Potential use of EGFR-targeted molecular therapies for tumor suppressor CYLD-negative and poor prognosis oral squamous cell carcinoma with chemoresistance.

BACKGROUND: Tumor suppressor CYLD dysfunction by loss of its expression, triggers malignant transformation, especially drug resistance and tumor invasion/metastasis. Although loss of CYLD expression is significantly associated with poor prognosis in a large variety of tumors, no clinically-effective treatment for CYLD-negative cancer patients is available. METHODS: We focused on oral squamous cell carcinoma (OSCC), and sought to develop novel therapeutic agents for CYLD-negative cancer patients with poor prognosis. CYLD-knockdown OSCC cells by using CYLD-specific siRNA, were used to elucidate and determine the efficacy of novel drug candidates by evaluating cell viability and epithelial-mesenchymal transition (EMT)-like change. Therapeutic effects of candidate drug on cell line-derived xenograft (CDX) model and usefulness of CYLD as a novel biomarker using patient-derived xenograft (PDX) model were further investigated. RESULTS: CYLD-knockdown OSCC cells were resistant for all currently-available cytotoxic chemotherapeutic agents for OSCC, such as, cisplatin, 5-FU, carboplatin, docetaxel, and paclitaxel. By using comprehensive proteome analysis approach, we identified epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, played key roles in CYLD-knockdown OSCC cells. Indeed, cell survival rate in the cisplatin-resistant CYLD-knockdown OSCC cells was markedly inhibited by treatment with clinically available EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib. In addition, gefitinib was significantly effective for not only cell survival, but also EMT-like changes through inhibiting transforming growth factor-ß (TGF-ß) signaling in CYLD-knockdown OSCC cells. Thereby, overall survival of CYLD-knockdown CDX models was significantly prolonged by gefitinib treatment. Moreover, we found that CYLD expression was significantly associated with gefitinib response by using PDX models. CONCLUSIONS: Our results first revealed that EGFR-targeted molecular therapies, such as EGFR-TKIs, could have potential to be novel therapeutic agents for the CYLD-negative OSCC patients with poor prognosis.

Wnt/ß­catenin signaling is a novel therapeutic target for tumor suppressor CYLD­silenced glioblastoma cells.

Tumor suppressor cylindromatosis (CYLD) dysfunction by its downregulation is significantly associated with poor prognosis in patients with glioblastoma (GBM), the most aggressive and malignant type of glioma. However, no effective treatment is currently available for patients with CYLD­downregulated GBM. The aim of the present study was to identify the crucial cell signaling pathways and novel therapeutic targets for CYLD downregulation in GBM cells. CYLD knockdown in GBM cells induced GBM malignant characteristics, such as proliferation, metastasis, and GBM stem­like cell (GSC) formation. Comprehensive proteomic analysis and RNA sequencing data from the tissues of patients with GBM revealed that Wnt/ß­catenin signaling was significantly activated by CYLD knockdown in patients with GBM. Furthermore, a Wnt/ß­catenin signaling inhibitor suppressed all CYLD knockdown­induced malignant characteristics of GBM. Taken together, the results of the present study revealed that Wnt/ß­catenin signaling is responsible for CYLD silencing­induced GBM malignancy; therefore, targeting Wnt/ß­catenin may be effective for the treatment of CYLD­negative patients with GBM with poor prognosis.

Glioma Cells Acquire Stem-like Characters by Extrinsic Ribosome Stimuli.

Although glioblastoma (GBM) stem-like cells (GSCs), which retain chemo-radio resistance and recurrence, are key prognostic factors in GBM patients, the molecular mechanisms of GSC development are largely unknown. Recently, several studies revealed that extrinsic ribosome incorporation into somatic cells resulted in stem cell properties and served as a key trigger and factor for the cell reprogramming process. In this study, we aimed to investigate the mechanisms underlying GSCs development by focusing on extrinsic ribosome incorporation into GBM cells. Ribosome-induced cancer cell spheroid (RICCS) formation was significantly upregulated by ribosome incorporation. RICCS showed the stem-like cell characters (number of cell spheroid, stem cell markers, and ability for trans differentiation towards adipocytes and osteocytes). In RICCS, the phosphorylation and protein expression of ribosomal protein S6 (RPS6), an intrinsic ribosomal protein, and STAT3 phosphorylation were upregulated, and involved in the regulation of cell spheroid formation. Consistent with those results, glioma-derived extrinsic ribosome also promoted GBM-RICCS formation through intrinsic RPS6 phosphorylation. Moreover, in glioma patients, RPS6 phosphorylation was dominantly observed in high-grade glioma tissues, and predominantly upregulated in GSCs niches, such as the perinecrosis niche and perivascular niche. Those results indicate the potential biological and clinical significance of extrinsic ribosomal proteins in GSC development.

Relationship between clinicopathological characteristics and CYLD expression in patients with cholesteatoma.

Middle ear cholesteatoma is a destructive disease in which inflammation plays an important role in development and progression, and there are currently no biomarkers predicting prognosis or recurrence. Cylindromatosis (CYLD), a tumor suppressor deubiquitinase, serves as a negative regulator of inflammation expressed in tissues including the middle ear. To determine the clinical significance of CYLD in acquired cholesteatoma, we evaluated CYLD expression in acquired cholesteatoma tissue by immunostaining and analyzed its correlation with clinicopathological characteristics. Our immunohistochemical analysis revealed that CYLD expression levels were varied in the tissues of acquired cholesteatoma patients. The relative expression levels of CYLD in cholesteatoma exhibited a significant correlation with the grade of otorrhea (R = 0.532, p = 0.039). Moreover, the period of epithelialization was also significantly associated with the relative expression levels of CYLD (R = 0.720, p = 0.002). In addition, CYLD expression tended to be lower in the group with recurrence. These results suggest that low CYLD expression correlates with postoperative recovery of acquired cholesteatoma, while potentially affecting the induction of recurrence. This is the first report showing that low CYLD expression correlates with accelerated disease recovery, and suggests a new aspect of CYLD as a prognostic predictor of acquired cholesteatoma.

Evaluation of differences and dosimetric influences of beam models using golden and multi-institutional measured beam datasets in radiation treatment planning systems.

PURPOSE: The beam model in radiation treatment planning systems (RTPSs) plays a crucial role in determining the accuracy of calculated dose distributions. The purpose of this study was to ascertain differences in beam models and their dosimetric influences when a golden beam dataset (GBD) and multi-institution measured beam datasets (MBDs) are used for beam modeling in RTPSs. METHODS: The MBDs collected from 15 institutions, and the MBDs' beam models, were compared with a GBD, and the GBD's beam model, for Varian TrueBeam linear accelerator. The calculated dose distributions of the MBDs' beam models were compared with those of the GBD's beam model for simple geometries in a water phantom. Calculated dose distributions were similarly evaluated in volumetric modulated arc therapy (VMAT) plans for TG-119 C-shape and TG-244 head and neck, at several dose constraints of the planning target volumes (PTVs), and organs at risk. RESULTS: The agreements of the MBDs with the GBD were almost all within ±1%. The calculated dose distributions for simple geometries in a water phantom also closely corresponded between the beam models of GBD and MBDs. Nevertheless, there were considerable differences between the beam models. The maximum differences between the mean energy of the energy spectra of GBD and MBDs were -0.12 MeV (-10.5%) in AcurosXB (AXB, Eclipse) and 0.11 MeV (7.7%) in collapsed cone convolution (CCC, RayStation). The differences in the VMAT calculated dose distributions varied for each dose region, plan, X-ray energy, and dose calculation algorithm. The ranges of the differences in the dose constraints were -5.6% to 3.0% for AXB and -24.1% to 2.8% for CCC. In several VMAT plans, the calculated dose distributions of GBD's beam model tended to be lower in high-dose regions and higher in low-dose regions than those of the MBDs' beam models. CONCLUSIONS: We found that small differences in beam data have large impacts on the beam models, and on calculated dose distributions in clinical VMAT plan, even if beam data correspond within ±1%. GBD's beam model was not a representative beam model. The beam models of GBD and MBDs and their calculated dose distributions under clinical conditions were significantly different. These differences are most likely due to the extensive variation in the beam models, reflecting the characteristics of beam data. The energy spectrum and radial energy in the beam model varied in a wide range, even if the differences in the beam data were <±1%. To minimize the uncertainty of the calculated dose distributions in clinical plans, it was best to use the institutional MBD for beam modeling, or the beam model that ensures the accuracy of calculated dose distributions.

Simulation analysis of optimized brachytherapy for uterine cervical cancer: Can we select the best brachytherapy modality depending on tumor size?

PURPOSE: To choose the optimal brachytherapeutic modality for uterine cervical cancer, we performed simulation analysis. METHODS AND MATERIALS: For each high-risk clinical target volume (HR CTV), we compared four modalities [classical conventional intracavitary brachytherapy (ConvICBT), Image-guided ICBT (IGICBT), intracavitary/interstitial brachytherapy (ICISBT), and interstitial brachytherapy (ISBT) with perineal approach] using dose-volume histograms using eight sizes of HR CTV (2 × 2 × 2 cm to 7 × 4 × 4 cm) and organs at risk model. RESULTS: In ConvICBT, the doses covered 90% of the HR CTV [D90(HR CTV)] decreased from 197% prescribed dose (PD) for the HR CTV size (2 × 2 × 2 cm) to 73% PD for 5 × 4 × 4 cm, whereas the other three modalities could achieve 100% PD for all HR CTV sizes. The minimum doses received by the maximally irradiated 2-cm(3) volumes for organs at risks of IGICBT demonstrated lower values than those of ConvICBT for the HR CTV size of 4 × 3 × 3 cm or smaller. ICISBT demonstrated lower values than those of IGICBT for 4 × 3 × 3 cm or larger. ISBT demonstrated lowest values for 5 × 4 × 4 cm or larger. CONCLUSIONS: HR CTV size of 4 × 3 × 3 cm seems to be a threshold volume in this simulation analysis, and IGICBT is a better choice for smaller HR CTV than the threshold volume. On larger HR CTV, ICISBT or ISBT is the better choice.

Preliminary results of MRI-assisted high-dose-rate interstitial brachytherapy for uterine cervical cancer.

PURPOSE: To investigate the effectiveness of our novel MRI-assisted high-dose-rate interstitial brachytherapy for uterine cervical cancer. METHODS AND MATERIALS: Between June 2005 and June 2009, 29 previously untreated patients with cervical cancer were enrolled (2 T2b, 2 T3a, 19 T3b, and 6 T4 tumors). We implanted MRI-compatible plastic catheters using our unique ambulatory technique. The total treatment doses were 30-36 Gy (6 Gy per fraction) combined with external beam radiotherapy. RESULTS: The median D90 (high-risk clinical target volume), D(2 cc) (bladder), and D(2 cc) (rectum) per fraction were 6.9, 5, and 4.6 Gy, respectively. The 3-year local control rates were 100%, 95%, and 83% for T2, T3, and T4 tumors, respectively. Grade 3 or 4 late complications occurred in 4 patients. CONCLUSIONS: Our preliminary evaluation of image-based high-dose-rate interstitial brachytherapy showed favorable local treatment results with an acceptable complication rate.

Comparison of calculated dose by helical tomotherapy treatment planning machine and measured dose of radiophotoluminescence glass dosimeter in lung lesions using Rando Phantom.

AIM: To examine the compatibility of the measured and calculated dose for the treatment of lung lesions by helical tomotherapy. MATERIALS AND METHODS: The administered dose was measured a total of 55 times at 22 points with a radiophotoluminescence glass dosimeter (RPLGD) inserted in the position of an anthropomorphic Rando Phantom. Two Gy were prescribed and calculated with a tomotherapy planning machine for a 3-cm diameter spherical planning target volume (PTV) created in the lung area. Compatibility (measured dose/calculated dose and σ value=(D(meas)-D(calc))/D(prescribed)) × 100 (%)) was analyzed according to dosimeter location. RESULTS: Deviations between measured and calculated doses for the lung lesion were within 4% for planning target volume, indicating that adequate dose delivery to the PTV was achievable. On the other hand, we found dose deviations up to 15% for the lower prescribed dose range (64% or less) for the measured dose/calculated comparison and a 6% deviation according to the σ value in or near inhomogeneous tissue. CONCLUSION: Although the measured dose satisfied the clinical requirement in almost all areas including PTV, we should note that there may be discrepancies between expected calculated dose and irradiated dose in or near inhomogeneous area.

Interstitial brachytherapy using virtual planning and Doppler transrectal ultrasonography guidance for internal iliac lymph node metastasis.

To expand the indications for high-dose-rate interstitial brachytherapy (HDR-ISBT) for deep-seated pelvic tumors, we investigated the usefulness of Doppler transrectal ultrasonography (TRUS) guidance and virtual planning. The patient was a 36-year-old female. She had right internal iliac lymph node oligometastasis of vaginal cancer 12 months after radical radiotherapy. The tumor could not be found by gray-scale TRUS and physical examination. Virtual planning was performed using computed tomography with template and vaginal cylinder insertion. We uploaded the images to our treatment planning software and reconstructed the contours of the clinical target volume (CTV) and right internal iliac vessel. Virtual needle applicators were plotted using the template holes for virtual planning. At the time of implantation, Doppler TRUS was used to prevent vessel injury by needle applicators. Applicators were implanted in accordance with virtual planning and Doppler TRUS could detect the right iliac vessel. The percentage of CTV covered by the prescribed dose was 99.8%. The minimum dose received by the maximally irradiated 0.1-cc volume for the right internal iliac vessel was 95% prescribed dose. Complete response was achieved, however, radiological findings showed marginal recurrence at 15 months after HDR-ISBT. Post-radiation neuropathy occurred as a late complication four months after treatment; however, the pain was well controlled by medication. We consider that virtual planning and Doppler TRUS are effective methods in cases where it is difficult to detect the tumor by physical examination and gray-scale TRUS, thereby expanding the indications for ISBT.

Daily CT measurement of needle applicator displacement during multifractionated high-dose-rate interstitial brachytherapy for postoperative recurrent uterine cancer.

We investigated daily needle applicator displacement during multifractionated high-dose-rate interstitial brachytherapy (HDR-ISBT) for postoperative recurrent uterine cancer. Eight patients with postoperative recurrent uterine cancer received HDR-ISBT with or without external beam radiotherapy using our unique ambulatory technique. To analyze displacement, we obtained daily computed tomography (CT) images for 122 flexible needle applicators at 21, 45, 69, and 93 hours after implantation. Displacement was defined as the length between the center of gravity of titanium markers and the needle applicator tips along the daily CT axis. For cases in which displacement was not corrected, we also calculated the dose that covered 90% of the clinical target volume (D90(CTV)) using a dose-volume histogram (DVH). Median caudal needle applicator displacement at 21, 45, 69, and 93 hours was 3, 2, 4, and 5 mm, respectively. More than 15 mm displacement was observed for 2% (2 of 122) and 17% (10 of 60) of needle applicators at 21 and 93 hours, respectively. Cases in which dwell positions were not changed to correct the treatment plan, 2 of 8 patients showed more than 10% reduction in D90(CTV) values compared with the initial treatment plan. Correction of dwell positions of the treatment source improves treatment DVH for multifractionated HDR-ISBT.

Assessment of daily needle applicator displacement during high-dose-rate interstitial brachytherapy for prostate cancer using daily CT examinations.

To improve treatment conformity for prostate cancer, we investigated daily applicator displacement during high-dose-rate interstitial brachytherapy (HDR-ISBT). Thirty patients treated with HDR-ISBT as monotherapy were examined. All patients received a treatment dosage of 49 Gy per 7 fractions over 4 days. For dose administration, we examined 376 flexible applicators (1128 points) using our unique ambulatory implant technique. Using CT images with a 3-mm slice thickness, we calculated the relative coordinates of the titanium markers and the tips of the applicators. We calculated the distance between the center of gravity of the markers and the tips of the catheters, and compared the distances measured on the day of implantation and the second, third, and fourth treatment days. The mean displacement distance for all applicators was 4.3 ± 3.4 mm, 4.6 ± 4.1 mm, and 5.8 ± 4.5 mm at 21, 45, and 69 hours after initial planning CT. We used a 15-mm margin for needle displacement and only 2 points of 2 patients (16 mm and 18 mm at 69 hours, 2/1128 = 0.2%) exceeded this range. Almost patients (87%) showed the largest displacement within the first 21 hours. The relative doses that covered 100% of CTV (D100(CTV)) values compared with the initial treatment plan were reduced to 0.96 ± 0.08, 0.96 ± 0.08 and 0.94 ± 0.1 at 21, 45 and 69 hours. However, the relative D90(CTV) values kept acceptable levels (1.01 ± 0.02, 1.01 ± 0.03 and 1.01 ± 0.03). Cranial margin of 15 mm seems to be effective to keep D90(CTV) level if we do not do corrective action.