Analysis of Genomic Instability and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR

The paradigm of genetic alterations being restricted to direct DNA damage after exposure to ionizing radiation has been challenged by observations in which effects of ionizing radiation arise in cells that in themselves receive no radiation exposure. These effects are demonstrated in cells that are the descendants of irradiated cells (radiation-induced genomic instability) or in cells that are in contact with irradiated cells or receive certain signals from irradiated cells (radiation-induced bystander effects). Bystander signals may be transmitted either by direct intercellular communication through gap junctions, or by diffusible factors, such as cytokines released from irradiated cells. In both phenomena, the untargeted effects of ionizing radiation appear to be associated with free radical-mediated processes. There is evidence that radiation-induced genomic instability may be a consequence of, and in some cell systems may also produce, bystander interactions involving intercellular signalling, production of cytokines and free radical generation. These processes are also features of inflammatory responses that are known to have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. Thus, radiation-induced genomic instability and untargeted bystander effects may reflect interrelated aspects of inflammatory type responses to radiation-induced stress and injury and contribute to the variety of the pathological consequences of radiation exposures.

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Genomic instability of microsatellite repeats and its association with the evolution of chronic myelogenous leukemia [see comments]

Blood ◽

10.1182/blood.v83.12.3449.3449 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3449-3456 ◽

Cited By ~ 90

Author(s):

C Wada ◽

S Shionoya ◽

Y Fujino ◽

H Tokuhiro ◽

T Akahoshi ◽

...

Keyword(s):

Genomic Instability ◽

Chronic Myelogenous Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

Genetic Alterations ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

Genetic Event ◽

Familial Predisposition ◽

Somatic Instability

Abstract Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor-suppressor genes. Investigation of genomic instability of microsatellites has indicated a new mechanism for human carcinogenesis in hereditary nonpolyposis colorectal cancer and sporadic cancer and this instability has been shown to be related to inherited predisposition to cancer. This study was conducted to determine whether such microsatellite instability is associated with the evolution of chronic myelogenous leukemia (CML) to the blast crisis. Nineteen CML patients clinically progressing from the chronic phase to accelerated phase or blast crisis and 20 other patients in the CML chronic phase were studied. By polymerase chain reaction assay, DNAs for genomic instability in five separate microsatellites in chromosome arms 5q (Mfd27), 17p (Mfd41), 18q (DCC), 3p (CI3–9), and 8p (LPL) were examined. Differences in unrelated microsatellites of chronic and blastic phase DNAs in 14 of 19 patients (73.7%) were demonstrated. Somatic instability in five microsatellites, Mfd27, Mfd41, DCC, CI3–9, and LPL, was detected in 2 of 19 (10.5%), 8 of 19 (42.1%), 11 of 19 (57.9%), 4 of 17 (23.5%), and 4 of 17 (23.5%) cases. In 10 of 19 cases (52.6%), genetic instability in at least two of five microsatellites was observed and was categorized as replication error (RER+) phenotype. CML evolution cases with myeloid, lymphoid, and mixed phenotypes and the blast crisis and accelerated phase showed somatic instability in a number of microsatellites. No alterations in leukemic cells at the chronic phase could be detected in any microsatellites. These data indicate instability of microsatellites (RER+) but not familial predisposition to possibly be a late genetic event in the evolution of CML to blast crisis. In the microsatellite of the DCC gene, complicated alterations in band patterns caused by instability as well as loss of heterozygosity (LOH) were observed in 13 of 19 cases (68.4%): instability in 9 cases, instability plus LOH in 2 cases, and only LOH in 2 cases. These highly frequent alterations in microsatellites, including instability and LOH, suggesting that secondary events due possibly to loss of fidelity in replication and repair machinery may be significantly associated with CML evolution.

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Breast Cancer: A Molecularly Heterogenous Disease Needing Subtype-Specific Treatments

Medical Sciences ◽

10.3390/medsci8010018 ◽

2020 ◽

Vol 8 (1) ◽

pp. 18 ◽

Cited By ~ 2

Author(s):

Ugo Testa ◽

Germana Castelli ◽

Elvira Pelosi

Keyword(s):

Breast Cancer ◽

Genomic Instability ◽

Hormone Receptors ◽

Early Stage ◽

Brca Mutation ◽

Clonal Evolution ◽

Metastatic Breast ◽

Genetic Alterations ◽

Telomere Maintenance ◽

Molecular Features

Breast cancer is the most commonly occurring cancer in women. There were over two-million new cases in world in 2018. It is the second leading cause of death from cancer in western countries. At the molecular level, breast cancer is a heterogeneous disease, which is characterized by high genomic instability evidenced by somatic gene mutations, copy number alterations, and chromosome structural rearrangements. The genomic instability is caused by defects in DNA damage repair, transcription, DNA replication, telomere maintenance and mitotic chromosome segregation. According to molecular features, breast cancers are subdivided in subtypes, according to activation of hormone receptors (estrogen receptor and progesterone receptor), of human epidermal growth factors receptor 2 (HER2), and or BRCA mutations. In-depth analyses of the molecular features of primary and metastatic breast cancer have shown the great heterogeneity of genetic alterations and their clonal evolution during disease development. These studies have contributed to identify a repertoire of numerous disease-causing genes that are altered through different mutational processes. While early-stage breast cancer is a curable disease in about 70% of patients, advanced breast cancer is largely incurable. However, molecular studies have contributed to develop new therapeutic approaches targeting HER2, CDK4/6, PI3K, or involving poly(ADP-ribose) polymerase inhibitors for BRCA mutation carriers and immunotherapy.

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Protein Oxidative Damage in UV-Related Skin Cancer and Dysplastic Lesions Contributes to Neoplastic Promotion and Progression

Cancers ◽

10.3390/cancers12010110 ◽

2020 ◽

Vol 12 (1) ◽

pp. 110

Author(s):

Antonella Tramutola ◽

Susanna Falcucci ◽

Umberto Brocco ◽

Francesca Triani ◽

Chiara Lanzillotta ◽

...

Keyword(s):

Dna Damage ◽

Stress Response ◽

Oxidative Damage ◽

Genomic Instability ◽

Structural Damage ◽

Protein Quality ◽

Genetic Alterations ◽

Skin Carcinogenesis ◽

Neoplastic Progression ◽

Damage Repair

The ultraviolet (UV) component of solar radiation is the major driving force of skin carcinogenesis. Most of studies on UV carcinogenesis actually focus on DNA damage while their proteome-damaging ability and its contribution to skin carcinogenesis have remained largely underexplored. A redox proteomic analysis of oxidized proteins in solar-induced neoplastic skin lesion and perilesional areas has been conducted showing that the protein oxidative burden mostly concerns a selected number of proteins participating to a defined set of functions, namely: chaperoning and stress response; protein folding/refolding and protein quality control; proteasomal function; DNA damage repair; protein- and vesicle-trafficking; cell architecture, adhesion/extra-cellular matrix (ECM) interaction; proliferation/oncosuppression; apoptosis/survival, all of them ultimately concurring either to structural damage repair or to damage detoxication and stress response. In peri-neoplastic areas the oxidative alterations are conducive to the persistence of genetic alterations, dysfunctional apoptosis surveillance, and a disrupted extracellular environment, thus creating the condition for transformant clones to establish, expand and progress. A comparatively lower burden of oxidative damage is observed in neoplastic areas. Such a finding can reflect an adaptive selection of best fitting clones to the sharply pro-oxidant neoplastic environment. In this context the DNA damage response appears severely perturbed, thus sustaining an increased genomic instability and an accelerated rate of neoplastic evolution. In conclusion UV radiation, in addition to being a cancer-initiating agent, can act, through protein oxidation, as a cancer-promoting agent and as an inducer of genomic instability concurring with the neoplastic progression of established lesions.

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Determination of genomic damage in neuroblastic tumors by arbitrarily primed PCR: MYCN amplification as a marker for genomic instability in neuroblastomas

Neuropathology ◽

10.1111/j.1440-1789.2006.00675.x ◽

2006 ◽

Vol 26 (3) ◽

pp. 165-169 ◽

Cited By ~ 5

Author(s):

Jorge Munoz ◽

Elisenda Vendrell ◽

Gemma Aiza ◽

Manuel Nistal ◽

Angel Pestana ◽

...

Keyword(s):

Genomic Instability ◽

Mycn Amplification ◽

Neuroblastic Tumors ◽

Genomic Damage ◽

Arbitrarily Primed Pcr

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CLL Progression Is Associated with Increased Clonal Diversity and Replication Stress

Blood ◽

10.1182/blood.v124.21.1977.1977 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1977-1977

Author(s):

Angelo Agathanggelou ◽

Anna Skowronska ◽

Nick Davies ◽

Marwan Kwok ◽

Samuel Clockie ◽

...

Keyword(s):

Dna Damage ◽

Disease Progression ◽

Genomic Instability ◽

Conflicts Of Interest ◽

Clonal Diversity ◽

Replication Stress ◽

Tp53 Gene ◽

Genetic Alterations ◽

Paired Samples ◽

G1 Cells

Abstract There is increasing evidence to suggest that genetic lesions accumulate during CLL progression, often involving mutations in DNA damage response genes ATM and TP53, or SF3B1. However, little is known about the mechanisms which contribute towards genomic instability and clonal diversification during CLL progression. Genomic instability is a major cause of subclonal diversification and can be driven by replication stress (RS). It has been shown that in solid tumours RS drives tumourigenesis through the following steps: presence of unreplicated DNA, accumulation of DNA damage, activation and functional loss of ATM/p53 and genomic instability. In this study we addressed whether high proliferation rates during CLL progression are associated with increased subclonal diversification and with RS. We compared samples from the indolent and progressive stages of CLL, both in individual patients and in patient cohorts. We determined clonal diversification by two complementary methods: multiplexed-FISH and next generation sequencing (NGS). We measured RS by an assay that recognizes a region of unreplicated DNA. Using multiplexed-FISH that identifies the location of multiple genetic alterations at the single cell level, we identified in 3/11 relapsed samples the occurrence of novel subclones with ATM (11q) or TP53 (17p) deletions that were not observed in the pre-treatment tumours. These findings were corroborated by targeted NGS of recurrently mutated genes in CLL which revealed novel subclones carrying ATM, TP53, NOTCH1, SF3B1 or BIRC3 mutations at the time of relapse in 9 out of 22 paired samples. Of particular interest was the acquisition of multiple mutations in the TP53 gene observed in four patients. These data demonstrate an intrinsic propensity for clonal diversification during CLL progression. Having demonstrated an association between disease progression and subclonal diversification in CLL, we next sought to identify a mechanism behind this phenomenon. RS results in under-replicated DNA which is sequestered into nuclear compartments marked by 53BP1 protein. Therefore, the appearance of 53BP1 bodies in G1-cells represents a marker of RS. We observed that 53BP1 bodies were not present in PBMCs from healthy individuals, and that 20 CLL samples from an indolent stage of disease contained a low level of this RS marker. This was in stark contrast to the elevated proportion of CLL cells with 53BP1 bodies observed in 13 progressive tumours (p<0.0001). Finally to confirm that replication stress increases during disease progression we analysed a cohort of individual patients at different stages during the evolution of their disease. Intriguingly, and consistent with our hypothesis, we found that the proportion of 53BP1 body positive cells was stable during the indolent phase of disease and only increased significantly when the tumour made the transition from indolent to the progressive stage (p=0.0009). Collectively, our data indicate that replication stress correlates with the stage of CLL progression and may provide a mechanism behind the observed subclonal diversification. Disclosures No relevant conflicts of interest to declare.

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Chromosomal Instability in Cultured Mouse Hematopoietic Cells Associated with Oxidative Stress

Blood ◽

10.1182/blood.v118.21.4819.4819 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4819-4819

Author(s):

Alice M. Liu ◽

William W. Qu ◽

Xia Liu

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Genomic Instability ◽

Chromosomal Instability ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Genetic Alterations ◽

In Vitro Test ◽

Hematopoietic Stem

Abstract Abstract 4819 Hematopoietic stem cells (HSCs) that give rise to all blood cell types are important vehicles for cell-based and gene therapies. After isolation from the bone marrow, HSCs are often cultured in laboratory settings for purposes of ex vivo expansion, gene transduction, and bone marrow transplantation for the treatment of various disorders of the blood and immune systems. While undergoing proliferation and differentiation in vitro, test tube and dish culturing can potentially induce genomic instability in HSCs due to prolonged culturing periods or the exposure to increased levels of oxygen. Here we demonstrate that in vitro culturing outside their bone marrow niches, HSCs may change even under very short durations of time. Lineage− Scal-1+ c-Kit+ (LSK) cells that are enriched with HSCs revealed significant levels of genomic instability in culture, as evidenced by the emergence of aneuploidy cells. To further determine the effects of in vitro culturing conditions, whole bone marrow cells were cultured in a hypoxic environment of 2–3% oxygen, mimicking conditions inside the body's bone marrow. In this case, cells proved to undergo less genetic alterations. Proper dosages of the antioxidant N-Acetyl-Cysteine (NAC) similarly decreased occurrences of chromosomal changes. Furthermore, in vitro normoxic culture-induced chromosomal instability was enhanced in aged hematopoietic cells compared to that in young hematopoietic cells due to noted increased oxidative stress in aged cells. These results reveal that in vitro cell culturing does indeed cause genomic instability in hematopoietic cells. Reduced oxygen levels and additions of antioxidants can be employed as a possible agent to lower oxidative stress and decrease chances of transformation. Additionally, since hematopoietic cells are commonly developed in laboratory settings before transplantation for patient treatment, our findings raise a concern for using cultured hematopoietic cells for therapeutic purposes. Note: Alice Liu and William Qu contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

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Genomic Instability Profiles at the Single Cell Level in Mouse Colorectal Cancers of Defined Genotypes

Cancers ◽

10.3390/cancers13061267 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1267

Author(s):

Vasilis S. Dionellis ◽

Maxim Norkin ◽

Angeliki Karamichali ◽

Giacomo G. Rossetti ◽

Joerg Huelsken ◽

...

Keyword(s):

Genomic Instability ◽

Large Scale ◽

Molecular Mechanisms ◽

Single Cells ◽

Genetic Alterations ◽

Nucleotide Substitutions ◽

Driver Genes ◽

Normal Cells ◽

Cancer Driver ◽

Intratumoural Heterogeneity

The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apclox/lox, LSL-KrasG12D, and Tp53lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.

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Genomic instability of microsatellite repeats and its association with the evolution of chronic myelogenous leukemia [see comments]

Blood ◽

10.1182/blood.v83.12.3449.bloodjournal83123449 ◽

1994 ◽

Vol 83 (12) ◽

pp. 3449-3456 ◽

Cited By ~ 5

Author(s):

C Wada ◽

S Shionoya ◽

Y Fujino ◽

H Tokuhiro ◽

T Akahoshi ◽

...

Keyword(s):

Genomic Instability ◽

Chronic Myelogenous Leukemia ◽

Blast Crisis ◽

Chronic Phase ◽

Genetic Alterations ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

Genetic Event ◽

Familial Predisposition ◽

Somatic Instability

Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor-suppressor genes. Investigation of genomic instability of microsatellites has indicated a new mechanism for human carcinogenesis in hereditary nonpolyposis colorectal cancer and sporadic cancer and this instability has been shown to be related to inherited predisposition to cancer. This study was conducted to determine whether such microsatellite instability is associated with the evolution of chronic myelogenous leukemia (CML) to the blast crisis. Nineteen CML patients clinically progressing from the chronic phase to accelerated phase or blast crisis and 20 other patients in the CML chronic phase were studied. By polymerase chain reaction assay, DNAs for genomic instability in five separate microsatellites in chromosome arms 5q (Mfd27), 17p (Mfd41), 18q (DCC), 3p (CI3–9), and 8p (LPL) were examined. Differences in unrelated microsatellites of chronic and blastic phase DNAs in 14 of 19 patients (73.7%) were demonstrated. Somatic instability in five microsatellites, Mfd27, Mfd41, DCC, CI3–9, and LPL, was detected in 2 of 19 (10.5%), 8 of 19 (42.1%), 11 of 19 (57.9%), 4 of 17 (23.5%), and 4 of 17 (23.5%) cases. In 10 of 19 cases (52.6%), genetic instability in at least two of five microsatellites was observed and was categorized as replication error (RER+) phenotype. CML evolution cases with myeloid, lymphoid, and mixed phenotypes and the blast crisis and accelerated phase showed somatic instability in a number of microsatellites. No alterations in leukemic cells at the chronic phase could be detected in any microsatellites. These data indicate instability of microsatellites (RER+) but not familial predisposition to possibly be a late genetic event in the evolution of CML to blast crisis. In the microsatellite of the DCC gene, complicated alterations in band patterns caused by instability as well as loss of heterozygosity (LOH) were observed in 13 of 19 cases (68.4%): instability in 9 cases, instability plus LOH in 2 cases, and only LOH in 2 cases. These highly frequent alterations in microsatellites, including instability and LOH, suggesting that secondary events due possibly to loss of fidelity in replication and repair machinery may be significantly associated with CML evolution.

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The Genetic Landscape of Human Glioblastoma and Matched Primary Cancer Stem Cells Reveals Intratumour Similarity and Intertumour Heterogeneity

Stem Cells International ◽

10.1155/2019/2617030 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 6

Author(s):

Chiara Pesenti ◽

Stefania Elena Navone ◽

Laura Guarnaccia ◽

Andrea Terrasi ◽

Jole Costanza ◽

...

Keyword(s):

Stem Cells ◽

Genomic Instability ◽

T Cell Activation ◽

Cell Activation ◽

Pearson Correlation ◽

Genetic Alterations ◽

Rapid Progression ◽

Genomic Aberration ◽

Genetic Characteristics

Glioblastoma (GBM) is the most malignant human brain tumour, characterized by rapid progression, invasion, intense angiogenesis, high genomic instability, and resistance to therapies. Despite countless experimental researches for new therapeutic strategies and promising clinical trials, the prognosis remains extremely poor, with a mean survival of less than 14 months. GBM aggressive behaviour is due to a subpopulation of tumourigenic stem-like cells, GBM stem cells (GSCs), which hierarchically drive onset, proliferation, and tumour recurrence. The morbidity and mortality of this disease strongly encourage exploring genetic characteristics of GSCs. Here, using array-CGH platform, we investigated genetic and genomic aberration profiles of GBM parent tumour (n=10) and their primarily derived GSCs. Statistical analysis was performed by using R software and complex heatmap and corrplot packages. Pearson correlation and K-means algorithm were exploited to compare genetic alterations and to group similar genetic profiles in matched pairs of GBM and derived GSCs. We identified, in both GBM and matched GSCs, recurrent copy number alterations, as chromosome 7 polysomy, chromosome 10 monosomy, and chromosome 9p21deletions, which are typical features of primary GBM, essential for gliomagenesis. These observations suggest a condition of strong genomic instability both in GBM as GSCs. Our findings showed the robust similarity between GBM mass and GSCs (Pearson corr.≥0.65) but also highlighted a marked variability among different patients. Indeed, the heatmap reporting Gain/Loss State for 21022 coding/noncoding genes demonstrated high interpatient divergence. Furthermore, K-means algorithm identified an impairment of pathways related to the development and progression of cancer, such as angiogenesis, as well as pathways related to the immune system regulation, such as T cell activation. Our data confirmed the preservation of the genomic landscape from tumour tissue to GSCs, supporting the relevance of this cellular model to test in vitro new target therapies for GBM.

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