Radiation therapy has been long utilized as localized cancer treatment. Recent studies have also demonstrated that it has a distant effect by the enhanced immunity, but it rarely occurs. The purpose of this study was to investigate whether X-ray irradiation combined with anti-PD-L1 and anti-CTLA-4 antibodies (P1C4) provides a higher probability of this distant effect as well as enhanced local antitumor efficacy for osteosarcoma. LM8 mouse osteosarcoma cells were inoculated into both legs of C3H mice assigned to one of four groups, namely no treatment (No Tx), P1C4, X-ray irradiation (RAD) to the leg of one side, and combination (COMB) groups. Survival and treatment-related immune molecular changes were analyzed. Administration of P1C4 produced a tumor growth delay on day 30 in 18% of the mice. In contrast, combination therapy produced the strongest tumor growth inhibition not only at the irradiated tumor but also at unirradiated tumor in 67% of the mice. Accordingly, lung metastasis in the COMB group was strongly reduced by 98%, with a significant survival benefit. Unirradiated tumor in mice in the COMB group significantly recruited CD8 + tumor-infiltrating lymphocytes with a moderate reduction of Treg, producing a significant increase in the CD8/ Treg ratio. These results suggest that radiation enhances the efficacy of P1C4 treatment against distant metastasis as well as local control in osteosarcoma. Our data suggest that radiation therapy combined with dual checkpoint blockade may be a promising therapeutic option for osteosarcoma.

Carbon ion radiotherapy has been utilized even for X-ray resistant tumors. However, control of distant metastasis remains a major challenge in carbon ion irradiation. We investigated whether carbon ion irradiation combined with dual immune checkpoint blockade therapy (anti-PD-L1 and anti-CTLA-4 antibodies [P1C4]) provides anti-tumor efficacy for both local and distant sites. A mouse osteosarcoma cell line (LM8) was inoculated into both hind legs of C3H mice assigned to four groups: no treatment (NoTX), P1C4, 5.3 Gy of carbon ion irradiation to one leg (Cion), and combination (Comb) groups. In the Comb group, tumor growth delay was observed not only in the irradiated tumors but also in the unirradiated tumors. Notably, a complete response of unirradiated tumors was observed in 64% of mice in the Comb group, while only 20% of mice in the P1C4 group showed a complete response. Significant activation of immune cells was observed in the Comb group, with an increase in CD8+/GzmB+ tumor-infiltrating lymphocytes (TILs) in the irradiated tumor, and of CD8+/GzmB+ and CD4+ TILs in the unirradiated tumor, respectively. Depletion of CD8 abolished the tumor growth delay in unirradiated tumors in mice treated by Cion and P1C4. Overall survival was significantly prolonged in the Comb group. HMGB-1 release from irradiated tumors was significantly increased after Cion both in vitro and in vivo. These data suggest that carbon ion therapy enhances P1C4 efficacy against osteosarcoma in both the primary tumor and distant metastases mediated by immune activation.

BackgroundThe purpose of this study is to report our clinical outcomes using intensity-modulated radiation therapy (IMRT) for adjuvant treatment of cervical cancer, compared with three-dimensional conformal radiation therapy (3DCRT), in terms of tumor control, complications and dose-volume histogram (DVH) parameters.MethodsBetween March 2008 and February 2014, 62 patients were treated with concurrent nedaplatin-based chemotherapy and whole-pelvic external beam radiation therapy (RT). Of these patients, 32 (52 %) received 3DCRT and 30 (48 %) received IMRT.ResultsThe median follow-up periods were 40 months (range 2–74 months). The 3-year overall survival rate (OS), locoregional control rate (LRC) and progression-free survival rate (PFS) were 92, 95 and 92 % in the IMRT group, and 85, 82 and 70 % in the 3DCRT group, respectively. A comparison of OS, LRC and PFS showed no significant differences between IMRT and 3DCRT. The 3-year cumulative incidences of grade 2 or higher chronic gastrointestinal (GI) complications were significantly lower with IMRT compared to 3DCRT (3 % vs. 45 %, p < .02) and in patients with V40 of the small bowel loops of ≤340 mL compared to those with >340 mL (3 % vs. 45 %, p < .001). Patients treated with IMRT had a higher incidence of grade 3 acute hematologic complications (p < .05). V40 and V45 of the small bowel loops or bowel bag were predictive for development of both acute and chronic GI complications.ConclusionsOur results suggest that IMRT for adjuvant treatment of cervical cancer is useful for decreasing GI complications without worsening outcomes.

This study characterized a new unshielded diode detector, the microSilicon (model 60023), for small-field photon beam dosimetry by evaluating the photon beams generated by a TrueBeam STx and a CyberKnife. Temperature dependence was evaluated by irradiating photons and increasing the water temperature from 11.5 to 31.3°C. For Diode E, microSilicon, microDiamond and EDGE detectors, dose linearity, dose rate dependence, energy dependence, percent-depth-dose (PDD), beam profiles and detector output factor (OFdet) were evaluated. The OFdet of the microSilicon detector was compared to the field output factors of the other detectors. The microSilicon exhibited small temperature dependence within 0.4%, although the Diode E showed a linear variation with a ratio of 0.26%/°C. The Diode E and EDGE detectors showed positive correlations between the detector reading and dose rate, whereas the microSilicon showed a stable response within 0.11%. The Diode E and microSilicon demonstrated negative correlations with the beam energy. The OFdet of microSilicon was the smallest among all the detectors. The maximum differences between the OFdet of microSilicon and the field output factors of microDiamond were 2.3 and 1.6% for 5 × 5 mm2 TrueBeam and 5 mm φ CyberKnife beams, respectively. The PDD data exhibited small variations in the dose fall-off region. The microSilicon and microDiamond detectors yielded similar penumbra widths, whereas the other detectors showed steeper penumbra profiles. The microSilicon demonstrated favorable characteristics including small temperature and dose rate dependence as well as the small spatial resolution and output factors suitable for small field dosimetry.

Abstract. Osteosarcoma is the most common primary bone malignancy in pediatric and adolescent populations. Recurrence and metastatic potential can be due to a subpopulation of cells with stem cell-like characteristics, such as tumor-initiating cells (TICs), which maintain the capacity to regenerate entire tumors. Targeting the TICs in osteosarcoma is a promising avenue for the development of new therapies for this devastating disease. TICs are usually quiescent with a low protein turnover, decreased metabolism, and downregulation of proteasome activity. Recently, cancer cells with low proteasome activity have been identified as TICs in several types of cancer. We stably infected two osteosarcoma cell lines, MG-63 and U2-OS, with an expression vector for a fusion protein between the green fluorescent protein, ZsGreen, and the C-terminal degron of the murine ornithine decarboxylase to monitor the 26S proteasome activity in living cells. We separated the osteosarcoma cells with low proteasome activity using fluorescence-activated cell sorting (FACS) and verified whether these ZsGreen + cells had TIC-like properties. The ZsGreen + cells showed enhanced sphere formation capacity and underwent asymmetric divisions into ZsGreen + and ZsGreen -cells, whereas ZsGreen -cells underwent only symmetric divisions into ZsGreen -cells. Moreover, the ZsGreen + cells were more chemo-and radioresistant. Thus, the present study demonstrated that chemoradiation-resistant TICs can be visualized by this system and suggested the rationale for further study of osteosarcoma stem cells. IntroductionOsteosarcoma is the most common primary bone malignancy in children and young adults. Osteosarcoma occurs in the long bones of the limbs, particularly in the distal femur and proximal tibia. Osteosarcoma is a locally aggressive tumor and tends to produce early distant metastases, particularly to the lung. Before 1970, amputation was the only treatment for osteosarcoma patients and 80% patients died of metastatic disease (1). Since the 1970s, the combination of limb-sparing surgery and conventional chemotherapy agents, including methotrexate (MTX), cisplatin (CDDP), and doxorubicin, has been used to treat osteosarcoma. However, the 5-year patient survival has plateaued at ~60-70% (2).Tumors are organized into a hierarchy of heterogeneous cell populations. Recurrence and metastatic potential may be due to a subpopulation of cells with stem cell-like characteristics, such as cancer stem cells (CSCs) or tumor-initiating cells (TICs), which maintain the capacity to regenerate entire tumors (3). Targeting the TICs in osteosarcoma may be a promising avenue to explore for the development of new therapies for this devastating disease.Increasing evidence of the existence of TICs in patients with osteosarcoma has been reported. Identification of osteosarcoma TICs has been performed using CD133 (4,5), side populations (6,7), PKH26 (8), ALDH1 (9,10), and the promoter reporter assays of hTERT (11)