Extracorporeal irradiation of tumorous calvaria: a case series.

OBJECT When intracranial tumors invade the overlying skull, gross resection typically includes removal of the involved bone. Methods used to repair the resulting structural defect in the cranium include artificial prostheses, allogeneic bone grafts, and autoclaving the autologous graft. The authors have previously reported a case involving high-dose extracorporeal ionizing radiation to treat the tumorous calvaria intraoperatively, followed by reimplantation of the treated bone flap. In this paper the authors report the long-term follow-up of that case, as well as results of using extracorporeal irradiation of tumorous calvaria (EITC) for an additional 20 patients treated similarly. METHODS The decision to undergo EITC was typically anticipated preoperatively, but determined intraoperatively, if upon inspection the bone flap was invaded by tumor. The bone flap was then delivered to the radiation oncology department, where a total dose of 120 Gy was delivered, using a clinical linear accelerator, over a period of approximately 15 minutes. After the intracranial tumor resection was completed, the irradiated craniotomy bone flap was reimplanted and the wound was closed in a standard fashion. A retrospective review of patients who had undergone EITC was performed for evidence of calvarial tumor recurrence or other complications. RESULTS Since the originally reported case, 20 additional patients have received EITC during craniotomy for invasive tumors. Eighteen (86%) of 21 patients were diagnosed with meningioma: 12 (67%) with WHO Grade I, 5 (28%) with WHO Grade II, and 1 with WHO Grade III (6%). The remaining 3 patients presented with dural-based B-cell lymphoma with extensive adjacent bone invasion (n = 2) and metastatic adenocarcinoma of the lung (n = 1). Follow-up of the 21 patients ranged from 1 to 132 months, with a mean of 41 months and a median of 23 months. No patients have experienced tumor recurrence, infection associated with the treated calvaria, or evidence of bone flap resorption. CONCLUSIONS Calvaria reconstructions represent an important component in structural and cosmetic outcome following craniectomy for tumorous bone. The authors' long-term experience with EITC has been excellent with no local tumor recurrence or complications. Therefore, EITC represents an excellent and efficient option for cranial reconstruction in such patients.

The Influence of Hypoxia and pH on Bioluminescence Imaging of Luciferase-Transfected Tumor Cells and Xenografts.

Bioluminescence imaging (BLI) is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS) intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment.

Nicotine enhances proliferation, migration, and radioresistance of human malignant glioma cells through EGFR activation.

It has been suggested that continued tobacco use during radiation therapy contributes to maintenance of neoplastic growth despite treatment with radiation. Nicotine is a cigarette component that is an established risk factor for many diseases, neoplastic and otherwise. The hypothesis of this work is that nicotine promotes the proliferation, migration, and radioresistance of human malignant glioma cells. The effect of nicotine on cellular proliferation, migration, signaling, and radiation sensitivity were evaluated for malignant glioma U87 and GBM12 cells by use of the AlamarBlue, scratch healing, and clonogenic survival assays. Signal transduction was assessed by immunoblotting for activated EGFR, ERK, and AKT. At concentrations comparable with those found in chronic smokers, nicotine induced malignant glioma cell migration, growth, colony formation, and radioresistance. Nicotine increased phosphorylation of EGFR(tyr992), AKT(ser473), and ERK. These molecular effects were reduced by pharmacological inhibitors of EGFR, PI3K, and MEK. It was therefore concluded that nicotine stimulates the malignant behavior of glioma cells in vitro by activation of the EGFR and downstream AKT and ERK pathways.

Subcutaneous administration of D-luciferin is an effective alternative to intraperitoneal injection in bioluminescence imaging of xenograft tumors in nude mice.

Currently, intraperitoneal (IP) injection of D-luciferin is the preferred method of providing substrate for bioluminescent imaging (BLI); however it has a failure rate of 3-10% due to accidental intestinal injection. The present study evaluates the quality of BLI after subcutaneous (SC) injection of D-luciferin and demonstrates the effectiveness of SC injection in anatomically disparate tumor models. Mice bearing luciferase-expressing tumors underwent BLI after SC or IP injection of D-luciferin. The average time to maximal luminescence was 6 min (range 5-9 min) after SC injection and 8 min (range 5-8 min) after IP injection. Within 7 minutes of injection, SC and IP routes yielded similar luminescence in subcutaneous, intracranial, tongue, and lung xenograft tumor models. In a model of combined subcutaneous and intracranial xenografts, SC injection resulted in proportional luminescence at all sites, confirming that preferential delivery of substrate does not occur. While tumors were occasionally not visualized with IP injection, all tumors were visualized reliably with SC injection. Thus, SC injection of D-luciferin is a convenient and effective alternative to IP injection for BLI in nude mice. It may be a preferable approach, particularly for tumors with weaker signals and/or when greater precision is required.

Neural stem cell-preserving external-beam radiotherapy of central nervous system malignancies.

PURPOSE: Recent discoveries have implicated neural stem cells (NSC) as the source of plasticity and repair in the mature mammalian brain. Treatment-induced NSC dysfunction may lead to observed toxicity. This study evaluates the feasibility of NSC-preserving external beam radiotherapy. METHODS AND MATERIALS: A single computed tomography (CT) dataset depicting a right periventricular lesion was used in this study as this location reflects the most problematic geometric arrangement with respect to NSC preservation. Conventional and NSC preserving radiotherapy (RT) plans were generated for the same lesion using two clinical scenarios: cerebral metastatic disease and primary high-grade glioma. Disease-specific target volumes were used. Metastatic disease was conventionally treated with whole-brain radiotherapy (WBRT) to 3,750 cGy (15 fractions) followed by a single stereotactic radiosurgery (SRS) boost of 1,800 cGy to gross disease only. High-grade glioma was treated with conventional opposed lateral and anterior superior oblique beams to 4,600 cGy (23 fractions) followed by a 1,400 cGy (7 fractions) boost. NSC preservation was achieved in both scenarios with inverse-planned intensity modulated radiotherapy (IMRT). RESULTS: Cumulative dose reductions of 65% (metastatic disease) and 25% (high-grade glioma) to the total volume of the intracranial NSC compartments were achieved with NSC-preserving IMRT plans. The reduction of entry and exit dose to NSC niches located contralateral to the target contributed most to NSC preservation. CONCLUSIONS: Neural stem cells preservation with current external beam radiotherapy techniques is achievable in context of both metastatic brain disease and high-grade glioma, even when the target is located adjacent to a stem cell compartment. Further investigation with clinical trials is warranted to evaluate whether NSC preservation will result in reduced toxicity.

Audio-visual biofeedback for respiratory-gated radiotherapy: impact of audio instruction and audio-visual biofeedback on respiratory-gated radiotherapy.

PURPOSE: Respiratory gating is a commercially available technology for reducing the deleterious effects of motion during imaging and treatment. The efficacy of gating is dependent on the reproducibility within and between respiratory cycles during imaging and treatment. The aim of this study was to determine whether audio-visual biofeedback can improve respiratory reproducibility by decreasing residual motion and therefore increasing the accuracy of gated radiotherapy. METHODS AND MATERIALS: A total of 331 respiratory traces were collected from 24 lung cancer patients. The protocol consisted of five breathing training sessions spaced about a week apart. Within each session the patients initially breathed without any instruction (free breathing), with audio instructions and with audio-visual biofeedback. Residual motion was quantified by the standard deviation of the respiratory signal within the gating window. RESULTS: Audio-visual biofeedback significantly reduced residual motion compared with free breathing and audio instruction. Displacement-based gating has lower residual motion than phase-based gating. Little reduction in residual motion was found for duty cycles less than 30%; for duty cycles above 50% there was a sharp increase in residual motion. CONCLUSIONS: The efficiency and reproducibility of gating can be improved by: incorporating audio-visual biofeedback, using a 30-50% duty cycle, gating during exhalation, and using displacement-based gating.

Stereotactic body radiation therapy of lung tumors: preliminary experience using normal tissue complication probability-based dose limits.

OBJECTIVES: To assess the feasibility and toxicity of stereotactic body radiotherapy (SBRT) for patients with locally advanced or metastatic tumors in lung. METHODS: Twenty-five tumors in 17 patients were treated. All treatments were delivered in 3 daily fractions of 9 to 15 Gy per fraction. Normal tissue complication probability (NTCP) calculations (using the Lyman model) were performed to facilitate dose prescription, and doses were prescribed with a maximum allowable NTCP risk of pneumonitis of up to 20%, not to exceed 15 Gy per fraction. Planning target volumes were designed to allow for respiratory variation in tumor location. RESULTS: The median dose prescribed was 35 Gy (range, 24 to 45 Gy). Twenty-three of 25 tumors remained controlled at median follow-up of 14 months. Four patients experienced grade 1-2 acute toxicity. Late toxicity developed in 2 patients who received treatment to peri-hilar tumors, including one patient in whom bronchial stenosis developed with complete occlusion and lobar atelectasis 6 months after treatment. No patient had grade 3 or 4 radiation pneumonitis. CONCLUSIONS: SBRT prescribed within the confines of NTCP-restricted dosing on this protocol resulted in no radiation pneumonitis. Tissues other than lung parenchyma which are unaccounted for by NTCP may be dose-limiting when performing hypofractionated SBRT in the lung.

Inability of transforming growth factor-beta to cause SnoN degradation leads to resistance to transforming growth factor-beta-induced growth arrest in esophageal cancer cells.

It is well established that loss of a growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers including esophageal cancer. However, the molecular basis for the abrogation of this key homeostatic mechanism is poorly understood. In esophageal cancer cell lines that are resistant to TGF-beta-induced growth inhibition, TGF-beta also fails to decrease transcription of c-myc despite the presence of functional signaling components. Consequently, to gain a better understanding of the mechanisms leading to resistance to TGF-beta-induced growth arrest, the basis for the inability to decrease c-myc transcription was investigated. Regardless of sensitivity to TGF-beta-induced growth arrest, TGF-beta enhanced the ability of Smad3-protein complexes to bind c-myc regulatory elements. However, in a growth inhibition-resistant esophageal cancer cell line, the Smad3-protein complexes contained the SnoN oncoprotein. Furthermore, in esophageal cancer cell lines that are resistant to TGF-beta-induced growth arrest, TGF-beta does not cause degradation of SnoN. Analyses of the effect of modulating SnoN expression in both growth inhibition-sensitive and growth inhibition-resistant cell lines showed that degradation of SnoN is a prerequisite for both TGF-beta-induced repression of c-myc transcription and growth arrest. The data indicate that SnoN-Smad3 complexes do not cause repression of c-myc transcription but rather prevent functionality of active repressor complexes. Thus, these studies reveal a novel mechanism for resistance to TGF-beta-induced growth inhibition in esophageal cancer, namely the failure to degrade SnoN. In addition, they show that SnoN can block TGF-beta repression of gene transcription.

Molecular targeting in radiotherapy: epidermal growth factor receptor.

Radiation therapy is utilized as a treatment to cure or manage cancer; however, because of risk to local healthy tissue-and a modest success rate of some radiotherapy-strategies have been sought that would increase the therapeutic index of the treatment while reducing damage to surrounding tissue. Cell and tissue irradiation stimulates a series of biochemical and molecular signals; various components of this ionizing radiation (IR)-inducible signal transduction cascade can promote the survival of tumor cells. Identification of interactions between IR and a signaling pathway creates an opportunity to target those signaling intermediates to improve the outcome of radiotherapy. The epidermal growth factor receptor (EGFR, also termed ErbB1) is involved in normal development and differentiation of epithelial cells as well as in tumorigenesis. The EGFR is activated by IR, thus making this receptor and other members of the ErbB family important targets for radiosensitizing molecular interventions. Recent approaches have utilized monoclonal antibodies, small molecules, and transgenic technologies to undermine the kinase activity of EGFR.

Esophageal cancer: outcomes of surgery, neoadjuvant chemotherapy, and three-dimension conformal radiotherapy.

Neoadjuvant chemotherapy and radiation are being utilized with increasing frequency in the multimodal treatment of esophageal cancer, although their effects on morbidity, mortality, and survival remain unclear. The objective of this study was to determine the outcome of multimodal treatment in patients with localized esophageal cancer treated at a single institution. Between 1995 and 2002, 118 patients underwent treatment for localized esophageal cancer, utilizing surgery alone, chemoradiation alone, or surgery following neoadjuvant chemoradiation. There was no statistically significant difference in morbidity, mortality, or length of stay between the patients who received multimodal therapy when compared to surgery alone. A surgical resection after down-staging was possible in 9 out of 28 patients (32%) with a clinically non-resectable tumor (T4 or M1a). Forty-seven percent of the patients who received neoadjuvant therapy had a complete pathologic response with a 3-year survival of 59% as compared to only 20 months in those patients who did not achieve a complete response (P = 0.037). Neoadjuvant chemotherapy administered concomitantly with conformal radiotherapy can be performed safely in the treatment of esophageal cancer, without increasing the operative morbidity, mortality, or length of stay. The higher complete response rates to neoadjuvant treatment (as compared to other reports) may be due to the use of three-dimensional conformal radiation therapy or the novel use of weekly carboplatin and paclitaxel.

Transforming growth factor-beta is an endogenous radioresistance factor in the esophageal adenocarcinoma cell line OE-33.

Transforming growth factor (TGF)-beta has profound effects on epithelial cell differentiation and is capable of modulating the response to exposure to ionizing radiation. We recently reported that TGF-beta downregulates c-myc mRNA expression and inhibits the growth of OE-33 esophageal carcinoma cells in vitro. These studies investigate the role of TGF-beta in the in vitro radiation response of OE-33 and four other human esophageal cancer cell lines. TGF-beta enhanced radioresistance of OE-33 cells, but did not affect the radiosensitivity of either of the two other adenocarcinoma cell lines BIC1 and SEG1 or of squamous carcinomas KYSE and OE-21. The TGF-beta enhanced radioresistance phenotype was associated with induced G0/G1 cell cycle arrest and upregulation of the G1 cyclin-dependent kinase inhibitor p27kip1 as well as downregulation of c-myc protein expression. Comparison of the relative radiosensitivities of untreated cells suggested that OE-33 (SF2 = 0.71) cells were inherently more radioresistant than BIC1 or SEG1 cells (SF2 = 0.6 and 0.56, respectively). Conditioned medium obtained from unirradiated OE-33 cells enhanced radioresistance compared with fresh medium. This enhancement was abrogated by preincubation of conditioned medium with a neutralizing anti-TGF-beta antibody suggesting endogenous TGF-beta production by OE-33 cells. Enzyme-linked immunoabsorbent assays revealed that exposure to ionizing radiation increased TGF-beta production in all five cell lines. These results suggest that TGF-beta acts as an endogenous, radiation-inducible radioresistance factor in OE-33 esophageal carcinoma cells.

Tumor control probability predictions for genetic radiotherapy.

PURPOSE: Genetic radiotherapy, the combination of gene therapy and radiation therapy, for cancer treatment is evolving from laboratory studies to clinical trials. Genetic radiotherapy involves the viral infection of cells that change the sensitivity of transduced cells to radiation. Because there is no patient outcome data for genetic radiotherapy, prospective models are needed to determine the expected benefit of this new modality. Such a prospective model has been developed in this work. METHODS AND MATERIALS: An existing tumor control probability (TCP) calculation model developed for external beam radiotherapy was modified for genetic radiotherapy. Specifically, the (1) transduced fraction and (2) enhancement factor of the transduced cells were included in the model. Parametric studies of the effects of these two variables on TCP for head-and-neck cancer were performed. RESULTS: Using reasonable transduction fraction and enhancement factor values (0.8 and 1.4, respectively), the model predicts an increase in the TCP for genetic radiotherapy over radiotherapy alone by up to 15% for the same radiotherapy dose. The theoretical limit of TCP increase was calculated to be near 70%, which may occur with improved techniques that increase the transduced fraction or because of a strong bystander effect. To maintain existing TCP, dose reductions from 5 Gy (reasonable values) to >30 Gy (ideal case) are predicted for genetic radiotherapy over radiotherapy alone. CONCLUSIONS: Our results indicate that genetic radiotherapy has the potential to significantly improve tumor control over radiotherapy alone.

The effects of pentoxifylline on the survival of human glioma cells with continuous and intermittent stereotactic radiosurgery irradiation.

PURPOSE: In linac-based stereotactic radiosurgery, treatment is delivered intermittently via multiple individual small radiotherapy arcs. The time lapses between the individual arcs permit greater damage repair and increased tumor cell survival in comparison with continuous irradiation. Because pentoxifylline (PTX) has been reported to prevent radiation-induced cell cycle arrest at the G2/M checkpoint, where damage repair is critically linked to cell survival, we hypothesized that PTX would exert a favorable radiosensitization effect by reducing the recovery observed during intermittent stereotactic radiosurgery. METHODS AND MATERIALS: The human glioma cell line T98G was used to study the effects of continuous vs. intermittent irradiation with or without PTX. Cell cycle patterns were studied using flow cytometry. Clonogenic assays of single cells and spheroid outgrowth assays provided a quantitative measure of PTX-mediated radiosensitization. The PTX effect upon cells in low oxygen conditions was also studied in vitro after enzymatic oxygen scavenging. RESULTS: Flow Cytometry: T98G cells exposed to both continuous and intermittent irradiation exhibit similar arrest at the G2/M checkpoint. The addition of 2 mM PTX significantly reduced the radiation-induced G2/M block in both irradiation schemes. Clonogenic Assays: The same PTX concentration applied before a continuous dose of 12 Gy, two intermittent doses of 6 Gy, or three intermittent doses of 4 Gy, all given within a 1-h interval, consistently caused radiosensitization. The drug enhancement ratios for PTX were 1.5, 2.7, and 6.0 for the continuous and two different intermittent dose schedules, respectively. Adding PTX after irradiation yielded lower enhancement ratios than pre-irradiation application. A similar pattern was observed after total doses of 4, 6, 9, or 12 Gy, as well. In low oxygen conditions, PTX was seen to have the same effects as in normoxic conditions. Spheroid Outgrowth Assays: The in vitro PTX effects were replicated in the spheroid outgrowth assays. CONCLUSION: In human glioma cells, PTX abrogates the radiation-induced G2/M block observed after either continuous radiation exposure or intermittent exposures modeling clinical linac-based radiosurgery. The PTX-mediated reduction of the G2/M block translates into radiosensitization, most notably during intermittent exposures, and is presumably a consequence of diminished DNA damage repair at the G2/M checkpoint, though other contributing effects cannot be ruled out. The radiosensitization effect of PTX is sustained under low oxygen conditions. These results support consideration of the clinical evaluation of PTX to enhance the efficacy of linac-based radiosurgery involving intermittent irradiation through multiple arcs.

Heterogeneity in the transforming growth factor beta response of esophageal cancer cells.

The role of the transforming growth factor beta (TGFbeta) pathway in the development and progression of esophageal cancers is poorly understood. As an initial step in clarification of this issue, the functional status of the TGFbeta pathway was evaluated in a panel of esophageal cancer derived cell lines. Both adenocarcinoma and squamous cell carcinoma derived lines were represented. Although the TGFbeta pathway was intact and functional in four of the five cell lines, only one of them was growth inhibited by TGFbeta. In one cell line, the loss of a growth inhibitory response to TGFbeta could be explained by decreased expression of Smad 4 and a general inability to activate TGFbeta responsive promoters. In the other three cell lines, TGFbeta was able to activate transcription of TGFbeta responsive promoters, but unable to downregulate transcription of c-myc. Taken together these findings indicate that a selective loss in the ability of TGFbeta to regulate expression of a key component of the growth inhibitory pathway may contribute to the poor prognosis of esophageal cancers.