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Background Imatinib has so far been the first-choice treatment in chronic myeloid leukemia with excellent results. However, only a proportion of patients achieve major molecular response – hence the need to find biological predictors of outcome to select the optimal therapeutic strategy now that more potent inhibitors are available. Design and methods We investigated a panel of 20 polymorphisms in 7 genes, potentially associated with the pharmacogenetics of imatinib, in a subset of 189 newly diagnosed chronic myeloid leukemia patients enrolled in the TOPS trial. Included in this analysis were polymorphisms in the transporters hOCT1, MDR1, ABCG2, OCTN1, and OATP1A2, and in the metabolizing genes CYP3A4 and CYP3A5. Results In the overall population, the OCTN1 C allele (rs1050152), a simple combination of polymorphisms in the hOCT1 gene and in the genes involved in imatinib uptake were significantly associated with major molecular response. The combination of polymorphisms in imatinib uptake was also significantly associated with complete molecular response. Analyses restricted to Caucasians highlighted the significant association of MDR1 CC (rs60023214) genotype with complete molecular response. Conclusions We demonstrate the usefulness of a pharmacogenetic approach for stratification of chronic myeloid leukemia patients in terms of likelihood to achieve major or complete molecular response on imatinib. This represents an attractive opportunity for therapy optimization, worth testing in clinical trials.

We developed a new method based on the single strand conformation polymorphism (SSCP) technique for the detection of a G23591A (Asp312Asn) polymorphism in exon 10 of the XPD gene. In the process we also identified a novel polymorphism 23623C-ins (IVS10+17C-ins) in intron 10 of the same gene. With this newly developed SSCP-based method of genotyping we could detect both polymorphisms in the same assay and thus consequently determine the haplotype. In order to determine the population frequency of the novel polymorphism and the haplotype frequency, 302 healthy individuals were genotyped. The allelic frequency of the 23623C-ins intronic polymorphism was 0.16, whereas the frequency of the variant allele for the G23591A polymorphism was 0.39. Forty-three individuals (14%) were heterozygous for both polymorphisms but none carried polymorphic variants for both G23591A and 23623C-ins on the same allele. The effect of the novel intronic insertion polymorphism, which is located 16 nt downstream of the 3'-end of exon 10 of the XPD gene and involves a mononucleotide C repeat sequence, on expression remains to be determined.

This report is part of a biomarker study conducted in an Italian population with exposure to environmental benzene ranging from 1.43 to 31.41 μg/m 3 (values from personal sampling). DNA damage induced by benzene is the crucial mechanism of its genotoxicity, which leads to chronic benzene poisoning, haematotoxicity and leukaemia. Therefore, genetic variation in DNA-repair genes may modulate susceptibility to benzene-induced DNA damage. In light of this, the effects of polymorphisms in DNA-repair genes ( APEX1 , hOGG1 , NBS1 , XPD , XRCC1 , and XRCC3 ) on micronucleus (MN) formation as a biomarker of early biological effects were evaluated. A significantly higher median MN frequency was recorded in traffic wardens than in controls. However, none of the analysed polymorphisms was significantly associated with the median MN frequency. A gene–gender interaction was observed for the APEX1 genotype. The APEX1 variant genotype was associated with significantly lower median MN frequency in men , not in women. Statistical analysis did not reveal any association between the score of the protective alleles – hypothetically pushing the pathway towards optimal DNA-damage repair – and MN. Even though there are some limitations in the study, our results indicate that the general population may be exposed to benzene concentrations higher than the threshold level for air-quality standards in the European Union of 10 μg/m 3 . Furthermore, urban traffic wardens are exposed to significantly higher levels of benzene than individuals spending most of the time indoors. This higher exposure may contribute to DNA damage, suggesting that benzene might be implicated both as an environmental and occupational risk factor in leukaemia and other haematological diseases. In conclusion, this study suggest the need for (i) regular monitoring of traffic wardens for possible exposure to benzene, as a precautionary step to reduce the associated health risks, and (ii) more comprehensive studies in order to better elucidate the involvement of APEX1 genotypes in benzene genotoxicity.

The DNA repair protein XPD is involved in the transcription-coupled nucleotide excision repair of DNA lesions induced by many tobacco and environmental carcinogens. Common polymorphisms in XPD exon 10 (G>A, Asp312Asn) and exon 23 (A>C, Lys751Gln) have been identified, and lung cancer cases homozygous for the variant allele in either exon have been reported to have a reduced repair capacity against benzo[ a ]pyrene-induced DNA damage. We therefore investigated a possible effect of these variant alleles on the p53 mutation frequency and spectrum among 97 Swedish lung cancer patients. Transversions were found to occur more frequently among patients with at least one variant allele than among wild type homozygotes. The XPD variant alleles may thus be associated with reduced DNA repair proficiency. This finding is not in accord with the previous report that associated the exon 23 wild type allele with increased frequency of X-ray-induced chromatid aberrations.

During their working and living activities, humans are constantly exposed to different environmental genotoxic agents. Biological consequences of the exposure are accumulation of different mutations and DNA damage that can lead to disruption of genetic material. Numerous endogenous genotoxic agents can also cause DNA damage. Loss of normal cell function can cause cell death or can result in different health disorders, including teratogenic and cancerogenic effects (Jeggo & Lavin, 2009). There is an increasing concern about mutagenic and cancerogenic effects of genotoxic agents and their influence on individuals who are exposed to them by accident, or by living/working lifestyle (Au, 1991; Carrano & Natarajan, 1988; Kassie et al., 2000). Exposure to ionising radiation, as a well known genotoxic agents (Balasem & Ali, 1991; Erexon et al., 1991; Fenech et al., 1990; He et al., 2000; Jeggo & Lavin, 2009), can leed to different DNA damage, such as oxidative damage, base and sugar modification in DNA, apurinic/apirimidinic places, single/double strand chromosomal breaks, adduct creation, inter/intra DNA cross linking and other types of damage (Hall & Giaccia, 2006). Numerous studies deal with ionising radiation exposure of individuals who are chronically professionally exposed to low doses (Andreassi, 2009; Au, 1991; Carrano & Natarajan, 1988; Kassie et al., 2000). The results showed the increase in chromosomal damage during chronically low dose exposure (Barquinero et al., 1993; Boutcher, 1985; Cardoso et al., 2001; Jha, 1991; Nowak & Jankowski, 1991), but without confirmed relationship between the received dose and the intensity of DNA damage (Bolus, 2001; Coates et al., 2004; Morgan, 2003; Mothersill et al., 2000, 2001; Seymour & Mothersill, 2000). The influence of chronically low dose exposure has been considered mutagenic and cancerogenic (Fachini et al., 2009). Although it is possible to estimate the influence of absorbed dose and the effect on the

An integrated approach based on environmental and biological monitoring, including the analysis of biomarkers of exposure [excretion of S-phenylmercapturic acid (S-PMA)], early biological effects [micronucleus (MN) frequency] and susceptibility (genetic polymorphisms), was applied to characterize benzene exposure in a group of 70 traffic policemen and 40 employees of the city of Bologna, Italy. Median personal benzene exposure was 6.55-fold higher for traffic policemen than for controls (P < 0.0001). This higher exposure was confirmed by a significant, 2.53-fold higher S-PMA excretion in traffic policemen compared with that observed for indoor workers (P < 0.0001). Median MN frequency was also significantly higher in policemen compared with indoor workers (P = 0.001), emphasizing the genotoxic effect potentially associated with benzene exposure. With regard to biomarkers of susceptibility, the analysis revealed that high epoxide hydrolase (mEH) (predicted) enzyme activity was significantly correlated with a lower median MN frequency (P = 0.003). A gene–gender interaction was observed for the glutathione-S-transferase M1 (GSTM1) genotype. The GSTM1-null genotype was associated with a significantly higher median MN frequency in men, not in women. Statistical analysis did not reveal any association between the presence of the protective allele, pushing the pathway towards benzene detoxification, and MN frequency or S-PMA excretion. Even though there are some limitations in the study, our results indicate that policemen are exposed to higher levels of benzene than individuals spending most of the time indoors. This higher exposure may contribute to DNA damage, suggesting an increase health risk from traffic benzene emission. Finally, a more comprehensive study is warranted in order to better elucidate the involvement of EPHX1 genotypes combination in benzene genotoxicity.

Cilj ovog istraživanja bio je procijeniti razinu oštećenja DNA u leukocitima periferne krvi prije, neposredno nakon i 120 min nakon ozračivanja dozom zračenja od 2 Gy te usporediti razine nastalih oštećenja između kontrolne skupine i skupine medicinskih radnika izloženih niskim dozama ionizirajućeg zračenja na svojim radnim mjestima. Istražen je utjecaj polimorfi zma u genima koji sudjeluju u popravku DNA; *hOGG1, XRCC1 i MGMT na razinu izmjerenih oštećenja. Istraživanjem je obuhvaćeno 40 zdravih ispitanika obaju spolova (20 kontrola i 20 izloženih). Razina oštećenja DNA mjerena je komet-testom u alkalnim uvjetima, pri čemu su za svakog ispitanika i svaku vremensku točku analizirane vrijednosti dužine repa, % DNA u repu i repnog momenta kometa. Rezultati pokazuju da su vrijednosti % DNA u repu i repni moment kometa bili statistički značajno viši u izloženoj populaciji prije, neposredno nakon i 120 min nakon ozračivanja. Pokazano je da nosioci polimorfnih alela ovih gena u izloženoj skupini imaju statistički značajno više razine oštećenja DNA, kako naspram homozigota pripadne skupine, tako i naspram cijele kontrolne skupine te da početno oštećenje DNA značajno negativno korelira s ukupnom dozom zračenja koju su primili tijekom radnog vijeka. Dobiveni rezultati upućuju na vrijednost kombiniranja alkalnoga komet-testa i genotipizacije izloženih pojedinaca u genima za popravak DNA, što bi moglo pridonijeti prepoznavanju subpopulacija sklonijih nakupljanju oštećenja DNA, a time i sklonijih riziku od razvoja tumorskih bolesti., Increasing exposure to ionising radiation raises a great concern about potential DNA damage in occupationally exposed individuals. Polymorphisms of DNA repair genes can determine individual sensitivity and DNA damage response to low doses of ionising radiation. The objective of this study was to assess DNA damage in leukocytes at baseline, immediately after and 120 min after exposure to gamma-radiation of 2 Gy, to compare DNA damage between the control group of subjects and subjects occupationally exposed to low-dose gamma-radiation, and to determine the relationship between hOGG1 (8-oxoG specifi c DNA glycosylase/AP-Lyase, Ser326Cys), XRCC1 (X-ray repair cross-complementing protein-group 1, Arg194Trp), and MGMT(O6-methylguanine-DNA methyltransferase, Leu84Phe) gene and DNA damage. The study enrolled 40 healthy subjects (20 controls and 20 occupationally exposed subjects), whose leukocytes were exposed to ionising radiation and tested for DNA damage (tail length, percentage od DNA in comet tail, and tail moment) using the alkaline version of the comet assay. Our results show that tail DNA percentage and tail moment were signifi cantly higher in the exposed group at baseline, immediately after, and 120 min after exposure to 2 Gy. The exposed subjects carrying polymorphic alleles had signifi cantly higher DNA damage than homozygous carriers of the same gene and controls. Combined use of the alkaline comet assay and genotyping of DNA repair genes could help discover sensitive occupationally exposed individuals who can accumulate higher DNA damage and are at higher risk of developing tumours.

Susceptibility to DNA damage varies among individuals and sensitivity to bleomycin (BLM) may reflect the inter-individual differences. BLM sensitivity in part may be explained by inherited differences in DNA repair genes. We investigated the association between genetic polymorphisms in the GSTT1, GSTM1, XPD, XRCC1 and XRCC3 genes and the levels of spontaneous and BLM-induced DNA damage in peripheral blood lymphocytes from 200 healthy, unexposed individuals. The investigation of BLM sensitivity on cancer- or disease-free subjects and not occupationally exposed to known mutagen represents the strengths of the present study, as the detection of genetic damage is not biased by any disease- and occupational-related factor. The micronucleus (MN) assay was used to detect the spontaneous and BLM-induced genetic damage whereas, genotype analysis was carried out using methods based on polymerase chain reaction. Poisson regression analysis showed that subject's age, gender and smoking status had no effect on the spontaneous and BLM-induced MN frequencies. Genotype analysis revealed a clear association between GSTT1-null and XPD polymorphisms and both spontaneous and BLM-induced MN frequencies, whereas the effect of the XRCC1 polymorphism was marginally significant only with regard to spontaneous MN frequency. Genotype analysis did not reveal a clear association between the other studied SNPs (GSTM1 and XRCC3) and MN frequencies. Poisson regression analysis revealed no association between the score of protective alleles and the frequency of spontaneous MN. However, an increased number of protective alleles was significantly associated with a lower frequency of BLM-induced MN (P=0.0003). This finding highlights the genetic basis for BLM sensitivity, which could be a valid and useful surrogate for identifying genotypes that might increase susceptibility in population exposed to carcinogens. Further investigations in a large sample size and including more SNPs, reflecting the complexity of DNA repair machinery, might lead to the identification of a genetic profile responsible for the susceptibility to genotoxicants, with a far-reaching long-term impact on primary prevention and early detection of disease associated genes.

Mutagen sensitivity assay, by measuring chromosome damage induced by an in vitro treatment of peripheral lymphocytes with bleomycin, has been proposed as a biomarker for assessing cancer susceptibility. Recently, a single nucleotide polymorphism (SNP A1450G) of the gene for bleomycin hydrolase (BLHX), a specific neutral cysteine protease able to metabolise bleomycin, was proposed as a plausible candidate to variation in mutagen sensitivity. To shed more light on the effect of BLHX genotype on the expression of chromosome damage induced in vitro by bleomycin, we determined mutagen sensitivity for 45 non-smoker healthy volunteers. The level of bleomycin-induced chromosome damage was assessed as frequencies of micronuclei (MN) in cytokinesis-blocked lymphocytes. The subjects were genotyped for the BLHX gene, to determine the possible effect of this polymorphism on mutagen sensitivity. No difference in the spontaneous value of MN was detected between the homozygotes wild-type (A/A) and the carriers of variant alleles A/G heterozygotes or G/G homozygotes (MN/1000 binucleated (BN) cells: 6.69 ± 2.53 and 6.37 ± 4.87, respectively). A substantial effect of BLHX polymorphism in predetermining individual mutagen sensitivity status was observed: subjects with the BLHX A/A genotype displayed significantly lower mean levels of bleomycin-induced MN frequency than the carriers of A/G or G/G variant alleles combined (12.00 ± 3.76 MN/1000 BN vs. 16.37 ± 8.86 MN/1000 BN, respectively; P = 0.029). The multiple regression analysis, including BLHX genotype and age, confirmed the significant effect of BLHX variant alleles (A/G, G/G) on the chromosome damage induced by bleomycin ( P = 0.01), whereas age correlated only with the spontaneous MN frequency.

Previous studies have shown frequent mutations in the BRAF (V-raf murine sarcoma viral oncogene homolog B1) or NRAS (neuroblastoma RAS viral [V-ras] oncogene homolog) genes in cutaneous melanoma, but the relationship between these alterations and tumor cell proliferation has not been examined in human melanoma. In our study of 51 primary nodular melanomas and 18 paired metastases, we found mutations in BRAF (codon 600, previously denoted 599) in 15 primary tumors (29%) and eight metastases (44%). The figures for NRAS mutations were 27% and 22%, respectively. Mutations in BRAF and NRAS genes were mutually exclusive in all but one case, and were maintained from primary tumors through their metastases. Mutations, however, were not associated with tumor cell proliferation by Ki-67 expression, tumor thickness, microvessel density, or vascular invasion, and there were no differences in patient survival. Although BRAF and NRAS mutations are likely to be important for the initiation and maintenance of some melanomas, other factors might be more significant for proliferation and prognosis in subgroups of aggressive melanoma.

Understanding the risks deriving from protracted exposure to low doses of ionizing radiation has remarkable societal importance in view of the large number of work settings in which sources of IR are encountered. To address this question, we studied the frequency of micronuclei (MN), which is an indicator of DNA damage, in a population exposed to low levels of ionizing radiation and in matched controls. In both exposed population and controls, the possible influence of single nucleotide polymorphisms in XRCC1 , XRCC3 and XPD genes on the frequency of micronuclei was also evaluated. We also considered the effects of confounding factors, like smoking status, age and gender. The results indicated that MN frequency was significantly higher in the exposed workers than in the controls [8.62 ± 2.80 versus 6.86 ± 2.65; P = 0.019]. Radiological workers with variant alleles for XRCC1 or XRCC3 polymorphisms or wild-type alleles for XPD exon 23 or 10 polymorphisms showed a significantly higher MN frequency than controls with the same genotypes. Smoking status did not affect micronuclei frequency either in exposed workers or controls, while age was associated with increased MN frequency in the exposed only. In the combined population, gender but not age exerted an influence on the yield of MN, being higher in females than in males. Even though there is a limitation in this study due to the small number of subjects, these results suggest that even exposures to low level of ionizing radiation could have genotoxic effects and that XRCC3 , XRCC1 and XPD polymorphisms might contribute to the increased genetic damage in susceptible individuals occupationally exposed to chronic low levels of ionizing radiation. For a clear conclusion on the induction of DNA damage caused by protracted exposure to low doses of ionizing radiation and the possible influence of genetic polymorphism in DNA repair genes larger studies are needed.

Among the toxic chemicals present in the ambient air of urban centres, benzene raises particular concern due to its haematoxicity and leukaemogenic hazards, probably related to clastogenic factors. However, little is known about the health risks associated with environmental – rather than industrial – exposure to benzene. We analysed micronucleus (MN) frequencies in peripheral lymphocytes by use of the cytokinesis-block technique, and haematological parameters among 49 traffic police and 36 indoor workers (controls) in the city of Bologna. The analysis of urban air provided by a municipal air-quality monitoring station indicated that the levels of environmental benzene were often above the recommended threshold level (10 μg/m 3 ) whereas other pollutants – nitrogen oxides, polycyclic aromatic hydrocarbon compounds, total suspended particulate matter, carbon monoxide, sulfur dioxide – did not exceed the maximum atmospheric concentration established for air-quality standards. Mean levels of individual airborne benzene exposure – as measured by personal devices worn during 4-h morning work-shifts – were six-fold higher in the traffic police than in controls ( P = 0.001). While no significant difference in haematological parameters was found between the two groups, MN frequency was significantly higher among the traffic police than in indoor workers ( P = 0.001). Among the study population, MN frequency was found to increase with age, but no influence was observed for gender or smoking. Although it cannot be excluded that the increase of MN frequency observed in traffic police could also depend, apart from benzene, on the complex mixture of pollutants encountered in urban air, our data indicate that elevated personal benzene exposure could represent a genetic risk. The analysis of biomarkers of genetic damage in subjects particularly exposed to environmental benzene deserves careful study.

The ambient air of urban centres is polluted with potentially toxic chemicals mostly arising from the combustion or fuels used for transport. Among these compounds, benzene raises particular concern due to its haematoxicity and leukaemogenic risks. Although limits of benzene in air have been established in the European Union (5 μg/m3), individual exposure levels—and therefore risk estimates—cannot merely be extrapolated from environmental concentrations. Molecular epidemiology can facilitate health risk assessment by investigating the relationship between exposure to environmental pollutants and quantification of biomarkers that lie on the pathway of carcinogenesis upstream of clinical disease. We review the available for biomarker studies regarding health risks linked to environmental benzene exposure, and make some suggestions for future work.

A limited number of genes have been identified that explain heritable risks of breast cancer (BC). We searched for low-penetrant genes in an association study using two populations: 223 Finnish unselected patients and 172 Polish familial cases, both with locally collected healthy controls. Candidate genes included DNA repair genes, methylenetetrahydrofolate reductase (MTHFR) and cyclin D1 genes. The frequencies for single nucleotide polymorphisms (SNPs) were measured in the following genes: NBS1, XPC, XPD, XRCC1, XRCC3, MTHFR, and cyclin D1. Odds ratios (ORs) were calculated to the wild-type genotype. The positive findings in the Finnish series were repeated in the Polish series. Significant findings among Finns were associations to XPC exon 15, XPD exon 10 and XRCC3 exon 7, the latter of borderline significance. None of these results could be repeated in the Polish series. The XPC result among Finns was probably an artifact of the control group deviating from the Hardy-Weinberg Equilibrium (HWE). The attempt to repeat the result for the XPD polymorphism among Poles was probably not valid because the control group deviated from the HWE. We conclude that within statistical power of the present study, none of the tested polymorphisms associated with BC, with the probable exception of XPD.

A limited number of genes have been identified that explain heritable risks of breast cancer (BC). We searched for low-penetrant genes in an association study using two populations: 223 Finnish unselected patients and 172 Polish familial cases, both with locally collected healthy controls. Candidate genes included DNA repair genes, methylenetetrahydrofolate reductase (MTHFR) and cyclin D1 genes. The frequencies for single nucleotide polymorphisms (SNPs) were measured in the following genes: NBS1, XPC, XPD, XRCC1, XRCC3, MTHFR, and cyclin D1. Odds ratios (ORs) were calculated to the wild-type genotype. The positive findings in the Finnish series were repeated in the Polish series. Significant findings among Finns were associations to XPC exon 15, XPD exon 10 and XRCC3 exon 7, the latter of borderline significance. None of these results could be repeated in the Polish series. The XPC result among Finns was probably an artifact of the control group deviating from the Hardy-Weinberg Equilibrium (HWE). The attempt to repeat the result for the XPD polymorphism among Poles was probably not valid because the control group deviated from the HWE. We conclude that within statistical power of the present study, none of the tested polymorphisms associated with BC, with the probable exception of XPD.

We determined mutations in the BRAF, N-ras, and CDKN2A genes in 27 histologically diverse melanocytic nevi and corresponding surrounding tissues from 17 individuals. Mutations in the BRAF and N-ras gene were found in 22 nevi (81%) from 16 individuals (94%). The predominant BRAF mutation T1799A (V600E) was detected in 18 nevi; 1 nevus had a novel A1781G (D594V) mutation in the same gene and 3 nevi had mutations in codon 61 of the N-ras gene. In 4 individuals both nevi carried a BRAF mutation, whereas in 2 other individuals 1 nevus showed a BRAF mutation and the second nevus had an N-ras mutation. In 2 individuals normal skin distant from nevi showed a BRAF mutation. No mutations were detected in the CDKN2A gene. The mutations in the BRAF and N-ras genes, in this study, were not associated with histologic type, location, skin type, size, or numbers of nevi. Our results suggest that mutations in the BRAF gene and to some extent in the N-ras gene represent early somatic events that occur in melanocytic nevi. We hypothesize the dual effect of solar ultraviolet irradiation on melanoma, through mutagenesis and by increasing the number of melanocytic nevi, many of which carry a BRAF or N-ras mutation.

Chromosome aberrations frequency was estimated in peripheral lymphocytes from hospital workers occupationally exposed to low levels of ionizing radiation and controls. Chromosome aberrations yield was analyzed by considering the effects of dose equivalent of ionizing radiation over time, and of confounding factors, such as age, gender and smoking status. Frequencies of aberrant cells and chromosome breaks were higher in exposed workers than in controls (P = 0.007, and P = 0.001, respectively). Seven dicentric aberrations were detected in the exposed group and only three in controls, but the mean frequencies were not significantly different. The dose equivalent to whole body of ionizing radiation (Hwb) did appear to influence the spectrum of chromosomal aberrations when the exposed workers were subdivided by a cut off at 50 mSv. The frequencies of chromosome breaks in both subgroups of workers were significantly higher than in controls (< or =50 mSv, P = 0.041; >50 mSv, P = 0.018). On the other hand, the frequency of chromatid breaks observed in workers with Hwb >50 mSv was significantly higher than in controls (P = 0.015) or workers with Hwb < or =50 mSv (P = 0.046). Regarding the influence of confounding factors on genetic damage, smoking status and female gender seem to influence the increase in chromosome aberration frequencies in the study population. Overall, these results suggested that chromosome breaks might provide a good marker for assessing genetic damage in populations exposed to low levels of ionizing radiation.

The RAS-RAF-mitogen-activated protein kinase pathways mediate the cellular response to growth signals. In melanocytes, BRAF is involved in cAMP-dependent growth signals. Recently, activating mutations in the BRAF gene, were reported in a large proportion of melanomas. We have studied mutations in the BRAF gene and their association with clinical parameters.We analyzed exons 1, 11, and 15 of the BRAF gene and exons 1 and 2 of the N-ras gene for mutations in 38 metastatic melanomas by PCR-single-strand conformation polymorphism and direct sequencing. Kaplan-Meier survival and multivariate analyses were used to correlate mutations with various clinical parameters.Mutations in exon 15 of the BRAF gene were detected in 26 (68%) melanomas. In 25 cases, mutation involved the "hot spot" codon 600(2)of the BRAF gene. Three melanomas without a BRAF mutation carried amino acid substituting base changes at codon 61 of the N-ras gene. In a multivariate proportional hazard (Cox) model, BRAF mutation, along with the stage of metastatic melanomas, showed a statistically significant hazard ratio of 2.16 (95% confidence interval 1.02-4.59; chi(2) for the model 6.94, degrees of freedom 2, P = 0.03) for diminished duration of response to the treatment. In a Kaplan-Meier survival model, cases with BRAF mutation showed longer disease-free survival (median of 12 months) than cases without mutation (median of 5 months), although this association was not statistically significant (Log-rank test P = 0.13).Our results, besides confirming the high frequency of BRAF mutations in metastatic melanomas, also underline the potential importance of these mutations in disease outcome.

The DNA repair proteins XPD and XRCC1 are involved in the nucleotide and base excision repair of DNA lesions induced by many tobacco and environmental carcinogens. Common variant alleles at the XPD (312Asn, 751Gln) and XRCC1 (399Gln) loci have been identified and associated with increased risk for lung cancer. We therefore investigated a possible effect of these variant alleles on the frequency and spectrum of p53 mutations in the tumors of 97 Swedish lung cancer patients (56 never-smokers and 41 age-, gender-, and hospital-matched ever-smokers). The p53 gene was mutated in 4 never-smokers (7%) and 11 ever-smokers (27%). Smoking-related transversion-type mutations predominated over transitions among smokers (8:3), but not among never-smokers (1:3). None of the variant alleles altered the overall frequency of p53 mutation. Transversions, however, were marginally increased among patients with at least one XPD variant allele compared with patients who were wild-type homozygotes (73% vs. 25% for the Asp312Asn polymorphism, P = 0.095; 78% vs. 33% for Lys751Gln, P = 0.085). Five of six women or six of seven smokers who carried at least one XPD 751Gln allele had p53 transversion. The XRCC1 variant allele did not show any effect on the p53 mutation. We conclude that the XPD variant alleles may be associated with an increased frequency of smoking-related p53 mutations in lung tumors, presumably due to reduced DNA repair proficiency.

The DNA repair protein xeroderma pigmentosum complementation group D (XPD) is involved in the nucleotide excision repair of DNA lesions induced by many tobacco and environmental carcinogens. In order to study the functional impact of the common polymorphisms in XPD exon 10 (G > A, Asp312Asn) and exon 23 (A > C, Lys751Gln), we have genotyped 185 Swedish lung cancer cases (97 smokers and 88 never-smokers) and 162 matched population controls (83 smokers and 79 never-smokers). Presence of one or two variant alleles was associated with increased risk for lung cancer among never-smokers only, in particular younger (

In the context of a medical surveillance program aimed at preventing cancer risk from exposure to ionizing radiation, we investigated chromosomal damage in peripheral lymphocytes from 37 hospital workers exposed to low levels of ionizing radiation and 37 controls. The micronuleus (MN) assay was used as a biomarker of genetic damage. The influence of confounding factors like smoking status, age and gender was investigated by multiple regression analysis. The results indicated that, overall, MN frequency was higher in exposed workers than in controls, although the difference was not statistically significant. Interestingly, smoking status significantly raised MN frequency among the exposed workers but not among controls. This suggests that smoking can influence chromosomal damage induced in humans by ionizing radiation. Among both exposed workers and controls, MN frequency was found to increase with age. Female gender influenced the increase in MN frequency in the exposed group. Our results suggest that the effect of cigarette smoking should be carefully factored into genetic monitoring studies assessing the risks associated with low level radiation exposure.

The availability of multiple options for chronic myeloid leukemia (CML) treatment is not paralleled by the availability of biological predictors of outcome allowing to identify patients (pts) who are more likely to benefit from dasatinib or nilotinib rather than imatinib (IM). We thus investigated a panel of 20 SNPs in ABCB1, ABCG2, SLC22A1, OCTN1, CYP3A4 and CYP3A5 genes that can be hypothesized to influence IM transport and metabolism in 189 newly diagnosed CML pts enrolled in the TOPS phase III trial (Cortes et al, J Clin Oncol 2010). Median age was 46 yrs; male to female ratio was 103:86; 156 (83%) pts were Caucasian and 23 (12%) were Asian; low, intermediate and high Sokal risk pts were 84 (44.4%), 65 (34.4%) and 40 (21.2%), respectively. Baseline demographic/clinical features did not differ significantly from those of the entire population enrolled. Treatment outcomes (major molecular response [MMR]; complete molecular response [CMR]) were compared according to i) each candidate genotype ii) summary measures based on combinations of SNPs in the same gene and iii) summary measures based on combinations of SNPs in functionally related genes (uptake; efflux). Additive models, with SNPs represented as number of major alleles, were used. Significant results are summarized in the Table below and will be presented in detail. Cumulative incidence plots were also analysed, with similar results. Our data suggest that in CML pts, genotyping should be taken into account in an attempt to further individualize treatment, with the aim of enhancing efficacy in terms of MMR and CMR achievement. On the basis of our findings, stratification of pts according to genotypes may be proposed for selection between IM and second generation TKIs, and represents an attractive opportunity for new clinical trials. Supported by Novartis, TOPS Correlative Studies Network Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2011-5448

Abstract 670 The availability of multiple options for chronic myeloid leukemia (CML) treatment is not paralleled by the availability of biological predictors of outcome allowing to identify patients (pts) who are more likely to benefit from dasatinib or nilotinib rather than imatinib (IM). Pharmacogenetics has proven a potential source of biomarkers given the known influence of polymorphisms in key genes encoding drug transporters and metabolizing enzymes on drug delivery – hence effectiveness. In CML, only two studies had so far explored this field, but both were conducted in heterogeneous populations including pts at different stages of disease, not all receiving IM first-line. We thus aimed to investigate a panel of 20 single nucleotide polymorphisms (SNPs) in ABCB1, ABCG2, SLC22A1, OATP1A2, OCTN1, CYP3A4 and CYP3A5 genes that can be hypothesized to influence IM transport and metabolism in 189 newly diagnosed CML pts enrolled in the TOPS phase III trial (Cortes et al, J Clin Oncol 2010). Pts selection was exclusively based on availability of written informed consent and sufficient amount of archived material. Median age was 46 years; male to female ratio was 103 to 86; 156 (83%) pts were Caucasian and 23 (12%) were Asian; low, intermediate and high Sokal risk pts were 84 (44.4%), 65 (34.4%) and 40 (21.2%), respectively. Baseline demographic/clinical features did not differ significantly from those of the overall population. Treatment outcomes (complete cytogenetic response [CCyR]; major molecular response [MMR] and complete molecular response [CMR]) were compared according to i) each candidate genotype ii) summary measures based on combinations of SNPs in the same gene and iii) summary measures based on combinations of SNPs in functionally related genes (uptake; efflux). CC genotype in OCTN1 had a favorable impact on the achievement of MMR at 12 months (MMR@12m; P = 0.03). With respect to the summary measures, combination of SNPs in the SLC22A1 gene was significantly correlated with MMR@12m (P = 0.03). When considering summary measures of uptake and efflux, the former was found to be associated with both MMR@12m and CMR@12m (P = 0.003 and P = 0.01, respectively). A separate analysis limited to Caucasian pts (n=156) yielded similar results (Table 1). In addition, the analysis in the Caucasian subgroup evidenced a significant association between the CC genotype in ABCB1 rs60023214 and MMR@12m (P = 0.005) (Table 1). Cumulative incidence plots based on the Kaplan-Meier method were also analyzed in the overall population and in Caucasians, with comparable results. Representative plots are shown in Figure 1. There was evidence for difference among MMR cumulative incidence curves for 2 single SNPs and 2 score measures. Presence of the major allele in OCTN1 (CC) and of the minor allele in CYP3A4 rs2740574 (GG) were associated with increased MMR rate (P = 0.028 and P = 0.042, respectively, in the overall population and P = 0.027 and P = 0.038, respectively, in Caucasians). Similarly, an increase in the number of favorable alleles in the SLC22A1 gene was associated with increased MMR rate (P = 0.030 and P = 0.043 in the overall population and in Caucasians, respectively). In addition, the combination of favorable alleles in the genes involved in IM uptake was associated with increased rates of both MMR and CMR (P = 0.004 and P = 0.015, respectively, in the overall population and P = 0.005 and P = 0.009, respectively, in Caucasians). Our results suggest that SNP genotyping might be helpful in selecting pts who are more likely to benefit from first-line use of more potent inhibitors. Further assessment of the SNPs here identified in larger series of pts is warranted. Table 1 . Summary of significant associations Overall population (n=189) Caucasians (n=156) Gene, rs Outcome P Hazard ratio[95% CI] P Hazard ratio[95% CI] OCTN1 - rs1050152 MMR@12m 0.03 0.78 (0.63, 0.98) 0.03 0.77 (0.60, 0.97) ABCB1 (MDR1) - rs60023214 CMR@12m 0.06 0.71 (0.49, 1.02) 0.005 0.56 (0.37, 0.84) SLC22A1 (hOCT1) - SNP combination MMR@12m 0.03 1.24 (1.02, 1.50) 0.04 1.24 (1.00, 1.54) Uptake - SNP combination MMR@12m 0.003 1.21 (1.06, 1.37) 0.005 1.24 (1.06, 1.44) Uptake - SNP combination CMR@12m 0.01 1.30 (1.05, 1.61) 0.01 1.37 (1.08, 1.74) Download : Download high-res image (197KB) Download : Download full-size image Figure 1: . Cumulative incidence of pts achieving MMR and CMR according to the # of major alleles in uptake-related gene (SLC22A1, OATP1A2, OCTN1) SNPs Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures: Hughes: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria. White: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Saglio: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Rosti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Hatfield: Novartis: Employment. Martinelli: Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

6554 Background: Imatinib mesylate (IM) is the first-choice treatment in CML, with excellent response rates. Major and complete molecular responses are the current goals of therapy, but not all pat...

Abstract 3283 Poster Board III-1 IM is the first-choice treatment in CML and allows to obtain excellent response rates. Some pts, however, experience suboptimal response or resistance, which highlights the need to find biological predictors of outcome in order to guide therapy optimization. Since blood and tissue concentrations of drugs may be influenced by variations between individuals (single nucleotide polymorphisms, SNPs) in genes encoding drug metabolizing enzymes and drug transporters, we have investigated if SNPs influencing the delivery if IM to target cells may account for differences in response. To test that hypothesis, we have genotyped a panel of SNPs known to affect the activity of the cytochrome p450 family isoforms and of the transporters that have been implicated in IM metabolism and transport in a subset of pts enrolled in the TOPS trial -; a randomized phase III study of IM 400 mg/d vs IM 800 mg/d in newly diagnosed CML (Cortes et al, ASH 2008). Included in this analysis were 18 SNPs in 7 genes: hOCT1 (ins/del, rs12208357, rs683369, rs4646277, rs4646278, rs2282143); ABCB1 (rs60023214, rs1128503, rs10245483, rs3213619, rs1128501), ABCG2 (rs2231137, rs2231142); OCTN1 (rs1050152); OATP1A2 (rs11568563); CYP3A5 (rs28365083, rs776746); CYP3A4 (rs28371759). Genotype distributions were tested for Hardy-Weinberg equilibrium (HWE). Additive models were used to assess association between individual SNPs and three response outcomes: major molecular response (MMR) at 12 months, optimal CgR and optimal response as defined by European LeukemiaNet (ELN) recommendations (Baccarani, Blood 2006). Summary measures based on SNPs from the same gene and those related functionally (uptake and efflux) were defined as the number of alleles, across the genes of interest, hypothesized to be associated with favorable response. Association of individual SNPs and summary variables with response was assessed with logistic regression models and likelihood ratio and exact tests. A total of 166 pts were genotyped, out of which 133 whites from Italy (N=21), the U.S.A. (N=66) and Australia (N=46); 22 Asian from Korea (N=17), Italy (N=1), and Australia (N=4); and 11 non-white and non-Asian from the U.S.A (N=6) and Australia (N=5). Selection of pts was based exclusively on availability of a written informed consent for correlative substudies and of a sufficient amount of archived DNA material. The response rates by 12 months among the subset of pts analyzed were 51% for MMR, 87% for optimal cytogenetic response according to ELN, and 85% for optimal response according to ELN. Our analyses have identified four monomorphic, non-informative SNP ( hOCT1: rs4646278, rs2282143; MDR1: rs1128501; and CYP3A4: rs28371759). The results have shown, when analyzing the patient population as a whole, that no association could be found between individual SNPs and any of the response variables considered. However, among whites, but not other pts, we have observed a trend of association between achievement of MMR and two SNPs in the hOCT1 and CYP3A5 genes. Namely, the wild type allele for hOCT1 rs683369 was associated with a higher response rate compared to heterozygote and homozygote mutants (60% vs 36%, p=0.057). Also, the mutant allele for CYP3A5 rs28365083 was associated with a higher response rate (p=0.058). In the subset of pts from the U.S.A, there was a evidence of association between achievement of MMR and three SNPs in the hOCT1 and MDR1 genes. Those with the wild type allele for hOCT1 ins/del had a lower MMR rate compared to those carrying at least one minor allele (48% vs 81%, p=0.08). The wild type for hOCT1 rs683369 was associated with a higher response rate compared to heterozygote and homozygote mutants (65% vs 36%, p=0.05). MDR1 rs60023214 (p=0.08) and the MDR1 gene summary measure of total number of favorable alleles among all MDR1 SNPs (p=0.08) were correlated with MMR, with improved response in the wild type group. Our study suggests for the first time a role of ethnicity in determining correlations between SNP-based molecular signature and response to treatment. If confirmed by larger studies, this suggests that ethnicity should be taken in consideration to better interpret pharmacogenetic analyses in CML and could explain the differences in results of the studies published so far. Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures: White: Novartis and Britol-Myers Squibb: Research Funding. Baccarani:Novartis: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau.

We determined mutations in the BRAF, N-ras, and CDKN2A genes in 27 histologically diverse melanocytic nevi and corresponding surrounding tissues from 17 individuals. Mutations in the BRAF and N-ras gene were found in 22 nevi (81%) from 16 individuals (94%). The predominant BRAF mutation T1799A (V600E) was detected in 18 nevi; 1 nevus had a novel A1781G (D594V) mutation in the same gene and 3 nevi had mutations in codon 61 of the N-ras gene. In 4 individuals both nevi carried a BRAF mutation, whereas in 2 other individuals 1 nevus showed a BRAF mutation and the second nevus had an N-ras mutation. In 2 individuals normal skin distant from nevi showed a BRAF mutation. No mutations were detected in the CDKN2A gene. The mutations in the BRAF and N-ras genes, in this study, were not associated with histologic type, location, skin type, size, or numbers of nevi. Our results suggest that mutations in the BRAF gene and to some extent in the N-ras gene represent early somatic events that occur in melanocytic nevi. We hypothesize the dual effect of solar ultraviolet irradiation on melanoma, through mutagenesis and by increasing the number of melanocytic nevi, many of which carry a BRAF or N-ras mutation.

Circulating miR-126-3p levels were determined in 136 healthy subjects (CTRs) aged 20-90 years and 193 patients with type-2 diabetes mellitus (T2DMs) aged 40-80 years, to explore the combined effect of age and glycemic state on miR-126-3p expression. Moreover, intra/extracellular miR-126-3p levels were measured in human endothelial cells (HUVECs) undergoing senescence under normo/hyper-glycemic conditions. Plasma miR-126-3p was significantly higher in the oldest compared with the youngest CTRs ( 75 years; relative expression: 0.27±0.29 vs. 0.48±0.39, p=0.047). Age-based comparison between CTRs and T2DM demonstrated significantly different miR-126-3p levels only in the oldest (0.48±0.39 vs. 0.22±0.23, p