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Influence of Cardio-Respiratory Fitness on Physical Performance in Elite Youth Soccer- Juniper Publishers
Large components of competitive soccer match-play are comprised of low intensity activity , as a result, superior levels of running economy (RE) may result in the preservation of energy during periods of low-intensity exercise. Work by Ziogas et al.  revealed that players who competed in a higher division had a superior RE when running at 12 km/h- 1 (Division A = 75.8%, Division B = 78.7%, Division C = 80.5% VO2max). This demonstrates that measures of RE (expressed as a percentage of VO2max) may be able to discriminate between playing level, but provides no indication to the extent to which RE may impact upon physical performance during soccer matchplay. Moreover, Stroyer et al.  assessed the RE and the activity patterns of young soccer players during match-play, however the relationship between the two measures was not reported. As a result, the contribution that RE and determinants of RE may play with regard to effective soccer-match play, particularly in youth populations, where there is an increased reliance upon aerobic energy production [5,12], requires investigating. Furthermore, due to the intensified , exhaustive and intermittent [1,3] nature of soccer, other determinants of RE may also provide an indication toward an individual’s ability to perform highintensity activities during soccer match-play. Di Paco et al.  demonstrated an improved ventilatory efficiency for a given exercise intensity in soccer players of a superior fitness level. Consequently, the purpose of this study was to determine the relationship between: VO2max, RE and determinants of RE, and physical measures associated with elite youth soccer performance during soccer match-play within a group of highly trained youth soccer players. It was hypothesized that superior measures of VO2max, RE and parameters of RE would be significantly correlated with physical performance measures during elite youth soccer match-play.
Upon arrival to the laboratory and following the necessary screening procedures, participants were fitted with a Polar Heart rate monitor (Polar Electro, Kempele, Finland) and facemask (Hans Rudolph, Hans Rudolph, Kansas City, USA), which was connected to an online gas analysis system (Cortex Meta Max 3B, Cortex Biophysik GmbH, Leipzig, Germany). The online gas analyser was calibrated prior to each visit according to the manufacturer’s instructions, using a known gas concentration and a 3-L syringe for manual volume calibration of the ventilation sensors. Following a standardised 10min warm up and a full description of the test and safety procedures, participants began to run at a speed of 8km/h at a 1% incline  on a motorised treadmill (HP Cosmos, Pulsar, Sportgerate GmbH, Nussdorf, Germany). The speed of the treadmill was increased by 1km/h every two minutes, this continued until participants reached 90% of their age predicted heart rate max (207-(0.7 x age)) . At this point, the treadmill speed remained constant whilst the incline of the treadmill was increased by 1% every minute until volitional exhaustion; this procedure was employed to avoid over-striding and potential early termination and inaccurate assessment of participant’s VO2max. This method has been successfully used before to elicit VO2max during treadmill running in young athletes . Rolling 15 sec averages were calculated for the final minute of the test to verify if a plateau was present, with the highest 15 sec average during the test being regarded as VO2max . It is well documented that children can exercise to exhaustion without exhibiting a VO2 plateau . Nevertheless, test criteria for a maximal effort, were the achievement of at least two of the following three performance values: 1) respiratory exchange ratio (RER) above 1.00, 2) plateau in VO2 despite an increasing speed (< 2mL∙kg-1∙min-1; 10) 3) heart rate ±10 beats/min of age predicted maximal heart rate . The VO2max protocol resulted in 100% percent of the participants reaching an RER value of greater than 1.00 and a heart rate value within ±10beats/min of age predicted maximal heart rate, while 88% of the participants presented a plateau in VO2 despite an increase in speed. As all players did fulfil two of the stated criteria, the term VO2max was preferred .
The gaseous exchange threshold (GET) was identified using the V-slope method (VE (ordinate), VO2 (abscissa). Two regression lines were created based upon the relationship between VCO2 and VO2. The intercept point between the two regression lines was then visually identified, with the VO2 value at the intercept (GETS) being extrapolated to the abscissa . To identify the speed at GET, a regression equation was formulated for VO2 and running velocity, for each individual. The individual’s VO2 at GET was then inputted into the individual’s respective regression equation to calculate the running velocity at GET. The V-slope method has been shown to be a viable and reliable method for detecting and identifying the gaseous exchange threshold in children . The GET was assessed by two individual researchers (experienced in the detection of GET), demonstrating 82% agreement (14 out of 17). For the remaining three participants, a third researcher was approached to verify the GET. Following this test, running velocities corresponding to 80% GET and 95% GET were calculated for the assessment of running economy. On the subsequent visit to the laboratory, and following a minimum of 24 hours recovery, participants’ RE was assessed at three progressive sub-maximal intensities (one absolute: 8km/h and two relatives: 80% GET and 95% GET exercise intensities), with 2 min passive recovery between each intensity. Prior to testing, participants were fitted with a Polar Heart rate monitor and face-mask, which was connected to a pre-calibrated online gas analysis system. Following a standardised 10min warm-up and a full description of the test procedures, participants began to run at the lowest exercise intensity (either 8km/hour the running velocity equivalent to 80% GET), with the gradient of the treadmill set at 1% . Participants ran at this speed for 4 min, ensuring that a steady state was maintained for the final minute . A 2min passive recovery was then permitted, in which participants’ heart rate recovered to pre-exercise levels. Pilot testing demonstrated that a 2min passive recovery was shown to provide sufficient time for players to recover to resting heart rate levels, ensuring there were no ensuing effects on the subsequent exercise bouts. During this recovery period, the treadmill speed was increased to the next progressive speed (higher intensity of 8km/h-1 or 80% GET). Again, participants exercised at this speed for 4min, attaining a steady state, before undertaking a further 2min recovery. Following the final 2min of passive recovery, participants completed another 4min period of sub-maximal running at the highest intensity (95% GET).
Extraction of the appropriate measures for assessing RE included applying rolling 15sec averages for the final minute of each sub-maximal exercise intensity to ensure that a steady state had been attained. It has been shown that male youth populations will attain a steady state following 3min of sub-maximal exercise . Current results revealed that all participants (100%) attained a steady state (< 2mL•kg-1•min-1 (10), in the final minute of exercise) for each sub-maximal exercise intensity. Following the identification of a steady state, RE was defined as the average VO2 during the final minute of each exercise bout . Relative oxygen consumption (VO2) was obtained for each exercise intensity as well as determinants of RE: minute ventilation (VE) and ventilatory equivalent (VEVO2). For the Yo-Yo IR1 test, cones were placed 20m apart, with a 5m recovery zone marked out at one end. The Yo-Yo IR1 test requires participants to run 2 x 20m shuttle runs at increasing speeds, interspersed with 10 seconds of active recovery. The pace of the test was controlled by audio signals emitted from a CD player (Sony CFD-V7, Sony, Tokyo, Japan). For the maximal Yo-Yo IR1 test, players were required to run until volitional termination of the test or, when they have twice failed to meet the designated cones in time with the audio signal, at which point they are removed from the test. The test score is the distance covered at the point they withdraw from the test. All players had previous experience of completing the Yo-Yo IR1 test and were therefore familiarised to the testing. During the test, players were allowed to consume fluids ad libitum. Previous evidence supports the use of the Yo-Yo IR1 test as a valid measure of soccer-specific fitness, particularly within youth populations .
For the assessment of participants’ performance during soccer match-play, three separate, sterile (controlled, replicable and include the same composition of players in the same positions) 11 v 11 matches were conducted and analysed using 10Hz GPS (Catapult, Melbourne, Australia) and Polar Heart rate monitors. Each match was conducted on the same third generation outdoor artificial pitch with the same pitch dimensions utilised within competitive match-play within this population (90 x 50m) and at the same time of day. Matches were comprised of 2 x 20min halves with a 5min rest interval between halves. The composition of the teams and positions remained the same for all three matches and each participant was assigned their own GPS for all matches. Matches were performed on three separate occasions with a minimum of 24hrs between matches.
Following each match, the GPS data was downloaded and analysed using Catapult Software (Catapult, Melbourne, Australia) and specially designed Microsoft Excel spreadsheets. Following this, an average of the 3 games was calculated for each player for each variable. Data was recorded for the whole match, each 20min half and into successive 5min epochs (e.g. 0-5min, 1-6min, 2-7min, 3-8min, etc), to establish and quantify the peak 5min epoch, the subsequent 5 min epoch to the peak 5 min epoch and the mean of the cumulative 5min epochs throughout the match. These were then averaged over the 3 sterile matches. Previous research has used discrete 5min epochs (0-5min, 5-10min, etc.) to identify the peak 5min interval during soccer match-play . However, the adoption of such criteria may result in missing the true peak 5min epoch, as this may occur between the pre-determined, discrete 5min epochs. Consequently, matches were analysed using successive 5min epochs (0-5min, 1-6min, 2-7min, etc.) for the identification of the most intense 5min epoch. Information recorded included total distance (TD), metres per min (m/min), relative high speed running distance metres (HSR), relative high speed efforts (HSReff), and relative very high speed running distance metres (VHSR), relative very high speed efforts (VHSReff) and relative sprint distance metres (S). Relative HSR running was regarded as distance covered above 50% of maximal linear velocity, relative VHSR was regarded as any distance covered above 70% of maximal linear velocity and relative Sprint as anything above 90% maximal linear velocity. The same thresholds were used for HSReff and VHSReff and an effort was regarded as any occurrence when such a speed was attained and sustained for greater than 0.2s. Maximal velocity was defined as the maximal velocity obtained during a 20m sprint from a standing start and obtained from the respective GPS devices which the player would wear during the competitive and sterile soccer matches.
A Shapiro Wilks test found the data to be normally distributed; therefore parametric statistical calculations were applied. Partial correlations, controlling for maturation, were performed on the whole sample to assess the relationship between measures of cardio-respiratory fitness and physical soccer-based measures. To aid interpretation of the results, confidence intervals (90%) for correlations were calculated and the magnitude of the correlations were determined using the modified scale by Hopkins  (r <0.1, trivial; 0.1-0.3, small; 0.3-0.5, moderate; 0.5-0.7, large; 0.7-0.9, very large; >0.9, nearly perfect; and 1 perfect).All statistical analysis was performed using SPSS version 21.0 (IBM SPSS statistics for Windows, IBM, Armonk, New York) and Microsoft Excel (Microsoft Excel 2013, Microsoft, Redmond, Washington) with the level of significance (alpha) set at 0.05.
Note: VO2max = Maximal oxygen consumption; GET = Gaseous exchange threshold; VEVO2 = Ventilatory equivalent; RER = Respiratory exchange ratio; %HRmax = percentage of maximum heart rate; TD = Total Distance; HSR = High speed running; VHSR; Very high-speed running. Partial correlations were controlled for using self-assessed Tanner Stage values, * = significant correlation (P< 0.05).
Note: VO2max = Maximal oxygen consumption; GET = Gaseous exchange threshold; VEVO2 = Ventilatory equivalent; RER = Respiratory exchange ratio; %HRmax = percentage of maximum heart rate; TD = Total Distance; HSR = High speed running; VHSR; Very high-speed running. Partial correlations were controlled for using self-assessed Tanner Stage values, * = significant correlation (P< 0.05).
Similar to the present study, Krustrup et al.  reported significant relationships between measures of relative VO2max and high-intensity distance covered during soccer match-play (r = 0.81), in elite female soccer players. Rebelo et al. , however, reported no significant relationships between measures of VO2max and time spent in absolute speed zones during soccer-match play (r =-0.13-0.25; P>0.05). However, it must be noted that Rebelo et al.  assessed players’ physical performance during soccer match-play using time-motion analysis, one individual at a time and over a variety of games, thus exacerbating the issues of inter-match variability associated with assessing physical soccer performance during soccer match-play . In the current study, all players were assessed simultaneously, using GPS analysis and during sterile soccer matches, in order to account for the complexities of variance involved in assessing physical performance during soccer match-play.
The current results demonstrate that traditional measures of RE (VO2 or %VO2max) when exercising at an absolute intensity (8km/h) are related to measures of physical performance during soccer match-play. However, when RE is measured at a relative (80%GET and 95%GET) exercise intensity, traditional measures of RE are not related to variables associated with superior physical soccer performance during match-play, within a group of highly trained youth soccer players. The use of RE as a measure to assess soccer-specific fitness is rarely adopted, this is probably due to the high-intensity, intermittent characteristics of soccer match-play. Despite this, research has advocated the need to improve soccer players’ levels of RE via training . In support of this, the current results demonstrated that a superior RE during an absolute intensity of 8 km/h was significantly related to measures of HSR during soccer match-play. The current findings, however, may be a result of the different internal load which an absolute intensity of 8 km/h equates to for each player, although such a finding may still be relevant as physical performance during competitive match-play does not account for players’ relative fitness levels, maturity status or size (e.g. leg length) either. Consequently, the use of absolute sub-maximal intensities may be preferable when utilising traditional measures of RE to distinguish between physical performance during youth soccer match-play. Conversely, when relative exercise intensities (80% and 95% GET) were employed to assess RE, no relationships between traditional measures of RE and physical measures of performance during soccer matchplay were evident. When, however, alternative determinants of RE are evaluated with respect to their relationship with physical soccer performance different relationships emerge.
While results from the present study provide little support for relationships between measurements of RE and physical performance during soccer-match-play, a significant inverse relationship between VEVO2 and measures associated with physical soccer performance was demonstrated. Work by Franch et al.  reported an average decrement of 11 L.min-1, in VE, while exercising at an intensity equivalent to 85% VO2max, following 6 weeks of intensive endurance-based interval training. In addition, these same authors reported that other determinants of running economy such as: stride length, stride frequency, respiratory exchange ratio and muscle fibre distribution were unaltered. As a result, the responsiveness of VE to high intensity intermittent training, like that which is experienced during soccer, may have a positive impact on physical performance during soccer matchplay; with players at a higher level demonstrating an enhanced ventilatory efficiency (VEVO2) for a given exercise intensity  and therefore increased levels of physical performance during soccer match-play.
There were some limitations associated with this study. Firstly, correlation analyses do not infer causality. The ‘thirdvariable problem’ must be considered, as other measured or un-measured variables may affect the results. In an attempt to account for this, partial correlations were employed to control for the effects of maturation. Furthermore, the techniques used to assess maturity status within this study should also be considered. Indeed, the Tanner stage method of estimating maturity status does not provide information on the point at which a player entered a particular stage of maturation or how long an individual has been in a particular stage of maturation. Finally, relative thresholds were used to assess players’ physical performance during soccer match-play. While this partly accounts for differences in growth and maturation, future research may look to examine the impact of such relationships when utilising both relative and absolute speed thresholds within the GPS analysis, when evaluating physical performance during soccer match-play. Such research will allow practitioners and researchers to establish the most appropriate methods for quantifying youth players’ physical performance in both competition and training.
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Diagnostic Value of High Resolution Neckultrasongraghy, Fine Needle Aspiration Cytology and BRAFV600E Mutation in Diagnosis of Malignant Thyroid Nodules- Juniper Publishers
Patients and methodology: 50 patients from Kasr alainy endocrine outpatient clinic with solitary or multiple thyroid nodules are included, All patients subjected to full history and examination, thyroid profile, high resolution neck High resolution neck ultrasound (U/S), Fine needle Aspiration Cytology (FNAC), u/S guided FNAC and BRAFV600E analysis on FNAC using DNA sequencing then high resolution melt curve analysis (HRMA) for confirmation.
Results: The incidence of BRAFV600E mutation among papillary thyroid cancer (PTC) patients was 55.6%. it’s sensitivity 42.9%, specificity was 100% in diagnosing PTC. Sensitivity of high resolution neck ultrasonograghy in detecting malignancy was 88.2%, specificity 72.7%. Addition of ultrasonography to BRAFV600E analysis increased ultrasound sensitivity in detecting PTC preoperative to 92%. There was a positive correlation between most suspicious ultrasonography findings and presence of BRAFV600E mutation (increased AP/T diameter, Increase intra-nodular blood flow, cervical lymphadenopathy, absent or incomplete halo (all p value<0.001), irregular border p value 0.004, micro calcifications p value 0.007.
Conclusion: Adding BRAF-V600E analysis to work up of suspicious thyroid nodule would increase the sensitivity of preoperative diagnosis of PTC especially in cases of indefinite pathological findings. HRMA is simple, low cost tool for BRAF-V600E analysis in comparison to DNA sequencing method.
Keywords:Thyroid nodule; Thyroid cancer; High resolution neck ultrasound (U/S); Fine needle aspiration cytology (FNAC); BRAFV600E mutation
Abbreviation:(U/S): High Resolution Neck Ultrasound; FNAC: Fine Needle Aspiration Cytology, PTC: Papillary Thyroid Cancer; HRMA: High Resolution Melt Curve Analysis
High resolution u/S is the most sensitive diagnostic imagingtechnique for the detection of the thyroid nodules , it cancharacterize suspicious nodules by some sonographic , but itdepends on the skills and accuracy of the operator .
FNAC is the most important step in the work up of thethyroid nodule; Diagnostic accuracy has improved using USneedle localization due to a decreased number of inadequatespecimens and false negative results . The pathology reportfrom FNAB according to Bethesda may be read as non-diagnostic,benign, indeterminate lesion (atypia or follicular lesion ofundetermined significance), Follicular neoplasm or suspiciousfor follicular neoplasm, Suspicious for malignancy and malignant[8,9]. Benign lesions on FNAB have an approximate 3% risk ofmalignancy (vary with patient population), and may be followedclinically with ultrasound or with a repeat FNAB , which, ifalso benign, decreases the risk of a false negative to 1.3% ,the only malignant pathology reliably diagnosed through FNABis papillary thyroid carcinoma, as features such as ‘OrphanAnnie’ nuclei, nuclear grooves, intra-nuclear inclusions, andpsammoma bodies can be sufficient for a diagnosis . Benignand malignant follicular neoplasms and oncocytic (Hurthle cell)adenomas and carcinomas cannot be distinguished on the basisof cytology alone, as tissue architecture is required to make thediagnosis of malignancy through observation of capsular orangio-lymphatic invasion .
Indeterminate cytology findings vary from 15% to 25%. Indeterminate FNAC results and cyto-diagnostic errors areunavoidable due to overlapping cytological features particularlyamong hyperplastic adenomatous nodules, follicular neoplasmsand follicular variants of papillary carcinoma . Thecorrelation of u/S features with FNAB results helps to overcomethe limitations of FNAB alone  even when cytological resultswere the same, the malignancy rate was higher when noduleshad suspicious u/S findings , So, u/S features and FNABresults are complementary to each other .
From that comes the importance of a new diagnostic toolto solve the problem, to overcome the limitations of FNA and toavoid unnecessary surgery. Several molecular factors have beenadded to improve the diagnostic accuracy of u/S-guided FNAB[2,18] one of the promising genetic factors is the analysis of theV600E mutation [19,20].
- Full clinical history including age, sex, past historyof head and neck irradiation and positive family history ofthyroid cancer.
- Thyroid examination.
- Thyroid profile (FT3, FT4, and TSH).
- High resolution neck ultrasound.
- u/S guided FNAC.
- BRAFV600E analysis.
- Hypo-echogenicity. Micro- or interrupted rimcalcifications, Irregular Margins.
- Absence of Halo or incomplete halo.
- Increased Intra-nodular flow.
- Increase antro- posterior to transverse diameter(AP/T) diameter.
- Significant increase in size over time.
- Invasion of anterior strap muscles
- Presence of abnormal cervical lymphadenopathy.
DNA extraction: This was performed using ThermoScientific Gene JET Whole Blood Genomic DNA Purification MiniKit supplied by Thermo Scientific Fermentas. The extractedDNA then subjected to the algorithm for molecular detectionof BRAFV600E mutation. The aim of the current study is thedetection of the most common mutation in the BRAF gene.V600E in DNA samples collected from thyroid nodules.
We used DNA sequencing method for BRAF V600E mutationdetection, unfortunately all samples were BRAF V600E mutationnegative, so we tried more accurate method (HRMA), then DNAsequencing for validation of the mutant BRAF results had done.
Detection of PCR amplification products: Fluorescentstained DNA was detected by using Agarose Gel Electrophoresisand Ultra-Violet Light Trans-illumination.
Purification of PCR product: This was performed usingQIAquick® PCR Purification Kit supplied by QIAGEN® (Table 2).
Sequencing coding sequence and intronic boundaries ofExon 2 and Exon 3 in INS gene Cycle Sequencing: Using theApplied Biosystems* (ABI) PRISM® BigDye® Cycle SequencingReady Reaction Kit.
Second purification: Removal of excess Dye DeoxyTMterminators from sequencing reactions by using Centri-Sepcolumns (Princeton separations) Terminator v3.1 CompletedDNA long Read capillary electrophoresis on the ABI 3500.
www.ncbi.nlm.nih.gov) and the CLCBIOsequence viewer 6 program (www.clcbio.com ).
High resolution melt curve analysis: The HRMA reactionwas done according to the protocol of the kit MeltDoctor TMHRM Master Mix Applied Biosystems, USA.
DNA sequencing for validation of the BRAF mutantresults: The samples with mutant results were processed afterHRM analysis by pipetting the reaction from the reaction tubesfollowed by purification of the products.
Statistical analysis: Pre-coded data was entered on thecomputer using “Microsoft Office Excel Software” program(2010) for windows. Data was then transferred to the StatisticalPackage of Social Science Software program, version 21 (SPSS) tobe statistically analyzed.
Data was summarized using range, mean, standard deviationand median for quantitative variables and frequency andpercentage for qualitative ones. Comparison between groups wasperformed using independent sample t-test or Mann Whitneytest for quantitative variables and Chi square or Fisher’s exacttest for qualitative ones. P values less than 0.05 were consideredstatistically significant, and less than 0.01 were consideredhighly significant.
50 nodules were included but unfortunately because ofrepetition of genetic analysis for BRAF V600E gene mutations byDNA sequencing then again by HRMA methods that leads to that3 samples were not enough to complete genetic analysis so BRAFV600E analysis completed only in 47 samples.
We found a positive correlation with significant p valuebetween some suspicious sonographic criteria and malignancyand some suspicious sonographic criteria and BRAF V600Emutation as increased (AP/T) diameter, absent or interruptedhalo, micro-calcifications, increase intra-nodular blood flow,presence of suspicious LNs and irregular border with increasedboth sensitivity and specificity (Table 4 & 5).
Six malignant nodules were found to harbor BRAF V600Egene mutation, five of them were papillary thyroid carcinomaand one was follicular neoplasm by FNAC with P value 0.001 andnothing was found in benign nodules so there was significantcorrelation between BRAF V600E gene mutation and papillarythyroid cancer. Overall, sensitivity of ultrasound in detectingmalignancy was found to be 88.2%, specificity 72.7%, Sensitivityof US and BRAF V600E mutation combined was 92% (Table 6).
The incidence of BRAF-V600E gene mutation in our patientswith PTC was 55.6%; previous meta-analyses have publishedvalues for the overall prevalence of the BRAF-V600E mutationin PTC ranging from 29 to 83%. [10,22,23], this wide rangemay be due to variations in PTC subtype, subjects’ geographicalbackgrounds, and research methodology.
No difference in BRAFV600E gene mutation incidencebetween different age groups and this shows agreement withAh Young Park et al. , but regarding our result of absenceof significant association of BRAFV600E gene mutation withspecial sex that shows disagreement with Ah Young Park et al.study that shows increase mutation in male sex, this may be dueto the small number of cases involved in our study, also 90% ofour cases are females that cannot reflect the actual prevalence.
Our study contained five nodules (10%) with undeterminedcytology, four of them have suspicious US features and all werenegative regarding BRAF-V600E gene mutation, we can’t excludemalignancy in these cases by absence of BRAF-V600E genemutation alone as BRAF-V600E mutation is found only in nearly40% of PTC cases .
The presence of BRAF-V600E mutation in one case offollicular neoplasm out of five cases shows agreement with onlyone case report Pennelli et al. , this conflicting result maybe due to inadequate cytological diagnosis and interpretationas follicular variant of papillary cancer may be interpreted as amicro follicular lesion in FNA , so this case need follow upand pathological confirmation post thyroidectomy to make sureof this result.
In this present study BRAF-V600E mutation was negativein benign cases, this result shows agreement with other studies[10,11] and BRAF-V600E mutation is believed to be specific forPTC because no report has been issued of this mutation in benignnodules .
The sensitivity of BRAF-V600E gene mutation in detectingPTC was 42.9%, specificity was 100%, positive predictive value100%, and accuracy 83%, this result shows agreement with Kimet al that shows BRAF-V600E mutation alone has a specificityof 100% and a positive predictive value of 100% for diagnosingmalignancy in PTC [9,29].
Nam et al.  reported significant improvements in thesensitivity, negative predictive value, and diagnostic accuracy ofFNA cytology for diagnosing malignancy by adding BRAF-V600Eanalysis to both US and FNAC .
Several studies have found a positive correlation betweenthe BRAF-V600E mutation and suspicious sonographic featuresof thyroid nodules , other studies show inconsistent results;Kwak, et al. found that only marked hypo echogenicity wasassociated with BRAF-V600E positivity, Hwang et al. found thatonly calcification was associated with BRAF-V600E positivity[29,31] both these studies predominantly evaluated smallpapillary thyroid micro carcinomas, with mean tumor sizesof 6-9mm3, the small size was likely a significant confoundingfactor in characterizing these lesions by preoperative US.
Our study shows positive correlation between mostsuspicious US findings and BRAF-V600E gene mutation andshows that AP/T≥1, absent or incomplete halo, irregular border,micro-calcifications and Increase intra-nodular blood flow havepositive correlation with positivity of BRAF-V600E mutationthese results show agreement with Kabaker et al.  thatshowed association of BRAF-V600E positivity with most knownsuspicious u/S finding including taller than wide shape, illdefinedmargin, hypo-echogenicity, micro-calcifications andabsent halo (p value<0.001) .
This study also showed that BRAF-V600E gene mutation issignificantly present in cases with cervical lymphadenopathythis result shows agreement with many studies that correlateBRAFV600E gene mutation with the frequency of lymph nodemetastases [10,22] Park et al.  study concluded that theBRAF-V600E mutation was associated extra-thyroidal extension,central and lateral lymph node metastasis, and advanced tumorstage (P<0.0001, so BRAFV600E analysis preoperative may be ofvalue as prognostic marker and affect surgical decision regardingtype of surgery.
DNA sequencing method shows high cost, time consumingand low sensitivity for the clinical screening of BRAF V600Emutation and this shows agreement with other study  thatshows accuracy of DNA sequencing is low when dealing withcytological specimens where the cells in question may be aminor population among the vast majority of background nonneoplasticcells, Furthermore, this method needs expensiveequipment which may not be economical for all the patients, In contrast HRMA has been proved to be applicable, costefficientand very sensitive scanning method that allows rapiddetection of DNA sequence variations without cumbersomepost–polymerase chain reaction (PCR) methods, which is notachievable by direct sequencing . Because targeted therapyfor thyroid cancers with multikinase inhibitors is under activedevelopment [36,37], the detection of mutations in the FNACmaterial may be helpful in the future to guide mutation-specifictargeted therapies that can be initiated preoperatively or inthose patients who are not surgical candidates. This study hassome limitations as small number of patients included, somegenetic mutations should be included as RAS and RET and RET/PTC rearrangement.
u/S finding, BRAFV600E gene mutation analysisis a new hopeful diagnostic and prognostic tool that helps toovercome limitations of high resolution neck ultrasonographyand fine needle aspiration biopsy, Adding BRAF-V600E analysisto u/S and FNA would increase the sensitivity of preoperativediagnosis of PTC especially in cases of indefinite nodules. HRMAis an excellent, simple, low cost tool for BRAF-V600E analysis incomparison to DNA sequencing method.
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