As Twitter has grown in popularity to 313 million monthly active users, researchers have increasingly been using it as a data source for tobacco-related research.The objective of this systematic review was to assess the methodological approaches of categorically coded tobacco Twitter data and make recommendations for future studies.Data sources included PsycINFO, Web of Science, PubMed, ABI/INFORM, Communication Source, and Tobacco Regulatory Science. Searches were limited to peer-reviewed journals and conference proceedings in English from January 2006 to July 2016. The initial search identified 274 articles using a Twitter keyword and a tobacco keyword. One coder reviewed all abstracts and identified 27 articles that met the following inclusion criteria: (1) original research, (2) focused on tobacco or a tobacco product, (3) analyzed Twitter data, and (4) coded Twitter data categorically. One coder extracted data collection and coding methods.E-cigarettes were the most common type of Twitter data analyzed, followed by specific tobacco campaigns. The most prevalent data sources were Gnip and Twitter’s Streaming application programming interface (API). The primary methods of coding were hand-coding and machine learning. The studies predominantly coded for relevance, sentiment, theme, user or account, and location of user.Standards for data collection and coding should be developed to be able to more easily compare and replicate tobacco-related Twitter results. Additional recommendations include the following: sample Twitter’s databases multiple times, make a distinction between message attitude and emotional tone for sentiment, code images and URLs, and analyze user profiles. Being relatively novel and widely used among adolescents and black and Hispanic individuals, Twitter could provide a rich source of tobacco surveillance data among vulnerable populations. As Twitter has grown in popularity to 313 million monthly active users . SimilarInternet users also use Twitter [Tobacco-related tweets can reach a relatively young and ethnically diverse audience. Pew Research Center studies have found that, in the United States, a third of teenagers use Twitter , while 2 Twitter . TherefoResearch utilizing Twitter data is fairly novel without established standards across studies. Thus, it could be advantageous to establish what methods are being used and their strengths and weaknesses. Standards for reporting social media data are needed to be able to compare methods and results across studies . This reRecognize Tobacco in Its Many Forms [A literature search was conducted in July 2016 using the databases PsycINFO, Web of Science, PubMed, ABI/INFORM, Communication Source, and the journal Tobacco Regulatory Science. Searches included a Twitter term and a tobacco term: (Twitter OR tweet) AND (tobacco OR nicotine OR...) . Tobaccony Forms . SearcheOne coder independently reviewed all titles and abstracts and selected 43 articles from the initial 274 that referenced any of the tobacco products and Twitter. Full text of the articles was then reviewed. Explicit inclusion criteria were determined a priori to reduce coder bias when selecting articles. Articles were included in the review if they met the following criteria: (1) original research in a peer-reviewed journal, (2) focused on tobacco, (3) analyzed Twitter data, and (4) coded data categorically . If an article was excluded for failing to meet multiple inclusion criteria, it was counted in the higher-order criteria for exclusion. A total of 16 articles were excluded, so 27 articles were included in the review . We exclOne author read each article to extract methodological information on data collection , coding methods , and coded categories. This information is presented in detail in In total, 22 of the 27 articles discussed the sources they used for their sample of Twitter messages, while 5 articles did not. Twitter provides 3 primary sources of data: Twitter’s Search application programming interface (API), Twitter’s Streaming API, and Twitter’s Firehose. An additional method is Twitter’s REST API, which allows tracking specific users by their username. One study collected data using Twitter’s REST API.Twitter’s Search API is free to use and provides a maximum of 3200 past tweets  with a limit of 180 searches every 15 minutes ,15. TwitMore useful to researchers is Twitter’s Streaming API, which provides all tweets related to the search terms up to a limit of 1% of the Twitter database for that time period. If the selected search terms are infrequently used across Twitter , all tweets related to the search terms will be available. However, if the selected search terms are commonly used, tweets related to those terms will be available up to a limit of 1% of the current Twitter database. Twitter’s Streaming API is free, publicly available data . A totalProviding the greatest access to data, Twitter’s Firehose has real-time access to 100% of Twitter content. Twitter’s Firehose formerly was handled by multiple data providers . After the acquisition of Gnip in 2014, Twitter transitioned to only allowing access to Twitter’s Firehose through Gnip for a fee from August 2015 onward . A totalTwitter data were collected across the studies from 2008 to 2015. The time span of Twitter data collected within an individual study ranged from 1 day to 5 years with a median of 14 weeks .Among the 27 studies, 41% (n=11) analyzed messages related to e-cigarettes, 19% (n=5) related to other tobacco products, and 22% (n=6) about specific tobacco campaigns . The remKim and colleagues  proposedSearch keyword selection was tailored to the studies’ specific topics . To searThe second step of the framework is to apply the search filter and split data into retrieved and unretrieved sets. The third step is to assess the search filter on its ability to distinguish between relevant and irrelevant messages . PrecisiCoding methods for the studies included hand-coding, machine learning, or a combination of the two. Hand-coding involves one or more human coders categorizing data. When 2 or more coders independently code data, a coding agreement score  between the coders can be calculated. In contrast, machine learning uses an algorithm for a computer to learn how to code data. However, human-coding is used for an initial subset of data to help refine the algorithm to improve its accuracy. Coding categories may be determined a priori based on prior research or they may be developed inductively through the process of coding. Studies used hand-coding only (n=17), machine learning + hand-coding (n=8), and machine learning only n=2; . Of the The data collected in the hand-coded studies ranged from a collection period of 1 day to 1 year, while the machine learning studies ranged from 1 month to 5 years of Twitter data. Across the articles that used hand-coding, the number of coders per tweet ranged from 1 to 6. Coding agreement was reported by 20 articles: coding agreement percentage , kappa scores , Cronbach alpha , and both coding agreement percentage and kappa scores (n=1). Kappa values can be interpreted as poor (<.20), fair (.21-.40), moderate (.41-.60), good (.61-.80), and very good (.81-1.00) . CronbacURLs in tweets can provide information that changes the context or meaning of a tweet. Following URLs to their respective webpages can be time-consuming, but it can increase coding accuracy. Machine learning algorithms can analyze the text within URLs but may require human coders to follow them to their respective webpages. A total of 15 articles reported whether they followed URLs  , countryStudies analyzing tobacco-related Twitter data have grown in number in recent years. Although we searched for articles published from 2006 to 2016, articles meeting inclusion criteria for this review were published from 2011 to 2016, with 85% (23/27) of the publications occurring in 2014-2016. Widely used among adolescents and black and Hispanic individuals, Twitter could provide a rich source of tobacco-related data among these groups. One of the benefits of Twitter research is the ability to focus on emerging issues and products that are not yet addressed in surveillance or epidemiological research. Collecting tobacco data in real time via Twitter could be a useful tool for tobacco surveillance, which could help inform tobacco control policies and social media campaigns.Twitter data can change rapidly as they are being posted daily . If a stThe primary sources of data were Gnip and Twitter’s Streaming API, which offer different strengths and weaknesses. If a study wants access to all tobacco-related tweets, then Gnip may be more effective. However, if a study is interested in tweets about a specific tobacco campaign or has a constrained budget, then Twitter’s Streaming API may be a better data source. Alternatively, if a study is focusing on multiple social media sites , then using Radian6, Simply Measured, or Sysomos Heartbeat may be appealing because of their cross-platform analysis.The results of this review suggest that there are some gaps in the types of tobacco products studied by Twitter analyses. E-cigarettes were the product that was addressed by most studies, while none of the studies focused on smokeless tobacco, snus, bidis, or kreteks. The focus on e-cigarettes over other products could be due in part to their recent rise in popularity and recent debates about policies. From 2011 to 2014, e-cigarette use among high school students in the United States significantly increased from 1.5% to 13.4% . SimilarFuture Twitter studies could benefit from a standard of reporting data collection methods. Only 4 of the articles in this review reported such rigorous methods of selecting their search keywords as suggested by Kim and colleagues’  search fThe methods of coding were hand-coding, machine learning, or a combination of the two. Machine learning can code larger quantities of data at a quicker rate than hand-coding, but human coders may have greater discretion at coding for the complexities and subtlety of language such as humor, irony, or sarcasm. For example, algorithms developed to detect irony only retrieved 54%-57% of tweets coded as irony by multiple independent human coders . Hand-coThe sentiment of tweets could help evaluate whether the responses to pro- and antitobacco efforts are positive or negative as a way of understanding social norms about these products. Clarity and comparability across studies could be improved if a distinction is made between attitude and emotion when coding for sentiment. For example, a tweet could be pro-vaping but have a negative emotional tone or it could be anti-vaping but have a positive tone. Only 1 article clarified the meaning by making an explicit distinction between coding for emotional sentiment and message attitude . This isThe three most common themes used for coding were advertisements or marketing; health, safety, harms; and use. Surveillance of these themes could be beneficial to understand whether tobacco advertisements are being circulated on Twitter with the potential of reaching underage individuals, whether the content is making unproven claims about the health and safety of their products, or promoting the use of their products to vulnerable populations such as youth or ethnic minorities.The most common user account descriptions that the studies coded for were personal accounts, industry, news, unclassified, and bots. Determining the user description and demographic information for the accounts that tweet about tobacco could help determine whether tobacco companies, pro-vaping advocacy groups, or antitobacco efforts are circulating with a potential to reach certain groups . Researchers could also monitor how news organizations are presenting tobacco-related information to the public.It may be difficult to determine the demographics of the person tweeting or of the audience exposed to the tweets, which could be especially problematic when studies want to focus on vulnerable populations . A Twitter account could be run by an individual, multiple people , or bots. To reduce bias, bots should be identified and the tweets from these accounts removed from analysis or identified as tweets originating from automated accounts . Only 4 It is recommended that tobacco Twitter studies adopt methodological standards of reporting and data quality assessment. Important information to consider reporting include data sources , the date range of tweets collected, the number of tweets coded, whether retweets were coded, whether coders followed URLs, whether images were coded, the categories coded, the decision criteria for each category, the number of unique Twitter accounts, and the types of Twitter accounts. Studies that use hand-coding should also consider reporting the number of independent coders, the number of tweets coded across coders, and their coding agreement, while it is important for machine learning studies to detail the development and refinement of their algorithms. Providing this information is likely to increase comparability across studies and the ability to replicate results.Depending on research goals, studies may want to sample Twitter’s databases multiple times, especially if they collect from Twitter’s Search API or Streaming API, which put limitations on the amount of data that can be collected per sample. Sampling at multiple times is also important for studies that have access to Twitter’s Firehose through Gnip, considering that tweets are in real time, which could lead to fluctuations based on real-world events . Following messages over time could also help establish trends in the content of posts. However, some studies may be interested in short-term reactions to an event, which requires different sampling methods than following trends. For example, if a study is interested in short-term reactions to the FDA’s new regulations for e-cigarettes, then a single sample before and after the implementation of the new regulations could be sufficient.When coding for sentiment, researchers could improve clarity by making a distinction between whether they are coding for message attitude or for emotional tone . A clearIf relevant to the research questions, Twitter studies may want to code images and URLs. This added step can be time-consuming, but doing so could change the context or meaning of a tweet. For example, an image could help determine if a tweet should be coded as humor or sarcasm, while following a URL could help determine if the tweet is an advertisement. Failing to code images and URLs could result in missing significant content that could affect coding accuracy and skew results.Analyzing Twitter user profiles could provide context for tweets. The same pro–e-cigarette tweet could hold very different significance for a study’s results if it is from a vape shop versus an adolescent. However, the limitations of analyzing user profiles should be recognized and steps taken to improve accuracy. It should be noted that user profiles may be misleading  or profiles of bots. Bots should be identified and potentially removed to reduce bias and improve the quality of data . We cannA limitation of Twitter data is that it does not provide much information on the effects of tweets on behavior. For example, tweeting about tobacco use does not necessarily mean that the person tweeting uses tobacco . AdditioThere are several limitations of this systematic review. First, the results are limited to the databases and search keywords selected, which could have resulted in incomplete retrieval of identified research. Second, this review is limited to its inclusion criteria and the decision rules of the single, independent coder who selected the articles and extracted the data to be included in the review. Individual bias was limited by explicit inclusion, exclusion, and data extraction criteria. However, some studies that were included or excluded for this review may have varied given different inclusion criteria or a different coder. Finally, this review is limited to methodology of categorically coded tobacco Twitter data. Different methodological results and recommendations may have been made if the topic of Twitter data had been different  or if the review had focused on different outcomes .Categorically coded Twitter research can be used for certain insights that other survey research does not provide: emerging issues, popular content in real time, changes over time, how tobacco companies and pro-vaping advocacy groups use social media to increase message exposure in the population , how tobacco control policies and campaigns can most effectively use social media, arguments by groups that may be incorporated into media message design, and quick reactions to antitobacco media campaigns and regulations. There are several approaches that researchers are taking to this end, each having its own set of strengths and weaknesses. Standards for data collection and coding should be developed to more easily compare and replicate tobacco-related Twitter results. Additional recommendations, dependent on one’s research goals, include the following: sample Twitter’s databases multiple times, make a distinction between message attitude and emotional tone for sentiment, code images and URLs, analyze user profiles, and identify and remove bots.


Pathological results were classified as pCR, good (GPR) or poor (PRR). In breast and metastatic ALNs, high levels of CD163+ TIMs were significantly associated with a pCR. In blood, high levels of neutrophils were significantly associated with pCR in metastatic ALNs, whilst the % of mDC1 and pDC and expression of HLA-DR, mDC1 CD40, and CD83 were significantly reduced. NAC significantly reduced tumour DCs but increased blood DCs. PPRs to NAC had significantly reduced HLA-DR, CD40, and CD86 expression. Our study demonstrated novel findings documenting the differential but important contributions of innate immunity to pCRs in patients with LLABCs undergoing NAC.The tumour microenvironment consists of malignant cells, stroma, and immune cells. The role of adaptive immunity in inducing a pathological complete response (pCR) in breast cancer with neoadjuvant chemotherapy (NAC) is well studied. The contribution of innate immunity, however, is poorly documented. Breast tumours and axillary lymph nodes (ALNs) from 33 women with large and locally advanced breast cancers (LLABCs) undergoing NAC were immunohistochemically assessed for tumour-infiltrating macrophages (TIMs: M1 and M2), neutrophils (TINs), and dendritic cells (TIDCs) using labelled antibodies and semiquantitative methods. Patients' blood neutrophils ( Anticancer immune mechanisms play an important role in the induction, development, and dissemination of malignant disease in man –4. Both + and CD8+) and low levels of T regulatory cells (Tregs: FOXP3+ (forkhead box protein 3) and CTLA-4+ (cytotoxic T lymphocyte antigen-4)) and subsequent pCRs with NAC in primary breast tumours and ALN metastases [The role of adaptive immunity , and suppressor cytokines) in women with breast cancer undergoing NAC has been investigated in primary breast tumours, but much less so in tumour-draining ALNs. Significant associations have been documented between high levels of infiltration by T effector cells  has been shown to be associated with a reduced DFS and OS in human cancers  [Polymorphonuclear leukocytes (neutrophils) play a crucial role in dealing with invading pathogens and assist in wound healing. They release a range of toxic substances (reactive oxygen species and proteinases) to deal with invading microorganisms . Increaselanoma) –33. TherDendritic cells (DCs) are derived from haematopoietic progenitor cells in the bone marrow, monocytes being major precursor cells. They are crucial antigen-presenting cells, initiating and directing adaptive immune responses . Variousβ (TGF-β)), hypoxia, and prostaglandins (PGE2), act as chemoattractants and induce DC recruitment into the tumour microenvironment [+ DCs [Various factors, including tumour stroma-released chemokines and cytokines  and high levels of TILs and CD8+ T cells in the primary and ALN metastatic tumours were significantly associated with subsequent pCRs [+ : FOXP3+ T cell ratios in the primary breast tumours and metastatic ALNs (tumour-free paracortex) were significantly associated with pCRs, highlighting the close and complex interrelationships between NAC and adaptive immunity. Furthermore, we recently described the important relationship between NK cells , a key component of innate immunity, and NAC and pCR responses [We have recently documented, in women with LLABCs undergoing NAC, that a significant reduction of both circulating and tumour-infiltrating Tregs  and locally advanced breast cancers , enrolled in a study of NAC (108 patients enrolled between 2008 and 2011), were studied . HistoloThe NAC trial evaluated the effect of the addition of capecitabine (X) to docetaxel (T) preceded by adriamycin and cyclophosphamide (AC). Pathological responses were assessed in the excised surgical specimens after NAC. Established and previously published grading criteria were used to define histopathological responses in the breast , 48. GooISRCTN00407556.The study was given approval by the Leicestershire, Northamptonshire & Rutland Research Ethics Committee 1: Reference number 07/H0406/260; Favourable Opinion 24/01/2008. All patients enrolled in the study gave informed consent to participate in and to publish the results of the study. The study registration is n = 30) and following completion of NAC (n = 16). These blood samples were from the same patients whose tumour and nodal specimens were investigated immunohistochemically for CD1a+ DCs. Venous blood samples were also collected from 10 age-and sex-matched healthy female donors (HFDs) to establish normal values. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on Ficoll-Hypaque, washed and made up in RPMI with 10% foetal calf serum (FCS)  and antibiotics (TCM), and stored at −80°C for further analysis.Venous blood samples were collected before  and the pDChigh  subsets and their costimulatory molecules were documented in 2 × 106 cells/100 μl of PBMCs and stained for cell surface markers for 30 minutes (mins) with 5 μl fluorescein isothiocyanate (FITC) anti-human LIN1, 5 μl Texas red conjugate (ECD) anti-human HLA-DR, 5 μl Pacific Blue anti-human CD11c, 5 μl allophycocyanin (APC) Cy7 anti-human CD40, 5 μl Pe-Cy7 anti-human CD80, 5 μl APC anti-human CD86, 5 μl Alexa 700 anti-human CD197, 2.5 μl Percp-fluor-710 anti-human CD1c (BDCA-1) for mDCshigh, and 5 μl CD303-PE (BDCA-2) for pDCshigh which were added to the corresponding tubes and incubated for 30 mins at 4°C. The PBMC pellet was washed twice in phosphate-buffered saline. The PBMCs were then resuspended in 400 μl of 0.5% paraformaldehyde fixative solution for flow cytometric analysis on a Beckman Coulter FC500.The mDC1Blood neutrophil counts (pre- and post-NAC) in 108 women with LLABC were collected and used in the analysis to evaluate association with NAC-induced pCR in the breast and tumour-draining ALNs.μm tissue sections. Briefly, paraffin-embedded tissue sections were dewaxed and rehydrated using xylene and graded alcohol. Citrate buffer, pH 6.0, at 98°C was added for 20 mins for antigen retrieval. After serial blocking, the sections were incubated with the primary monoclonal antibody (MAb) against CD68 , 1 : 300 dilution for 30 mins at room temperature (RT); MAb against CD163 , prediluted for 30 mins at RT; MAb against CD1a , 1 : 200 dilution for 15 mins at RT; MAb against CD66b , 10 μg/ml for 30 mins at RT; MAb against IDO , 0.75 μg/ml for 15 mins at RT; and MAb against VEGF , 1 : 50 dilution for 30 mins at RT. The Novolink™ polymer detection system, Leica RE7280-K with polymeric horseradish peroxidase- (HRP-) linker antibody conjugates and diaminobenzidine (DAB) chromogen, was used for enzyme-substrate labelling. Finally, the sections were counterstained with haematoxylin, dehydrated, and mounted in DPX mounting medium. Positive and negative staining controls were carried out with tonsil sections except for CD163 (liver sections) and IDO . Negative staining controls were demonstrated by omitting the primary antibody. Positive and negative controls were simultaneously performed with every IHC staining run. All primary antibodies used in this study have been validated and certified by the commercial suppliers for IHC assessment of paraffin-embedded specimens.Immunohistochemical assessments of immune cell subsets and expression of indoleamine 2,3-dioxygenase (IDO) and vascular endothelial growth factor (VEGF) were performed in 4 + or CD163+ cells, which was considerably higher than the average level of CD68+/CD163+ cells, was defined as hotspots (TF hotspot). All sections were evaluated at a distance away from areas of necrosis. The TF hotspots of the two view fields with the highest measurements at ×200 magnification were averaged out (CD68 or CD163 TF mean). The average infiltration (CD68 or CD163 TF mean) was semiquantitatively graded as no/weak (grade 1), moderate (grade 2), strong/robust (grade 3), and massive infiltration (grade 4). Tumours classified as 1 included totally negative specimens as well as specimens containing some scattered positively stained cells along the tumour margin. Tumours were classified as 2 when CD68 or CD163 staining was continuous along the tumour margin but did not extend from the tumour front for more than one cell layer on average. CD68 or CD163 staining that, on average, extended two to three cell layers from the tumour margin over the whole section was classified as 3; CD68 or CD163 staining extending more than three cell layers from the tumour margin in all fields was classified as 4. For statistical analysis, grades 1 and 2 were defined as low level of infiltration whereas grades 3 and 4 were defined as high level of infiltration [Whole tissue sections were studied rather than microarrays (to minimise sampling bias). The majority of macrophages present in breast tumours were located along the border of tumour nests. Immunostaining for TIMs was evaluated along the tumour front (TF) over the whole section (7–10 view fields per section). This evaluation followed previously published studies documenting the level of TIMs –51. Tumoltration –51.+ and CD66b+ cells in the breast tumours, the average numbers of brown membrane-stained cells regardless of the intensity were counted in 5 high-power fields (400x HPFs). Positively stained cells in contact with tumour cells or within the tumour cell nests were defined as “intratumoural” whereas positively stained cells in the interstitial stroma surrounding tumour nests were defined as “peritumoural/stromal.” Evaluation of infiltrations in post-NAC specimens was undertaken on residual tumour nests, and in the case of pCR (complete disappearance of invasive tumour cells in the specimen), in the tumour bed. The latter was characterised histologically as a hyalinised, amorphous area with haemosiderin deposits [To evaluate the presence and extent of CD1adeposits , 53.+ and CD163+) in ALNs were quantified as the average % of all cells in 5 HPFs in nonmetastatic medullary areas of ALNs. The average number of cell counts in 5 HPFs in nonmetastatic areas with the greatest accumulations of positively stained cells on scanning at low magnification was determined for CD66b+ and CD1a+ cells. These quantitative evaluations followed the methodological scoring for documenting various immune cell subsets present in ALNs in patients with breast cancer [Positively stained macrophages  by a factor representing the intensity of immune reactivity , giving a maximum score of 300. A score of <50 was considered negative, and a score of 50–100 was considered weakly positive (1+). A score of 101–200 was regarded as moderately positive (2+) and a score of 201–300 as strongly positive (3+). Negative and 1+ were considered as low expression whereas 2+ and 3+ were considered as high expression , 56.All scored assessments were performed without the knowledge of the patients' clinical and pathological parameters. The pathological responses were assessed by a consultant breast pathologist. The scoring systems used in establishing the biological markers present in breast tumours and metastatic tumours in ALNs, and ALNs in this investigation, followed previously published studies documenting the scoring systems employed (described above).U test (between two variables/groups)) was used to compare the groups based on pathological responses. The Pearson chi-square test was performed to compare the binomial data (negative/low versus high) on level of TIMs and expression of IDO/VEGF between groups. To evaluate and compare the related sample data between pre-NAC and post-NAC groups, as well as primary breast tumours and metastatic tumours in ALNs, the related sample Wilcoxon signed-rank test and related sample McNemar test were performed for comparing the number of cell counts and the expression of IDO/VEGF, respectively. A probability value  of equal to or less than 0.05 (2 tailed) was considered statistically significant.Statistical analyses were performed with the IBM SPSS statistics software, version 21 . Where the data did not follow a normal distribution, a nonparametric test  was calculated by assuming the common standard deviation of circulating blood Tregs as 0.5. As our findings are derived from several assays of different parameters, the sample size of at least 7 in each group may not be optimal for some of the tests. In addition, multiple hypothesis testing may lower the significance of our findings.The sample size of this study was based on a cohort of patients from a previous study in which circulating Tregs were documented pre- and post-NAC . A samplhttps://doi.org/10.1155/2017/1049023). The responses to NAC and the tumour recurrence and survival at 4 years of follow-up are documented.The patient and tumour characteristics of the 33 patients studied are shown in Table A , respectively, following 8 cycles of NAC. There was, however, no significant association between CD68+ TIMs and GPR and pCR with NAC . As the levels of CD68+ TIMs in primary tumours showed no association with a pCR following NAC, we elected not to study this subset in the ALN metastases  . There was, on the other hand, no intra- or peritumoural alteration in the level of CD66b+ TINs  . Figures6b+ TINs .+ and CD163+) in tumour-draining ALNs, comparing metastatic (tumour-free areas) with nonmetastatic, in women with LLABCs undergoing NAC  following NAC. There was no association, however, with the expression of IDO  or nonmetastatic (data not shown).+ TIMs were significantly associated with ER status (p = 0.046) and tumour grade (p = 0.004). In addition, both parameters were significantly associated with tumour pCRs following NAC  (n = 33) studied (p = 0.001)  . Thus, E= 0.001) .n = 108) were associated with a significant pCR in metastatic ALNs following 8 cycles of NAC. There were no comparable changes in the primary breast cancers, nor any significant associations with nodal status, DFS and OS (+ neutrophils in patients following 8 cycles of NAC (data not shown). There was a significant reduction in the circulating levels of PMN neutrophils (p = 0.001) after NAC (High levels of circulating PMN neutrophils pre-NAC in women with LLABCs (S and OS . There wfter NAC . All patn = 30) was significantly reduced (1.26 ± 0.20%) compared with healthy, age-matched female donors (HFDs (n = 9): 1.70 ± 0.30) (p = 0.034). The % of mDC1shigh was also significantly reduced compared with HFDs , as was the % pDCshigh  (The % of DCs in the blood of women with LLABCs (= 0.041) .+ and pDC+ subsets, compared with HFDs (47.30 ± 8.00% versus 65.00 ± 3.10% (p = 0.045), 48.12 ± 9.50% versus 65.52 ± 5.00% (p = 0.002), resp.)  .high, compared with HFDs (35.01 ± 8.00% versus 44.87 ± 6.50% (p = 0.011), 3.18 ± 0.40% versus 4.04 ± 0.30% (p = 0.047), resp.). The expression of CD80, CD86, and LNHR CD197 on the mDC1shigh were unaltered  were comparable with those that had a poor pathological response (n = 7) (1.64 ± 0.25 versus 1.29 ± 0.25) (p = 0.024)  . The % l= 0.024) . There w= 0.024) .high (p = 0.048) and pDCshigh (p = 0.017) and expression of HLA-DR , when compared with the blood levels of HFDs. Women whose tumours showed a GPR had no alterations in circulating levels  and CD86 (p = 0.001), compared with HFDs. These findings were not seen in women whose tumours had GPRs  following NAC in patients whose tumours had a PPR  and expression of HLA-DR (mDC1s and pDCs), CD40, and CD86 (mDC1s) and failure to achieve a GPR with NAC. Thus, women whose breast cancers failed to undergo a GPR with NAC showed significantly reduced, inactive, and immature DC subsets in the circulation.+ TIMs (M2 macrophage phenotype) in both the primary breast cancer and metastatic ALNs were significantly associated with pCRs in malignant tissue following NAC. TIMs are derived from circulating monocytes and primitive bone marrow progenitors [β and IL-10. TIMs also secrete arginase 1 in the tumour microenvironment, reducing L-arginine in situ and inhibiting CTL production and function [In the current study reported, the CD163genitors . They argenitors . Once emgenitors , 60, 61.function –64. Bingfunction , 65.+ TIMs in LLABCs are a predictor for a good pathological response and pCR but not necessarily for DFS and OS. In our study, there was a significant association between TIMs and tumour ER status and grade and between pCR and tumour ER status and grade. Highly proliferative, triple-negative breast cancers are significantly associated with high rates of pCR but have a poor DFS and OS [A pCR following NAC in breast cancer has been shown in some studies to be a surrogate marker for a good prognosis and survival , 66. CorS and OS , 46, 68.S and OS . A recenS and OS .γ (IFN-γ) and bacterial products, produce proinflammatory cytokines, express IL-12, enhance adaptive immunity, and induce lysis of tumour cells [+ TIMs, the M1CD68+ TIMs had no demonstrable association with a pCR. Heys et al. studied TIM subsets in breast cancer, using CD68  expression of suppression of cytokine signalling (SOCS) 1 and SOCS 3 [γ and Toll-like receptor 4, whilst SOCS 3 (M1 phenotype) inhibits signal transduction and activation of transcription 3 (STAT 3) signalling. High levels of SOCS 3+ TIMs in LLABCs were associated with a pCR with NAC. There was, however, no association between SOCS 1+ TIMs and pathological response to NAC [M1 TIMs are activated by interferon-ur cells –21. In cd SOCS 3 . SOCS 1 e to NAC . Thus, M+ M2 macrophages was associated with a favourable nodal status in patients with breast cancer. They did not investigate TIMs in metastatic SLNs [+ TIMs in metastases in tumour-draining ALNs, prior to NAC and the subsequent pCR following 8 cycles of NAC. Our study, on the other hand, found no significant difference in the level of CD163+ macrophages in metastatic (tumour-free areas) versus nonmetastatic medullary areas in ALNs. The significant association between CD163+ TIMs in primary breast cancers and metastatic ALNs and pCRs with NAC has not been previously reported.In sentinel lymph nodes (SLNs), Mansfield et al. found that the presence of sinusoidal CD163tic SLNs . In our In patients with cancer, blood neutrophils tend to be immature and produce low levels of free radicals . IncreasThere were significantly reduced blood levels of PMN neutrophils following NAC, albeit all patients were given granulocyte colony-stimulating factor following randomisation.+ TINs have been found previously to be significantly associated with poor DFS and OS in a variety of nonbreast solid cancers [+ TINs were not significantly associated with pathological responses to NAC. They were also resistant to NAC. The TINs were present in small numbers in the tumour microenvironment, and only a small cohort (n = 16) of patients was studied. A larger series is required to confirm these findings. There are, however, no published articles documenting their relevance in women with LLABCs undergoing NAC.Tumour entry of PMN neutrophils is induced by chemotactic molecules secreted by intratumoural TIMs and cancer cells , 28. Hig cancers –32. In o+ TINs and pCR with NAC. Moreover, there was no difference in the level of infiltration by CD66b+ neutrophils in the paracortical areas of metastatic (tumour-free areas) and nonmetastatic ALNs. Such findings have not previously been documented.In tumour-draining metastatic ALNs, there was also no significant association between the level of infiltration by CD66b+ DCs and concurrently circulating DCs, mDC1shigh and pDCshigh. In solid cancers, evidence suggests that DCs in the tumour-microenvironment are present in small numbers, are immature, and poorly activated [In our study, we investigated tumour-infiltrating CD1activated , 71, 72.ctivated . In breactivated .+ TIDCs in breast cancer [+ TIDCs in LLABCs and the pathological responses with NAC. Our findings showed no significant association between the levels of CD1a+ TIDCs and pCR in either the primary tumours or metastatic ALNS. There is evidence that DCs in breast cancer (primary tumours and ALNs) are not only poorly activated and immature but may be also immunosuppressive, inhibiting the generation of CTLs and inducing T cell tolerance [in vitro induced the secretion of TGF-β and enhanced the generation of CD4+CD25+ FOXP3+ Tregs [+ DCs although it had no effect on TIMs and TINs. NAC, by significantly reducing suppressor cells (Tregs and myeloid-derived suppressor cells) within the tumour microenvironment, may have contributed to the immune-associated tumour cell death [Although the presence of TIDCs has been documented to be associated with a better clinical outcome in a number of human solid cancers, this is not the case with CD1at cancer , 73–75. olerance , 41, 43.olerance , 77. Dumll death , 57. NACThe percentages of circulating levels of DCs, mDC1s and pDCs, were significantly reduced in women with LLABCs when compared with healthy females. In addition, the expression of HLA-DR, CD40, and CD83 molecules on the surface of mDC1s were also significantly reduced, as was HLA-DR expression of pDCs. The expression of the costimulatory molecules CD80/86 was unchanged compared with healthy females. We did not carry out any functional assays to ascertain specific effector functions such as efficacy in antigen presentation and secretion of cytokines. NAC, in particular anthracyclines and taxanes, is known to disrupt tumour cells, exposing expression of calreticulin and release of tumour-associated antigens (TAAs) , 79. Thihigh and pDCshigh and reduced expression of HLA-DR, CD40, and CD83 on mDC1shigh and HLA-DR on pDCshigh in those patients whose tumours showed a PPR to NAC. In addition, post-NAC patients whose tumours had a GPR had a significantly increased expression of HLA-DR, CD40, CD86, and the LNHR CD197. These significant associations suggest a possible and important interaction between DCs and TAA presentation to naïve T cells and immune-induced tumour cell death during NAC. The findings documented, to the best of our knowledge, have not been published previously.There was a significant difference in the level of circulating DCs between those patients whose tumours showed a good pathological response  and those whose tumours had a poor pathological response  to 8 cycles of NAC. Following NAC, the blood DC levels in the GPR group were increased and were significantly higher than the levels documented in healthy females. There was also a significantly reduced % of circulating mDC1s+ CTLs [γ, IL-2, and IL-6, as well as enhancing NK cell cytotoxicity [+ Tregs and to restore T effector and NK cell function in patients with disseminated disease [Many chemotherapeutic drugs produce short-lived inhibitory effects on innate and adaptive immune cells. Some , however, can enhance or suppress specific aspects of the immune mechanism and activate immune-mediated tumour cell death, contributing to the good pathological responses documented in primary cancers and metastatic ALNs , 79–85, + CTLs , 86, 87.+ CTLs , 89. Taxtoxicity , 90. Mor disease .Our study has provided further knowledge and understanding of the relevance and contribution of certain components of innate immunity to tumour cell death in both the primary breast cancer and metastases in tumour-draining ALNs in women with LLABCs undergoing NAC. High levels of TIMs (M2) appear to induce/enhance pCRs in primary and ALN metastatic breast cancers probably through their association with high tumour grade and negative ER status. High level of expression of VEGF and the resultant increased vascularity results in enhanced delivery of chemotherapeutic agents to the tumour cell milieu. Circulating levels of DCs and expression of HLA-DR and costimulatory molecules were significantly reduced in patients with LLABCs. This diminished number of activated DCs and thus decreased capacity to present TAAs (released by NAC and innate NK cells) to naïve adaptive T cells results in reduced generation of CTLs. This trend was significantly reversed in patients in whom NAC induced a pCR. These various immune mechanisms highlight the close and important interaction between innate and adaptive anticancer immunity.+ and CD8+) and regulatory (FOXP3+ and CTLA-4+) cells and NK cells in immune-mediated breast cancer cell death in women with LLABCs undergoing NAC. Our novel findings have further increased our knowledge and documented the important contribution of innate immunity to tumour cell death in women with LLABCs undergoing NAC. The significant associations between the beneficial pathological responses (GPR and pCR) in the tumour microenvironment (primary and ALN metastases) and key innate immune cells  complement our findings with NK cells and are an important contribution to the understanding of putative anticancer immune responses in NAC, resulting in immune-mediated tumour cell death.There is a significant body of evidence documenting the important contribution by circulating and tumour-infiltrating T effector .


Decorin (DCN), an extracellular matrix (ECM) protein, belongs to the small leucine-rich proteoglycan family. As a pluripotent molecule, DCN regulates the bioactivities of cell growth factors and participates in ECM assembly. Accumulating evidence has shown that DCN acts as a ligand of various cytokines and growth factors by directly or indirectly interacting with the corresponding signalling molecules involved in cell growth, differentiation, proliferation, adhesion and metastasis and that DCN especially plays vital roles in cancer cell proliferation, spread, pro-inflammatory processes and anti-fibrillogenesis. The multifunctional nature of DCN thus enables it to be a potential therapeutic agent for a variety of diseases and shows good prospects for clinical and research applications.DCN, an extracellular matrix (ECM) protein that belongs to the small leucine-rich proteoglycan family, is widely distributed and plays multifunctional roles in the stroma and epithelial cells. Originally, DCN was known as an effective collagen-binding partner for fibrillogenesis  and to m Mammalian DCN, whose complex gene contains 8 exons, is located in chromosome 12q21-q22 and comprises a molecular weight of 92.5-kDa with a coding gene that is of 1080-bp in length. The synthesis and secretion of DCN mainly occur in the rough endoplasmic reticula and Golgi apparatuses of fibroblasts, smooth muscle cells, and macrophages. DCN comprises a 42-kDa conserved protein core that is involved in protein/protein interactions and a single glycosaminoglycan (GAG) chain that is covalently attached to a serine residue near the N terminus consisting of either chondroitin or dermatan sulfate , 8. StruFibrosis is a pathological process that occurs in chronic injury, intricate inflammation or long-standing metabolic disease  , 25 thatIn the process of fibrogenesis, transforming growth factor-β (TGF-β) is undoubtedly the most powerful pro-fibrotic cytokines, which can activate fibroblasts, prevent their apoptosis and force them to overproduce matrix components, such as collagen types I, III, and IV and fibronectin . FurtherHowever, the mechanism of anti-fibrosis involves not only the interaction between DCN and TGF-β but also other matrix molecules and cytokines. It has increasingly been accepted that DCN regulates the production of extracellular matrix components (such as fibronectin and thrombospondin-1), inhibits collagen I maturation, and stimulates collagenase. In no-mutation animal models, disruption of the DCN gene leads to skin fragility and abnormal collagen morphology, characterized by uncontrolled lateral fusion of fibrils . More reAdditionally, as a component of the ECM, soluble SLRPs function as endogenic ligands of Toll-like receptor 2/4 (TLR2/4) and trigger acute inflammatory responses  and innaFurthermore, by down-regulating the bioactivity of TGF-β1, DCN decreases the abundance of oncogenic microRNA (miR-21), a transcriptional inhibitor of a tumour suppressor named programmed cell death protein 4 (PDCD4), in a TLR2/4 independent manner  Figure . and ultInterestingly, studies in a triple-negative orthotopic breast carcinoma xenograft models, showed an unexpected and paradoxical role for the DCN protein core in inhibiting genes that were necessary for immunomodulatory responses . TherefoEarly genetic studies have demonstrated that deficiency of DCN is permissive for tumour development. These studies tested DCN-null mice with a high-fat diet and showed that these mice have a higher-risk of developing spontaneous intestinal tumours than the control mice; moreover, DCN and p53 double-KO mice showed aggressive T-cell lymphomas with a significantly faster rate of progression than the p53-null mice . GeneticOwing to its direct pan-inhibition of numerous key pathways emerging from receptor tyrosine kinase signalling, DCN has been considered as a “guardian from the matrix” .2+ increase, with concomitant induction of p21 gene expression, up-regulation of p21 protein (an inhibitor of the cell cycle) and conversion of caspase3 [More specifically, monomeric DCN binds to RTKs and evokes receptor dimerization, transient autophosphorylation, caveolin-1-mediated internalization, and, eventually, degradation within lysosomes . EGFR, bcaspase3  , hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and connective tissue growth factor (CTGF) . Ma et aIn contrast, in the tumour microenvironment, DCN shows an obvious inhibitory function in angiogenesis. When DCN is overexpressed, the tumour growth rate strongly decreases, and tumour angiogenesis is dramatically inhibited. Analyses based on immunohistochemistry, Northern blotting and protein imprinting have revealed that tumour cells expressing DCN show reduced levels of vascular endothelial growth factor . Thus, iin vivo and in vitro studies have strongly suggested that DCN blocks the growth and distant metastasis of breast cancer cells by down-regulating EGFR expression and interfering with the formation of EGFR/ErbB2 dimers [Specifically, the newly formed DCN/Met heterodimer continuously degrades the major oncogenes, namely, β-catenin and Myc, via the 26s proteasome after undergoing phosphorylation  Figure . Moreove2 dimers . Thus, rPerforming its function as “a guardian from the matrix”, DCN slows the spread and metastasis of tumour cells by indirectly inducing vascular endothelial cell autophagy; the possible mechanisms include endothelial autophagic complex formation and reduced synthesis of autophagy inhibitors. DCN is capable of evoking a prolonged autophagic program in a Peg3 -dependent manner. Importantly, autophagy functions by targeting the tumour microenvironment rather than acting solely on the actual tumour . Peg3, aIn tumorus, autophagy serves as inhibitor of tumour initiation by clearing misfolded proteins, ROS (reactive oxygen species) and other factors . Once stAdditionally, DCN promotes the expression of FAS/FASL, induces BMP2k (BMP-2 inducible protein kinase) gene expression, and impacts the tumour microenvironment; through attenuation of TGF-β, DCN stabilizes E-cadherin and other proteins in order to inhibit the development and metastasis of tumour cells . BecauseTumour gene-virus treatment , the basRecent evidences have shown that, for solid tumors, connective tissue and ECM may have a prominent role in inhibiting viral spread after their administration , and the+ T cells and proficient viral spreading within tumour tissues. This therapeutic mechanism of a cytokine plus DCN is a promising cancer immunotherapeutic approach for overcoming tumour-induced immunosuppression.Moreover, DCN acts as an adjuvant for overcoming TGF-β-mediated immunosuppression. Yun et al  aimed toDCN, a prototypical small leucine-rich proteoglycan, was initially defined as structural constituent of the ECM and as a participant in fibrillogenesis. Soon afterwards, it was demonstrated that DCN was essential for anti-fibrosis and pro-inflammatory processes. Furthermore, emerging studies have suggested that DCN is a pan-RTK inhibitor that possesses anti-tumour capabilities involved in cell growth, differentiation, survival and metastasis. Therefore, DCN is promising therapeutic agent for a variety of diseases and shows good prospects for clinical applications as an anti-tumour therapy. In recent years, a recombinant therapeutic approach with DCN and an oncolytic adenovirus has made significant progress in animal models. However, as part of the clinical application process, many problems need to be addressed by researchers, including the reduced ability of viral replication in the hypoxic microenvironment of the solid tumour and the limited distribution of the virus. Furthermore, extensive preclinical studies are needed because recombinant DCN has not met the standard of clinical drugs. In the future, our research will focus on solving these problems and lead to new diagnostic and individualized treatment approaches for cancers.


We aimed to study the potential influence of tumour blood flow –obtained from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)- in the metabolomic profiles of endometrial tumours.Liquid chromatography coupled to mass spectrometry established the metabolomic profile of endometrial cancer lesions exhibiting high (n=12) or low (n=14) tumour blood flow at DCE-MRI. Univariate and multivariate statistics  classifier and hierarchical clustering) and receiver operating characteristic (ROC) curves were used to establish a panel for potentially discriminating tumours with high versus low blood flow.Tumour blood flow is associated with specific metabolomic signatures. Ortho-PLS-DA and RF classifier resulted in well-defined clusters with an out-of-bag error lower than 8%. We found 28 statistically significant molecules . Based on exact mass, retention time and isotopic distribution we identified 9 molecules including resolvin D and specific lysophospholipids associated with blood flow, and hence with a potentially regulatory role relevant in endometrial cancer.Tumour flow parameters at DCE-MRI quantifying vascular tumour characteristics are reflected in corresponding metabolomics signatures and highlight disease mechanisms that may be targetable by novel therapies. Endometrial cancer (EC) is the most common gynaecologic malignancy in the high-income countries, and its incidence has increased by 21% since 2008 .Angiogenesis is an essential mechanism for tumour growth, invasion and metastatic spread in many cancers types . Moreoveb) based on dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and low tumour Fb is observed in EC patients with reduced recurrence /progression-free survival [In EC, increased microvascular proliferation correlates with a more aggressive phenotype -8. Furthsurvival , 9. Altosurvival .Cancer was primarily considered a proliferative disorder; however, altered cell metabolism has emerged as a “hallmark of cancer”, since growing tumour cells rewire their metabolic program to meet the biosynthetic and bioenergetic demands of constant cell growth and phenotype changes. Metabolomics analysis provides direct evidences of biochemical context-dependent changes that can be correlated with the pathologic state of a cell, tissue or organ . Therefob metabolic biomarkers may highlight preoperative functional tumour characteristics with potential relevance for preoperative risk assessment and for identification of EC patients with expected poor outcome.The present study explores the different metabolomic tumour signatures potentially characterizing EC lesions exhibiting high versus low tumour blood flow at DCE-MRI. The identification of high versus low Fb using a non-targeted metabolomics approach. We employed multivariate statistics to study changes in whole sample metabolome both in unsupervised-principal component (PCA) and hierarchical clustering analysis- and in supervised approaches-partial least discriminate analysis (PLS-DA), ortho-PLS-DA and Random Forest (RF) [The main objective of this work was to determine metabolomics differences based on tumour Fest (RF) .Patients had a mean age of 68 years and 20/26 (77%) of tumours had endometrioid histology Table . In totab clustered better than lesions exhibiting low tumour blood flow. Once we discarded the presence of outliers and checked the technical quality of the technique we applied ortho-PLSDA as a specific tool for clustering .In the present work PCA analysis was chosen as an unsupervised method Figure . This ang Figure . This ang Figure . Becauseg Figure . Specifib. Moreover, resolvin D was identified as a potential tumour Fb biomarker. The ROC curve of resolvin D  confirmed the robustness of this metabolite to predict high tumour Fb  with a potential identity were used , lysophosphatydilserine (14:0), lysophosphatydilinositol (20:0) and lysophosphatydilcholine (20:3) in EC tumour progression. These results reinforce the idea of LPL metabolism as a disease modifier in EC progression. Furthermore, this study has revealed that tissue resolvin D could be a good predictor of high tumour Fb . Resolvin D is known to be involved in the resolution of inflammation processes and shows potent effects in protecting against neovascularization and hypoxia in some pathological diseases [Previous studies have suggested a specific metabolomics signature of EC compared to normal tissue , but thereatment . Among areatment . In our diseases . Thus, odiseases .All in all, the present work delves into the different signalling molecules involved in tumour progression. These achievements can serve to better understand EC metabolism helping to develop, in a near future, novel and more personalized therapies. Moreover, our results support the promising role of DCE-MRI as preoperative non-invasive imaging tool depicting tumour vascular features relevant for the corresponding metabolomic tumour signature that may be targetable in EC therapy.Endometrial tissue from hysterectomy specimens was collected from 26 patients surgically treated for endometrial carcinoma. All patients were diagnosed and treated at the same university hospital  written informed consent was obtained from all patients for the collection of imaging data and specimens for biomarker studies included in an institutional review board-approved protocol (Rek Vest 2009/2315).2, slice thickness = 5 mm, number of averages (NA) = 1). The temporal resolution was 2.49 s. and sequential images were acquired from 30 s before i.v. administration of contrast medium to 6.3 min after contrast injection. At 2 min post contrast medium administration, a pause of 33 s was utilized to acquire axial oblique T1-weighted gradient-echo contrast-enhanced images.Preoperative pelvic magnetic resonance imaging (MRI) was performed in all patients on a 1.5 Tesla MRI scanner  using a six-channel body coil and a standardized imaging protocol , 9 20 mgb) are shown in Figure Regions of interests (ROIs) were drawn within the endometrial tumour by a radiologist (ISH with > 7 years of experience with pelvic MRI) who was blinded for tumour stage, histological diagnosis and patient outcome. Care was taken to avoid including necrotic and haemorrhagic areas of the tumours in the ROIs. The dynamic image series were analysed using an in-house implementation of the adiabatic approximation model of Johnson and Wilson . Represehttps://metlin.scripps.edu/index.php) which includes accurate masses and MS/MS spectrum for 961.829 molecules.Tissue samples (50–70 mg) were homogenized using 20 volumes of cold methanol as previously described . MetabolAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Written informed consent was obtained from all patients for the collection of imaging data and specimens for biomarker studies; included in an institutional review board-approved protocol (Rek Vest 2009/2315). This article does not contain any studies with animals performed by any of the authors.


There is an error in the funding statement. The number of the National Institutes of Health grant "GM074942" should instead be "GM094585".


The TTL was related to pT stage (p = 0.01) and tumour size (p < 0.01) in low-grade tumours. Multivariate logistic regression showed independent correlation of molecular positivity with gender, tumour grade and number of fresh LN [AUC = 0.71 (95 % CI = 0.62–0.79)]. Our results show that lymph node CK19 mRNA detection correlates with classical high-risk factors in stage I–II colon cancer patients. Total tumour load is a quantitative and objective measure that may help to better stage early colon cancer patients.Stage I–II (pN0) colorectal cancer patients are surgically treated although up to 25 % will eventually die from disease recurrence. Lymph node (LN) status is an independent prognostic factor in colorectal cancer (CRC), and molecular tumour detection in LN of early-stage CRC patients is associated with an increased risk of disease recurrence and poor survival. This prospective multicentre study aimed to determine the relationship between LN molecular tumour burden and conventional high-risk factors in stage I–II colon cancer patients. A total of 1940 LN from 149 pathologically assessed pN0 colon cancer patients were analysed for the amount of tumour cytokeratin 19 (CK19) messenger RNA (mRNA) with the quantitative reverse transcription loop-mediated isothermal amplification molecular assay One-Step Nucleic Acid Amplification. Patient’s total tumour load (TTL) resulted from the sum of all CK19 mRNA tumour copies/μL of each positive LN from the colectomy specimen. A median of 15 LN were procured per case . Molecular positivity correlated with high-grade (The online version of this article (doi:10.1007/s00428-016-1990-1) contains supplementary material, which is available to authorized users. Surgical resection with no adjuvant therapy is recommended for most stage I–II colorectal cancer (CRC) patients, except for selected high-risk stage II patients given the significant impact of chemotherapy on stage III disease , 2. AlthThe use of additional techniques, i.e. immunohistochemistry (IHC) or reverse transcriptase polymerase chain reaction, makes it possible to find LN tumour burden not detected with conventional HE analysis in 25 to 50 % of CRC patients, due to both increased sensitivity and the more extensive study than usually permitted by histological sections , 10–17.Although the prognostic value of LN molecular tumour detection in early-stage CRC is controversial –18, therMost studies have focused in dichotomic (positive-negative) or semi-quantitative scales (isolated tumour cells (ITC), micro- and macrometastases) to assess molecular results , 19, 20.In this multicentre prospective study, we tried to correlate the TTL, as determined by OSNA, with classical clinical and pathologic high-risk factors, in an effort to determine whether the TTL could be used as an additional factor to better select stage I–II patients at risk of recurrence. Such an approach is now widely used in the treatment of breast cancer , 30, 31.This is a prospective observational study including 10 institutions. Inclusion criteria were patients over 18 years old, with primary histologically confirmed colon cancer, cN0 preoperative diagnosis and positive CK19 IHC of the primary tumour. Exclusion criteria included rectal tumours, non-invasive pTis and pT0 tumours, positive LN on HE, synchronous tumours or other malignancies, cN1, gross adipose tissue involvement by the tumour, metastatic cancer, neo-adjuvant chemotherapy, familial adenomatous polyposis, carcinomas on inflammatory bowel disease and presence of stent-type intraluminal devices.Fresh LN procurement from the mesocolon fat was performed within 50 min after surgical excision. When immediate LN dissection was not possible, the surgical specimen was kept up to 3 h in the refrigerator at 4 °C until LN dissection was done. During the LN harvesting process, the dissection area was kept cold by putting a thick layer of chopped ice under an elevated metallic surface and covered with a clean filter paper for LN dissection . Incubation for 20 min with the primary CK19 antibody  was performed in the AutostainerLink 48 (Dako). Membranous staining with or without cytoplasm staining of ≥10 % of the tumour cells was defined as positive IHC in colon carcinomas . Analysis was performed using the RT-LAMP isothermal amplification method with the RD-100i automated gene-amplification system (Sysmex). The amount of CK19 mRNA amplified was detected by a change in turbidity upon precipitation of magnesium pyrophosphate  [Logistic regression analysis was used to predict the OSNA outcome. The backward stepwise algorithm was used to determine the best-fitting model. The classification avidity of the model was assessed by the ROC curve and a 10-fold cross-validation technique was applied for the model validation. The Cohen’s kappa was used to assess the degree of agreement between OSNA outcome and model prediction. All analyses were performed using V.3.1.0) .We analysed 3512 LN from 211 colon cancer patients recruited in 10 hospitals between June 2012 and December 2013. We excluded 27 non-invasive tumours (9 pT0 and 18 pTis), 34 cases with HE-positive LN, and one patient with synchronous tumours. Finally, 149 stage I–II colon cancer patients met the study selection criteria. All primary tumours showed positivity for CK19 IHC. Demographic, clinical, pathological, and lymph node characteristics of the study sample and correlation with CK19 mRNA results are summarized in Table Among the 2483 LN procured from 149 cases, slightly over 78 % of them were freshly isolated and submitted for OSNA analysis (1940 fresh LN vs 543 FFPE LN). Hence, we obtained a median of 15 LN per case, of which 12 LN were analysed by OSNA and HE.p = 0.012) and had a higher total LN weight per case (p < 0.01) than negative ones .CK19 mRNA positivity was observed in 76/149 patients (51 %). Most of those positive cases; i.e. 80 %, had only 1 to 3 positive LN. Thus, among all OSNA LN analysed, 9,8 % (190/1940) were positive. Association analysis showed that OSNA-positive cases harboured more freshly procured LN , with a trend between histological type and tumour size (p = 0.11). The logistic regression model including OSNA results as dependent variable and tumour size as independent variable adjusted by histological type or grade showed a no significant relation between tumour size and OSNA response .High-grade tumours were significantly larger than their low-grade counterparts , there was a positive trend between CK19 mRNA copies/μL and pT stage; while TTL in pT1 cases was 1280 CK19 mRNA copies/μL and 1790 CK19 mRNA copies/μL in pT2, it rose to 3080 CK19 mRNA copies/μL in pT3 tumours . Cumulative TTL showed that 29 % of patients (22/76) had more than 5000 CK19 mRNA copies/μL. Although not significant (p = 0.01) and tumour size  . When stratified by tumour grade, it showed that among low-grade tumours, TTL increased with tumour stage (pT) (7) Table . This trAt the time of writing of this manuscript, all patients had a follow-up of 2 years ; 8.7 % (13/149) patients recurred . Four of the cases were OSNA positive, with a median TTL of 4375 CK19 mRNA copies/μL ; 2 of the 3 dead patients were OSNA positive.This study quantifies the amount of total tumour load within the lymph nodes of stage I–II colon cancer patients, and suggests that LN molecular detection of CK19 mRNA could become an objective indicator of risk in such individuals , 28, 35.Our findings confirm the presence of undetected nodal tumour burden in early-stage colon cancer using the OSNA method. Compared to previous studies, we have the highest percentage of cases with CK19 mRNA detection in LN (i.e. 51 %) –25, 28. CRC lymph nodes are usually small, especially in early stages of the disease, but may still contain tumour burden, as size is not a good preoperative indicator of LN staging, or a predictor of the presence of tumour –45. AlthOur results show that the presence of CK19 mRNA in regional LN was associated with other classical high-risk factors such as mucinous/signet ring types, histologic high-grade, tumour size and male gender. Moreover, it is noteworthy the trend observed between TTL and pT stage, as pT3 had the highest TTL (3080 CK19 mRNA copies/μL) and rate of CK19 mRNA detection (37/66).We also observed a different behaviour between low and high-grade tumours, with a significant orderly increase of TTL in regional LN with the increase of pT stage and tumour size in low-grade tumours. This phenomenon may be accounted for by the fact that low-grade tumours tend to follow an orderly sequence of accumulation of molecular alterations as they grow and acquire infiltrative proprieties . In contThe biologic significance of LN molecular tumour detection is of leading importance. Although the sole presence of small amounts of tumour cells in regional LN does not necessarily imply poor prognosis, it may indicate a yet undefined risk of disease recurrence. This would explain why, with historical recurrence records of 2.5 % in pT1 N0 tumours, we have found that almost 60 % had some CK19 mRNA tumour burden in their LN , 49. In Quantifying the amount of tumour burden, not just the presence or absence of tumour, in regional lymph nodes is therefore important in prognosticating from molecular results, as has been stated in CRC , 20, 35 Our study has some drawbacks; firstly, a follow-up period of 2 years is a limited time to establish the amount of CK19 mRNA copies that would have clinical significance; thus, the OSNA results could not be correlated with prognosis. Nevertheless, this study was not designed to determine the role of the molecular results as a prognostic factor, but to find out whether the OSNA results correlated with other classical CRC high-risk factors. Secondly, in order to evaluate the histological high-risk factors, we focused on histologically pN0 specimens. To assess the potential predictive value of the TTL, colectomy specimens should be studied regardless of their LN status, thus comparing histological and molecular evaluation methods and their ability to predict disease recurrence.In conclusion, this study identifies the presence of undetected tumour burden in LN of early colon cancer patients. TTL correlates with other CRC classical high-risk factors and may be placed among them. We thereby support CK19 mRNA TTL as a fast and reliable approach to help better stage early colon cancer. Long-term follow-up and validation studies are needed to obtain a predictive prognostic scale for stage I–II colon cancer patients based on the patient’s TTL.AC, adenocarcinoma; AUC, area under the curve; CK19, cytokeratin 19; CRC, colorectal cancer; FFPE, formalin-fixation paraffin-embedding; HE, Haematoxylin and Eosin; IHC, immunohistochemistry; IQR, interquartile range; ITC, isolated tumour cells; LN, lymph node; OSNA, one-step nucleic acid amplification; pT, tumour stage; ROC, receiver-operator characteristic; RT-LAMP, reverse transcription loop-mediated isothermal amplification; TTL, total tumour loadESM 1Material used for fresh lymph node retrieval. (a) Image of the dissection area, with a thick layer of chopped ice beneath an elevated metallic surface. By placing filter paper above, the fresh LN dissection could be performed on a clean surface. (b) Microcentrifuge tubes were also kept cold by holding them in chopped ice (PDF 160 kb)ESM 2Case distribution regarding TTL values and pT stage. The distribution of TTL (CK19 mRNA copies/μL) shows different median values for each pT stage. Copies: CK19 mRNA copies/μl; Cum (%): cumulative percentage; n: number of cases (PDF 108 kb)


The optimal control of blood volume without fluid overload is a main challenge in the daily care of intensive care unit (ICU) patients. Accordingly this study focused on the identification of biomarkers to help characterize fluid overload status.7). Blood and urine samples were taken daily and sodium and water balance strictly calculated resulting in a total cumulative assessment of ∆Na+ and ∆H2O. Furthermore, plasmatic biomarkers ) and Sequential Organ Failure Assessment (SOFA) scores were measured at D2, D5 and D7. Blood volumes were measured with 51Cr fixed on red blood cells at D2 and D7.Sixty-seven patients were studied from ICU admission to day 7  contains supplementary material, which is available to authorized users. The maintenance of optimal blood volume without the development of a positive fluid balance is a major challenge in the daily care of patients suffering from acute traumatic, subarachnoid hemorrhage or infectious inflammatory disorders –10. IndeThe accurate monitoring of fluid balance is therefore crucial in guiding fluid resuscitation. However, there is no gold standard method for complete measurement and the use of input/output charts in intensive care unit (ICU) patients is notorious for being incomplete and inaccurate. Consequently, accurate and reproducible methods to improve the monitoring of fluid balance are essential.Several plasmatic biomarkers may contribute to arbitrating conflict between the control of optimal blood volume and the development of fluid overload: these include stress hormones, such as cortisol and catecholamines; hormones involved in volume regulation by sodium chloride or water retention, such as the renin-angiotensin II-aldosterone system ; vasopreEtude des marqueurs iNnovants de la VOLémie (ENVOL study) was to identify a reliable surrogate biomarker capable of predicting blood volumes and/or cumulative sodium and water balance (∆Na+ and ∆H2O). We also tried to evaluate any relationship between extracellular volume and blood volume measurements. For these purposes, we precisely characterized changes in extracellular and blood volumes and changes in several plasmatic biomarkers involved in volume regulation during the first 7 days of ICU stay.The first aim of the Comité de protection des personnes Ile de France VII) approved the study on December 2011 (reference 11-045). Written informed consent was systematically obtained from all participants included in the study or from a relative, in accordance with French legal ethics.This prospective, 7-day observational study was conducted between March 2012 and September 2014 in the 38-bed Department of Anesthesiology and Intensive Care at Bicêtre University Hospital, in Le Kremlin-Bicêtre, France. The Institutional Review Board of the hospital , aneurysmal subarachnoid hemorrhage (SAH), severe non-cerebral trauma (NCT) or postoperative peritonitis with septic shock (PPS). Patients were included if they required continuous mechanical ventilation on day 2. SBT was defined as brain trauma with a Glasgow coma score of <9. Patients with SAH were included in the presence of a score ≥4 on the World Federation of Neurosurgical Societies (WFNS) scale . Patient0)), and on days 2, 5 and 7  [+, K+, Cl-, urea, creatinine and osmolarity. The urinary electrolytes, creatinine, urea and osmolarity were measured in the morning and the total 24-h output was used to calculate the previous day’s loss of Na+, K+, urea and the creatinine clearance. Weight was carefully measured using weighing beds and recorded on D2 and D7. The baseline weight was that recorded by the patient or a relative.During the ICU stay, general and demographic data were collected, including age, sex, weight, height, simplified index of illness severity after the first 48 h (Simplified Index of Gravity (IGS) II) and admission date. Sequential Organ Failure Assessment (SOFA) scores were measured upon admission (day 0 (D and D7) . Mean arThe daily intravenous fluid administration was based on the monitoring of heart rate, arterial pressure, blood lactate concentration, serial echocardiograms, cardiac filling pressures and output and signs of fluid responsiveness in ventilated patients , 27.+ and ∆H2O). A >36 g ∆Na+ or >4 L ∆H2O was defined as fluid overload [Sodium and fluid balances were calculated daily in order to estimate changes in extracellular space. The previous day’s inputs and outputs of sodium and water were calculated each morning. All other losses were measured, including from ileostomies and external ventricular drainage when present. Sodium losses were measured from all liquids and deducted from sodium intake. The difference between water administration from enteral nutrition and daily crystalloids or colloid infusion and water loss was calculated. Insensitive losses were adjusted for body temperature. The sodium and water gains or losses were calculated daily and added to the previous day’s measurements as cumulative fluid balance (∆Naoverload –18. The 2 and D7 (±1 day), using 10 mL of the patient’s red blood cells (RBC) labeled with radioactive chromium (51Cr-RBC). We re-injected a known quantity of radioactive RBC intravenously and collected two arterial samples 10 and 30 minutes later. From the radioactivity of these samples we derived RBCV in mL/kg using the patient’s body weight recorded before admission [7, we also measured PV by intravenously injecting a small amount of albumin labeled with radioactive iodine , and collected arterial samples at 10 and 30 minutes and at 2 h [125I is 45 ± 10 mL/kg, slightly larger than that measured with 51Cr-RBC [The red blood cell volume (RBCV) was measured on Ddmission . The artdmission . On D7, d at 2 h . The nor51Cr-RBC .2, D5 and D7. MR-proADM, Pro-ANP, renin, angiotensin II, aldosterone, cortisol, norepinephrine and epinephrine, copeptin, pro-endothelin and EPO were measured for potential interference with extracellular or blood volumes. All biologic biomarkers were analyzed together after inclusion in November 2014.Plasma biomarkers were analyzed on DBecause our original aim was to assess the correlation of the biomarkers with intravascular volumes, the study sample size was calculated considering that brain natriuretic peptide (BNP) and EPO are good surrogate markers of intravascular volumes. We then used published values of BNP and EPO concentrations in similar patients , 30 to c2, D5 and D7 values were performed on the log transformed data using the Tukey test after analysis of variance (ANOVA). As our objective was also the measurement of extracellular volume, we also studied fluid overload. For this purpose we considered 4 L and 36 g as ∆H2O and ∆Na+ , respectively, for the cutoff of response variables [Because biomarkers were approximately log-normally distributed, comparisons between Dariables –18.2O and ∆Na+ and SOFA score were performed on the same day as the biomarkers were assessed. Because of the large number of variables that were candidates for inclusion in multivariate analysis, we first used random forest regression [We also calculated the performance of biomarkers to predict a SOFA score >9. The calculation of ∆Hgression  to selec2O and ∆Na+) and SOFA score. The optimal sensitivity/specificity cutoff for predictive variables was calculated, using the non-weighted Youden index. The statistical significance was set at P <0.05. Data are reported as means ± standard deviation (SD), medians (25–75 percentiles) or counts and percentages or 95 % confidence interval (95 % CI).Receiver operating characteristic (ROC) curves were constructed to calculate the performance of the biomarkers in predicting fluid and sodium overload , SAH (n = 20), NCT (n = 20) and PPS (n = 6) were studied. General demographic data, the number of patients studied in each group and SOFA scores on D, SAH  on D2 was observed in 43 patients (64 %) for ∆Na+ and 36 for ∆H2O (54 %), and on D7 in 18 (27 %) for ∆Na+ and 33 (49 %) for ∆H2O. This sodium and water positive balances on D2 and D7 were observed in all groups, with a higher increase in sodium and water in the NCT and PPS than in the SBT and SAH groups. For example, on D2, a cumulative increase in Na+ of 70 ± 32 and 77 ± 28 g was measured in the NCT and PPS group, in contrast to 43 ± 24 and 28 ± 24 g in the SBT and SAH groups  = 7.2∆H2O(L)-4.0; r2 = 0.67; P < 0.0001). As a known indicator of extracellular space, plasma concentration of proteins and variation in weight were related to ∆Na+ and ∆H2O, though these relationships were weak . Like plasma proteins, hemoglobin was weakly related to ∆Na+ (r2 = 0.15) or ∆H20 (r2 = 0.23), P < 0.001.The cumulative sodium and water gains are reported as ∆Naoup Fig. . A fluid51Cr in 62 patients on D2 and in 63 patients on D7 . TBV, RBCV and PV are shown in Fig. 2 (45 %) and 31 on D7 (49 %) were in the normal range. Hypovolemia (TBV <20 %) was present in 34 patients (55 %) on D2 and 32 patients (51 %) on D7. Low RBCV was observed in all but three transfused patients  on D7. We found no significant relationship between TBV or RBCV and ∆Na+ or ∆H2O  and 10 patients on D7 (16 %) were below the 20 % range. Importantly, we found no relationship between PV and ∆Na+ or ∆H2O  and was not related to PV . On D7, PV was measured by 125I-albumin in 58 patients. There was a statistically significant relationship between PV calculated from 51Cr-RBC and PV directly measured with 125I-albumin (P < 0.0001). The slope of the Deming regression was 0.852 (0.610–1.08) with an r2 value of 0.75.The distribution of PV was in the normal range Fig. . Only 132 and decreased significantly on D5 and D7 . MR-proADM and EPO decreased significantly on D7. Cortisol, aldosterone, pro-ANP and pro-endothelin remained unchanged. Plasma norepinephrine concentration was not reliable because it was infused as a treatment.Detailed kinetics of all biomarkers are shown in Table P = 0.01 and P = 0.03) and MR-proADM to ∆H2O (P < 10-5). SOFA was related to ∆Na+ (P < 10-5), MR-proADM (P < 10-5), and EPO (P = 0.03) . MR-proADM has good discriminative properties with an area under the curve (AUC) of 0.838 (0.780–0.888) for ∆H2O and an AUC of 0.823 (0.764–0.880) for ∆Na+ , sensitivity 0.865 (0.787–0.933)) for ∆Na+ and 1.125 nmol/L ) for ∆H2O. Furthermore, MR-proADM predicted a SOFA score >9 with an AUC of 0.750 (0.658–0.830) and a threshold at 1.035 nmol/L (specificity 0.635 (0.560–0.711), sensitivity 0.762 (0.643–0.881)).We constructed ROC curves with MR-proADM because it was the only predictor covariate common to the three variables tested (∆HP < 10-5) but the ROC curve constructed for a 50 % decrease in RBCV revealed a low AUC (0.70 (0.60–0.78)).We also tested the relationships between biomarkers and measured blood volumes. It is noteworthy that among all biomarkers, only EPO was related to RBCV (2 and D7. We found that MR-proADM thresholds of 0.865 nmol/L for ∆Na+ and 1.125 nmol/L for ∆H20 were predictive of fluid and of salt overloads, respectively. Moreover, MR-proADM was related to the concurrent SOFA score.This study revealed that MR-proADM, a biomarker of endothelial permeability , 23, mayExcessive sodium and fluid balance is a risk factor for morbidity and mortality in critically ill patients –9, 33. AWhile MR-proADM seems to be a particularly reliable indicator of sodium and fluid balance, it does not exclusively indicate capillary permeability. Indeed, MR-proADM is a stable fragment of pro-adrenomedullin which reflects levels of the rapidly degraded active peptide adrenomedullin . In addiIn the ICU, fluid balance is evaluated by the use of input/output charts and by the daily measure of weight. However, it is time consuming and difficult to perform accurately. In addition, the present study identified a weak relationship between weight and fluid balance. Measurement of patient’s weight in inflammatory disorders may be biased by practical issues but also by loss of muscle mass, interfering with the estimate of fluid overload. Moreover, we found that the usual markers of extracellular volume, such as plasma proteins and hemoglobin also had a weak relationship with sodium or water balance. Thus, the monitoring of sodium and fluid balance is currently a difficult task using tools that have their own limits. MR-proADM concentration, on the other hand, offers a measure of sodium balance and extracellular space, which could be used as a good surrogate to improve fluid balance monitoring. Possibly, it will be useful in the future to test a score centered on MR-proADM but also taking into account simple values such as basal weight or plasma proteins, to easily obtain an even better measurement.+ or ∆H2O are not predictors of blood volume. We found no relationship between fluid balance and plasma volume. This was particularly unexpected, because plasma volume expansion is the main justification for fluid infusion. Yet, plasma was the only volume that was in the normal range on D2 and D7. The absence of correlation between PV and fluid balance supports the hypothesis of possible trapping of Na+ and water in the interstitial volume [No marker accurately estimated the TBV, PV or RBCV. Moreover, ∆Nal volume , 37. Tig51Cr and directly measured with 125I-albumin at D7 strengthened our results. The distribution volume of albumin may be larger than that obtained with RBC, especially when the capillary permeability is pathologically increased. On D7 the difference was weak, suggesting that the capillary permeability was nearly repaired. While it would have been worthwhile to examine this comparison on D2, this was precluded by the interactions between 125I and some of the measurements of biomarkers.The good correlation between the measurements of PV deducted from RBCV with 51Cr is a recognized method but requires time [The observation of a decreased RBCV is common and explains low TBV. The measurement of RBCV with res time . As hemares time . EPO is 2, D5 and D7, long after the admission of patients into the ICU, often after the peak of the disease manifestation. The distribution of extracellular volume may be different at D0 during initial resuscitation. The precise timing of measurements may also be a critical factor. While our study observed the changes that took place on D2 and D7 after an acute event, further studies are needed to fill the gaps.The present study has some limitations. First, we found no marker that describes blood volume. We need a better understanding of all physiologic determinants, including capillary permeability, hormonal influences and low RBCV and interactions among theses determinants. On the other hand, we identified a biomarker of cumulative salt and water balance, which could be an interesting tool for fluid management in the ICU. Second, we made our study measurements on DOur study was observational. Patients who were included reflect the daily practice of our service. However, the group of patients as a covariate did not influence the prediction of fluid balance or blood volume, suggesting that volume abnormalities are independent of the pathology. Other studies will need to confirm our results.A positive sodium balance is an important negative prognostic factor. We found that MR-proADM is a valuable surrogate to evaluate sodium and fluid overload in the first week after an acute and critical inflammatory illness. MR-proADM could improve the fluid balance monitoring and guide fluid resuscitation and administration of vasopressors or diuretics. After this first step, further studies are needed to test the ability of MR-proADM monitoring to control or prevent fluid overload and organ failure.


The pancreatic pseudocyst is a pancreatic fluid collection which classically develops due to acute or chronic pancreatitis. A 68-year-old male with the remote history of alcohol abuse presented with abdominal pain secondary to acute pancreatitis. The first computed tomography (CT) of the abdomen showed acute necrotizing pancreatitis. He was initially treated conservatively. Repeat CT of the abdomen after two weeks revealed a peripancreatic fluid collection of 20x12x10 cm. One month later, he became septic following biliary stent placement. Repeat CT of the abdomen showed an enlarging pseudocyst of 25x20x14 cm (estimated 7000 mL of fluid). Percutaneous CT-guided cyst drainage was performed and only three liters of infected fluid could be drained which eventually grew Enterococcus faecalis. Due to lack of improvement, he underwent laparotomy with pancreatic necrosectomy, pseudocyst debridement, and cholecystectomy. The patient did well postoperatively and until one-year follow-up visit. The largest pancreatic pseudocyst in the literature (about 9500 mL) was reported in 1882. To our knowledge, this case is the second largest pseudocyst in the literature which was successfully managed by surgical resection.  The pancreatic pseudocyst is a pancreatic fluid collection which classically develops four weeks after acute pancreatitis, with an approximate incidence rate of 5-15% . It can A 68-year-old Caucasian male with a remote history of heavy alcohol abuse who presented with acute abdominal pain and vomiting secondary to acute pancreatitis. The initial computed tomography (CT) of the abdomen showed acute necrotizing pancreatitis with no loculated peripancreatic fluid collection. The ultrasound of the abdomen showed a sludge-filled gallbladder. The patient was initially treated conservatively with no significant improvement. Follow-up CT of the abdomen after two weeks revealed a peripancreatic fluid collection of 20x12x10 cm (estimated 2400 mL of fluid). The prolonged hospital course was complicated by multi-organ failure, mechanical ventilation use, total parenteral nutrition (TPN) administration, and Candida fungemia. No surgical intervention was needed. One month later, the patient underwent endoscopic retrograde cholangio-pancreatography (ERCP) with balloon extraction of choledocholithiasis and stent placement. Subsequently, he became encephalopathic and septic. Repeat CT scan of the abdomen showed marked interval increase in the size of the pancreatic pseudocyst to 25x20x14 cm (estimated 7000 mL of fluid) , fluid infection, hemorrhage, and fistula formation ,5. TheraThe term giant pancreatic pseudocyst is traditionally used when the size is greater than 10 cm . HoweverThe pancreatic pseudocyst is one of the consequences of pancreatitis. It can result in serious complications which may require surgical intervention. Serial abdominal imaging is an important tool to monitor these cysts. Spontaneous resolution may occur, but pseudocyst may continue growing in size in some cases. This case is reporting the second largest pancreatic pseudocyst in the literature after the cyst that was found in 1882.


In the original publication of this article the correspondence authors’ e-mail has been missed out.The missing e-mail addresses are provided in this erratum.


Some of our colleagues believe that everything should be tested in prospective, randomized clinical trials. Some of our colleagues also believe that so-called pragmatic trials can help improve the care of critically ill patients. The designs of such studies are based on the postulate that patients are all the same and will be globally either improved or harmed by one strategy compared with another. This may be true for some interventions but is certainly not true for all, including choice of crystalloid solution.The difference between saline solutions and balanced solutions is that saline solutions contain 154 mEq/L of sodium and chloride (to be normotonic) and balanced solutions contain anions other than chloride. Hence, liberal administration of saline solutions results in hyperchloremia , 3. WithThe choice of intravenous fluid should be based on consideration of certain baseline factors, but also on elements that arise during therapy. For example, at baseline, administration of saline may be indicated in the presence of hypochloremia whereas a hypotonic fluid may be warranted in the presence of hypernatremia. Importantly, the situation may evolve with time as the fluid administered may induce metabolic changes leading to a different “best” fluid choice. Giving several liters of 0.9 % saline will result in hyperchloremia, so continuing this fluid strategy is not rational.Let us imagine a clinical trial in which saline is compared with a balanced solution Fig. . In the In scenario B, administration of saline solutions is continued, regardless of the test results showing increasing hyperchloremia. Here it is likely that the trial would demonstrate worse outcomes in the saline arm. The overall conclusion would be that saline solutions should be banned or that hyperchloremia should be prevented—these findings could have been predicted without doing the trial! Moreover, the trial would be criticized as it deviates from clinical practice.Such a study may also have important “negative” clinical and economic consequences. In scenario A, one can argue that, with no differences between the study arms, one should select the cheapest solution and abandon use of costlier, balanced solutions, at least initially. This may result in administration of large amounts of sodium chloride, a condition that was not actually tested in the trial. In scenario B, saline solutions may be banned altogether and only balanced solutions used, thus unduly increasing health care costs. And saline solutions may then no longer be available for specific indications, such as traumatic brain injury (for its normotonicity) or metabolic alkalosis.We rather propose improved education of the medical community regarding the chemical composition of crystalloid solutions and better understanding of how solutions should be selected depending on each patient’s individual situation. We as clinicians should check blood chloride levels exactly as we do sodium and potassium levels and adapt our fluid choices accordingly.The real issue for which a fluid trial could be of use is to look at potential beneficial/detrimental effects of added solutes, such as acetate, gluconate, and even calcium or magnesium supplementation, but this would require a particular design.We should be pleased that we have the choice of several different intravenous solutions, just as we are happy to be able to have a choice of liquids to drink. Any prospective, randomized controlled trial on different types of oral fluid would certainly indicate that excessive amounts of most drinks, especially when they contain alcohol, high sugar, or stimulants, are bad for the body. This does not mean that we should only drink water. But we should vary the types of liquid we drink and drink all liquids in moderate, responsible amounts. Similarly, we should not limit our intravenous fluid choices to just one or two types and should not give any one type in excess.Undoubtedly, the safest intravenous solution in a patient without major metabolic abnormalities would have a composition close to that of human plasma. It has been said that bicarbonate is unstable when in solution, but this is not true as bicarbonate solutions are readily available from the shelf; actually, renal substitution fluids for continuous hemofiltration contain a mixture of electrolytes, including bicarbonate. Some people already administer these solutions as regular intravenous solutions but the 5-liter bag size complicates this practice. The industry is hesitant to launch such a solution, in anticipation of the costly clinical trials that may be required by the authorities. We argue that a fluid with such a natural composition does not need extensive clinical trials. The scientific community should request that such solutions be developed and insist that the authorities minimize the requirements needed to commercialize such a solution.There are only a few indications for repeated saline administration: metabolic alkalosis (for the high chloride input) and hyponatremia (for the high sodium input), as well as severe brain injury for its normotonic composition. When such conditions are not present, administration of saline solutions should usually be restricted to not more than 1 liter per 24-h period.Comparing two intravenous solutions with different compositions makes no sense… unless electrolyte measurements are not available.None.