Level Of Genetic Instability Research Articles

Receptor Status of Tumor Cells and Mechanisms of Disorders of Tissue Homeostasis in Carcinogenesis of Serous Ovarian Cancer

The aim of the study is to define the value of sex hormone receptors expression level in serous ovarian cancer tumor cells for different variants of genetic instability of the cellular substrate and to evaluate its pathogenetic role in the biological of the tumor. Material and methods . The study of surgical and biopsy material from 89 patients with serous ovarian cancer was performed in histological preparations stained with hematoxylin and eosin and by the Felgen method. Subsequently, the degree of histological differentiation, mitotic regime and ploidy of tumor cells was evaluated, as well as immunohistochemical analysis of the expression in tumor cells of the tumor suppressor mtp53, progesterone and estrogen receptors. Two study groups were distinguished. The first group consisted of 62.9% of patients with negative p53 IGH status. The second group included 37.1% of cases with positive IGH expression of p53 in cells of serous ovarian cancer. Results. The first group of the studied tumors was characterized by significant morphological heterogeneity. On the contrary, in the group of serous carcinomas where a pronounced expression of oncogen p53 was diagnosed with a pronounced tendency to a low degree of histological differentiation of the parenchymal component of the tumor showed much greater morphological similarity and uniformity. At the same time, tumors in this group were characterized by the maximum indicators of ploidy of tumor cells, which, in our opinion, indicates an increase in cellular atypism and pronounced genetic instability against the background of high proliferative activity. With a significantly different level of genetic instability between the carcinomas of the study groups, the expression of estrogen receptors showed an inverse dependence on the level of cell proliferative activity and the amount of genetic material in the cancer cell. A negative correlation of the level of expression of sex hormone receptors with the degree of tumor differentiation regardless of the level of genetic abnormalities was revealed. Conclusion. It has been shown that the potential of tumor progression and the clinically more aggressive behavior of a neoplastic tissue substrate largely depends on the level of genetic instability in the epithelium of a cancer tumor, but at the same time, the altered regulatory mechanisms of cellular interactions (dysfunction of paracrine and endocrine regulation) in the tumor type cells leading to a violation of tissue homeostasis.

Correlations between Risk Factors for Breast Cancer and Genetic Instability in Cancer Patients-A Clinical Perspective Study.

Molecular epidemiological studies have identified several risk factors linking to the genes and external factors in the pathogenesis of breast cancer. In this sense, genetic instability caused by DNA damage and DNA repair inefficiencies are important molecular events for the diagnosis and prognosis of therapies. Therefore, the objective of this study was to analyze correlation between sociocultural, occupational, and lifestyle risk factors with levels of genetic instability in non-neoplastic cells of breast cancer patients. Total 150 individuals were included in the study that included 50 breast cancer patients submitted to chemotherapy (QT), 50 breast cancer patients submitted to radiotherapy (RT), and 50 healthy women without any cancer. Cytogenetic biomarkers for apoptosis and DNA damage were evaluated in samples of buccal epithelial and peripheral blood cells through micronuclei and comet assay tests. Elder age patients (61–80 years) had higher levels of apoptosis (catriolysis by karyolysis) and DNA damage at the diagnosis (baseline damage) with increased cell damage during QT and especially during RT. We also reported the increased frequencies of cytogenetic biomarkers in patients who were exposed to ionizing radiation as well as for alcoholism and smoking. QT and RT induced high levels of fragmentation (karyorrhexis) and nuclear dissolution (karyolysis) and DNA damage. Correlations were observed between age and karyorrhexis at diagnosis; smoking and karyolysis during RT; and radiation and karyolysis during QT. These correlations indicate that risk factors may also influence the genetic instability in non-neoplastic cells caused to the patients during cancer therapies.

Abstract P6-07-04: Distinct repertoires of somatic mutations affecting driver genes in mucinous and neuroendocrine carcinomas of the breast

Abstract Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic type, which accounts for approximately 2% of all invasive breast cancers (IBCs) and is characterized by clusters of tumor cells floating in large amounts of extracellular mucin. MCB comprises two main subtypes based on architectural and cytologic features: mucinous A (paucicellular) and mucinous B (hypercellular). Although MCBs are low-grade ER-positive/HER2-negative tumors of luminal molecular subtype, these cancers lack concurrent losses of 16q and gains of 1q, the hallmark genetic features of low-grade ER+/HER2- breast cancers, and have low levels of genetic instability. Neuroendocrine carcinoma of the breast (NCB) accounts for 2% - 5% of IBCs and displays morphologic features similar to those of neuroendocrine tumors of other organs. Previous transcriptomic analyses have revealed that NCBs and mucinous B, but not mucinous A, breast cancers display similar gene expression profiles. The aims of this study were to determine whether MCBs and NCBs share a similar repertoire of somatic genetic alterations and if these aberrations are distinct from those reported in common forms of ER+/HER2- IBCs. Material and methods: DNA extracted from microdissected MCBs (n=7 mucinous A, n=6 mucinous B), NCBs (n=14) and adjacent normal tissues were subjected to massively parallel sequencing targeting all exons of 254 genes most frequently mutated in IBC or related to DNA repair. Somatic point mutations were identified using MuTect and somatic insertions and deletions (indels) were defined using Strelka and Varscan2. We retrieved mutations in the same 254 genes in common forms of ER+/HER2- IBC (n=252) from The Cancer Genome Atlas (TCGA). Results: The most frequently mutated genes in MCBs were GATA3 (31% of cases, 4/13, all frame-shift indels), followed by KMT2C (MLL3) and MAP3K1 (both 23%). GATA3 and KMT2C (29%) were the most frequently mutated genes in mucinous A cancers, whereas MAP3K1 (33%) was the most frequently mutated gene in mucinous B cancers. ARID1A mutations were found in three of 14 (21%) NCBs, of which 2 were truncating mutations. A comparative analysis of the repertoire of somatic mutations found in mucinous A, mucinous B and NCBs did not reveal any statistically significant differences. As compared to common forms of ER+/HER2- IBCs, MCBs were found to have a significantly lower frequency of PIK3CA (8% vs 42%, p=0.02) mutations, which was particularly evident in mucinous A cancers (0% vs 42%, p=0.04). NCBs displayed significantly higher frequencies of somatic mutations affecting ARID1A (21% vs 2%, respectively, p=0.006), FOXA1 (14% vs 2%, respectively, p<0.05) and a lower frequency of PIK3CA somatic mutations (14% vs 42%, respectively, p<0.05) than common forms of ER+/HER2- IBCs. Conclusion: The frequency of mutations affecting bona fide breast cancer genes differed among mucinous A, mucinous B and NCBs. The repertoire of somatic mutations found in MCBs and NCBs differed from that of common forms of ER+/HER2- IBCs, in particular by the low frequency of somatic mutations affecting PIK3CA. Citation Format: Piscuoglio S, Ng CKY, Marchio C, Eberle CA, Guerini-Rocco E, Mariani O, Vincent-Salomon A, Reis-Filho JS, Weigelt B. Distinct repertoires of somatic mutations affecting driver genes in mucinous and neuroendocrine carcinomas of the breast. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-04.

Two-tier system on the origin of epithelial ovarian carcinomas and associated molecular biological basis

Ovarian epithelial carcinomas are the most common lethal gynecological malignancies. Ovarian carcinomas are divided into Types I and II based on different morphologies, genetic alterations, and biomarker expression. Low-grade micropapillary serous carcinoma are Type I tumors. Type I tumors are slow growing, generally confined to the ovary at diagnosis, and with better prognosis. These tumors develop from well established precursor lesions that are termed "borderline" tumors. Type 1 tumors are genetically stable and are characterized by mutations in a number of different genes including KRAS, BRAF, PTEN, and beta-catenin. Type II tumors are rapidly growing and highly aggressive neoplasms, for which well defined precursor lesions have not been described. They may arise in the fimbrial epithelium of the oviduct with advanced stage, more aggressive behavior, and worse prognosis. High-grade serous carcinoma belongs to Type II tumors. This group of tumors has a high level of genetic instability and is characterized by TP53 mutation. Hence, ovarian cancer comprises a heterogeneous group of tumors with distinctly different histological characteristics, molecular genetic features, and clinical course.

Genetic Instability in Peripheral Lymphocytes as Biological Marker for Non-Small Cell Lung Cancer Patients in the South Indian State of Andhra Pradesh

This study aims, first, at evaluating the DNA and chromosomal damage in non-small cell lung cancer (NSCLC) patients from the South Indian state of Andhra Pradesh, and then at correlating these results with possible confounding factors that might potentially play a role in causing genetic damage. The study included 246 NSCLC patients (177 men and 69 women) and 250 healthy controls (180 men and 70 women) for the analysis of DNA and chromosomal damage using the comet assay and micronucleus test. Both DNA and chromosomal damage were found to be increased in NSCLC patients compared to healthy controls, and the extent of the damage was higher in males than female patients. The smoking status had a profound effect on the extent of DNA and chromosomal damage in NSCLC patients. The degree of genetic damage correlated with the stage of the disease. However, the histological status had no effect on the extent of DNA and chromosomal damage among NSCLC patients. We here report, for the first time, that the NSCLC patients selected form the Andhra Pradesh population had increased DNA damage and higher mean micronucleus frequencies in peripheral lymphocytes, indicating a strong background level of genetic instability.

HER2 amplification and overexpression between primary and liver metastatic breast cancer: Significance and implication.

150 Background: The systemic management of metastatic breast cancer (MBC) is mostly based on the ER or HER2 status of the primary tumor. However, the hormonal status or the amplificaction/overexpression HER2 may change in every metastatic site because of the effects of the long-term treatment of metastatic cancer with endocrine therapy, chemotherapy, or targeted agents. The purpose of this study was to investigate the frequency of change in HER2 expression in primary and distant metastatic tumors (especially in liver) in HER2+ breast cancer patients. Methods: We retrospectively analyzed the results of 31 consecutive metastatic breast cancer patients that were seen in our center over 4 months from February 2014 through May 2014. All patients were rebiopsied after consultation and samples were sent for standard immunohistochemistry (IHC) for ER, PR, and HER2 and formalin-fixed, paraffin-embedded (FFPE) samples were sent for genomic (Foundation Medicine) and proteomic analysis (Theranostics). All results from the metastatic samples were compared to the baseline IHC and/or FISH results for HER2. Results: A change in HER2 status was observed in 26% of the cases. 16% of cases underwent a negative to positive conversion in HER2 status while 10% of cases underwent a positive to negative conversion. It is notable that all 5 patients that underwent a negative to positive conversion in HER2 status had biopsies taken from metastatic disease in the liver. Overall, 45% of patients with metastatic disease in the liver had a negative to positive conversion in HER2 status. Conclusions: The results of this study emphasize the significance of confirming HER2 expression in a recurrence lesion. This discordance may be due to the increasing level of genetic instability occurring throughout disease progression that can significantly influence the alterations of the HER2 gene. If feasible, HER2 reassessment in metastatic lesions should be carefully taken into account, especially for metastases coming from non-HER2 amplified breast cancer. Although HER2 status is usually appraised in primary tumor, knowledge of the HER2 status in metastases may be of potential value for therapeutic decision making.

Impact of ABL kinase domain mutations on the outcome of patients with chronic myeloid leukemia (CML).

6588 Background: Patients with CML who develop resistance to imatinib commonly have mutations in the BCR-ABL kinase domain (KDM). Studies looking at outcomes in patients with P-loop versus non-P-loop mutations within the ABL-Kinase Domain have produced conflicting results. Methods: The Total Cancer Care (TCC) database was used to identify patients with CML treated at Moffitt Cancer Center (MCC). Descriptive data were reported, chi square test was used for categorical variables, and Kaplan Meier curves were used for OS and PFS. Log rank test was used to compare survival times between groups. Results: Between 1992 and 2011, 540 CML patients were treated at MCC. Of those, 51% were male and 71% were under the age of 60. Sixty percent (n=322) were diagnosed after 2001. Of the 540 patients, 6.5% (n=35) were found to have mutations of which 26 were detected in patients diagnosed after 2001. Of the 35 patients, 74% (n=26) had single mutations and 26% (n=9) had compound mutations. P-loop mutations were seen in 17% (n=6) and 43% (n=15) had T315I mutations. Patients with KDM progressed to accelerated or blast phase in 46% (n=16) of cases compared to 27% (n=136) without mutations (p=0.03). Median OS was 126 months, 109 months, and not reached in patients with P-loop, T315I, and non-P-loop mutations respectively (p=0.17). The corresponding median PFS was 85 months, 89 months, and not reached (p=0.20). In patients with one mutation median OS was not reached compared to 105 months in patients with compound mutations (p=0.27). After 2001, patients with KDM had a median PFS of 75 months and OS of 126 months while neither was reached in the non-mutation cohort (p=0.007, p=0.26 respectively). Median PFS in patients with single mutations was 85 months versus 10 months in those with compound mutations (p=0.037). Patients with KDM had additional Ph+ clones on cytogenetics in 49% of cases compared with 19% of cases in the non-mutation group (P < 0.005). Conclusions: T315I and P-loop KDM predict PFS and OS in CML patients, and convey a trend for worse prognosis. The presence of additional Ph+ clones in patients with BCR-ABL KDM indicates a higher level of genetic instability and clonal evolution, which may be the contributing factor to poor outcomes.

Abstract P6-04-08: Microarray Characterization of 24 Cases of Pure Mucinous Carcinoma of the Breast

Abstract Background: Mucinous carcinoma of the breast has been shown to be a distinct histological entity with a better clinical prognosis than invasive ductal carcinoma, NOS. Only a few cases have thus far been studied by molecular analysis. The purpose of this is study is to further evaluate the genomic changes in mucinous carcinoma of the breast. Design: 24 pure mucinous breast carcinomas with available fresh frozen tissue were retrieved from our files. The cases were reviewed for histologic grade (SBR), mitoses, percent mucin, intracytoplasmic mucin, type A or B mucinous carcinoma, and estrogen receptor (ER) and progesterone receptor (PR) expression. Follow-up was available for 13 patients and included 4 recurrences. Representative sections of the tumors were dissected from the normal tissue present in the blocks and were assayed by Agilent (Santa Clara, CA, USA) human genome CGH microarray kit 105A. Analysis was performed using Agilent (Santa Clara, CA, USA) genomic workbench 5.0 software. Results: The 24 mucinous carcinomas were composed of 19 type A and 5 type B mucinous carcinomas, including 21 with >60% mucin and 3 with <60% mucin. Seven cases contained intracytoplasmic mucin. Tumors were grade 1 in 20 cases and grade 2 in 4 cases. Mitotic count per 10hpfs was 0 or 1-2 in 19 cases and >3 in 5 cases. ER was positive in all cases, and PR was positive in all but 4 cases. Analysis of the genome arrays showed that 17 of the cases had a complex level of genetic instability with three or more (up to 15) of the chromosomes showing instability while four show only one or two chromosomes affected. On the other hand, there are no abnormalities found in four of the cases. The most frequently affected chromosome is chromosome 1 which is involved in 12 of the cases, with three cases showing 1p partial loss/full loss, and nine cases showing 1q partial gain/full gain. Gains were also commonly seen in 8p, 8q, 11q, 16p, 17q and 20q. Losses were most frequently seen in 6q, 11q, 16q and 17p. Four of five Type B tumors exhibited a complex pattern, and all four grade 2 tumors had a complex pattern. Of the tumors that recurred, three of four had a complex pattern of genetic instability. Conclusions: Evaluation of pure mucinous carcinomas studied showed a high level of genomic complexity with multiple chromosomal changes in most cases. Tumors that were grade 2 and mucinous type B tumors tended to be of higher complexity than grade 1 and type A mucinous tumors. The most commonly affected chromosomes include chromosomes 1, 6, 8, 11, 16, and 17. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-08.

Abstract 3265: Immortalization and comprehensive characterization of a human ovarian adenocarcinoma cell line derived from an Ashkenazi Jewish patient

Abstract We have established and characterized a new ovarian cancer cell line derived from stage IV serous ovarian adenocarcinoma metastatic tissue from a 47 year old Ashkenazi Jewish patient. The patient developed ovarian cancer at age 44 and died of the disease at age 48. The tissue was obtained after an extensive secondary cytoreductive intraperitoneal surgery. Prior to the surgery the patient underwent primary cytoreduction followed by several systemic therapies with recurrences. A culture of the metastatic tissue was transfected with Simian Virus 40 Large T-Antigen and telomerase. The cell line has been in culture on and off during 3 years. The identity of the cell line was confirmed by Short Tandem Repeat genotyping on genomic DNA extracted from early and late passages as well as from frozen metastatic tissue. The cell line was further characterized by array-Comparative Genomic Hybridization and gene expression microarray; and by Spectral Karyotyping and Fluorescent In Situ Hybridization. The results of these analyses were compared, when appropriate, with those of frozen metastatic tissue. A low level of genetic instability was detected in the cell line, but the karyotypes seem to be near diploid and relatively stable, after several years of cultivation. We believe that this novel ovarian cancer cell line could contribute to the understanding of the molecular genetic basis of the disease in this advanced stage, and be representative of the tumors of a great number of patients that become resistant to the majority if not all known cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3265.

Genetic Instability in Cancer: An Optimal Control Problem

Carcinogenesis (cancer generation) relies on a sequence of mutations that transforms cells, leading to their unchecked growth. An important phenomenon in cancer is genetic instability, or an increased rate of mutations, with a higher associated cell death rate. Given the trade-off between an increase in mutant cells by higher mutation rate and higher loss of mutants through a higher death rate, we ask the question: what mutation rate is most advantageous for cancer? To seek an answer to this question, we investigate an optimal control problem of normal and mutant cell growth where the abnormal mutation rate plays the role of a time-dependent control. The analysis of this problem shows that the best “strategy” for the fastest time to cancer is to start with a high level of genetic instability initially, and then to switch to a low level of genetic instability. The exact shape of the optimal mutation rate as a function of time depends on how genetic instability contributes to the death rate of cells. If the death rate is a linear or an increasing concave function of the mutation rate, the optimal mutation rate is bang-bang, which changes from its highest to its lowest value with a finite jump discontinuity. However, if the death rate is an increasing convex function of the mutation rate, then the optimal control is a continuous decreasing function of time. Two known mechanisms of cancer initiation have been considered, an activation of an oncogene and an inactivation of a tumor-suppressor gene. Mathematical justification of the results of the first, a one-step process, is reported herein.

Integrative Molecular Profiling of Triple Negative Breast Cancers Identifies Potential Therapeutic Targets Including Amplifications of FGFR2.

Abstract Triple negative breast cancers (TNBCs) have a relatively poor prognosis emphasising the need to identify new subtype specific target therapies. Based on the concept of oncogene addiction, we searched for potential therapeutic targets by identifying genes consistently over-expressed when amplified in TNBC. Fifty six TNBCs were subjected to high resolution tiling path microarray-based comparative genomic hybridisation (aCGH); out of these cases, 24 were also subjected to genome-wide microarray-based mRNA expression analysis. TNBCs showed a high level of genetic instability, with recurrent regions of amplification (>4 copies) included multiple regions on 1q and 8q, 3q25, 10p14, 10q26, 13q34, 15q26 and 19q12-19q13. Integration of aCGH and expression data revealed 38 genes that were significantly overexpressed when amplified. This list includes known oncogenes and potential therapeutic targets, such as MCL1 (1q21.2), FGFR2 (10q26.3), BUB3 (10q26.3), RAB20 (13q34), PKN1 (19p13.12), and NOTCH3 (19p13.12). To validate FGFR2 as a therapeutic target, we screened a panel of cell lines, by western blotting and aCGH, and identified two TNBC cell lines with FGFR2 amplification. In these cell lines FGFR2 was constitutively active in a ligand independent manner, and RNA interference-mediated silencing of FGFR2 selectively decreased survival of cell lines harbouring FGFR2 amplification. Likewise FGFR2 amplified cell lines were highly sensitive to FGFR tyrosine kinase inhibitor PD173074 (IC50 <20nM). Treatment with PD173074 induced apoptosis in amplified cell lines, as did treatment with PI3 kinase inhibitors LY294002 and BEZ-235 suggesting that apoptosis resulted from inhibition of AKT signalling. Examination of publically available CGH data sets confirmed FGFR2 amplification in 4% (5/124 95%CI 1.3-9.2%) of TNBC, with no cases of FGFR2 amplification in other subtypes (0/150, p=0.02). Our results suggest that FGFR2 amplification is a therapeutic target in a small subset of TNBCs. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3147.

Three NewBLMGene Mutations Associated with Bloom Syndrome

Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.

Barrett's Esophagus in 2008: an Update

With the rising incidence and overall poor prognosis of esophageal adenocarcinoma (EA) there is great interest in furthering our understanding of Barrett's esophagus, the precursor lesion for most cases of EA. The best available evidence from true population-based analysis suggests that the prevalence of Barrett's is 1.6%. In addition, nearly half of the patients with Barrett's are asymptomatic. Several risk factors for development of Barrett's have been identified including gastro-esophageal reflux disease (GERD), central obesity, H. pylori eradication, and male gender. The precise incidence of progression from Barrett's to esophageal adenocarcinoma is not known, but it probably is less than 0.5% per year, and our ability to predict who is at highest risk for progression remains poor. The degree of dysplasia is currently used as a marker for risk of progression to cancer though there is increasing evidence that biomarkers and level of genetic instability may provide better predictive measures. Intensive acid-suppression and COX-2 inhibition are potential strategies to reduce the risk of progression, though definitive studies are needed. Endoscopic surveillance remains the mainstay of management for non-dysplastic and low grade dysplasia Barrett's. The advent of various endoscopic ablative therapies has provided a promising alternative to surgery for Barrett's patients with high grade dysplasia (HGD).

A novel role of DNA fragmentation factor as a tumor suppressor through maintaining genomic stability

10023 Background: DNA fragmentation is a hallmark of apoptosis. However, the biological function of apoptotic DNA fragmentation remains unclear. Genes encoding the nuclease responsible for DNA fragmentation, DFF40/CAD and DFF45/ICAD, are deleted, mutated, or aberrantly expressed in many human tumor types. Abnormalities in this gene are associated with aggressive tumors and poor prognosis in cancer patients. Tumor-specific DFF45 gene mutations were identified in human tumors, indicating the involvement of this gene in tumor development. Methods: We studied genetic instability, cellular transformation and tumorigenesis in cells and mice deficient in DNA fragmentation. We applied two methods to inhibit DNA fragmentation, overexpressing a modified ICAD which inhibits DNA fragmentation during apoptosis in stable cell lines and disrupting cad gene in transgenic mice. Genetic instability was studied by gene amplification assay, mutation assay and cytogenetic analysis including chromosomal aberrations and translocations. Cellular transformation was studied by soft agar assay using CAD-/- MEF cells. Tumorigenesis in DFF/CAD-knockout mice was studied by radiation carcinogenesis and two-stage skin chemical carcinogenesis. Results: (1) Human and mouse cancer cell lines with their CAD activity inhibited exhibited significantly more genetic instability under spontaneous or mutagen (ionizing radiation) induced conditions. These are reflected as elevated frequencies of chromosomal aberrations, gene amplifications, and gene mutations; (2) Mouse cells with targeted disruption of the cad gene exhibit a similar, increased level of genetic instability when the host animal is irradiated; (3) Mechanistically, CAD maintains genetic instability through the removal of cells with DNA damage, a role that is similar to genomic “gatekeepers” like p53; (4) CAD-null mouse embryo fibroblasts exhibited enhanced cellular transformation; (5) Significantly enhanced susceptibility to chemical and radiation-induced tumor development was observed in mice with targeted disruption of the CAD gene, indicating that it plays a role in suppressing tumor development. Conclusions: CAD plays an important role in maitaining genetic stability and suppressing tumor development. No significant financial relationships to disclose.

TCR trans-Rearrangements: Biological Significance in Antigen Recognition vs the Role as Lymphoma Biomarker

V(D)J rearrangements occur within loci of TCR and BCR genes, thus generating the diversity of the AgR repertoire. In addition, interlocus V(D)J rearrangements occur, giving rise to so-called "trans-rearrangements." Such trans-rearrangements increase the diversity of the immune receptor repertoire and can be expressed as functional chimeric TCR proteins on the surface of T cells. Although chimeric receptors are not pathogenic per se, the frequency of AgR trans-rearrangements correlates with the level of genetic instability and thus could be used as a predictive biomarker for lymphoma risk.

Overexpression of aurora B kinase (AURKB) in primary non-small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability

Aurora kinases are key regulators of chromosome segregation during mitosis. We have previously shown by microarray analysis of primary lung carcinomas and matched normal tissue that AURKB (22 out of 37) and AURKA (15 out of 37) transcripts are frequently over-represented in these tumours. We now confirm these observations in a second series of 44 carcinomas and also show that aurora B kinase protein levels are raised in the tumours compared to normal tissue. Elevated levels of expression in tumours are not a consequence of high-level amplification of the AURKB gene. Using a coding sequence polymorphism we show that in most cases (seven out of nine) tumour expression is predominantly driven from one AURKB allele. Given the function of aurora B kinase, we examined whether there was an association between expression levels and genetic instability. We defined two groups of high and low AURKB expression. Using a panel of 10 microsatellite markers, we found that the group showing the higher level of expression had a higher frequency of allelic imbalance (P=0.0012). Analysis of a number of other genes that are strongly and specifically expressed in tumour over normal lung, including SERPINB5, TERT and PRAME, showed marked allelic expression imbalances in the tumour tissue in the context of balanced or only marginally imbalanced relative allelic copy numbers. Our data support a model of early carcinogenesis wherein defects in the process of inactivation of lung stem-cell associated genes during differentiation, contributes to the development of carcinogenesis.

Enhancement of genetic instability in human B cells by Epstein-Barr virus latent infection.

The level of genetic instability, as assessed by micronucleus (MN) formation, was higher in Epstein-Barr virus (EBV)-converted B-cell lines with one copy of the EBV genome integrated in each cell than in the parental, EBV-negative, B lymphoma cells. MN induced by EBV latency, as analysed by in situ hybridization, contained mainly centromeric regions, indicating that the presence of EBV affects the segregation of entire chromosomes. The instability was inhibited by treatment with antioxidants. Flow cytometric analysis indicated that there was a higher basal level of peroxides in EBV(+) cells. Direct oxidative stress caused by hydrogen peroxide (which is known to be both apoptogenic and mutagenic) enhanced the number of MN only in an EBV-converted clone. These cells were also resistant to apoptosis, as expected, suggesting that in the parental EBV cells apoptosis may efficiently eliminate cells with genetic damage. These results show for the first time a direct involvement of EBV in the induction of genetic instability, suggesting that it could contribute to tumour progression.

IDENTIFICATION OF COPIA-LIKE RETROTRANSPOSABLE ELEMENT BY APPLE

Until now attempts to use the existing molecular techniques (RFLP, RAPD or AFLP) for the identification of numerous apple fruit colour mutants (from Jonagold or Gala varieties for instance) always failed. In previous reports, the AFLP technique allowed us to detect occasional polymorphisms between some Jonagold mutants and their mother-variety. However, it was demonstrated that such variations, although not artefactual, were not correlated with the fruit colour genotype and are likely to be linked to a certain level of genetic instability within vegetatively propagated lines. In order to isolate cDNA fragments that might be differentially expressed in relation with the fruit colour phenotype, a differential display technique was used to compare mRNA populations extracted from the fruit epidermis of mutants versus the original Jonagold variety. Candidate fragments were obtained using the differential display protocol of Sokolov and Prockop (1994). After their recovery from gels, those fragments were cloned and sequenced. One of them appeared to share strong sequence similarities with the reverse transcriptase gene of several copia-like retrotransposons. These transposable elements have been previously described in diverse plant species like cotton, pea, tobacco, maize or wheat. A Southern blot analysis performed on DNA from several apple varieties clearly demonstrated that the corresponding fragments were present in numerous copies within the genome of all tested cultivars. A genomic library was screened using the reverse transcriptase fragment in order to isolate fulllength copies of this element. Their characterization and sequence analysis are in progress. The demonstration of the existence of a transposable element in the apple genome could be a major step in the understanding of the origin of the genetic variation in this species. This information could also be used to develop new molecular marker methodologies, that could be particularly useful for mutant identification and cultivar conformity testing in apple.

Transcription increases the deletion frequency of long CTG.CAG triplet repeats from plasmids in Escherichia coli.

Induction of transcription into long CTG.CAG repeats contained on plasmids in Escherichia coli is shown to increase the frequency of deletions within the repeat sequences. This elevated genetic instability was detected because active transcription into the triplet repeat influenced the growth transitions of the host cell, allowing advantageous growth for cells harboring plasmids with deleted repeat sequences. The variety of deletion products observed in separate cultures suggests that transcription altered the metabolism of the DNA in a manner that produced random length changes in the repeat sequence. For cultures containing plasmids without active transcription into the triplet repeat, or those maintained in exponential growth, deletions occurred within the repeat at a lower frequency (5-20-fold lower). In these incubations the extent of deletions was proportional to the number of cell divisions and many repeat lengths were observed within each culture, suggesting that the decrease in average repeat length at long incubation times was due to multiple small deletions. These observations show that deletions within long CTG.CAG repeats contained on plasmids in E.coli occur via more than one pathway and their level of genetic instability is altered by the enzymatic processes occurring upon the DNA.

Isolation and characterization of a mutator strain of Streptomyces ambofaciens ATCC23877 exhibiting an increased level of genetic instability

In Streptomyces ambofaciens ATCC23877, 0.7% of pigment-defective mutants (Pig−) can be observed in the progeny of wild-type colonies. A mutator (Mut−) strain was isolated from the offspring of the wild-type strain. The Mut− strain produced colonies that sported nonpigmented papillae. Furthermore, the frequency of Pig− colonies obtained in the progeny of this strain was fivefold higher than in the wild-type strain. This strain showed the same level of sensitivity to ultraviolet light and mitomycin C as the wild-type strain. This Mut− phenotype was found to be reversible at high frequency (3 × 10−3). Genomic analysis using pulsed-field gel electrophoresis (PFGE) showed that the Pig− mutants arisen from the Mut− strain were less frequently rearranged (32% were deleted) compared with the mutants arising from the wild type (59% were deleted). Moreover, the Pig− papillae mutants possessed no visible rearrangement as revealed by PFGE analyses.Key words: Streptomyces, genetic instability, mutator strain, papillae.