Most sporadic cancers arising in early childhood are of unknown etiology and represent devastating diagnoses. Encouragingly, the optimization of multiagent cytotoxic regimens and the development of new multimodal oncologic strategies as well as improved diagnostic procedures increased the average 5-year survival probabilities of childhood cancer patients from less than 30% before 1960 to up to 80% today [1], [2], [3]. However, this strong clinical benefit to pediatric patients in long-term survival is dampened by an elevated risk to develop therapy-associated late sequelae, primarily caused by the genotoxic impact of the two cornerstones of cancer therapy: radiation therapy (RT) and chemotherapy (CT). 60-90% of long-term childhood cancer survivors develop one or more chronic health conditions and 20-80% experience severe therapy-associated adverse health consequences during adulthood, with a second primary neoplasm (SPN) representing one of the most deleterious late complications of primary cancer treatment [4]. The risk of SPNs is known to depend on lifestyle and genetic factors, the entity and treatment of the first primary neoplasm (FPN), and particularly the age at treatment.

Compared to adult tumor patients with a 1.2- to 3-fold increased risk of SPNs, survivors of an FPN in childhood or adolescence have a 6- to 10-fold elevated risk of SPNs due to increased intrinsic tumor susceptibility from genetic predisposition, genotoxic exposures at a developmental stage, and long life expectancy after effective tumor control [3], [5], [6], [7]. However, the principal determinants and etiology for the vast majority of sporadic childhood cancers as well as an inherent proneness to iatrogenic SPNs have not yet been elucidated. Less than 10% of early-onset malignancies have been ascribed to known inherited germline or de novo familial mutations in high-penetrance predisposing genes [8], [9], [10], [11], [12]. Very rare prototypical genetic cancer predisposing syndromes of compromised DNA repair and chromosomal instability like ataxia telangiectasia, Nijmegen breakage syndrome, Werner syndrome, Bloom syndrome (BLM), and Fanconi anemia (FA) highly predispose children to different FPNs as well as subsequent SPNs [11], [13], [14]. In the general population, intrinsic cancer risk is more likely to be associated with pathogenic germline variants in low- or intermediate-penetrance genes such as BRCA1 and 2 or mismatch repair genes and their interaction [15], [16], [17]. The susceptibility to sporadic early-onset FPNs or subsequent therapy-related SPNs is commonly associated with a genetically determined failure for the proper repair of endogenous and exogenous DNA damage; the latter induced, e.g., by ionizing radiation (IR) or cytostatic agents in cancer therapy. High doses of IR applied during RT, as well as several CT drugs such as alkylating agents, etoposide, or cyclophosphamide, are established risk factors for SPNs [3]. However, these antineoplastic strategies differ in their mode of action and induce varying spectra of SPNs.

Since genomic instability fostered by compromised DNA repair is a characteristic feature and driver of carcinogenesis, elevated cytogenetic damage in healthy surrogate tissues is considered a potent indicator of cancer risk [18]. A large body of studies has shown an association between sporadic chromosomal instability or impaired repair of ex vivo IR-induced DNA damage and proneness to various primary tumor entities [19], [20] but increased susceptibility to SPNs in long-term cancer survivors has received almost no attention. Therefore, we have previously conducted a study on this highly relevant issue showing no general difference in spontaneous or IR-induced chromosomal aberrations in primary skin fibroblasts from long-term survivors of a pediatric FPN with or without an SPN compared with tumor-free donors [21].

Besides prompt induction of DNA damage by IR, replication stress is a main source of genomic instability and a driver of tumor development [22]. It is triggered by various factors, e.g., DNA lesions or adducts induced by chemical compounds, non-IR and IR, reactive oxygen species, or secondary products of cellular metabolism [23]. In cancer therapy, it is the main mechanism of action of many systemic CT agents [24], but also IR-induced DNA damage during RT may trigger delayed replication stress and genomic instability [25], [26]. Therefore, it stands to reason that increased intrinsic susceptibility to endogenous or exogenous replication stress and the consequent genomic instability are associated with a higher risk for sporadic FPNs or therapy-associated SPNs.

To address this issue, the present study examined spontaneous and replication stress-induced DNA damage in a carefully matched case-control study nested in a cohort of long-term survivors of childhood cancer who were successfully treated for a sporadic FPN and either developed an SPN or not as well as tumor-free controls. For this purpose, we first established and adapted the cytokinesis block micronucleus (CBMN) assay in a set of fibroblasts with genetic DNA repair deficiency and cancer predisposition syndromes to monitor replication stress-induced cytogenetic damage. We then sought to determine whether patients with sporadic early-onset cancer are generally more susceptible to mild replication stress than the general population and whether there are differences between former childhood cancer patients who did or did not develop an SPN associated with DNA-damaging cancer therapies. This study will provide relevant information on potential impairments of molecular mechanisms of the DNA damage response that increase the intrinsic susceptibility to sporadic childhood cancer or therapy-associated SPNs, with the potential as predictive biomarkers for the stratification of high-risk patients at a first cancer diagnosis.