Replication stress drives chromosomal instability in fibroblasts of childhood cancer survivors with second primary neoplasms.

New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary neoplasms (SPNs) as a late consequence of genotoxic cancer therapies with the highest risk among former childhood cancer patients. Since risk factors and predictive biomarkers for therapy-associated SPN remain unknown, we examined the sensitivity to mild replication stress as a driver of genomic instability and carcinogenesis in fibroblasts from 23 long-term survivors of a pediatric first primary neoplasm (FPN), 22 patients with the same FPN and a subsequent SPN, and 22 controls with no neoplasm (NN) using the cytokinesis-block micronucleus (CBMN) assay. Mild replication stress was induced with the DNA-polymerase inhibitor aphidicolin (APH). Fibroblasts from patients with the DNA repair deficiency syndromes Bloom, Seckel, and Fanconi anemia served as positive controls and for validation of the CBMN assay supplemented by analysis of chromosomal aberrations, DNA repair foci (γH2AX/53BP1), and cell cycle regulation. APH treatment resulted in G2/M arrest and underestimation of cytogenetic damage beyond G2, which could be overcome by inhibition of Chk1. Basal micronuclei were significantly increased in DNA repair deficiency syndromes but comparable between NN, FPN, and SPN donors. After APH-induced replication stress, the average yield of micronuclei was significantly elevated in SPN donors compared to FPN (p = 0.013) as well as NN (p = 0.03) donors but substantially lower than for DNA repair deficiency syndromes. Our findings suggest that mild impairment of the response to replication stress induced by genotoxic impacts of DNA-damaging cancer therapies promotes genomic instability in a subset of long-term cancer survivors and may drive the development of an SPN. Our study provides a basis for detailed mechanistic studies as well as predictive bioassays for clinical surveillance, to identify cancer patients at high risk for SPNs at first diagnosis.

Spontaneous and Radiation-Induced Chromosome Aberrations in Primary Fibroblasts of Patients With Pediatric First and Second Neoplasms.

The purpose of the present study was to investigate whether former childhood cancer patients who developed a subsequent secondary primary neoplasm (SPN) are characterized by elevated spontaneous chromosomal instability or cellular and chromosomal radiation sensitivity as surrogate markers of compromised DNA repair compared to childhood cancer patients with a first primary neoplasm (FPN) only or tumor-free controls. Primary skin fibroblasts were obtained in a nested case-control study including 23 patients with a pediatric FPN, 22 matched patients with a pediatric FPN and an SPN, and 22 matched tumor-free donors. Clonogenic cell survival and cytogenetic aberrations in Giemsa-stained first metaphases were assessed after X-irradiation in G1 or on prematurely condensed chromosomes of cells irradiated and analyzed in G2. Fluorescence in situ hybridization was applied to investigate spontaneous transmissible aberrations in selected donors. No significant difference in clonogenic survival or the average yield of spontaneous or radiation-induced aberrations was found between the study populations. However, two donors with an SPN showed striking spontaneous chromosomal instability occurring as high rates of numerical and structural aberrations or non-clonal and clonal translocations. No correlation was found between radiation sensitivity and a susceptibility to a pediatric FPN or a treatment-associated SPN. Together, the results of this unique case-control study show genomic stability and normal radiation sensitivity in normal somatic cells of donors with an early and high intrinsic or therapy-associated tumor risk. These findings provide valuable information for future studies on the etiology of sporadic childhood cancer and therapy-related SPN as well as for the establishment of predictive biomarkers based on altered DNA repair processes.