Manifestation Of Genomic Instability Research Articles

Extrachromosomal DNA in the cancerous transformation of Barrett’s oesophagus

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1–6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett’s oesophagus. These data included 206 biopsies in Barrett’s oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case–control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett’s-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.

Abstract 5227: Cross-trait genome-wide association meta-analyses of clonal hematopoiesis and solid tumor risk

Abstract Clonal hematopoiesis (CH) is an age-related phenomenon where hematopoietic stem cells and their progeny acquire specific somatic mutations in a small, well-defined set of leukemogenic genes conferring them with fitness advantage leading to clonal proliferation. Mosaic chromosomal alterations (mCAs) are somatic structural variants that are manifestations of genomic instability and a sign of CH when they occur in blood. The recent availability of large-scale cohorts with blood exome sequence and genotype data has powered genome-wide association studies (GWAS) investigating the germline genetic contribution to CH and mCA risk. However, phenotypes of somatic mutation acquisition in driver genes, clonal selection and expansion, and chromosomal mosaicism are processes that are not confined to blood and contribute to neoplastic transformation across tissue types. GWAS of solid tumor susceptibility have so far identified hundreds of germline variants associated with cancer risk and it is likely that a subset of these variants contributes to solid tumor risk by affecting these somatic phenotypes in the corresponding tissues of origin and that some of the variants exert these somatic effects across tissues. We aimed to identify this subset of solid tumor risk variants by undertaking fixed effects inverse-variance weighted meta-analyses combining breast, prostate, ovarian, and endometrial cancer GWAS (total n = 237,483 cases/317,006 controls; no UK Biobank samples included) with UK Biobank-based CH (10,203 “cases”/173,918 controls) and mCA (66,011 “cases”/378,188 controls) risk GWAS in pairs where each pair involved one solid tumor type and one of either CH or mCA (for example, breast cancer and mCA constituted a pair). These analyses collectively identified 61 independent (r2<0.05) lead variants at combined cross-trait genome-wide significance (P<5 × 10−8) across 22 genomic loci where each lead variant was associated with each trait individually at P<10−3, had the same direction of allelic effect across traits, and little evidence of heterogeneity in effect (I2<40%). New loci (>1 Mb away from previously reported risk loci for a given trait) counts were: 14 new loci in the context of mCA risk, 6 new loci for CH risk, 2 for breast cancer risk, and one each for prostate, ovarian, and endometrial cancer risks. In silico functional annotation of these loci suggested lead variant-target genes that could be assigned to the discrete pathways of mitotic spindle assembly (INCENP, CENPN, ZWILCH, MAD1L1), p53 signaling (TP53, MDM4, SPI1), DNA damage repair (ATM, RAD51C, CHEK2, USP28, PARP8), and myeloid oncogenesis (WT1, HOXA9, L3MBTL3). Our novel approach combining the latest and largest blood somatic genomic trait GWAS with large-scale solid tumor risk GWAS thus yielded clear mechanistic insights into the shared inherited genetic basis of solid tumor risk and somatic genomic traits. Citation Format: Victoria Gray, George Richenberg, Pedro Quirós, Matthew Freedman, Paul Pharoah, Simon Gayther, Michelle Jones, George Vassiliou, Kate Lawrenson, Siddhartha Kar. Cross-trait genome-wide association meta-analyses of clonal hematopoiesis and solid tumor risk. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5227.

Detection of DNA Microsatellites Using Multiplex Polymerase Chain Reaction Aboard the International Space Station

Abstract As human exploration extends further into deep space, it is critical to understand the cellular impacts of spaceflight in order to ensure the safety of future astronauts. Extended exposure to cosmic radiation and microgravity has been shown to cause genetic damage and impair cellular DNA repair mechanisms, which together can lead to genomic instability. In particular, microsatellite instability (MSI), in which dysfunction in DNA mismatch repair (MMR) causes alterations in tandemly repeated “microsatellite” sequences, is a manifestation of genomic instability that has been associated with certain cancers. In this study, we establish the feasibility of an on-orbit multiplex polymerase chain reaction (PCR)-based assay to detect mutations in cancer-related microsatellites. Multiplex PCR was used to amplify five quasimonomorphic microsatellites in space and on Earth from both wild-type and MMR-deficient human cell lines. These data provide proof of concept of simultaneous amplification of multiple DNA sequences in space, expanding in-flight research and health-monitoring capabilities.

Delayed effects of accelerated heavy ions on the induction of HPRT mutations in V79 hamster cells

Fundamental research on the harmful effects of ionizing radiation on living cells continues to be of great interest. Recently, priority has been given to the study of high-charge and high-energy (HZE) ions that comprise a substantial part of the galactic cosmic ray (GCR) spectra that would be encountered during long-term space flights. Moreover, predictions of the delayed genetic effects of high linear energy transfer (LET) exposure is becoming more important as heavy ion therapy use is increasing. This work focuses mainly on the basic research on the delayed effects of HZE ions on V79 Chinese hamster cells, with emphasis on the induction of HPRT mutations after prolonged expression times (ET). The research was conducted under various irradiation conditions with accelerated ions 18O (E=35.2MeV/n), 20Ne (E=47.7MeV/n and 51.8MeV/n), and 11B (E=32.4MeV/n), with LET in the range from 49 to 149 keV/μm and with 60Co γ-rays. The HPRT mutant fractions (MF) were detected in irradiated cells in regular intervals during every cell culture recultivation (every 3days) up to approximately 40days (70–80 generations) after irradiation. The MF maximum was reached at different ET depending on ionizing radiation characteristics. The position of the maximum was shifting towards longer ET with increasing LET. We speculate that the delayed mutations are created de novo and that they are the manifestation of genomic instability. Although the exact mechanisms involved in genomic instability initiation are yet to be identified, we hypothesize that differences in induction of delayed mutations by radiations with various LET values are related to variations in energy deposition along the particle track. A dose dependence of mutation yield is discussed as well.

Massive Parallel Sequencing and Mutations Analysis of Mismatch Repair Genes in Recurrent Childhood Acute Lymphoblastic Leukemia Cases-Identification of Possible Molecular Signature of Poor Clinical Outcome

[Introduction] Mechanism of recurrence is fully characterized and is significant to improve prognosis in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and T lymphoblastic leukemia (T-ALL).. We have analyzed recurrent cases of childhood BCP-ALL and T-ALL. [Procedure] We analyzed clinical samples of 18 cases with BCP-ALL and 3 cases with T-ALL, who had been treated in our institution. We have investigated gene status of IKZF1, CRLF2, CDKN2A/2B, JAKs, IL7RA, and TP53 using RT-PCR and MLPA methods and IGH/TCR on genomic PCR. Whole exome analysis was done using Ion AmpliSeq Exome in seven cases. [Result] We analyzed clinical samples of 21 cases with ALL, who had been treated in our institution. The cases were 18 cases with BCP-ALL and 3 cases with T-ALL. We have investigated gene status of IKZF1, CRLF2, CDKN2A/2B,JAKs,IL7RA, NOTCH (for T-ALL), and TP53. Whole exome analysis was done in two cases, particularly for a case from which we have established cell line. < Result > Eight BCP-ALL cases with P2RY8-CRLF2 chimeric transcript: four cases of which had P2RY8-CRLF2 chimeric transcript only at initial diagnosis; two cases obtained transcript at recurrence. This was confirmed employing LD-PCR genomic analysis. Ten BCP-ALL cases had deletion of IKZF1; two showed deletion at relapse and a case demonstrated deletion at diagnosis only. Two BCP-ALL cases showed mutation of IL7RA. One of T-ALL cases from which we have established cell line showed mutation of MSH2 and more than 200 non-synonymous mutations on whole exome analysis. One T-ALL cases showed mutation of JAK3 at diagnosis. One BCP-ALL case had MLH1 mutation < Discussion > The present study has suggested acquisition of complex genetic change at different point of evolution may work in recurrence of ALL. The present study has indicated P2RY8-CRLF2 works not as simple growth advantage but rather as manifestation of genomic instability. This may be also illustrated by recurrent cases with T-ALL having mutation of MSH2 and BCP-ALL having mutation of MLH1.Mutation of mismatch repair gene may be driver of gene mutation acquisition and consequently alterations of CREBBP and RAS or IKZF1, TP53, mismatch repair genes, or emergence of P2RY-CRLF2 chimeric transcript may be prognostically relevant in childhood acute lymphoblastic leukemia. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetic effects and possibilities of their transgenerational transfer in the generations of persons living in radionuclide polluted areas after the Chernobyl accident

The specific features of a manifestation of genomic instability, as shown by the karyological indicators of the micronucleus test using buccal epithelial cells, were investigated. To assess an immune response to the mutagenic action of ionizing radiation, peripheral blood lymphocytes with the phenotypic characteristics of CD95+ subpopulations were quantified using flow-through cytofluorometry, by estimating the possible consequences of transferring the mutagenic effect of ionizing radiation to posterity. A total of 112 first- and second-generation patients residing in the areas polluted with radionuclide cesium-137 (more than 556 kBq/m2) were examined in the aspect of family. The data obtained in the investigation employing the models for individual families may anticipatorily consider certain cytogenetic indicators in second-generation children as predictors of carcinogenesis and the possibility of their transgenerational transfer in the generations of persons exposed to radiation.

Intraclonal recovery of ‘slow clones’—a manifestation of genomic instability: Are mitochondria the key to an explanation?

Intraclonal recovery following X-irradiation in an in vitro study of L5178Y-S murine leukaemic cells is reviewed. This phenomenon was first described in 1994 occurring in the slowly growing clones ('slow clones') present among the survivors in irradiated cell populations. An attempt to explain these experimental data is given in terms of the present knowledge of the role of mitochondria in nontargeted radiation effects, with the focus on genomic instability and mtDNA-related epigenetic modifications of the nuclear genome. An understanding of this intraclonal recovery may be important in preventing tumour regrowth following radiotherapy, as well as in decreasing the risk of secondary radiation-induced malignancies.

Emerging evidence for the role of genomic instability in male factor infertility

Male infertility is a common and complex pathology affecting0about 7% of men of reproductive age. Given its complexity, the underlying etiology for male infertility is often unknown. A growing amount of evidence suggests genomic instability may be an important factor in some cases of male factor infertility. While some specific manifestations of genomic instability, such as increased sperm aneuploidy rates and increased somatic translocations and inversions in infertile men, are well established, other facets of genomic instability associated with male infertility have not been thoroughly investigated. A limited body of recent work has identified a potential association between microsatellite instability and spermatogenic failure. In addition, mutations in mismatch repair and tumor suppressor genes, which could potentially lead to genomic instability, have been identified in some infertile men and animal models. In addition, results of two epidemiologic studies suggest spermatogenic defects might be just one aspect of a more systemic problem, possibly due to increased genomic instability. In this review we discuss well-established links between genomic instability and male infertility, as well as some of the emerging but less established data to support this relationship. We also propose some important areas of future research toward a more complete understanding of the underlying mechanisms for male infertility.

Genomic instability in the epidermis induced by atomic bomb (A‐bomb) radiation

Radiation etiology is suggested in the occurrence of basal cell carcinoma (BCC) of the skin among atomic bomb (A-bomb) survivors. Any genotoxicity, including ionizing radiation, can induce a DNA damage response (DDR), leading to genomic instability (GIN), which allows the accumulation of mutations during tumorigenesis. In this study, the authors evaluated the presence of GIN in the epidermis of survivors as a late effect of A-bomb radiation. In total, 146 BCCs, including 23 cases arising from nonexposed skin, were identified in survivors from 1968 to 1999. The incidence rate (IR) of BCC was calculated with stratification by distance in kilometers from the hypocenter (< or =1.5 km, 1.6-2.9 km, and > or =3 km). Nineteen epidermal samples surrounding BCC at the nonexposed sites were collected and tested for p53 binding protein 1 (53BP1) expression with immunofluorescence. 53BP1 rapidly forms nuclear foci at the sites of DNA double strand breaks (DSBs). Because 1 manifestation of GIN is the induction of endogenous DSBs, the level of 53BP1-focus formation (DDR type) can be considered as a marker for GIN. : The incidence rate of BCC increased significantly as exposure distance approached the hypocenter. Of the 7 epidermal samples from the proximal group (< or =1.5 km), 5 samples predominantly expressed DDR and an abnormal type of 53BP1 expression. In contrast, 4 of 5 samples from the distal group (> or =3 km) and all samples from the control group predominantly expressed the stable type of 53BP1 expression in the epidermis. : The current results demonstrated the endogenous activation of DDR in the epidermis surrounding BCC in the proximal group, suggesting the presence of a GIN in the survivors as a late effect of A-bomb radiation, which may indicate a predisposition to cancer.

An investigation of microRNAs mapping to breast cancer related genomic gain and loss regions

Various regions of amplification or loss are observed in breast tumors as a manifestation of genomic instability. To date, numerous oncogenes or tumor suppressors on some of these regions have been characterized. An increasing body of evidence suggests that such regions also harbor microRNA genes with crucial regulatory roles in cellular processes and disease mechanisms, including cancer. Here, we investigated 35 microRNAs localized to common genomic gain and/or loss regions in breast cancers. To examine amplification or loss of these microRNAs as a result of genomic instability, we performed semiquantitative duplex polymerase chain reaction in 20 breast cancer cell lines, 2 immortalized mammary cell lines, and 2 normal DNA controls. A comprehensive DNA fold number change data for 35 microRNA genes on chromosomal gain/loss regions are presented in breast cancer cells. A 23% (8/35) of the investigated microRNAs showed significant fold number increases (greater than fourfold) compared to GAPDH in one or more of the breast cell lines. Although no homozygous deletions were detected, fold number decreases indicating potential loss regions were observed for 26% (9/35) of the investigated microRNAs. Such fold number changes may point out some of these microRNAs as potential targets of the genomic instability regions as oncogene and tumor suppressor candidates.

Long-term and transgeneration effects after g-irradiation of BALB/c male-mice in doze range 1- 6 Gy

Study of the long-term and transgeneration effects after exposure of ionizing radiation, as manifestation of genomic instability, is very important for predication of emergency radiation situations’ consequences. We were investigated exposure of γ-radiation in doze range 1 – 6 Gy on example of amounts of the single-starnd DNA breaks (SSB) in the spleen lymphocytes (SL) of irradiated mice and changes in RAPD- and ISSR-patterns of their progeny. Male mice BALB/c strain were acute irradiated by γ-radiation at doses 1, 2, 3 and 6 Gy on gamma-unit GYPOS (dose rate- 4,5 Gy/min, source- Cs-137). For the study of long-term effects, mice were killed in an 11 months after irradiation. The SSB-level was evaluated using the alkaline comet-assay as described by Singh et al. [1998] with additional exposure of the hydrogen peroxide. The DNA comets analyzed with a fluorescence microscope AxioPhote by using the method of visual estimation [Collins et al., 1995]. For study of transgeneration effects after irradiation and comparison of sensitivity of different stages of spermatogenesis males were crossed with females the same strain in two weeks (post meiotic stage) and in three months (pre meiotic stage) after an irradiation. The offspring in both cases contained in standard conditions and were killed in the 3-4 weeks of age. DNA was isolated from liver by using DNA Prep TM (IZOGEN Lab). Amplification was performed by using lyophilizated PCR-mix DNA CoreTM (IZOGEN Lab) on thermocycler PT-48 (TDL Company). Products of amplification were separated in 1,5 % agaroze gel and visualized by ethydium bromide. For detection effects of irradiation was used RAPD- and ISSR-assays. Analysis of offspring patterns carried out on the basis of comparison with patterns, with the purpose of registration of new, not parental bands, as case of mutation. We are counting mutation frequency per animal in the group and processed these data statistically. Our results are show, that the SL of the animals irradiated dozes 1 – 3 Gy were more resistance to H 2 O 2 exposure, but, beginning at dose 1 Gy, mutation frequencies in irradiated offspring is significantly distinguished from the control group. At the same time, change of mutation frequencies is independent of increasing of the irradiation dose. The comparison of sensitivity on different stages of spermatogenesis are indicated, that post-irradiated changes in RAPD- and ISSR-patterns of pre and post meiotic cells have similar character

Long-term effects of irradiation in the progeny of mammalian cells

It is shown that gamma-irradiation has remote consequences for mammalian cells cultivated in vitro. Many generations in the progeny of cells surviving acute and chronic irradiation at high and low doses are characterized by a number of abnormalities, including delayed cell death, the formation of micronuclei and giant cells, an increased frequency of sister chromatid exchanges, a reduced potential for repair, the loss of adaptive response, and increased radiosensitivity. These phenomena are regarded as manifestations of genomic instability induced by ionizing radiation.

Remote Consequences of Irradiation for the Progeny of Mammalian Cells

It is shown that γ-irradiation has remote consequences for mammalian cells cultivated in vitro. Many generations in the progeny of cells surviving acute and chronic irradiation at high and low doses are characterized by a number of abnormalities, including delayed cell death, the formation of micronuclei and giant cells, an increased frequency of sister chromatid exchanges, a reduced potential for repair, the loss of adaptive response, and increased radiosensitivity. These phenomena are regarded as manifestations of genomic instability induced by ionizing radiation.

Absence of the microsatellite mutator phenotype in human bronchial epithelial cells transformed by alpha particles

The immortalized human bronchial epithelial cell line, BEP2D, can become malignantly transformed following its irradiation with alpha particles. Exposed cells progress through a latent period of eight to ten weeks prior to exhibiting malignant properties. The molecular basis for this delay is unclear, however it is thought to involve a manifestation of genomic instability brought on by ionizing radiation. In this study we addressed whether the microsatellite mutator phenotype which arises in cells that lack mismatch repair function could have played a role in the radiation-induced transformation of BEP2D cells. Three cell lines, including BEP2D and two transformed clonal derivatives, H2BT2L and R30T1L, were examined for the presence of the microsatellite mutator phenotype using a selectable reporter system developed in our laboratory. This reporter system is based on the ability of stably transduced cells to restore the reading frame of a hygromycin B phosphotransferase transferase gene rendered out of frame by the insertion of a (CA)(13) repeat tract immediately downstream of the ATG start codon. The parental BEP2D cell line had a relatively low hygromycin B resistant colony formation frequency of 2.36 x 10(-4). This value was similar to the frequencies of two malignantly transformed derivatives H2BT2L (0.996 x 10(-4)) and R30T1L (1.87 x 10(-4)). We therefore conclude that the H2BT2L and R30T1L cell lines are not deficient in their hMSH2, hMLH1, hPMS2 or hMSH3 mismatch repair gene functions, and that other factors orchestrated the alpha particle induced malignant transformation of H2BT2L and R30T1L cells.