Biomarkers Of Genomic Instability Research Articles

Abstract 3430: Mosaic loss of the Y chromosome is associated with prostate cancer risk

Abstract Mosaic loss of the Y chromosome (mLOY), a type of clonal hematopoiesis, is the most frequent chromosomal alteration observed in leukocytes of aging men. mLOY is a putative biomarker of genomic instability with evidence of associations with risk of some solid tumors, but more studies are needed to refine associations and identify potential mechanisms for cancer risk. Prostate cancer (PCa), the most common non-cutaneous cancer in males, is also associated with aging. To further investigate potential relationships between mLOY and PCa, we examined DNA derived from leukocytes of male participants in two large biobanks: the UK Biobank (UKBB; N = 210,103) and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO, N = 26,795). Of the 236,898 male participants without previous cancer diagnosis, 14,253 (6.0%) were diagnosed with incident PCa after DNA collection. High-density genotyping array data collected during the studies were used to create virtual karyotypes for detecting mLOY in whole blood samples utilizing a phase-based detection method in MoChA software. Analyses were restricted to men without cancer at genotyping and multivariable regression models were adjusted for age, smoking status, and genetic similarity. Men with detectable mLOY were older (mean = 62 vs 56 years) and more likely to be smokers (14.8% vs 9.7%) than men without mLOY. A total of 3,848 (27.0%) men with PCa and 42,835 (19.2%) men free of PCa had detectable mLOY. mLOY clonal fraction ranged from 0.01% to 72.1% of total leukocytes. 18.7% of PCa cases and 13.6% of men free of PCa had high clonal fraction mLOY, defined as greater than 10% of leukocytes with mLOY. Fixed effect meta-analysis of multivariable models from UKBB and PLCO produced evidence for a positive association between mLOY in leukocytes and incident PCa; (Odds Ratio (OR) = 1.062, 95% Confidence Interval (CI) [1.020, 1.107], P-Value (p) = 0.004). The effect estimate was the same in men with low clonal fraction of mLOY (OR = 1.062, 95% CI [1.012, 1.113], p = 0.014) and men with high clonal fraction of mLOY (OR = 1.065, 95% CI [0.998, 1.137], p = 0.057). Age at PCa diagnosis was not associated with mLOY or the clonal fraction of mLOY. A sub-analysis of separate participants from PLCO with mLOY detected in buccal cells (n = 18,011) also showed a small positive effect between mLOY and PCa risk, but the relationship did not reach statistical significance (OR = 1.036, 95% CI [0.877, 1.224], p = 0.673)). Our investigation of the relationship between mLOY and PCa in two large prospective studies provides additional evidence for an association between mLOY detected in the blood and increased PCa risk, meriting additional studies of potential biologic mechanisms underlying the relationship. Citation Format: Rebecca L. Kelly, Weiyin Zhou, Kara M. Barnao, Corey D. Young, Aubrey K. Hubbard, Sairah M. Khan, Wen-Yi Huang, Stephen J. Chanock, Mitchell J. Machiela. Mosaic loss of the Y chromosome is associated with prostate cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3430.

Abstract IA020: Micronuclei are a nexus of DNA damage signaling and genomic instability

Abstract Micronuclei have classically been considered inert biomarkers of genomic instability arising from failed DNA damage repair and chromosome segregation errors in mitosis. Work from several groups including our own have now demonstrated that micronuclei are active contributors to the cellular response to DNA damage by driving chromothripsis and through induction of anti-viral gene expression programs mediated by the cGAS-STING pathway. We now show that the specific DNA damage type leading to micronuclei formation influences the capacity for cGAS localization and activation. This arises in part due to discrete chromatin features, present prior to DNA damage induction, that are retained in micronuclei several days after their formation. Together these data demonstrate that micronuclei connect the pre-damage chromatin status and the chromatin context of micronuclei-inciting lesions to the long-term signaling outcomes important for anti-tumor immunity. This finding implies that micronuclei represent archives for the nature of DNA repair failure and downstream signaling consequences. Based on this premise we will present our efforts in micronuclear proteomics where we uncover common features of micronuclei including splicing deficiencies. Such deficiencies presage exacerbated MN-associated DNA damage that is upstream of chromothripsis. These studies have direct implications for how cells sense the aftermath of DNA damage and how micronuclei contribute to ongoing genomic instability. Citation Format: Shane M. Harding. Micronuclei are a nexus of DNA damage signaling and genomic instability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA020.

Effects of dietary restriction on genome stability are sex and feeding regimen dependent.

Preserving genome stability is essential to prevent aging and cancer. Dietary restriction (DR) is the most reproducible non-pharmacological way to improve health and extend lifespan in various species. Whether DR helps to preserve genome stability and whether this effect is altered by experimental variables remain unclear. Moreover, DR research relies heavily on experimental animals, making the development of reliable in vitro mimetics of great interest. Therefore, we tested the effects of sex and feeding regimen (time-restricted eating, alternate day fasting and calorie restriction) on genome stability in CF-1 mice and whether these effects can be recapitulated by cell culture paradigms. Here, we show that calorie restriction significantly decreases the spontaneous micronuclei (MN), a biomarker of genome instability, in bone marrow cells of females instead of males. Alternate day fasting significantly decreases cisplatin-induced MN in females instead of males. Unexpectedly, daily time-restricted eating significantly exacerbates cisplatin-induced MN in males but not in females. Additionally, we design several culture paradigms that are able to faithfully recapitulate the key effects of these DR regimens on genome stability. In particular, 30% reduction of serum, a mimetic of calorie restriction, exhibits a strong ability to decrease spontaneous and cisplatin-induced MN in immortalized human umbilical vein endothelial cells. We conclude that the effects of different DR regimens on genome stability are not universal and females from each diet regimen sustain a more stable genome than males. Our results provide novel insight into the understanding of how DR influences genome stability in a sex and regimen dependent way, and suggest that our in vitro DR mimetics could be adopted to study the underlying molecular mechanisms.

Use of liquid-based cytology samples reveals genomic instability and cell death in patients undergoing orthodontic treatment.

To examine the use of liquid-based exfoliative cytology to determine the presence of genomic instability and cell death in the oral mucosa of patients with orthodontic appliances. Fifty-four oral mucosa samples were collected from 18 patients and divided into three stages: T0, before fixation of orthodontic appliances; T1, 25 days after appliance fixation; T2, 90 days after appliance fixation. All samples were Papanicolaou-stained and observed by microscopy (1,000 cells/sample) to ascertain the frequency of micronucleated cells (MN) and nuclear abnormalities (nuclear buds (NB), binucleated (BN), condensed chromatin (CC), karyorrhexis (KR), and karyolysis (KL)). Differences were analyzed statistically using the Mann-Whitney, Wilcoxon, Kruskal-Wallis and chi-squared tests. After placement of orthodontic appliances, significant differences were observed for genomic instability biomarkers (MN and NB) and cell death (CC, KR and KL) (P < 0.05). Female patients and older patients exhibited a higher frequency of MN. Liquid-based cytology has revealed that orthodontic appliances induce genomic instability and cell death in epithelial tissue of the oral mucosa, facilitating sample preservation and yielding more than one preparation per sample. Future studies should investigate whether such cell damage can be reversed through cell repair or whether cell alterations evolve and lead to disease.

Cytogenetics in Fanconi Anemia: The Importance of Follow-Up and the Search for New Biomarkers of Genomic Instability.

Fanconi Anemia (FA) is a disease characterized by genomic instability, increased sensitivity to DNA cross-linking agents, and the presence of clonal chromosomal abnormalities. This genomic instability can compromise the bone marrow (BM) and confer a high cancer risk to the patients, particularly in the development of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). The diagnosis of FA patients is complex and cannot be based only on clinical features at presentation. The gold standard diagnostic assay for these patients is cytogenetic analysis, revealing chromosomal breaks induced by DNA cross-linking agents. Clonal chromosome abnormalities, such as the ones involving chromosomes 1q, 3q, and 7, are also common features in FA patients and are associated with progressive BM failure and/or a pre-leukemia condition. In this review, we discuss the cytogenetic methods and their application in diagnosis, stratification of the patients into distinct prognostic groups, and the clinical follow-up of FA patients. These methods have been invaluable for the understanding of FA pathogenesis and identifying novel disease biomarkers. Additional evidence is required to determine the association of these biomarkers with prognosis and cancer risk, and their potential as druggable targets for FA therapy.

Somatic loss of the Y chromosome is associated with arsenic exposure among Bangladeshi men.

Arsenic exposure increases the risk of several cancers in humans and contributes to genomic instability. Somatic loss of the Y chromosome (LoY) is a potential biomarker of genomic instability and cancer risk. Smoking is associated with LoY, but few other carcinogens have been investigated. We tested the cross-sectional association between arsenic exposure and LoY in leukocytes among genotyped Bangladeshi men (age 20-70 years) from the Health Effects of Arsenic Longitudinal Study. We extracted the median of logR-ratios from probes on the Y chromosome (mLRR-chrY) from genotyping arrays (n = 1364) and estimated the percentage of cells with LoY (% LoY) from mLRR-chrY. We evaluated the association between arsenic exposure (measured in drinking water and urine) and LoY using multivariable linear and logistic regression models. The association between LoY and incident arsenic-induced skin lesions was also examined. Ten percent of genotyped men had LoY in at least 5% of cells and % LoY increased with age. Among men randomly selected for genotyping (n = 778), higher arsenic in drinking water, arsenic consumed and urinary arsenic were associated with increased % LoY (P = 0.006, P = 0.06 and P = 0.13, respectively). LoY was associated with increased risk of incident skin lesions (P = 0.008). Arsenic exposure was associated with increased LoY, providing additional evidence that arsenic contributes to genomic instability. LoY was associated with developing skin lesions, a risk factor for cancer, suggesting that LoY may be a biomarker of susceptibility in arsenic-exposed populations. The effect of arsenic on somatic events should be further explored in cancer-prone tissue types.

Abstract 5689: Effects of homologous recombination-related gene mutation subtypes on gene instability and the efficacy of platinum-containing regiments in triple-negative breast cancer

Abstract Background: Platinum-containing chemotherapy is treatment with high efficacy in homologous recombination (HR) deficient cancers that arise in carriers of mutations in the BRCA1/2 genes. However, previous reports have shown that TNBC patients carrying no BRCA1/2 mutations also could benefit from platinum therapy. This study aimed to explore the association between HR-related gene mutations and genomic instability, and further evaluate the effectiveness of platinum-containing chemotherapy preliminarily. Methods: A total of 386 TNBC patients were screened from a surgical cohort (NCT01150513) and a metastatic cohort. Finally, 189 TNBC patients with clinical and tumor sequencing data availability were included, including 149 patients treated with radical surgery and adjuvant chemotherapy. All tumor samples were collected during the surgical operation and subjected to NGS for evaluating homologous recombination deficiency (HRD) score and 15 HR-related gene pathogenic or likely pathogenic mutations (including ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51D, RAD51C, RAD54L). HRD score was an algorithmic assessment of three biomarkers of genomic instability as loss of heterozygosity, telomeric-allelic imbalance, large-scale state transitions and based on over 10,000 single-nucleotide polymorphisms in the human genome. Results: In 189 TNBC cohort, 40 patients (21.1%) had BRCA1/2 deleterious mutations, with additional 26 patients (13.8%) carrying mutations in HR-related genes other than BRCA1/2. Compared with BRCA1/2-intact patients, those carrying BRCA1/2m displayed a significantly higher HRD score (median, 35.5 vs 20.0; P=0.02). However, patients with HRm obtained a similar HRD score with HRwt patients (median, 28.0 vs 21.0, P=0.83). TNBC patients with PALB2m (n=8) had a small trend higher HRD score compared with BRCA1/2m (median HRD score 39.0 and 35.5), and TNBC patients with RAD51 family (n=7), ATM (n=5) and other HR-related gene mutations had a numerical trend lower HRD score compared with BRCA1/2m (median HRD score 22.0, 4.0, 14.5 and 35.5). Of the 149 patients in surgical cohort, 74 received platinum-containing, and 75 underwent platinum-free adjuvant chemotherapy. We analyzed the associations between HR-related gene mutations and disease-free survival (DFS), and found that patients with RAD51Bm (n=3), RAD51Cm (n=1) or PPP2R2Am (n=1) displayed worse DFS for patients treated with adjuvant chemotherapy (P =0.012, P&amp;lt;0.0001 or P&amp;lt;0.0001, respectively). Conclusions: In this study, we found that different HR-related genes mutation subtypes may exert distinct effects on genomic instability, and mutations in HR-related genes beyond the context of BRCA1/2 may serve as a biomarker for platinum-based adjuvant therapy for TNBC patients, which warrants further studies. Citation Format: Xue Wang, Yimeng Chen, Feng Du, Jian Yue, Yiran Si, Xiaochen Zhao, Lina Cui, Bei Zhang, Ting Bei, Peng Yuan, Binghe Xu. Effects of homologous recombination-related gene mutation subtypes on gene instability and the efficacy of platinum-containing regiments in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5689.

Efficacy of platinum-based chemotherapy in metastatic breast cancer and HRD biomarkers: utility of exome sequencing

Metastatic breast cancer (MBC) is frequently managed by platinum-based chemotherapy during the disease course. The real benefit of these treatments is uncertain at advanced stages of the disease and in non-triple-negative subtypes. Since homologous recombination deficiency (HRD) could inform about tumor sensitivity to DNA-damaging agents, we aimed to determine biomarkers of genomic instability, and their link with platinum efficacy. In this single-center study, we report BRCA1/2 mutational status, HRD score and signature 3 levels, all obtained by tumor exome sequencing, in 86 patients with various subtypes of MBC and who received platinum-based chemotherapy. Overall response rate, disease control rate, PFS and PFS2/PFS1 ratio were evaluated to assess platinum-based chemotherapy efficacy. Among the 86 tumor samples analyzed, 7 harbored BRCA1/2 mutations. We found a subset of BRCA-proficient MBC with high HRD score or high S3 levels, comparable to BRCA-mutated tumors. However, these patients with high HRD score or high S3 tumor level do not seem to benefit more from platinum-based chemotherapy than the others, in terms of response rates and/or PFS, regardless of BC molecular subtype. By multivariate analysis, only the absence of liver metastases was independently associated with significantly better PFS on platinum-based chemotherapy. However, some of our exploratory analyses reveal that certain methods, when optimized, seem to associate with platinum benefit. Tumor exome sequencing methodology for quantifying HRD has to be approached systematically, and further validated and standardized prior to its clinical use. Further studies are warranted to confirm these results to guide platinum use in MBC.

Elucidating the genetic architecture underlying IGF1 levels and its impact on genomic instability and cancer risk

Background: Insulin-like growth factor-1 (IGF1) has been implicated in mitogenic and anti-apoptotic mechanisms that promote susceptibility to cancer development and growth. Previous epidemiological studies have described phenotypic associations between higher circulating levels of IGF1 in adults with higher risks for breast, prostate, ovarian, colorectal, melanoma and lung cancers. However, such evidence is prone to confounding and reverse causality. Furthermore, it is unclear whether IGF1 promotes only the survival and proliferation of cancerous cells, or also the malignant transformation of healthy cells. Methods: We perform a genome-wide association study in 428,525 white European ancestry individuals in the UK Biobank study (UKBB) and identify 831 independent genetic determinants of circulating IGF1 levels, double the number previously reported. Results: Collectively these signals explain ~7.5% of the variance in circulating IGF1 levels in EPIC-Norfolk, with individuals in the highest 10% of genetic risk exhibiting ~1 SD higher levels than those in the lowest 10%. Using a Mendelian randomization approach, we demonstrate that genetically higher circulating IGF1 levels are associated with greater likelihood of mosaic loss of chromosome Y in leukocytes in men in UKBB (OR per +1 SD = 1.038 (95% CI: 1.010-1.067), P=0.008) and 23andMe, Inc. (P=6.8×10-05), a biomarker of genomic instability involved in early tumorigenesis. Genetically higher IGF1 is also associated with higher risks for colorectal (OR = 1.126 (1.048-1.210), P=1.3×10-03) and breast cancer (OR= 1.075 (1.048-1.103), P=3.9×10-08), with similar effects on estrogen positive (ER+) (OR = 1.069 (1.037-1.102), P=2.3×10-05) and estrogen negative (ER-) (OR = 1.074 (1.025-1.125), P=3.9×10-08) subtypes. Conclusions: These findings give an insight into the genetic regulation of circulating IGF1 levels and support a causal role for IGF1 in early tumorigenesis and risks for breast and colorectal cancers.

X chromosome aneuploidy and micronuclei in fertile mares

Abnormalities of chromosomes are an important and well documented cause of disorders of sexual development, fertility problems and congenital anomalies in mammals. Detection of low-level 63,X/64,XX mosaicism during routine cytogenetic evaluation is a challenge because its clinical significance is not yet fully clear. This study describes the prevalence and levels of 63,X mosaicism for a cohort of fertile mares and compares the results with eight problem mares for which no clinical cause of sub-fertility was found. The study design allowed for the analysis of micronuclei which are biomarkers of genomic instability and can disturb cell divisions, drive cancer development or cause congenital diseases.Although 27% of the fertile mares were identified to be 63,X mosaics, the results showed that the rates of abnormal cells were very low (1–3%). Levels of abnormal cells in problem mares with 63,X mosaicism were similar or higher. The average rate of micronuclei in the blood of the fertile mares was ∼1%, well below the baseline (5%) which was proposed for peripheral blood of normal healthy humans. We found weak to modest, but not significant, correlations between the age of fertile mares and 63,X cells (Kendall’s tau b = 0.2905; p > 0.05) as well as the rate of micronuclei (Kendall’s tau b = 0.1896; p > 0.05). Likewise, the correlation between presence of a 63,X cell line and micronuclei rate was not significant (Kendall’s tau b = 0.3201; p > 0.05). The presence of 63,X cells in rates greater than 3% may indeed indicate a higher risk for sub-fertility and eventually for associated health problems in such mares. Detection and elimination of mares with high level of X aneuploidies from breeding may have a positive effect on the fertility within the general horse population. This data may support the evaluation of problem mares with mosaic karyotypes involving the X chromosome.

Getting wise about lost Ys.

A new study has identified genetic determinants of mosaic loss of chromosome Y (LOY) in leukocytes that are also associated with increased risk of various non-haematological cancers, suggesting LOY in blood is a biomarker of genome instability in other tissues.

Genetic predisposition to mosaic Y chromosome loss in blood.

Mosaic loss of chromosome Y (LOY) in circulating white blood cells is the most common form of clonal mosaicism1–5, yet our knowledge of the causes and consequences of this is limited. Using a newly developed approach, we estimate that 20% of the UK Biobank male population (N=205,011) has detectable LOY. We identify 156 autosomal genetic determinants of LOY, which we replicate in 757,114 men of European and Japanese ancestry. These loci highlight genes involved in cell-cycle regulation, cancer susceptibility, somatic drivers of tumour growth and cancer therapy targets. We demonstrate that genetic susceptibility to LOY is associated with non-haematological health outcomes in both men and women, supporting the hypothesis that clonal haematopoiesis is a biomarker of genome instability in other tissues. Single-cell RNA sequencing identifies dysregulated autosomal gene expression in leukocytes with LOY, providing insights into why clonal expansion of these cells may occur. Collectively, these data highlight the utility of studying clonal mosaicism to uncover fundamental mechanisms underlying cancer and other ageing-related diseases.

Abstract SY31-03: Predictive biomarkers in circulating tumor cells to direct management metastatic castration-resistant prostate cancer (mCRPC)

Abstract Tissue and cfDNA NGS profiling is improving our ability to detect and clinically validate the significance of molecular alterations that drive tumor growth resulting in actionable therapeutic strategies that can improve outcomes for subsets of patients and accelerate the regulatory approval of targeted agents. Confounding our knowledge base are three critical aspects: 1) intra patient and intra-tumor heterogeneity for which the clustering of alterations and signaling pathways cannot be easily disassociated without cellular context, 2) epigenetic and functional cellular biology alterations which perturb genomic pathways presumed to drive tumor growth, and 3) understanding the clinical significance of “variants of uncertain significance” (with respect to changes in protein function) (1) (https://www.ncbi.nlm.nih.gov/pubmed/26582918). Our objective is to improve the outcomes for men with mCRPC starting by identifying unmet needs at decision points in management, credentialing biomarkers to better inform the decision, followed by the analytical and clinical validation of the assay and the biomarker in a sequence of trials in which samples are obtained at the decision point and associated with outcomes, and showing that use of the test result to direct management improves patient outcomes compared to non-use of the test. Our focus is on predictive biomarkers of sensitivity and resistance assessed at the level of the single circulating tumor cell (CTC) through Functional Cell Profiling (FCP) analyzed by 1) protein characterization &amp; localization, 2) cellular and sub-cellular morphology aggregated with machine learned CTC profiles, and 3) low pass whole genome copy number changes. Establishing a phenotype genotype relationship enables a rapid turnaround of test results to inform decision making for an individual patient. Building on the initial results reported by Investigators at JHU (2) showing the association of the presence of the ARV7 (androgen receptor splice variant 7) in CTC using a PCR based assay predicted for resistance to androgen receptor signaling inhibitors (ARSI) but not taxane based chemotherapy, we applied a protein based assay that enabled distinguishing the localization of the splice variant, nuclear vs. cytoplasmic that showed increased specificity, resistance to ARSI, and clinical utility based on a treatment specific interaction that showed in a survival benefit for treatment with a taxane for patients with AR-V7+ CTC (3, 4). The assay, now marketed, has also received a favorable coverage determination from CMS. Later, building on the regulatory precedent for image based phenotyping that enabled rapid cervical cancer screening, we analyzed CTCs based on protein expression and localization and digital pathology features similar to what is applied in facial recognition analyses, to define phenotypically unique cell type (5), studied independently for the relationship to sensitivity to standard of care (SOC) drugs. The aim was to enable rapid test turnaround to enable real time treatment selection decisions. Such profiling has identified unique cell types associated with resistance to ARSi’s and taxanes, and single cell genomic analyses confirmed differences between cell types, providing insight into the underlying mechanisms. More recently we have focused on the development of a phenotypic biomarker of genomic instability, an important driver of tumor progression, that utilizes morphology to identify CTCs with an abnormal number of single cell Large Scale Transitions (LSTs). The resultant biomarker, phenotypic LST (pLST), was then examined in patient CTCs prior to and shortly after treatment with an ARSI or taxane. Those determined to be pLST biomarker positive have worse OS than those who don’t. An important aspect of liquid biopsy analysis is the biologic characterization of the cells that persist, i.e. that are resistant de novo, and which emerging during therapy that have acquired resistance to treatment. In the case of CTCs, we have validated through 5 prospective phase III trials that the elimination of all CTCs on therapy portends a better clinical response than those who have residual CTCs (6). In this context, pLST CTC biomarker positivity ~1 mo after the start of therapy portends a poor outcome whereby fewer than 15% of patients survive beyond 1 yr. Importantly however, is that these pLST+ cells are/were eliminated in patients treated with a PARP inhibitor and had improved PSA response rates7, suggesting that specifically targeting genomic instability is a valid approach where other standard of care drugs fail. Functional Cell Profiling or FCP has also provided an improved understanding of the tumor heterogeneity common in many patients, occurrence of discordance between phenotype and genotype, and the greater biologic learnings of single cell analysis. The framework has allowed us to postulate a novel strategy for analyzing CTCs through FCP for prediction of therapeutic benefit and subclonal disease resistance. Characterizing CTCs in individual patients may provide insight into disease evolution, inform treatment decisions for individual patients and guide new drug development. Longitudinal monitoring of phenotypic CTC subtypes in multiple clinical trials is currently underway.

Imaging features of genomic instability in circulating tumor cells (CTCs) from metastatic triple-negative breast cancer (metTNBC) patients (pts) who received PIKTOR.

e14527 Background: A subset of TNBCs have homologous recombination deficiency (HRD) with upregulation of compensatory DNA repair pathways. A combination of TAK-228 and TAK-117 (PIKTOR), investigational oral TORC1/2 and PI3Kα selective inhibitors, respectively, is hypothesized to increase genomic instability (GI) and decrease double strand DNA repair. CTCs from metTNBC pts are being characterized for imaging features that may correlate GI in blood obtained at baseline (BL) prior to beginning PIKTOR, and at disease progression (PD) on PIKTOR. Pts are treated with a cisplatin-based regimen after PD on PIKTOR. Methods: Following IRB-approved informed consent, metTNBC pts received 4 mg PO TAK-228 and 200 mg PO TAK-117 QDx3d QW until PD. Blood samples collected at BL and at PD were sent to Epic Sciences for CTC analyses including enumeration, cell morphology, phenotypic heterogeneity, and GI analysis via a previously developed GI prediction algorithm based on cell phenotypes (Jendrisak et al AACR 2018). Results: The 9 pts treated with PIKTOR to date had mets in lymph nodes (n = 8), lung (n = 5), chest wall (n = 2), and bone (n = 2). Median number of prior regimens for metTNBC was 2; median duration of PIKTOR treatment was 8 wks (range, 5-15). CTCs were detected in 62.5% (5/8) BL and 100% (9/9) PD samples. 2 pts had high phenotypic CTC heterogeneity at both BL and PD (pt#36: 0.69 to 0.64; pt#80: 0.93 to 1.41), 1 pt had increased heterogeneity (pt#75: 0 to 1.68) and 1 pt had decreased heterogeneity (pt#67: 1.26 to 0) post-PIKTOR. 1 pt had predicted phenotypic GI (pGI+) CTCs detected at both BL and PD (pt#80), 1 pt had pGI+ CTCs at BL only (pt#36) and 1 pt at PD only (pt#75). Higher expression of the epithelial marker, cytokeratin (CK), and larger cell size were observed in the PD samples. Conclusions: Following treatment with and PD on PIKTOR, higher CK expression, higher phenotypic heterogeneity, and more pGI+ CTCs were observed in a subset of pts, suggesting that these TN CTCs may have developed epithelial transition and greater GI with PIKTOR treatment. Accrual continues and CTC GI biomarkers will be correlated with pts’ response to subsequent cisplatin-based therapy.

Association of telomere length with chronic exposure to ionizing radiation among inhabitants of natural high background radiation areas of Ramsar, Iran

Purpose: There are few areas in the world where ionizing radiation (IR) dose received by the public from radon gas and other radioactive elements are much higher than recommended limits. Telomere length is a potential biomarker of genomic instability due to oxidative stress from IR exposure. In this study, we investigated the impact of chronic exposure to environmental IR on relative telomere length (RTL) in white blood cells (WBC) among inhabitants of high background radiation areas (HBRA) of Ramsar, Iran.Materials and methods: One hundred and four individuals from HBRAs of Ramsar and 104 age-matched subjects from normal background radiation areas (NBRA) of Iran were enrolled in the study. The RTLs of WBC DNA samples were measured by a quantitative polymerase chain reaction (q-PCR) approach.Results: Mean RTL in HBRA and NBRA groups did not show any significant difference (1.21 ± 0.71 versus 1.22 ± 0.66, p = .306). After controlling for all demographic variables, less than 1% of the variances in RTL values were related to background radiation exposure.Conclusion: We conclude that chronic exposure to natural IR has no statistically significant effect on RTL among the inhabitants of HBRAs of Ramsar compared with a control group.

The Role of Micronucleus Assay to Detect Genetic Instability in Respiratory Cancer Patients.

Asbestos represents the main risk factor for malignant pleural mesothelioma (PM) even if only a minority of exposed people develop this tumor, suggesting a significant role for genetic susceptibility. This study aims to evaluate micronuclei (MN) frequency as a biomarker of genome instability in peripheral blood lymphocytes of PM and lung cancer (LC) patients when compared with healthy controls (HCs) and patients with nonneoplastic respiratory diseases (RDs). Lymphocyte cytokinesis-block MN assay was carried out on 317 subjects. Mutagen sensitivity, measured by quantifying MN frequency after an in vitro challenge with ionizing radiation, was evaluated in 252 subjects. A significant increase in MN frequency was observed in cancer patients compared to HCs, with a mean ratio (MR) of 1.35 and 1.36 at baseline and 1.43 and 1.38 after irradiation in PM and LC patients, respectively. A positive (synergistic) interaction between asbestos exposure and disease status was observed for MN frequency after irradiation in PM patients with possible exposure to asbestos (MR = 1.62). The evidence of increased genetic damage in cancer cases confirms lymphocyte cytokinesis-block MN assay as a sensitive predictor of the role of genetic instability in carcinogenic processes.

The frequencies of micronuclei, nucleoplasmic bridges and nuclear buds as biomarkers of genomic instability in patients with urothelial cell carcinoma

Bladder urothelial cell carcinoma (UCC) is an increasingly prevalent cancer worldwide, and thus, gaining a better understanding of its identifiable risk factors is a global priority. This study addressed this public health need with the understanding that cancer-initiating events, such as chromosome breakage, loss and rearrangement, can be reasonably used as biomarkers to evaluate an individual’s cancer risk. Overall, forty bladder cancer patients and twenty controls were evaluated for genomic instability. To the best of the investigators’ knowledge, this is the first study to perform micronucleus (MN) assays simultaneously in urothelial exfoliated cells (UEC), buccal exfoliated cells (BEC), and peripheral blood lymphocytes (PBL) in first-diagnosed, non-smoker bladder UCC patients. Additionally, the frequency of nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in PBL was evaluated. The MN frequencies in UEC, BEC, and PBL, as well as the frequencies of NPBs and NBUDs, were significantly higher in patients than in controls. In conclusion, MN assays, particularly in UEC, may be used to identify individuals who are at high risk of developing UCC, as single or as additional triage test to UroVysion FISH test. Our results further validate the efficacy of biomarkers, such as MN, NPBs, and NBUDs, as predictors of genomic instability.

Gold nanoparticles - an optical biosensor for RNA quantification for cancer and neurologic disorders diagnosis.

PurposeThe objective of this study is to develop a facile tool for the absolute detection and quantification of nucleic acid transcripts, using a gold nanoparticle-based optical biosensor. Topoisomerase 1 (TOP1) and tyrosyl DNA phosphodiesterase 2 (TDP2) were among the nucleic acid transcripts of choice due to their role as genomic instability biomarkers and their implication in various cancers and neurologic disorders. This opens the door to develop a simple tool that can be used for diagnosing and monitoring treatment response for such diseases, overcoming the requirements for high cost, time, and complexity of the existing technologies for the absolute quantification of transcripts of interest.Materials and methodsThe TOP1 and TDP2 mRNA transcripts were first captured specifically using magnetic nanoparticles that were functionalized with TOP1- and TDP2-specific probes, respectively. The captured mRNA was then directly detected and quantified using the gold aggregating gold (GAG) assay, without the need for amplification as in existing technologies used for the quantification of transcripts.ResultsA linear correlation exists between the GAG assay and the qPCR for the quantification of the TOP1 and TDP2 mRNA transcripts (101–104 copies). The detection limit of the GAG assay in mRNA quantification was up to 10 copies per reaction. Wild-type and TDP2-deficient cell lines confirmed the assay specificity and reproducibility in distinguishing between different transcripts.ConclusionThe GAG assay can be utilized as an inexpensive, rapid, simple, and sensitive tool for the absolute quantification of RNA for different applications, instead of the laborious, expensive, and sophisticated real-time PCR.

Genotoxic damage in end-stage renal disease

End-stage renal disease (ESRD) patients are at increased risk for cardiovascular events and cancer, possibly due to genomic instability associated with renal disease and/or its therapy. Prognostic biomarkers of genomic instability may prove useful for initiating appropriate intervention strategies. We conducted a case-control study, performing the single-cell gel electrophoresis assay (circulating leukocytes) and the micronucleus cytome assay (buccal epithelial cells). Cases (ESRD patients; n = 55) were on weekly/fortnightly dialysis therapy and controls (n = 39) were healthy adults. The patients had significantly elevated levels of DNA damage and micronucleated cells. DNA damage showed higher validity and sensitivity than did chromosome damage, for discriminating patients from controls. The patient group showed significant increases in cell proliferation, cytokinetic defects, and cell death, and a decrease in repair index. Correlations were seen between genetic damage and both time-on-medication and time-on-dialysis; between condensed chromatin cells and sex; and between pyknotic cells and dietary pattern. Following stratification by age, gender, and dialysis frequency, significantly elevated DNA damage and MN frequency were seen in the fortnightly dialysis patients, perhaps due to accumulated uremic toxicants. DNA and chromosome damage may be useful prognostic biomarkers for initiating timely interventions against co-morbidities in ESRD patients.

The association between micronucleus, nucleoplasmic bridges, and nuclear buds frequency and the degree of uterine cervical lesions

Background and aim: The loss of genomic stability plays an important role in carcinogenesis. Therefore, it is imperative to use certain biomarkers of DNA damage due to genomic instability in order to predict cancer risk. The aim of this study was the evaluation of genomic instability in patients with cervical lesions.Materials and methods: We investigated the genetic damages in 80 subjects: 40 patients with high-grade squamous intraepithelial lesions (HSIL), 20 patients with invasive squamous cervical cancer (SCC) and 20 healthy women with a biomarker in two different tissues; the micronucleus (MN) test in peripheral blood lymphocytes (PBL), and in buccal exfoliated cells (BEC). This study also examined the frequency of other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear bunds (NBUDs) in PBL.Results: The frequency of MN in BEC, MN in PBL, NPB in PBL and NBUD in PBL were significantly higher (p < 0.001), in patients compared to controls. The DNA damages in BEC and PBL were correlated positively with histological grade of cervical lesions.Conclusion: Although larger studies are needed, our data support the predictive value of MN, NPB and NBUD as biomarkers of genomic instability for evaluation of risk level of cancer diseases.