To evaluate efficacy and toxicity clinical in the intensified treatment of locally advanced squamous cell carcinoma of the head and neck (SCCHN) with the combination of chemotherapy, the EGFR antibody cetuximab, and intensity-modulated radiation therapy (IMRT) in a concomitant boost concept.REACH is a prospective, bi-centric phase II trial of carboplatin/5-FU and cetuximab weekly combined with IMRT. Primary endpoint is locoregional control, secondary endpoints include acute radiation effects and adverse events. Evaluation of disease response is carried out according to the Response Evaluation Criteria in Solid Tumors (RECIST); toxicity is assessed using NCI CTC v 3.0.Treatment was tolerated moderately well, acneiforme erythema occurred in 74.1% (grade II/III), mucositis grade III in 28.6%, and radiation dermatitis grade III in 14.3%. Higher-grade side-effects resolved quickly until the first follow-up post treatment. Objective response rates were promising with 28.6% CR at first follow-up and 92.9% thereafter.The combination of standard carboplatin/5-FU and cetuximab is feasible and results in promising objective response rates. The use of an IMRT concomitant boost is practicable in a routine clinical setting resulting in only moderate overall toxicity of the regimen.ISRCTN87356938. Long-term disease control for patients with advanced squamous cell carcinoma of the head and neck (SCCHN) is still challenging. For locally advanced or unresectable SCCHN without evidence of distant metastases, combined radiochemotherapy is the only curative treatment possibility. In the MARCH meta-analysis, concurrent chemoradiotherapy results in an absolute survival benefit of 6.5% at 5 years with the most effective regimen being simultaneous chemoradiation -3. This Intensified radiotherapy such as altered fractionation schedules also lead to a significant improvement of local control but also overall survival if radiation therapy is performed as a single therapy modality with the highest benefit in hyperfractionated radiotherapy schedules . Adding While intensified treatment regimen - whether as intensified chemoradiation or altered fractionation radiotherapy - have been shown to improve outcome ,6, it isVarious options exist: first of all, the use of more tolerable chemotherapy combinations, second the integration of molecular targeted drugs and third the use of modern concepts of radiotherapy.Staar et al. combined carboplatin with 5-FU and hyperfractionated accelerated radiotherapy in a randomized phase III trial  and presModern radiotherapy techniques such as intensity-modulated radiotherapy (IMRT) and image guidance (IGRT) have rapidly found acceptance in the routine treatment of SCCHN. Intensity-modulated radiotherapy (IMRT) has been shown to reduce acute and late toxicity  and prevREACH combines all of these approaches: systemic treatment is intensified by the combination of chemotherapy and EGFR antibody treatment (carboplatin/5-FU and cetuximab) while local treatment is intensified by a concomitant boost concept in IMRT technique.Aim of the trial is to evaluate disease control, toxicity, and feasibility of this intensified triple-therapy.Patients with pathologically confirmed, locally advanced non-metastatic squamous cell cancer of oropharynx, hypopharynx, or larynx were eligible for the trial. Age between 18 and 70 years, Karnofsky performance score of > 70%, and adequate bone marrow, liver, and renal function were also required. Exclusion criteria were nasopharyngeal carcinoma, prior chemotherapy, radiotherapy, or exposure to EGFR pathway targeting therapy .Work-up included complete panendoscopy, diagnostic CT scans of the neck and chest, abdominal ultrasound, and bone scan. In the absence of contraindications, all patients received diagnostic MRI scans for treatment planning and follow-up.The trial was reviewed and approved by the University of Heidelberg Medical School Ethics Committee; informed consent was obtained from all patients prior to inclusion.2 body surface followed by weekly infusions with 250 mg/m2. All patients received 8 mg dexamethasone and 4 mg dimetinden prior to each application of cetuximab. Missed out doses of cetuximab during the treatment course are to be omitted. Cetuximab-induced skin reactions are treated according to standard in-house protocols and recommendations of the vendor with topic or systemic antibiotics as indicated.Patients received the loading dose cetuximab one week prior to RT (d1) at the recommended dose of 400 mg/m2 body surface as a one-hour intravenous infusion, 5-FU at 600 mg/m2 of body surface as continuous infusion over 23 hours. Patients are provided with standard antiemetic prophylaxis and hydration according to institutional protocols.Carboplatin and 5-FU are given on days 8-12 and 36-40 (corresponding to radiotherapy-week 1 and 5). Carboplatin is prescribed at 70 mg/m®, ITV), planning examinations include CT-scan and contrast enhanced MRI for 3D image correlation. Target volumes are delineated in accordance with current guidelines and recommendations [Patients are immobilized using individual thermoplastic head masks incl. shoulder fixation (HeadStepndations -18.All patients are treated with inversely-planned intensity-modulated radiotherapy (IMRT) either at a 6 MV linear accelerator in step-and-shoot technique or at a 6 MV tomotherapy unit under regular image guidance.A dose of 50.4 Gy in daily fractions of 1.8 Gy (Monday to Friday) is prescribed to the neck (CTV 2). From study day 29 onwards, patients receive an additional fraction at 1.5 Gy per day in a classical concomitant boost regimen ,20 to a Regular follow up is carried out 6 weeks post treatment, 3 months (4-5 months post completion of therapy) thereafter, and then in 6 monthly intervals including fibreoptic examination and local imaging with MRI. In the presence of contra-indications, local imaging is carried out with contrast-enhanced CT scans.REACH is a prospective, bi-centric, single-arm phase II trial of combined radiochemoimmunotherapy with weekly cetuximab, carboplatin and 5-FU (according to the Staar protocol ) and IMRPrimary endpoint of the trial is local-regional control (LRC); secondary endpoints are disease-free survival (DFS), progression-free survival (PFS), overall survival (OS), acute and late radiation effects, adverse events. Control and survival data are calculated from administration of cetuximab loading dose.Physical examination and monitoring of adverse events as well as routine hematologic and chemical blood analysis is performed weekly throughout the treatment period. Toxicity is assessed using NCI CTC v 3.0.Evaluation of disease response is carried out according to the Response Evaluation Criteria in Solid Tumors (RECIST)  6 weeks,Between August 2009 and March 2012, twenty-one patients were accrued to the REACH trial. Three patients subsequently had to be excluded from the trial: two patients had to be excluded due to poor compliance prior to treatment start, another patient developed an anaphylactic reaction to the trial medication on first exposure to cetuximab and had to discontinue treatment within the trial. Eighteen patients with a median age of 57 years (43 - 69) are treated within the protocol, fourteen patients have completed treatment. Most patients had very advanced disease not accessible to surgical treatment. Fifteen out of 18 patients were male, all patients had a history of heavy smoking. Fourteen patients completed trial treatment as scheduled. Median dose to the cervical lymph nodes was 50 Gy (49 - 51), the primary and involved lymph nodes received a median total dose of 70 Gy (69 - 74 Gy). Median follow-up from cetuximab loading dose excluding patients still under treatment (n = 14) is 17.4 months (6.5 - 30 months). Baseline characteristics are displayed in Table Treatment was generally tolerated well with the most common side effects being the typical, cetuximab-induced acneiforme skin rash, mucositis, dysphagia, dermatitis, and xerostomia. No chemotherapy cycle had to be postponed or dose-reduced due to toxicity, acneiforme rash was mostly moderate and did not lead to treatment interruptions or dose changes. Twelve out of 18 patients received a prophylactic feeding tube prior to therapy start, however, only one patient was feeding tube dependent during therapy. A list of observed side effects can be found in Table There were six serious adverse events, three events were judged unrelated to the trial treatment: one patient has deceased due to systemically progressive disease (CTC °V), one patient was hospitalized for treatment of tonsillitis (CTC °III) 6 months after completion of therapy, and one patient developed septic shock of unknown genesis more than 6 months post completion of therapy, and had a protracted, complicated hospital stay. One patient was diagnosed with an abdominal wall abscess under therapy, which had also occurred several times in the past prior to trial treatment; relatedness to the trial medication is therefore questionable but cannot be excluded. Another patient developed an iliac artery embolism and consequently had to undergo surgery. This patient did not have any history of embolic events, no cardiac arrhythmias or any other predisposing factors. Relationship of this event with the trial treatment is therefore possible. There was one anaphylactic reaction to cetuximab at first exposure leading to discontinuation of trial treatment. All patients recovered from the adverse events.Acute toxicity commonly observed in patients receiving chemoradiation is resolving rapidly so far, residual mucositis on the first follow-up (6 weeks post treatment) was seen in 7/14 patients. Dysphagia was generally resolving fast, 3 patients so far had evidence of dysphagia at the second follow-up. One patient with large mediastinal lymph node metastasis was still feeding-tube dependent though this is not attributable to study treatment. There is no higher-grade xerostomia, no evidence of higher-grade late effects have been found so far , altered fractionation (accelerated/hyperfractionated treatment), and systemic agents (concomitant radiochemotherapy with carboplatin/5-FU and the EGFR antibody cetuximab) in order to improve local control and ultimately patient outcome.2 q3 weeks with a more favourable toxicity profile and subsequently higher patient compliance [While treatment intensity with both the use of radiochemotherapy, immunotherapy, and concomitant boost is high, protocol treatment is tolerated reasonably well and without treatment delays. The combination of carboplatin and 5-FU has been shown to achieve similar results as cisplatin 100 mg/mmpliance . Bone mampliance . Mucositmpliance . Mucositmpliance ,23-27. Hmpliance -30. Alsompliance ,27). Agampliance -30 suppoAddition of cetuximab to chemoradiation with carboplatin/5-FU so far lead to a rather moderate, increased skin toxicity: acneiforme rash CTC grade II/III was present in 10/14 patients (71.4%) and correlates well with the observations by Bonner and colleagues ,9 using Response rates in the triple combination regimen are promising so far: two patients only had stable disease on their first follow-up after treatment corresponding to an overall objective response rate of 85.7% . One patient with stable disease had developed complete remission 4-5 months post completion of treatment, the other patient with stable disease on the first follow-up was diagnosed with distant metastases and has deceased 6 months post completion of the REACH regimen. REACH response rates are in the range of rates reported by other groups evaluating the combination of chemotherapy and cetuximab in the definitive setting ,31,33 evWe had six reported serious adverse events, for three of them there is (anaphylactic reaction) or may be a relationship to the trial medication , all of these patients recovered uneventfully from the adverse event, hence discontinuation of the trial was not warranted.So far, five phase II trials using chemotherapy, cetuximab and mostly IMRT are reported. Apart from Pfister et al., all of them were completed as scheduled and did not observe any unexpected toxicity -30,33, hUnfortunately patient accrual to the REACH trial is rather slow reflecting the fact that treatment of locally advanced SCCHN is still primarily surgical in Germany. However, functional outcome in of conservative treatment strategies as reflected in the REACH trial is excellent, apart from the patient with distant progressive disease and mediastinal mass, treatment related dysphagia has rapidly recovered post treatment. To date, there has been no case of swallowing dysfunction.The combination of our standard radiochemotherapy regimen with carboplatin/5-FU and cetuximab is feasible resulting in promising objective response rates. Also the use of a classical concomitant boost concept in intensity-modulated technique has been shown to be practicable in a routine clinical setting resulting in only moderate overall toxicity of the regimen. However, patient accrual needs to be improved.JD is a member of the Merck KGaA advisory board. All other authors declare that they have no competing interests.Merck Serono has reviewed the publication, views and opinions described do not necessarily reflect those of Merck Serono.ADJ, JK, CS, and MWM are responsible for patient accrual, ADJ, AVN, KP, MWM for patient treatment, ADJ, JK, CS, and MWM for patient follow-up. MWM and JD were responsible for concept and design of the trial. ADJ, AVN, and KL coordinated and organized the trial. All authors read and approved the final manuscript.Supported by a project grant from Merck KGaA, Darmstadt, Germany


Toxoplasma encephalitis immune reconstitution inflammatory syndrome(TE-IRIS) is rare and usually occurs in an unmasking, rather than paradoxical form. To the best of our knowledge, only two cases of paradoxical TE-IRIS and nine cases of unmasking TE-IRIS have been previously described. We present two additional cases of histopathology-consistent paradoxical TE-IRIS, after early initiation of antiretroviral therapy (ART), and review the literature on TE-IRIS. Three of the four reported cases of paradoxical TE-IRIS were associated with early (within one week) initiation of ART, an issue that was not addressed in the 2009 US Department of Health and Human Services guidelines for the treatment of opportunistic infections. Toxoplasma IgG levels were undetectable. His chest X-ray revealed a right middle lobe infiltrate and three sputum samples for acid fast bacilli (AFB) were negative. Respiratory symptoms resolved with ceftriaxone and azithromycin. An MRI of his brain revealed two ring-enhancing lesions in the right precentral and occipital temporal areas  or prophylaxis, presented with shortness of breath, productive cough and fever for one week, as well as progressive weakness of his left upper extremity over the previous two weeks. His exam was significant for weakness (4/5) in his left-hand grip. His CD4 count was 8 (1%) cells/µl and his viral load was 547,000 copies/ml. Seruml areas . The patToxoplasma therapy, showed enlargement of the two prior lesions and development of a third lesion (Toxoplasma gondii tachyzoites and numerous bradyzoites (Toxoplasma treatment and ART were continued and the patient was started on corticosteroids (dexamethasone 4 mg every 4 hours tapered to prednisone 10 mg per day over 2 weeks), with gradual improvement but not complete resolution of his symptoms and signs.Over the next two weeks, the patient’s function deteriorated: his left deltoid muscle strength weakened to 2/5 (previously 5/5), he developed a left facial droop and left hip flexor and quadriceps weakness were 3/5 (previously 5/5). He consented to a lumbar puncture and his CSF revealed 6 white blood cells (WBC) per mm3 (96% lymphocytes and 4% monocytes), glucose of 41 g/L and protein of 92 g/L. EBV PCR in the CSF was positive. On hospital day ten, he suffered a tonic-clonic seizure. A repeat MRI on day 14 of empiric anti- lesion . After tyzoites , as wellyzoites . A repeaA 51 year-old man presented with two weeks of fevers, weight loss, frontal headaches, unsteady gait and upper extremity left-sided weakness. Three weeks prior, he had been diagnosed with AIDS and was not on ART or prophylaxis. On exam, he had left deltoid and hand-grip weakness (3/5) and left hip flexor and quadriceps extremity weakness (4/5), as well as decreased light sensation in his left arm.Toxoplasma IgG was positive. An MRI revealed multiple ring-enhancing lesions in his frontoparietal region (His CD4 count was 152 (13%) cells/µl and his viral load was 433,000 copies/ml. region . CSF shoT. gondii by immunohistochemistry .The patient experienced improvement in his weakness and gait to near-baseline and was discharged to rehabilitation. Ten days after initiation of treatment, the patient’s headache and left-sided weakness returned, in addition to a new left-sided facial droop. A repeat MRI showed no significant change . A repeaemistry  and a CDemistry . His symToxoplasma encephalitis being the most common cause of focal brain lesions in AIDS patients in resource-rich countries1, TE-IRIS is rare3 due to the elegant mechanisms of immune evasion byT. gondii4. A delicately controlled balance between host immune response and parasite immune evasion characterizes the life cycle ofT. gondii. Bradyzoites survive in a dormant intracellular state in a parasitophorous vacuole that, typically, evades intracellular acidification. The tissue cysts are kept in check by a Th1 predominant immune response5 characterized by macrophages and dendritic cells, producing IL-12, triggering CD4 cells to release IL-2, IFN-γ, IL-6 and TNF-α, thereby stimulating a reactive oxygen and nitric oxide-mediated cytotoxic response.DespiteT.gondii growth and abscess formation with necrotic centers and hyperemic edges, which appear as the characteristic multiple ring-enhancing lesions on MRI  there is also evidence supporting delayed initiation of ART. In a retrospective review of 514 patients with HIV and tuberculosis meningitis, there was significant mortality in those started on ART and anti-tuberculosis therapy simultaneously9. In a randomized, double-blinded, placebo-controlled prospective study of tuberculosis meningitis there was significant morbidity associated with immediate initiation of ART10. Recently, the prospective COAT  trial, designed to look at the optimal timing of ART initiation in cryptococcal meningitis, was stopped early due to increased mortality with early initiation of ART11. Since TE-IRIS is very rare, there is a paucity of data regarding the timing of ART initiation; therefore this issue is not addressed in the current US Department of Health and Human Services guidelines12.For most opportunistic infections, there is increasing evidence to support early initiation of ARTet al. report three cases of unmasking TE-IRIS and 65 cases of TE that did not experience paradoxical TE-IRIS despite virologic and immunologic response2. All three patients had unmasking TE-IRIS (none of them had been diagnosed with TE before the initiation of ART) and they all developed IRIS more than a month after the initiation of ART. None of the 65 patients with TE who did not develop IRIS had received ART before two weeks of anti-toxoplasma treatment . To our knowledge, we describe only the third and fourth cases of paradoxical TE-IRIS in the literature13. In both of our patients, as well as the case of paradoxical TE-IRIS presented by Tremont-Lukats3, ART was started simultaneously or within the first week of anti-toxoplasmic therapy. In the case of paradoxical TE-IRIS described by Cabralet al.13, ART was delayed for twenty days after initiation of anti-toxoplasmic therapy .Immune reconstitution inflammatory response syndrome cases are classified as either unmasking (unmasking of an occult infection not known at the time of ART initiation) or paradoxical . In their case series of TE-IRIS, Martin-Blondel14, the worsening symptoms led to a brain-biopsy. In both patients, histopathology showed a vigorous cytotoxic immune response, consistent with TE-IRIS4. Both patients improved with continuation of anti-parasitic treatment, ART and high-dose steroids. In these two cases and one of the two previous cases of paradoxical TE-IRIS, initiation of ART took place simultaneous or within the first week of anti-parasitic therapy. While generalizations cannot be made with only four cases, until further data evaluates the issue, we recommend very close monitoring if ART is started before at least two weeks of anti-toxoplasma therapy. Moreover, our cases illustrate that, for CNS toxoplasmosis in AIDS patients who have been recently started on ART, after performing a biopsy, the threshold to administer steroids for possible IRIS should be low, if there is any evidence of clinical deterioration.In conclusion, we present two patients with AIDS and CNS toxoplasmosis, who had disease progression, despite empiric anti-toxoplasmic therapy and a 1-log decrease in their HIV viral load with ART. Since the overwhelming majority of patients with toxoplasmic encephalitis improve after two weeks of anti-toxoplasma therapyInformed consent for publication of the clinical details and clinical images was obtained from the patients. In general, earlier HAART improves outcome particularly in those with the lowest CD4 counts. However, opportunistic infections of the CNS such as TB and cryptococcosis are an exception, and timing of HAART requires special consideration. The optimal timing of HAART in the setting of toxoplasma encephalitis has not been studied. This well written and informative case report illustrates that initiating HAART (very) early after beginning anti-toxoplasma therapy might be deleterious, deserving further studies.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is a well written and interesting observation that is helpful for doctors treating HIV-infected patients. Although the number of patients is low with TE-IRIS, one could argue to delay HAART until the patient has been treated for 2 weeks with anti-toxoplasmosis treatment, which is an important message.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is a useful addition to the literature and serves as a cautionary note to clinicians. There are some minor issues: the grading scale for weakness is the MRC scale and the viral load referred to in the first patient relates to HIV.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for your comments. Yes, the strength scale is MRC & yes the "viral load" was meant to imply "HIV viral load."


Petersen-MCI) affect measured impairment rates and the performance of short cognitive tests such as the Mini Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA), particularly in cerebrovascular disease. We therefore evaluated the effect of different operational definitions on measured rates of Petersen-MCI and on reliability of short cognitive tests in patients with TIA and stroke.Mild cognitive impairment (MCI) is at least as prevalent as dementia after transient ischaemic attack (TIA)/stroke and is increasingly recognised as an important outcome in observational studies and randomised trials. However, there is no consensus on how impairment should be defined, and numerous different criteria exist. Previous studies have shown that different criteria for cognitive impairment impact on prevalence rates in epidemiological studies. However, there are few data on how operational differences Consecutive patients underwent the MMSE, MoCA and neuropsychological battery ≥1 year after TIA or stroke in a population-based study. MCI was defined using the Petersen method and subclassified as single or multiple domain, both with  and without (modified) subjective memory impairment. Different cut-offs  on a given test relative to published norms were compared together with use of single versus multiple tests to define domain impairment.91 non-demented subjects completed neuropsychological testing  at a mean of 3.1 ± 1.9 years after the index event. Rates of cognitive impairment ranged from 14/91 (15%) for MCI-original at >2 SD cut-off to 61/91 (67%) MCI-modified at >1 SD cut-off, and the proportion of MCI that was multiple domain varied, e.g. 24/46 (52%) versus only 5/27 (20%) at 1 versus 2 SD cut-off for MCI-modified. Requirement for subjective memory complaint approximately halved estimates , whereas use of multiple tests versus a single test to define a cognitive domain had less impact. In general, diagnostic accuracy was higher, and optimal cut-offs lower, on MMSE and MoCA for multiple-domain versus single-domain MCI, but the MoCA appeared superior for detecting MCI-modified, whereas the MMSE performed well in detecting MCI-original.Even within established criteria for MCI, differences in operational methodology result in 4-fold variation in MCI estimates. Optimal MMSE and MoCA cut-offs are lower, and reliability more similar, when criteria for MCI are more stringent. Our findings have implications for sample size and adjusted relative risk calculations in randomised trials and for comparisons between studies. In the first year after stroke, around a quarter of patients will have dementia, rates being lowest in those with first ever stroke and highest in those with recurrent events ,2. Mild However, even within established criteria for cognitive impairment such as those described by Petersen and colleagues , there iLack of consensus on how to operationalise established criteria is important since it is likely to result in lack of comparability between studies and conflicting results regarding the performance of short cognitive tests which is problematic since such short tests are increasingly used as outcome measures in large pragmatic studies and clinical trials. We hypothesised that lower thresholds for defining impairment would result in higher measured rates of cognitive impairment in patients with TIA and stroke, higher optimal cut-offs on short cognitive tests and superiority of the Montreal Cognitive Assessment (MoCA) , which iPatients were participants in the Oxford Vascular Study (OXVASC 2002 -), a prospective population-based cohort study of all acute vascular events occurring within a defined population of about 91,000 ,16. The The NINDS-CSN Harmonization Standards  recognis(i) Trail Test (parts A and B) ,21;(ii) Symbol Digit Modalities Test ;(iii) Boston Naming Test (30-item version) ,23;(iv) Rey-Osterrieth complex figure copy ,25;(v) Hopkins Verbal Learning Test-Revised ,27;(vi) letter  amnestic single domain: objective impairment of memory only;(ii) amnestic multiple domain: memory and at least one other cognitive domain impaired;(iii) non-amnestic single domain: one single domain other than memory impaired;(iv) non-amnestic multiple domain: at least two cognitive domains impaired but not memory.We examined the effect of different operational methodologies as follows:(i) different thresholds  for defwww.karger.com/doi/10.1159/000355496); test combinations for a given cognitive domain were selected on the basis of the NINDS-CSN Harmonization Standards recommendations [(ii) using abnormal performance on a single test versus combinations of tests to define impairment in a given cognitive domain ,9  requiring multiple-versus single-domain impairment;(iv) with  and without (Petersen-modified criteria) requirement for a subjective memory complaint .For all designations of cognitive impairment, subjects had no impairment of basic functional activities of daily living as measured by the Barthel index, and did not fulfil the DSM-IV dementia diagnostic criteria .An additional point for low education (≤12 years) was added to the MoCA score in line with the original publication . ReliabiNinety-one non-demented patients completed MMSE, MoCA and Neuropsychological Battery assessments at a mean of 3.1 ± 1.9 years after the index event. TIA (n = 42) and stroke (n = 49) patients were similar in age , male sex  and education <12 years .Rates of cognitive impairment ranged from 14/91 (15%) for the most stringently defined cognitive impairment  to 61/91 (67%) for the least stringently defined impairment  versus 12/42 (29%), with OR = 3.06, 95% CI = 1.28-7.36, p = 0.01 for MCI-modified, 1.5 SD cut-off and single test definition, and 12/49 (24%) versus 5/42 (12%), with OR = 2.40, 95% CI = 0.77-7.50, p = 0.13 for MCI-original, 1.5 SD cut-off online . MultiplAUC were generally higher for the MoCA than the MMSE for detecting MCI where subjective memory complaint was not required (MCI-modified), whereas the MMSE was more reliable at identifying MCI-original. More stringent cut-offs 1.5 or 2 SD) for defining MCI usually resulted in higher AUC. AUC were also higher for identifying multiple-domain MCI than for identifying single-domain MCI and were highest for multiple-domain MCI versus no MCI, i.e. when the difference in cognitive performance between the two groups being compared was greatest  in patients with cerebrovascular disease and on optimal cut-offs on short cognitive tests. We observed that the measured rate of MCI after TIA and stroke was highly dependent on operational methodology with lowest rates seen when cut-offs were most stringent, in keeping with studies in Parkinson's disease [Previous studies have described variable estimates of (vascular) dementia and cognitive impairment criteria ,9,10,11,Amnestic single-domain impairment, common in memory research cohorts, was rare, in keeping with findings from clinical practice , whereasRegardless of operational definition, multiple-domain cognitive impairment was generally detected more reliably than single-domain impairment by short cognitive tests particularly by the MMSE perhaps unsurprisingly, since the MMSE was developed to detect moderate/severe cognitive impairment. The MMSE also performed well in detecting MCI-original (subjective memory complaint required) suggesting sensitivity to Alzheimer-type impairment presumably owing to an emphasis on memory, language and orientation. When subjective memory complaint was not a requirement, MCI rates were much higher and the MoCA performed well in detecting both single- and multiple-domain impairment consistent with its sensitivity to mild deficits not detected by the MMSE ,20. ThesGiven the reliability of short cognitive tests for detection of multi-domain impairment, short tests may be equally as good as the formal Neuropsychological Battery in identifying those at high risk of conversion to dementia. Further, it has been shown that some subjects with global cognitive impairment defined on short tests do not have MCI by formal criteria but are nevertheless at high risk of dementia . If shorOur results provide some explanation for conflicting results from previous studies regarding optimal cut-offs and relative performance of the MMSE and MoCA for detection of cognitive impairment after stroke. Godefroy et al.  performeThe relatively low MoCA cut-offs and similar MMSE and MoCA performance in the studies by Godefroy et al.  and DongOur study has some limitations. First, we used a relatively small sample of stable patients with TIA and stroke, and patients with dysphasia and severe stroke were not included. Second, we did not determine the effect of informant report of cognitive decline on the measured rate of impairment. Third, we were not able to examine the effects of different psychological tests and norms within a given domain owing to the need to be pragmatic in testing older and sometimes frail patients, but this is likely to be an additional factor in comparing results between studies. Finally, we selected tests according to the Harmonisation Standards recommendations  which arIn conclusion, even within established criteria for MCI, differences in operational definitions resulted in 4-fold variation in MCI estimates. In addition, optimal MMSE and MoCA cut-offs were lower, and reliability of the two tests was more similar, when requirements for MCI were more stringent. Our findings have implications for measuring cognitive impairment outcomes in randomised trials and observational studies in terms of sample size and adjusted relative risk calculations and choice of cut-offs on short cognitive tests. Our data should also facilitate the interpretation of reported MCI rates from across different studies.Competing interests: none declared.Supplementary dataClick here for additional data file.


Long-term asthma management falls short of the goals set by international guidelines. The Internet is proposed as an attractive medium to support guided self-management in asthma. Recently, in a multicenter, pragmatic randomized controlled parallel trial with a follow-up period of 1 year, patients were allocated Internet-based self-management (IBSM) support (Internet group [IG]) or usual care (UC) alone. IBSM support was automatically terminated after 12 months of follow-up. In this study, IBSM support has been demonstrated to improve asthma-related quality of life, asthma control, lung function, and the number of symptom-free days as compared to UC. IBSM support was based on known key components for effective self-management and included weekly asthma control monitoring and treatment advice, online and group education, and communication (both online and offline) with a respiratory nurse.The objective of the study was to assess the long-term effects of providing patients 1 year of IBSM support as compared to UC alone.Two hundred adults with physician-diagnosed asthma  from 37 general practices and 1 academic outpatient department who previously participated were invited by letter for additional follow-up at 1.5 years after finishing the study. The Asthma Control Questionnaire (ACQ) and the Asthma Quality of Life Questionnaire (AQLQ) were completed by 107 participants (60 UC participants and 47 IG participants). A minimal clinical important difference in both questionnaires is 0.5 on a 7-point scale.At 30 months after baseline, a sustained and significant difference in terms of asthma-related quality of life of 0.29 (95% CI 0.01-0.57) and asthma control of -0.33 (95% CI -0.61 to -0.05) was found in favor of the IBSM group. No such differences were found for inhaled corticosteroid dosage or for lung function, measured as forced expiratory volume in 1 second.Improvements in asthma-related quality of life and asthma control were sustained in patients who received IBSM support for 1 year, even up to 1.5 years after terminating support. Future research should be focused on implementation of IBSM on a wider scale within routine asthma care.International Standard Randomized Controlled Trial Number (ISRCTN): 79864465; http://www.controlled-trials.com/ISRCTN79864465 (Archived by WebCite at http://www.webcitation.org/6J4VHhPk4). Asthma is a common chronic disease with a prevalence of approximately 6% among adults . It is cProvision of Internet technology has been proposed as an appealing medium for asthma management -14. IndeIn this paper, we aim to assess the long-term effects of providing patients 1 year of IBSM support as compared to UC alone.Two hundred patients who previously participated in a 12-month multicenter, nonblinded, pragmatic randomized controlled parallel trial were invited for additional follow-up 1.5 years after finishing the study. Full details of the study methodology and subjects for the Self-Management of Asthma Supported by Hospitals, ICT, Nurses and General practitioners (SMASHING) study have been published elsewhere . Briefly1) by using a handheld electronic spirometer . After this session, patients were asked to report during a 2-week period FEV1 (daily), day and night symptom score (daily), and to fill in at least once weekly an Asthma Control Questionnaire (ACQ) ) scores in favor of the IG  and 5. N1  and 7. PThis study indicates that provision of IBSM support for 1 year leads to sustained benefits in terms of asthma control and asthma-related quality of life as compared with usual care, even up to 1.5 years after terminating support. IBSM support was additional to usual care and consisted of education, an action plan, self-monitoring, and regular medical review. Therefore, this study illustrates that sustained health improvements in health can be achieved by a structured care approach and self-management support as outlined by the Chronic Care model . To our The original SMASHING study had several strengths, which have been discussed at length elsewhere . BrieflyEven though IG patients had better outcomes in terms of asthma control and asthma-related quality of life as compared with UC patients, this difference was not accompanied with a higher ICS dosage for IG patients. Whether this can be attributed to the ability of the individual IG patient to tailor treatment to his or her need remains unclear. Moreover, the propensity to change in-treatment steps could have been reduced as guidance by IBSM support was terminated.To what extent demonstrated long-term benefits can be attributed to the mode of delivery, beyond incorporating components  for effective self-management, remains unclear. More specifically, whether the IBSM approach has led to improved self-management skills can only be postulated as we did not collect data on self-management outcomes, such as self-efficacy or compliance. If self-management skills have been improved, this is due to more intensive support in the start-up phase, since at 12 months after baseline only about 60% of the patients were still using the program on their own initiative . This ilFrom a patient perspective, there is an increasing demand to use modern technology in the management of chronic disease. In asthma, this is illustrated by a growing number of available apps for asthma “self-management” . UnfortuGiven the sustained benefits, cost-effectiveness becomes more favorable for IBSM, since major intervention costs, such as equipment and education sessions, apply to the first year, while maintenance costs can be spread over a longer period and could be improved by an increased number of users.For successful adoption of IBSM within routine care and into a patient’s daily life, several preconditions need to be identified and addressed among stakeholders ,35. For A comprehensive Internet-based self-management approach leads to sustained benefits in terms of asthma-related quality of life and asthma control, even up to 1.5 years after terminating support, particularly for patients with asthma that was not well controlled at baseline. Future research is needed to gain insight on long-term outcomes, cost-effectiveness, and strategies for integration of self-management support by modern technology in real-life settings.


Electroconvulsive therapy (ECT) is an effective therapy for several psychiatric disorders, including severe major depression, mania and certain forms of schizophrenia. It had been proposed that ECT acts by modulating local plasticity via the stimulation of neurogenesis. In fact, among antidepressant therapies, ECT is the most robust enhancer of neurogenesis in the hippocampus of rodents and non-human primates. The existence of ECT-triggered neurogenesis in other brain areas, particularly in those adjacent to the other main locus of neurogenesis, the subventricular zone (SVZ), had so far remained unknown. Here we show that ECT also strongly enhances neurogenesis in frontal brain areas, especially in the rostro-medial striatum, generating specific, small-size calretinin-positive interneurons. We provide here the first evidence that ECT stimulates neurogenesis in areas outside the hippocampus. Our data may open research possibilities that focus on the plastic changes induced by ECT in frontal limbic circuitry. ECT is the therapy of choice for treatment-refractory depression, as well as for treatment of acute mania and catatonic schizophrenia The occurrence of neurogenesis in other brain areas following ECT is unknown. The anterior rodent SVZ is, in addition to the hippocampal dentate gyrus, a primary site of adult neurogenesis Three-month old male Sprague-Dawley rats  were housed under standard conditions and received ECT (once daily for 10 day)  via earclip electrodes after pretreatment with an electrode gel, as described previously Rats receiving 10 electroconvulsive seizures were injected intraperitoneally (i.p.) with 100 mg/kg BrdU twice per day (at 9∶00 a.m. and 5 p.m.) for 5 days beginning on Day 8 of the ECT treatment and continuing 3 days following the last ECT see . AnimalsTo evaluate intermediate stages of neurogenesis, we used an established marker for identifying newborn migrating neurons, doublecortin (DCX) Labelled cells were quantified from coronal sections in the medial rostral striatum. Every sixth section from coordinates 3.70 to 1.70 mm relative to Bregma was quantified, representing four sections per brain. Cell counting was performed by an investigator blind to the treatment using a LEICA confocal laser scanning microscope (LEICA TCS-NT), similar to the procedure described previously P<0.05. Data are expressed as the mean±S.E.M.Statistical analysis of changes in the number of BrdU/DCX, BrdU/CR, DCX or CR-positive cells after ECT treatment was performed after testing the normality of the distribution of the data, using the Mann-Whitney U test, and the statistical significance level was determined at vs. 10.17±1.08; p = 0.002). A few scattered BrdU/DCX-positive cells were detected in the lateral septum and nucleus accumbens, but not in adjacent cortical areas. In rats treated with a single electroconvulsive seizure, the increase in the number of DCX/BrdU-positive cells after 10 days was significantly higher in ECT-treated rats than in sham-treated animals , but clearly less than the level obtained after the application of 10 electroconvulsive seizures.ECT administered to adult rats for 10 days  evoked avs. sham-treated animals both at 4  and 6 weeks post-ECT . Few CR/BrdU cells with similar morphological characteristics  were also present in the nucleus accumbens and lateral septum following ECT . A quantDue to its short half-life time of a few hours, BrdU incorporation may reflect only a glimps of the stimulatory effect of ECT on neurogenesis. Therefore, we also determined whether ECT increased the total number of neuroblasts and CR-positive interneurons in the medial striatum. We found that, similar to neuroblasts incorporating BrdU, ECT-treated rats demonstrated a strong increase in the total number of DCX-positive cells . SimilarThe present results demonstrate for the first time the occurrence of an unexpected and robust ECT-induced neurogenesis outside the hippocampus, opening new avenues for research focusing on plastic changes induced by ECT in limbic areas. Previously, enhanced gliogenesis and proliferation of endothelial cells, following ECT, but no neurogenesis, had been reported in the frontal cortex, suggesting a lack of effect of ECT on SVZ neurogenesis Our findings could be of importance regarding plastic changes induced by ECT in frontal limbic circuitry, especially in areas adjacent to the SVZ, such as perigenual regions. The medial striatum is part of the limbic system, with afferents from the medial prefrontal and cingulate cortices


To assess associations between subcutaneous neurofibromas (SC-NFs) and internal neurofibromas in patients with neurofibromatosis type 1 (NF-1) and to determine whether the association between SC-NFs and peripheral neuropathy was ascribable to internal neurofibromas.Prospective multicentre case-control study. Between 2005 and 2008, 110 NF-1 adults having two or more SC-NFs were individually matched for age, sex and hospital with 110 controls who had no SC-NF. Patients underwent standardized MRI of the spinal cord, nerve roots and sciatic nerves and an electrophysiological study. Analyses used adjusted multinomial logistic regression (ORa) to estimate the risk of the presence of internal neurofibromas or peripheral neuropathies associated with patients presented 2 to 9 SC-NFs, at least 10 SC-NFs as compared to patients without any .Cases had a mean age of 41 (± 13) years; 85 (80%) had two to nine SC-NFs and 21 (19%) at least ten SC-NFs. SC-NFs were more strongly associated with internal neurofibromas in patients with ten or more SC-NFs than in patients with fewer NF-SCs . The association with SC-NFs was stronger for diffuse, intradural, and > 3 cm internal neurofibromas than with other internal neurofibromas. Axonal neuropathy with slowed conduction velocities (SCV) was more strongly associated with having at least ten SC-NFs  than with having fewer SC-NFs . Bivariate analyses showed that the association between axonal neuropathy with SCV and sciatic neurofibromas was mediated by the association between SC-NFs and sciatic neurofibromas.The at-risk phenotype of NF-1 patients (i.e. NF-1 patients with SC-NFs) is ascribable to associations linking SC-NFs to internal neurofibromas at risk for malignant transformation and to axonal neuropathies with slowed conduction velocities. Axonal neuropathies with SCV are particularly common in patients with at least ten SC-NFs.ORPHA86301 Neurofibromatosis type 1 (NF-1 MIM 162200]) is a common autosomal dominant disorder associated with increased morbidity and mortality[200) is aSubcutaneous neurofibromas (SC-NFs) were independently associated with mortality among adults with NF-1 in two independent populations from France  and NortThe primary objective of this prospective matched case-control study was to characterise the association between SC-NFs and internal neurofibromas in patients routinely investigated with magnetic resonance imaging (MRI), the most sensitive method for detecting internal neurofibromas and assessing their features . The secondary objective was to determine whether peripheral neuropathy was associated with having internal neurofibromas along the nerve roots. Peripheral neuropathy was detected using a routine electrophysiological study.This case-control study was conducted prospectively from February 2005 to December 2008 in three hospital centres in France. For a type 1 error of 0.05, 100 cases and 100 controls provided 90% power for detecting odds ratios (ORs) greater than 3 for factors having a 20% prevalence in the general population of adults with NF-1. We decided to select 110 cases and 110 controls to allow for some patients being excluded secondarily . Controls were matched individually to cases on age (± 3 years), sex, and hospital. The study was approved by the Ile-de-France IX  ethics committee, and all cases and controls gave their informed consent.The cases were identified among adults with NF-1 in our NF-network database. They met diagnostic criteria for NF-1 established at the National Institutes of Health Consensus Development Conference (n = 1 099) . Among pAmong these 192 potential cases, 110 cases were selected at random for study inclusion.For each case, we looked for a control individually matched on age, sex, and centre in the NF-network database. Potential controls were probands or sporadic NF-1 patients who were at least 17 years old and had no SC-NFs and no contraindications to MRI. When we found several controls appropriate for the same case, we selected one at random. Thus, 110 controls were selected.Data, including demographic information  and clinical features were collected during routine clinical assessments at the neurofibromatosis clinics by one dermatologist at each centre  Table . The skiAll cases and controls had a standardized MRI study of the spinal cord, nerve roots, and sciatic nerve. MRI was performed using a field strength of 1.5-T. T1- and T2-weighted nonenhanced sequences  including short-tau inversion-recovery (STIR) sequences were acquired using a slice thickness of 4 mm with no gap. The image matrix was 512/256. Three coronal images  were acquired to characterize the internal neurofibromas. MRI data were reviewed by a senior radiologist (PB) who was unaware of the clinical features. We recorded the presence of internal neurofibromas, their type , their distribution , their size (< 3 cm or ≥3 cm), and their location .All cases and controls underwent a standardized electrophysiological study to look for peripheral neuropathy. The investigation included sensory/motor nerve conduction study for the superficial/deep peroneal nerves and the sural/tibial nerves at both lower limbs and for the median and ulnar nerves at one upper limb. The electrophysiological data were reviewed by a senior physiologist (JPL) who was unaware of the clinical features. Patients were classified as having either no neuropathy  or an axonal distal, sensory-predominant polyneuropathy . Among axonal neuropathies, we differentiated those with and without slowed conduction velocities (SCVs). Neuropathies with and without SCVs shared the same presentation in terms of action potential amplitude reduction, but neuropathies with SCVs additionally showed moderate but diffuse decrease of motor nerve conductions, very homogeneously, without any increased temporal dispersion or conduction block. Therefore, we assumed that neuropathies with SCVs were primarily axonal because they did not present any relevant criteria in favour of a demyelinating process . We alsop values < 0.05 were considered statistically significant. Quantitative variables are reported as mean ± standard deviation (SD) and qualitative variables as number (%).Data were double-keyboarded and analysed using STATA software version 11 . All tests were two-tailed and First, we compared cases and controls using univariate analysis (chi-square test or Fisher exact test as appropriate). Then, logistic regression models were used to estimate ORs adjusted for matching variables (aORs) with their 95% confidence intervals (95%CIs) for each characteristic of internal neurofibromas . Similarly, we estimated aORs with their 95%CIs separately for each type of peripheral neuropathy and radiculopathy. Then, to test our hypothesis that the association between SC-NFs and peripheral neuropathy was related to internal neurofibromas along the nerve roots, bivariate analyses including peripheral neuropathies and internal neurofibromas were performed. Finally, to assess whether the number of SC-NFs was associated with internal neurofibromas and/or peripheral neuropathy, cases were classified into two groups based on whether they had two to nine SC-NFs or at least ten SC-NFs. The, risk associated with each of these categories versus the controls (no SC-NFs) was estimated using adjusted multinomial logistic regression (aOR). Control patients were the referential category.p = 0.03) and at least one plexiform neurofibroma (p = 0.04). Cases had fewer large café-au-lait spots compared to controls but the difference was not statistically significant (p = 0.07).Between 2005 and 2008, 110 cases were matched for age, sex, and hospital to 110 NF-1 controls. Four cases and eight controls were excluded secondarily because their MRI scans were not interpretable. Table Table Radiculopathies were not significantly associated with the presence of SC-NFs Table .p = 0.18). The association between axonal neuropathy with SCVs and SC-NFs persisted only in the subgroup with sciatic neurofibromas . Thus, the association between SC-NF and axonal neuropathy with SCVs was due to the presence of internal sciatic neurofibromas.The joint analysis identified no interactions between variables associated with the presence of SC-NFs. A strong association was found between the presence of sciatic neurofibromas and that of axonal neuropathy with SCVs . In the bivariate analysis including axonal neuropathy with SCVs and sciatic neurofibromas, axonal neuropathy with SCVs was no longer associated with SC-NFs  compared to patients with two to nine SC-NFs   is a common disease with an increased propensity for developing both benign and malignant tumors . NF1 hasThe main concern in the long-term clinical management of adults with NF-1 is the identification of patients at high risk for MPNSTs developed from pre-existing internal neurofibromas. At present, no effective treatment is available for MPNST untreatable by surgery. New treatments are usually approved based on proof of efficacy in a controlled trial. When conducting controlled trials, an important consideration is patient selection based on severity of illness. Controlled trials in adults with NF-1 should target patients who have a high-risk phenotype defined by the presence of SC-NF. Patients with a high risk of internal neurofibromas must be therefore identified as potential new therapies are being developed. For instance, there are at least seven phase II/III trials in which the internal neurofibromas are being treated by targeted therapy such as ranibizumab, niltotinib... . In addition, we believe that targeted therapy will be of greater efficacy on non-transformed internal neurofibromas. At present time, an annual careful clinical examination is recommended for all patients with NF1 in order to detect clinical symptoms associated with MPNSTs such as pain, neurological deficit and enlargement of a pre-existing peripheral nerve sheath tumour . RoutineThe internal validity of this study depends on many factors, including the unbiased recruitment of cases and controls and the accuracy of the information obtained about the presence of internal neurofibromas. MRI, an extremely reliable method for detecting internal neurofibromas, was performed routinely in all our cases and controls. Assessment bias was minimized by having the MRI data reviewed by senior radiologist (PB) who was unaware of the clinical data. Assessment bias was minimized by having the electrophysiological data reviewed by senior physiologist (JPL) who was unaware of the MRI data. Clinical information regarding the neuropathy was not available. However, according to our experience the diffuse symmetric peripheral neuropathies related to NF-1 are asymptomatic or pauci-symptomatic with minor sensory manifestations .As with all hospital-based case-control studies, selection bias cannot be excluded. However, the prevalence of the various clinical features in the cases were similar to those in the controls (except for the number of cutaneous neurofibromas) and to those reported previously in NF-1 patients, indicating that our study population was representative of NF-1 patients . RegardiNF1 and surrounding gene compare to patients with mutations restricted to the NF1 genes. A recent study confirmed in a large cohort that NF1 microdeletion patients have a significantly higher incidence of learning disabilities and facial dysmorphism than patients with intragenic NF1 mutation [In summary, this study shows a strong association between the presence of internal neurofibromas and the presence of SC-NF. The increased risk in NF-1 patients with subcutaneous neurofibromas (high risk phenotype) can be ascribed in part to the associations between SC-NFs and internal neurofibromas (prone to transformation to MPNSTs) and the presence of a neuropathy with SCVs. The prevalence of neuropathy with SCVs is particularly high in patients with at least ten SC-NFs. To better understand the patho-physiological processes in the high risk phenotype of NF-1 patients, the next step would be to perform genotype-phenotype correlation studies. A more severe clinical phenotype has been reported in NF1 patients carrying genomic microdeletions involving mutation . HoweverNF1 implied in the internal NF development. One of the major aims of such studies would be to identify key targets for novel drugs' development.Further studies are therefore needed to find others genes beside aOR: adjusted Odds Ratio; CI: Confident Interval; MPNST: Malignant peripheral Nerve sheath tumor; MRI: magnetic resonance imaging; NF-1: Neurofibromatosis 1; SC-NF(s): Sub-Cutaneous Neurofibroma(s); SCVs: Slow Conduction Velocities; SD: standard deviationES, SBG, LVA, SF, JPL, AD, PB, PC, SB, PW have non-financial interests that may be relevant to the submitted work.PW conceived and designed the study, and is guarantor for the study. SBG contributed to the concept and design, and supervised analysis and interpretation of the data. ES performed the data analyses and wrote the initial draft of the article, to which all the authors contributed. All authors had full access to the data in the study and can take responsibility for the integrity of the data and the accuracy of the data analysis. All authors have given final approval for the final version to be published. The NF-France network contributed to the patients' inclusion.


Morphine is the most effective pain-relieving drug, but it can cause unwanted side effects. Direct neuraxial administration of morphine to spinal cord not only can provide effective, reliable pain relief but also can prevent the development of supraspinal side effects. However, repeated neuraxial administration of morphine may still lead to morphine tolerance.To better understand the mechanism that causes morphine tolerance, we induced tolerance in rats at the spinal cord level by giving them twice-daily injections of morphine (20 µg/10 µL) for 4 days. We confirmed tolerance by measuring paw withdrawal latencies and maximal possible analgesic effect of morphine on day 5. We then carried out phosphoproteomic analysis to investigate the global phosphorylation of spinal proteins associated with morphine tolerance. Finally, pull-down assays were used to identify phosphorylated types and sites of 14-3-3 proteins, and bioinformatics was applied to predict biological networks impacted by the morphine-regulated proteins.Our proteomics data showed that repeated morphine treatment altered phosphorylation of 10 proteins in the spinal cord. Pull-down assays identified 2 serine/threonine phosphorylated sites in 14-3-3 proteins. Bioinformatics further revealed that morphine impacted on cytoskeletal reorganization, neuroplasticity, protein folding and modulation, signal transduction and biomolecular metabolism.Repeated morphine administration may affect multiple biological networks by altering protein phosphorylation. These data may provide insight into the mechanism that underlies the development of morphine tolerance. Morphine is primarily used to treat severe pain caused by acute injuries and chronic diseases. However, systematic administration of morphine can cause many side effects, including impairment of mental and physical functions, psychological dependence, addiction, and tolerance An animal model in which morphine is repeatedly injected into the spinal cord has been used to mimic the direct neuraxial administration of morphine in patients and to study morphine tolerance at the spinal cord level The development of morphine tolerance is thought to be associated with dysregulated phosphorylation of proteins for two reasons. First, morphine exerts its pharmacologic effects by acting at opioid receptors Phosphoproteomics is used to study the phosphorylation of many proteins (the phosphoproteome), rather than individual proteins in a biological sample All animal experiments were carried out with the approval of the Institutional Animal Care and Use Committee at the National Defense Medical Center, Taiwan, and were consistent with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain.Male Sprague-Dawley rats (250–300 g) were used in this study. As shown in Nociception tests were carried out at baseline  and on day 5  as shown in Final and baseline PWLs were compared and used to calculate maximal possible analgesic effect (MPAE) of morphine. MPAE was calculated by the formula: [/(cutoff time – baseline PWL)] x 100% g for 15 min at room temperature to remove insoluble debris. Protein concentration in the supernatant was determined by a modified Bradford method After nociception testing, the animals were killed and lumbar enlargement segments of spinal cord were harvested. The spinal tissues were immediately lysed and homogenized with a sonication probe in 0.5 mL of lysis buffer . The lysates were centrifuged at 15000 The spinal proteins (250 µg) from control and morphine-treated groups were loaded onto IPG strips  for simultaneous rehydration. Isoelectric focusing was performed by using a voltage-time program of 50 V for 12 h, 500 V for 1 h, 1000 V for 1 h, and 7000 V to give a total of 140,000 V-h. Immediately after focusing, the IPG strips were equilibrated for 15 min in equilibration buffer  containing 1% dithiothreitol, and then for 15 min in equilibration buffer containing 2.5% iodoacetamide. The second dimension separation was carried out at 15°C with a vertical electrophoresis system  in 1 mm 12.5% acrylamide gels run at 20 mA/gel.After electrophoresis, the 2-DE gels were subjected to fluorescence staining with Pro-Q Diamond phosphoprotein dye  for detection of protein phosphorylation levels I) of each protein spot were estimated by the software based on the positions of standard markers and standard pI positions, respectively.Spot detection, gel matching, and spot quantification were carried out with 2-DE gel analysis software . To correctly estimate phosphorylation levels, we normalized the phosphorylation intensities of an individual protein revealed by Pro-Q Diamond dye to the total expression level of the same protein revealed by SYPRO Ruby fluorescent dye http://www.uniprot.org/) by using the MS-Fit on-line search engine (http://prospector.ucsf.edu/). For each PMF search to identify a protein, the mass tolerance was set at 150 ppm, and one missed tryptic cleavage was allowed.Proteins whose phosphorylation level differed significantly between control and morphine-treated groups were excised from the 2-DE gels and digested in the gel with trypsin. The digested proteins were subjected to MALDI-TOF MS analysis  to obtain the peptide mass fingerprint (PMF). Each mass spectrum was obtained from the average of signals generated from at least 500 laser shots. The PMFs were processed by using Flexanalysis™ and Biotools™ software  and were used to search the UniProt database (To identify the types and sites of phosphorylation in the most significantly changed protein (i.e. 14-3-3 proteins), we performed pull-down assays at both peptide and protein levels. For pull-down assay of a phophopeptide, the selected protein was in-gel digested into many peptides, and phosphopeptides of the digested protein were precipitated by using Phos-trap™ magnetic beads  according to the manufacturer's protocol For pull-down assay of phophoproteins, we homogenized spinal cord with RIPA buffer  and precipitated phosphoproteins from the lysate by using a mixed mouse monoclonal anti-phospho-serine and anti-phospho-threonine antibody . Precipitated proteins were then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Western blotting was carried out sequentially by electroblotting proteins onto polyvinylidene difluoride membranes, incubating the membranes with a mouse monoclonal anti-14-3-3 antibody , and probing with anti-mouse HRP-conjugated secondary antibody. Bands were visualized with an ECL detection kit . The lysates also were subjected to SDS-PAGE and Western blotting to check the total amount of 14-3-3 protein and glyceraldehyde 3-phosphate dehydrogenase, which served as input controls.To further estimate the impact of morphine on biological networks, the bioinformatics tool STRING  was used to elucidate biological networks regulated by the proteomics-identified phosphoproteins p<0.05. The data are presented as the mean ± SD.Student's t-test was used for the statistical comparison of data from control and morphine-treated groups. Differences were considered significant at p>0.05), whereas the final PWLs on day 5 were significantly shortened in morphine-treated rats as compared to those in control rats (p<0.05). Because PWL inversely correlates with the extent of pain sensation, the shortened PWLs indicate a reduced analgesic effect of morphine. To ensure the development of morphine tolerance, we also quantified the degree of morphine antinociception by calculating MPAEs. As shown in p<0.05). Again, this quantitative data confirmed that the analgesic effect of morphine was reduced after repeated morphine injections and verified morphine tolerance.PWLs and MPAEs were measured to ensure the development of morphine tolerance in rats after repeated injections of morphine. As shown in Comparative 2-DE-based proteomic analysis revealed a marked difference in phosphorylation pattern of spinal cord proteins between control and morphine-treated rats. Patterns of protein phosphorylation are shown by the representative 2-DE gels stained with ProQ Diamond phosphorylation detection kits , and patThe magnified images in Gfap , a glia Chaperones are involved in maintaining proper conformations of and modulating activities of other proteins. After repeated injection of morphine, phosphorylation levels of one endoplasmic reticulum (ER)-specific chaperone Hspa5;  [28] andScaffold proteins are key regulators of many signaling pathways. Repeated treatment with morphine reduced the phosphorylation level of Phb , a scaffThe phosphorylation levels of three metabolic enzymes were altered, including two glycolysis enzymes, Eno2  and AldoPull-down assays were performed to identify the types and sites of phosphorylation in the most significantly changed protein (i.e. 14-3-3 proteins) in 2-DE. Phosphopeptide-enriching magnetic beads induced the precipitation of two major phosphopeptides  in the injected spinal area. This finding is consistent with previous reports showing that systematic treatment of morphine dysregulates metabolic enzymes and alters the functional state of carbohydrate metabolism in both the CNS and peripheral organs In conclusion, our proteomics data showed that repeated intrathecal injection of morphine dysregulated the phosphorylation of 10 proteins in the spinal cord. Bioinformatics analysis revealed five functional networks of proteins that are affected and known to be involved in cytoskeletal reorganization, neuroplasticity, protein folding and modulation, signal transduction, and biomolecular metabolism . These p


Main contributors to adverse outcomes in severely burned pediatric patients are profound and complex metabolic changes in response to the initial injury. It is currently unknown how long these conditions persist beyond the acute phase post-injury. The aim of the present study was to examine the persistence of abnormalities of various clinical parameters commonly utilized to assess the degree hypermetabolic and inflammatory alterations in severely burned children for up to three years post-burn to identify patient specific therapeutic needs and interventions.Patients: Nine-hundred seventy-seven severely burned pediatric patients with burns over 30% of the total body surface admitted to our institution between 1998 and 2008 were enrolled in this study and compared to a cohort non-burned, non-injured children. Demographics and clinical outcomes, hypermetabolism, body composition, organ function, inflammatory and acute phase responses were determined at admission and subsequent regular intervals for up to 36 months post-burn. Statistical analysis was performed using One-way ANOVA, Student's t-test with Bonferroni correction where appropriate with significance accepted at p<0.05. Resting energy expenditure, body composition, metabolic markers, cardiac and organ function clearly demonstrated that burn caused profound alterations for up to three years post-burn demonstrating marked and prolonged hypermetabolism, p<0.05. Along with increased hypermetabolism, significant elevation of cortisol, catecholamines, cytokines, and acute phase proteins indicate that burn patients are in a hyperinflammatory state for up to three years post-burn p<0.05.Severe burn injury leads to a much more profound and prolonged hypermetabolic and hyperinflammatory response than previously shown. Given the tremendous adverse events associated with the hypermetabolic and hyperinflamamtory responses, we now identified treatment needs for severely burned patients for a much more prolonged time. Despite significant advances in therapeutic strategies, e.g., improving resuscitation, enhancing wound coverage, appropriate infection control, and improving treatment of inhalation injury, severe burns remain a devastating injury affecting nearly every organ system and leading to significant morbidity and mortality Several studies have indicated that these metabolic phenomena post-burn occur in a timely manner, suggesting two distinct patterns of metabolic regulation following injury th day and required on average 4 operations. During acute hospitalization, 32% of the patients suffered from inhalation injury, minor infections occurred in 43% of the patients, sepsis occurred in 10%, multi-organ failure in 16%, and 8% of our patients died  for height at admission, while the percentage of burned children that fell below the 50th percentile for height was significantly greater for up to two years post-burn, indicating a profound growth delay in this patient population. Forty-two percent of the patients included this study were below the mean for weight at admission, while the percentage of burned children that fell below the 50th percentile for weight was significantly greater for up to three years post burn, p<0.05. Data demonstrate that it takes approximately 1–2 years for pediatric burn patients to grow again and improve their height and weight percentile.On admission, the patient population fell within essentially the normal distribution pattern for both height and weight. Thirty-six percent of the patient population fell below the 50Analysis of CO, CI and HR revealed marked alterations in response to burn. CO increased immediately post-burn and remained significantly elevated over 12 months before gradually decreasing to values of non-burned controls . CI was Analysis of liver size determined by ultrasounds demonstrated markedly increased liver size in response to the initial burn trauma for up to three years, p<0.05 . ThroughUrinary norepinephrine, epinephrine, and cortisol increased markedly immediately after burn trauma. Urinary norepinephrine increased 10-fold during the early phase post-burn and remained significantly elevated up to 540 days post-burn when compared with non-burned control patients, p<0.05 . UrinaryWe found that almost all cytokines measured within this study were significantly altered in response to burn injury . Dramati2-macroglobulin concentrations displayed significantly decreased values for up to 60 days post-burn before gradually increasing to levels of non-burned controls, p<0.05  and aspartat-aminotransferase (AST) significantly increased immediately upon burn trauma and remained significantly elevated for the remaining of the study period, p<0.05 . Serum AAll serum hormones measured within this study demonstrated significant alterations in response to burn trauma. Serum levels of both, insulin growth factor (IGF)-I and insulin-like growth factor binding protein-3 (IGF BP-3), decreased significantly immediately post-burn and remained diminished for most of the remaining study period, compared to levels of non-burned controls, p<0.05 . Human gThe importance of this study is that it clearly demonstrated that burn induced metabolic and inflammatory changes persisted for 3 years after the injury. The relevance of post-burn hypermetabolism and inflammation is that they induce insulin resistance for 3 years Even though the metabolic alterations after severe burn injury are similar to any major trauma, severe burns are characterized by a hypermetabolic response that is more severe and sustained than any other form of trauma Persistently increased glucose and insulin levels as shown in this study are of serious clinical concern since hyperglycemia has been frequently linked to impaired wound healing 1-acidglycoprotein, and CRP demonstrated significant increases for up to nine months post-burn. Serum constitutive hepatic proteins, in contrast, such as retinol-binding protein, pre-albumin and transferrin, were found to be significantly decreased for up to six months post-injury. This decrease could be due to decreased production, increased consumption or increased loss due to capillary leakage. These proteins represent commonly utilized markers for general homeostasis indicating the severity and intensity of the prolonged post-burn dysbalance Other significant observations in this large prospective trial include substantially affected expression of acute phase proteins. Particularly haptoglobin, αrhGH) in trauma patients have been demonstrated in various settings. Besides enhancing immune function rhGH stimulates protein synthesis and attenuates the nitrogen loss after injury and improves clinical outcomes rhGH modulates the hepatic acute phase response by increasing constitutive hepatic proteins, decreasing acute phase proteins, modulating cytokine expression, and increasing IGF-I concentrations rhGH administration in trauma patients has been shown to increase mortality in a prospective, randomized, double-blind study by Takala and colleagues rhGH has been restricted. Administration of IGF-I may thus represent a promising therapeutic alternative, since recent studies could demonstrate that IGF-I, in combination with its principle binding protein, improved muscle protein synthesis, hepatic acute phase and inflammatory response, and the immune system As recently demonstrated by Jeschke et al. Determination of serum osteocalcin and parathyroid hormone levels also demonstrated significant decreases for nine and up to 36 months, respectively, associated with profound decreases in BMC and BMD. As shown by Klein and others Analysis of various cardiac parameters showed marked alterations in response to burn. Percent of normal CO as well as CI were significantly elevated during the first 180 days post-burn, accompanied by a massive tachycardia with 120–180% predicted HR for the whole time period studied. Elevated levels of plasma catecholamines instigate the cardiac stress post-burn. Plasma catecholamine levels are elevated up to nine months post-burn, but the derangements in cardiac physiology last up to three-years after the initial trauma. Elevated catecholamines increase myocardial oxygen delivery, myocardial oxygen consumption and cause focal degeneration of the myocardium and hypertrophy. In excess, they cause cardiac deficiency, local myocardial hypoxia, and cardiac death Therapeutic advancements in the acute phase post-burn, such as early excision and closure of the burn wound, more appropriate infection control and anti-catabolic therapeutic intervention, including beta-adrenergic blockade with propranolol, growth hormone, insulin-like growth factor, oxandrolone, testosterone, and insulin have substantially contributed to significant improvement of morbidity and mortality rates in burn patients during acute hospitalization. However, based on this study, we suggest that a severe burn is not an acute illness but rather a chronic health problem. We thus believe that burn patients should be carefully monitored for at least 3–4 years in order to reverse these complex metabolic alterations post-burn.All thermally injured pediatric patients with burns covering more than 30% of their TBSA were admitted to our institution between 1998 and 2008, required at least one surgical intervention, and consented to the University of Texas Medical Branch Institutional Review Board-approved experimental protocol were included in this study. Patient, parent or legal guardian provided with written consent for participation in the study.On admission, the extent and degree of burn was assessed and recorded on a standard Lund and Browder chart by the attending burns surgeon present. Information also recorded at the time of admission included burn related (date and mechanism) as well as demographic data (age and gender). All patients were treated in our pediatric burns intensive care unit according to standardized protocols.2 TBSA burned +2000 cc/m2 TBSA lactated Ringer's solution given in increments over the first 24 hours. Within 24 hours of admission, all patients underwent total burn wound excision and the wounds covered with available autograft skin and any remaining open areas were covered with homograft. After the first operative procedure, it took 5–10 days until the donor site was healed and patients were taken back to the operation theater. This procedure was repeated until all open wound areas were covered with autologous skin material.Patients were resuscitated if needed according to the Galveston formula with 5000 cc/m2 body surface +1500 kcal/m2 area burn or we assessed the need by measuring the resting energy expenditure (REE), multiplied by 1.4 with weekly adjustments as previously published All patients underwent the same nutritional treatment according to a standardized protocol. The intake is calculated as 1500 kcal/m5 colony forming units per gram tissue. Sepsis was defined as a positive blood culture or pathologic tissue culture identifying the pathogen during hospitalization or at autopsy, in combination with at least 3 of the following: leucocytosis or leucopenia , hyperthermia or hypothermia (>38.5 or <36.5°C), tachycardia (>150 BPM in children), refractory hypotension (systolic BP <90 mmHg), thrombocytopenia , hyperglycemia (serum glucose >240 mg/dl), and enteral feeding intolerance  as previously published Patient demographics  and concomitant injuries such as inhalation injury, sepsis, morbidity, and mortality were recorded. Minor infection was defined as a positive tissue culture with more than 10Results obtained during the three-year period were divided into fifteen different time phases: 0 to7, 8 to 10, 11 to 16, 17 to 22, 23 to 28, 29 to 34, 35 to 40, 41 to 60, 61 to 90, 91 to 180, 181 to 270, 271 to 365, 366 to 540, 541 to 730 and 731 to 1,100 days post-burn. Data presented include a number of 31 to 307 different measurements at each time point. If any patient had more than one measurement performed during the respective time period, results were averaged to give a single mean result for each patient at each time period. One-hundred seven non-burned children, who consented for research studies and required blood and/or 24-hour urine collections, were used as normal cohort.As part of our routine clinical practice, all patients underwent REE measurements weekly during acute hospitalization and during admissions for reconstructive operations for up to three years post-burn. REE was measured using a Sensor-Medics Vmax 29 metabolic cart  as previously published Heights and weights were measured during acute hospitalization subsequent stays for reconstructive purposes and were plotted on standard growth charts M-Mode echocardiograms were completed as follows: at the time of the study, none of the patients presented with or previously suffered from other concomitant diseases affecting cardiac function, such as diabetes mellitus, coronary artery disease, long-standing hypertension, or hyperthyroidism. Study variables included: resting CO, cardiac index (CI), stroke volume (SV), resting heart rate (HR) and left ventricular ejection fraction (LVEF). SV and CO were adjusted for body surface area and expressed as indexes. All cardiac ultrasound measurements were made with the Sonosite Titan echocardiogram with a 3.5 MHz transducer. Recordings were performed with the subjects in a supine position and breathing freely. M-Mode tracings were obtained at the level of the tips of the mitral leaflets in the parasternal, long axis position and measurements were performed according to the American Society of Echocardiography recommendations. Left ventricular volumes determined at end diastole and end systole were used to calculate EF, SV, CO and CI. Three measurements were performed and averaged for data analysis Twenty-four-hour urine collections were taken regularly throughout acute hospital stay and during admissions for reconstructive operations and rehabilitation services. These samples were collected and chilled by the bedside prior to transport to our clinical lab for processing using HPLC techniques as previously published Blood was collected from the burn patients at the time of admission, pre-operatively, and every Monday and Thursday at 6:00 AM, as well as during subsequent stays for surgical and rehabilitation services for serum cytokine and hormone analysis per hospital protocol. Blood was drawn in a serum-separator collection tube and centrifuged for 10 minutes at 1320 rpm; the serum was removed and stored at −70°C until assayed.Serum hormones were determined using HPLC and ELISA techniques. The Bio-Plex Human Cytokine 17-Plex panel was used with the Bio-Plex Suspension Array System  to profile expression of Interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12p70, IL-13, IL-17, granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1-beta (MIP-1β), and tumor necrosis factor (TNF). The assay was performed according to the manufacturer's instructions. Acute phase and constitutive protein levels were measured using BN II Plasma Protein Analyzer  by a nephelometric technique, according to manufacturer's instructions.The study was reviewed and approved by the Institutional Review Board of the University Texas Medical Branch, Galveston, Texas. Prior to the study, each subject, parent or child's legal guardian signed a written informed consent form. Analysis of variance (ANOVA) with post hoc Bonferroni correction, paired and unpaired Student's t-test, Chi-square analysis, and Mann-Whitney tests were used where appropriate. Data are expressed as means±SD or SEM, where appropriate. Significance was accepted at p<0.05.www.clinicaltrials.gov: #NCT00239668 and #NCT00673309. A steering committee consisting of academic investigators designed the study and monitored its conduct. Data were collected by the investigators and analyzed by scientists. All the authors had access to the data, participated in the data analysis and interpretation, and wrote the manuscript. All authors vouch for the accuracy and completeness of the data and the statistical analysis. All authors participated in the writing of the manuscript and approved the final manuscript before submitting it for publication.This study was registered at


Of the 111 patients included in the analysis, 78 (70.3%) had hormonal therapy (HT) and 33 (29.7%) had chemohormonal therapy (CHT) recommendations pre-testing. The Recurrence Score was significantly higher in those with a pre-testing CHT recommendation compared with those with a pre-testing HT recommendation . Post-testing, treatment of 24 patients (21.6%) was different from their pre-testing recommendation. The difference between CHT recommendation rate pre-testing and the rate of CHT received post-testing was nonsignificant for the entire cohort and for patients’ subgroups  . Following classification of the cohort into two Recurrence Score subcategories , changes in treatment decisions  were reported for 16.5% of low-intermediate and 34.4% of high-intermediate patients. Post-testing, the rate of CHT decreased (by 58%) in the low-intermediate subcategory and increased (by 64%) in the high-intermediate subcategory . In logistic regression analyses, the Recurrence Score subcategory was the only significant predictor of changes in treatment decisions . The only significant difference between the two subsets of patients with such a change  was the Recurrence Score . These findings demonstrate that intermediate Recurrence Score results provide clinically relevant information and impact treatment decisions in ER + early breast cancer.This retrospective study evaluated the impact of intermediate Recurrence Score The study was approved by the institutional review board of Rambam Health Care Campus.type DX testing were documented on requisition of the assay.Clinicopathological characteristics, Recurrence Score results, and treatments received were obtained from patients’ records. Physicians’ treatment recommendations prior to OncoT-test/Mann–Whitney–Wilcoxon (MWW) and Chi-squared/Fisher’s exact tests were used, as appropriate, to compare continuous and categorical parameters, respectively, between patients treated with hormonal therapy alone and those treated with chemohormonal therapy. McNemar's test was used to assess whether the difference between the proportions of patients with hormonal and chemohormonal therapy recommendations (pre-testing) and the proportion of patients who received hormonal and chemohormonal therapy (post-testing) was significant. Logistic regression analyses were used to assess the probability of a treatment change from pre-testing hormonal therapy recommendation to post-testing treatment with chemohormonal therapy and from pre-testing hormonal therapy recommendation to post-testing treatment with chemohormonal therapy as a function of patient’s age, tumor size, tumor grade, and the Recurrence Score result. All analyses were conducted using SAS statistical software version 9. 2 ; P <0.05 was considered significant.Descriptive statistics were used to summarize clinicopathologic characteristics and Recurrence Score results. type DX testing in Israel  were recommended hormonal therapy alone and 33 patients (29.7%) were recommended chemohormonal therapy. These two groups were not statistically significantly different with respect to age , and had statistically significant differences with respect to tumor size , nodal involvement , grade distribution , and surgery type . In comparison to patients with a pre-testing recommendation of hormonal therapy alone, those with a pre-testing recommendation of chemohormonal therapy had larger tumors, and there were higher proportions of node-positive patients, grade 3 tumors, and patients who had undergone mastectomy  .After knowing the Recurrence Score results, 24 patients  received treatment that was different from their pre-testing treatment recommendation. Of the 78 patients who were originally recommended hormonal therapy alone, 11 received chemohormonal therapy ; of the 33 patients who were originally recommended chemohormonal therapy, 13 received hormonal therapy alone  . The overall change rate in treatment decisions  was similar in the younger (≤50 years) and older (>50 years) age subgroups , and in patients with smaller (≤15 mm) and larger (>15 mm) tumors . Of the 16 patients with grade 1 tumors, only 1  had a treatment decision change ; in patients with grade 2 and grade 3 tumors, the overall change rate was more pronounced and similar .Treatment changes were also analyzed by age , tumor size , and grade (for patients with grade 2 and 3 tumors). In all the analyzed subgroups, the net change in chemotherapy use  was nonsignificant . In the high-intermediate-Recurrence Score subcategory, the changes were primarily in the opposite direction , and consequently, the overall rate of chemohormonal therapy increased significantly after knowing the Recurrence Score results .To explore whether clinicians perceive intermediate Recurrence Score patients in a uniform manner, the cohort was divided into two intermediate Recurrence Score subcategories , only main effects were considered for the logistic regression analyses. For patients with a pre-testing hormonal therapy recommendation, the only significant predictor of a treatment change was high-intermediate Recurrence Score subcategory . For patients with a pre-testing chemohormonal therapy recommendation, multivariate logistic regression analysis was not technically possible because there were only 33 such patients. For these patients, univariate analysis was performed and demonstrated that the low-intermediate Recurrence Score subcategory was a significant predictor of a treatment change .To assess the impact of the clinicopathologic parameters and the Recurrence Score results on treatment changes, we modeled the probability of a change between treatment recommendation and actual treatment received as a function of age , tumor size , grade , and the Recurrence Score subcategory  for patients with pre-testing hormonal therapy recommendations and for patients with pre-testing chemohormonal therapy recommendations. Possible interaction between the effect of the Recurrence Score subcategory and patients’ age, tumor size, or tumor grade (each separately) on the probability of a treatment change was examined by the Breslow Day test. As the effect of the Recurrence Score subcategory was consistent in all subgroups , and the Breslow Day tests were nonsignificant   revealed no statistically significant differences in clinicopathologic parameters including patients’ age, tumor size, nodal status, tumor grade, and type of surgery performed between the groups   of the cohort. The observed changes were in both directions  with no significant net change in chemotherapy use . Unlike low- and high-Recurrence Score patients, for whom the impact of the Recurrence Score results on treatment decisions has been shown to be consistent across studies  , higher intermediate Recurrence Score results were significantly associated with a decision to treat with chemohormonal therapy, suggesting that clinicians evaluate the Recurrence Score as a continuous parameter and do not consider all intermediate-Recurrence Score patients as having the same risk of recurrence. Also, in an analysis of treatment change by Recurrence Score subcategory (using a cutoff of 25, which is the cutoff used in the ongoing Oncotype DX phase 3 TAILORx and RxPONDER trials that were designed to assess chemotherapy benefit in node-negative patients with Recurrence Score results between 12 and 25 and node-positive patients with results ≤25, respectively (ClinicalTrials.gov website Our study evaluated the factors that may influence changes in treatment decisions in intermediate-Recurrence Score patients. Traditional parameters such as patient’s age, tumor size, and tumor grade, did not have a statistically significant impact on the change in chemotherapy use from pre- to post-Oncotype DX testing has been shown to increase clinicians’ confidence in their treatment decisions (Lo et al. OncoThe limitations of this study include its retrospective design and a relatively small sample size. In addition, since this was a single-center study, its ability to represent treatment patterns in the general breast cancer patient population in Israel may be limited.In summary, our findings demonstrate that intermediate Recurrence Score results are not perceived in a uniform manner by clinicians, but rather provide clinically relevant information that impacts adjuvant treatment decision-making in patients with ER + early invasive breast cancer.