Evaluation ofPIG‐A‐mutated granulocytes and ex‐vivo binucleated micronucleated lymphocytes frequencies after breast cancer radiotherapy in humans

Abstract

Although the PIG-A gene mutation frequency (MF) is considered a good proxy to evaluate the somatic MF in animals, evidence remains scarce in humans. In this study, a granulocyte PIG-A-mutant assay was evaluated in patients undergoing radiation therapy (RT) for breast cancer. Breast cancer patients undergoing adjuvant RT were prospectively enrolled. RT involved the whole breast, with (WBNRT) or without (WBRT) nodal area irradiation. Blood samples were obtained from participants before (T0) RT, and T1, T2, and T3 samples were collected 3 weeks after the initiation of RT, at the end of RT, and at least 10 weeks after RT discontinuation, respectively. The MF was assessed using a flow cytometry protocol identifying PIG-A-mutant granulocytes. Cytokinesis-blocked micronucleated lymphocyte (CBML) frequencies were also evaluated. Thirty patients were included, and five of them had received chemotherapy prior to RT. The mean (±SD) PIG-A MFs were 7.7 (±12.1) per million at T0, 5.2 (±8.6) at T1, 6.4 (±8.0) at T2 and 3.8 (±36.0) at T3. No statistically significant increases were observed between the PIG-A MF at T0 and the MFs at other times. RT significantly increased the CBML frequencies: 7.9 ‰ (±3.1‰) versus 33.6‰ (±17.2‰) (p < .0001). By multivariate analysis, the CBML frequency was correlated with age at RT initiation (p = .043) and irradiation volume at RT discontinuation (p = .0001) but not with chemotherapy. RT for breast cancer therapy failed to induce an increase in the PIG-A MF. The PIG-A assay in humans needs further evaluation, in various genotoxic exposures and including various circulating human cells.

The PIG‐A gene mutation assay in human biomonitoring and disease

“…One study measured granulocyte PIG‐A mutant levels in breast cancer patients undergoing radiotherapy but found no change in mutant cell number. However, the authors state that as the irradiation administered concerned only a part of the sternum and ribs, the probability of a granulocyte precursor gaining a mutation could have been too low to significantly impact blood cell PIG‐A mutation levels (Bonetto et al, 2021). Another study measuring erythrocyte mutant cell levels in a cohort of patients undergoing chemotherapy ± radiotherapy could not evaluate the effect of treatment as no pre‐treatment blood samples were obtained.…”

“…A longer time frame for observation of granulocyte mutation induction is possible when mutations are induced in bone marrow residing precursor cells (myeloblasts). Although granulocyte mutant cell levels are present at approximately the same frequency as erythrocyte mutant cells in healthy volunteers (Rondelli et al, 2013), using granulocytes to measure PIG‐A mutation requires blood pre‐processing and a more complex flow cytometry gating strategy (Bonetto et al, 2021; Peruzzi et al, 2010; Rondelli et al, 2013). Peripheral blood mononuclear cells (PBMCs) have also been interrogated for PIG‐A mutation status.…”

“…More research is required to assess the effectiveness of detecting genotoxic exposures in humans using the PIG‐A assay, especially in different blood cell types (Bonetto et al, 2021). International collaborative projects are required and are anticipated to provide further evidence of the application of the human PIG‐A assay.…”

The PIG-A gene mutation assay in human biomonitoring and disease

“…One study measured granulocyte PIG-A mutant levels in breast cancer patients undergoing radiotherapy but found no change in mutant cell number. However, the authors state that as the irradiation administered concerned only a part of the sternum and ribs, the probability of a granulocyte precursor gaining a mutation could have been too low to significantly impact blood cell PIG-A mutation levels (Bonetto et al, 2021). Another study measuring erythrocyte mutant cell levels in a cohort of patients undergoing chemotherapy +/-radiotherapy could not evaluate the effect of treatment as no pre-treatment blood samples were obtained.…”

“…However, the short life span of granulocytes (1-2 days) provides only a short window in which mutant cell levels can be accurately measured following a mutagenic event. Although granulocyte mutant cell levels are present at approximately the same frequency as erythrocyte mutant cells in healthy volunteers (Rondelli et al, 2013), using granulocytes to measure PIG-Amutation requires blood pre-processing and a more complex flow cytometry gating strategy (Bonetto et al, 2021;Peruzzi et al, 2010;Rondelli et al, 2013). Peripheral blood mononuclear cells (PBMCs) have also been interrogated for PIG-A mutation status.…”