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While nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene polymorphism Arg139Cys (rs116855232) is known to be a risk factor for thiopurine-induced severe leukopenia, association with the NUDT15 gene polymorphism Arg139His (rs147390019) has not yet been clarified. In addition, the accuracy of TaqMan PCR to assess these two polymorphisms has not been investigated. In this study, we evaluated TaqMan PCR for detection of the NUDT15 single-nucleotide polymorphisms (SNPs) and examined the clinical impact of Arg139His on thiopurine-induced leukopenia. First, we demonstrated that a TaqMan PCR assay successfully detected the Arg139His polymorphism of NUDT15 in clinical samples. Next, the NUDT15 gene polymorphisms (Arg139Cys and Arg139His) were separately analyzed by TaqMan Real-Time PCR in 189 patients from August 2018 to July 2019. The incidences of leukopenia within 2 years were 16.2, 57.9, and 100% for arginine (Arg)/Arg, Arg/cysteine (Cys), and Arg/histidine (His), respectively. The leukopenia was significantly increased in Arg/Cys and Arg/His compared with Arg/Arg. This retrospective clinical study indicated that, in addition to Arg139Cys, Arg139His may be clinically associated with a high risk of leukopenia. Pharmacogenomics will help in selecting drugs and determining the individualized dosage of thiopurine drugs.

Additional file 1: Fig. S1. TCF/β-catenin-dependent transcriptional activities of mouse ES cells stably expressing scLRP6S and scFZD8S. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8S following stimulation with BSA-FL are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean of triplicates and standard deviation from one representative experiment. Fig. S2. TCF/β-catenin-dependent transcriptional activities in mouse ES cells stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8L cells following stimulation with CHIR99021 (a WNT activator) or BSA-FL at indicated concentrations are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation. Fig. S3. TCF/β-catenin-dependent transcriptional activities of mouse embryonic fibroblasts (MEFs) expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in MEFs expressing mock or scLRP6S/scFZD8L following stimulation with BSA-FL at the indicated concentrations are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation from triplicate measurements. Fig. S4. Western blotting for non-canonical Wnt activation in scLRP6S and scFZD8L-expressing cells. Protein expression levels of Ror2 and β-actin in mock- or scLRP6S/scFZD8L-expressing ES cells (A) or NIH3T3 cells (B) treated with Wnt5a, Wnt3a or BSA-FL are shown. Fig. S5. BSA-FL diffuses into EBs. Representative fluorescence microscopy images in EB sections from ES/mock and ES/scLRP6S/scFZD8L cells treated with vehicle or BSA-FL. Nuclei are counterstained with DAPI showing BSA-FL deposition possibly in the plasma membrane. The right panels are enlarged images of the boxed area of the BSA-FL-treated EBs. Scale bars = 50 μm. Fig. S6. Incidence of spontaneously beating EBs derived from three independent ES cell lines expressing scLRP6S and scFZD8L. The incidence of spontaneously beating EBs stimulated by 0.1 μg/ml BSA-FL, 50 ng/ml Wnt3a, or vehicle for 24 h from day 2 of differentiation is shown. Time course of the incidence of spontaneously beating EBs differentiated from ES/scLRP6S/scFZD8L stimulated by 0.1 μg/ml BSA-FL, 50 ng/ml Wnt3a, or vehicle for 24 h from day 2 after EB formation. Fig. S7. BSA-FL enhances cardiac differentiation of EBs expressing scLRP6S and scFZD8L. Representative flow cytometry plots of cardiac troponin T (cTnT, indicated by FITC) vs. phycoerythrin (PE) for differentiated ES/mock and ES/scLRP6S/scFZD8L treated with vehicle, Wnt3a or BSA-FL. Fig. S8. TCF/β-catenin-dependent transcriptional activities in mouse ES cells stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/mock and ES/scLRP6S/scFZD8L cells following BSA-FL treatment with or without IWP-4 or XAV939, WNT inhibitors, are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The values of mock-transduced cells treated with vehicle were set to 1. The data represent the mean and standard deviation. Fig. S9. TCF/β-catenin-dependent transcriptional activities in mouse ES cells expressing single chimeric receptors. TCF/β-catenin-dependent transcriptional activities measured by luciferase reporter assay in mouse ES cells transiently transfected with plasmids encoding various chimeric LRP6 or FZD8. The values in mock-transfected cells treated with vehicle were set to 1. The data represent the mean of duplicates and standard deviation from one representative experiment. Fig. S10. Activity of BSA-FL and Wnt3a after incubation at 37 °C. TCF-dependent transcriptional activities were measured by luciferase reporter assay in mouse ES cells transiently transfected with scLRP6S and scFZD8L. The cells were stimulated with 0.1, 1, or 10 μg/ml BSA-FL or 50 ng/ml Wnt3a that were incubated at 37 °C for 0, 1, 2, 4, or 7 days. Luciferase activities of samples stimulated by each ligand with no incubation were set to 100. Fig. S11. TCF/β-catenin-dependent transcriptional activities in three independent mouse ES cell lines stably expressing scLRP6S and scFZD8L. TCF/β-catenin-dependent luciferase activities in ES/scLRP6S/scFZD8L following indicated BSA-FL treatment are shown. Luciferase luminescence intensities were normalized to Renilla luciferase luminescence intensities derived from co-transfected pSV40-Rluc. The data represent the mean and standard deviation. Table S1. List of primer sequences used for plasmid construction. Table S2. List of primer sequences used for real-time qPCR.

Total body irradiation (TBI) with ovarian shielding is expected to preserve fertility among hematopoietic stem cell transplant (HSCT) patients with myeloablative TBI-based regimens. However, the radiation dose to the ovaries that preserves ovarian function in TBI remains poorly understood. Furthermore, it is uncertain whether the dose to the shielded organs is associated with relapse risk. Here, we retrospectively evaluated the relationship between fertility and the dose to the ovaries, and between relapse risk and the dose to the pelvic bones. A total of 20 patients (median age, 23 years) with standard-risk hematologic diseases were included. Median follow-up duration was 31.9 months. The TBI prescribed dose was 12 Gy in six fractions for three days. Patients' ovaries were shielded with cylinder-type lead blocks. The dose-volume parameters (D98% and Dmean) in the ovaries and the pelvic bones were extracted from the dose-volume histogram (DVH). The mean ovary Dmean for all patients was 2.4 Gy, and 18 patients recovered menstruation (90%). The mean ovary Dmean for patients with menstrual recovery and without recovery were 2.4 Gy and 2.4 Gy, respectively, with no significant difference (P = 0.998). Hematological relapse was observed in five patients. The mean pelvis Dmean and pelvis D98% for relapse and non-relapse patients were 11.6 Gy and 11.7 Gy and 5.6 Gy and 5.3 Gy, respectively. Both parameters showed no significant difference (P = 0.827, 0.807). In conclusion, TBI with ovarian shielding reduced the radiation dose to the ovaries to 2.4 Gy, and preserved fertility without increasing the risk of relapse.

The aim of this study was to evaluate the dose-volume histogram parameters for late hematuria and rectal hemorrhage in patients receiving radiotherapy after radical prostatectomy.