Eric T. Shinohara

Lithium treatment prevents neurocognitive deficit resulting from cranial irradiation.

Curative cancer treatment regimens often require cranial irradiation, resulting in lifelong neurocognitive deficiency in cancer survivors. This deficiency is in part related to radiation-induced apoptosis and decreased neurogenesis in the subgranular zone of the hippocampus. We show that lithium treatment protects irradiated hippocampal neurons from apoptosis and improves cognitive performance of irradiated mice. The molecular mechanism of this effect is mediated through multiple pathways, including Akt/glycogen synthase kinase-3beta (GSK-3beta) and Bcl-2/Bax. Lithium treatment of the cultured mouse hippocampal neurons HT-22 induced activation of Akt (1.5-fold), inhibition of GSK-3beta (2.2-fold), and an increase in Bcl-2 protein expression (2-fold). These effects were sustained when cells were treated with lithium in combination with ionizing radiation. In addition, this combined treatment led to decreased expression (40%) of the apoptotic protein Bax. The additional genes regulated by lithium were identified by microarray, such as decorin and Birc1f. In summary, we propose lithium treatment as a novel therapy for prevention of deleterious neurocognitive consequences of cranial irradiation.

DNA-Dependent Protein Kinase Is a Molecular Target for the Development of Noncytotoxic Radiation–Sensitizing Drugs

DNA-dependent protein kinase (DNA-PK)-defective severe combined immunodeficient (SCID) mice have a greater sensitivity to ionizing radiation compared with wild-type mice due to deficient repair of DNA double-strand break. SCID cells were therefore studied to determine whether radiosensitization by the specific inhibitor of DNA-PK, IC87361, is eliminated in the absence of functional DNA-PK. IC87361 enhanced radiation sensitivity in wild-type C57BL6 endothelial cells but not in SCID cells. The tumor vascular window model was used to assess IC87361-induced radiosensitization of SCID and wild-type tumor microvasculature. Vascular density was 5% in irradiated SCID host compared with 50% in C57BL6 mice (P < 0.05). IC87361 induced radiosensitization of tumor microvasculature in wild-type mice that resembled the radiosensitive phenotype of tumor vessels in SCID mice. Radiosensitization by IC87361 was eliminated in SCID tumor vasculature, which lack functional DNA-PK. Irradiated LLC and B16F0 tumors implanted into SCID mice showed greater tumor growth delay compared with tumors implanted into either wild-type C57BL6 or nude mice. Furthermore, LLC tumors treated with radiation and IC87361 showed tumor growth delay that was significantly greater than tumors treated with radiation alone (P < 0.01 for 3 Gy alone versus 3 Gy + IC87361). DNA-PK inhibitors induced no cytotoxicity and no toxicity in mouse normal tissues. Mouse models deficient in enzyme activity are useful to assess the specificity of novel kinase inhibitors. DNA-PK is an important target for the development of novel radiation-sensitizing drugs that have little intrinsic cytotoxicity.

RADIATION THERAPY IS ASSOCIATED WITH IMPROVED SURVIVAL IN THE ADJUVANT AND DEFINITIVE TREATMENT OF INTRAHEPATIC CHOLANGIOCARCINOMA

PURPOSE Intrahepatic cholangiocarcinomas (IHC) are rare tumors for which large randomized studies regarding the use of radiation are not available. The purpose of this study was to examine the role of adjuvant and definitive radiation therapy in the treatment of IHC in a large group of patients. METHODS AND MATERIALS This is a retrospective analysis of 3,839 patients with IHC collected from the Surveillance, Epidemiology, and End Results (SEER) database. The primary endpoint was overall survival (OS). RESULTS Patients received either surgery alone (25%), radiation therapy alone (10%), surgery and adjuvant radiation therapy (7%) or no treatment (58%). The median age of the patient population was 73 years (range, 22-102 years); 52% of patients were male and 81% were Caucasian. Median OS was 11 (95% confidence interval [CI], 9-13), 6 (95% CI, 5-6), 7 (95% CI, 6-8), and 3 months for surgery and adjuvant radiation therapy, surgery alone, radiation therapy alone, and no treatment, respectively. The OS was significantly different between surgery alone and surgery and adjuvant radiation therapy (p = 0.014) and radiation therapy alone and no treatment (p < 0.0001). Use of surgery and adjuvant radiation therapy conferred the greatest benefit on OS (HR = 0.40; 95% CI, 0.34-0.47), followed by surgery alone (hazard ratio [HR], 0.49; 95% CI, 0.44-0.54) and radiation therapy alone (HR, 0.68; 95% CI, 0.59-0.77) compared with no treatment, on multivariate analysis. Propensity score adjusted hazard ratios (controlling for age, race/ethnicity, stage, and year of diagnosis) were also significant (surgery and adjuvant radiation therapy vs. surgery alone (HR, 0.82; 95% CI, 0.70-0.96); radiation therapy alone vs. no treatment (HR, 0.67; 95% CI, 0.58-0.76)). CONCLUSIONS The study results suggest that adjuvant and definitive radiation treatment prolong survival, although cure rates remain low. Future studies should evaluate the addition of chemotherapy and biologics to the treatment of IHC.

Radiosensitization of lung cancer by nutlin, an inhibitor of murine double minute 2.

p53 plays a critical role in cell cycle arrest and induction of apoptosis. Certain malignancies carry wild-type p53, which is frequently down-regulated by murine double minute 2 (MDM2) overexpression. Availability of a small-molecule inhibitor against MDM2, nutlin, has made it feasible to evaluate the anti-MDM2-based therapeutic strategies. The rationale for the current study is that functional p53 has been linked with improved responses to radiation treatment. Hence, this study evaluates the use of nutlin, a small-molecule inhibitor that blocks the interaction of p53 and MDM2, in sensitizing cancer cells to radiation. Expression of MDM2, p53, and p21 in both p53 wild-type and p53-defective lung cancer cell lines was examined. Clonogenic and 7-amino-actinomycin D studies were used to determine possible mechanisms of cell death. The combined effect of MDM2 inhibition and radiation on cell cycle was also studied. We found that radiosensitization by nutlin occurs in lung cancer cells with wild-type p53. There were increased apoptosis and cell cycle arrest following administration of nutlin and radiation. Furthermore, the combination of nutlin and radiation decreased the ability of endothelial cells to form vasculature, as shown by Matrigel assays. Our data suggest that nutlin is an effective radiosensitizer of p53 wild-type cells. The radiosensitizing effect seems to be at least partially due to induction of apoptosis and cell cycle arrest. In addition, nutlin may be an effective radiosensitizer of tumor vasculature. [Mol Cancer Ther 2006;5(2):411–7]

Radiotherapy Is Associated With Improved Survival in Adjuvant and Palliative Treatment of Extrahepatic Cholangiocarcinomas

PURPOSE Extrahepatic cholangiocarcinomas (EHC) are rare tumors of the biliary tree because of their low incidence, large randomized studies examining radiotherapy (RT) for EHC have not been performed. The purpose of this study was to examine the role of adjuvant and palliative RT in the treatment of EHC in a large patient population. METHODS AND MATERIALS This was a retrospective analysis of 4,758 patients with EHC collected from the Surveillance, Epidemiology, and End Results database. The primary endpoint was overall survival. RESULTS Patients underwent surgery (28.8%), RT (10.0%), surgery and RT (14.7%), or no RT or surgery (46.4%). The median age of the patient population was 73 years (range, 23-104), 52.5% were men, and 80.7% were white. The median overall survival time was 16 months (95% confidence interval [CI] 15-17), 9 months (95% CI 9-11), 9 months (95% CI 9-10), and 4 months (95% CI 3-4) for surgery and RT, surgery, RT, and no RT or surgery, respectively. The overall survival was significantly different between the surgery and surgery and RT groups (p < .0001) and RT and no RT or surgery groups (p < .0001) on the log-rank test. The propensity score-adjusted analyses of surgery and RT vs. surgery (hazard ratio, 0.94; 95% CI, 0.84-1.05) were not significantly different, but that for RT vs. no RT or surgery (hazard ratio, 0.61; 95% CI, 0.54-0.70) was significantly different. CONCLUSION These results suggest that palliative RT prolongs survival in patients with EHC. The benefit associated with surgery and RT was significant on univariate analysis but not after controlling for potential confounders using the propensity score. Future studies should evaluate the addition of chemotherapy and biologic agents for the treatment of EHC.

Inhibition of signal transducer and activator of transcription 3 activity results in down-regulation of Survivin following irradiation

Signal transducer and activator of transcription 3 (Stat3) and Survivin are constitutively up-regulated in various human tumor cells. We previously found Survivin to be significantly reduced in response to radiation in human umbilical vein endothelial cells (HUVEC) but not in tumor cell lines. In this study, we examined the effect of Stat3 on Survivin expression in irradiated HUVECs and breast cancer cells. We also studied how inhibition of Stat3 and Survivin activity affects cell survival and angiogenesis following irradiation. We determined that Survivin was significantly increased by overexpression of an active Stat3 (Stat3-C). Following irradiation, the level of phospho-Stat3 Tyr705, but not phospho-Stat3 Ser727, was reduced in HUVECs, whereas it remained unchanged in irradiated breast cancer cells. Correspondingly, Stat3 DNA-binding activity following irradiation was specifically down-regulated in HUVECs but not in breast cancer cells. Mutation of Tyr705 abolished radiation-induced down-regulation of Survivin. Clonogenic and endothelial cell morphogenesis assays suggested that DN-Stat3 and DN-Survivin together resulted in the greatest radiosensitization of MDA-MB-231, decreasing angiogenesis and cell survival. In summary, Stat3 modulates Survivin, and both are potential therapeutic targets for radiation sensitization in breast cancer. [Mol Cancer Ther 2006;5(11):2659–65]

Increasing Sensitivity to Radiotherapy and Chemotherapy by Using Novel Biological Agents that Alter the Tumor Microenvironment

Sensitivity to radiation and chemotherapy can be influenced by factors extrinsic to the cancer cell. For example, severely hypoxic cells require 2-3 times the radiation dose as do well-oxygenated cells to achieve similar cell killing. Apart from the tumor cells, neighboring cells such as endothelial cells may influence radiosensitivity. Irradiation can lead to expression of molecules that may increase radio/chemoresistance, for example vascular endothelial growth factor (VEGF), a secreted protein that regulates angiogenesis, or hypoxia inducible factor-1 alpha (HIF-1 alpha), a master transcription factor that regulates gene expression in hypoxia. Hence, response to cytotoxic therapy may be improved by modulating the tumor microenvironment (TME). Several agents in clinical use may do this. Some of these target vasculature, either directly or indirectly by disrupting VEGF and/or HIF-1 signaling. Many of these agents have been shown to increase radio/chemosensitivity in preclinical models. A confounding factor in terms of radiosensitization is the variable effect of these drugs on tumor oxygenation. Some of these agents ablate the vasculature, thereby increasing hypoxia. Others may normalize tumor vasculature, leading to increased blood flow and oxygenation, thereby potentially increasing radiosensitivity and the delivery of chemotherapy. Inhibitors of EGFR signaling and the PI3K/Akt pathway can also cause similar vascular changes in preclinical models, perhaps by indirectly inhibiting VEGF secretion. In summary, agents are currently available in the clinic that might modulate the TME in a way that could improve radio/chemosensitivity. The challenge is to show that this occurs in human patients and then use this information to optimize cancer therapy.

Active integration: new strategies for transgenesis

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed “Active Transgenesis” to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac’s specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

The Role of Radiation Therapy in Malignant Thymoma: A Surveillance, Epidemiology, and End Results Database Analysis

Introduction: The potential benefits and long-term complications of radiotherapy treatment for malignant thymoma are unclear. This is a retrospective analysis of outcome in patients with malignant thymoma from the Surveillance, Epidemiology, and End Results database between 1973 and 2005. Methods: Of the 1987 patients identified, 1334 were analyzed. Patients were categorized according to the Masaoka staging system as stage I to IIA, IIB or III to IV. The primary end points were overall survival, cardiac mortality, and the development of secondary malignancies. Results: Patients received surgery and radiation (50%), surgery alone (26%), radiation alone (12%), or no treatment (12%). The median follow-up time for survivors was 65 months (range, 1–361 months). There was no significant increase in the 12-year cumulative incidence rate of death from heart disease (10.2% radiation versus 7.5% no radiation, p = 0.83) or incidence of secondary malignancies (11.7% versus 12.4%, p = 0.70) with radiation. Compared with surgery alone, adjuvant radiation significantly improved overall survival in patients with stage III to IV disease (p = 0.04) and demonstrated a nonsignificant trend in patients with stage IIB disease (p = 0.09). However, after excluding patients surviving less than 4 months to account for surgical mortality, the benefit with radiation was no longer significant (stage IIB: p = 0.45, stage III–IV: p = 0.44). Conclusions: Radiation does not seem to increase the risk of cardiac mortality or secondary malignancy in patients with malignant thymoma. Although the routine use of postoperative radiotherapy in malignant thymoma does not appear warranted, high-risk patients may benefit from adjuvant radiation. This study can help to design prospective trials to further establish the role of radiotherapy in malignant thymoma.

Interdigitating and follicular dendritic cell sarcomas: a SEER analysis.

Objectives: Follicular dendritic cell sarcoma (FDCS) and interdigitating dendritic cell sarcoma (IDCS) are rare neoplasms of dendritic cell origin. Because of the rarity of these diagnoses, optimal management is unclear. Methods: In this study, we reviewed the data on FDCS and IDCS available in the Surveillance, Epidemiology, and End Results database. Fifty-four patients with FDCS and 20 with IDCSs were identified between the years 2001 and 2008. Results: Median follow-up was 28 months. Sixty-one percent of FDCS patients and 55% of IDCS patients presented with localized disease. Of the FDCS patients with localized disease, 31/33 (94%) underwent surgical resection. Fifty-five percent (6/11) of localized IDCS patients underwent surgical resection. Radiation therapy was given to 30% of patients. Overall survival was significantly better for patient with FDCS compared to those with IDCS. Median survival was 35 months in patients with IDCS and was not reached in patients with FDCS. There was a trend toward improved overall survival in FDCS patients with localized disease. IDCS patients with localized disease had a significantly improved overall survival compared with those with distant disease with 2-year overall survival of 72% versus 33%, respectively (P=0.05). Conclusions: These data demonstrate that most patients with localized disease are treated similar to a soft tissue sarcoma with primary surgical resection with or without radiation. No chemotherapy data were available in the Surveillance, Epidemiology, and End Results database. The role of chemotherapy and radiation therapy remains unclear.