Subsequent primary neoplasms after childhood cancer therapy - design and description of the German nested case-control study STATT-SCAR.

BACKGROUND: Subsequent primary neoplasms (SPN) are among the most severe late effects and the second most frequent cause of death in childhood cancer patients. In this paper we introduce method and properties of the STATT-SCAR study (Second Tumor After Tumor Therapy, Second Cancer After Radiotherapy), which is a joint nested matched case-control study to evaluate the impact of chemotherapy (STATT) as well as radiotherapy (SCAR) on the risk of developing a SPN. METHODS: Based on the cohort of the German childhood cancer registry (GCCR), we selected patients diagnosed with a first neoplasm before age 15 or younger between 1980 and 2014. We selected those with a SPN at least half a year after the first neoplasm, and matched up to four controls to each case. Therapy data were acquired from various sources, including clinical study centers and treating hospitals. To analyze the impact of radiotherapy, organ doses were estimated by using reconstructed treatment plans. The effect of chemotherapy was analyzed using substance groups summarized after isotoxic dose conversion. RESULTS: 1244 cases with a SPN were identified and matched with 4976 controls. Treatment data were acquired for 83% of all match groups (one case and at least one control). Based on preliminary analyses, 98% of all patients received chemotherapy and 54% of all patients were treated with radiotherapy. CONCLUSIONS: Based on our data, detailed analyses of dose response relationships and treatment element combinations are possible, leading to a deeper insight into SPN risks after cancer treatments. TRIAL REGISTRATION: The study is registered at the German clinical trial register (DRKS) under number DRKS00017847 [45].

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.

Hypermethylation of RAD9A intron 2 in childhood cancer patients, leukemia and tumor cell lines suggest a role for oncogenic transformation.

Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or an unhealthy lifestyle. However, risk factors might trigger the oncogenic transformation of cells. Among other regulatory signals, hypermethylation of RAD9A intron 2 is responsible for the increased expression of RAD9A protein, which may play a role in oncogenic transformation. Here, we analyzed the RAD9A intron 2 methylation in primary fibroblasts of 20 patients with primary cancer in childhood and second primary cancer (2N) later in life, 20 matched patients with only one primary cancer in childhood (1N) and 20 matched cancer-free controls (0N), using bisulfite pyrosequencing and deep bisulfite sequencing (DBS). Four 1N patients and one 2N patient displayed elevated mean methylation levels (≥ 10 %) of RAD9A. DBS revealed ≥ 2 % hypermethylated alleles of RAD9A, indicative for constitutive mosaic epimutations. Bone marrow samples of NHL and AML tumor patients (n=74), EBV (Epstein Barr Virus) lymphoblasts (n=6), tumor cell lines (n=5) and FaDu subclones (n=13) were analyzed to substantiate our findings. We find a broad spectrum of tumor entities with an aberrant methylation of RAD9A. We detected a significant difference in mean methylation of RAD9A for NHL versus AML patients (p ≤0.025). Molecular karyotyping of AML samples during therapy with hypermethylated RAD9A showed an evolving duplication of 1.8 kb on Chr16p13.3 including the PKD1 gene. Radiation, colony formation assays, cell proliferation, PCR and molecular karyotyping SNP-array experiments using generated FaDu subclones suggest that hypermethylation of RAD9A intron 2 is associated with genomic imbalances in regions with tumor-relevant genes and survival of the cells. In conclusion, this is the very first study of RAD9A intron 2 methylation in childhood cancer and Leukemia. RAD9A epimutations may have an impact on leukemia and tumorigenesis and can potentially serve as a biomarker.

Identification of Genetic Predispositions Related to Ionizing Radiation in Primary Human Skin Fibroblasts From Survivors of Childhood and Second Primary Cancer as Well as Cancer-Free Controls: Protocol for the Nested Case-Control Study KiKme.

BACKGROUND: Therapy for a first primary neoplasm (FPN) in childhood with high doses of ionizing radiation is an established risk factor for second primary neoplasms (SPN). An association between exposure to low doses and childhood cancer is also suggested; however, results are inconsistent. As only subgroups of children with FPNs develop SPNs, an interaction between radiation, genetic, and other risk factors is presumed to influence cancer development. OBJECTIVE: Therefore, the population-based, nested case-control study KiKme aims to identify differences in genetic predisposition and radiation response between childhood cancer survivors with and without SPNs as well as cancer-free controls. METHODS: We conducted a population-based, nested case-control study KiKme. Besides questionnaire information, skin biopsies and saliva samples are available. By measuring individual reactions to different exposures to radiation (eg, 0.05 and 2 Gray) in normal somatic cells of the same person, our design enables us to create several exposure scenarios for the same person simultaneously and measure several different molecular markers (eg, DNA, messenger RNA, long noncoding RNA, copy number variation). RESULTS: Since 2013, 101 of 247 invited SPN patients, 340 of 1729 invited FPN patients, and 150 of 246 invited cancer-free controls were recruited and matched by age and sex. Childhood cancer patients were additionally matched by tumor morphology, year of diagnosis, and age at diagnosis. Participants reported on lifestyle, socioeconomical, and anthropometric factors, as well as on medical radiation history, health, and family history of diseases (n=556). Primary human fibroblasts from skin biopsies of the participants were cultivated (n=499) and cryopreserved (n=3886). DNA was extracted from fibroblasts (n=488) and saliva (n=510). CONCLUSIONS: This molecular-epidemiological study is the first to combine observational epidemiological research with standardized experimental components in primary human skin fibroblasts to identify genetic predispositions related to ionizing radiation in childhood and SPNs. In the future, fibroblasts of the participants will be used for standardized irradiation experiments, which will inform analysis of the case-control study and vice versa. Differences between participants will be identified using several molecular markers. With its innovative combination of experimental and observational components, this new study will provide valuable data to forward research on radiation-related risk factors in childhood cancer and SPNs. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/32395.

Development of screening questions for doctor-patient consultation assessing the quality of life and psychosocial burden of glioma patients: an explorative study.

PURPOSE: Psychosocial screening for glioma patients is challenging because many patients suffer from neurocognitive deficits, which may impair assessment. This study's aim was to exploratively develop three screening questions for unmet needs to prospectively be applicable in patient-doctor consultation. METHODS: Patient interviews, a survey for health-care professionals and a weighted scoring procedure were developed for this study. Six main areas were defined according to main areas of validated questionnaires (psyche, cognition, body, role functioning, social support, unmet needs). Patients and health-care professionals rated the importance of these areas and corresponding items, patients additionally stated whether the issues addressed affected them. RESULTS: A total of 50 patients were included, and 36 health-care professionals participated in the online survey. The three areas (psyche, body and cognition) considered to be most relevant by both, health-care professionals and patients, generated three screening questions. If the patient was affected by the issue addressed with a screening question, a subordinate question from that area that our patient sample considered most important could additionally be asked. The elaborated screening questions are the following: (1) main area psyche: "Has your mood worsened?", (2) main area body: "Do physical changes put a strain on you?", and (3) main area cognition: "Has your memory capacity worsened?" CONCLUSION: These questions represent a basis for further research regarding their application in neuro-oncological clinical routine.

Comparison of time and dose dependent gene expression and affected pathways in primary human fibroblasts after exposure to ionizing radiation.

BACKGROUND: Exposure to ionizing radiation induces complex stress responses in cells, which can lead to adverse health effects such as cancer. Although a variety of studies investigated gene expression and affected pathways in human fibroblasts after exposure to ionizing radiation, the understanding of underlying mechanisms and biological effects is still incomplete due to different experimental settings and small sample sizes. Therefore, this study aims to identify the time point with the highest number of differentially expressed genes and corresponding pathways in primary human fibroblasts after irradiation at two preselected time points. METHODS: Fibroblasts from skin biopsies of 15 cell donors were exposed to a high (2Gy) and a low (0.05Gy) dose of X-rays. RNA was extracted and sequenced 2 h and 4 h after exposure. Differentially expressed genes with an adjusted p-value < 0.05 were flagged and used for pathway analyses including prediction of upstream and downstream effects. Principal component analyses were used to examine the effect of two different sequencing runs on quality metrics and variation in expression and alignment and for explorative analysis of the radiation dose and time point of analysis. RESULTS: More genes were differentially expressed 4 h after exposure to low and high doses of radiation than after 2 h. In experiments with high dose irradiation and RNA sequencing after 4 h, inactivation of the FAT10 cancer signaling pathway and activation of gluconeogenesis I, glycolysis I, and prostanoid biosynthesis was observed taking p-value (< 0.05) and (in) activating z-score (≥2.00 or ≤ - 2.00) into account. Two hours after high dose irradiation, inactivation of small cell lung cancer signaling was observed. For low dose irradiation experiments, we did not detect any significant (p < 0.05 and z-score ≥ 2.00 or ≤ - 2.00) activated or inactivated pathways for both time points. CONCLUSIONS: Compared to 2 h after irradiation, a higher number of differentially expressed genes were found 4 h after exposure to low and high dose ionizing radiation. Differences in gene expression were related to signal transduction pathways of the DNA damage response after 2 h and to metabolic pathways, that might implicate cellular senescence, after 4 h. The time point 4 h will be used to conduct further irradiation experiments in a larger sample.

Correction to: Molecular karyotyping and gene expression analysis in childhood cancer patients.

The correct Author names are presented in this paper.

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.

Radiotherapy for childhood cancer and subsequent thyroid cancer risk: a systematic review.

Most of the pooled analyses and reviews reported an association between radiotherapy for childhood cancer and an increased thyroid cancer risk. Up to now this article presents the first systematic literature review on this association combined with a critical assessment of the methodological quality of the included articles. PubMed and Web of Science databases were searched for relevant articles until May 2016. We included peer-reviewed cohort and case-control studies that investigated an association between radiotherapy for childhood cancer and the occurrence of subsequent thyroid cancer. A systematic overview is presented for the included studies. We identified 17 retrospective cohort studies, and four nested case-control studies, representing 100,818 subjects. The age range at first cancer diagnosis was 0-25.2 years. Considerable variability was found regarding study sizes, study design, treatment strategies, dose information, and follow-up periods. 20 of the 21 identified studies showed increased thyroid cancer risks associated with childhood radiation exposure. The large majority showed an increased relative risk or odds ratio confirming the association between radiotherapy and thyroid cancer although the variation in results was large. Additionally to a pooled analysis that has been published recently, we systematically included 17 further studies, which allowed us to cover information from countries that were not covered by large-scale childhood cancer survivor studies. The methodological limitations of existing studies and inconsistencies in findings across studies yielded a large study heterogeneity, which made a detailed comparison of study results difficult. There is a need to strengthen standardisation for reporting.

Second Malignancies Following Childhood Cancer Treatment in Germany From 1980 to 2014.

BACKGROUND: Because of improvements in cancer treatment, more than 80% of all children with cancer now survive at least five years from the time of diagnosis. As a result, late sequelae of cancer and its treatment have become more common, particularly second malignancies. We studied the current incidence of second malignancies among childhood cancer survivors in Germany. METHODS: This study is based on the cohort of the German Childhood Cancer Registry (Deutsches Kinderkrebsregister, DKKR). Persons given the diagnosis of a first malignancy at any time in the years 1980-2014 who were no more than 14 years old at the time of diagnosis and survived at least six months thereafter were included in the study. Cumulative incidences and hazard ratios were calculated, and comparisons with the general population were made with the aid of standardized incidence ratios (SIR). RESULTS: Among the 47 650 survivors included in the study, there were 1262 cases of second malignancies. After a follow-up interval of up to 35 years, the cumulative incidence of second malignancies was 8.27% (95% confidence interval [7.51; 9.03]). Second malignancies were more common in female patients (hazard ratio 1.29, [1.16; 1.44]) and in those who had had a systemic cancer as their initial malignancy (hazard ratio 1.22 [1.09; 1.36]). The SIR compared to the general population for the period 1955-2014 was 7.08 [6.42; 7.9] for female patients and 5.83 [5.27; 6.42] for male patients. CONCLUSION: The cumulative incidence of second malignancies is 5.4% at 25 years and 8.3% at 35 years; these figures may be slight underestimates. The DKKR is an epidemiologic registry containing no data about treatment, so the effect of treatment on the risk of second malignancies could not be studied. The acquisition and evaluation of treatment data for the overall cohort is currently one of the main tasks for research on the late sequelae of childhood cancer. This may enable conclusions to be drawn about whether treatment strategies that have been introduced to lessen the risk of a second malignancy actually have the desired effect.

Mitochondrial BAX Determines the Predisposition to Apoptosis in Human AML.

Purpose: Cell-to-cell variability in apoptosis signaling contributes to heterogenic responses to cytotoxic stress in clinically heterogeneous neoplasia, such as acute myeloid leukemia (AML). The BCL-2 proteins BAX and BAK can commit mammalian cells to apoptosis and are inhibited by retrotranslocation from the mitochondria into the cytosol. The subcellular localization of BAX and BAK could determine the cellular predisposition to apoptotic death.Experimental Design: The relative localization of BAX and BAK was determined by fractionation of AML cell lines and patient samples of a test cohort and a validation cohort.Results: This study shows that relative BAX localization determines the predisposition of different AML cell lines to apoptosis. Human AML displays a surprising variety of relative BAX localizations. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITD status, and leukocytosis. Analysis of a validation cohort of 80 elderly patients treated with myelosuppressive chemotherapy confirmed that relative BAX localization correlates with probability of disease progression, FLT3-ITD status, and leukocytosis. Relative BAX localization could therefore be helpful to identify elderly or frail patients who may benefit from cytotoxic therapy.Conclusions: In this retrospective analysis of two independent AML cohorts, our data suggest that Bax localization may predict prognosis of patients with AML and cellular predisposition to apoptosis, combining the actual contribution of known and unknown factors to a final "common path." Clin Cancer Res; 23(16); 4805-16. ©2017 AACR.

Background gamma radiation and childhood cancer in Germany: an ecological study.

The relationship of low-dose background gamma radiation and childhood leukaemia was investigated in a number of studies. Results from these studies are inconclusive. Therefore, in the present study 25 years of German childhood cancer data were analyzed using interpolated background annual gamma dose rate per community in an ecological study. The main question was leukaemia; as exploratory questions we investigate central nervous system (CNS) tumours, thyroid carcinomas and diagnoses less likely to be related to radiation. A Poisson regression model was applied and a fractional polynomial model building procedure. As the main sensitivity analysis a community deprivation index was included as a potential confounder. It was found that outdoor background gamma annual dose rates in Germany range roughly from 0.5-1.5 mSv/a with an average of 0.817 mSv/a. No association of annual ambient gamma dose rates with leukaemia incidence was found. Amongst the exploratory analyses, a strong association was found with CNS tumour incidence [rate ratio for 1.5 vs 0.5 mSv/a: 1.35; 95% confidence interval (1.17, 1.57)]. The community level deprivation index was not a confounder. It is concluded that the present study did not confirm an association of annual outdoor ambient gamma dose rate and childhood leukaemia, corresponding to some studies and contrasting others. An association with CNS incidence was found in the exploratory analyses. As this is an ecological study no causal interpretation is possible.

Prevalence of cardiovascular late sequelae in long-term survivors of childhood cancer: A systematic review and meta-analysis.

BACKGROUND: Cardiovascular diseases are well-known late effects of childhood cancer and research on these late effects is a highly important emerging field. We conducted a systematic review with a meta-analysis to give an overview of the current evidence and the prevalence of late cardiovascular events. PROCEDURE: We included publications in which the study populations were children and adolescents who survived cancer. Outcome was defined as all cardiovascular clinical and subclinical endpoints or diagnoses appearing at least one year after cancer diagnosis. A systematic overview is presented for all included studies. A quantitative meta-analysis was conducted for hypertension and stroke. RESULTS: Sixty-four papers were included in the review. The age range at cancer diagnosis was 0-24 years; age at the end of follow-up ranged from 7 to 71 years. Prevalence of cardiovascular late effects varied from 0% for stroke up to 70% for subclinical hypertension. Large heterogeneity was found regarding study size, study design, definition of endpoints, and investigation/examination method. The weighted average prevalence was 19.7% for hypertension and 2.3% for stroke. As no specific results for gender, cancer therapy, or age at cancer diagnosis were present in most papers, a detailed comparison and pooled analysis was difficult. CONCLUSION: This review showed the vast range of cardiovascular late effects after childhood or adolescent cancer therapy. The differences between the papers prevented drawing a conclusive picture of the prevalence of cardiovascular late effects. Large cohort studies and better reporting are needed to improve the knowledge on this topic.

Increased liver carcinogenesis and enrichment of stem cell properties in livers of Dickkopf 2 (Dkk2) deleted mice.

Dkk2 a antagonist of the Wnt/ß-catenin-signaling pathway was shown to be silenced in diverse cancers. More recent data indicate that Dkk family members may also possess functions independent of Wnt-signaling during carcinogenesis. The detailed biological function of Dkks and its relevance for liver cancer is unknown. We analyzed the effects of a genetic deletion of Dkk2 (Dkk2-/-) in a hepatocarcinogenesis model using DEN/Phenobarbital. Untreated Dkk2-/- animals, showed considerable atypia with variation of hepatocyte size and chromatin density. In livers of Dkk2-/- mice nodule formation was seen at 9 months of age with focal loss of trabecular architecture and atypical hepatocytes and after DEN induction Dkk2-/- mice developed significantly more liver tumors compared to controls. Whole transcriptome analysis of untreated Dkk2-/- liver tissue revealed a Dkk2-dependent genetic network involving Wnt/ß-Catenin but also multiple additional oncogenic factors, such as e.g. Pdgf-b, Gdf-15 and Hnf4a. Dkk2-/- tumor cells showed a significant deregulation of stemness genes associated with enhanced colony forming properties. Integration of the Dkk2-/- signature into human data was strongly associated with patients survival. Dkk2 deletion results in alterations of liver morphology leading to an increased frequency of liver cancer. The associated genetic changes included factors not primarily related to Wnt/ß-Catenin-signaling and correlated with the clinical outcome of HCC-patients.

Radiotherapy and subsequent thyroid cancer in German childhood cancer survivors: a nested case-control study.

BACKGROUND: Radiotherapy is associated with a risk of subsequent neoplasms (SN) in childhood cancer survivors. It has been shown that children's thyroid glands are especially susceptible. The aim is to quantify the risk of a second neck neoplasm after primary cancer radiotherapy with emphasis on thyroid cancer. METHODS: We performed a nested case-control study: 29 individuals, diagnosed with a solid SN in the neck region, including 17 with thyroid cancer, in 1980-2002 and 57 matched controls with single neoplasms were selected from the database of the German Childhood Cancer Registry. We investigated the risk associated with radiotherapy exposure given per body region, adjusted for chemotherapy. RESULTS: 16/17 (94.1 %) thyroid SN cases, 9/12 (75 %) other neck SN cases and 34/57 (59.6 %) controls received radiotherapy, with median doses of 27.8, 25 and 24 Gy, respectively. Radiotherapy exposure to the neck region increased the risk of the other neck SNs by 4.2 % (OR = 1.042/Gy (95 %-CI 0.980-1.109)) and of thyroid SN by 5.1 % (OR = 1.051/Gy (95 %-CI 0.984-1.123)), and radiotherapy to the neck or spine region increased the thyroid risk by 6.6 % (OR = 1.066/Gy (95 %-CI 1.010-1.125)). Chemotherapy was not a confounder. Exposure to other body regions was not associated with increased risk. CONCLUSIONS: Radiotherapy in the neck or spine region increases the risk of thyroid cancer, while neck exposure increases the risk of any other solid SN to a similar extent. Other studies showed a decreasing risk of subsequent thyroid cancer for very high doses; we cannot confirm this.