Noriyuki Okonogi

Radiotherapy-induced anti-tumor immunity contributes to the therapeutic efficacy of irradiation and can be augmented by CTLA-4 blockade in a mouse model.

Purpose There is growing evidence that tumor-specific immune responses play an important role in anti-cancer therapy, including radiotherapy. Using mouse tumor models we demonstrate that irradiation-induced anti-tumor immunity is essential for the therapeutic efficacy of irradiation and can be augmented by modulation of cytotoxic T lymphocyte (CTL) activity. Methods and Materials C57BL/6 mice, syngeneic EL4 lymphoma cells, and Lewis lung carcinoma (LL/C) cells were used. Cells were injected into the right femurs of mice. Ten days after inoculation, tumors were treated with 30 Gy of local X-ray irradiation and their growth was subsequently measured. The effect of irradiation on tumor growth delay (TGD) was defined as the time (in days) for tumors to grow to 500 mm3 in the treated group minus that of the untreated group. Cytokine production and serum antibodies were measured by ELISA and flow cytometry. Results In the EL4 tumor model, tumors were locally controlled by X-ray irradiation and re-introduced EL4 cells were completely rejected. Mouse EL4-specific systemic immunity was confirmed by splenocyte cytokine production and detection of tumor-specific IgG1 antibodies. In the LL/C tumor model, X-ray irradiation also significantly delayed tumor growth (TGD: 15.4 days) and prolonged median survival time (MST) to 59 days (versus 28 days in the non-irradiated group). CD8(+) cell depletion using an anti-CD8 antibody significantly decreased the therapeutic efficacy of irradiation (TGD, 8.7 days; MST, 49 days). Next, we examined whether T cell modulation affected the efficacy of radiotherapy. An anti-CTLA-4 antibody significantly increased the anti-tumor activity of radiotherapy (TGD was prolonged from 13.1 to 19.5 days), while anti-FR4 and anti-GITR antibodies did not affect efficacy. Conclusions Our results indicate that tumor-specific immune responses play an important role in the therapeutic efficacy of irradiation. Immunomodulation, including CTLA-4 blockade, may be a promising treatment in combination with radiotherapy.

Cranial irradiation induces bone marrow-derived microglia in adult mouse brain tissue.

Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV–GFP mice, aged 8–12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

Carbon-ion beams induce production of an immune mediator protein, high mobility group box 1, at levels comparable with X-ray irradiation

X-ray radiotherapy activates tumor antigen-specific T-cell responses, and increases in the serum levels of high mobility group box 1 (HMGB1) induced by X-ray irradiation play a pivotal role in activating anti-tumor immunity. Here, we examined whether carbon-ion beams, as well as X-rays, can induce HMGB1 release from human cancer cell lines. The study examined five human cancer cell lines: TE2, KYSE70, A549, NCI-H460 and WiDr. The proportion of cells surviving X- or carbon-ion beam irradiation was assessed in a clonogenic assay. The D10, the dose at which 10% of cells survive, was calculated using a linear–quadratic model. HMGB1 levels in the culture supernatants were assessed by an ELISA. The D10 dose for X-rays in TE2, KYSE70, A549, NCI-H460 and WiDr cells was 2.1, 6.7, 8.0, 4.8 and 7.1 Gy, respectively, whereas that for carbon-ion beams was 0.9, 2.5, 2.7, 1.8 and 3.5 Gy, respectively. X-rays and carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of A549, NCI-H460 and WiDr cells at 72 h post-irradiation with a D10 dose. Furthermore, irradiation with X-rays or carbon-ion beams significantly increased HMGB1 levels in the culture supernatants of all five cell lines at 96 h post-irradiation. There was no significant difference in the amount of HMGB1 induced by X-rays and carbon-ion beams at any time-point (except at 96 h for NCI-H460 cells); thus we conclude that comparable levels of HMGB1 were detected after irradiation with iso-survival doses of X-rays and carbon-ion beams.

A novel strategy of radiofrequency hyperthermia (neothermia) in combination with preoperative chemoradiotherapy for the treatment of advanced rectal cancer: a pilot study

The safety of weekly regional hyperthermia performed with 8 MHz radiofrequency (RF) capacitive heating equipment has been established in rectal cancer. We aimed to standardize hyperthermia treatment for scientific evaluation and for assessing local tumor response to RF hyperthermia in rectal cancer. Forty‐nine patients diagnosed with rectal adenocarcinoma were included in the study. All patients received chemoradiation with intensity‐modulated radiation therapy 5 days/week (dose, 50 Gy/25 times) concomitant with 5 days/week for five times of capecitabine (1700 mg/m2 per day) and once a week for five times of 50 min irradiations by an 8 MHz RF capacitive heating device. Thirty‐three patients underwent surgery 8 weeks after treatment. Three patients did not undergo surgery because of progressive disease (PD) and 13 refused. Eight (16.3%) patients had a pathological complete response (ypCR) after surgery. Among patients without surgery, 3 (6.1%) had clinical complete response (CR) and 3 (6.1%) had local CR but distant PD (CRPD). Ninety percent of ypCR + CR patients were shown in 6.21 W min−1 m−2/treatment or higher group of average total accumulated irradiation output with 429°C min−1 m−2 or higher group of total accumulated thermal output. However, a patient with CRPD was in the higher total accumulated thermal output group. We propose a new quantitative parameter for the hyperthermia and demonstrated that patients can benefit from mild irradiation with mild temperature. Using these parameters, the exact output, optimal thermal treatment, and contraindications or indications of this modality could be determined in a multi‐institutional, future study.

Visualization of complex DNA double-strand breaks in a tumor treated with carbon ion radiotherapy.

Carbon ion radiotherapy shows great potential as a cure for X-ray-resistant tumors. Basic research suggests that the strong cell-killing effect induced by carbon ions is based on their ability to cause complex DNA double-strand breaks (DSBs). However, evidence supporting the formation of complex DSBs in actual patients is lacking. Here, we used advanced high-resolution microscopy with deconvolution to show that complex DSBs are formed in a human tumor clinically treated with carbon ion radiotherapy, but not in a tumor treated with X-ray radiotherapy. Furthermore, analysis using a physics model suggested that the complexity of radiotherapy-induced DSBs is related to linear energy transfer, which is much higher for carbon ion beams than for X-rays. Visualization of complex DSBs in clinical specimens will help us to understand the anti-tumor effects of carbon ion radiotherapy.

Radio-sensitization effect of an mTOR inhibitor, temsirolimus, on lung adenocarcinoma A549 cells under normoxic and hypoxic conditions

The mammalian target of rapamycin (mTOR) correlates with cell survival under hypoxia and regulates hypoxia-inducible factor-1α (HIF-1α), a key protein in hypoxia-related events. However, the role of mTOR in radio-resistance has not been fully investigated. Therefore, the effect of mTOR on the radio-resistance of cancer cells under hypoxia was evaluated using the mTOR inhibitor temsirolimus. Clonogenic survival was examined in the A549 human lung adenocarcinoma cell line under normoxia or hypoxia, with or without temsirolimus. An oxygen enhancement ratio (OER) was calculated using the D10 values, the doses giving 10% survival. Western blotting was performed to investigate the effect of temsirolimus on mTOR and the HIF-1α pathway under normoxia and hypoxia. A549 cells showed a radio-resistance of 5.1 and 14.2 Gy, as indicated by D10 values under normoxia and hypoxia, respectively; the OER was 2.8. The cell survival rates under hypoxia and with temsirolimus remarkably decreased compared with those under normoxia. The D10 values of the cells under normoxia and hypoxia were 4.8 and 5.4 Gy, respectively (OER = 1.1). mTOR expression was suppressed by temsirolimus under both normoxia and hypoxia. HIF-1α expression decreased under hypoxia in the presence of temsirolimus. These results suggest that temsirolimus can overcome the radio-resistance induced by hypoxia. When the fact that mTOR acts upstream of HIF-1α is considered, our data suggest that the restoration of radiation sensitivity by temsirolimus under hypoxia may be associated with the suppression of the HIF-1α pathway. Temsirolimus could therefore be used as a hypoxic cell radio-sensitizer.

Changes in Bone Mineral Density in Uterine Cervical Cancer Patients After Radiation Therapy

PURPOSE To prospectively investigate the changes in bone mineral density (BMD) after pelvic radiation therapy in patients with uterine cervical cancer. METHODS AND MATERIALS Of 52 cervical cancer patients who received pelvic RT in our university hospital between 2009 and 2011, 46 patients without recurrence and who were followed up for more than 12 months were included in the study. The BMD of the irradiated region and nonirradiated regions, serum estradiol, tartrate-resistant acid phosphatase-5b, and N-terminal cross-linking telopeptide of collagen 1 were measured before, at 3 months after, and at 12 months after RT. The patient cohort was divided into 2 groups according to estradiol level before RT, and the groups were defined as postmenopausal (<40 pg/mL) and premenopausal (≥40 pg/mL). RESULTS The mean BMDs within the irradiation field (lumbar vertebra 5) in the postmenopausal and the premenopausal groups were 0.825 and 0.910 g/cm(2) before RT and 0.746 and 0.841 g/cm(2) 12 months after RT, respectively. Significant decreases were observed in both groups (P<.05 and P<.01, respectively). In addition, in the premenopausal group the mean BMDs of the nonirradiated regions at thoracic vertebrae 9-12 and lumbar vertebrae 2-4 were 0.753 and 0.958 g/cm(2) before RT and were significantly decreased to 0.706 and 0.921 g/cm(2) 12 months after RT (P<.01 and P<.05, respectively). Estradiol significantly decreased 3 months after RT, whereas tartrate-resistant acid phosphatase-5b and N-terminal cross-linking telopeptide of collagen 1 continued to increase over time in the premenopausal group. CONCLUSIONS A decrease in BMD in the irradiated region after RT was observed within 1 year, regardless of menopausal status. Furthermore, in premenopausal patients, pelvic RT caused a decrease in systemic BMD.

HLA class I expression and its alteration by preoperative hyperthermo-chemoradiotherapy in patients with rectal cancer.

Objective Enhancing immunologic responses, including human leukocyte antigen (HLA) class I expression on tumor cells and recognition and elimination of tumor cells by tumor-specific cytotoxic T lymphocyte (CTL), is considered a novel concept of radiotherapy. The present study examined patients who underwent preoperative hyperthermo-chemoradiotherapy (HCRT) for locally advanced rectal cancer to assess the correlation between HLA class I expression and clinical outcome. Materials and Methods Seventy-eight patients with locally advanced rectal adenocarcinoma who received preoperative HCRT were enrolled. The median age of the patients was 64 years (range, 33–85 years) and 4, 18, and 56 patients had clinical stage I, II and III disease, respectively. Formalin-fixed and paraffin-embedded tissues excised before and after HCRT were subjected to immunohistochemical analysis with an anti-HLA class I-A, B, C antibody. HLA class I expression was graded according to tumor cell positivity. Results In pre-HCRT, the number of specimens categorized as Grade 0 and 1 were 19 (24%) and 58 (74%), respectively. Only 1 patient (1%) showed Grade 2 expression. However, 6 (8%), 27 (35%), 7 (9%), and 12 (15%) post-HCRT specimens were graded as Grade 0, 1, 2, and 3, respectively. There was a significant increase in HLA class I expression in post-HCRT specimens (p<0.01). However, neither pre- nor post-HCRT HLA class I expression affected overall survival and distant metastasis-free survival in clinical stage III patients. Univariate analysis revealed that Post-HCRT HLA class I expression showed a significant negative relationship with LC (p<0.05). Nevertheless, multivariate analysis showed that there was no correlation between HLA class I expression and clinical outcome. Conclusion HCRT increased HLA class I expression in rectal cancer patients. However, multivariate analysis failed to show any correlation between the level of HLA class I expression and prognosis.

Carbon-ion radiotherapy for locally advanced cervical cancer with bladder invasion

The purpose of this study was to evaluate the efficacy and toxicities of carbon-ion radiotherapy (C-ion RT) for locally advanced cervical cancer with bladder invasion by a subset analysis of pooled data from eight prospective clinical trials at the National Institute of Radiological Sciences. Between June 1995 and January 2014, 29 patients with locally advanced cervical cancer with bladder invasion were identified. The median age was 56 years old (range 31–79 years old). The median tumor size at diagnosis on magnetic resonance imaging was 6.7 cm (range 3.5–11.0 cm). Histologically, 20 patients had squamous cell carcinoma and 9 had adenocarcinoma. C-ion RT was performed as a dose-escalation study in the initial trials. All patients received prophylactic whole-pelvic or extended-field irradiation and local boost. The total dose to the cervical tumor was 52.8–74.4 Gy (relative biological effectiveness) in 20 or 24 fractions. Weekly cisplatin (40 mg/m2/week, five cycles) was concurrently given to four patients. The median follow-up of all patients was 28.6 months (range 8.8–238.6 months). Grade 2 or higher late complications in the bladder were observed in eight patients, with seven developing vesicovaginal fistula. Six patients had Grade 2 or higher complications in the rectosigmoid colon. The 3-year overall survival rate was 47%, the 3-year local control rate was 66%, and the 3-year disease-free survival rate was 28%. In this study, C-ion RT showed favorable local control with reasonable toxicities, but the results were still unsatisfactory. We have the expectation of improvement of therapeutic effects by using C-ion RT with concurrent chemotherapy.

Output-limiting symptoms induced by radiofrequency hyperthermia. Are they predictable?

Abstract Background: During radiofrequency (RF) hyperthermia treatment, hot-spot phenomena may occur and prevent treatment continuation if the output is not lowered. We previously reported a significant correlation between the initial energy output at which output-limiting symptoms occurred and patient status. Patients with a complete clinical response had significantly increased temperature, while some patients with partial clinical response and stable disease had increased temperature, depending on the occurrence of output-limiting symptoms. To predict the initial energy output at which output-limiting symptoms occur, we performed multiple regression analysis with the parameters of patients’ physical status. Materials and methods: Hyperthermia alone or concomitant with chemotherapy and/or radiotherapy was applied in 62 patients with malignant disease for a total of 310 treatments with a Thermotron RF-8 between December 2011 and April 2014. Results: No output-limiting symptoms were shown in 65.5% of 310 treatments. Pain (29.7%), micturition desire (1.9%), skin discomfort (0.6%), subcutaneous induration (1.6%), cold sensation (0.6%), and nausea (0.3%) were reported in the 310 treatments. A good predictive equation for initial energy output at which output-limiting symptoms occur was determined with two parameters, initial time of an output-limiting symptom onset, and thickness of the fat of the abdominal wall. Multiple regression analysis showed an adjusted R2 = 0.99 and variance inflation factor < 2. Conclusions: We present a good predictive equation for initial energy output at which output-limiting symptoms occur. It is critical to prevent RF hyperthermia-induced output-limiting symptoms and establish new prevention strategies.