Polymorphisms in MDM2 and TP53 Genes and Risk of Developing Therapy-Related Myeloid Neoplasms.

One of the most severe complications after successful cancer therapy is the development of therapy-related myeloid neoplasms (t-MN). Constitutional genetic variation is likely to impact on t-MN risk. We aimed to evaluate if polymorphisms in the p53 pathway can be useful for predicting t-MN susceptibility. First, an association study revealed that the Pro variant of the TP53 Arg72Pro polymorphism and the G allele of the MDM2 SNP309 were associated with t-MN risk. The Arg variant of TP53 is more efficient at inducing apoptosis, whereas the Pro variant is a more potent inductor of cell cycle arrest and DNA repair. As regards MDM2 SNP309, the G allele is associated with attenuation of the p53 apoptotic response. Second, to evaluate the biological effect of the TP53 polymorphism, we established Jurkat isogenic cell lines expressing p53Arg or p53Pro. Jurkat p53Arg cells presented higher DNA damage and higher apoptotic potential than p53Pro cells, after treatment with chemotherapy agents. Only p53Pro cells presented t(15;17) translocation and del(5q). We suggest that failure to repair DNA lesions in p53Arg cells would lead them to apoptosis, whereas some p53Pro cells, prone to cell cycle arrest and DNA repair, could undergo misrepair, generating chromosomal abnormalities typical of t-MN.

Analysis of α-particle-induced chromosomal aberrations by chemically-induced PCC. Elaboration of dose-effect curves.

In a similar way to high-dose exposures to low-LET radiations, cells show difficulties reaching mitosis after high-LET radiation exposure. For this reason, techniques have been proposed that are able to analyze chromosome aberrations in interphase by prematurely condensing the chromosomes (PCC-techniques). Few dose-effect curves for high-LET radiation types have been reported, and none for α-particles. The aim of this study was to evaluate, by chemically-induced PCC, the chromosome aberrations induced by several doses of α-particles. Monolayers of peripheral lymphocytes were exposed to an α-source of Americium-241 with a mean energy entering the cells of 2.7 MeV. Lymphocytes were exposed to 10 doses, from 0-2.5 Gy, and then cultured for 48 h. Colcemid and Calyculin-A were added at 24 and 1 h before harvesting, respectively. During microscope analysis, chromosome rings and extra chromosome pieces were scored in G2/M-PCC and M cells, while dicentric chromosomes were only scored in M cells. As the dose increased, fewer cells were able to reach mitosis and the proportion of G2/M-PCC cells increased. Chromosome rings were hardly observed in M cells when compared to G2/M-PCC cells. Extra fragments were more frequent than rings in both G2/M-PCC and M cells, but with lower frequencies than in G2/M-PCC cells. The distribution of dicentrics and extra fragments showed a clear overdispersion; this was not so evident for rings. The dose-effect curves obtained fitted very well to a linear model. Damaged cells after α-particle irradiation show more difficulties in reaching mitosis than cells exposed to γ-rays. After α-particle irradiation the frequency of all the chromosome aberrations considered increased linearly with the dose, and α-particles clearly produced more dicentrics and extra chromosome pieces with respect to γ-rays. After α-particle exposure, the existence of extra chromosome fragments in PCC cells seems to be a good candidate for use as a biomarker for dose assessment. However, the observed frequencies of different types of chromosomal aberrations could be influenced by some methodological aspects; for this reason, and in order to avoid possible methodological bias, standardization of the technique will be desirable.

A New Model for Biological Dose Assessment in Cases of Heterogeneous Exposures to Ionizing Radiation.

In biological dosimetry by dicentric analysis, an exposure to radiation is considered non-homogeneous if the dicentric cell distribution shows overdispersion with respect to Poisson distribution. Traditionally, when this occurs, all non-homogeneous exposures are considered as partial-body exposures, assuming that there is only a mixture of irradiated and nonirradiated cells. The methods to estimate the dose in the irradiated fraction and the initial fraction of irradiated cells are based on separating which part of the cells without aberrations comes from the nonirradiated or irradiated fractions. In this study we show a new approach based on a mixed Poisson model, which allows for a distinction to be made between partial and heterogeneous exposures. To validate this approach blood samples from two donors, a male and a female, irradiated at different doses, were mixed at a 1:1 proportion to simulate partial and heterogeneous exposures. The results show a good agreement between the observed proportion of male and female cells and the proportion estimated by the model. Additionally, a good agreement was observed between the delivered doses, the initial fraction of cells and the ones estimated by the model. This good agreement was also observed after very high-dose irradiation (up to 17 Gy), when the lymphocyte cultures were treated with caffeine. Based on these results, we propose the use of this mixed Poisson model for a more accurate assessment of non-homogeneous exposures.

A new model of biodosimetry to integrate low and high doses.

Biological dosimetry, that is the estimation of the dose of an exposure to ionizing radiation by a biological parameter, is a very important tool in cases of radiation accidents. The score of dicentric chromosomes, considered to be the most accurate method for biological dosimetry, for low LET radiation and up to 5 Gy, fits very well to a linear-quadratic model of dose-effect curve assuming the Poisson distribution. The accuracy of this estimation raises difficulties for doses over 5 Gy, the highest dose of the majority of dose-effect curves used in biological dosimetry. At doses over 5 Gy most cells show difficulties in reaching mitosis and cannot be used to score dicentric chromosomes. In the present study with the treatment of lymphocyte cultures with caffeine and the standardization of the culture time, metaphases for doses up to 25 Gy have been analyzed. Here we present a new model for biological dosimetry, which includes a Gompertz-type function as the dose response, and also takes into account the underdispersion of aberration-among-cell distribution. The new model allows the estimation of doses of exposures to ionizing radiation of up to 25 Gy. Moreover, the model is more effective in estimating whole and partial body exposures than the classical method based on linear and linear-quadratic functions, suggesting their effectiveness and great potential to be used after high dose exposures of radiation.

Suitability of scoring PCC rings and fragments for dose assessment after high-dose exposures to ionizing radiation.

Assessment of radiation doses through measurement of dicentric chromosomes may be difficult due to the inability of damaged cells to reach mitosis. After high-dose exposures, premature chromosome condensation (PCC) has become an important method in biodosimetry. PCC can be induced upon fusion with mitotic cells, or by treatment with chemicals such as calyculin A or okadaic acid. Several different cytogenetic endpoints have been measured with chemically induced PCC, e.g., via scoring of extra chromosome pieces or ring chromosomes. The dose-effect curves published with chemically induced PCC show differences in their coefficients and in the distribution of rings among cells. Here we present a study with calyculin A to induce PCC in peripheral blood lymphocytes irradiated at nine different doses of γ-rays up to 20Gy. Colcemid was also added in order to observe metaphase cells. During microscopical analysis the chromosome aberrations observed in the different cell-cycle phases (G2/M-PCC, M/A-PCC and M cells) were recorded. The proportion of G2/M-PCC cells was predominant from 3 to 20Gy, M cells decreased above 1Gy and M/A-PCC cells remained constant at all doses and showed the highest frequencies of PCC rings. Depending on the cell-cycle phase there was a difference in the linear coefficients in the dose-effect curves of extra fragments and rings. Poisson distribution among PCC rings was observed after calyculin A+colcemid treatment, facilitating the use of this methodology also for partial body exposures to high doses. This has been tested with two simulated partial exposures to 6 and 12Gy. The estimated doses in the irradiated fraction were very close to the real dose, indicating the usefulness of this methodology.

The use of caffeine to assess high dose exposures to ionising radiation by dicentric analysis.

Dicentric analysis is considered as a 'gold standard' method for biological dosimetry. However, due to the radiation-induced mitotic delay or inability to reach mitosis of heavily damaged cells, the analysis of dicentrics is restricted to doses up to 4-5 Gy. For higher doses, the analysis by premature chromosome condensation technique has been proposed. Here, it is presented a preliminary study is presented in which an alternative method to analyse dicentrics after high dose exposures to ionising radiation (IR) is evaluated. The method is based on the effect of caffeine in preventing the G2/M checkpoint allowing damaged cells to reach mitosis. The results obtained indicate that the co-treatment with Colcemid and caffeine increases significantly increases the mitotic index, and hence allows a more feasible analysis of dicentrics. Moreover in the dose range analysed, from 0 to 15 Gy, the dicentric cell distribution followed the Poisson distribution, and a simulated partial-body exposure has been clearly detected. Overall, the results presented here suggest that caffeine has a great potential to be used for dose-assessment after high dose exposure to IR.

Induction of incomplete and complex chromosome aberrations by 30 kVp X rays.

X rays of 26-30 kVp are routinely used for mammography screening. For radioprotection purposes, a quality factor (Q) of 1 is assumed for all photon energies, but it is thought that the relative biological effectiveness (RBE) increases as the photon energy decreases. The analysis of radiation-induced chromosome aberrations is one of the most widely used methods to study the interaction between radiation and DNA. Here we present a FISH study on metaphases from peripheral blood samples irradiated with three different X-ray energies (30, 80 and 120 kVp). The study comprises two FISH approaches: one using pantelomeric and pancentromeric probes to evaluate the induction of incomplete chromosome aberrations and the other using mFISH to evaluate the induction of complex chromosome aberrations. The results indicate that exposure to 30 kVp X rays resulted in a modest increase in the induction of incomplete elements and complex aberrations compared to 80 and 120 kVp X rays.

Identification of human specific gene duplications relative to other primates by array CGH and quantitative PCR.

In order to identify human lineage specific (HLS) copy number differences (CNDs) compared to other primates, we performed pair wise comparisons (human vs. chimpanzee, gorilla and orangutan) by using cDNA array comparative genomic hybridization (CGH). A set of 23 genes with HLS duplications were identified, as well as other lineage differences in gene copy number specific of chimpanzee, gorilla and orangutan. Each species has gained more copies of specific genes rather than losing gene copies. Eleven of the 23 genes have only been observed to have undergone HLS duplication in Fortna et al. (2004) and in the present study. Then, seven of these 11 genes were analyzed by quantitative PCR in chimpanzee, gorilla and orangutan, as well as in other six primate species (Hylobates lar, Cercopithecus aethiops, Papio hamadryas, Macaca mulatta, Lagothrix lagothricha, and Saimiri sciureus). Six genes confirmed array CGH data, and four of them appeared to have bona fide HLS duplications (ABCB10, E2F6, CDH12, and TDG genes). We propose that these gene duplications have a potential to contribute to specific human phenotypes.

Genetic changes including gene copy number alterations and their relation to prognosis in childhood acute myeloid leukemia.

We studied a series of 68 subjects diagnosed with childhood acute myeloid leukemia (AML) using conventional cytogenetics and fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) to analyze mutations in FLT3 and NPM1 genes, and/or array comparative genomic hybridization (CGH). Cytogenetic/FISH abnormalities were observed in 71% of subjects, FLT3-ITD mutations in 15%, and NPM1 mutations in 13%. The array CGH alterations (average 3.6 per case) were observed in 96% of the tested subjects. The most frequent alterations were gains of 8q24.3 and 11p15.5-p15.4 in 16% of the samples. Six genes (AKT1, RUNX1, LTB, SDC1, RUNX1T1, and JAK2) from the imbalanced regions have been reported to be involved in AML, whereas other 30 cancer genes, not previously reported in an AML context, were identified as imbalanced. They probably correspond to non passenger alterations that cooperate with the recurrent translocations. The clinical data and genetic changes were tested to find out the possible association with prognosis. Genomic instability (four or more genomic imbalances) was correlated with poor patient outcome (p = 0.029).

Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint.

Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of gamma-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

Persistence of radiation-induced chromosome aberrations in a long-term cell culture.

The aim of the present study was to evaluate the persistence of chromosome aberrations induced by X rays. FISH painting and mFISH techniques were applied to long-term cultures of irradiated cells. With painting, at 2 Gy the frequency of apparently simple translocations remained almost invariable during all the culture, whereas at 4 Gy a rapid decline was observed between the first and the second samples, followed by a slight decrease until the end of the culture. Apparently simple dicentrics and complex aberrations disappeared after the first sample at 2 and 4 Gy. By mFISH, at 2 Gy the frequency of complete plus one-way translocations remained invariable between the first and last sample, but at 4 Gy a 60% decline was observed. True incomplete simple translocations disappeared at 2 and 4 Gy, indicating that incompleteness could be a factor to consider when the persistence of translocations is analyzed. The analysis by mFISH showed that the frequency of complex aberrations and their complexity increased with dose and tended to disappear in the last sample. Our results indicate that the influence of dose on the decrease in the frequency of simple translocations with time postirradiation cannot be fully explained by the disappearance of true incomplete translocations and complex aberrations. The chromosome involvement was random for radiation-induced exchange aberrations and non-random for total aberrations. Chromosome 7 showed the highest deviations from expected, being less and more involved than expected in the first and last samples, respectively. Some preferential chromosome-chromosome associations were observed, including a coincidence with a cluster from radiogenic chromosome aberrations described in other studies.

Mitotic delay in lymphocytes from BRCA1 heterozygotes unable to reduce the radiation-induced chromosomal damage.

Double strand breaks (DSB) are critical lesions involved in the formation of chromosomal aberrations. In response to DNA damage, the cell has mechanisms of repair and cell-cycle control to maintain the genome integrity in which BRCA1 gene is implicated. In the present study an evaluation of the radio-induced damage in G(2) phase of the cell cycle in lymphocytes from BRCA1 heterozygotes is presented. For this purpose Calyculin-A-based premature chromosome condensation (PCC) combined with mitotic arrest has been applied to examine with conventional cytogenetics the damage in G(2) and M phase cells, and to evaluate the G(2)-to-M phase transition. Irradiated peripheral blood lymphocytes from seven heterozygote females (BRCA1(+/-)) and seven control females (BRCA1(+/+)) have been analyzed. The mean proportion of G(2) cells in BRCA1(+/-) was significantly higher than in BRCA1(+/+), indicating a higher G(2) delay after IR exposure in cells from BRCA1(+/-) females. On the other hand, whereas the mean frequency of chromatid breaks (chtb) in G(2) cells was not statistically different between both groups, the mean frequency of chtb in M cells of the BRCA1(+/-) group was significantly higher than in the BRCA1(+/+) one. Moreover, the mean proportion of M cells with aberrations was significantly higher in BRCA1(+/-) than in BRCA1(+/+) suggesting that in spite of the higher G(2) delay of BRCA1(+/-) more damaged cells are able to pass the G(2)-to-M transition.

Cytogenetic damage induced by radiotherapy. Evaluation of protection by amifostine and analysis of chromosome aberrations persistence.

PURPOSE: To evaluate the cytogenetic damage induced by radiotherapy, the effect of concomitant amifostine and the persistence of translocations and dicentrics after the treatment. MATERIALS AND METHODS: Blood samples from 16 head and neck cancer patients were obtained at different times, just before treatment, at the 1st and 22nd sessions, at the end of radiotherapy, and one, four and 12 months later. Solid stain and fluorescent in situ hybridization (FISH) techniques were applied to analyse chromosome aberrations. RESULTS: In all the analysis the frequencies of dicentrics plus rings were slightly lower in the group of patients receiving concomitant amifostine, but in each sampling point the differences were not significant. The persistence of translocations and dicentrics one year after radiotherapy was very similar, with a decline of more than 50%. For all the chromosome aberrations considered, a negative correlation between their initial yield and the percentage of this yield remained 12 months after radiotherapy was observed (p < 0.05). CONCLUSION: No significant protection by amifostine against radiation-induced chromosome damage was observed in head and neck cancer patients treated only with radiotherapy. In these cases, the persistence of translocations and dicentrics during the first year after radiotherapy is similar and related to their initial yield.

Genomic imbalances in Schistosoma-associated and non-Schistosoma-associated bladder carcinoma. An array comparative genomic hybridization analysis.

Carcinoma of the urinary bladder is the most common malignancy in many tropical and subtropical countries due to endemic infection by Schistosoma hematobium (bilharzia). In the current study, we performed a high-resolution analysis of gene copy number amplifications using array comparative genomic hybridization to compare DNA copy number changes in pools of Schistosoma-associated (SA) and non-Schistosoma-associated (NSA) bladder cancer (BC). Many DNA copy number changes were detected in all studies, with multiple gains and losses of genetic material. The most frequent alterations were gains on 5p15.2 approximately p15.33, 8q13.1, and 11q13, and losses on 8p21.3 approximately p22 and 22q13. Even when SA pools showed no Schistosoma-specific gene copy number profiling as compared to NSA pools, some genes seemed to be gained (ELN on 7q11.23) and some lost (PRKAG3 on 2q35 and PRDM6 on 5q23.2) in SA-SCC. The following genes were gained in all histopathologic categories: SRC (20q11.23), CEBPB (20q13.13), and GPR9 (Xq13.1). Our study did not provide clear evidence of differences in carcinogenesis of SA-BC and NSA-BC.

Disruption of the CNTNAP2 gene in a t(7;15) translocation family without symptoms of Gilles de la Tourette syndrome.

Caspr2 is a member of neurexin superfamily, members of which are transmembrane proteins that mediate cellular interactions in the nervous system. Recently, truncation of the CNTNAP2 gene coding for the Caspr2 protein has been suggested to be associated with the Gilles de la Tourette syndrome, a neurological disorder characterized by motor and vocal tics, and behavioral anomalies. In this study, we describe a familial balanced reciprocal translocation t(7;15)(q35;q26.1) in phenotypically normal individuals. The 7q35 breakpoint disrupts the CNTNAP2 gene, indicating that truncation of this gene does not necessarily lead to the symptoms of the complex Gilles de la Tourette syndrome.

Genetic abnormalities and clinical outcome in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the elderly population. Under conventional cytogenetic (CC) analysis, approximately 50% of CLL cases show clonal aberrations. Using fluorescent in situ hybridization (FISH), the percentage of patients with abnormalities rises to almost 80%, the most frequent being 13q14, ATM, and TP53 deletions and trisomy 12. The aim of this study was to establish the incidence of genetic changes in B-CLL patients using CC and FISH and to evaluate the prognostic implications. Of the 65 patients analyzed, genetic aberrations were found in 36.7% with CC and in 68.4% with FISH. The frequencies of abnormalities were as follows: 13q deletion, 42.1%; trisomy 12, 19.2%; ATM deletion, 17.5%; and TP53 deletion, 8.7%. Significant differences were observed when the overall survival was correlated with Rai stage (P = 0.000). FISH abnormalities were correlated with age, sex, morphology, white blood cell count, CD38 expression, Rai stage, disease status, and survival. Significant differences were obtained with age (P = 0.05) and disease status (P = 0.01). Deletion of 13q was the most frequent abnormality (36.6%) among old patients (> or =60); trisomy 12 was the most frequent (31.3%) in younger patients (<60). Half of the patients with stable disease showed 13q deletion, and the most frequent abnormality in patients with progressive disease was ATM deletion (22.2%).

Microdeletion and microduplication 22q11.2 screening in 295 patients with clinical features of DiGeorge/Velocardiofacial syndrome.

The 22q11.2 region is susceptible to chromosomal rearrangements, leading to various types of congenital malformation and mental retardation. The most common anomaly is 22q11.2 microdeletion, associated with DiGeorge/Velocardiofacial syndrome (DG/VCFS). Recently the microduplication 22q11.2 syndrome has been identified. Some clinical features in patients with this new chromosomal disorder present a substantial overlap with DG/VCFS. The aim of this hospital-based study was to evaluate the incidence of deletions and duplications on 22q11.2 in patients with DG/VCFS features. We investigated a group of 295 patients with widely variable manifestations associated with DG/VCFS. Along with the clinical diagnoses different anomalies were noted such as conotruncal cardiac anomaly, velopharyngeal insufficiency, characteristic facial dysmorphic features, language impairment, developmental delay/learning difficulties, and immunologic anomalies or thymic hypoplasia. Laboratory studies included conventional cytogenetic and FISH testing. Metaphase and interphase cells were analyzed for the presence of 22q11.2 microdeletion or microduplication. There were 12 patients who carried 22q11.2 microdeletion and no microduplication in the region was identified. Other chromosomal anomalies were reported in five patients with an overlapped DG/VCFS phenotype. All patients with 22q11.2 microdeletion showed a characteristic phenotype of DG/VCFS. We did not identify 22q11.2 microduplication, suggesting that this is a rare event in patients with DG/VCFS features.

Common pattern of unusual chromosome abnormalities in hereditary papillary renal carcinoma.

In this study, we summarized cytogenetic and comparative genomic hybridization (CGH) results, mutation analysis of the MET gene, and immunohistochemistry results of tumors from three patients in the same family who were affected by hereditary papillary renal carcinoma (HPRC). All three patients showed germline mutations in the tyrosine kinase domain of the MET proto-oncogene, and developed bilateral and multiple papillary renal tumors. DNA mutation analysis showed an increased dosage of the mutant allele in six tumors from two patients but not in two tumors from the third patient. In addition to the recurrent chromosomal alterations found in papillary renal carcinomas, cytogenetic analyses revealed the presence of an identical chromosomal translocation, t(2;15)(q13;p11), in two different tumors from the same patient. Moreover, the same pattern of autosomal trisomies (+7, +12, +13, +17) was detected by CGH analysis in tumors from different siblings. Taking into account that the presence of an identical structural chromosomal aberration in two tumors and the gain of chromosome 13 are unusual chromosomal changes in this type of tumor, we can conclude that our results confirm those of other authors and suggest that the genetic predisposition to HPRC might predispose the acquisition of genomic alterations in specific chromosomes or chromosomal regions.

Analysis of kidney tumors by comparative genomic hybridization and conventional cytogenetics.

Comparative genomic hybridization (CGH) and conventional cytogenetic karyotyping were used to screen for losses and gains of DNA sequences along chromosomes in ten renal tumors (RCC) of different histologic types (clear-cell RCC, papillary RCC, and one oncocytoma). Loss of 3p was the most common change in clear-cell RCC. All papillary tumors, either adenomas or carcinomas revealed gains of chromosomes 7 and 17q without limitation to size and grade. Homozygotic loss of the pseudoautosomal Xp or Yp region was detected in three RCC tumors. A dicentric (Y;14) was present as the sole chromosome abnormality in the oncocytoma. Both techniques showed concordant results in tumors with homogeneous karyotype. However, in tumors with several composite clones some discrepancies were observed, especially in cases of clear-cell RCC where chromosomal abnormalities present in a low number of metaphases could not be detected by CGH.