Protein phosphatase 2A (PP2A) inhibitor CIP2A indicates resistance to radiotherapy in rectal cancer.

Preoperative (chemo)radiotherapy, (C)RT, is an essential part of the treatment of rectal cancer patients, but tumor response to this therapy among patients is variable. Thus far, there are no clinical biomarkers that could be used to predict response to (C)RT or to stratify patients into different preoperative treatment groups according to their prognosis. Overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) has been demonstrated in several cancers and is frequently associated with reduced survival. Recently, high CIP2A expression has also been indicated to contribute to radioresistance in head and neck squamous cell carcinoma, but few studies have examined the connection between CIP2A and radiation response regarding other malignancies. We have evaluated CIP2A protein expression levels in relation to tumor regression after preoperative (C)RT and survival of rectal adenocarcinoma patients. The effects of CIP2A knockdown by siRNA on cell survival were further investigated in colorectal cancer cells exposed to radiation. Patients with low-CIP2A-expressing tumors had more frequently moderate or excellent response to long-course (C)RT than patients with high-CIP2A-expressing tumors. They also had higher 36-month disease-specific survival (DSS) rate in categorical analysis. In the multivariate analysis, low CIP2A expression level remained as an independent predictive factor for increased DSS. Suppression of CIP2A transcription by siRNA was found to sensitize colorectal cancer cells to irradiation and decrease their survival in vitro. In conclusion, these results suggest that by contributing to radiosensitivity of cancer cells, low CIP2A protein expression level associates with a favorable response to long-course (C)RT in rectal cancer patients.

Dose monitoring in pediatric and young adult head and cervical spine CT studies at two emergency duty departments.

PURPOSE: As the number of pediatric computed tomography (CT) imaging is increasing, there is a need for real-time radiation dose monitoring and evaluation of the imaging protocols. The aim of this study was to present the imaging data, patient doses, and observations of pediatric and young adult trauma-and routine head CT and cervical spine CT collected by a dose monitoring software. METHODS: Patient age, study date, imaging parameters, and patient dose as volume CT dose index (CTDIvol) and dose length product (DLP) were collected from two emergency departments' CT scanners for 2-year period. The patients were divided into four age groups (0-5, 6-10, 11-15, and 16-20 years) for statistical analysis and effective dose determination. The 75th percentile doses were evaluated to be used as local diagnostic reference levels (DRLs). RESULTS: Six hundred fifteen trauma head, 318 routine head, and 592 trauma cervical spine CT studies were assessed. All mean CTDIvol values were statistically lower in hospital B (40.3 ± 12.3, 30.03 ± 11.1, and 6.9 ± 3.1 mGy, respectively) than in hospital A (53.0 ± 12.9, 43.2 ± 8.7, and 18.3 ± 7.3 mGy, respectively). Statistically significant differences were observed on scanning length between hospitals and between CTDIvol values when protocol was updated. The 75th percentiles of trauma cervical spine in hospital B can be used as local DRL. Non-optimized protocols were also revealed in hospital A. CONCLUSION: Dose monitoring software offers a valuable tool for evaluating the imaging practices and finding non-optimized protocols.

LOW-DOSE CT PROTOCOL OPTIMIZATION FOR THE ASSESSMENT OF ACUTE APPENDICITIS: THE OPTICAP PHANTOM STUDY.

The aim was to evaluate effects of voltage, noise input (NI) and iterative reconstruction (IR) on radiation dose and image quality in order to establish a contrast enhanced low-dose protocol for assessment of acute appendicitis. An anthropomorphic abdominal phantom mimicking contrast enhanced abdomen was scanned with 80, 100 and 120 kV, standard and strong IR and 11 NIs (66 protocols). A total of 14 test tubes of increasing iodine dilutions and one tube with an appendicolith were evaluated within the phantom. The dose, HUs, noise, contrast-to-noise ratio (CNR) and figure of merit (FOM) were determined. Visual quality scores were assessed by two readers. A clinically used voltage-IR combination (120 kV, standard IR) was used as a reference. Overall, 100 kV with standard IR (p = 0.002) and 80 kV with both IRs (p < 0.001) showed higher CNR than the reference, but noise was most pronounced at 80 kV (p < 0.001). The highest FOM was found in the 100 kV protocols (p < 0.001). The reference and 100 kV with standard IR had highest image quality scores, where the 100 kV protocol enabled a distinct dose reduction. Lowering the voltage seems to be a more favorable tool than IR changes in optimizing the dose in contrast enhanced abdominal CT. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01022567.

Compass model-based quality assurance for stereotactic VMAT treatment plans.

PURPOSE: To use Compass as a model-based quality assurance (QA) tool for stereotactic body radiation therapy (SBRT) and stereotactic radiation therapy (SRT) volumetric modulated arc therapy (VMAT) treatment plans calculated with Eclipse treatment planning system (TPS). MATERIALS AND METHODS: Twenty clinical stereotactic VMAT SBRT and SRT treatment plans were blindly selected for evaluation. Those plans included four different treatment sites: prostate, brain, lung and body. The plans were evaluated against dose-volume histogram (DVH) parameters and 2D and 3D gamma analysis. The dose calculated with Eclipse treatment planning system (TPS) was compared to Compass calculated dose (CCD) and Compass reconstructed dose (CRD). RESULTS: The maximum differences in mean dose of planning target volume (PTV) were 2.7 ±â€¯1.0% between AAA and Acuros XB calculation algorithm TPS dose, -7.6 ±â€¯3.5% between Eclipse TPS dose and CCD dose and -5.9 ±â€¯3.7% between Eclipse TPS dose and CRD dose for both Eclipse calculation algorithms, respectively. 2D gamma analysis was not able to identify all the cases that 3D gamma analysis specified for further verification. CONCLUSIONS: Compass is suitable for QA of SBRT and SRT treatment plans. However, the QA process should include wide set of DVH-based dose parameters and 3D gamma analysis should be the preferred method when performing clinical patient QA. The results suggest that the Compass should not be used for smaller field sizes than 3 × 3 cm2 or the beam model should be adjusted separately for both small (FS ≤ 3 cm) and large (FS > 3 cm) field sizes.

Excess of Radiation Burden for Young Testicular Cancer Patients using Automatic Exposure Control and Contrast Agent on Whole-body Computed Tomography Imaging.

BACKGROUND: The aim of the study was to assess patient dose from whole-body computed tomography (CT) in association with patient size, automatic exposure control (AEC) and intravenous (IV) contrast agent. PATIENTS AND METHODS: Sixty-five testicular cancer patients (mean age 28 years) underwent altogether 279 whole-body CT scans from April 2000 to April 2011. The mean number of repeated examinations was 4.3. The GE LightSpeed 16 equipped with AEC and the Siemens Plus 4 CT scanners were used for imaging. Whole-body scans were performed with (216) and without (63) IV contrast. The ImPACT software was used to determine the effective and organ doses. RESULTS: Patient doses were independent (p < 0.41) of patient size when the Plus 4 device (mean 7.4 mSv, SD 1.7 mSv) was used, but with the LightSpeed 16 AEC device, the dose (mean 14 mSv, SD 4.6 mSv) increased significantly (p < 0.001) with waist cirfumference. Imaging with the IV contrast agent caused significantly higher (13% Plus 4, 35% LightSpeed 16) exposure than non-contrast imaging (p < 0.001). CONCLUSIONS: Great caution on the use of IV contrast agent and careful set-up of the AEC modulation parameters is recommended to avoid excessive radiation exposure on the whole-body CT imaging of young patients.

Comparison of Effective Dose and Image Quality for Newborn Imaging on Seven Commonly Used CT Scanners.

This study compares the image quality and the patient doses on seven different computed tomography (CT) scanners for newborn chest imaging. The dose was measured by using an anthropomorphic newborn phantom and thermoluminescence dosemeters (TLDs). The effective dose was estimated separately based on a dose-length-product display, TLD measurements and the ImPACT CT dose calculation software. The image quality was assessed using a signal-to-noise ratio and a contrast-to-noise ratio (CNR). In order to compare the different scanners, a figure of merit (FOM) based on the rate of CNR2 and computed tomography dose index (CTDIvol) was calculated. The organ doses within the scan area ranged between 0.3 and 2.9 mGy and they depended on the organ and used scanner. The highest effective dose (1.1 mSv) was observed on Aquilion 32 and the lowest effective dose was observed on the Aquilion One (0.22 mSv). The lowest organ doses and highest FOM were observed on the Optima 660. With the Aquilion One and the Definition Dual Flash the examination was 71-90% faster when compared with other scanners. Newer devices equipped with novel dose-saving methods provide a lower dose, as well as take better advantage of the radiation in the image formation.

HSV-1 Infection Modulates the Radioresponse of a HPV16-positive Head and Neck Cancer Cell Line.

BACKGROUND: The combined effects of Human papillomavirus (HPV) and Herpes simplex type 1 (HSV-1) infections and their effects on cancer cell radioresistance are unexplored. MATERIALS AND METHODS: An HPV16-positive hypopharyngeal carcinoma cell line (UD-SCC-2) was infected with wt-HSV-1 at low multiplicity of infection (MOI) and irradiated with 2 Gy at 24 h postinfection. Viability assays and quantitative reverse-transcriptase PCR for HPV16 E6, E7, nuclear factor kappa B1, B-cell CLL/lymphoma 2 (BCL2), and caspases 3, 8 and 9 at 24, and 72 h, as well as immunocytochemistry for BCL2, caspase 3, cyclin E, mouse double minute 2 homolog (MDM2), HSV-1 and Ki-67 were performed at 144 h postirradiation. RESULTS: At 144 h, cell viability was significantly lowered by irradiation only in uninfected cells. Infection combined with irradiation resulted in increased expression of E6, E7, BCL2 and NF-κB1 at 144 h. Simultaneously, E6 and E7 were down-regulated in non-irradiated infected cells. Irradiation and infection with 0.00001 MOI separately up-regulated caspase 3 but infection with 0.0001 MOI halved its expression in irradiated cells. CONCLUSION: HSV-1 infection modulates radioresistance of HPV16-positive hypopharyngeal carcinoma cells.

Comparison of physical quality assurance between Scanora 3D and 3D Accuitomo 80 dental CT scanners.

BACKGROUND: The use of cone beam computed tomography (CBCT) in dentistry has proven to be useful in the diagnosis and treatment planning of several oral and maxillofacial diseases. The quality of the resulting image is dictated by many factors related to the patient, unit, and operator. MATERIALS AND METHODS: In this work, two dental CBCT units, namely Scanora 3D and 3D Accuitomo 80, were assessed and compared in terms of quantitative effective dose delivered to specific locations in a dosimetry phantom. Resolution and contrast were evaluated in only 3D Accuitomo 80 using special quality assurance phantoms. RESULTS: Scanora 3D, with less radiation time, showed less dosing values compared to 3D Accuitomo 80 (mean 0.33 mSv, SD±0.16 vs. 0.18 mSv, SD±0.1). Using paired t-test, no significant difference was found in Accuitomo two scan sessions (p>0.05), while it was highly significant in Scanora (p>0.05). The modulation transfer function value (at 2 lp/mm), in both measurements, was found to be 4.4%. The contrast assessment of 3D Accuitomo 80 in the two measurements showed few differences, for example, the grayscale values were the same (SD=0) while the noise level was slightly different (SD=0 and 0.67, respectively). CONCLUSIONS: The radiation dose values in these two CBCT units are significantly less than those encountered in systemic CT scans. However, the dose seems to be affected more by changing the field of view rather than the voltage or amperage. The low doses were at the expense of the image quality produced, which was still acceptable. Although the spatial resolution and contrast were inferior to the medical images produced in systemic CT units, the present results recommend adopting CBCTs in maxillofacial imaging because of low radiation dose and adequate image quality.

CIP2A is an Oct4 target gene involved in head and neck squamous cell cancer oncogenicity and radioresistance.

Radiotherapy is a mainstay for treatment of many human cancer types, including head and neck squamous cell carcinoma (HNSCC). Thereby, it is clinically very relevant to understand the mechanisms determining radioresistance. Here, we identify CIP2A as an Oct4 target gene and provide evidence that they co-operate in radioresistance. Oct4 positively regulates CIP2A expression both in testicular cancer cell lines as well as in embryonic stem cells. To expand the relevance of these findings we show that Oct4 and CIP2A are co-expressed in CD24 positive side-population of patient-derived HNSCC cell lines. Most importantly, all Oct4 positive HNSCC patient samples were CIP2A positive and this double positivity was linked to poor differentiation level, and predicted for decreased patient survival among radiotherapy treated HNSCC patients. Oct4 and CIP2A expression was also linked with increased aggressiveness and radioresistancy in HNSCC cell lines. Together we demonstrate that CIP2A is a novel Oct4 target gene in stem cells and in human cancer cell lines. Clinically these results suggest that diagnostic evaluation of HNSCC tumors for Oct4 or Oct4/CIP2A positivity might help to predict HNSCC tumor radioresistancy. These results also identify both Oct4 and CIP2A as potential targets for radiosensitation.

Isocentric integration of intensity-modulated radiotherapy with electron fields improves field junction dose uniformity in postmastectomy radiotherapy.

BACKGROUND: In postmastectomy radiotherapy (PMRT), the dose coverage of the planning target volume (PTV) with additional margins, including the chest wall, supraclavicular, interpectoral, internal mammary and axillar level I-III lymph nodes, is often compromised. Electron fields may improve the medial dose coverage while maintaining organ at risk (OAR) doses at an acceptable level, but at the cost of hot and cold spots at the electron and photon field junction. To improve PMRT dose coverage and uniformity, an isocentric technique combining tangential intensity-modulated (IM)RT fields with one medial electron field was implemented. MATERIAL AND METHODS: For 10 postmastectomy patients isocentric IMRT with electron plans were created and compared with a standard electron/photon mix and a standard tangent technique. PTV dose uniformity was evaluated based on the tolerance range (TR), i.e. the ratio of the standard deviation to the mean dose, a dice similarity coefficient (DSC) and the 90% isodose coverage and the hot spot volumes. OAR and contralateral breast doses were also recorded. RESULTS: IMRT with electrons significantly improved the PTV dose homogeneity and conformity based on the TR and DSC values when compared with the standard electron/photon and tangent technique (p < 0.02). The 90% isodose coverage improved to 86% compared with 82% and 80% for the standard techniques (p < 0.02). Compared with the standard electron/photon mix, IMRT smoothed the dose gradient in the electron and photon field junction and the volumes receiving a dose of 110% or more were reduced by a third. For all three strategies, the OAR and contralateral breast doses were within clinically tolerable limits. CONCLUSION: Based on these results two-field IMRT combined with an electron field is a suitable strategy for PMRT.

The combined effects of irradiation and herpes simplex virus type 1 infection on an immortal gingival cell line.

BACKGROUND: Oral mucosa is frequently exposed to Herpes simplex virus type 1 (HSV-1) infection and irradiation due to dental radiography. During radiotherapy for oral cancer, the surrounding clinically normal tissues are also irradiated. This prompted us to study the effects of HSV-1 infection and irradiation on viability and apoptosis of oral epithelial cells. METHODS: Immortal gingival keratinocyte (HMK) cells were infected with HSV-1 at a low multiplicity of infection (MOI) and irradiated with 2 Gy 24 hours post infection. The cells were then harvested at 24, 72 and 144 hours post irradiation for viability assays and qRT-PCR analyses for the apoptosis-related genes caspases 3, 8, and 9, bcl-2, NFκB1, and viral gene VP16. Mann-Whitney U-test was used for statistical calculations. RESULTS: Irradiation improved the cell viability at 144 hours post irradiation (P = 0.05), which was further improved by HSV-1 infection at MOI of 0.00001 (P = 0.05). Simultaneously, the combined effects of infection at MOI of 0.0001 and irradiation resulted in upregulation in NFκB1 (P = 0.05). The combined effects of irradiation and HSV infection also significantly downregulated the expression of caspases 3, 8, and 9 at 144 hours (P = 0.05) whereas caspase 3 and 8 significantly upregulated in non-irradiated, HSV-infected cells as compared to uninfected controls (P = 0.05). Infection with 0.0001 MOI downregulated bcl-2 in non-irradiated cells but was upregulated by 27% after irradiation when compared to non-irradiated infected cells (P = 0.05). Irradiation had no effect on HSV-1 shedding or HSV gene expression at 144 hours. CONCLUSIONS: HSV-1 infection may improve the viability of immortal cells after irradiation. The effect might be related to inhibition of apoptosis.

A dosimetric phantom study of dose accuracy and build-up effects using IMRT and RapidArc in stereotactic irradiation of lung tumours.

BACKGROUND AND PURPOSE: Stereotactic lung radiotherapy (SLRT) has emerged as a curative treatment for medically inoperable patients with early-stage non-small cell lung cancer (NSCLC) and the use of intensity-modulated radiotherapy (IMRT) and volumetric modulated arc treatments (VMAT) have been proposed as the best practical approaches for the delivery of SLRT. However, a large number of narrow field shapes are needed in the dose delivery of intensity-modulated techniques and the probability of underdosing the tumour periphery increases as the effective field size is decreased. The purpose of this study was to evaluate small lung tumour doses irradiated by intensity-modulated techniques to understand the risk for dose calculation errors in precision radiotherapy such as SLRT. MATERIALS AND METHODS: The study was executed with two heterogeneous phantoms with targets of Ø1.5 and Ø4.0 cm. Dose distributions in the simulated tumours delivered by small sliding window apertures (SWAs), IMRT and RapidArc treatment plans were measured with radiochromic film. Calculation algorithms of pencil beam convolution (PBC) and anisotropic analytic algorithm (AAA) were used to calculate the corresponding dose distributions. RESULTS: Peripheral doses of the tumours were decreased as SWA decreased, which was not modelled by the calculation algorithms. The smallest SWA studied was 2 mm, which reduced the 90% isodose line width by 4.2 mm with the Ø4.0 cm tumour as compared to open field irradiation. PBC was not able to predict the dose accurately as the gamma evaluation failed to meet the criteria of ±3%/±1 mm on average in 61% of the defined volume with the smaller tumour. With AAA the corresponding value was 16%. The dosimetric inaccuracy of AAA was within ±3% with the optimized treatment plans of IMRT and RapidArc. The exception was the clinical RapidArc plan with dose overestimation of 4%. CONCLUSIONS: Overall, the peripheral doses of the simulated lung tumours were decreased by decreasing the SWA. To achieve adequate surface dose coverage to small lung tumours with a difference less than 1 mm in the isodose line radius between the open and modulated field, a larger than 6 mm SWA should be used in the dose delivery of SLRT.

Identification and regulation of a stage-specific stem cell niche enriched by Nanog-positive spermatogonial stem cells in the mouse testis.

The ability of spermatogonial stem cells to acquire embryonic stem cell (ESC) properties in vitro has recently been of great interest. However, studies focused on the in vivo regulation of testicular stem cells have been hampered because the exact anatomical location of these cells is unknown. Moreover, no specialized stem cell niche substructure has been identified in the mammalian testis thus far. It has also been unclear whether the adult mammalian testis houses pluripotent stem cells or whether pluripotency can be induced only in vitro. Here, we demonstrate, for the first time, the existence of a Nanog-positive spermatogonial stem cell subpopulation located in stage XII of the mouse seminiferous epithelial cycle. The efficiency of the cells from seminiferous tubules with respect to prolonged pluripotent gene expression was correlated directly with stage-specific expression levels of Nanog and Oct4, demonstrating the previously unknown stage-specific regulation of undifferentiated spermatogonia (SPG). Testicular Nanog expression marked a radioresistant spermatogonial subpopulation, supporting its stem cell nature. Furthermore, we demonstrated that p21 acts as an upstream regulator of Nanog in SPG and mouse ESCs, and our results demonstrate that promyelocytic leukemia zinc finger is a specific marker of progenitor SPG. Additionally, we describe a novel method to cultivate Nanog-positive SPG in vitro. This study demonstrates the existence and location of a previously unknown stage-specific spermatogonial stem cell niche and reports the regulation of radioresistant spermatogonial stem cells.

Increased beam attenuation and surface dose by different couch inserts of treatment tables used in megavoltage radiotherapy.

The use of solid carbon fiber table materials in radiotherapy has become more common with the implementation of image-guided radiotherapy (IGRT), since the solid materials give less imaging artifacts than the so-called tennis racket couchtops. The downside of the solid carbon fiber couch inserts is that they increase the beam attenuation, resulting in increased surface doses and inaccuracies in determine the dose in the patient. The purpose of this study was to evaluate the interaction of 6 and 15 MV photons with eight different couch inserts. The presented results enable direct comparison of the attenuation properties of the studied couchtops. With a direct posterior beam the maximum attenuations reach 3.6% and 2.4% with 6 and 15 MV, respectively. The measured maximum attenuation by a couchtop with an oblique gantry angle was 10.8% and 7.4% at 6 and 15 MV energies, respectively. The skin-sparing effect was decreased substantially with every couchtop. The highest increases in surface doses were recorded to be four- and threefold, as compared to the direct posterior open field surface doses of 6 and 15 MV, respectively. In conclusion, the carbon fiber tabletops decrease the skin-sparing effect of megavoltage photon energies. The increased beam attenuation and skin doses should be taken into account in the process of treatment planning.

A method to improve target dose homogeneity of craniospinal irradiation using dynamic split field IMRT.

PURPOSE: Craniospinal irradiation (CSI) is technically very challenging and field edge matching is needed because of the mechanical limitations of standard linear accelerators. We assessed the feasibility of intensity-modulated radiotherapy (IMRT) in CSI to overcome the standard feathering and dose inhomogeneities associated with the standard feathering technique in the junction areas. MATERIALS AND METHODS: The use of IMRT in CSI was studied with five patients CT scanned in the supine position. Isocentric treatment plans of three dimensional conventional radiotherapy (3D-CRT) and split field IMRT (sfIMRT) with dynamic intrafractional feathering were created with the same field setup and the resulted dose distributions were compared. The effect of treatment inaccuracy was simulated with an intentional shift of +/-3mm with both treatment plans. Dosimetric verification of the sfIMRT treatment plan was performed with radiographic films placed in a phantom. RESULTS: The sfIMRT treatment plans resulted in a better dose coverage and uniformity in the target volume. The +/-3mm shift had only a minor effect on the dose distribution of the sfIMRT treatment plan whereas with the 3D-CRT the shift resulted in an error of +/-38% of the calculated dose in the spinal cord. The measured dose distribution of the sfIMRT treatment plan correlated well with the calculations. CONCLUSIONS: Improved dose homogeneity in the target volume was achieved with the sfIMRT compared to the conventional 3D-CRT treatment plan. With the sfIMRT technique only a single treatment plan is required to deliver the total treatment dose and the resulting dose distribution is also less volatile for technical uncertainties of the treatment.

The EGFR inhibitor gefitinib suppresses recruitment of pericytes and bone marrow-derived perivascular cells into tumor vessels.

Drugs that target EGFR have established anti-tumor effect and are used in the clinic. Here we addressed whether inhibition of EGFR tyrosine kinase activity by gefitinib in tumor microenvironment affected tumor angiogenesis or vasculogenesis. A syngeneic tumor model of mice with grafted GFP-labeled bone marrow cells was used to analyze the effects of gefitinib on different cellular components of tumor vasculature. To characterize tumor cell-independent stromal effects of EGFR targeting, the mice were injected with B16 melanoma cells not expressing significant quantities of EGFR, and treated with gefitinib for seven days, a period not sufficient for significant reduction in total tumor volume. Numbers of vessels as well as cell surface areas covered by markers of endothelial, pericyte and bone marrow-derived progenitor cells were quantified by image analysis of tumor sections. Quantitative analysis of immunohistochemical data demonstrated that gefitinib decreased the coverage of small CD31-positive vessels with NG2-positive pericytes, as well as reduced the recruitment of perivascular GFP-positive bone marrow-derived progenitor cells within the tumor tissue. These results suggest that inhibition of EGFR activity in tumors has vascular effects in the absence of direct effect on tumor cells. EGFR targeting may lead to suppressed mobilization of pericytes needed for vessel stabilization, as well as of bone marrow-derived perivascular progenitor cells. These findings introduce novel cellular mechanisms by which EGFR targeted drugs may suppress tumor growth.

Concurrent cetuximab, cisplatin, and radiation for squamous cell carcinoma of the head and neck in vitro.

BACKGROUND AND PURPOSE: For locoregionally advanced HNSCC, chemoradiotherapy with cisplatin or another platinum compound is considered as one of the standard treatment regimes. Cisplatin has improved the loco-regional control, but also increased especially the acute side effects. Cetuximab blocks ligand binding and receptor activation by binding to the extracellular domain of the EGFR. The blockade of EGFR signaling in combination with cytotoxic drugs or with radiotherapy could be a novel effective management with a relatively favourable toxicity for HNSCC. In the present study we have examined in vitro a potentially novel effective management for HNSCC, cetuximab combined with cisplatin and radiotherapy. MATERIALS AND METHODS: Seven head and neck SCC cell lines were studied. Cetuximab concentrations of 0.22-8.20 nM and cisplatin concentrations of 0.038-0.220 microg/ml were used. In order to test the concurrent use of cetuximab, cisplatin and radiation, the cells were treated with the desired drug concentrations immediately after irradiation, plated into 96-well culture plates, and incubated for 4 weeks. The number of positive wells was counted. The PE was calculated and fraction survival data were fitted to the LQ model. AUC value was obtained with numerical integration. The types of interaction were analyzed. RESULTS: Cetuximab and cisplatin constantly induced an additive or supra-additive effect when combined with irradiation in the seven HNSCC cell lines tested. CONCLUSIONS: We evaluated concurrent cetuximab, cisplatin, and radiation for HNSCC cell lines. Preliminary efficacy results are encouraging, and further development of this targeted combined modality paradigm is warranted.

Concomitant chemoirradiation with vinorelbine and gefitinib induces additive effect in head and neck squamous cell carcinoma cell lines in vitro.

BACKGROUND AND PURPOSE: The study was performed to measure the radiosensitizing effect of vinorelbine together with gefitinib in head and neck squamous cell carcinoma (HNSCC) cell lines in vitro. MATERIALS AND METHODS: Three recently established HNSCC cell lines, originating from larynx and oral cavity tumors, were tested in this study. Vinorelbine concentration of 0.5 nM was used, corresponding to the IC(70) value of cell lines. Gefitinib concentrations of 0.10-0.35 microM were used, corresponding to IC(70)- and IC(50)-values of each cell line, causing 30% and 50% inhibition in clonogenic survival, respectively. Vinorelbine was added to the medium and the cells were plated in 96-well culture plates in this solution. 24h later the cells were irradiated in plates with 4 MeV photons generated by a linear accelerator, producing radiation doses 0.75-7.5 Gy. Immediately after irradiation the desired concentrations of gefitinib were added, whereafter the plates were incubated at 37 degrees C with 5% CO(2). After four weeks, the number of wells containing coherent living colonies, consisting of 32 cells or more, was counted. The plating efficiency was calculated and the fraction survival data were fitted to the linear quadratic model, F=exp[-(alphaD+betaD(2))]. The area under the survival curve (AUC) value was obtained with numerical integration. ErbB receptor expression of the HNSCC cell lines was analyzed by Western blotting. RESULTS: The growth-inhibitory effect of simultaneous vinorelbine and gefitinib concomitant with radiation was supra-additive in cell line UT-SCC-33 and additive in cell lines UT-SCC-19A and -34. CONCLUSIONS: HNSCC is in vitro constantly sensitive to the combination of vinorelbine and gefitinib, which have together an additive effect in concomitant use with irradiation. Further studies are warranted to evaluate the concomitant use of vinorelbine and gefitinib with irradiation in clinical studies. In addition, both drugs are available in an oral formulation allowing effortless administration schedules.

Concurrent use of vinorelbine and gefitinib induces supra-additive effect in head and neck squamous cell carcinoma cell lines.

PURPOSE: Squamous cell carcinoma of the head and neck (HNSCC) remains a clinical challenge because of the high rate of locoregional disease recurrence. Standard treatment includes surgery, radiation, chemoradiation or a combination of these approaches. New therapies are needed to achieve improved survival, quality of life and organ function in these patients. A novel molecular targeted therapy incorporated into our current treatment strategies may have a significant role in the treatment of HNSCC. The aim of this study was to evaluate the sensitivity of HNSCC cell lines to vinorelbine combined with gefitinib in vitro. METHODS: Six recently established cell lines were used: UT-SCC-9, -11, -19A, -29 and -34 (laryngeal SCC) and UT-SCC-33 (oral cavity SCC). Chemosensitivity was tested using the 96-well plate clonogenic assay. The vinorelbine concentrations used varied between 0.4 and 1.0 nM and the gefitinib concentrations varied between 0.05 and 1.6 muM. Survival data were fitted to the LQ model, and the area under the curve (AUC) value was obtained with numerical integration. The type of interaction was determined by comparing the AUC ratio of the two drugs to the survival fraction (SF) of gefitinib alone. RESULTS: In the current study the combination of vinorelbine and gefitinib had a clear supra-additive or additive cytotoxic effect on the HNSCC cell lines. CONCLUSIONS: This finding is encouraging as a proof of the possible benefit of combing an EGFR targeting compound with a cell cycle specific drug and warrants further studies of available combinations in vitro.

Signaling via ErbB2 and ErbB3 associates with resistance and epidermal growth factor receptor (EGFR) amplification with sensitivity to EGFR inhibitor gefitinib in head and neck squamous cell carcinoma cells.

PURPOSE: The epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) has shown antitumor activity in clinical trials against cancers, such as non-small cell lung cancer and head and neck squamous cell carcinoma (HNSCC). Research on non-small cell lung cancer has elucidated factors that may predict response to gefitinib. Less is known about molecular markers that may predict response to gefitinib in HNSCC patients. EXPERIMENTAL DESIGN: We analyzed possible associations of responsiveness to gefitinib with molecular markers of the EGFR/ErbB receptor family signaling pathway using 10 established HNSCC lines in vitro. IC50 of gefitinib sensitivity was determined using clonogenic survival assays. ErbB signaling was assessed by Western and real-time reverse transcription-PCR analyses of EGFR, ErbB2, ErbB3, and ErbB4 expression levels as well as by phosphorylation analysis of pEGFR, pErbB2, pErbB3, pAkt, and pErk. EGFR sequences encoding kinase domain and EGFR gene copy numbers were determined by cDNA sequencing and real-time PCR, respectively. Finally, responsiveness to gefitinib was compared with responsiveness to the anti-EGFR antibody cetuximab (Erbitux). RESULTS: Expression levels of pErbB2 (P = 0.02) and total ErbB3 protein (P = 0.02) associated with resistance to gefitinib. Combining gefitinib with pertuzumab (Omnitarg), an antibody targeting ErbB2 heterodimerization, provided additional growth-inhibitory effect over gefitinib alone on relatively gefitinib-resistant HNSCC cell lines. The same markers did not predict resistance to cetuximab. In contrast, a similar trend suggesting association between EGFR gene copy number and drug sensitivity was observed for both gefitinib (P = 0.0498) and cetuximab (P = 0.053). No activating EGFR mutations were identified. CONCLUSIONS: EGFR amplification may predict sensitivity to gefitinib in HNSCC. However, other EGFR/ErbB receptor family members than EGFR may contribute to resistance to gefitinib. ErbB2 and ErbB3 may have potential as predictive markers and as therapeutic targets for combination therapy in treatment of HNSCC with gefitinib.