiple sequential FISH analyses with up to six probes were performed to define samples with multiple cytogenetic alterations. MYD88 mutations were studied by Sanger sequencing. All cases but one displayed at least one genetic alteration (96%). Nine patients exhibited a single genetic mutation and 12 combined several alterations (52%). We observed a split for BCL2, BCL6, or MYC in 1/23, 6/23, and 3/23 of cases, respectively. No double-hit lymphoma was observed. CDKN2A deletion was detected by FISH in only 5/23 cases. BLIMP1 and/or 6q deletion was observed at a higher rate in 10/20 of cases. No correlation between rearrangement and immunohistochemical expression was found for BCL2 or MYC. FISH tracking of sequential hybridizations showed that several alterations were carried by the same nuclei. The p.L265P MYD88 mutation was found in 11/18 (61%) of cases. Contrary to most cutaneous lymphomas that rarely harbor primary genetic alteration of their nodal histological equivalent, primary cutaneous large B-cell lymphoma, leg type seems to be a 'cutaneous counterpart' of activated B-cell-like diffuse large B-cell lymphoma with a similar cytogenetic profile and a high rate of MYD88 oncogenic L265P mutation. This also suggests a common lymphomagenesis with NF-jB activation, strong IRF4 expression and terminal B-cell differentiation blockage. Our data support the use of therapies targeting NF-jB, as most patients displayed disease progression and resistance to conventional therapies.</p>