The criteria for stopping Delphi studies are often subjective. This study aimed to examine whether consensus and stability in the Delphi process can be ascertained by descriptive evaluation of trends in participants' views.A three round email-based Delphi required participants (n = 12) to verify their level of agreement with 8 statements, write comments on each if they considered it necessary and rank the statements for importance. Each statement was analysed quantitatively by the percentage of agreement ratings, importance rankings and the amount of comments made for each statement, and qualitatively using thematic analysis. Importance rankings between rounds were compared by calculating Kappa values to observe trends in how the process impacts on subject's views.Evolution of consensus was shown by increase in agreement percentages, convergence of range with standard deviations of importance ratings, and a decrease in the number of comments made. Stability was demonstrated by a trend of increasing Kappa values.Following the original use of Delphi in social sciences, Delphi is suggested to be an effective way to gain and measure group consensus in healthcare. However, the proposed analytical process should be followed to ensure maximum validity of results in Delphi methodology for improved evidence of consensual decision-making. The Delphi technique is suggested to be an effective way to gain and measure group consensus in healthcare . Delphi Although originally used as a methodological tool in social sciences, recently there has been a rise in the use of Delphi in healthcare research -9. DelphDelphi studies have been used to develop and identify consensus by experts on a given topic. As interest has grown in the analysis of the data produced by this method, authors have attempted to clarify the relevant concepts.It has been suggested that consensus is the same as agreement and that agreement can be determined by:1) the aggregate of judgements  ,2) a move to a subjective level of central tendency ,3) or alternatively, by confirming stability, which is "the consistency of answers between successive rounds of the study ."The first of these occurs within each Delphi round. The second and third occur between rounds. Researchers have been inconsistent in their use of these concepts. In addition some researchers support the use of pre-determined levels of consensus to reduce research bias  whilst oet al.[2) to test for stability. However, this cannot be considered to test stability in Delphi studies as it will determine "the independence of the rounds from responses found in them" [2 to determine individual stability prior to group stability was developed from the work of Dajani et al., and therefore this should also be viewed with caution. Since these works few examples of χ2 as a stability measure appear in Delphi literature. More recently Greatorex & Dexter used means and standard deviations (SD) for comparing movement between Delphi rounds as a measure of both stability and convergence [Scheibe et al. suggest et al.. Dajani et al. propose in them"  not the in them"  using χ2vergence .In 1998 the NHS Health Technology Assessment group produced a detailed report on the requirements of effective consensus development methods [In order to reduce the subjectivity in stopping criteria used in Delphi studies, this study aimed to examine whether consensus and stability in the Delphi process can be ascertained by descriptive evaluation of trends in participants' views. We evaluated the evolution of consensus and stability by examining agreement percentages, importance rankings (based on simple descriptive statistics) and Kappa values. These were used to explore how quantitative results could inform Delphi users, firstly on the production of central tendency/consensus, and secondly on stability, thus reducing subjectivity in reporting Delphi results. The use of simple descriptive statistics, as used in previous work , makes tAn email-based Delphi study was undertaken and comparative statistical testing applied. Ethical approval was obtained from University of Teesside and informed consent obtained from each participant. A convenience heterogeneous sample n = 12) of volunteer nursing, occupational therapy and physiotherapy students. A three round email-based Delphi study was undertaken following a pilot, which checked whether the statements were clear, unambiguous and non-leading. Statements were taken directly from the paper by McCallin  which re2 of voluBetween rounds views were analysed using Colazzi's 7-stage thematic analysis, modified from Holloway & Wheeler . Quantit1. Percentage response rates,2. Percentages for each level of agreement for each statement to compensate for varying response rates,3. Median, range and their associated group rankings using the importance ratings,4. Mean (SD) and their associated group rankings using the importance ratings,5. Weighted Kappa (K) values to compare chance-eliminated agreement between rounds.For the following round, statements were rephrased if appropriate with the aim of moving towards consensus, based on the level of agreement and the majority theme highlighted. Successive rounds consisted of statements (some rephrased) percentage agreement levels, anonymous feedback from the previous round to show the range of views received, and the importance rankings using the median rank values. Participants were requested to read the feedback before responding again to statements.Scaling methods of agreement and importance were adapted from Sim & Wright  and haveet al. [Weighted Kappa (K) statistics were calculated for the within-subject level of agreement in their importance rankings between two rounds, not the level of agreement between participants. K values show a chance-corrected proportional agreement . A weighet al.  and inteet al. , Table 3et al.  for a fuet al.  stated aThe results section summarises the Delphi in terms of how consensus and stability evolved through rounds 1 to 3 by looking at the: -• Agreement percentages,• Importance rankings,• Statement evolution,• Theme production,• Kappa values.Agreement values were affected in the second round by a reduced response of 83% (10 of 12).The main themes highlighted remained constant throughout the three rounds but decreased in duplication over the rounds. The range interval and SD decreased to low levels (difference of 2 and 0.60 respectively). By the end this statement had the lowest range and SD, suggesting greatest stability and consensus, also it had a low number of comments (3). When the median and mean were equal the range, 1–3, and SD, 0.60 were low. Importance ranking was always high (second place).There was a variety of themes generated (6) in round 1, suggesting divergence, changing to 3 themes in round 2, then 2 in round 3, suggesting convergence.For statement 2 there was an increase in agreement between round 1 and 2, then divergence rather than convergence occurred between rounds 2 and 3. Based on the SD there was less consensus for this statement in round 3.New themes were generated in round three, also suggestive of instability. Interestingly, the two non-responders from round 2 disagreed with the third round statement affecting the percentage of agreement. This was 1 of only 2 statements showing disagreement in round 3 and showing an increase in disagreement between any rounds. Mean and median ranks both show this statement became less important to the participants, yet there was always a 2-interval difference between these ranks.Four themes were highlighted in round one. Despite the change in agreement between rounds two and three only one comment was given in round three, the least number of comments for any statement throughout the Delphi. An increase in those strongly agreeing (from 8% to 80%) was observed for round 2, as shown in Table New themes were generated in round 3 suggesting stability had not yet occurred.For this statement agreement percentages and ranges did not vary appreciably still showing disagreement in round 3, suggesting stability. Although only changing by 0.06 between rounds 2 and 3, the SD suggests there is still convergence. The statement became less important, ranked 3 to 5.Round 1 produced 5 themes. In round 3 all responses reflected different themes, previous themes and new themes suggesting the views of participants had not been exhausted. Agreement increased over the 3 rounds, inversely to the ranking of importance. Both the median and the mean define a move towards lesser importance with the progression of rounds. The range intervals of 6, 5 and 5, over the rounds suggest consensus was weak yet stability had occurred but the SD was still lowering suggesting continued convergence.In round 1 there were 2 themes, which were reflected again in rounds 2 and 3, although the amount of participants responding decreased to 3 in both round 2 and round 3. This statement was consistently high on agreement and importance always ranked first. However, range interval of 4, and SD of 1.17, were not the lowest observed in round 3, suggesting the possibility of further convergence.Only 4 comments were obtained in round 1 all reflecting agreement. Round 2 instigated only three comments, still reflecting agreement. This was the only statement for which the number of comments increased in round 3 these included new themes, suggesting instability. However, agreement increased for this statement. Both mean and median values showed the importance ranking lower but the range and SD both showed a move towards consensus.In general, K-values increased between rounds when rounds progressed, and when adjacent rounds were compared as opposed to round 1 Vs round 3. Statement 7, round 1 Vs round 2 showed the least agreement, K = 0.313, yet was consistently ranked the most important. This apparent anomaly is discussed below. Statement 4 had the second lowest SD by round 3 yet never achieved a K > 0.5. Statement 1, 3 and 7 got the 3 highest Kappa values, comparing round 2 and 3.The Delphi results show a change in participants' views towards consensus and stability as indicated by a trend towards:• an increase in percentage agreements• convergence of importance rankings• increase in Kappa values• a decrease in comments as rounds progressedAn increase was observed in percentage agreements for all statements over the 3 rounds with only 2 statements Tables  and 8 shet al.[et al.[Oscillatory movements at individual and group level were consistent with those described by Scheibe et al.. A devial.[et al..The median and mean values for importance show the group aggregate rank, whereas ranges and SD show the spread, i.e. disagreement  of the gWhile a decrease in range generally reflected a decrease in SD, there was no direct relationship between them. For example, between rounds 2 and 3, in statement 6 the SDs decreased from 1.65 and 1.56 but the ranges stayed constant, at 3–8. This highlights the different information each provides, SDs give an indication of the aggregate judgement where as ranges summarise the outliers views. Also between statements, equal ranges were represented by different SDs. For example statements 2 and 3 had a range of 1–8 in round 1, but standard deviations of 2.50 and 2.27, respectively. These findings expand on Greatorex & Dexter's conclusion  that eac• there may be increased potential for bias as researchers will need to make individual judgements on acceptable convergence levels for each statement,• Alternatively, preconceived levels of convergence to determine consensus, as suggested by Williams & Webb , would bWhen interpreting the mean and median importance rankings, some between-test validity can be demonstrated. For example, when the mean and median were equal the lowest ranges and SDs were observed Tables  and 7. Abetween rounds in Kappa rather than values within a particular round. Also Kappa-crosstabs plot individuals' rank from one round against their rank in the next round, therefore K-values measure the value of agreement for individuals between two rounds, not agreement between different participants within a round. It was interesting to observe the apparent contradiction, between the results for Kappa and percentage agreement for statement 7. This may have been due to the small range of answers as compared with other statements, because Kappa is affected by range. This highlights the need to use a range of descriptive statistics in Delphi analysis.As expected, the greatest difference between K-values was between round 1 and 3 Table , with K-Generally the number of comments decreased in each round. However, statement 8 Table  did not New themes were generated in round 3 for the 4 statements of least importance Tables  and 11, Statements 1 and 4 which received fewer comments in round 3 had the lowest SDs. Because quantitative analysis aggregates participants' judgements, outlying individual judgements are not represented. However, the reduction in comments does support the lowering of range intervals and SD, substantiating the evolution of consensus and validating quantitative data with qualitative data.The results presented here demonstrate two points that can be used to improve Delphi studies in future. Firstly, the results have demonstrated that the mean and SD, when combined with the range and medians, can be used to show whether convergence has occurred, by a movement towards central tendency. This is in agreement with the findings of Greatorex and Dexter . The amoThere are limitations to this study. This study used a small sample size therefore the results should be treated with some caution, and followed up by a larger study. However this offered the greatest opportunity for analysing all qualitative data, because an increased sample size would have led to saturation of data, and it is not uncommon for Delphi studies to use this type of sample size.Using standard descriptive statistics and Kappa calculations in conjunction with thematic analysis and the number of comments generated, it was possible to demonstrate movement towards consensus and stability in this Delphi study. Following the original use of Delphi in social science, Delphi is suggested to be an effective way to gain and measure group consensus in healthcare . There iThe author(s) declare that they have no competing interests.JLF and EAH designed the study. JLF collected the data. All authors participated in the analysis and interpretation of the data, and the drafting, progress and revision of the manuscript.The pre-publication history for this paper can be accessed here:


Treatment of unstable thoracolumbar fractures is controversial regarding short or long segment pedicle screw fixation. Although long level fixation is better, it can decrease one motion segment distally, thus increasing load to lower discs.We retrospectively analyzed 31 unstable thoracolumbar fractures with partial or intact neurology. All patients were operated with posterior approach using pedicle screws fixed two levels above and one level below the fracture vertebra. No laminectomy, discectomy or decompression procedure was done. Posterior fusion was achieved in all. Post operative and at final follow-up radiological evaluation was done by measuring the correction and maintenance of kyphotic angle at thoracolumbar junction. Complications were also reported including implant failure.Average follow-up was 34 months. All patients had full recovery at final follow-up. Average kyphosis was improved from 26.7° to 4.1° postoperatively and to 6.3° at final follow-up. And mean pain scale was improved from 7.5 to 3.9 postoperatively and to 1.6 at final follow-up, All patients resumed their activity within six months. Only 4 (12%) complications were noted including only one hardware failure.Two levels above and one level below pedicle screw fixation in unstable thoracolumbar burst fracture is useful to prevent progressive kyphosis and preserves one motion segment distally. The thoracolumbar junction is the most common site of spinal injuries. The surgical treatment of unstable fractures and fracture dislocations of thoracolumbar spine remains controversial . The goaHere we present our results in unstable thoracolumbar fracture patients who were treated with pedicle screw fixation two level above and one level below the fracture vertebra, in order to preserve motion segment below the level of fracture. The purpose was to study the effectiveness of pedicle screw fixation, two levels above and one level below the fractured vertebra, in order to prevent postoperative kyphosis and high implant failure rate.We retrospectively reviewed the results of unstable thoracolumbar fractures with partial or intact neurology in consecutive 31 patients who were operated between June 2004 and June 2006 at our institute by a single spine surgeon  performed all surgeries. At surgery, the patients were placed in a hyperextended prone position with the abdomen hanging free, thus preventing excessive intraoperative bleeding and achieving a significant initial reduction of the spinal fracture. All patients were operated with single stage posterior surgery using pedicle screw instrumentation, two levels above and one level below the fractured vertebra Figs.  and 2. AAll patients were managed with immobilization in a custom-molded thoracolumbosacral brace for three months postoperatively. All patients with age more than 40 years were investigated in form of DEXA  to evaluate BMD  as a routine protocol in our clinic for the treatment of osteoporosis. The patients were followed with serial physical examinations and radiographs. With the use of lateral radiographs centered over the fracture level, kyphosis or lordosis was measured from the superior end-plate of the intact vertebra cephalad to the fracture to the inferior end-plate of the vertebra caudad to the fracture. Progression was considered to be absent, minor, or major. Minor progression was defined as kyphosis measuring 5 to 10° more at the time of follow-up than it had on the immediate postoperative radiographs. Major progression was defined as an increase of 10° or more compared with the measurement on the initial postoperative radiographs. Denis pain score were also compared statistically for each patient, immediate postoperatively and at the final follow-up, with preoperative pain score.Average follow-up was 34 ± 8.2 months . Mean injury surgical interval was 3.5 days ranging from minimum 6 hours to maximum 14 days. Table The average pre-operative kyphotic angle was 26.7° which improved to 4.1° in the immediate post-operative period suggesting 84.6% correction  . Two patients (patient 12 and 19) had screw loosening. One was 63-years-old male and other was 59-years-old female. They were osteoporotic patients which were confirmed with bone densitometry. Therefore screw loosening in them was thought to be due to osteoporosis. None of them had any deterioration in their functional activity. One 40 years male (patient 18) had breakage of right L1 screw; however there was no movement noted on flexion-extension radiogram and patient was symptomless. No treatment was done for the hardware failure in that patient. Finally, one 42-years-old male (patient 35) developed superficial wound infection which was treated with repeated dressings and parenteral antibiotics. His further follow-up was uneventful. All patients were able to return to their previous activity within six months of surgery and none of the patient had deterioration in neurology on regular follow-up. At final follow-up there was no instability detected on the flexion-extesion radiogram in all patients.Posterior transpedicular screw fixation initially was reported by Boucher in 1959 . Since tAlthough SS pedicle instrumentation has been considered as a superior method, ,23 severAs all of the patients in our study had partial or intact neurology at the time of presentation, decision to operate was taken if they had kyphosis angle more than 20-degree and/or anterior vertebral body height more than 50%; which suggested unstable fractures. Average preoperative kyphosis angle was 26.7° preoperatively which improved to 4.1° immediate postoperatively and maintained at 6.3° at final follow-up. Our results suggested a success similar to LSPF construct reported in the literature. And we agree that fixing the fracture two levels above prevents progressive kyphosis development. Additionally evaluation of pain scale for all the patients suggested that our fixation strategy had been successful in improving the pain scales at final follow-up and all the patients were able to go back to their previous level of activity after the operation.Butt et al  recentlyAs our patients had partial neurological injury or intact neurology, we could preserve the lamina and other posterior structures in all patients. We feel that further study comparing the effectiveness of this construct in patients with laminectomy would be helpful to confirm the success of this technique. We think this could be the only weak factor in our study and we recommend further study in laminectomized patients in future. Additionally, we could not evaluate the union in all patients because all subjects did not undergo for CT scan at final follow-up. However, there was no instability noted on flexion-extension radiogram at final follow-up.Our study reported prevention of progressive kyphosis development with the use of two levels above and one level below pedicel screw construct in unstable thoracolumbar burst fractures in patients with partial or intact neurology. Additionally it is helpful to preserve one motion segment distally at lumbar level and improving the pain scale at final follow-up.The authors declare that they have no competing interests. Each author certifies that he has no commercial associations  that might pose a conflict of interest in connection with the submitted article.HNM has contributed in conception and design of data, analysis and interpretation of data, drafting the manuscript and revising it critically, KJC has contributed in conception and design of data, drafting the manuscript and given the final approval of manuscript, IWS has contributed in drafting the manuscript and data analysis, HSY has contributed acquisition of data and revising it critically, JHH has contributed in acquisition of data, revising the manuscript critically and given the final approval, HKK has contributed in drafting the manuscript and designing of data and revising it critically, KCN has contributed in acquisition of data and analysis and interpretation of data, and JHY has contributed in acquisition and analysis of data. All authors read and approved the final manuscript.


Risk of complex disorders is thought to be multifactorial, involving interactions between risk factors. However, many genetic studies assess association between disease status and markers one single-nucleotide polymorphism (SNP) at a time, due to the high-dimensional nature of the search space of all possible interactions. Three ensemble methods have been recently proposed for use in high-dimensional data . An intuitive way to detect an association between genetic markers and disease status is to use variable importance measures, even though the stability of these measures in the context of a whole-genome association study is unknown. For the simulated data of Problem 3 in the Genetic Analysis Workshop 15 (GAW15), we examined the variability of both rankings and magnitude of variable importance measures using 10 variables simulated to participate in gene × gene and gene × environment interactions. We conducted 500 analyses per method on one randomly selected replicate, tallying the rankings and importance measures for each of the 10 variables of interest. When the simulated effect size was strong, all three methods showed stable rankings and estimates of variable importance. However, under conditions more commonly expected to be encountered in complex diseases, random forests and generalized boosted regression showed more stable estimates of variable importance and variable rankings. Individuals endeavoring to apply statistical learning methods to detect interaction in complex disease studies should perform repeated analyses in order to assure variable importance measures and rankings do not vary greatly, even for statistical learning algorithms that are thought to be stable. The use of statistical learning methods to detect interactions between genetic and environmental risk factors is fuelled by the necessity of using methods developed for use with high-dimensional data ), in which explicitly considering all possible two-way, three-way, or higher-order interactions is computationally not feasible. Ensemble methods, or methods that consider multiple models such as classification trees, may be a more efficient way to detect interactions in a high-dimensional space. A simple and intuitive approach for selection of 'interesting' variables that may be involved in interaction is to use the associated importance measure for each variable to rank them  and to LogicReg package) [randomForest) [gbm package) [gbm) with decision stumps (decision trees with a single split) as the base classifier. Boosting algorithms can be seen as gradient-descent algorithms that seek to find a weak classifier that most reduces the error or loss function [The statistical methods assessed in this study included Monte Carlo logic regression  -6, randomForest) -9, and gpackage) -12. Logipackage) . The ranfunction . The conBecause the focus of this study was to examine the stability of variable importance rankings and scores for three ensemble methods on variables involved in simulated interactions, we requested the generating model for the simulated data before beginning analysis. After reviewing the generating model, we included data from all chromosomes containing SNPs simulated with gene × gene or gene × environment interaction: chromosome 6 (674 SNPs), chromosome 8 (442 SNPs), chromosome 16 (204 SNPs), plus lifetime smoking status and sex. On chromosomes 16 and 8 no linkage disequilibrium (LD) was simulated between the disease loci and observed SNPs, so the simulated disease loci were included in our data for analysis. Gene × gene interaction was simulated between the DRB1 locus on chromosome 6 and locus A on chromosome 16; the minor allele at locus A (frequency = 0.30) acted in a dominant fashion to increase risk with the DRB1*4 allele . An interaction between locus C on chromosome 6 and sex was simulated with the C allele (frequency = 0.50), acting log-additively to increase risk of RA in females. Women with one copy of the C allele were modeled to have 2.1-fold increased risk versus women with the cc genotype; women with two copies of the C allele showed 4.41-fold increased risk. Locus B on chromosome 8 was modeled to interact with lifelong smoking status in a dominant fashion; smokers with at least one copy of the B allele  had 1.5-fold higher risk of RA versus smokers with the bb genotype. To approximate a case-control study we used control data paired with a single randomly selected affected offspring from each family. One hundred replicates were created by the workshop organizers, no data sets had missing values, and no genotyping error or misclassification of sex and smoking status was assumed. All genotypes were recoded into a pair of binary variables: one as two-allele dominant and the other as two-allele recessive. Smoking status and gender were retained as binary predictors.r2 = 0.50) and because the generating model for simulation for the DRB1 disease locus was tri-allelic, we retained both SNPs as variables of interest. To assess the variability of ranking and magnitude of importance measures, we randomly selected a single replicate (number 19) and repeated our analyses using the three ensemble methods 500 times, tallying the ranking and influence measures of each of our 10 variables of interest. GBM tallies rankings only for variables with a non-zero importance score, so any variable with an importance score of 0 does not have a corresponding rank.The variables of interest  included sex, smoking status, and the disease loci on chromosomes 8 and 16. Because of the strong LD between SNPs 153 and 154 on chromosome 6 , all three methods place these SNPs near the top of the importance rankings Table . In factImportance scores (as measured by the interquartile range of importance scores for each method) were nearly identical for all variables with non-zero importance scores using GBM, with the largest difference between the first quartile and third quartile score being 0.2 Table . MCLR shOverall, the variability of importance rankings and scores using variables involved in modeled interactions was fairly consistent for all methods evaluated for the chromosome 6-sex interaction, which had a much larger effect size than the other two interactions (chromosome 8 × smoking and chromosome 6 × chromosome 16). With regards to the smaller effect sizes, GBM and RF seemed less variable in rankings and importance scores than MCLR, and GBM was superior to RF in stability of rankings and importance measures, which is not surprising because boosting is an averaging process across an additive expansion of trees .It should be noted that these results are specific to this simulation model, and might be very different under alternative simulation models. In particular, the very large effect size for the chromosome 6 locus might paint an overly optimistic picture of the stability of variable importance rankings and scores. This implies that the disease locus on chromosome 8  in particular may serve as a good benchmark for the variability of statistical learning rankings and importance scores as applied to complex multifactorial diseases when larger effect sizes are also present. In this particular case, it appears that GBM and RF show more stable estimates of variable rankings and importance measures. However, the comparison between RF/GBM and MCLR may not be completely fair, considering that the Gini index used for RF measures the reduction in impurity and GBM attempts to reduce the loss function across the additive expansion of trees, and MCLR is simply tallying variables included in the model across the Markov chain.Especially in situations in which the expected effect size is modest, researchers who use statistical learning methods to detect SNP × SNP (or gene × gene) interaction in complex disease studies should perform repeated analyses to determine the variability of importance measures and rankings, even for statistical learning algorithms that are thought to be stable.The author(s) declare that they have no competing interests.


The method introduced by Knox for evaluation of space-time clustering has been applied to 872 cases of childhood (0-14 year old) leukaemia diagnosed in Greece over the 10 year period 1980-89. Greek towns are characterised by substantial population mixing due to internal migration, whereas there is relative isolation in mountainous rural areas. Predetermined space (5 km) and time (1 year) limits were used on the basis of previous reports in order to define the clustering cell. There is highly significant evidence for clustering of childhood leukaemia in Greece as a whole, the observed number of pairs that are close in both spaces and time exceeding the expected number by 5.2% (P = 0.004). The excess is particularly evident for leukaemia cases in 0 to 4-year-old children, among whom the observed number of pairs that are close in both space and time exceeded the expected number by 9.4% (P = 0.004). There is no evidence of space-time clustering for leukaemia cases older than 5 years. The overall pattern is descriptively similar in urban and semiurban areas and is especially marked for acute lymphoblastic leukaemia at the childhood peak ages (2-4 years) with an excess of 19% (P = 0.0006). In the rural population there is evidence for clustering of cases belonging to older and broader age groups, a phenomenon compatible with a delay in the development of herd immunity against putative infectious aetiological agents. The findings of the present study provide support for the hypothesis that a substantial proportion of cases of childhood leukaemia may arise as a rare sequel to exposure to an agent or agents, most probably viral in nature.


Histopathology57, 27–38 Image analysis as an adjunct to manual HER-2 immunohistochemical review: a diagnostic tool to standardize interpretationDobson L, Conway C, Hanley A, Johnson A, Costello S, O’Grady A, Connolly Y, Magee H, O’Shea D, Jeffers M & Kay E (2010) Accurate determination of HER-2 status is critical to identify patients for whom trastuzumab treatment will be of benefit. Although the recommended primary method of evaluation is immunohistochemistry, numerous reports of variability in interpretation have raised uncertainty about the reliability of results. Recent guidelines have suggested that image analysis could be an effective tool for achieving consistent interpretation, and this study aimed to assess whether this technology has potential as a diagnostic support tool.in situ hybridization (FISH) testing. An excellent concordance rate of 95% was observed between FISH and the automated review across 136 informative cases.Across a cohort of 275 cases, image analysis could accurately classify HER-2 status, with 91% agreement between computer-aided classification and the pathology review. Assessment of the continuity of membranous immunoreactivity in addition to intensity of reactivity was critical to distinguish between negative and equivocal cases and enabled image analysis to report a lower referral rate of cases for confirmatory fluorescence This study has validated that image analysis can robustly and accurately evaluate HER-2 status in immunohistochemically stained tissue. Based on these findings, image analysis has great potential as a diagnostic support tool for pathologists and biomedical scientists, and may significantly improve the standardization of HER-2 testing by providing a quantitative reference method for interpretation. HER-2 gene or overexpression of its protein product in cell membranes is seen in 10–30% of invasive breast cancer and is associated with increased disease recurrence and poor prognosis.Targeted therapeutics or personalized medicine regimes are driving a new era of integrated diagnostics and therapeutics, particularly in oncology. Many anticancer antibodies have been approved in association with companion tests for biomarker expression to identify the most responsive patients, ensuring that accurate evaluation of biomarker status has become particularly acute in the clinical laboratory. The role that biomarkers can play is exemplified by HER-2: a prognostic, predictive and therapy selection factor for patients with breast cancer. Amplification of the in situ hybridization (FISH).in situ hybridization (CISH) or silver-enhanced in situ hybridization, which do not rely on fluorescent microscopy, represent alternatives to FISH in terms of HER-2 oncogene analysis. However, although these methods have been determined to give comparable results to FISH, they are not yet widely utilized in diagnostic pathology.13The therapeutic relevance of HER-2 status demands highly reliable and robust testing to identify tumours that overexpress this protein.In contrast, qualitative IHC testing is advocated as the primary assay for identifying candidates for trastuzumab because it is readily available, easily performed in most clinical pathology laboratories and has many advantages over FISH or CISH in terms of economics, as well as being highly amenable to automation.20Recently, high rates of discordance between IHC reviewed at high-volume HER-2 reference centres and low-volume regional laboratories has cast doubt on the reliability of results.Thus there is an urgent need to develop more sensitive image analysis tools that may be utilized with any of the prevalent HER-2 antibody kits in clinical pathology. Towards this goal, we have developed a HER-2 image analysis algorithm that may be applied to slides immunohistochemically stained with Dako HercepTest® , Ventana Pathway®  or Leica Oracle™  HER-2 antibodies. In particular our approach employs a novel quantification base, determining the continuity or extent of circumferential membranous immunoreactivity, a parameter overlooked by other algorithms despite being an important factor for differentiating HER-2 classification. It is hypothesized that by considering this parameter, the number of ambiguous cases may be reduced by enabling better separation between negative and equivocal groups. This multi-site study set out to assess whether this HER-2 algorithm could improve the diagnostic accuracy of IHC scoring, with the performance of the algorithm measured against both manual review and FISH evaluation as the accepted standard.A total of 448 consecutive cases were selected for this study from the archives of Beaumont Hospital and Adelaide and Meath Hospital, two HER-2 reference laboratories in Dublin, Ireland, of which 425 were successfully stained, reviewed and digitized. All cases were formalin fixed, paraffin embedded and processed in the routine diagnostic laboratory of the institute of origin according to standardized protocols.n = 144) and Leica Oracle™ HER-2 (n = 140) antibodies according to the manufacturer’s instructions. Those cases supplied from Adelaide and Meath Hospital were analysed for HER-2 protein expression with Ventana Pathway® HER-2 (4b5) (n = 141) according to the manufacturer’s instructions. In all cases, suitable negative and positive control slides were treated in a similar manner to ensure appropriate staining.The cases supplied from Beaumont Hospital were assessed for HER-2 protein expression using Dako HercepTest®  in the facility of origin. All procedures were performed in accordance with the manufacturer’s recommended protocol.A representative cohort of cases was selected for FISH testing for verification purposes. Of the 425 cases supplied, 219 were analysed for Immunohistochemically stained full-face sections were digitized by SlidePath using a NanoZoomer Digital Pathology (NDP) System . The NDP system utilizes charge-coupled device time delay integration technology to achieve scans with a spatial resolution of 0.46 μm/pixel. Scanning time at 20× was approximately 3 min for a 20 × 20 mm biopsy specimen. Images were approximately 55–487 Mb per whole section biopsy specimen and were archived using SlidePath’s Digital Slideserver, a secure, web-enabled digital slide management system.At each site, HER-2 protein expression was reviewed by a Consultant Pathologist. For those cases where FISH analysis was carried out, gene amplification status was reviewed by a Biomedical Scientist. All cases were classified according to the new American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) and UK guideline recommendations for HER-2 testing as detailed in in situ regions excluded from analysis  based on the distribution of staining absorbance and membrane continuity for each category. In addition, a constraint was included that automatically defined any case with <1% immunopositive pixels in selected regions as negative or 0/1+. These computational steps were then incorporated into the algorithm, resulting in an output of a dedicated HER-2 classification, along with a percentage confidence in that score.For 180 of the 275 remaining test cases the manufacturer control cell line material was also available for analysis. Cell lines provide consistency in terms of both the quantity of material and the gradation of protein expression, and when used as part of a validated system have applications in internal quality assurance, providing a standard against which a laboratory can gauge against day-to-day drift in assay sensitivity.Sigma Plot Version 8  and SPSS Version 15 (SPSS Inc.) were used to perform statistical analyses including concordance and Cohen’s κ statistics. The Landis and Koch Kappa interpretation scale was used to evaluate the level of κ agreement.27The sensitivity and specificity of both the automated and manual review were calculated using FISH evaluation as the gold standard, where:The concordance between image analysis evaluation of HER-2 status and manual review by a Consultant Pathologist was blindly assessed on a cohort of 275 cases stained with Dako HercepTest®, Leica Oracle™ and Ventana Pathway®. Statistical analysis established that there was agreement in the classification of 250 of the 275 cases, representing a concordance of 91% between the pathology and image analysis reviews . Kappa wAs expected, analysis of the corresponding cell-line control material determined that those slides stained using automated systems exhibited less variance than those prepared manually. Nonetheless, normalization to compensate for variance had no impact on the classification of HER-2 by image analysis.n = 136) were also analysed by FISH, the ‘gold-standard’ method of HER-2 evaluation. The concordance rate between HER-2 gene amplification and IHC review was determined to be excellent for both image analysis and the pathology review, demonstrating that image analysis can robustly and accurately classify HER-2 status .Receiver–operating characteristic curve analysis was used to compare the accuracy of the manual and image analysis methods with FISH evaluation as the standard . The areet al.,et al. and Wang et al.A number of other image analysis systems are commercially available for use as decision support tools in the clinical setting. Nevertheless, The disparity in the accuracy of the image analysis systems may be attributed to a variety of factors. In the first instance, the HER-2 algorithm reviewed here has the capacity to identify and eliminate artefactual staining which may be observed when automated staining systems are employed or improper use of such equipment occurs . WithoutMoreover, it is evident from HER-2 gene amplification are used to identify patients for whom trastuzumab may be of benefit for treatment of breast cancer in the metastatic or adjuvant disease settings.32High levels of HER-2 protein expression or The last 10 years have seen enormous advances in the capabilities of image analysis systems applied to tissue sections with complex computer algorithms used to interpret the images.The approach detailed in this study has aimed to address the inherent deficiencies in other systems. In the first instance, the algorithm under review measures the continuity of membranous immunoreactivity as well as the intensity of reactivity, and has demonstrated that consideration of both parameters enables accurate distinction of HER-2 status. Furthermore, this HER-2 algorithm has been validated to perform with some of the most prevalent HER-2 antibodies on the market. Although the HER-2 guidelines for testing do not stipulate the use of a particular antibody, the Dako HercepTest® and Ventana Pathway® are recommended as FDA-approved kits, and the Leica Oracle™ HER-2 antibody is also frequently employed in laboratories that have demonstrated concordance with a validated method.et al. using the AQUA platform, or Wang et al. and Tawfik et al. using the ACIS system.HER-2 gene-amplified cases with a false-positive rate of 0%.Our findings demonstrate that image analysis can accurately and robustly classify HER-2 status. A concordance rate of 91% was observed in comparison with manual review by a pathologist, and the significant value of image analysis was exemplified by a 4% reduction in the reporting of equivocal cases. This represents a decrease in the number of cases requiring confirmatory FISH testing and thus a potential cost saving for clinical laboratories. Moreover, the concordance of image analysis with gene amplification status as the standard was observed to be 95%, which represents better correlation and accuracy with FISH than the manual interpretation of IHC. The data from this study are substantially greater than reported by existing systems in FDA approval documentation and independently by Camp et al.Although it is generally accepted that the standard assessment of IHC will remain the manual pathology review, our findings suggest that integration of image analysis into the diagnostic workflow could significantly enhance the reproducibility of scoring, particularly in those laboratories where there is lack of experience in interpreting HER-2 staining. However, aside from providing assistance for interpretation, image analysis could be utilized as an internal resource to qualify the quality of IHC, introducing an unprecedented level of internal laboratory quality assurance. Both the ASCO/CAP and UK guidelines recommend the use of control material, which should be used consistently by each laboratory with each run of tests.Undoubtedly, the recent reports of poor observer variability regarding the evaluation of HER-2 in the clinical setting justify the development of software tools to help standardize interpretation, particularly in equivocal cases. Based on this study, Tissue IA has been validated as a consistent scoring tool with excellent levels of concordance with manual scoring and FISH, advocating the use of Tissue IA as a decision support system for pathologists to assist in the diagnosis of disease. Further independent studies to demonstrate the accuracy of the system have been initiated.SlidePath applications are not cleared by the FDA, Health Canada, or in the EU for diagnostic or clinical use. All applications are intended solely for use in the research or educational setting, such as university or pharmaceutical development. These applications are described as Research Applications or Research Use Only.


An association between the occurrence of calcified discs, visible on radiographic examination (CDVR), and disc extrusions has been suggested in published literature over the past 10-20 years, mainly from Nordic countries. It has also been postulated that dogs without CDVR would not develop disc extrusions. Furthermore, inheritance of CDVR has been calculated and it has been postulated that, by selecting dogs for breeding with few, or no CDVR, the prevalence of disc extrusions in the Dachshund population may be reduced.The prevalence of radiographic detectable intervertebral disc calcifications was calculated from one hundred surgeries for disc extrusion, performed in 95 Dachshunds, in order to determine if the disc causing clinically significant IVDD, had radiographic signs of calcification at the time of confirmed disc extrusion. Inclusion criteria, for each dog, included a complete physical, orthopedic and neurologic examination, radiographs of the entire vertebral column, a myelogram or magnetic resonance imaging examination indicating extradural spinal cord compression, and finally a surgical procedure confirming the diagnosis of a disc extrusion. In addition to descriptive statistics, age correlation with number of calcifications visible at radiographic examination and with CDVR at the surgery site was examined.We found that disc extrusions occur as frequently in discs that are found to have radiographic evidence of calcification as those discs that do not have signs of radiographic calcification, and that IVDD  requiring surgery does occur in the absence of any calcified discs on radiographic examination. We found that calcified discs were more frequent in our Dachshund population compared to previous studies suggesting that disc calcification might be a serious risk factor for developing disc extrusion. Further studies are needed to show, conclusively, if selection of breeding dogs based on CDVR in the Dachshund will reduce the incidence of IVDD. The presence of the calcifications of intervertebral disc should be evaluated with caution, as only part of the calcifications will be detected and the real extent of the disc degeneration may be underestimated. Intervertebral disc disease (IVDD) in dogs is one of the most common disorders in veterinary neurology. The chondrodystophic (hypochondroplastic) breeds are affected more frequently and in the Dachshunds the breed prevalence of IVDD is around 19% . DegenerSome authors claim an association between the occurrence of CDVR and disc extrusions -14. It hTo the author's knowledge, evidence that dogs with higher number of CDVR are predisposed to disc extrusion, is still missing. It is also not proven that only the CDVRs cause disc extrusion and that discs without CDVR do not cause disc extrusion. Therefore it is still questionable if selection of Dachshunds with less CDVR will reduce the incidence of the IVDD in the Dachshund population in the future.We postulate that intervertebral discs with or without CDVRs can be affected by disc extrusions as the majority of the intervertebral discs are degenerated and calcified, as proven by histopathological studies ,5. The aDachshunds treated surgically for intervertebal disc extrusion at the Referral Animal Neurology Hospital, Aisti, Helsinki, from 2005-2008, were included in the study. Inclusion criteria, for each dog, included a complete physical, orthopedic and neurologic examination, radiographs of the entire vertebral column, a myelogram or magnetic resonance imaging examination indicating extradural spinal cord compression, and finally a surgical procedure confirming the diagnosis of a disc extrusion.A complete neurological examination, including neuroanatomic localization, was performed in each dog according to Lorenz and Kornegay . Neurolo® 1 mg/ml) 10-20 μg/kg intramuscularly (IM) and methadone (L-Polamivet®) 0.1-0.2 mg/kg IM for the radiographic examination. If sedation was inadequate for perpendicular positioning of the vertebral column to the film, anesthesia was induced with propofol (Propofol®) and maintained on isofluran (Isofluran®) and O2. Lateral and ventrodorsal survey radiographs were taken of the entire vertebral column, from C1 to S3. Eight exposures, 4 lateral and 4 ventrodorsal, using 15 × 30 cm flexible phosphor screen  were taken of each column and digitally processed with Kodak direct view CR500. When sedation was adequate for optimal radiographic imaging, anesthesia was not induced before performing the MRI or the myelogram.Dogs were sedated with medetomidine  together and the decisions if the discs were deemed calcified or not, were made in consensus. The images were magnified and contrast was changed in order to achieve optimal impression of the area of interest.Two of the authors (JJ and RV) evaluated the radiographs on E-film  at a dose of 0.3 ml/kg. A lumbar puncture (L5-L6) was performed when the neurologic examination indicated a thoracic or lumbar localization and a cisternal puncture was performed when the neurologic examination indicated a C1-T2 localization. Radiographs were obtained immediately after injection in lateral, ventrodorsal, and oblique views (45°). Myelography interpretation was performed according to Sharp and Wheeler [A myelogram was performed by subarachnoid injection of contrast media, Iohexol (Omnipaque Wheeler .Magnetic resonance imaging (MRI) was performed with an ESAOTE 0.18 T equipment. Dogs were positioned in left lateral recumbency in a human elbow or shoulder coil. Sagittal and transverse images were acquired using T1W  and T2W  sequences. Extruded disc material was characterized by low signal intensity within the epidural space in T1 and T2 weighted images . MRI wasFollowing diagnostic imaging, the dogs proceeded directly to surgery. Surgical approach depended on the localization of the affected disc. A ventral decompression (slot) was performed when the disc extrusion was located in the cervical area, or a left or right sided hemilaminectomy was performed when the disc extrusion was located in the thoracic and lumbar area as described by Sharp and Wheeler ,19. DiscIn addition to descriptive statistics, age correlation with number of CDVR and with CDVR at the surgery site was examined. For analysis, three age groups were defined: dogs < 5 years of age, 5-7 years of age and 8 years and older. Non-parametric Kruskal-Wallis test followed by Tukey test with ranked sums for pair wise comparisons was used to compare the difference in the number of CDVR between age groups. Differences in proportions of dogs having CDVR at surgery site between age groups pair wise were tested using chi-square test.The number of CDVR was divided in two groups: 0-4 CDVR and 5 or more CDVR.Statistical analyses were performed with Stata 10/IC  software. Statistical software WINKS SDA 6.0  was used for Kruskal-Wallis test. The level of significance was set at 5% (p = 0.05).Ninety five dogs were included in the study. Five dogs had two surgeries during the time of the study and their data were evaluated twice as two separate examinations making together 100 patients in the study population. The five Dachshunds, involved in two surgeries, had their second extrusion involving a disc separate from the one previously affected. The median age of affected dogs at the time of examination was 74 months . The study group included 44 female and 51 male Dachshunds. One female and four male dogs were operated and included twice in the study population. The neuroanatomic lesion localization corresponded to the area of disc extrusion in all cases. The neuroanatomic localization was for 6 dogs at C1-C5, for 88 dogs at T3-L3 and for 6 dogs at the L4-S3 spinal cord segments. Hyperesthesia and paraparesis with decreased to absent postural reactions in the pelvic limbs were the most frequent complaint. The neurological status was assessed, and grade I was found in 3 dogs, grade II in 46 dogs, grade III in 24 dogs, grade IV in 13 dogs and finally grade V in 14 dogs. Subsequently, 97% of the Dachshunds of our study showed neurological deficits related to their disc extrusion.other dogs had an intervertebral disc present between the first and second sacral segments making a total number of 27 intervertebral discs per dog. None of these additional discs were calcified on radiographic examination; therefore they were not included in further calculations. A block vertebrae and consequently an absent intervertebral disc was found in four dogs (between T13-L1 in one and between L6-L7 in three dogs). Therefore, in our study population, a total of 2600 disc spaces were evaluated.Two dogs had eight lumbar vertebrae, two Calcified discs were found in 87 of the 100 radiographic examinations of spines , decreased in two  and increased in one (8/9) dog in the repeated radiographic examination. The time period between the two surgeries was less than 12 months in 3 dogs , 16 months in one and 32 months in another patient. The disc next to the previous operation site was extruded in three cases . In the other two cases a more remote disc caused the second extrusion .It has been postulated that it is usually intervertebral discs with CDVR that are subjected to Hansen type I herniation . ConversMultiple studies have suggested that screening the vertebral columns of Dachshunds intended for breeding, by radiographic examination, may be valuable in reducing the incidence of disc extrusion ,9-14. AcAbsence of CDVR on radiographic examination does not mean that the disc is neither degenerated nor calcified. It has been conclusively demonstrated that radiographic examination is considerably less sensitive than histopathology in detection of calcifications within the intervertebral disc calcifications . HistopaThe occurrence of intervertebral disc calcifications is not constant throughout the hypochondroplastic dog's life. CDVR seem to be best visualized at a younger age and later decline in frequency as the hypochondroplastic dog matures . Hansen We found a bimodal anatomical occurrence of CDVR, with one minor peak at the transition from cervical to thoracic spine, and a larger peak at the transition from thoracic to lumbar spine Figure .. This bFgf4) [Chondroid metaplasia is a unique degenerative process of intervertebral discs seen only in hypochondroplastic dog breeds ,2,22. ThFgf4) . SubsequFgf4) ,10,12. TFgf4) ,24.It still needs to be proven that selection of breeding dogs based on CDVR in the Dachshund has a potential to reduce the incidence of IVDD in the Dachshund population. Absence of radiological disc calcifications does not exclude degenerative changes in the disc nor does it exclude disc calcification.Intervertebral disc calcification is undoubtedly a sign of severe disc degeneration and is a serious risk factor for the development of IVDD. Our results indicate that CDVR were more frequent in our study of Dachshunds surgically treated for disc extrusions compared to previous studies ,13,14,21In conclusion, we found that disc extrusions occur as frequently in discs that are found to have radiographic evidence of calcification as those discs that do not have signs of radiographic calcification, and that IVDD requiring surgery does occur in the absence of any calcified discs on radiographic examination. We found that calcified discs were more frequent in our Dachshund population compared to previous studies suggesting that disc calcification might be a serious risk factor for developing disc extrusion. Further studies are needed to show, conclusively, if selection of breeding dogs based on CDVR in the Dachshund will reduce the incidence of IVDD. The presence of the calcifications of intervertebral disc should be evaluated with caution, as only part of the calcifications will be detected and the real extent of the disc degeneration may be underestimated.The authors declare that they have no competing interests.CR drafted the manuscript, JJ and RV carried out the evaluations of the radiographs, SC designed the study, RV performed the statistical analysis and CR, JJ, RV and SC were all involved in the clinical procedures involved in the study.All authors read and approved the final manuscript.


By univariate analysis, we noted that the survival curves by intraepithelial CD8+ T cells became separated only after 1 to 2 years postoperation. Multivariate analyses revealed that the beneficial effect of this factor becomes significant only after a longer (more than 2 year), but not after a shorter (less than 2 year) follow-up period. Furthermore, the number of intraepithelial CD8+ T cells was significantly higher in patients alive for more than 5 years than in patients who either died of cancer after a curative operation or patients who underwent a noncurative operation. Patients' cancer-specific death long after a curative operation is thought to be caused by the growth of micrometastases in other organs or near the primary sites. The effects of intraepithelial CD8+ T cells, therefore, may be mediated by suppression of micrometastasis, rather than suppression of growth in the primary tumour. In conclusion, our data support a hypothesis on the presence of systemic immunosurveillance against micrometastasis of cancer cells.T-cell infiltration into human cancer tissues can be a manifestation of host immune responses to cancer cells. The present study was undertaken to explore the clinicopathological significance of intraepithelial CD8 After considerable controversy, recent studies have supported the hypothesis that the presence of immunosurveillance has an effect on tumour development . Tumour-+ T cells (designated as CD8+T cells within cancer cell nests in this paper) has a more significant impact on patients' survival than that of CD8+ T cells in other locations within the tumour. Recently, + and CD57+ cells along the invasive margin have a significant impact on better disease-free survival in patients with colorectal cancer in a study of 93 patients. CD8+ T cells distributed among cancer cells or along the invasive margin are positively associated with apoptosis of cancer cells in hepatocellular (+ T cells (defined as CD8+T cells among cancer cells or those in contact with cancer cells), as well as CD8+ or CD4+ lymphocytes in other cancer locations, were positively correlated with the biological malignancy of cancer, associating lymphocyte responses with a poor prognosis. Furthermore, 10–20% of human colorectal carcinoma is associated with microsattelite instability (MSI), and cases with MSI are characterised by more frequent infiltration by lymphocytes, increased rate of apoptotic cancer cells, and better survival than cases with microsatellite stability (MSS) , anti-hMLH1, and anti-hMSH2. After deparaffinisation, sections were pretreated by autoclave heating for 5 min. Dextran-polymer method with EnVison plus (Dako) was used as the secondary antibody.+ T cells were defined as CD8+ T cells located within cancer cell nests, not including those in the stroma, and their distribution was quantified as described previously . The observation periods were divided at 2 years in the multivariate analyses, because the number of patients who died of the primary disease became equal with this dichotomy.The method was described previously . The lack of the expression of mismatch repair proteins was detected in 36 of 366 cases. Five cases were excluded due to lack of reactivity for MMR proteins in non-neoplastic cells. This lack was not associated with a significant increase in the number of intraepithelial T cells (P=0.21 by Mann–Whitney test). The number of intratumoral CD8+ T cells positively correlated with peritumoral TILs .Figure 1A T cells  and perihe stage , and it + T cells (divided at the median) revealed a distinctive feature: the two curves were nearly the same during the first 1 year, and became gradually differentiated after 2 years postoperation  and invasion pattern. The impact (Odds ratio) of intraepithelial CD8+ T cells was smaller than that of stage-related factors, which may be caused by the negative correlation with stage , patients who died of the primary disease after a curative operation , and patients who died of the primary disease after non-curative operation . As shown in Figure 3+ T cells in group A was significantly higher than those in groups B and C, without any significant differences between groups B and C. These data were consistent with the notion that intraepithelial CD8+ T cells are a prognostic factor, and further suggested that the decrease of these T cells in stage IV may not be a consequence of systemic immunosuppression by widely spread cancer .To check the above-mentioned results from a different angle, we compared the number of intraepithelial CD8+ T cells in 21 representative cases of well or moderately differentiated adenocarcinoma, of which only one case showed the lack of MMR proteins. Peritumoral T cells refers to T cells located along the invasive margin, which were usually more abundant than intraepithelial T cells (+ T cells showed a much higher labelling index for Ki-67 (7% in median) than peritumoral CD8+ T cells did (1% in median), indicating presumable tumour-specific reactivity of intraepithelial T cells.We next estimated the proliferative activity of T cells by their labelling index for Ki-67 by double-labelling immunohistochemistry , and that this effect was most evident when the patients were divided into two groups at the median number of these T cells. Of particular importance, we newly demonstrated that the effects of these immune cell factors became manifest only after a long follow-up period.The present paper confirmed that the number of intraepithelial CD8+ T cells. We initially observed Epstein–Barr virus-associated gastric cancer to find an abundance of intraepithelial CD8+ T cells . Also, the number of peritumoral CD68+ macrophages and that of peritumoral neutrophils were not significant even under univariate analysis . Therefore, we focused on intraepithelial CD8+ T cells in the present study (371 cases).First, it is necessary to summarise why we focused on intraepithelial CD8+ T cells attack tumour cells only at the primary site, our results are difficult to explain, because our data showed that the patients' survival was affected by the degree of T-cell responses in the resected primary tumour. Thus, other explanations are required. Here we hypothesise effective suppression of micrometastasis in distant organs or near the primary site in cases with higher numbers of these CD8+ T cells. In other words, our data suggest the presence of a systemic immunosurveillance mechanism functioning at sites other than primary tumour, and that this immune activity is in good correlation with the number of CD8+ T cells in the primary tumour. Inversely, only insufficient immune mechanisms would have been functioning in cases without such intensive CD8+ T-cell responses. These considerations are consistent with the present observation that the number of intraepithelial CD8+ T cells is equally low in the cases that died of cancer after a curative operation and in patients who underwent a noncurative operation due to unresectable macrometastases. The latter two situations could be similar from the standpoint of the insufficient host immune responses to control the metastasis.The main causes of death after curative operation are metachronous metastases in distant organs, lymph nodes, or peritoneal cavity and recurrence at the primary site. These ominous consequences are generally caused by the growth of micrometastases that were considered to be present at the time of operation. So long as we assume that intraepithelial CD8+ T cells we observed suggests the presence of certain antigenic stimuli to these lymphocytes . An incoherence emerges with this consequence theory; that is, patients who died of cancer after curative operation, in this scenario, would be expected to have a higher number of intraepithelial CD8+ T cells because their metastatic foci are clinically undetectable, being unable to cause systemic immunosuppression at the time of operation , as a result of frame shift mutation, is related to a prominent tissue infiltration of T cells in colorectal cancer (+ T cells at longer follow-up periods. This has lead us to the hypothesis that these CD8+ T cells may be a indicator of the presence of systemic immunosurveillance that can suppress micrometastasis in other sites as well as tumour suppression in the primary sites. Considering possible clinical applications, further studies are required to analyse these presumptive immune effector mechanisms.In conclusion, our present study demonstrated the effect of intraepithelial CD8


Here a carbon sequestration strategy is proposed in which certain dead or live trees are harvested via collection or selective cutting, then buried in trenches or stowed away in above-ground shelters. The largely anaerobic condition under a sufficiently thick layer of soil will prevent the decomposition of the buried wood. Because a large flux of CO2 is constantly being assimilated into the world's forests via photosynthesis, cutting off its return pathway to the atmosphere forms an effective carbon sink.To mitigate global climate change, a portfolio of strategies will be needed to keep the atmospheric CO-1, and currently about 65 GtC is on the world's forest floors in the form of coarse woody debris suitable for burial. The potential is largest in tropical forests (4.2 GtC y-1), followed by temperate (3.7 GtC y-1) and boreal forests (2.1 GtC y-1). Burying wood has other benefits including minimizing CO2 source from deforestation, extending the lifetime of reforestation carbon sink, and reducing fire danger. There are possible environmental impacts such as nutrient lock-up which nevertheless appears manageable, but other concerns and factors will likely set a limit so that only part of the full potential can be realized.It is estimated that a sustainable long-term carbon sequestration potential for wood burial is 10 ± 5 GtC y2($50/tC), lower than the typical cost for power plant CO2 capture with geological storage. The cost for carbon sequestration with wood burial is low because CO2 is removed from the atmosphere by the natural process of photosynthesis at little cost. The technique is low tech, distributed, easy to monitor, safe, and reversible, thus an attractive option for large-scale implementation in a world-wide carbon market.Based on data from North American logging industry, the cost for wood burial is estimated to be $14/tCO The cumulative fossil fuel emission since 1800 is 330 GtC, but only about half of that remains in the atmosphere; the remainder absorbed by carbon sinks in the ocean and on land [Atmospheric COestation . The emi-1 by 2050 in the absence of climate change policies, according to a range of emissions scenarios [2 concentration for the Special Report on Emissions Scenarios (SRES) A2 emissions scenario is between 450–600 ppmv by 2050, and 700–1000 ppmv by 2100, and global mean surface temperature may increase between 1.5–5.5°C [2 concentrations reach between 450 and 600 ppmv [Fossil fuel emissions are projected to reach 9–20 GtC ycenarios . Dependi600 ppmv .2 concentration below 450–600 ppmv poses an unprecedented challenge to humanity. There are two main approaches: (1) to reduce emissions; (2) to capture CO2 and store it, i.e., sequestration. Since our economy depends heavily on fossil fuel, which comprises more than 80% of primary energy use, to reduce carbon emissions requires drastic changes in energy use efficiency and the use of alternative energy sources that are generally not economically competitive at present [2 below a dangerous level.Keeping the atmospheric CO present ,10. Even2 capture, either from the atmosphere or at industrial sources; (2) storage. Capture out of the atmosphere is assumed to be much more expensive because of the low CO2 concentration in the atmosphere relative to N2 and O2. For this reason, most current proposals seek to combine capturing CO2 with power generation, with several pilot power plants planned or underway [2 include pumping it into deep ocean where CO2 may react with water under the high pressure to form methane hydrates [2 lakes, burying carbon inside deep ocean sediments where conditions are even more stable than ocean bottom [2 in geological formations such as old mines and deep saline aquifers [Carbon sequestration involves two steps: (1) COunderway . The prohydrates  or staysn bottom . The tecaquifers . There i2 capture is done by the natural process of photosynthesis, and storage is low tech and distributed, thus attractive in two important aspects: cost and safety.Here I suggest a biospheric carbon sequestration approach in which wood from old or dead trees in the world's forests is harvested and buried in trenches under a layer of soil, where the anaerobic condition slows the decomposition of the buried wood. This can be supplemented by selective cutting of other suitable trees. On the storage side, high-quality wood can also be stored in shelters for future use. In this technique, COThe possibility of carbon sequestration via wood burial stems from the observation that natural forest is typically littered with dead trees Fig. . It is h-1 are temporarily sequestered by land vegetation . In a steady state, the death rates of these carbon components equal to their respective decomposition rates and add up to NPP such that the net land-atmosphere carbon flux is near zero (NPP = Rh). If we can stop or slow down a part of the decomposition pathway, we have the hope to sequester CO2 at a rate that may rival the current fossil CO2 emission of 8 GtC y-1. Since woody material is most resistant to decomposition due to its lignin-cellulose fiber structure which also minimizes nutrient lock-up (below), I will focus on this carbon pool.Globally, approximately 60 GtC y-1. Then the key question is how NPP is partitioned into the three main carbon pools: leaf, wood, and root. Leaves grow and fall in a deciduous forest each year, but may last a few years in an evergreen forest. Fine woody material such as twigs and small branches may break and fall often, but tree trunks and major branches have a lifespan of decades to centuries and longer. Thus, even though wood biomass is much larger than leaf biomass, its long lifetime suggests a production rate that is much smaller than otherwise. Root biomass can be large and the death rate is also substantial as roots constantly grow to search for nutrient and water. A 'naïve' first guess could be that NPP is partitioned equally into these three pools, leading to a 20 GtC y-1 wood growth rate, thus 20 GtC y-1 wood death rate at steady state. Since fine woody debris decompose more quickly and more difficult to handle, coarser material such as trunks and major branches are more suitable for burial. Assuming half of the woody material is coarse, then about 10 GtC y-1 dead wood may be available for burial, thus leading to a 10 GtC y-1 carbon sink. Assuming an average residence time of 10 years for dead trees on the forest floor, about 100 GtC (10 GtC y-1 times 10 years) in the form of coarse woody debris would be already on the forest floor. These dead wood materials are under various stages of decay, but even if half of that can be collected and buried, it provides a substantial readily available carbon sink.Two major questions need to be first answered concerning the potential of this method: what is the production rate of dead wood, and how much is there in the world's forests? Unfortunately, there is a general lack of knowledge of dead wood on the forest floor, and this carbon pool is often neglected in carbon budget accounting. Since death rate is fundamentally limited by growth rate, the dead wood production rate can not exceed the world total NPP of 60 GtC yThe proposal is to (1) collect dead trees on the forest floor and (2) selectively log live trees. Then the tree trunks are either buried in the trenches dug on the forest floor  or suitable landfills, or logs piled up above ground sheltered away from rain Fig. . The burTo quantify the size of this potential carbon sink, the global dynamic vegetation and terrestrial carbon model VEGAS ,18,19 wa-1, of which 19 GtC y-1 goes into dead leaf, 17 GtC y-1 into dead wood, and 21 GtC y-1 to dead root structures. Since fine wood  decomposes quickly, is more difficult to handle , and may occupy more burial space, only coarse wood will be considered as suitable for burial. Forestry literature generally makes a distinction between fine and coarse woody debris, typically using 10 cm stem diameter to separate the two classes. Unfortunately, the relative contribution to the total wood death from fine and coarse wood is difficult to quantify, in part due to the different lifetime . It is sometimes unclear how these pools and fluxes are defined and what the reported numbers represent in forestry literature. I thus somewhat arbitrarily designate the fine:coarse ratio of death rate to be 7:10 so that the coarse wood death rate is 10 GtC y-1.The modeled global NPP is 57 GtC yThe spatial distribution of coarse wood death rate is shown in Fig. -1 carbon sequestration potential, temperate forest has 3.7 GtC y-1, while the boreal region has 2.1 GtC y-1. Since the model considers only potential vegetation (no agriculture) the temperate regions may have substantially smaller potential.The carbon sequestration potential of coarse wood for various geographical regions is given in Table -1, with major contribution from the Amazon rainforest. Africa follows with 1.9 GtC y-1. Russia has a potential of 1.2 GtC y-1 due to its vast expanse of boreal forest. The conterminous US has a potential of 0.8 GtC y-1 with its extensive broadleaf and mixed forests along the East Coast and the South, and the mountainous West. Canada has a 0.7 GtC y-1 potential from its mixed and boreal forests. Of the 0.9 GtC y-1 potential for China, probably only a fraction can be realized because much of the country's forests has long been converted into cropland. However, a successful reforestation program could boost the size of this fraction.At a regional scale Table , South A-1, with the uncertainty range coming from the tree lifespan estimates. Based on [-1 as the coarse woody debris production rate. A comparison is listed in Table -1 model estimate, i.e., a range of 5–15 GtC y-1.The coarse wood death rate estimated by the model is the result of plant functional type and climate dependent carbon allocation strategy that is not well constrained in current generation of global vegetation models . ObservaBased on , MatthewBased on  estimate-1 potential, I assumed natural vegetation, which by itself would be an overestimate because some of the potential forest area has been converted to cropland. Since current world forest area is 3 times that of cropland, and a significant part of cropland corresponds to potential grassland and even desert rather than potential forest, the degree of overestimation is modest. On the other hand, the actual potential could be higher due to other factors such as selective cutting (below), planting fast growing tree species, and burying smaller-sized wood. In addition, reforestation, deforestation and climate change in the future will complicate any attempt at a precise estimate including land use. Thus, the choice in using potential vegetation was made here.In estimating the 10 GtC yAs a legacy of past tree death, a significant amount of dead wood has accumulated in the world's forests in various stages of decay Fig. . I used -1 estimated above will be available.The implication of this large existing carbon pool is that in the initial stage of wood burial, more than the sustainable rate of 10 GtC y-1 dead wood production rate could also be enhanced by active forest management. Instead of waiting for the trees to die, one can also harvest relatively mature trees via techniques such as selective cutting. At first sight, this seems to be a carbon source as live trees take up CO2. However, if trees are selected properly, it may lead to an overall sink because younger forest tends to be more productive, and somewhere in the development stage, productivity significantly exceeds respiration and decomposition loss [The 10 GtC yion loss . Since tThe implementation of a wood burial scheme will involve three major steps:(1) Enabling access to the forest if not already in place;(2) Site selection, trench digging for burial or building a shelter for above ground storage;(3) Selective tree cutting or the collection of dead wood followed by trimming, shortening and burial or storage, repeated at an appropriate return interval.-2 y-1  would accumulate about 100 tonne of carbon per year for a typical coarse wood production rate of 0.1 kgC my-1 Fig. . At a re5 m Fig. . The surThe actual trench size and distribution need to balance several factors such as cost of digging trench, transporting deadwood, minimizing disturbance to the forest, and selecting the location that most effectively prevents decomposition. Onsite burial is preferred wherever possible to minimize transportation cost. Transportation may be needed where soil is too shallow to dig trenches of sufficient depth. Since soil condition can vary greatly even within a small area such as soil moisture content variation associated with topography, care needs to be taken in site selection.Depending on the dead wood accumulation and decay rates, this process can be repeated every few (1–10) years, but the burial sites will be different each time. The main criterion for choosing return interval will be a balance between the cost of each operation and the need not to let the dead trees rot away. If selective cutting is the main operation mode so that there is little natural tree death (trees are cut before they die), the dominant factor will be the density of suitable trees to remove. In the case of plantation, it may be a good strategy to clear cut small sections (group cutting) for its low cost, allowing trees to grow back as secondary succession.Compared to above-ground shelter storage, trench burial is a better choice for fallen trees as they are typically already in the process of decomposition, so they are less useful as lumber wood. On the other hand, shelter storage preserves lumber wood for easy use should future demand increases.The technology required for collecting or selectively cutting trees is low tech and has been around for thousands of years. Most modern large-scale logging is done by machines in many places such as Europe and North America. The road system for access is already in place in many of these regions such as the US 'Forest Highway' system. Half of the world's forests are already within 10 km, and three quarters are within 40 km of major transportation infrastructure . Since t2 measurements [2 observations [Such a distributed system can be run with little government intervention except for monitoring, as long as economic incentive is provided through schemes such as carbon trading. In North America, much of the forested land is privately owned. The potential for carbon sequestration will have a positive impact on the logging industry and many land owners and the economy in many regions. The accounting and monitoring of the carbon sinks can be done by certified engineers when logging companies return for each round of harvest. This can be supplemented by larger-scale monitoring systems such as eddy correlation flux measurement , source/urements  assistedrvations . The vas-1 carbon sequestration potential is to be utilized, nearly all the world's forests will need to be managed. Then a main question would be the accessibility to the remote forest regions. Firstly, extremely steep mountainous regions or boggy wetland will be difficult to access. Secondly, there are practically no roads to the deep tropical forests. Moreover, a proposal of building a network of roads in the heart of a rainforest will raise major environmental concerns such as loss of biodiversity. On the other hand, economic incentives will continue to stimulate such road expansion. Even in this case, the issue of law enforcement for illegal deforestation, and more broad governance issues need to be first ensured before countries in these regions reach a point-of-no-return. In the near future, a beneficial practice is to bury rather than to burn the trees in the regions with ongoing deforestation.If a major portion of the estimated 10 GtC y-1. Difficulty in accessing steep terrains where forests are typically better preserved will further reduce this number. In fact, giving the cost of road construction and environmental concerns, it is desirable to manage more efficiently a smaller fraction of the available forests through methods such as selective cutting or burying part of the finer woody debris, than disturbing a larger fraction at lower per unit area carbon sequestration rate.If the cores of the tropical rainforests are to be left intact which accounts for about 20% of the total carbon sequestration potential  sequestered, with an uncertainty range of $25–$100/tC.The scale of the climate change problem dictates that any mitigation strategy, whether being alternative energy source, carbon sequestration technique, or geo-engineering approach, has to be cost effective when operated on a large scale. Data from the US logging industry indicate that a typical cost for harvesting 1 tonne of lumber wood is about $20 . Since l2 capture and geological storage  cost for wood burial is lower than the $20–270/tCO2 for power plant CCS. The large range in power plant CCS is due to the increasing cost as cheaply available old mines run out. In the case of wood burial, there is no practical storage capacity limitation. A major cost of industrial CCS is the capturing of CO2 from flu gas, while wood burial is free air capture with near-zero cost because it is done by the natural process of photosynthesis.It is illuminating to compare this with power plant CO2 during 2005–2007. In comparison, the voluntary Chicago Climate Exchange (CCX) price has been around $3–4/tCO2. Although the wood burial cost is somewhat higher than the current market price, it is expected that future climate mitigation policy will result in higher prices for carbon. When implemented at global scale, many factors will vary from location to location such as technology and labor costs. The cheapest will be the forests that are already under intense management where roads and machinery are in place. The price may increase as the total area of forests utilized this way increases. The operation of machinery will consume some fossil fuel and emit CO2. These factors need to be evaluated.It is also interesting to compare this cost with the pioneering European Union Emission Trading System (EUETS) carbon cap-and-trade market price. The EUETS price has fluctuated between €1–33/tCO-1) is carried out in the next few decades, say, by 2050, the scale of such a world-wide operation would be enormous, as illustrated in the scenario below.Even if only half of the estimated potential  one week to dig a trench, collect/cut and bury wood over a 100 hectare area, 200,000 crews (2 million workers) and sets of machinery would be needed. This estimate is admittedly simplistic and the task could be quite labor-intensive if it is to be carried out in dense or steep-sloped natural forests.If each trench has a 500 tC capacity , the rate (in terms of either mass or volume) at which we bury wood needs to be comparable to the rate we burn fossil fuel. If wood burial is used as part of a portfolio, the operation could be scaled down accordingly.The scale of such an operation may be difficult to imagine at first sight, but the enormous scale of the CO-1 sequestration rate. This is only 0.5% of world total Gross Domestic Product (GDP) of $48 trillion in 2006, compared to the projected 5–20% GDP potential economic damage from climate change [The plausibility of this operation may be more easily comprehended from an economical point of view. A $50/tC cost for wood burial corresponds to $250 billion per year at a 5 GtC yBecause of the low oxygen condition below soil surface, the decomposition of buried wood is expected to be slow. This is supported by the observation of extremely slow decomposition of woody material such as furniture in landfills where wood products are found to be well preserved after many years of burial by Micales and Skog . Indeed,2 as the anthropogenic CO2 'pulse' is absorbed into the deep ocean and the carbonate sediments . Thus the way wood is buried will determine the decomposition rate, and can be managed to desired effect. Long term monitoring and research of representative sites will be useful for finding optimal burying methods.One potential drawback of wood burial is that nutrient in wood will be locked away. The same drawback also applies to other methods of large-scale vegetation use such as biofuels. This is a serious concern because nutrients may already be a limitation for plant growth in some forest ecosystems. Plants recycle a major part of the nutrient in dead material. This is especially so in the tropical rainforests where the recycling is so efficient that most of the nutrient is locked in live and dead trees rather than in the soil. If a major fraction of the nutrient becomes locked up by buried wood, the forest growth could be severely limited after some decades so that the strategy becomes unsustainable. Here I use nitrogen as an indicator of nutrient for analysis.Fortunately for our purpose, the nutrient content in wood is much smaller than in leaves and fine roots. For instance, typical carbon to nitrogen ratio (C:N) is 20:1 for leaves, but 200:1 for wood ,32. ThisThe magnitude of this potential problem can be viewed in two ways. First, because leaf turnover rate is comparable to wood turnover (above), but the C:N ratio is 10 times larger, so that the nitrogen recycling rate in leaves and fine wood is more than 90% faster than that in the coarse wood, even though the total amount of nitrogen in wood may not be too different from that in leaves. The fact that tropical rainforest is extremely quick at 'grabbing' whatever nutrient is on the forest floor suggests the great ability of forest at utilizing what is on the ground.-1 of carbon is to be buried, a C:N ratio of 200 implies that about 50 MtN y-1 (Mega tonnne or 1012 gram of nitrogen per year) will be locked up in the buried wood. Although 50 MtN y-1 is a nontrivial amount, this is only a fraction of both the global natural nitrogen fixation rate of 110 MtN y-1 and the anthropogenic N (mainly from fossil fuel burning and fertilizer use) deposition rate of 140 MtN y-1 [The ultimate question is whether internal fixation and external input are fast enough to compensate for the loss rate due to burial lockup. If 10 GtC y MtN y-1 . In addi MtN y-1 . Our curDead wood, whether standing (snags) or down, plays an important role in forest ecology, acting as habitat for animals such as cavity-nesting birds, plants and microbial lives. To minimize the impact, it may be desirable not to completely clean the forest floor, but leave a fraction to maintain these important ecological functions.Although modern forest logging practice has shown that disturbance can be kept at minimum, there is no guarantee it will be the case when practiced world-wide. If not executed properly, it may harm forest regeneration capability, biodiversity and cause significant loss of soil carbon. One method is to have ecological monitoring and carbon accounting conducted together by certified agencies or institutes following carefully crafted international standards.The soil carbon pool is a dynamic balance between dead vegetation input and decomposition. If the deadwood input to soil is reduced, the soil carbon pool will decrease somewhat. It is difficult to quantify this possible loss at present. Regardless of the extent of this soil carbon loss, equilibrium will be reached after sometime so that the cumulative effect of a sustainable wood burial will eventually exceed the initial loss.-1 [-1 coarse wood production rate, there will be large additional capacity for carbon sequestration. Indeed, the burial scheme may be carried out most naturally as an expansion of the existing logging capacity. In addition, if old furniture and building lumbers are buried rather than left to decay in open dumps, they will still serve the purpose of carbon sink. This has already been practiced to some extent in landfills.Wood has been a major resource for humans ever since our ancestors learned to use fire and sticks. Current world total wood consumption is about 0.9 GtC y-1 . Obvious-1 wood production rate also provides an (approximate) upper limit on how much biofuel can be produced, and the caveats discussed here such as nutrient lock-up also apply.Research is ongoing in cellulosic biofuel where cellulose in woody material is converted to fuel . Should One possibility is that if roads are built into remote forests, it will make it easier for deforestation. What has happened in the Brazilian Amazon over the last 3–4 decades where deforestation  follows road construction cautions against the implementation of wood harvest and burial in such regions. For this and many other environmental concerns, a considerable fraction should be preserved and left completely natural. A wise strategy would be intense management of suitable land to achieve higher efficiency while preserving as many forests in their natural states as possible.There may be the concern that wood burial (or any other effective carbon sequestration scheme) will hinder the motivation to reduce emissions and the development of alternative energy. While this is a legitimate and important concern, there is currently a major mismatch between the urgency of the climate problem and the slow pace of the transition toward a carbon-neutral economy due to technological, economical and political hurdles. Carbon sequestration should only be used to 'buy time' so that the society has sufficient lead time to adjust while avoiding dangerous climate change.Reforestation is a widely embraced carbon sequestration technique ,38. Howe2 emissions  declare that they have no competing interests.This is a single-authored paper.