Introduction: Neurofeedback training aims at learning self-regulation of brain activity underlying cognitive, emotional or physiological functions. Despite of promising investigations on neurofeedback as a tool for cognitive rehabilitation in neurological diseases, such as after stroke, there is still a lack of research on feasibility and efficiency of neurofeedback in this field.Methods: The present study aimed at investigating behavioral and electrophysiological effects of 10 sessions of sensorimotor rhythm (SMR) neurofeedback in a 74-years-old stroke patient (UG20). Based on previous results in healthy young participants, we hypothesized that SMR neurofeedback leads to a decrease in sensorimotor interferences and improved stimulus processing, reflected by changes in event-related potentials (ERPs) and electrophysiological coherence. To assess whether UG20 benefited from the training as much as healthy persons of a similar age, a healthy control group of N = 10 elderly persons was trained as well. Before and after neurofeedback training, participants took part in a multichannel electroencephalography measurement conducted during a non-verbal and a verbal learning task.Results: Both UG20 and the healthy controls were able to regulate their SMR activity during neurofeedback training. Moreover, in a non-verbal learning task, changes in ERPs and coherence were observed after training: UG20 showed a better performance in the non-verbal learning task and a higher P3 amplitude after training than before, and coherence between central and parietal electrodes decreased after training. The control group also showed a behavioral improvement in the non-verbal learning task and tendencies for higher P3 amplitudes and decreased central-parietal coherence after training. Single-case analyses indicated that the changes observed in UG20 were not smaller than the changes in healthy controls.Conclusion: Neurofeedback can be successfully applied in a stroke patient and in healthy elderly persons. We suggest that SMR neurofeedback leads to a shutting-down of sensorimotor interferences which benefits semantic encoding and retrieval. Electroencephalography (EEG)-based neurofeedback is a promising tool for cognitive improvement and rehabilitation. While most traditional cognitive trainings consist of specific tasks that aim at improving cognitive functions, neurofeedback is aimed at directly regulating the brain activity underlying cognitive functioning. An important possible future area of application for neurofeedback is cognitive rehabilitation of neurological diseases, such as stroke , as neurDuring sensorimotor rhythm (SMR)-based neurofeedback, the power of SMR, a frequency band ranging from 12 to 15 Hz, is extracted from the EEG signal. SMR is an oscillatory rhythm recorded over central scalp regions, that is supposed to be originating from the thalamic nuclei, specifically from ventroposterior lateral and reticular nuclei , 2000. SHitherto, there is still a lack of studies assessing the efficiency and feasibility of neurofeedback protocols in stroke patients. In two studies reported by Figure 4) indicated that UG20 mainly suffered from short- and long-term memory deficits, while other cognitive functions  remained unaffected. Thus, we decided to apply SMR neurofeedback training in UG20, as this training has proven to be specifically effective in improving memory performance. Neurofeedback was conducted for 10 sessions in the course of 3 weeks at UG20s home. During neurofeedback training, UG20 did not receive medications that affected vigilance or attention and did not take part in other forms of cognitive training. UG20 received an expense allowance of 7 Euro/hour. UG20 provided written informed consent before participation and consented to publication of his data.UG20  had suffered a stroke (ICD-10 diagnosis I63.1) due to basilar artery thrombosis (ICD-10 diagnosis I65.1) with lesions in right cerebellum, bilateral hippocampus, right mesencephalon, left occipital lobe, bilateral splenium 4.5 months before start of SMR training. At the time of stroke onset, UG20 was retired after working for several years as department head of an inventory department. He had completed 12 years of education. After the stroke, UG20 stayed in neurorehabilitation for 3 months, then he was released back home. Since his stroke, UG20 has suffered from memory deficits (amnestic syndrome), while his score of 29 in the mini-mental state examination MMSE,  did not N = 10 healthy elderly participants . Healthy participants were recruited from the general population, by public bulletins and newspaper announcements and gave their written informed consent before participation. Exclusion criteria were any current or previous psychiatric or neurological disorders and history of severe head injuries. Controls received an expense allowance of 7 Euro/hour. The ethics committee of the University of Graz, Austria, approved all aspects of the present study (reference numbers GZ. 39/21/63 ex 2011/12 and GZ. 39/22/63 ex 2011/12) and the study was in line with the code of ethics of the World Medical Association, Declaration of Helsinki. Participants provided written informed consent before participation.As a reference for the evaluation of training-related changes in cognitive performance and EEG signal in UG20, we trained a control sample of mean + 1 SD), while the threshold of SMR power was adapted automatically after each run on the basis of the previous run (mean). When the SMR band power exceeded the predefined threshold, the color of the bar changed from red to green. When participants were able to move the SMR bar above its threshold while keeping the artifact bars below their thresholds for more than 250 ms, they were rewarded by getting points shown at the bottom of the screen. Participants were not given any instruction of how to control the feedback bars but were only told to stay mentally focused and physically relaxed.Electroencephalography signals for SMR training were recorded at channel Cz, digitized at 256 Hz and filtered with a 0.5 Hz high-pass and a 60 Hz low-pass filter. A 10-channel system  and a g.USBamp 16-channel standard amplifier  were used for data recording. The ground electrode was located at the right mastoid, the reference at the left mastoid. Vertical electrooculogram (EOG) was recorded by two electrodes on top and below the left eye (bipolar montage). Each participant performed 10 training sessions (approximately 50 min each) on different days. Sessions were conducted 2–5 times a week and consisted of 10 min of preparation, a 3-min baseline trial in which the participants saw moving bars while instructed to relax themselves without trying to control the bars voluntarily, and six 3-min feedback runs. In these runs, participants were instructed to increase SMR power while reducing electromyography (EMG) and EOG artifacts. Three bars were presented on a computer screen: the height of the bar in the middle reflected real-time SMR power , while the height of the left bar reflected eye-blink artifacts  and the height of the right bar EMG artifact power (EEG band power between 21 and 35 Hz which indicates movements and other high-frequency disturbances). The data recorded in the baseline trial was used to calculate individual thresholds for the three bars. For the artifact bars, thresholds were kept constant over all neurofeedback runs  band power was extracted by means of a procedure provided by the Vision Analyzer software . The resulting power values were averaged over all artifact free segments of each training run. To assess training effects, SMR power was averaged per run of training across all different sessions. SMR power values were z-transformed to ensure comparability across sessions and subjects. To analyze more closely the time course of SMR power over the training runs averaged over all sessions, we conducted linear regression analyses . As during successful neurofeedback, a within-session increase of the feedback frequency power is expected, the slope of the regression line was used as an indicator of neurofeedback performance see .Figure 1 for a depiction of task procedure). In the NVLT, participants saw geometric or irregular shapes consisting of black lines on a white rectangle presented on a black background screen. After seeing each shape, participants had to indicate by button press whether they had seen this figure before or not. The VLT followed the same procedure, but instead of figures, pseudo-words (neologisms) were used as stimuli. Neologisms were presented in black letters (2 cm font size) on a white rectangle over a black background screen.The same protocol was followed for UG20 and the healthy control sample: before and after neurofeedback training, 60-channel EEG measurements were carried out while participants were presented with a non-verbal (NVLT) and a verbal (VLT) memory task adapted from UG20 additionally performed a comprehensive neuropsychological test battery before and after neurofeedback training to evaluate the cognitive profile of the stroke patient in more detail. This test battery included standardized neuropsychological tests to assess attention . The results of this regression analysis indicated that participants showed consistent linear increase in SMR power over the training runs . When analyzing the time course of SMR power over the training runs separately for each participant of the SMR group, 8 out of 10 participants  showed a positive gradient of the learning curve. Of the two remaining participants, one showed a flat learning curve gradient and one a negative gradient. A single-case analysis (t(9) = 1.93, p = 0.09, d = 1.22], indicating that his ability to alter SMR power was at least as good but not statistically different from that of healthy controls. The increase in SMR power observed in UG20 across training runs indicated successful training of SMR in the stroke patient.As shown in analysis  indicateFigure 3). Healthy participants showed improvements in the non-verbal learning task after training as well, which were significant in a t-test . In the verbal learning task, no change in performance was observed in the controls . To further assess the improvement observed in UG20, the pre–post difference in his test scores was compared to the pre–post difference of healthy controls applying single-case analysis according to the approach of t(9) = -0.127, p = 0.451, d = -0.08], indicating that UG20 improved at least as much as the controls in the NVLT after the training.After neurofeedback training, UG20 improved in the non-verbal task, while performance in the verbal learning task remained unaltered after training .In the neuropsychological test battery assessed before and after neurofeedback training, stroke patient UG20 showed significant performance improvements in non-verbal short-term memory  and working memory (CBTT backward task) tasks when comparing the pre- and post-assessment. The performance in several scales of the CVLT assessing verbal short- and long-term memory performance also improved . In the forward task of the CBTT and List A of the CVLT assessing short-term memory performance, UG20 showed a decreased performance after neurofeedback training compared to the pre-assessment. In sum, UG20 showed significant improvements in memory tasks, whereas attentional and executive functions did not change when comparing the results of the pre- and post-assessment : P3 amplitude was enhanced post-training compared to pre-training. N1 amplitude in the non-verbal task was slightly increased in UG20 after training as well. In the verbal learning task, no clear pre–post differences in P3 or N1 amplitudes could be observed in UG20 .In t(9) = 1.91, p = 0.08, d = 1.21, bootstrappingp = 0.078; see Figure 5]. When comparing the N1 amplitude between pre- and post-test, no differences were found . In the verbal task, there were neither pre–post differences in P3 amplitudes  nor in N1 amplitudes .The healthy group showed a tendency for a higher P3 amplitude in the post-compared to the pre-measurement during stimulus processing of the non-verbal task  between these values, thus indicating a tendency of amplitude increase of similar extent as observed in the controls. Also, there was no significant difference between the patient’s and the healthy participants’ change in N1 according to single-case analysis based on t(9) = -0.016, p = 0.494, d = -0.016), indicating no significant change in N1 in the patient.A single-case-analysis  comparinFigure 7).Coherence analyses for the non-verbal task indicated a decrease in coherence between electrodes Cz and CPz during the non-verbal task for UG20. In the verbal learning task, coherence decreased as well (see t(9) = 1.92, p = 0.09, d = 1.21, bootstrappingp = 0.06]. For the other two electrode pairs, coherence did not differ significantly after training when compared to before training . For the verbal task, no differences in coherence were observed .For healthy controls, coherence during the non-verbal learning task between electrodes Cz and CPz showed a marginally significant decrease after NF compared to pre-training values : the decrease in UG20s coherence values was larger than the decrease in the healthy elderly. In the verbal learning task, according to single-case analysis, the decrease in coherence was also significantly larger in UG20 than in healthy controls . There were no significant differences between pre–post coherence changes of UG20 and the controls in the other two electrode pairs and in the verbal learning task .Single-case analysis  for the The present study investigated the applicability of SMR-based neurofeedback training in one stroke patient (UG20) and a healthy elderly control sample and assessed behavioral and electrophysiological effects of the training. Results indicated that participants learned to increase SMR voluntarily during neurofeedback. After 10 sessions of SMR up-training, UG20 and the control group showed behavioral improvements in a non-verbal learning memory task, an increase in P3 amplitudes and a decrease in electrophysiological coherence during this task. Single-case Crawford analyses indicated that the changes observed in UG20 were as large as the changes in the age-matched controls, suggesting that UG20 benefited as much from the training as the control group. These results will be discussed in more detail below.In a first step, we assessed whether UG20 was able to increase SMR activity voluntarily through neurofeedback training. Regression analysis showed that during the neurofeedback training sessions, UG20s SMR activity increased linearly, indicating that he was able to voluntarily increase SMR activity. The control group also showed consistent increases in SMR power over the training runs averaged, indicating successful up-training of SMR activity. This finding is consistent with previous studies demonstrating the applicability of slow cortical potentials-, alpha- and theta-based neurofeedback paradigms in healthy older persons . A singlBefore and after neurofeedback training, UG20 was confronted with a verbal learning task and a non-verbal learning task. UG20 showed an improvement in the non-verbal learning task after neurofeedback training. The control group’s results mirrored those of UG20, as they showed a significant improvement of performance in the non-verbal learning task as well. A single-case Crawford analysis indicated that UG20 improved his non-verbal memory performance as much after the training as the healthy elderly group. These results are well in line with studies demonstrating improved memory abilities after SMR neurofeedback training . FurtherWhile UG20 as well as healthy elderly controls showed improvements in their non-verbal memory performance after SMR based neurofeedback training, no change in performance was observed in the verbal learning task. This finding contrasts with our previous study , where wThe results of the neuropsychological test battery suggest that SMR neurofeedback training had mainly effects on memory functions in stroke patient UG20. Before the start of the neurofeedback training, UG20 showed severe memory deficits (T-scores < 40). After SMR neurofeedback, UG20 showed a higher performance in different memory functions compared to the pre-assessment. For instance, working memory performance as assessed with the CBTT backward task was below average during the pre-assessment. After SMR neurofeedback training, performance in the CBTT backward task was in a normal range. Short-term memory performance (VVM2 “city map 1” and CVLT List B) was also far below average before the start of the neurofeedback training. During the post-assessment, performance in these short-term memory tasks was only slightly lower than the normal range. Attentional and executive functions did not change due to SMR neurofeedback training. This result is in line with previous findings in healthy people that indicated that SMR neurofeedback training leads to memory improvements . Of noteWhile completing the non-verbal and verbal learning tasks, our participants underwent 60-channel EEG measurements enabling us to assess ERPs during stimulus processing. In line with the behavioral results, in UG20 changes in EEG parameters after neurofeedback training were only apparent in the non-verbal learning task: in this task, he showed higher P3 amplitudes after training compared to the pre-measurements. N1 amplitude was also slightly higher in UG20 after training than before. In the healthy elderly group, a similar pattern was observed: analyses of ERPs showed a tendency for higher P3 amplitudes in the post-compared to the pre-measurements during presentation of correctly identified target stimuli. However, there was no change in N1 amplitude after training in the control group. Single-case analyses showed that the pre–post change in P3 amplitude in UG20 was not significantly different from the pre-post-change observed in healthy controls. Further, the change in N1 amplitude visible in UG20 was not larger than the change observed in the healthy elderly group. Thus, while neither UG20 nor the control group showed a reliable difference in N1 amplitude after training, they showed a tendential increase in P3 amplitude after training. The observed increase in P3 amplitude is consistent with previous results  and indiFurthermore, coherence analyses were carried out assessing electrophysiological coherence between the feedback region (electrode Cz) and parietal areas. In UG20, coherence between Cz and CPz decreased strongly after training compared to before and this change was apparent in both the verbal and the non-verbal learning task. This reduced coherence between motor and parietal areas indicates lower functional connectivity between these brain areas after training. Thus, activity in these regions was less synchronized after training, which could be a further indicator of a reduction of sensorimotor interferences achieved by SMR neurofeedback training. In the control group, coherence in the non-verbal learning task was marginally decreased after training compared to pre-training, and there were no changes in coherence in the verbal learning task. Single-case analyses indicated that electrophysiological coherence during the verbal and non-verbal learning task decreased more strongly in UG20 than in the healthy participants, further corroborating that SMR training was effective in UG20. Of note, compared to our previous results in healthy young participants , the conThe behavioral and electrophysiological results of our study partially replicate a previous study of our group on SMR neurofeedback in healthy young participants . In thisIn the present case-study, we could show that a stroke patient and healthy elderly controls were able to self-regulate their SMR activity during 10 sessions of SMR neurofeedback training. Moreover, observed behavioral improvements after training in a non-verbal learning task were accompanied by increased P3 amplitudes and a decreased coherence between motor and parietal areas during this task. These results offer support for the hypothesis that SMR neurofeedback enhances the inhibition of sensorimotor interferences, thereby facilitating stimulus processing and the formation of deep semantic associations. Furthermore, the improvements we observed in the UG20 were not smaller than those in the healthy controls, suggesting that neurofeedback might be a suitable tool for stroke patients and should be investigated further in future studies. Research on neurofeedback for stroke rehabilitation is of special importance as positive effects of traditional cognitive trainings in this domain remain disputed . Also, tJR: participated in the design of the study and data acquisition, analyzed, and interpreted data; and prepared the manuscript. SK: participated in the study design, analyzed data, and critically revised the manuscript. DS: acquired data, contributed to study design, carried out patient recruitment and the patient’s clinical assessment. PG: contributed to study coordination, patient recruitment, data collection, and medical care of the patient. GW: participated in the study design and coordination, performed statistical analysis and critically revised the manuscript. CN: participated in conception and supervision of the study, interpreted data and contributed to manuscript revision. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over 50% of patients with head and neck squamous cell carcinoma (HNSCC) present with locoregionally advanced disease. Those at intermediate-to-high risk of recurrence after definitive therapy exhibit advanced disease based on tumour size or lymph node involvement, non-oropharynx primary sites, human papillomavirus (HPV)-negative oropharyngeal cancer, or HPV-positive oropharynx cancer with smoking history (>10-pack-years). Non-surgical approaches include concurrent chemoradiotherapy, induction chemotherapy followed by definitive radiotherapy or chemoradiotherapy, or radiotherapy alone. Following locoregional therapies , no standard intervention exists. Overexpression of epidermal growth factor receptor (EGFR), an ErbB family member, is associated with poor prognosis in HNSCC. EGFR-targeted cetuximab is the only targeted therapy that impacts overall survival and is approved for HNSCC in the USA or Europe. However, resistance often occurs, and new approaches, such as targeting multiple ErbB family members, may be required. Afatinib, an irreversible ErbB family blocker, demonstrated antiproliferative activity in preclinical models and comparable clinical efficacy with cetuximab in a randomized phase II trial in recurrent or metastatic HNSCC. LUX-Head & Neck 2, a phase III study, will assess adjuvant afatinib versus placebo following chemoradiotherapy in primary unresected locoregionally advanced intermediate-to-high-risk HNSCC.Patients with primary unresected locoregionally advanced HNSCC, in good clinical condition with unfavourable risk of recurrence, and no evidence of disease after chemoradiotherapy will be randomized 2:1 to oral once-daily afatinib (40 mg starting dose) or placebo. As HPV status will not be determined for eligibility, unfavourable risk is defined as non-oropharynx primary site or oropharynx cancer in patients with a smoking history (>10 pack-years). Treatment will continue for 18 months or until recurrence or unacceptable adverse events occur. The primary endpoint measure is duration of disease-free survival; secondary endpoint measures are disease-free survival rate at 2 years, overall survival, health-related quality of life and safety.Given the unmet need in the adjuvant treatment of intermediate-to-high-risk HNSCC patients, it is expected that LUX-Head & Neck 2 will provide new insights into treatment in this setting and might demonstrate the ability of afatinib to significantly improve disease-free survival, compared with placebo.NCT01345669.ClinicalTrials.gov The online version of this article (doi:10.1186/1745-6215-15-469) contains supplementary material, which is available to authorized users. The most common type of malignant tumour of the head and neck is squamous cell carcinoma, with an estimated global incidence of approximately 600,000 cases per year . Locoreget al.[For oropharyngeal squamous cell carcinoma (OPSCC), high-risk patients with locoregionally advanced disease have been previously defined as patients with HPV-negative tumours who either have a ≤10 pack-year smoking history with T4 tumours  Staging Classification for Head and Neck Cancers)  or have et al.. Human pet al., 12, witet al.. Human pet al.. HoweverP < 0.001 and overall survival hazard ratio = 0.31, P = 0.002) [p16 is a tumour suppressor and cell-cycle regulator that is upregulated in HPV-positive OPSCC and often lost in HPV-negative OPSCC. In OPSCC, p16 expression is well established as a surrogate marker of HPV infection and is also associated with prognosis , 15. Ind= 0.002) .et al.[et al. in 2012 [P < 0.001), a worse 3-year progression-free survival rate , significantly more locoregional relapses  and significantly more second primary tumours  versus patients with HPV-positive OPSCC. Therefore, because of the available data on the prognostic factors in OPSCC, both patients with p16-negative locoregionally advanced OPSCC and patients with non-OPSCC locoregionally advanced disease were considered to have high-risk locoregionally advanced HNSCC.Overall survival in patients with high-risk locoregionally advanced OPSCC is low; in the Radiation Therapy Oncology Group 0129 trial reported by Ang  in 2012 , similaret al.[Following diagnosis of primary locoregionally advanced HNSCC, standard treatment options are concurrent chemoradiotherapy, radiotherapy alone, induction chemotherapy followed by radiotherapy, or concurrent chemoradiotherapy , with ciet al., an absoet al..The use of chemotherapy after locoregional treatment has been studied in only a limited number of randomized trials in HNSCC. Early studies examining the impact of chemotherapy in the maintenance setting demonstrated modest, but minimal, benefits, and these did not translate into a survival benefit overall –20. A siP = 0.029) [Epidermal growth factor receptor  is expressed in over 90% of HNSCCs , with hi= 0.029) .Cetuximab, an EGFR-targeting monoclonal antibody, is the only targeted treatment approved in the USA, Japan and Europe for HNSCC, where it is approved in combination with radiotherapy for the treatment of locoregionally advanced disease , 26. In P = 0.005), as well as the secondary endpoints of overall survival  and progression-free survival  [n = 442) phase III study, in which cetuximab plus platinum/5-fluorouracil was compared with platinum/5-fluorouracil alone, did show an impact on survival, demonstrating an improvement from 7.4 months for the control arm to a median of 10.1 months for the cetuximab arm [In patients with untreated locoregionally advanced HNSCC, cetuximab plus radiotherapy compared with radiotherapy alone showed a statistically significant improvement in the primary endpoint of locoregional control  . Cetuxim= 0.006) . A largeExtensive cross-talk between all ErbB-dependent signalling pathways in HNSCC, as well as the numerous molecular and genetic aberrations present, contribute to the development of cetuximab resistance . AcquireP < 0.001) in the first-line EGFR mutation-positive setting [Afatinib is an oral, irreversible ErbB family blocker that completely and irreversibly blocks signalling by EGFR, HER2 and ErbB4. It also blocks transphosphorylation of ErbB3 , 34. Afa setting . In more setting .in-vitro combination experiments demonstrated additive activity of afatinib when combined with standard chemotherapy [Preclinical activity of afatinib in HNSCC has been observed in human HNSCC FaDu mouse models , whilst otherapy . Furtherotherapy . In stagotherapy . FurtherGastrointestinal and dermatological side effects are common in patients receiving EGFR tyrosine kinase inhibitors, and have a large impact on patients’ quality of life. Afatinib has a manageable tolerability profile in clinical trials, with toxicity dominated particularly by gastrointestinal and dermatological side effects. Recent pooled data analyses suggest that adverse events associated with afatinib can be effectively managed in patients with solid tumours , 44.The dosage for afatinib was initially established during monotherapy trials, with a maximum tolerated daily dose of either 40 or 50 mg , 46. LUXThe LUX-Head & Neck 2 trial (NCT01345669) investigates the efficacy and tolerability of afatinib compared with placebo when given as adjuvant therapy after chemoradiotherapy, in patients with primary unresected HNSCC prior to chemoradiotherapy and no evidence of disease  post-chemoradiotherapy. The hypothesis being tested is that prolonged, irreversible, small-molecule inhibitor-mediated ErbB family blockade will improve disease-free survival outcomes. The study aims to select patients with unfavourable prognosis according to disease stage, tumour site and smoking status or history.The primary objective of this study is to evaluate the superiority of afatinib as adjuvant therapy versus placebo in terms of disease-free survival (primary endpoint) following chemoradiotherapy in patients with primary unresected locoregionally advanced  HNSCC. Secondary endpoints include the disease-free survival rate at 2 years, overall survival rate, health-related quality of life and safety. Voluntary biomarker assessments were also conducted.The LUX-Head & Neck 2 trial is a randomized, multicentre, double-blind, placebo-controlled, phase III trial. Patients will undergo magnetic resonance imaging, positron emission tomography or computed tomography approximately 8 to 12 weeks after completion of chemoradiotherapy and, if there is no evidence of disease, patients will be randomized no later than 24 weeks post-chemoradiotherapy to either afatinib or placebo in the ratio 2:1. Randomization will be conducted centrally with a validated random number-generating system at Boehringer Ingelheim, verified by a trial-independent statistician, and implemented via an interactive internet and voice-response system. Access to the randomization code will be supervised by the clinical trial support group; those directly involved in the conduct and analysis of the trial will not have access to the randomization schedule prior to database lock. Stratification will be based on patients’ nodal status  [This trial is expected to randomize 669 patients to treatment worldwide (446 in the afatinib arm and 223 in the placebo arm) and will be carried out in compliance with the protocol, and in accordance with the principles laid down in the Declaration of Helsinki, the International Conference on Harmonisation Guideline for Good Clinical Practice and applicable regional-specific regulatory requirements. The study protocol has been reviewed by an independent ethics committees ), and at randomization have chemoradiotherapy-induced adverse events classified as less than or equal to grade 2 using the Common Terminology Criteria for Adverse Events. Patients are required to have no evidence of disease after chemoradiotherapy . Patients must also have adequate bone marrow, liver and kidney function. The main exclusion criteria are prior treatment with EGFR-targeted small molecules, EGFR-targeted antibodies or any investigational agents for HNSCC, patients with a smoking history of ≤10 pack-years and with primary tumour site of base of tongue or tonsil, and primary cancer of nasopharynx, sinuses or salivary glands.Eligible patients must be ≥18 years of age, have an ECOG performance status of 0 or 1 and have histologically or cytologically confirmed locoregionally advanced HNSCC at the time of randomization . Patients are required to have unresected disease prior to chemoradiotherapy and an unfavourable risk of recurrence. Patients can have unresected disease for the following reasons: (1) they may have technically unresectable disease owing to tumour fixation, or invasion to either base of the skull, cervical vertebrae, nasopharynx or fixed lymph nodes; (2) there may be low surgical curability  [Disease-free survival, the primary endpoint measure, is defined as the time from randomization until documented tumour recurrence or second primary tumour or death of any cause, whichever occurs first. The definition of recurrence or second primary tumour is the appearance of any new lesions without any evident benign aetiology, assessed by imaging of the head and neck and chest by the investigator and an independent central review. The primary analysis will be based on the investigator’s assessment. Tumour recurrence will be further classified into subtypes of local, regional or distant recurrence. Disease-free survival rate at 2 years, the key secondary endpoint, will be calculated in each group using the Kaplan-Meier method. Overall survival is defined as time from the date of randomization until death.The incidence and intensity of adverse events will be evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0. All adverse events, serious and non-serious, occurring during the course of the trial , regardless of relatedness to study medication, will be collected, documented and reported by the investigator. Serious and non-serious adverse events occurring later than 28 days after the last administration of study medication will only be reported if they are considered relevant by the investigator. All adverse events, including those persisting after the end of the study medication, must be followed up until they have resolved or have been sufficiently characterized. Safety endpoints are the overall incidence and intensity of adverse events, gastrointestinal events , skin reactions  and change from baseline in all laboratory tests.Health-related quality of life will be assessed using the following patient-reported outcome measures: European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (QLQ-C30); European Organisation for Research and Treatment of Cancer Quality of Life Head and Neck cancer module (QLQ-H&N35); and EQ-5D health status questionnaire. Questionnaires will be completed at randomization and at each tumour assessment time point until disease recurrence. Questionnaires will be filled in by the patients at the clinic before any clinical assessment and treatment, and before provision of any new information about their disease status.Participation in the biomarker analysis is voluntary and is not a prerequisite for participation in the trial. Separate informed consent must be provided by patients for this part of the study, in accordance with local ethical and regulatory requirements. Biomarker analyses will be performed using archival tumour tissue; fresh tumour biopsies are not possible as there is no residual disease in participating patients. Exploratory biomarkers to be assessed are p16 status, ErbB ligands, ErbB receptor expression, EGFR mutational or activation status and ErbB downstream signalling markers.This trial is powered to detect a prolonged median disease-free survival of 6.2 months with afatinib over an assumed disease-free survival of 15.8 months in the placebo arm [dKM) will be calculated as the difference between the two Kaplan-Meier estimates. Overall survival will be analyzed using the stratified log-rank test, the Kaplan-Meier method and the stratified Cox proportional hazards model, in the same manner as for disease-free survival.Disease-free survival will be analyzed using the stratified log-rank test with nodal status (N0 to N2a versus N2b to N3) and ECOG performance status (0 versus 1) being the stratification factors. The Kaplan-Meier method will be used to estimate the 25th percentile, median and 75th percentile disease-free survival for each treatment group and the stratified Cox proportional hazards model will be used to derive the hazard ratio between the two treatment groups. The difference in the disease-free survival rate at 2 years between afatinib and placebo  is assessing afatinib versus methotrexate in patients with recurrent or metastatic HNSCC who have progressed with platinum-based therapy . The priNovel activity of afatinib has been shown in preclinical models of head and neck cancer , 41 and The trial was initiated in October 2011 and is currently recruiting patients in Argentina, Australia, Austria, Belgium, Brazil, Canada, Chile, the Czech Republic, Denmark, Finland, France, Germany, Greece, Hungary, India, Israel, Italy, Japan, Mexico, Portugal, Russia, Spain, Sweden, Switzerland, the Netherlands, the UK and the USA Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Below are the links to the authors’ original submitted files for images.


The possibility of incorporating generics into combination antiretroviral therapy and breaking apart once-daily single-tablet regimens (STRs), may result in less efficacious medications and/or more complex regimens with the expectation of marked monetary savings. A modeling approach that assesses the merits of such policies in terms of lifelong costs and health outcomes using adherence and effectiveness data from real-world U.S. settings.A comprehensive computer-based microsimulation model was developed to assess the lifetime health  and economic outcomes in HIV-1 infected patients initiating STRs compared with multiple-table regimens including generic medications where possible (gMTRs). The STRs considered included tenofovir disoproxil fumarate/emtricitabine and efavirenz or rilpivirine or elvitegravir/cobicistat. gMTRs substitutions included each counterpart to STRs, including generic lamivudine for emtricitabine and generic versus branded efavirenz.Life expectancy is estimated to be 1.301 years higher (discounted 0.619 QALY gain) in HIV-1 patients initiating a single-tablet regimen in comparison to a generic-based multiple-table regimen. STRs were associated with an average increment of $26,547.43 per patient in medication and $1,824.09 in other medical costs due to longer survival which were partially offset by higher inpatients costs  with gMTRs treatment. Overall, STRs presented incremental lifetime costs of $16,335.91 compared with gMTRs, resulting in an incremental cost-effectiveness ratio of $26,383.82 per QALY gained.STRs continue to represent good value for money under contemporary cost-effectiveness thresholds despite substantial price reductions of generic medications in the U. S. Innovations in antiretroviral therapy (ART) have dramatically altered the natural history of HIV infection, transforming it into a manageable chronic disease . ART hasIncreasing life expectancy contribute to increased total lifetime costs of HIV care, posing challenges for funders despite annual costs of treating someone with HIV having remained fairly constant over the years . Many diAt first glance, these strategies seem appealing because they appear to present significant economic savings. Caution is warranted in interpreting these results, however, because some of these economic analyses are focused mainly on ART costs, ignoring other important costs such as inpatient and outpatient costs, or even wider economic societal implications, that may account for up to 45% of the total lifetime costs of HIV care –10. MoreThere is evidence that antiretroviral pill burden and treatment adherence are key determinants of the risk of hospitalization and inpatients costs , 12. PatAlso of concern is the justification of monetary savings being considered at the expense of potential health losses without adequately addressing the fact that societal value for a health loss is regarded to be substantially higher than the societal value for a health gain, i.e. monetary saving required to move from optimal to sub-optimal therapy are higher than the maximum willingness to pay for a health gain obtained by moving from lower to a higher health status .The present study includes a computer-based microsimulation model of HIV disease progression based on adherence and effectiveness data from real-world settings in the US to comprehensively assess the long-term health and economic outcomes of HIV infected patients initiating single-tablet regimens (STRs) compared with multiple-tablet regimens including generic medications when possible (gMTRs).In a simulation study two treatment strategies were considered for HIV-1 infected adult patients initiating first-line ART : a brandThe expected long-term, comparative economic and clinical impact of the proposed treatment strategies were analyzed using a microsimulation cost-effectiveness model for HIV treatment. Given the chronic and progressive nature of the disease, the model considered a lifetime time horizon. The economic impact was measured in terms of the expected total per-person cost to the U.S. health system, whereas the clinical impact or effectiveness was measured in terms of corresponding expected quality-adjusted life-years (QALY). Following U.S. recommendations, an annual 3% discount rate was applied to both costs and effectiveness. Deterministic sensitivity analysis was used to assess parameter uncertainty, where possible.At the core of the microsimulation model lies a state-transition model , in whicPatients enter the model in the “non-suppressed” state (transition 0), where they initiate first-line therapy. Within this state, a monthly probability of virologic suppression (VS), applied during the first 6 months of therapy, determines if and when a patient moves to the “suppressed” state, and can be considered a responder to therapy (transition 1). Patients in the “non-suppressed” state that do not achieve VS within the first 6 months of therapy move to the “non-suppressed” state of the subsequent therapy line at the next scheduled medical visit (transition 2).Once transitioned to the “suppressed” state, from the 7th month of therapy onwards, patients are subject to a monthly probability of virologic failure after suppression has been achieved (VF). Upon VF, patients move to the “rebound” state (transition 3), where they stay until the next scheduled medical visit, upon which they move to the “non-suppressed” state of the subsequent therapy line (transition 4).Irrespective of their state of virologic suppression, patients are continuously subject to a monthly probability of discontinuation due to reasons other than VF (transitions 5). Upon discontinuation while in the “suppressed” state, patients immediately transition to the “suppressed” state of the subsequent therapy line. For the remaining non-virologically suppressed states, patients transition to the “non-suppressed” state of the subsequent therapy line. Finally, patients are also continuously subject to a monthly probability of death  and first-line treatment strategy (STR vs. gMTR) dependent adherence levels. Every patient is simulated under both an STR and a gMTR first-line strategy . Differential adherence between STRs and gMTRs is assumed to influence virologic response for first-line therapy only. Subsequent therapy lines are the same for all patients, irrespective of first-line STR or gMTR strategy. At each monthly cycle, the characteristics associated with each patient, including age, HIV-1 RNA viral load, CD4+ T cell count, quality of life (QoL), and health care and drug costs, are tracked and updated. An influence diagram relating the different model components can be found in 3  , present3 . Mean ag3  7]..3  phase II or III randomized clinical trials, with preference to phase III studies; 2) patient characteristics in terms of the number of previous therapy lines that best fitted the therapy line in question; 3) follow-up data in at least two points in time, with preference for the most recent studies with more available time-points and longer follow-up .First-line gMTR, EFV+TDF+3TC has been studied in clinical trial setting in Study 903 , 24 and The first-line therapy virologic suppression and failure probabilities presented in Monthly probabilities for discontinuation due to other reasons were determined from the clinical trials used for the calibration of virologic response probabilities, considering discontinuations for all reasons (including adverse events) except virologic failure (lack of efficacy) and death .Monthly probabilities of death were determined by multiplying CD4+ T cell count dependent standardized mortality ratios (SMR) to gender and age specific base mortality rates reported by the Center for Disease Control and Prevention (CDC) national vital statistics  38]..38].While in the “suppressed” state , patient3 and by an annual rate of 0.17 otherwise [For patients in the “rebound” state, viral load is reset to the highest level ever observed since treatment initiation (≥ 50 RNA cps/mL), after which it keeps increasing on a log10 scale, by an annual rate of 0.11 while CD4+ T cell counts are greater than or equal to 200 cells/mmtherwise . CD4+ T therwise .For patients in the “non-suppressed” state that will never suppress, viral load and CD4+ T cell count evolution is equivalent to that of patients in the “rebound” state. For the remaining patients, viral load and CD4+ T cell count are equivalent to the evolution discussed for patients in the “suppressed” state.Costs accounted for in the model include ART costs, hospitalizations (inpatient) costs, and other medical expenses . All cost, 2015; Unit ART costs were based on data from First Databank,   and are The model simulated 200,000 hypothetical individuals initiating ART  using real-world evidence. All results represent the averages of each group of hypothetical individuals (STR vs. gMTR) and are discounted at 3.0% per annum.After incorporating real-world evidence, over the lifetime of a patient an additional discounted 0.619 QALYs (14.466 vs. 13.847) were estimated to be gained with STRs compared with gMTRs . Single-Undiscounted life expectancy after ART initiation was estimated to be 22.4 years for STR and 21.1 years, with a benefit of 1.3 years in favor of the STR treatment. The undiscounted QALY gain for STR over gMTR was 1.3 QALY for a marginal lifetime cost of $41,188.85 resulting in an undiscounted ICER of $31,910.87 per QALY gained.A deterministic sensitivity analysis was conducted to determine the factors that had the greatest influence on the ICER. The range of variation to each parameter is described in the As shown in the Tornado diagram, the ICER increased when baseline CD4 cell count, gEFV price discount, percentage of patients suppressed at 48 weeks for EFV-based gMTR and odds ratio of ≥95% adherence are assumed higher than in the base case. The opposite is true for a higher percentage decrease in hospitalizations on STR vs. MTR, higher standardized mortality ratios and higher quality of life utilities.th-line therapy, a successive sequence of further therapy lines with efficacy, safety and cost parameterization equal to that of the 6th-line, results in a dominant scenario for STR as compared to gMTR: an increase of 0.49 discounted QALY, accompanied by $14,348 in discounted total cost savings.Considering second-line therapy effectiveness equal to that considered in Walensky et al. (2013)  decreaseThe cost-effectiveness results were most sensitive to baseline CD4+ T cell count, generic EFV price (% discount from branded EFV price) and 48 weeks virologic suppression rates for EFV-based gMTR. Sensitivity analysis to the values of other variables like the odds ratio of ≥95% adherence between STR and MTR, hospitalizations rates, second line-treatment effectiveness, standardized mortality ratios, quality of life utilities and costs revealed minor uncertainty in the range of 2% to 12% relative to baseline ICER.3 to 500–1000 cells/mm3, respectively.The assumption about the value of baseline CD4 count had the most impact in the variability of the ICER of STR over gMTR . It rangThe base case analysis already incorporated a substantial discount of 75% on the price of generic relative to branded efavirenz. Varying the price generic efavirenz up to a 95% discount was associated with a maximum ICER of $31,421/QALY . This vaIn this study we introduce a comprehensive model to assess the lifetime health and economic outcomes of HIV infected patients initiating STRs compared with MTRs including generic medications where possible. STRs considered are those composed of TDF/FTC and efavirenz or rilpivirine or elvitegravir/cobicistat. MTRs considered included the counterparts to STRs substituting emtricitabine by generic lamivudine (g3TC) and branded efavirenz by the generic. The underlying motivation was to understand the value for money of branded STRs in comparison to MTRs composed of generic medications available in the U.S. market.It is estimated that initial treatment for HIV infection with STRs would result in an enhanced life expectancy of 1.3 years (undiscounted) in comparison to use gMTRs. Lifetime health care costs are estimated to be higher with STR and partially offset by higher inpatients costs expected with gMTR treatment. We estimate a mean increment of $16,336 in lifetime health care costs with STR treatment over gMTR corresponding into an incremental cost of $26,384 per QALY. A sensitivity analysis to the key parameters of the model showed low level of uncertainty with ICER ranging from $13,790 to $33,211 per QALY.Our estimates of discounted lifetime medical costs of $547,540 in HIV-infected individuals starting up-to-date STRs are comparable with other recently published model based estimates  using a similar set of assumptions in patients initiating standard of care . Also, mOur study is subject to some limitations. The gMTR comparator in our study includes generic efavirenz and lamivudine in substitution of branded efavirenz and emtricitabine of EFV/TDF/FTC, RPV/TDF/FTC and EVG/COBI/TDF/FTC single-tablet regimens. In the literature, no direct evidence was found comparing these STRs and an MTR of RPV or EVG/COBI. In this case a simplifying assumption was made assuming the same relative measure of efficacy and safety between gEFV+TDF+g3TC and EFV/TDF/FTC (Study GS-236-102 and Study 903) , 23, 24,Although the gMTR comparators are not recommended options by the DHHS Panel on Antiretroviral Guidelines for Adults and Adolescents , as the Reported efficacy was assumed valid irrespective of the high levels of adherence observed and different measurement techniques used in studies GS-236-102 and 903. In first- and subsequent-line therapies no pooling of efficacy and safety data was performed if different studies were identified reporting on the same ART regimen. Virologic failure probabilities calibrated on our study are relatively low as compared to virologic failure probabilities of other first- and subsequent-line regimens. This can be expected to influence the average time spent in first-line therapy and in consequence, lifetime results.Because little is known about the adherence-effectiveness relationship in integrase inhibitor (INI) based regimens for modeling purpose this relationship was based on results of populations mainly treated with EFV-based therapy (>60%) and variable NRTI-backbones , 49. DifThe STR associated benefit on adherence over MTR is based on Sweet et al. 2014)  and Sax   and SaxPatients discontinuing sixth-line therapy due to virologic failure or other reasons move to a seventh-line, where they are modelled as being in an ART-free non-suppressive state, with HIV-1 RNA viral load levels increasing from their highest value ever observed, and CD4+ T cell counts decreasing from their lowest value ever observed, until death. One might question the assumption used to model this seventh-line, in that it is not clear if patients failing multiple therapy-lines in reality move to an ART-free state. Furthermore, depending on the average time spent in previous therapy-lines and age reached when entering this seventh-line, its influence on overall results might be larger than intended. This was dealt with in a sensitivity analysis, assuming a successive sequence of further therapy lines with efficacy, safety and cost parameterization equal to that of the sixth-line therapy.Due to the computational expensiveness of the model, traditional probabilistic sensitivity analysis through a large number of model runs at different random input values was deemed unfeasible. Solutions to this problem have been suggested in the literature, a common one of which is through regression meta-modelling . The appConsistent with other model estimates , 53 and Most notably, real-word evidence suggests cost-savings in HIV-infected patients starting ART with STR in addition to health gains , 54, 56.In contrast to this evidence and to our results is the study by Walensky et al. (2013) estimating that the incremental cost-effectiveness for first-line branded STR in United States is over $100,000/QALY relative to gMTR. The authors suggest that starting or switching to generic based regimens would initially yield annual savings approaching $1 billion for programs that fund HIV treatment in the United States, at the expense of a slightly less effective generic alternative (1 QALY loss per patient). But because non-medication costs and the costs for other medications were not included, and adherence to ART was not explicitly modeled, we anticipate that these results may be subject to a high degree of uncertainty and potentially underestimate the total costs associated with gMTR. In fact, breaking down once-daily fixed-dose ART into their single components and switching emtricitabine by less efficacious lamivudine  may leadUnder these circumstances, until a once-a-day STR generic version is available it is likely that current gMTR possibilities may still be unattractive under contemporary cost-effectiveness thresholds  , and mayIt should be noted that at the time of analysis in the U.S. no generic fixed dosed combinations (FDCs) were marketed yet. The current analysis is only applicable to scenarios where switching from an STR to generics means a therapy complication. In countries where generic FDCs are available, differences between generic and non-generic STRs will mainly be due to costs differences between both.In conclusion, our modeled analysis demonstrates that STRs provided to HIV-1-infected treatment-naive individuals is cost-effective compared with gMTRs. We hope the findings from this study will provide further insight and help guide decisions.S1 Table(DOCX)Click here for additional data file.


The mouse model of laser-induced choroidal neovascularization (CNV) has been used in studies of the exudative form of age-related macular degeneration using both the conventional slit lamp and a new image-guided laser system. A standardized protocol is needed for consistent results using this model, which has been lacking. We optimized details of laser-induced CNV using the image-guided laser photocoagulation system. Four lesions with similar size were consistently applied per eye at approximately double the disc diameter away from the optic nerve, using different laser power levels, and mice of various ages and genders. After 7 days, the mice were sacrificed and retinal pigment epithelium/choroid/sclera was flat-mounted, stained with Isolectin B4, and imaged. Quantification of the area of the laser-induced lesions was performed using an established and constant threshold. Exclusion criteria are described that were necessary for reliable data analysis of the laser-induced CNV lesions. The CNV lesion area was proportional to the laser power levels. Mice at 12-16 weeks of age developed more severe CNV than those at 6-8 weeks of age, and the gender difference was only significant in mice at 12-16 weeks of age, but not in those at 6-8 weeks of age. Dietary intake of omega-3 long-chain polyunsaturated fatty acid reduced laser-induced CNV in mice. Taken together, laser-induced CNV lesions can be easily and consistently applied using the image-guided laser platform. Mice at 6-8 weeks of age are ideal for the laser-induced CNV model. Age-related macular degeneration (AMD) is a major cause of blindness and vision impairment in the elderly ,2. NeovaIn vitro endothelial cell culture models of CNV lack complex in vivo cellular interactions with photoreceptors, retinal pigment epithelium, pericytes, inflammatory cells and glial cells [in vivo model of CNV, first described in 1979 [in vivo, it has been limited in predicting efficacy of drugs other than those involving the VEGF pathway [al cells . A laser in 1979 . Althoug pathway , this mo pathway . OptimizA slit lamp is often used to administer laser photocoagulation ,9,11. ThC57BL/6J mice  were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. All animal studies were performed according to the protocols reviewed and approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital.Mice were anesthetized with a mixture of xylazine (6 mg/kg) and ketamine (100 mg/kg), and pupils were dilated with topical drops of Cyclomydril . Two minutes after pupil dilation, lubricating eye drops (Alcon Laboratories) were applied to the cornea. The fundus was viewed with an imaging camera, and laser photocoagulation was induced using the image-guided laser system . The fundus image as well as the aiming beam can be observed on the monitor screen. Four laser burns at equal distance from the optic nerve were induced one by one in each eye by a green Argon laser pulse with a wavelength of 532 nm, a fixed diameter of 50 μm, duration of 70 ms, and varying power levels from 180 mW to 360 mW. If necessary, an orienting laser shot can also be generated approximately three times of the diameter of the optic nerve to help determine the relative positions of the lesions in an eye. After laser photocoagulation, the eyes were gently rinsed with sterile saline to remove the lubricating eye drops and treated with an antibiotic ointment, erythromycin . Mice were then placed on a pre-warmed warming plate at 35°C after the laser treatment until they awakened.Mouse pupils were dilated with Cyclomydril drops after the mice were anesthetized by the xylazine-ketamine mixture described above. Spectral domain optical coherence tomography (SD-OCT) with guidance of bright-field live fundus image was performed using the image-guided OCT system  according to the manufacturer’s instruction and using the vendor’s image acquisition software to generate bright field images, angiograms, and OCT scans.FFA to determine leakage (not to determine lesion size) was performed with the retinal imaging microscope  6 days after laser photocoagulation. Mice were anesthetized, pupils dilated, and intraperitoneally injected with fluorescein AK-FLUOR  at 5 μg/g body weight. Fluorescent fundus images were taken with the retinal imaging microscope at 5 and 10 minutes after fluorescein injection. The fluorescent intensity of CNV lesions was graded using ImageJ  by masked researchers , and theMice were euthanatized 7 days after laser photocoagulation. Eyes were immediately enucleated and fixed with 4% paraformaldehyde  in PBS for 1 hour at room temperature. For histology study, eyes were embedded in Tissue-Tek O.C.T. Compound , sectioned, and stained with hematoxylin and eosin . For flaData are presented as mean ± SEM. Student’s t test was used to compare 2 groups of samples. For more than 2 groups of samples, one-way ANOVA was performed using Prism 6 . p ≤ 0.05 was considered as statistically significant.in vivo retinal structure may also be examined by OCT, if applicable, to determine the cross-sectional area of CNV lesions 7 days after laser burns. To measure the surface area of CNV lesions, the fluorescence-stained retinal pigment epithelium/choroid/sclera flat-mounts were imaged , eye dissection, choroid staining and imaging, and CNV lesion quantification . Most ope imaged  and quanWe found that one key aspect of generating reliable and consistent CNV lesions with the image-guided laser system is the initial adjustment of focus. First, the lens should be positioned approximately 5 mm away from the cornea of mouse eyes where the major retinal vessels can be clearly observed by adjusting the lens focus. The optic nerve should be positioned in the center of the visual field by adjusting the position and height of the mouse holder . Next, tOnce the fundus is in focus, both the retinal major vessels  and the large-size choroidal vessels (pink) can be observed clearly. Four laser burns per eye should be generated at equal distance from the optic nerve  at the 3, 6, 9 and 12 o’clock positions or in the center of 4 individual retinal quadrants . The disThe laser-induced CNV model in mice has been often characterized as variable and inconsistent . EstabliTo accurately evaluate the laser-induced CNV, some lesions should be excluded. Severe hemorrhages will cause much larger CNV lesions, whereas choroidal damage will yield a CNV lesion much smaller than the fellow CNV lesions in the same eye. First, choroidal hemorrhages encroaching on the lesion should be analyzed and classified carefully : (1) if Previous studies indicate that the optimal time to measure the area of CNV lesion is at day 7 or day 14 after photocoagulation , and thaLaser power from 180 mW to 360 mW with identical duration of 70 ms and wavelength of 532 nm was used for photocoagulation. The percentages of quantifiable lesions are shown in Previous studies suggest that both gender and age of animals influence the outcome of laser-induced CNV ,15,16. TPreviously we reported that dietary intake of omega-3 long-chain polyunsaturated fatty acid (LCPUFA) reduces pathological retinal angiogenesis in oxygen-induced retinopathy . Severalin vivo models, such as apolipoprotein E over-expression or superoxide dismutase 1 loss in knockout mice [The laser-induced CNV model in mice exhibits choroidal angiogenesis under conditions of burn-induced inflammation, modeling some aspects of neovascular AMD. This model produces lesions faster and more consistently than many other genetic mouse AMD out mice ,20,21, ain vivo and promotes wound healing in both human and animal models [We found that laser CNV lesions were optimum using mice of either gender weighing 15–23 g at 6–8 weeks of age. Older mice exhibit a larger and more variable CNV area, especially older female mice, which is consistent with previous reports ,15,16. Tl models –24. Yet l models . This diin vivo model for the exudative form of AMD, yet has limitations. Driven by a wound-healing reaction, the laser-induced CNV model involves high levels of acute reaction inflammation [The laser-induced CNV model is currently the most widely used ammation ,26, whicammation –29. In aOur studies used the image guided laser system to optimize laser-induced CNV. Compared to the conventional slit lamp system, the image-guided laser system may be more convenient . One perObtaining reliable and consistent results using the laser-induced CNV model requires careful experimental design and implementation, including eye integrity check, optimal laser induction, strict and consistent exclusion criteria, masking methodology, and dependable quantification techniques. Fine focusing of real-time fundus imaging with a uniform observation of nerve fibers is an essential prerequisite for consistent laser photocoagulation in the eye. Our recommendation of the laser parameters using the image-guided Micron IV laser system is a power of 240 mW and duration of 70 ms for C57BL/6J mice. Different strains require different optimizations.These parameters for C57BL/6J mice are consistent with other researchers’ experiences that laser shots yielding the optimal CNV lesions are those with the lowest power level and shortest duration time yet still capable of rupturing Bruch’s membrane . IncreasOur findings provide the optimal settings and conditions to make use of the image-guided laser system for the goal of improving the consistence and reproducibility of experimental results in the laser-induced CNV model in mice for AMD research.


No significant differences were found for the other MMPs assayed. MMP-9 concentrations are increased in pericontusional brain early post-TBI and may represent a potential therapeutic target to reduce hemorrhagic progression and vasogenic edema.Matrix metalloproteinases (MMPs) are extracellular enzymes that have been implicated in the pathophysiology of blood–brain barrier (BBB) breakdown, contusion expansion, and vasogenic edema after traumatic brain injury (TBI). Specifically, in focal injury models, increased MMP-9 expression has been observed in pericontusional brain, and MMP-9 inhibitors reduce brain swelling and final lesion volume. The aim of this study was to examine whether there is a similarly localized increase of MMP concentrations in patients with contusional TBI. Paired microdialysis catheters were inserted into 12 patients with contusional TBI (with or without associated mass lesion) targeting pericontusional and radiologically normal brain defined on admission computed tomography scan. Microdialysate was pooled every 8 h and analyzed for MMP-1, -2, -7, -9, and -10 using a multiplex immunoassay. Concentrations of MMP-1, -2, and -10 were similar at both monitoring sites and did not show discernible temporal trends. Overall, there was a gradual increase in MMP-7 concentrations in both normal and injured brain over the monitoring period, although this was not consistent in every patient. MMP-9 concentrations were elevated in pericontusional, compared to normal, brain, with the maximal difference at the earliest monitoring times . Repeated-measures analysis of variance showed that MMP-9 concentrations were significantly higher in pericontusional brain ( Hemorrhagic expansion of the contusion core and elevation of intracranial pressure (ICP) as a result of pericontusional edema are major contributors to secondary brain injury.F1 In particular, the subfamily of gelatinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), have been implicated as key mediators of proteolytic blood–brain barrier (BBB) disruption associated with traumatic injury,2 ischemia,3 and neuroinflammatory disorders.4Matrix metalloproteinases (MMPs) are a family of over 20 extracellular endopeptidases that cleave a wide range of protein substrates in diverse signaling pathways.5 Correspondingly, an MMP inhibitor (GM6001) reduced the degree of BBB leakage and the extent of edema. In a similar mouse model, MMP-9 knockout animals were found to have smaller final lesion volume and better functional recovery, compared to wild-type mice.2In the rat cortical contusion model, MMP-9 expression was increased in lesioned tissue, compared to contralateral uninjured brain, and was associated with local BBB permeability and edema.6 Further, in vivo microdialysis studies in traumatic brain injury (TBI) patients have also shown increased concentrations of MMP-9 and, possibly, MMP-2 early postinjury.8 However, it is unclear from human studies to date whether MMP-9 overexpression in TBI patients is localized to pericontusional brain, as found in animal models, or is a more generalized response to trauma.Tissue samples from patients requiring surgical resection of brain contusions have been shown to have significantly higher expression of MMP-9, compared with lobectomies performed for nontrauma indications.This study sought to address this question by monitoring the temporal and spatial concentration of selected MMPs  in TBI patients using paired microdialysis catheters inserted simultaneously within pericontusional and radiologically normal brain.9 Severity of injury based on the initial computed tomography (CT) scan was graded using the modified Marshall scoring system.10 All patients received sedation (with or without neuromuscular blockade) and mechanical ventilation, together with multi-modality monitoring, and were managed according to a standardized tiered therapy protocol.11 Recovery at 6 months was measured on the Glasgow Outcome Scale (GOS) and dichotomized as good (GOS 4 or 5) or poor (GOS 1–3) outcome.12All study protocols were approved by the East of England (Essex) NHS Research Ethics Committee (ref 11/EE/0075). Assent from patients' next of kin was obtained. Eligible patients were adults (>18 years) admitted post-TBI with contusions evident on computed tomography (CT) imaging and requiring neurointensive care treatment. Patients were classified as having severe injury if the presenting Glasgow Coma Scale (GCS) score was less than or equal to 8.Invasive neuromonitoring was inserted at two sites for each patient with the intention of having a microdialysis catheter within radiologically normal white matter and another within pericontusional brain, but avoiding the hemorrhagic core. On admission, a triple-lumen cranial access device  was placed in the right or left frontal region as standard in our unit. An ICP monitor , a brain-tissue oxygen probe , and a microdialysis catheter  perfused with 3.5% (w/v) human albumin solution  in central nervous system (CNS) perfusion fluid were introduced through the access device. After assent, up to three further invasive monitors, at least one of which was a microdialysis catheter  were placed in proximity to a contusion, either through a second cranial access device or twist drill holes. If the first set of monitors happened to be placed adjacent to a contusion, the second set were inserted in radiologically normal brain on the contralateral frontal region. In patients requiring an emergent craniotomy for an acute subdural hematoma (SDH), the pericontusional monitoring was placed adjacent to underlying contusions under direct vision at the end of surgery, tunneled through the scalp.Hourly microdialysates were pooled into 8-h samples. All samples were analyzed using the Milliplex Multi-Analyte Profiling Human MMP five-plex  analyte premixed kit , according to the manufacturer's instructions. All samples were assayed in duplicate wells (25 μL per well), and the mean of these results was used. Plates were read using a Luminex 200 analyzer  running STarStation software . Protein concentrations were calculated by reference to an eight-point spline fit curve for each MMP.t-test. All calculations were performed in R software  and considered significant at 5%.To mitigate the effects of different monitoring periods in each patient, the mean concentrations of each MMP in the first 72 h postinjury (<72 h) and in the subsequent 72 h (>72 h) were calculated for each patient. Repeated-measures analysis of variance (ANOVA) with MMP concentration as the dependent variable, and site of monitoring  and time (<72 h vs. >72 h) as the independent variables, was then applied for each MMP separately. Univariate subgroup comparisons were analyzed with the independent-samples Twelve patients  were enrolled . Paired Mean time-concentration plots across for each MMP assayed are shown in In contrast, MMP-9 concentrations were consistently higher in the injured brain, compared to radiologically normal brain. The highest concentrations of MMP-9 were observed at the earliest monitoring times  within injured brain. Thereafter, MMP-9 concentrations decreased, but remained higher, within injured brain, compared to radiologically normal brain, to 72 h postinjury and beyond.p=0.03), and were also significantly higher in the early monitoring period (<72 h), compared with later time points .Mean concentrations of the five MMPs in normal and injured brain in the periods <72 and >72 h postadmission are shown in p>0.05) in concentrations of any of the assayed MMPs when the cohort of patients was divided into two groups based either on presenting severity of TBI (GCS ≤8 vs. >8), whether or not the patient had an SDH evacuated, and functional outcome (GOS 1–3 vs. 4–5).There were no significant differences , growth factors , neurotransmitters and small molecules , and hormones .16 In the wider experimental brain injury literature, MMPs have been identified as mediators of lesion expansion and perilesional edema in models of ischemic stroke and intracerebral hemorrhage.18 Importantly, recent evidence has highlighted the crucial roles that MMPs have in the subacute and chronic reparative processes after brain injury, such as neurovascular remodeling and migration of cells from the subventricular zone to damaged tissue.20Our findings are in accord with pre-clinical evidence that MMP-9 expression is increased in perilesional brain after experimental brain contusion and, in these models, corresponds with the development of BBB permeability and expansion of final lesion volume.22 Vilalta and colleagues studied 4 patients with diffuse brain injury using microdialysis and found similar early elevation of cerebral MMP-9 concentrations.7 More recently, Roberts and colleagues conducted a single-site microdialysis study of MMP-1, -2, -3, -7, -8, and -9 in 8 TBI patients with a mixture of diffuse and focal injuries; similarly to the present study, they found that MMP-9 was increased early postinjury and thereafter declined.8Grosslete and colleagues and Zheng and colleagues have previously reported elevated levels of MMP-9 in cerebrospinal fluid (CSF) sampled acutely from TBI patients by ventriculostomy, with comparable temporal profiles demonstrating greatest concentrations in the first sample at 24 h postinjury followed by a decline, but remaining higher than control CSF for at least 72 h.23The present data both corroborate these temporal MMP-9 profiles in a larger cohort of TBI patients and add the important new finding that, in patients with predominantly focal or contusional injury, MMP-9 response is localized to perilesional brain. The paired catheter design means that it is far less likely that the observed changes in MMP-9 are attributable to artefact from catheter insertion, given that a similar effect would have been observed at both monitoring sites. The evidence from this study goes some way to supporting the hypothesis that the mechanisms of MMP-9-induced BBB disruption and edema elucidated in animal models are also relevant in human TBI. Indeed, the temporal pattern of MMP-9 changes is consistent with MRI studies of contusion expansion in humans.8 In the present study, patients exhibited variable MMP-2 responses, with no consistent spatiotemporal pattern emerging on averaging across the cohort. Interestingly, an earlier study of contusion resection tissue also found MMP-2 concentrations that varied widely, and the resulting overall difference in expression, compared to control patient samples, was modest.6 Together, these findings suggest that MMP-2 concentrations may be significantly elevated in only a subset of TBI patients; whether this is a function of injury severity or other patient-specific clinical factors will require larger studies to resolve. The relative importance of MMP-2 compared with MMP-9 in the pathogenesis of contusion expansion and brain edema is unclear, and there is conflicting evidence on the role of MMP-2 in exacerbating lesion volume from different brain injury models.24 However, because both MMP-2 and -9 are gelatinases and act on similar substrates, they may represent redundant pathways in post-traumatic proteolytic breakdown of the BBB. If interventions targeted at MMP activity are to be successful, a more complete understanding of the role of MMP-2 will be essential, particularly given that evidence from pre-clinical TBI models suggests that MMP-2 and MMP-9 show different responses to current therapies, such as hypothermia.25Both previous microdialysis studies have found evidence for increased cerebral expression of MMP-2 post-TBI, although Vialta and colleagues identified an early peak similar to MMP-9, whereas in the later study by Roberts and colleagues, MMP-2 concentration was initially low and increased at approximately 48 h postinjury, with a subsequent decrease.1 Although MMP-TIMP complexes would likely exceed the 100-kDa cutoff of the CMA 71 microdialysis catheter, it cannot be determined from the present data whether all MMP-9 assayed was enzymatically active; future studies to address this question will be challenging owing to low absolute amount of protein recovered with microdialysis. Third, the findings of the current study injury do not speak to whether MMP concentrations will display similar temporal profiles in patients with a predominantly diffuse axonal injury pattern on initial CT.This study has a number of limitations. First, no patient was monitored within the first 12 h of injury, and therefore the expression pattern of MMPs in the very acute stage remains uncertain. Second, MMP activity is regulated by endogenous tissue inhibitor of metalloproteinase (TIMP) proteins, which bind with latent and activated MMPs.26 Despite a number of randomized trials, therapeutic hypothermia has not been conclusively shown to improve outcome post-TBI, though its use to control ICP remains widespread.28 Conceivably, targeting hypothermia to patients with increased MMP-9 expression may be a method to select the subgroup of patients in which the benefits of this treatment outweigh its deleterious effects.Induced hypothermia has been shown to attenuate MMP-9 expression in rodent TBI and stroke models, but, as yet, this phenomenon has not been investigated in human patients.29 Of interest, tetracycline antibiotics, most notably doxycycline, inhibit MMP-2 and -9 through chelation of the zinc ion in the catalytic site.30 Doxycycline, at subantimicrobial doses, has been shown to be of benefit in periodontitis, likely through its effect on MMP-9 activity.31 On the same basis, doxycycline has also been suggested as a treatment to stabilize growth of abdominal aortic aneurysms, although the evidence to date is conflicting.32 Currently available MMP inhibitors are not selective for specific subtypes, but, at the same time, are not equally active on all members of the enzyme family; combined with redundancy in MMP pathways, this may be an explanation for the limited efficacy of MMP inhibitors in clinical studies to date.MMP inhibitors have been investigated as promising therapies for a number of diseases, most notably in metastatic cancer. However, despite successful pre-clinical findings, early-phase clinical trials in cancer patients have proven disappointing owing to lack of efficacy and excessive adverse effects.This study suggests that MMP-9 may be a therapeutic target to reduce lesion progression and brain swelling in contusional TBI. Investigating the efficacy of MMP inhibitors in this context will first require further detailed studies of the association between pericontusional MMP-9 concentration, contusion expansion and vasogenic edema, ICP and treatment intensity, and cerebral metabolism to establish robust measures of efficacy. Human studies to date indicate that the useful therapeutic window for MMP-9 inhibition post-TBI may extend up to 72 h postinjury, but it is likely that any agent will be most effective at minimizing brain edema and contusion expansion if administered as acutely as possible. Defining the appropriate duration of MMP inhibition will also need to consider the later role these enzymes have in postinjury CNS repair.


Co-incubation with a CCL2-blocking-antibody enhanced in vitro cell sensitivity to temozolomide. Conversely, recombinant CCL2 activated JNK in human tumor melanoma cells. In keeping with these results, the combination of a JNK-inhibitor with temozolomide was synergistic. By showing that down-regulation of CCL2-driven signals by SAHA and temozolomide via JNK contributes to reduce melanoma growth, we provide a rationale for the therapeutic advantage of the drug combination. This combination strategy may be effective because of interference both with tumor cell and tumor microenvironment.Target-specific agents used in melanoma are not curative, and chemokines are being implicated in drug-resistance to target-specific agents. Thus, the use of conventional agents in rationale combinations may result in optimization of therapy. Because histone deacetylases participate in tumor development and progression, the combination of the pan-inhibitor SAHA and temozolomide might provide a therapeutic advantage. Here, we show synergism between the two drugs in mutant BRAF cell lines, in association with decreased phosphorylation of cell survival proteins . In the spontaneous  Melanoma is an aggressive disease exhibiting a metastatic behavior and intrinsic resistance to treatment, features leading to poor prognosis. In spite of the development of target-specific agents  which have improved progression-free survival and responsiveness to immune intervention , metastatic melanoma is still incurable . ResistaIn melanoma, deregulation of survival pathways as a consequence of BRAF mutation is a frequent event . BecauseMalignant transformation is also driven by epigenetic mechanisms that involve histone deacetylases (HDAC) which remove acetyl groups from histones, thereby promoting chromatin condensation and acting as transcription repressors . Convershttp://www.clinicaltrials.gov). Novel therapeutic options are needed to improve the activity of such clinically available agents and to define alternative treatments for patients recurring after BRAF inhibitors  as well as for patients bearing tumors with wild-type BRAF. In this regard, HDAC inhibitors represent promising therapeutic agents because they are expected to act on tumor cells and their microenvironment as they are endowed with immunomodulatory properties [ClinicalTrials.gov Identifier: NCT01587352).The only cytotoxic agent approved by the US Food and Drug Administration for the treatment of melanoma is the triazine dacarbazine . Temozoloperties . Indeed,operties -22. It ioperties -25. Manyoperties . Suppresoperties . Althougoperties , its rolin vivo on the ret transgenic mouse model of spontaneous melanoma. Here, we describe the molecular correlates of the efficacy of the combination of SAHA and TMZ, and we propose that disruption of CCL2-driven signals by SAHA and TMZ may impair survival of human melanoma cells resulting in a synergistic drug interaction which in mice results in delayed disease onset.Based on this background, we hypothesize that SAHA could be useful in an attempt to improve the efficacy of the TMZ and we used a panel of human melanoma cell lines, expressing alterations found in tumors from patients resistant to BRAF inhibitors, to investigate the therapeutic potential of SAHA in combination with the clinically available TMZ. Given the immunomodulatory potential of SAHA, the combination was tested BRAFV600E mutation, and two BRAF wild-type cell lines, LM18 and LM23. The LM20 and 501Mel cell lines display intrinsic resistance to the BRAF inhibitor PLX4032. LM20 cells carry amplifications of CDNN1 and CTNNB1, and 501Mel cells display high MET expression [A panel of human melanoma cell lines well characterized for their molecular features was used in this study. They included A375, LM17, LM20, LM36, 501Mel exhibiting the pression  . ret transgene received SAHA, TMZ or both drugs . Mouse Phospho-RTK array analyses in tumors indicated a down-modulation of selected phospho-proteins after treatment   in the drug combination treated groups as compared to controls , we found a statistically significant association between the experimental groups and each of the considered continuous variable. A significant down-modulation was observed after exposure to the drug combination as compared to control animals for chemokines  Figure . Similars Figure . Becauses Figure .via the generation, recruitment and activation of CD11b+Gr1+ MDSC [+Gr1+ immature myeloid cells measured by flow cytometry tended to be reduced in the tumors upon all applied treatments as compared to the controls  remained similar in all considered groups  from a tumor developed by mice . When ths Figure . We tests Figure .ret transgenic mice to suppress tumor cell survival signals employing the pan-HDAC-inhibitor SAHA in combination with TMZ. The ret transgenic mouse model was chosen because it recapitulates the complexity of melanoma disease. In the used panel of melanoma cell lines, irrespectively of the inherent cell sensitivity to single agents, we found an improved effect of the drug combination as compared to single drugs, in association with the suppression of pro-survival signals. A feature of tumor cells exposed to the combination was the modulation of the phosphorylation profile of proteins involved in regulation of cell survival including AKT and members of the MAPK pathway. Of note, phosphorylation of JNK was reduced upon exposure to the combination in the cell lines in which the synergistic interaction occurred. Such an effect was associated with increased apoptosis in some cell lines. The synergistic interaction was more evident in cells characterized by BRAF mutation than in cell carrying wild-type BRAF, although the drug interaction on the latter cells was still favourable.Based on the need for rationally improving the activity of clinically available agents, in the present study we used various molecularly characterized human melanoma cells, including cell lines with intrinsic resistance to the BRAF inhibitor vemurafenib, and ret mice, which benefitted from combined treatment in terms of disease free-survival. The combination treatment led to a down-regulation of JNK activation in tumors, similarly to in vitro findings. When investigating the effect of the combination on the tumor inflammatory microenvironment, we found that levels of chemokines including CCL2 were significantly down-regulated in the primary tumors after the combined treatment compared to controls. Our data are reminiscent of what observed in a recent report in which down-regulation of CCL2 after treatment with the BRAF inhibitor PLX4720 was associated with reduced tumor growth [In addition, an improved response to the combination of SAHA and TMZ was evident in ccl2 mRNA [in vivo model, we further investigated the relationship between CCL2 and JNK. In cultured murine melanoma cells, we found that CCL2 was released in the medium suggesting that it is produced by cells and it might act in sustaining survival by autocrine/paracrine mechanisms in the tumor microenvironment. When we treated human melanoma cells with recombinant-CCL2, we observed that the chemokine could trigger JNK activation as phosphorylation of JNK1/2 was found. Under such conditions, we did not observe modulation of IKBα, known to be down-regulated following NF-kB activation, and of AKT, whose phosphorylation remained unchanged after recombinant-CCL2 treatment. Thus, a specific CCL2-driven JNK1/2 activation occurred in our model. Moreover, JNK appeared to sustain growth of melanoma cells exposed to TMZ, because upon pharmacological JNK-inhibition, a synergistic effect could be obtained, similarly to what observed with SAHA, further corroborating that JNK pathway impairment is implicated in inhibition of melanoma cell growth.The available evidence supports that CCL2, a chemotactic factor for monocytes and T lymphocytes plays key roles in tumorigenesis promotion in different tumor types , 31. Intcl2 mRNA . Becausein vivo model we observed a decrease in VEGF transcript after SAHA. Moreover, HDAC inhibition has been shown to suppress TGFβ−induced epithelial to mesenchymal transition [In addition to modulation of CCL2 by the combination, we observed down-regulation of key inflammatory mediators . HDAC inhibitors have been reported to affect the levels of the angiogenic cytokine VEGF in different tumor types by a down-regulation of gene transcription and activation of proteasome activity , 35. In ansition . Here, w+Gr1+ immature myeloid cells. Previously, we have intensively investigated the distribution and function of CD11b+Gr1+ cells in primary tumors and metastases from ret transgenic mice. We have found that these cells are highly immunosuppressive as reflected by a strong production of nitric oxide, enhanced expression of arginase-1 and their ability to inhibit T cell proliferation in an in vitro assay [It was previously shown that HDAC inhibitors (including SAHA) sustain the generation and activation of Tregs and MDSC , 38. Conro assay -50. Althro assay , and in in vivo model after combined treatment, together with a tendency of MDSC to decrease, suggested that the combination of SAHA and TMZ might be endowed with a direct effect on the tumor and with an indirect effect on the microenvironment, as MDSC are attracted by CCL2 to the tumor site [Our findings showing that the levels of CCL2 are decreased in our mor site .via JNK contributes to reduce growth of melanoma cells provide a rationale for the therapeutic advantage of the combination of SAHA and TMZ , was grown in DMEM medium containing 1 mM sodium piruvate plus 10% FBS. The cell line which displayed a fusiform morphology and expressed TRP2 (The human melanoma cell lines A375 (ATCC CRL1619), 501Mel and the recently established melanoma cell lines  were cultured in RPMI-1640 medium plus 10% FBS , 53. Allsed TRP2 , was chased TRP2 .TMZ, SAHA and the JNK-inhibitor SP600125  were primarily dissolved in DMSO and diluted in water. For in vivo studies, SAHA was dissolved in DMSO/ethanol/PBS (10:5:85) and administered in a volume of 10 mL/kg. Human CCL2/MCP-1 monoclonal neutralizing antibody   were used. Mice were treated with vehicle, SAHA, TMZ or their combination for 5 days and they were sacrificed 5 days later. Tumor cells were processed for total protein extraction. Briefly, protein lysates were prepared from frozen tumor samples pulverized by the Mikro-Dismembrator II  in Lysis Buffer 15  added with protease and phosphatase inhibitors. Equal amounts of lysates were separated by SDS-PAGE and then transferred to nitrocellulose filters. Filters were incubated with antibodies to phosho-PDGF receptor , phospho-RET , JNK2 and phospho-JNK1/2 .Samples were fractionated by SDS-PAGE and blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried non-fat milk and then incubated overnight at 4°C with antibodies to JNK2 and phospho-JNK1/2 (R&D System), AKT2 and phospho-AKT2 , NF-kB  and IKB-a . Anti-tubulin, or anti-vinculin antibodies  were used as control for the protein loading. Antibody binding to blots was detected by chemiluminescence . For Proteome Profiler Antibody Arrays, cell lysates were prepared as described by the manufacturer (R&D System). Labscan imaging technology and software  was used for Western immunoblot anlysis visualization. Band intensities were quantified by ImageJ software by pixel-integrated intensity.ret trangene under the control of mouse metallothionein-I promoter, kindly provided by Dr. T. Dragani, were obtained as described previously [ret transgene. Ret transgenic mice developed melanoma starting from 6 weeks. Six-seven week old 48 F1 mice carrying the ret transgene showing no signs of macroscopic tumors were used for antitumor activity studies and were assigned to four groups according to a randomization list. The mice were treated (qdx5/wx3w) with a) 50 mg/kg TMZ or b) 100 mg/kg SAHA or c) their combination or d) vehicle. All controls displayed macroscopic evidence of disease  within 44 days from the experiment start.C57BL/6 mice carrying the human eviously . Mice weTaqMan® Arrays  and qReal-Time PCR assay are described in Supplementary Methods.This method is reported in the Supplementary Methods section.Tumors, bone marrow, spleen and lymph nodes were harvested into cold PBS, cut into pieces and mechanically disaggregated. Single cell suspensions were obtained by mechanical disaggregation of the specimens that after harvesting from mice were immediately transferred into PBS, cut into pieces and the obtained cell suspensions filtered through 50 μm cell strainers (BD Biosciences). After counting, cells were viably frozen in 10% DMSO in FBS. Thawed cells were washed in PBS and treated with Fc-blocking solution  followed by the incubation with directly-conjugated monoclonal antibodies (mAbs): CD11b-PE (cat. 553311), Gr1-PE-Cy7 (cat. 552985) and CD45.2-PerCP-Cy5.5 (cat. 552950) from BD Biosciences. To stain FoxP3 cells were fixed and permeabilized using FoxP3 staining set (eBioscience) prior to the addition of the Foxp3-PE mAbs (cat.72-5775-40). Acquisition was performed by multi-color flow cytometry using a FACSCantoII with FACSDiva software (BD Biosciences), with dead cells exclusion based on scatter profile. FlowJo software  was used to analyze at least 100,000 events per sample.Snap frozen primary tumor and LN samples were mechanically disrupted and treated by lysis solution (Bio-Rad). Protein concentration in lysates was determined using Bradford assay (Bio-Rad). After sonication, samples were centrifuged at 4,500 g for 6 min at 4 °C. Protein concentration in the supernatant was determined using Bradford assay and adjusted to 1000 μg/ml using serum diluent (both Bio-Rad). Amounts of inflammatory factors in tissue lysates (obtained from tissues from mice treated for 5 days and sacrificed 5 days later) were measured by multiplex technology (Bio-Rad).Transforming growth factor (TGF)-β1 was measured using a single plex kit (Millipore) according to manufacturer's protocol.The effect of the drug combination in cellular studies was evaluated using the Chou and Talalay method  in which