Apoptosis for prediction of radiotherapy late toxicity: lymphocyte subset sensitivity and potential effect of TP53 Arg72Pro polymorphism.

We tested apoptosis levels in in vitro irradiated T-lymphocytes from breast cancer (BC) patients with radiotherapy-induced late effects. Previous results reported in the literature were revised. We also examined the effect of TP53 Arg72Pro polymorphism on irradiation-induced apoptosis (IA). Twenty BC patients, ten with fibrosis and/or telangiectasias and ten matched controls with no late reactions, were selected from those receiving radiotherapy between 1993 and 2007. All patients were followed-up at least 6 years after radiotherapy. Using the combination of both CD3 and CD8 antibodies the in vitro IA was measured in CD3, CD8 and CD4 T-lymphocytes, and CD8 natural killer lymphocytes (CD8 NK) by flow cytometry. The TP53 Arg72Pro genotype was determined by sequencing. Patients with late radiotherapy toxicity showed less IA for all T-lymphocytes except for the CD8 NK. CD8 NK showed the highest spontaneous apoptosis and the lowest IA. IA in patients with toxicity appears to be lower than the control patients only in TP53 Arg/Arg patients (P = 0.077). This difference was not present in patients carrying at least one Pro allele (P = 0.8266). Our data indicate that late side effects induced by radiotherapy of BC are associated to low levels of IA. CD8 NK cells have a different response to in vitro irradiation compared to CD8 T-lymphocytes. It would be advisable to distinguish the CD8 NK lymphocytes from the pool of CD8+ lymphocytes in IA assays using CD8+ cells. Our data suggest that the 72Pro TP53 allele may influence the IA of patients with radiotherapy toxicity.

Cell Senescence-Related Pathways Are Enriched in Breast Cancer Patients With Late Toxicity After Radiotherapy and Low Radiation-Induced Lymphocyte Apoptosis.

Background: Radiation-induced late effects are a common cause of morbidity among cancer survivors. The biomarker with the best evidence as a predictive test of late reactions is the radiation-induced lymphocyte apoptosis (RILA) assay. We aimed to investigate the molecular basis underlying the distinctive RILA levels by using gene expression analysis in patients with and without late effects and in whom we had also first identified differences in RILA levels. Patients and Methods: Peripheral blood mononuclear cells of 10 patients with late severe skin complications and 10 patients without symptoms, selected from those receiving radiotherapy from 1993 to 2007, were mock-irradiated or irradiated with 8 Gy. The 48-h response was analyzed in parallel by RILA assay and gene expression profiling with Affymetrix microarrays. Irradiated and non-irradiated gene expression profiles were compared between both groups. Gene set enrichment analysis was performed to identify differentially expressed biological processes. Results: Although differentially expressed mRNAs did not reach a significant adjusted p-value between patients suffering and not suffering clinical toxicity, the enriched pathways indicated significant differences between the two groups, either in irradiated or non-irradiated cells. In basal conditions, the main differentially expressed pathways between the toxicity and non-toxicity groups were the transport of small molecules, interferon signaling, and transcription. After 8 Gy, the differences lay in pathways highly related to cell senescence like cell cycle/NF-κB, G-protein-coupled receptors, and interferon signaling. Conclusion: Patients at risk of developing late toxicity have a distinctive pathway signature driven by deregulation of immune and cell cycle pathways related to senescence, which in turn may underlie their low RILA phenotype.

Understanding variations in the use of hypofractionated radiotherapy and its specific indications for breast cancer: A mixed-methods study.

BACKGROUND AND PURPOSE: Radiation oncology guidelines favour hypofractionated whole-breast radiotherapy (HWBRT) over more conventional schemes in the conservative treatment of breast cancer, but its adoption still varies in clinical practice. This study assessed the patterns of HWBRT adoption in Catalonia (Spain). MATERIAL AND METHODS: We used a mixed-methods approach based on an explanatory sequential design, first collecting and analysing quantitative data on HWBRT use (>2.5Gy per fraction) in 11 public radiotherapy centres (2005-2015) and then performing 25 semi-structured interviews with all department heads and reference radiation oncologist/s. RESULTS: Of the 34,859 patients fulfiling the study criteria over the study period, just 12% were hypofractionated, reaching a percentage of 29% in 2015 (p<0.001). Our analysis showed a narrowing age gap between patients receiving conventional fractionation and hypofractionation in centres leading adoption. However, there were important differences in clinicians' interpretation of evidence (e.g. regarding the perceived risk of long-term toxicity) and selection of patients for specific indications, both within and between departments. CONCLUSIONS: Differences observed in the rate of adoption of HWBRT could not be tackled only using a rational, evidence-based approach. Factors related to the management of radiotherapy departments play a major role in the diffusion of therapeutic strategies.

Individual patient data meta-analysis shows a significant association between the ATM rs1801516 SNP and toxicity after radiotherapy in 5456 breast and prostate cancer patients.

PURPOSE: Several small studies have indicated that the ATM rs1801516 SNP is associated with risk of normal tissue toxicity after radiotherapy. However, the findings have not been consistent. In order to test this SNP in a well-powered study, an individual patient data meta-analysis was carried out by the International Radiogenomics Consortium. MATERIALS AND METHODS: The analysis included 5456 patients from 17 different cohorts. 2759 patients were given radiotherapy for breast cancer and 2697 for prostate cancer. Eight toxicity scores (overall toxicity, acute toxicity, late toxicity, acute skin toxicity, acute rectal toxicity, telangiectasia, fibrosis and late rectal toxicity) were analyzed. Adjustments were made for treatment and patient related factors with potential impact on the risk of toxicity. RESULTS: For all endpoints except late rectal toxicity, a significantly increased risk of toxicity was found for carriers of the minor (Asn) allele with odds ratios of approximately 1.5 for acute toxicity and 1.2 for late toxicity. The results were consistent with a co-dominant pattern of inheritance. CONCLUSION: This study convincingly showed a significant association between the ATM rs1801516 Asn allele and increased risk of radiation-induced normal tissue toxicity.

Individual patient data meta-analysis shows no association between the SNP rs1800469 in TGFB and late radiotherapy toxicity.

BACKGROUND AND PURPOSE: Reported associations between risk of radiation-induced normal tissue injury and single nucleotide polymorphisms (SNPs) in TGFB1, encoding the pro-fibrotic cytokine transforming growth factor-beta 1 (TGF-ß1), remain controversial. To overcome publication bias, the international Radiogenomics Consortium collected and analysed individual patient level data from both published and unpublished studies. MATERIALS AND METHODS: TGFB1 SNP rs1800469 c.-1347T>C (previously known as C-509T) genotype, treatment-related data, and clinically-assessed fibrosis (measured at least 2years after therapy) were available in 2782 participants from 11 cohorts. All received adjuvant breast radiotherapy. Associations between late fibrosis or overall toxicity, reported by STAT (Standardised Total Average Toxicity) score, and rs1800469 genotype were assessed. RESULTS: No statistically significant associations between either fibrosis or overall toxicity and rs1800469 genotype were observed with univariate or multivariate regression analysis. The multivariate odds ratio (OR), obtained from meta-analysis, for an increase in late fibrosis grade with each additional rare allele of rs1800469 was 0.98 (95% Confidence Interval (CI) 0.85-1.11). This CI is sufficiently narrow to rule out any clinically relevant effect on toxicity risk in carriers vs. non-carriers with a high probability. CONCLUSION: This meta-analysis has not confirmed previous reports of association between fibrosis or overall toxicity and rs1800469 genotype in breast cancer patients. It has demonstrated successful collaboration within the Radiogenomics Consortium.