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Background Advanced lung adenocarcinoma (LAC) is one of the most lethal malignancies worldwide. The aim of this study was to evaluate the global survival in a real-life cohort of patients with LAC harboring driver genetic alterations. Methods A series of 1282 consecutive Sardinian LAC patients who underwent genetic testing from January 2011 through July 2016 was collected. Molecular tests were based on the clinical needs of each single case (EGFR-exon18/19/21, ALK, and, more recently, BRAF-exon15), and the availability of tissue (KRAS, MET, and presence of low-frequency EGFR-T790M mutated alleles at baseline). Results The mean follow-up time of the patients was 46 months. EGFR, KRAS, and BRAF mutations were detected in 13.7%, 21.3%, and 3% of tested cases, respectively; ALK rearrangements and MET amplifications were found respectively in 4.7% and 2% of tested cases. As expected, cases with mutations in exons 18–21 of EGFR, sensitizing to anti-EGFR tyrosine kinase inhibitors (TKIs) agents, had a significantly longer survival in comparison to those without (p

Supplemental Digital Content is available in the text., Gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract. We provide in the present article the molecular characterization of a series of primary GISTs in a cohort of Sardinian patients (Italy), with the aim to describe the patterns of KIT and PDGFRa mutations and the corresponding clinical features. Ninety-nine Sardinian patients with histologically-proven diagnosis of GIST were included in the study. Medical records and pathology reports were used to assess the demographic and clinical features of the patients and the disease at the time of the diagnosis. Formalin-fixed, paraffin-embedded tissue samples were retrieved for each case, and mutation analysis of the KIT and PDGFRa genes was performed. KIT and PDGFRa mutations were detected in 81.8% and 5% of the cases, respectively. The most common KIT mutation was W557_K558del in exon 11, while D842V in exon 18 was the most common PDGFRa genetic alteration; V561D was the only PDGFRa mutation found in exon 12. The global “wild-type” cases, with no mutations in either the KIT or PDGFRa genes, were 13 (13.1%). The mean survival of those patients was approximately 46.9 (±43.9) months. Globally, 86.9% of Sardinian patients with GIST had a KIT or PDGFRa mutation; the former were more frequent in comparison with other Italian cohorts, while PDGFRa mutations were rare. No statistical differences in survival between mutated and wild-type cases, and between KIT and PDGFRa mutated cases were detected in our study.

Background Advanced lung adenocarcinoma (LAC) is one of the most lethal malignancies worldwide. The aim of this study was to evaluate the global survival in a real-life cohort of patients with LAC harboring driver genetic alterations. Methods A series of 1282 consecutive Sardinian LAC patients who underwent genetic testing from January 2011 through July 2016 was collected. Molecular tests were based on the clinical needs of each single case (EGFR-exon18/19/21, ALK, and, more recently, BRAF-exon15), and the availability of tissue (KRAS, MET, and presence of low-frequency EGFR-T790M mutated alleles at baseline). Results The mean follow-up time of the patients was 46 months. EGFR, KRAS, and BRAF mutations were detected in 13.7%, 21.3%, and 3% of tested cases, respectively; ALK rearrangements and MET amplifications were found respectively in 4.7% and 2% of tested cases. As expected, cases with mutations in exons 18–21 of EGFR, sensitizing to anti-EGFR tyrosine kinase inhibitors (TKIs) agents, had a significantly longer survival in comparison to those without (p

The improvement of the immunotherapeutic potential in most human cancers, including melanoma, requires the identification of increasingly detailed molecular features underlying the tumor immune responsiveness and acting as disease-associated biomarkers. In recent past years, the complexity of the immune landscape in cancer tissues is being steadily unveiled with a progressive better understanding of the plethora of actors playing in such a scenario, resulting in histopathology diversification, distinct molecular subtypes, and biological heterogeneity. Actually, it is widely recognized that the intracellular patterns of alterations in driver genes and loci may also concur to interfere with the homeostasis of the tumor microenvironment components, deeply affecting the immune response against the tumor. Among others, the different events linked to genetic instability—aneuploidy/somatic copy number alteration (SCNA) or microsatellite instability (MSI)—may exhibit opposite behaviors in terms of immune exclusion or responsiveness. In this review, we focused on both prevalence and impact of such different types of genetic instability in melanoma in order to evaluate whether their use as biomarkers in an integrated analysis of the molecular profile of such a malignancy may allow defining any potential predictive value for response/resistance to immunotherapy.

Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation status plays a predominant role in the management of MM patients. The aim of this study was to compare BRAF mutational testing performed by conventional nucleotide sequencing approaches with either real-time polymerase chain reaction (rtPCR) or next-generation sequencing (NGS) assays in a real-life, hospital-based series of advanced MM patients. Consecutive patients with AJCC (American Joint Committee on Cancer) stage IIIC and IV MM from Sardinia, Italy, who were referred for molecular testing, were enrolled into the study. Initial screening was performed to assess the mutational status of the BRAF and NRAS genes, using the conventional methodologies recognized by the nationwide guidelines, at the time of the molecular classification, required by clinicians: at the beginning, Sanger-based sequencing (SS) and, after, pyrosequencing. The present study was then focused on BRAF mutation detecting approaches only. BRAF wild-type cases with available tissue and adequate DNA were further tested with rtPCR (Idylla&trade, ) and NGS assays. Globally, 319 patients were included in the study, pathogenic BRAF mutations were found in 144 (45.1%) cases examined with initial screening. The rtPCR detected 11 (16.2%) and 3 (4.8%) additional BRAF mutations after SS and pyrosequencing, respectively. NGS detected one additional BRAF-mutated case (2.1%) among 48 wild-type cases previously tested with pyrosequencing and rtPCR. Our study evidenced that rtPCR and NGS were able to detect additional BRAF mutant cases in comparison with conventional sequencing methods, therefore, we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy of BRAF detection.

Malignant melanoma (MM) is one of the deadliest skin cancers. BRAF mutation status plays a predominant role in the management of MM patients. The aim of this study was to compare BRAF mutational testing performed by conventional nucleotide sequencing approaches with either real-time polymerase chain reaction (rtPCR) or next-generation sequencing (NGS) assays in a real-life, hospital-based series of advanced MM patients. Consecutive patients with AJCC (American Joint Committee on Cancer) stage IIIC and IV MM from Sardinia, Italy, who were referred for molecular testing, were enrolled into the study. Initial screening was performed to assess the mutational status of the BRAF and NRAS genes, using the conventional methodologies recognized by the nationwide guidelines, at the time of the molecular classification, required by clinicians: at the beginning, Sanger-based sequencing (SS) and, after, pyrosequencing. The present study was then focused on BRAF mutation detecting approaches only. BRAF wild-type cases with available tissue and adequate DNA were further tested with rtPCR (Idylla™) and NGS assays. Globally, 319 patients were included in the study; pathogenic BRAF mutations were found in 144 (45.1%) cases examined with initial screening. The rtPCR detected 11 (16.2%) and 3 (4.8%) additional BRAF mutations after SS and pyrosequencing, respectively. NGS detected one additional BRAF-mutated case (2.1%) among 48 wild-type cases previously tested with pyrosequencing and rtPCR. Our study evidenced that rtPCR and NGS were able to detect additional BRAF mutant cases in comparison with conventional sequencing methods; therefore, we argue for the preferential utilization of the aforementioned assays (NGS and rtPCR) in clinical practice, to eradicate false-negative cases and improve the accuracy of BRAF detection.

Female adnexal tumors of probable Wolffian origin are rare gynecologic tumors with

Cutaneous malignant melanoma, whose incidence is increasing steadily worldwide, is the result of complex interactions between individual genetic factors and environmental risk factors. Ultraviolet radiation represents the most important environmental risk factor for the development of skin cancers, including melanoma. Sun exposure and early sunburn during childhood are the principal causes of cutaneous melanoma insurgence in adults, with double the risk relative to a nonexposed population. Consequently, ultraviolet protection has long been recognized as an important measure to prevent such a malignancy. Biological and epidemiological data suggest that vitamin D status could affect the risk of cancer and play a role in cancer prevention by exerting antiproliferative effects. Solar radiations are critical for vitamin D synthesis in humans; however, uncontrolled and intensive sun exposure is dangerous to skin health and may contribute toward the development of cutaneous malignant melanoma. An optimum balance between sun protection and exposure is thus advocated. Additional research is required to confirm the preventive role of vitamin D in melanoma incidence or a positive influence on patient outcome.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/

Sinonasal mucosal melanoma (SNM) is a rare and aggressive type of melanoma, and because of this, we currently have a limited understanding of its genetic and molecular constitution. The incidence among SNMs of somatic mutations in the genes involved in the main molecular pathways, which have been largely associated with cutaneous melanoma, is not yet fully understood. Through a next-generation sequencing (NGS) approach using a panel of 25 genes involved in melanoma pathogenesis customized by our group, we performed a mutation analysis in a cohort of 25 SNM patients. Results showed that pathogenic mutations were found in more than 60% of SNM cases at a somatic level, with strikingly 32% of them carrying deleterious mutations in the BRAF gene. The identified mutations mostly lack the typical UV signature associated with cutaneous melanomas and showed no significant association with any histopathological parameter. Oncogenic activation of the BRAF-depending pathway, which may induce immune tolerance into the tumour microenvironment (i.e., by increasing the VEGF production) was poorly associated with mutations in genes that have been related to diminished clinical benefit of the treatment with BRAF inhibitors. Screening for mutations in BRAF and other MAPK genes should be included in the routine diagnostic test for a better classification of SNM patients.

High frequency of mutations seems to determine a higher occurrence of neoepitope formation and, thus, tumor immunogenicity. A somatic hypermutated status could thus act as a predictive biomarker of responsiveness to immunotherapy with recent immune checkpoint inhibitors. Among several factors involved in determining the hypermutated status, such as inactivating mutations in the DNA polymerases as well as exposure to external (cigarette smoke, UV radiation, chemicals) and endogenous (reactive oxygen species) mutagens, a defective DNA mismatch repair system may give rise to genetic instability and, particularly, to microsatellite instability (MSI). The occurrence of MSI has been associated with increased load of mutations and expression of abundant peptides that serve as neoantigens to elicit an immune response within a context of a favorable tumor microenvironment. Here we describe methodological strategies to investigate for the presence of the MSI phenotype in cancer samples, through a combination of molecular approaches performed on paraffin-embedded tissues.

High frequency of mutations seems to determine a higher occurrence of neoepitope formation and, thus, tumor immunogenicity. A somatic hypermutated status could thus act as a predictive biomarker of responsiveness to immunotherapy with recent immune checkpoint inhibitors. Among several factors involved in determining the hypermutated status, such as inactivating mutations in the DNA polymerases as well as exposure to external (cigarette smoke, UV radiation, chemicals) and endogenous (reactive oxygen species) mutagens, a defective DNA mismatch repair system may give rise to genetic instability and, particularly, to microsatellite instability (MSI). The occurrence of MSI has been associated with increased load of mutations and expression of abundant peptides that serve as neoantigens to elicit an immune response within a context of a favorable tumor microenvironment. Here we describe methodological strategies to investigate for the presence of the MSI phenotype in cancer samples, through a combination of molecular approaches performed on paraffin-embedded tissues.

IntroductionAberrant DNA methylation is frequently observed in colorectal cancer (CRC), but the underlying mechanisms are poorly understood. Ten-Eleven Translocation (TET) dioxygenases and DNA repair enzyme Thymine DNA Glycosylase (TDG) are involved in active DNA demethylation by generating and removing, respectively, novel oxidized cytosine species. Mutations ofTET1andTDG, and alterations of the levels of oxidized cytosine species have been identified in human CRC cases, but the biological significance of the TET-TDG demethylation axis in intestinal tumorigenesis is unclear.Material and MethodsWe generatedApcMinmice with additional inactivation ofTet1and/orTdg, and characterized the methylome and transcriptome of intestinal adenomas by DREAM and RNA sequencing, respectively.ResultsTet1-and/orTdg-deficientApcMinmice show enhanced intestinal tumorigenesis in comparison to wild typeTet1andTdg ApcMinmice. Specifically,Tet1and/orTdg-deficientApcMinadenomas manifested increased size or features of erosion and stroma activation. Methylome analysis revealed progressive loss of global DNA hypomethylation in colonic adenomas fromTet1-andTdg-deficientApcMinmice, and hypermethylation of CpG islands inTet1-deficientApcMinmice. In addition, RNA sequencing showed upregulation of genes in inflammatory and immune response pathways inTet1-andTdg-mutant colonic adenomas compared to controlApcMinadenomas.ConclusionsTaken together, these findings demonstrate the important role of active DNA demethylation mediated by TET-TDG in reducing intestinal tumor formation, by modulating the epigenome and inflammatory/immune responses. This study highlights a novel mechanism of epigenetic deregulation during intestinal tumorigenesis with diagnostic, therapeutic and prognostic implications.

Background: Cutaneous melanoma is one of the most frequent malignancies of the skin in Caucasian populations. Patients who develop cutaneous melanoma are at increased risk of developing a second primary melanoma. The estimated incidence of multiple primary melanoma (MPM) ranges from 1.2% to 8.2% of cases, with a high preponderance of melanomas occurring metachronously. Objectives: The aim of this study was to describe dermoscopic, microscopic, clinical, and molecular correlations between first and subsequent melanomas in patients with metachronous MPMs. Materials & methods: Twenty-four paired melanomas from 12 MPM patients were evaluated for architectural characteristics based on dermoscopy and confocal microscopy, as well as for mutations in BRAF and NRAS genes by Sanger-based sequencing analysis. Specific scores used for classifying features of dermoscopy (global pattern; 7-point check list; ABCD Stolz score) and confocal microscopy (Segura and Pellacani) were compared with genetic and histological data. Results: Consistency in dermoscopic patterns between the primary and subsequent cutaneous melanomas were observed in about two thirds of cases, whereas concordant features based on confocal microscopy were found in only about two fifths of cases. The majority of patients (7/12; 58%) presented consistent BRAF/NRAS mutation patterns between first and subsequent primary melanomas. A significant association between BRAF mutations and Pellacani score was evident. Conclusions: Similarities between the index melanoma and subsequent cutaneous melanomas were observed with regards to dermoscopic features and, to a much less extent, confocal microscopy findings. Our data further indicate that the Pellacani score may be used to predict BRAF mutations.

Cutaneous melanoma is a common and aggressive human skin cancers. Much is actually known about the molecular mechanisms underlying melanoma pathogenesis. The aim of the study was to evaluate any possible correlation between mutations in main growth-controlling genes (BRAF, NRAS, CDKN2A) and copy number variations in frequently amplified candidate genes (MITF, EGFR, CCND1, cMET, and cKIT) during melanoma initiation and progression. A large series of primary and secondary melanoma tissue samples (N = 274) from 232 consecutively-collected patients of Italian origin as well as 32 tumor cell lines derived from primary and metastatic melanomas underwent mutation screening and fluorescence in situ hybridization (FISH) analysis. Overall, BRAF, NRAS, and CDKN2A were found mutated in 62.5%, 12.5% and 59% cell lines and in 47%, 16%, 12% tumor tissues, respectively. Quite identical mutation patterns between primary tumors and metastatic lesions were found for BRAF and NRAS genes; mutations of CDKN2A gene appeared to be instead selected during tumor progression. In cell lines, high rates of gene amplifications were observed (varying from 12.5% for cKIT to 50% for MITF); vast majority of cell lines (75%) presented at least one amplified gene. Conversely, prevalence of gene amplification was significantly and progressively decreasing in melanoma metastases (12%) and primary melanomas (4%). Our findings suggest that gene amplifications may be acquired during the late phases of melanoma evolution and mostly act as "passenger" or "non-causative" alterations.

Long non-coding RNAs cover large part of the non-coding information of the human DNA, which represents more than 90% of the whole genome. They constitute a wide and complex group of molecules with more than 200 nucleotides, which generally lack an open reading frame, and are involved in various ways in the pathophysiology of cancer. Their roles in the regulation of gene expression, imprinting, transcription, and post-translational processing have been described in several types of cancer. CASC2 was discovered in 2004 in patients with endometrial carcinoma as a potential tumor suppressor. Since then, additional studies in other types of neoplasia have been carried out, and both mechanisms and interactions of CASC2 in cancer have been better elucidated. In this review, we summarize the current knowledge on the role of CASC2 in the genesis, progression, and clinical management of human cancer.

The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitro sumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Nitroso-N-methylurea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.

Background Activation of oncogenes downstream the EGFR gene contributes to colorectal tumorigenesis and determines the sensitivity to anti-EGFR treatments. The aim of this study was to evaluate the prognostic value of KRAS, BRAF, NRAS and PIK3CA mutations in a large collection of CRC patients from genetically-homogeneous Sardinian population. Methods A total of 1284 Sardinian patients with histologically-proven diagnosis of colorectal carcinoma (CRC) and presenting with metastatic disease were included into the study. Genomic DNA was isolated from formalin-fixed, paraffin-embedded primary tumour tissue samples of CRC patients and screened for mutations in RAS and BRAF genes, using pyrosequencing assays, and in PIK3CA gene, using automated DNA sequencing assays. Results Overall, mutation rates were 35.6 % for KRAS, 4.1 % for NRAS, and 2.1 % for BRAF. Among available DNA samples, 114/796 (14.3 %) primary CRCs were found to carry a mutation in the PIK3CA gene. In this subset of patients analysed in all four genes, a pathogenetic mutation of at least one gene was discovered in about half (378/796; 47.5 %) of CRC cases. A mutated BRAF gene was found to steadily act as a negative prognostic factor for either time to progression as metastatic disease (from detection of primary CRC to diagnosis of first distant metastasis; p = 0.009) or partial survival (from diagnosis of advanced disease to the time of death or last control; p = 0.006) or overall survival (p

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50-90% of patients developing TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species, which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34(+) CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na(+)/K(+)ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.

e21554Background: The prevalence of mutations in main genes involved in tumorigenesis during melanoma progression remains a controversial issue. Different melanoma tissues from the same patients we...

e21047 Background: The prevalence of mutations in driver genes during progression in cutaneous and mucosal melanomas remains inconclusive. We investigated the prevalence and distribution of mutations in main candidate genes involved in melanomagenesis among different melanoma tissues using a next-generation sequencing (NGS) approach. Methods: Forty-eight tumor samples from 36 patients with mucosal melanoma (MM) and fifty-two tumor samples from 34 patients with cutaneous melanoma (CM) were collected, after obtaining patients’ written informed consent for tissue sampling. Genomic DNA was isolated from macrodissected tumor tissues containing at least 80% neoplastic cells and analyzed for mutations in 25 most common melanoma-associated oncogenes and tumor suppressor genes, using the IMI Diagnostic Melanoma Panel on the Ion Torrent platform (Life Technologies, USA). Results: A total of 100 tumor tissues from 70 melanoma patients were analyzed. BRAF mutations were detected in 21/34 (62%) CM patients and 13/36 (36%) mm patients. The second most prevalent mutations were found in K-/N-RAS (6/34; 18%) and cKIT (6/36; 17%) genes among CM and mm patients, respectively. No concomitant mutations of BRAF, RAS, and cKIT genes were detected. Among others, mutations were more frequently found in CCND1 (20%), ARID2 (16%), and NF1(12%) genes, considering the entire series of patients. Vast majority of patients who had paired samples of primary and secondary melanomas showed consistent mutation patterns between primary tumors and metastatic lesions. Similar frequencies of mutations in driver genes were seen across metastatic sites. Conclusions: In the era of targeted therapies, assessment of the spectrum and distribution of mutations in main molecular targets among patients with melanoma is needed. Our findings about the prevalence of mutations in driver genes in paired tumor lesions from patients with cutaneous and mucosal melanoma may be useful in the management of such diseases. The Italian Melanoma Intergroup (IMI) includes the following additional members who participated as investigators in this study: Mario Mandalà, Paola Queirolo, Ignazio Stanganelli, Vanna Chiarion Sileni, Pietro Quaglino, Anna Maria Di Giacomo.

Lung cancer is the leading cause of cancer-related deaths over the world, characterized by a very high mortality rate. Molecular technique development tries to focus on early detection of cancers by studying molecular alterations that characterize cancer cells. Worldwide lung cancer research has focused on an ever-increasing number of molecular elements of carcinogenesis at genetic, epigenetic and protein levels. The non-invasiveness is the characteristic that all clinical trials on cancer detection should have. Abnormal chest imaging and/or non-specific symptoms are initial signals of lung cancer that appear in an advanced stage of disease. This fact represents the cause of the low 5-year survival rate: over 90% of patients dying within 5 years of diagnosis. Since smokers have higher quantity of sputum containing exfoliated cells from the bronchial tree, and the sputum represents the most easily accessible biological fluid and its collection is non-invasive, analysis of this sample represents a good area of research in early lung cancer diagnosis. Continued cigarette smoking is the cause of chronic obstructive pulmonary disease (COPD), with an estimated attributable risk factor exceeding 80% in smoking affected individuals. Lung cancer is found in 40-70% of patients with COPD, particularly in severe disease, and it is a common cause of death in these patients. A large prospective trial of almost half a million non-smokers showed as lung cancer is also common in patients with COPD who have never smoked. This review describes issues related to early lung cancer screening using non-invasive methods.

Abstract 520 Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of BCR-ABL1 kinase-positive chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 15–25% of patients initially responding favorably to imatinib will develop acquired drug resistance, which in 40–90% of cases is caused by genomic instability resulting in the appearance of clones expressing TKI resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species (ROS) resulting in excessive oxidative DNA damage such as oxidized DNA bases (8-oxoguanine and 5-hydroxycytosine→uracil) (Nieborowska-Skorska et al., Blood, 2012). Unfaithful and/or inefficient repair of these lesions generates TKI resistant point mutations in BCR-ABL1 kinase. Oxidative DNA lesions may be removed by base excision repair (BER) or, if not removed, will create mismatches, which are repaired by mismatch repair (MMR). Since we found that MMR is inhibited in CML-CP (Stoklosa et al., Cancer Res., 2008), the activity of BER is critical to prevent the accumulation of point mutations. Using an array of specific substrates and inhibitors/blocking antibodies we found that two major glycosylases, uracil-DNA glycosylase UNG2 and 8-oxoguanine glycosylase (OGG1) responsible for the excision of uracil (product of oxidation of cytosine) and 8-oxoguanine (8-oxoG) from DNA, respectively, were inhibited in BCR-ABL1 –transformed cell lines and CD34+ CML cells. The inhibitory effect was even more pronounced in CML blast phase (CML-BP) in comparison to CML-CP, it depended on BCR-ABL1 kinase activity and was not accompanied by deregulation of nuclear expression and/or chromatin association of these glycosylases. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases such as TEL-ABL1, TEL-PDGFbetaR and NPM-ALK did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI we found that UNG2 activity diminished the number of oxidized DNA bases detected by modified comet assay and prevented accumulation of point mutations in reporter gene Na+/K+ATPase, which encode resistance to ouabain. In conclusion, we hypothesize that inhibition of UNG2 and OGG1, accompanied by reduced MMR activity is responsible for accumulation of TKI-resistant BCR-ABL1 kinase point mutations and perhaps also other point mutations facilitating malignant progression of CML. Disclosures: No relevant conflicts of interest to declare.