Types Of Genetic Alterations Research Articles

Abstract P6-09-08: Identification of ESR1 mutation in breast cancers using targeted ultra-deep sequencing data analysis

Abstract Introduction: Estrogen Receptor 1 (ESR1) gene encodes an estrogen receptor, which regulates cell proliferation and promotes tumor progression in estrogen receptor(ER)-positive breast cancer (BC). Therefore, endocrine therapy that inhibiting ER downstream signal, is the most effective treatment strategy in ER-positive BC. However, about 25% of patients with primary disease and almost all patients with metastases will present with or eventually develop endocrine resistance. And genetic alteration of ESR1 is now identified as the endocrine resistance mechanism. However, a few data from clinical trials or public data base exists and could not reflect real world clinic. Therefore, we aimed to identify the frequency and type of ESR1 genetic alterations in BCs through this large scaled study. Methods: We performed targeted ultra-deep sequencing (CancerSCAN™) using BC tissue specimens. This sequencing was covered entire coding area of ESR1 gene and also detected copy number alteration and translocation of ESR1. Results: Targeted ultra-deep sequencing of ESR1 was performed using 618 BC tissues. Of 618 tissue samples, 253(40.9%) were MBCs, 362(58.6%) were early BCs (EBCs) and 3 were not identified. In terms of subtypes, 220 ER-positive BCs, 122 ER-positive and HER2-positive BCs, 119 HER2-positive and 153 triple-negative BCs (TNBCs) were included. BCs from patients under 40 year-old were 277(44.8%)(Median: 43.0, range: 23.5 -75.6). ESR1 genetic alterations were identified in 21 BCs (5 EBCs and 16 MBCs). In EBCs, 3 cases were observed in TNBCs and 2 cases were in ER-positive BCs (2.6% and 1.2%, respectively). All five EBC were treatment naïve status. Of 16 cases of ESR1 alterations in MBCs, 10 cases of ESR1 alterations were detected in ER-positive BCs (17.6%), 5cases in ER and HER2-positive BCs(6.7%) and 1 in HER2-positive BCs (1.2%). All ER-positive MBCs were treated with more than one line of endocrine therapy. Most commonly detected genetic alteration was single nucleotide variant (SNV) (15 of 21, 71.4%). Thirteen were in ligand binding domain and two cases occurred in activation function-1 (AF-1) domain (P79A and G145S). D538G and V392I were most frequently mutated loci followed by Y537N (3, 3 and 2 cases, respectively) and only metastatic ER-positive BCs harbored ESR1 activating mutation. Four copy number (CN) amplification in 2 ER-positive and 2 ER and HER2-positive BCs, one CN deletion in TNBC and one ESR1 fusion in ER and HER2-positive BC were also detected (19.0%, 4.8% and 4.8%, respectively). In frame ESR1 fusion was occurred between ESR1 and NPHS1 genes. Conclusion: In this experimental study, ESR1 genetic alterations were frequently detected in ER-positive MBC but ER-negative or EBC also harbored. The type of genetic alterations varied including SNVs, CN alterations and translocation and ESR1-NPHS1 fusion is the novel genetic alteration that has not been reported. To identify the role of ESR1 genetic alteration in ER-negative BCs and novel translocation, further functional validation would be warranted (Clinical trials.gov Number :NCT02591966). Citation Format: Kim J-Y, Park K, Park W-Y, Nam SJ, Kim SW, Lee JE, Lee SK, Jung HH, Yu JH, Ahn JS, Im Y-H, Park YH. Identification of ESR1 mutation in breast cancers using targeted ultra-deep sequencing data analysis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-09-08.

Characteristics and Clinical Significance of CRLF2 Mutations in Adult Acute Lymphoblastic Leukemia

Cytokine receptor-like factor 2(CRLF2) plays an important role in the development of normal B lymphocytes, which can mediate early B cell proliferation and survival. The aim of this study was to investigate the mutations of CRLF2 and its clinical significance in adult patients with acute lymphoblastic leukemia(ALL). Exons of CRLF2 were amplified, then the DNA was purified and sequenced; the frequency, position, types and clinical significance of CRLF2 mutations were analyzed. 6 types of genetic alterations in CRLF2 were found, among them the R186S prompted better prognosis, while L86I, F232F and W255C associated with poor prognosis. CRLF2 mutations may play an important role in the development and progressions of adult patients with ALL, and these genetic abnormalities may associate with clinical outcome.

Características citopatológicas de la leucemia aguda en el Hospital de Especialidades Pediátricas de Chiapas, México

BackgroundChildhood acute leukemia cytological features are unknown in Chiapas, Mexico. Defining these features is important because this is a relatively isolated population with high consanguinity index, and these aspects could determine differences in responses to treatment and outcome. MethodsEighty-one childhood acute leukemia cases treated at the Hospital de Especialidades Pediátricas in Chiapas were characterized by morphology, immunophenotype, genotype, initial risk assignment and status at the time of the study. ResultsThe proportion of leukemic cell types found in this study was B cell, 75.3%; myeloid, 16%; T cell, 3.7% and NK 1.2%. In B cell leukemia, genetic alterations were present in 40.6% of cases and had a specific outcome regardless of initial risk assessment. Cases with MLL gene alteration died within a month from diagnosis. Translocations were present in 17.5% B cases; t(1;19) was present in those with a favorable outcome. The t(12;21) translocation was related to initial remission and midterm relapse and dead. Hyperdiploidy was present in 20% of B cell cases with good outcome. In 38.5%of myeloid cases were translocations and karyotypic abnormalities. Short-term outcome in this group has been poor; 69% have died or abandoned treatment in relapse from 15 days to 37 months after diagnosis. ConclusionsRelative frequency of different types of acute leukemia in patients treated at a tertiary level pediatric hospital in Chiapas, Mexico, was similar to the one found in other parts of the country. Patients’ outcome, under a standardized treatment, differs according to the group, the subgroup and the presence and type of genetic alterations.

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Detection of Copy Number Alterations Using Single Cell Sequencing.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

MTE03.01 Basics of Molecular Biology for the Clinician

The rapid development of molecular biology in recent years has allowed us to understand the main molecular steps involved in the development and progression of lung cancer. The identification of molecular alterations in specific tumor genes that function as key drivers for neoplastic growth has laid the foundations for new therapeutic approaches with targeted agents. An accurate detection of target mutation is mandatory for an efficient treatment. The main limitation of targeted therapies is the occurrence of acquired resistance that makes cancer unresponsive to treatment. In many cases, through the acquisition of additional (secondary) mutations the tumor is able to acquire the heterogeneity which may enable it to adapt to various conditions of the microenvironment, including those determined by the effect of treatment with specific drugs. New generation drugs are constantly under development to overcome tumor resistance and increase survival of lung cancer patients. In this process, a constant monitoring of the mutational status of the tumor is required. Different types of genetic alterations are involved in tumor development, progression, and induction of resistance, including single nucleotide variants, indels, amplifications, fusions etc. Mutation detection before first line treatment is usually performed on tissue or cytological samples. Resected tumor samples, biopsies and cytological specimens are available in about 25%, 35% and 40% of NSCLC patients, respectively. At progression, a re-biopsy should be obtained to detect the emergence of resistance-inducing mutations. Transbronchial tissue biopsy is the most common sampling method used for re-biopsy. However, several factors limit the success rate of re-biopsy, such as the performance status of the patient, the difficulty of accessing some tumor sites, and the invasiveness of sampling methods. When the amount and/or quality of the biological material is insufficient for molecular analysis, circulating free DNA (cfDNA) can represent a valid alternative in selected patients. Liquid biopsies have several advantages over tissue or cells: they are less invasive, can be repeated over time, and have a more rapid turnaround time. However, there are some critical issues that must be considered: 1) the possibility to detect a mutation in cfDNA is dependent on several clinicopathological parameters, including tumor type, tumor burden, and particularly tumor stage (a locally advanced tumor has a significantly lower probability to spread mutant DNA in the blood than a metastatic tumor); 2) a large amount of wild-type DNA circulates in the plasma with only trace amounts of the mutant allele; therefore, the analysis of genetic aberrations in cfDNA is challenging, requiring well standardized pre-analytical/analytical protocols and dedicated techniques with high sensitivity and specificity. Different technologies/protocols are required for the detection of these genetic aberrations. Robust and sensitive molecular biology techniques are nowadays available to detect mutations in driver genes before initiating a targeted treatment or to identify the emergence of secondary mutations at disease progression. The use of multi marker assays, and in particular next generation sequencing, is progressively becoming popular, allowing on one hand to reduce the working time, costs per single assay, and the amount of nucleic acids required for testing and increasing, in the other hand, throughput and overall quality. Recently, semi-quantitative or quantitative detection methods for the assessment of genetic aberrations in cfDNA have been developed with a number of potential clinical implications. An accurate quantification of mutated alleles in cfDNA during the first days of treatment could: a) complement or replace more expensive and invasive methods to assess response in treated patients; b) represent a new way to compare the effectiveness of different drug; c) be an additional tool to evaluate the best treatment regimen for patients. In addition, a periodic quantification of the mutation burden during all the treatment time could allow an early detection of resistance-inducing mutations for possible changes to therapy. Targeted Therapies, Acquired resistance, Lung cancer molecular biology

Abstract 508: Prognostic value of circulating tumor DNA in advanced colorectal cancer patients: quantification of hypermethylated or mutant sequences using picoliter-droplet digital PCR

Abstract Background: Circulating tumor DNA (ctDNA) has been suggested as a new marker in different cancer types including colorectal cancer (CRC). Its prognostic role needs to be validated in prospective clinical studies using precise and robust procedures. In this context, picoliter-droplet digital PCR has arisen as a highly sensitive and quantitative approach offering a broad dynamic range of detection. Patients and methods: All consecutive advanced CRC patients receiving first-line chemotherapy were included in this monocentric prospective study. Plasma samples were collected before the 1st cycle of chemotherapy, then at 14 and 28 days after the beginning of the chemotherapy. Both, carcinoembryonic antigen (CEA) dosages and computed tomography (CT) were performed at baseline and every 8 weeks. For each patient, tumor DNA from biopsies was tested for the presence of KRAS, BRAF and TP53 mutations either by conventional qPCR or Next-Generation Sequencing. When no mutation was identified in the tumor, ctDNA was screened for hypermethylated sequences of WIF1 and NPY genes. CtDNA sequences (mutated or hypermethylated) were quantified (concentration, ng/mL) using picoliter-droplet digital PCR coupled to Taqman probes. Associations of ctDNA concentration with progression-free survival (PFS) and overall survival (OS) were analysed using a Cox proportional hazards model. Multivariate models were adjusted on age, gender, Kohne's score, CEA and type of genetic alteration. Results: Up to now 43 patients were included (mean age: 66±1.8years [62.6-69.6], gender ratio: M/F 1.15). At baseline ctDNA was detectable in 38/43 (88%) patients based on KRAS, BRAF or TP53 mutation (n = 27), or hypermethylation of the WIF1 or NPY genes (n = 16). Values ranged from 0 to 208 ng/mL, mean and median ctDNA concentration were 12.7 ng/mL and 2.05 ng/mL, respectively. After adjustment on prognostic covariates, the concentration of baseline ctDNA was significantly positively associated with a worse PFS (HR: 1.01 CI95% [1-1.02]; padj = 0.03) and OS (HR: 1.02 CI95% [1.01-1.035] padj = 0.004). The median PFS were 8.6 and 2.3 months in patients with less or more than 5 ng/mL of baseline ctDNA, respectively (HR: 6.7, CI95% 2-22; padj = 0.001). When ctDNA concentration was considered at 14 or 28 days, patients with a ctDNA ≥ 0.2 ng /mL have a significantly worse PFS (HR: 4.12 CI95% [1.41-12.0]; padj = 0.009) than the others independently of the baseline concentration of ctDNA. Conclusion: This study suggests that ctDNA is a strong prognostic factor highlighting the clinical relevance of quantifying circulating tumor DNA by picoliter-droplet digital PCR in first-line advanced colorectal cancer treatment. Citation Format: Fanny Garlan, Pierre Laurent-Puig, Nathalie Siauve, Audrey Didelot, Geraldine Perkins, Hélène Blons, Julien Taieb, Valerie Taly, Aziz Zaanan. Prognostic value of circulating tumor DNA in advanced colorectal cancer patients: quantification of hypermethylated or mutant sequences using picoliter-droplet digital PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 508.

Hyperhaploid uterine mesenchymal tumors—a novel genetic subgroup?

Hyperhaploid karyotypes have been described to occur in subsets of various solid tumors and leukemias. In these cases, monosomy is noted for most of the chromosomes while a few chromosomes still remain disomic. Evidence has emerged that at least in some tumor entities these remaining chromosomes are non-randomly selected. In addition, structural alterations can accompany the reduced chromosome number and secondary duplication of the chromosome complement is also a frequent finding. In this report, we describe hyperhaploidy in a case of an endometrial stromal nodule of a 50 year old woman who underwent hysterectomy because of symptomatic uterine fibroids. In addition, we review two other recently described cases of uterine mesenchymal tumors with that type of genetic alteration. Despite some histologic differences, striking similarities between these three cases exist with respect to the chromosomes were retained as disomic. Thus, the question arises if hyperhaploidy defines a novel genetic subgroup of uterine mesenchymal tumors.

Abstract P5-04-07: CDK8 protein complex as a potential biomarker and therapeutic target in breast cancer

Abstract Cyclin-dependent kinase 8 (CDK8) and its paralog CDK19 are transcriptional regulators that, in complex with CCNC, MED12 and MED13, mediate several carcinogenic signalling pathways such as NFκβ, TGFβ/BMP, WNT/β-catenin, HIF1A and serum growth factor network. Using immunohistochemical analysis, we found that CDK8/19 protein is overexpressed in invasive ductal carcinomas of the breast relative to non-malignant mammary tissues. TCGA database analysis showed that gene amplification is the most frequent type of genetic alterations of CDK8, CDK19, CCNC and MED13 in breast cancers, with MED13 appearing as one of the most frequently amplified genes in breast cancer (amplified in 9.7% of samples), whereas point mutations are more common in MED12. CDK8, CDK19 and CCNC expression was strongly increased but MED12 expression was decreased in tumors with mutant p53. Meta-analysis of transcriptome databases revealed that higher expression of CDK8, CDK19, CCNC and MED13 (but not MED12) is associated with shorter relapse-free survival (RFS) in the four molecular subtypes of breast cancer. The RFS correlations were much stronger in patients who underwent systemic adjuvant therapy than in untreated patients, suggesting that CDK8 and its interactive genes impact the failure of systemic therapy. This result is in agreement with the role of CDK8 as a mediator of the chemotherapy-induced paracrine network that promotes drug resistance and metastasis (Porter et al., PNAS, 109, 13799, 2012) and with our finding that a small-molecule CDK8/19 inhibitor augmented the efficacy of doxorubicin in a triple-negative breast cancer xenograft model. The expression levels of CDK8, CDK19, CCNC and MED13 in breast cancer samples were directly correlated with each other and with the expression of MYC but inversely correlated with estrogen receptor (ER)α expression. Since MYC is known to be a positive downstream mediator of the ER activity, we hypothesized that CDK8 may play a similar role, with an increase in CDK8 augmenting estrogen mitogenic signalling in tumors with decreased ER. Confirming this hypothesis, we have found that CDK8 inhibition by selective small-molecule CDK8/19 inhibitors or by shRNA knockdown suppresses estrogen-induced transcription in ER-positive breast cancer cell lines. CDK8/19 inhibition abrogates the mitogenic effect of estrogen on ER-positive cells and synergizes with the ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive cells with a CDK8/19 inhibitor significantly impeded the outgrowth of estrogen-independent cells, to a greater extent than did mTOR or HER2 targeted drugs. These results indicate that the expression of CDK8 and its interactive genes has a profound impact on the response to treatment in breast cancer and may provide novel biomarkers for relapse-free survival after adjuvant therapy. CDK8/19 inhibition may be useful to augment chemotherapy and hormone therapy of breast cancer and to prevent the development of tumors resistant to estrogen deprivation. Citation Format: McDermott MSJ, Györffy B, Chumanevich AA, Kaza V, Porter DC, Catroppo JF, Chen M, Oliver D, Shtutman M, Roninson IB, Broude EV. CDK8 protein complex as a potential biomarker and therapeutic target in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-07.

Association of polymorphisms with a family history of cancer and the presence of germline mutations in the BRCA1/BRCA2 genes.

IntroductionBreast cancer (BC) is an important public health problem worldwide. In Brazil, breast cancer is the most frequently diagnosed tumor and the leading cause of cancer death in women. Hereditary cancer represents approximately 5 to 10 % of BC cases. Even outside the hereditary cancer context, the presence of polymorphisms acting as genetic modifiers may contribute to a better or worse prognosis. Not much is known about the hereditary BC epidemiology in Brazil or about the influence of polymorphisms on hereditary predisposition.ObjectiveThis study examined the role of five different polymorphisms in four groups of women with BC: Group 1: women with a germline mutation in the BRCA1/2 genes; Group 2: women with variants of uncertain significance in BRCA1/2 and Group 3: women with no mutations in BRCA1/2.Patients and methodsThe women included in groups 1, 2 and 3 were patients from the Department of Oncogenetics of the Barretos Cancer Hospital who had undergone genetic testing because of a clinical suspicion of hereditary predisposition syndrome. The constitutive DNA was analyzed for the presence of polymorphisms at rs2981582 (FGFR2 gene); rs3803662 (TNRC9); rs889312 (MAP3K1); rs3817198 (LSP1 gene); and rs13281615 (8q24). The analyses were performed using PCR amplification and bi-directional sequencing.ResultsNo differences were identified in the frequency of the polymorphisms that were analyzed among the three groups. However, some associations were identified, such as the occurrence of bilateral breast cancer and homozygosity for the G allele in rs13281615 as well as the correlation between the SNPs rs2981582 and rs13281615 and the number of cancer cases in the family. Regarding the G allele of rs13281615, we observed that the proportion of individuals who were homozygous for this allele increased with the number of generations affected by cancer, regardless of the group where the patients were included. Concerning the rs2981582 we could observe that individuals from group 1 and homozygous CC had fewer cancer (and also fewer breast cancer) cases. Regarding the hormone receptors, we observed an increased frequency in C homozygotes (rs3803662) among estrogen receptor-negative individuals from groups 1 and 3. For rs2981582 (FGFR2), we observed an increased frequency of the T allele in women who were positive for the estrogen and progesterone receptors regardless of the BRCA1/2 mutational status (p = 0.020 and p = 0.014, respectively).ConclusionThe results presented here provide interesting data on the modifying effect of polymorphisms on a family history of cancer; this may be a variable to consider in the analysis of tumor diversity, and of the family history observed in families with hereditary breast cancer (even in those harboring the same type of genetic alteration).Electronic supplementary materialThe online version of this article (doi:10.1186/s13053-015-0042-1) contains supplementary material, which is available to authorized users.

The contribution of epithelial-mesenchymal transition to renal fibrosis differs among kidney disease models

The impact of the epithelial-mesenchymal transition (EMT) to the formation of renal fibrosis has been debated in several lineage-tracing studies, with conflicting findings. Such disparities may have arisen from varying experimental conditions such as different disease models, the mouse strain, and type of genetic alteration used. In order to determine the contribution of these factors to EMT, we generated four kidney disease models in several mouse strains genetically modified to express enhanced green fluorescence protein (EGFP) in cortical tubular epithelial cells under the control of the γ-glutamyl transpeptidase promoter. Using this approach, the EMT was visible and quantifiable based on a count of EGFP-positive interstitial cells in the fibrotic kidney sections of the four renal disease models found to be either EMT-prone or -resistant. The EMT-prone models consisted of unilateral ureteral obstruction and ischemic nephropathy in SJL mice. The EMT-resistant models consisted of ureteral obstruction in C57B/6 and F1(C57B/6 × SJL) mice, adriamycin nephrosis in 129 mice, and nephrotoxic serum nephritis in SJL mice. Analyses of these renal disease models suggest the emergence of EMT-derived fibroblasts arises in a disease-specific and strain-dependent manner. Thus, when considering molecular mechanisms and involvement of the EMT in renal fibrosis, it is important to take into account the experimental conditions, particularly the mouse strain and type of disease model.

Frequency and features ofTP53mutation in 30 Chinese patients with sporadic basal cell carcinoma

Basal cell carcinoma (BCC) is a prevalent form of nonmelanoma skin cancer. Although numerous studies in white populations suggest that mutations in the TP53 gene play an important role in the development of BCC, it is not clear whether this is also the case in East Asian populations such as in China. To investigate the frequency and the features of TP53 mutation in sporadic BCC in a Chinese population. In total, 30 patients with sporadic BCC, who had previously taken part in a study on PTCH1 mutations, were enrolled. BCC and control cells were obtained by laser-capture microdissection, and DNA was amplified and sequenced for analysis of TP53 mutations. In the 30 BCC samples, 6 TP53 point mutations were found (frequency of 20%), and 4 of these 6 mutations had ultraviolet (UV)-specific alterations. Combining these results with those of the previous study on PTCH1 mutations, we found that two patients with had three types of genetic alterations (each had two PTCH1 mutations and one TP53 point mutation). A further two patients each had one PTCH1 mutation and one UV signature TP53 mutation. In addition, the total number of UV-specific mutations of PTCH1 and TP53 accounted for 20% of the total patient group. The incidence of TP53 mutation in BCC in our Chinese subjects was lower than that reported for white populations. Many of the patients carried mutations of other genes in addition to of TP53. The majority of TP53 mutations were UV-induced specific alterations. However, the results of the two studies on TP53 and PTCH1 indicated that the incidence of UV-specific mutations is much lower in Chinese than in white populations.

Recombinogenic Conditions Influence Partner Choice in Spontaneous Mitotic Recombination

Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding yeast homolog of the mammalian common fragile sites, to examine recombination activities at these loci. We found that rates of URA3 inactivation of a hisG-URA3-hisG reporter at RSZ and non-RSZ loci were comparable under all conditions tested, including those that specifically promote chromosome breakage at RSZs (hydroxyurea [HU], mec1Δ sml1Δ, and high temperature), and those that suppress it (sml1Δ and rrm3Δ). These observations indicate that RSZs are not recombination hotspots and that chromosome fragility and recombination activity can be uncoupled. Results confirmed recombinogenic effects of HU, mec1Δ sml1Δ, and rrm3Δ and identified temperature as a regulator of mitotic recombination. We also found that these conditions altered the nature of recombination outcomes, leading to a significant increase in the frequency of URA3 inactivation via loss of heterozygosity (LOH), the type of genetic alteration involved in cancer development. Further analyses revealed that the increase was likely due to down regulation of intrachromatid and intersister (IC/IS) bias in mitotic recombination, and that RSZs exhibited greater sensitivity to HU dependent loss of IC/IS bias than non RSZ loci. These observations suggest that recombinogenic conditions contribute to genome rearrangements not only by increasing the overall recombination activity, but also by altering the nature of recombination outcomes by their effects on recombination partner choice. Similarly, fragile sites may contribute to cancer more frequently than non-fragile loci due their enhanced sensitivity to certain conditions that down-regulate the IC/IS bias rather than intrinsically higher rates of recombination.

The Sacred Disease: The Puzzling Genetics of Epileptic Disorders

In the September 12, 2013 issue of Nature, the Epi4K Consortium (Allen etal., 2013) reported sequencing 264patient trios with epileptic encephalopathies. The Consortium focused on genes exceptionally intolerant to sequence variations and found substantial interconnections with autism and intellectual disability gene networks.

KIF5B-RET fusions in lung adenocarcinoma

We identified in-frame fusion transcripts of KIF5B (the kinesin family 5B gene) and the RET oncogene, which are present in 1-2% of lung adenocarcinomas (LADCs) from people from Japan and the United States, using whole-transcriptome sequencing. The KIF5B-RET fusion leads to aberrant activation of RET kinase and is considered to be a new driver mutation of LADC because it segregates from mutations or fusions in EGFR, KRAS, HER2 and ALK, and a RET tyrosine kinase inhibitor, vandetanib, suppresses the fusion-induced anchorage-independent growth activity of NIH3T3 cells.

Abstract IA6: Gene fusions in lung cancer

Abstract Gene fusions represent one type of genetic alteration recently detected in lung cancer that results from chromosomal rearrangements. A fusion joining the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes was detected as a transforming gene in a small subset of lung adenocarcinomas. Importantly, patients with these fusions show responsiveness to ALK kinase inhibitors. Additional studies have identified multiple EML4-ALK gene fusion variants and the histopathological characteristics of the lung tumors identified. Various methods utilized to detect lung gene fusions have included retrovirus-mediated expression screening with 3T3 cell transformation assays, FISH, mRNA/protein expression, RACE-coupled PCR sequencing, massively parallel paired-end and transcriptome sequencing. Additional novel gene fusion events in lung cancer have recently been identified using these and newly defined approaches in small percentages of primary lung tumors. Potential mechanisms underlying the production and triggers for gene fusion events will be discussed.

Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine) at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine) at codon 57. In addition, we found in the same patient's sample a silent polymorphism at codon 11 (Ala11Ala) of exon 1.

Deregulation of Ion Channel and Transporter Encoding Genes in Pediatric Gliomas

Brain tumors, including the majority gliomas, are the leading cause of cancer-related death in children. World Health Organization has divided pediatric brain tumors into different grades and, based upon cDNA microarray data identifying gene expression profiles (GEPs), it has become evident in the last decade that the various grades involve different types of genetic alterations. However, it is not known whether ion channel and transporter genes, intimately involved in brain functioning, are associated with such GEPs. We determined the GEPs in an available cohort of 10 pediatric brain tumors initially by comparing the data obtained from four primary tumor samples and corresponding short-term cultures. The correspondence between the two types of samples was statistically significant. We then performed bioinformatic analyses on those samples (a total of nine) which corresponded to tumors of glial origin, either tissues or cell cultures, depending on the best “RNA integrity number.” We used R software to evaluate the genes which were differentially expressed (DE) in gliomas compared with normal brain. Applying a p-value below 0.01 and fold change ≥4, led to identification of 2284 DE genes. Through a Functional Annotation Analysis (FAA) using the NIH-DAVID software, the DE genes turned out to be associated mainly with: immune/inflammatory response, cell proliferation and survival, cell adhesion and motility, neuronal phenotype, and ion transport. We have shown that GEPs of pediatric brain tumors can be studied using either primary tumor samples or short-term cultures with similar results. From FAA, we concluded that, among DE genes, pediatric gliomas show a strong deregulation of genes related to ion channels and transporters.

Tumor hypoxia and genetic alterations in sporadic cancers

The cancer genome contains many gene alterations. How cancer cells acquire these alterations is a matter for discussion. One hypothesis is that cancer cells obtain mutations in genome stability genes at an early stage of tumor development, which results in genetic instability and generates a gene pool that enhances cellular proliferation and survival. Another hypothesis puts its emphasis on the natural selection of gene mutations for fitness. Recent data for systematic cancer genome sequencing shows that mutations in stability genes are rare in human sporadic cancers. Instead, many “passenger” mutations that do not drive the carcinogenesis process have been found in the cancer genome. Both the hypotheses mentioned above fall short in explaining recent data. Recently, many studies demonstrate the role of the tumor microenvironment, especially hypoxia and reoxygenation, in genetic instability. In this review, literature will be presented which supports a third hypothesis, i.e. that hypoxia/re-oxygenation induces genetic instability.

Integrative analysis of gene expression and copy number alterations using canonical correlation analysis

BackgroundWith the rapid development of new genetic measurement methods, several types of genetic alterations can be quantified in a high-throughput manner. While the initial focus has been on investigating each data set separately, there is an increasing interest in studying the correlation structure between two or more data sets. Multivariate methods based on Canonical Correlation Analysis (CCA) have been proposed for integrating paired genetic data sets. The high dimensionality of microarray data imposes computational difficulties, which have been addressed for instance by studying the covariance structure of the data, or by reducing the number of variables prior to applying the CCA. In this work, we propose a new method for analyzing high-dimensional paired genetic data sets, which mainly emphasizes the correlation structure and still permits efficient application to very large data sets. The method is implemented by translating a regularized CCA to its dual form, where the computational complexity depends mainly on the number of samples instead of the number of variables. The optimal regularization parameters are chosen by cross-validation. We apply the regularized dual CCA, as well as a classical CCA preceded by a dimension-reducing Principal Components Analysis (PCA), to a paired data set of gene expression changes and copy number alterations in leukemia.ResultsUsing the correlation-maximizing methods, regularized dual CCA and PCA+CCA, we show that without pre-selection of known disease-relevant genes, and without using information about clinical class membership, an exploratory analysis singles out two patient groups, corresponding to well-known leukemia subtypes. Furthermore, the variables showing the highest relevance to the extracted features agree with previous biological knowledge concerning copy number alterations and gene expression changes in these subtypes. Finally, the correlation-maximizing methods are shown to yield results which are more biologically interpretable than those resulting from a covariance-maximizing method, and provide different insight compared to when each variable set is studied separately using PCA.ConclusionsWe conclude that regularized dual CCA as well as PCA+CCA are useful methods for exploratory analysis of paired genetic data sets, and can be efficiently implemented also when the number of variables is very large.