Pseudomonas aeruginosa (P. aeruginosa) isolates from cystic fibrosis (CF) patients and compared to 80 non-CF P. aeruginosa. Phospholipids gave the highest signal intensity; 17 rhamnolipids and 18 quorum sensing molecules were detected, demonstrating that REIMS has potential for the study of virulence-related metabolites. P. aeruginosa isolates obtained from respiratory samples showed a higher diversity, which was attributed to the chronic nature of most respiratory infections. The analytical sensitivity of REIMS allowed the detection of a metabolome that could be used to classify individual P. aeruginosa isolates after repeated culturing with 81% accuracy, and an average 83% concordance with multilocus sequence typing. This study underpins the capacities of REIMS as a tool with clinical applications, such as metabolic phenotyping of the important CF pathogen P. aeruginosa, and highlights the potential of metabolic fingerprinting for fine scale characterisation at a sub-species level.Rapid evaporative ionisation mass spectrometry (REIMS) is a novel technique for the real-time analysis of biological material. It works by conducting an electrical current through a sample, causing it to rapidly heat and evaporate, with the analyte containing vapour channelled to a mass spectrometer. It was used to characterise the metabolome of 45  Pseudomonas aeruginosa is a ubiquitous, gram-negative bacterium which is readily eliminated by a healthy immune system. However, it can cause both acute and chronic infections, with substantial morbidity and mortality, in a number of clinical scenarios: acute in intubated patients, those with burns, cancer, immunosuppression, and chronic in cystic fibrosis (CF) and other forms of chronic suppurative lung disease1. Indeed, up to 60% of adult CF patients are chronically infected with P. aeruginosa which is associated with a poorer prognosis2. The genome of P. aeruginosa is one of the largest in the bacterial kingdom3, 10% of it being dedicated to environmental adaptation4. Virulence and persistence-associated mechanisms include a complex quorum sensing communication system which controls the transition to the more resistant biofilm mode of growth5. Amongst numerous varieties of secreted toxins, the bio-surfactant rhamnolipids play a multilevel role, facilitating access to nutrients and exhibiting adverse effects on host immune cells or microbial competitors6. P. aeruginosa is thus equipped with a wide range of adaptive responses through versatile metabolic strategies, allowing it to adjust to varying nutrient and oxygen availability, host immune factors and infecting competitors. This makes it particularly suited to the environmental niche of the CF airway7.9. Currently, the main MS platforms which have been introduced use matrix assisted laser desorption ionisation (MALDI) coupled to a time-of-flight (ToF) mass spectrometer. The analysis is conducted on pure bacterial cells embedded in a UV-absorbent matrix which aids ionisation. Microbial identification is based upon the detection of protein fingerprints, mainly of ribosomal origin10. Clinical platforms such as the Microflex LT  and the Vitek MS (bioMerieux) are widely available and MALDI-ToF is now recognised as a standard method for the identification of microorganisms. Whilst these systems accurately classify P. aeruginosa, other genera prove problematic and overall species level accuracy is 80–90%9. Besides, the embedding matrix required for MALDI analysis substantially limits its ability to study lower molecular weight compounds because of the presence of high matrix signal below the mass over charge ratio (m/z) 1000. Quorum sensing molecules (QSMs)11 and rhamnolipids12 have previously been detected using MALDI-ToF, but these methods required extraction from bacterial cultures and a chemical derivatisation or labelling step.During the last decade, MS has established itself in clinical laboratories as a quick, reliable, and low-cost method for microbial identification13. The main methods for detecting high-risk strains rely on molecular typing of DNA, including pulsed-field gel electrophoresis (PFGE), variable-number tandem repeats (VNTR), multilocus sequence typing (MLST), and whole genome sequencing (WGS)14. MLST is based upon the DNA sequencing of multiple, highly conserved, housekeeping genes15. Currently, the commercially available MS platforms cannot provide strain level classification of microbial species. Nevertheless, MALDI-MS has shown some potential when performed on bacterial protein extracts. To some extent, MALDI-ToF achieved the differentiation of outbreak strains of Acinetobacter baumannii16, Staphylococcus aureus lineages17, and five P. aeruginosa high-risk clones18. Fleurbaaij et al. recently showed that the ultrahigh resolution and increased dynamic range obtained with a Fourier transform ion cyclotron resonance detector allowed the classification of strains of P. aeruginosa in accordance with cluster analysis based on amplification fragment length polymorphism (AFLP), which was not achieved with a ToF detector19. Unfortunately, this technology remains excessively expensive and cumbersome, making it unsuitable for clinical implementation. Typing methods typically detect changes in slowly evolving regions of the genome however, finer scale characterisation may be a useful adjunct when investigating rapidly evolving characteristics such as virulence determinants.In clinical microbiology, strain level identification is important in understanding epidemiology, population structure, molecular evolution, and in the control of nosocomial and cross-infectionsin situ method for tumour margin detection20, it has recently been adapted to the study of microbial isolates cultured on agar plates21. This method has previously been shown to accurately classify clinically important yeast and bacteria to species level23. REIMS operates by subjecting microbial biomass grown on a standard agar plate to electric current using electrosurgical bipolar forceps. This causes the biomass to rapidly heat, resulting in the evaporation of particles containing ionised metabolites and structural lipids. The gas phase particles are then introduced into the mass spectrometer using the instrument’s vacuum system23. Typically, the REIMS spectrum of microorganisms comprises a wide range of structural lipids, originating from cell membranes24. The phospholipid profile detected in the 600 to 900 m/z range provides robust, accurate classification to Gram, genus, and species level23.Rapid evaporative ionisation mass spectrometry (REIMS) is an ambient ionisation MS technique which allows the real-time analysis of biological material. Originally developed as an intra-operative, P. aeruginosa cultures. We evaluated P. aeruginosa intra-species metabolic diversity and looked for virulence-associated metabolites. Additionally, we tested the capacities of REIMS for fine scale P. aeruginosa characterisation at a sub-species level.Unlike other MS methods, REIMS provides valuable metabolic information without sample extraction. Here we have explored the potential of REIMS for the direct analysis of P. aeruginosa colonies is presented in Fig. m/z range, deriving mostly from phosphatidyletanolamines (PE) and phosphatidylglycerols (PG). Amongst the most intense phospholipid features, PA(18:1/16:0), PE(18:1/16:1), PE(18:1/16:0), PG(18:1/16:1), PG(18:1/16:0), PG(19:1/16:0), PG(18:1/17:0), PG(18:0/17:1), PG(18:1/18:1), PG(19:1/17:1) were identified using tandem MS  based on their fragmentation pattern , mixed with 2-heptyl-4-hydroxyquinoline n-oxide (HQNO), usually presented the most intense peak at m/z = 258.1472. PQS is produced during the logarithmic phase of growth26, and levels in the airways of infected CF patients have been shown to correlate with infection stage27.Twelve ern Fig. . Both chm/z range 330–710, as shown in mass spectral annotations in Fig. P. aeruginosa growth in harsh environments. Rhamnolipids trigger inflammatory reactions in the lungs and inhibit ciliary function in the host6. They have been shown to be required for chronic infection to become established29, and high levels correlate with clinical decline in CF patients29. Additionally, they exhibit antimicrobial activity against a wide range of bacteria and fungi; potentially aiding P. aeruginosa’s dominance in the adult CF airway. Rhamnolipids also make a noteworthy contribution to PQS activity by enhancing the solubility of PQS in aqueous solutions, thus easing its diffusion from cell to cell30. They are involved in swarming motility, and biofilm development by regulating cell-surface physicochemistry, and further cell detachment and dispersion31. The overall surfactant properties are determined by the composition of the rhamnolipid profile which relies on strain variations and carbon source availability32. Rha-C10-C10 and Rha-Rha-C10-C10 are usually reported to be the most abundant compounds32. Yet, within the present population of P. aeruginosa isolates, the di-rhamnolipids Rha-Rha-C10, Rha-Rha-C10-C10, and Rha-Rha-C10-C12 appeared to be highly prevalent. This indicates disparity in the expressed DNA of our sample set, and/or differences in the growing environment and available nutrients, in comparison to the previous studies.Likewise, a total of 17 rhamnolipids were identified in the P. aeruginosa collected from CF patient samples were compared to P. aeruginosa isolated from non-CF patients which included respiratory samples of patients with bronchiectasis and non-respiratory samples . Given the high number of features detected with REIMS for every single analysis, it is necessary to employ statistical tools to reduce the dimensionality of the data in order to allow interpretation. Principal component analysis (PCA) is an unsupervised procedure commonly used to condense the information within a dataset into a reduced number of principal components which allow to visualise the highest variance of the results. Partial least square-discriminant analysis (PLS-DA) on the other hand is a supervised method. Whereas unsupervised PCA summarizes the information in an impartial manner, supervised PLS-DA seeks to represent the data according to the variables which allow best the separation of the classes of interest. PCA and PLS-DA plots in Fig. P. aeruginosa isolates. Although the areas overlap, isolates from the airways of CF and bronchiectasis patients showed substantially higher metabolic diversity compared to those collected from non-respiratory tract samples in Fig. m/z range which is typical for phospholipids. They allowed a relative separation between CF from non-respiratory isolates , and eight types had at least two isolates  was predicted below 0.5 in accordance with its type (ST_0633), 60% of the replicates were accurately predicted above the 0.83 average, and 40% were above 0.9. In the case of ST_0179 and ST_0395, MS data revealed differences in the quorum sensing and phospholipid regions respectively, thereby highlighting the higher resolution of the information provided by REIMS. In the case of ST_0633, and other poorly predicted types with single isolates, the low classification scores are likely to be due to insufficient reproducibility, especially in the case of mucoid strains.To assess the utility of REIMS for high resolution strain level characterisation, 45 isolates were typed by MLST. Whilst MLST typing methods differ from REIMS as it provides information on slowly evolving genomic regions, the data allows a better understanding of the genetic relationships within the isolates examined. This phylogenetic ‘backbone’ allows us to then explore the additional information offered by REIMS as a finer scale characterisation tool. Of the 45 P. aeruginosa isolates from respiratory samples displayed a substantially higher diversity in their metabolic profile, suggesting that REIMS is able to detect subsequent changes in isolates exposed to different conditions. It also allowed the detection and identification of lower mass metabolites, including virulence-associated QSMs and rhamnolipids. These are key factors for an infection to progress to chronicity, yet the study of this type of metabolites is limited by the performance, the cost, and the complexity of available technologies. The routine detection of these metabolites is not possible with the current clinical MALDI-ToF platforms, and REIMS could be useful in the assessment of pathogen virulence, potentially assisting in risk stratification.m/z, including phospholipids as well as low molecular weight metabolites, such as QSMs and rhamnolipids, which have been shown to vary between strains of P. aeruginosa34. Some isolates grouped under the same type showed a similar metabolome, whereas others displayed spectral differences, particularly in the quorum sensing and phospholipid ranges. Whilst traditional typing tools, such as MLST and VNTR, rely on slowly evolving genomic regions, REIMS could be used as an adjunct to provide fine scale classification, based on more rapidly evolving metabolic expression. It has been suggested that ‘resistance is often concentrated in a few strains’ which are more frequently observed within hospital isolates35. These strains can be found worldwide and have been dubbed ‘high- risk clones’36. Further research is needed to demonstrate if REIMS can be used to identify virulence-associated variations which would allow the rapid identification of such high-risk clones.In addition, REIMS classified strains with 83% concordance to MLST types, suggesting that metabolic fingerprinting can provide some strain level information. Indeed, the Random Forest model utilised the whole range of 21. The population structure at the time of analysis, i.e. whether the bacteria were growing as single colonies or as a lawn, also impacted signal intensities in the quorum sensing and rhamnolipid ranges (unpublished data). Therefore, for the purpose of this study, growing time and medium were standardised, and biomass of each sample was analysed from various sites on the plates to account for the natural metabolic diversity within one isolate. Nevertheless, it will be interesting to investigate these changes more precisely in the future, as REIMS improves our understanding of how P. aeruginosa evolves and adapts to its environment. Furthermore, the mucoid isolates ; 45 from non-respiratory tract samples, as part of routine clinical identification at Charing Cross Hospital . These P. aeruginosa isolates were cultured from samples collected from wounds, blood cultures, urine, drain fluid, and ear, vaginal, perineum, rectum, and skin swabs. After initial collection and culturing, all isolates were stored at −80 °C in CryoVials , following manufacturer’s guidelines, until cultured for REIMS analysis.CF bacterial isolates had originally been cultured by the Clinical Microbiology Laboratory of the Royal Brompton Hospital  from sputum and cough swabs collected from CF patients attending routine clinical visits. The isolates had been first identified through culture on selective cetrimide agar according to standard laboratory practice. Additional isolates of 23. In brief, single colonies were placed onto a 24-spot steel plate and embedded in an acidic solution of α-cyano-4-hydroxycinnamic acid as a UV-adsorbent matrix. Samples were analysed after drying following the manufacturer’s instructions. Data were processed using the Bruker Biotyper software (version 3.0) and library (version 5.0) for taxonomic classification of the isolates. The identity of the 135 P. aeruginosa isolates were confirmed.The identity of all microbial species was verified using the Microflex LT MALDI-ToF instrument  as previously describedP. aeruginosa isolates from CF patients were grown and analysed ten times on different days, six grew mucoid more than once  were used as a sampling probe. A biomass covering approximately 2 mm2 of one probe was collected directly from the medium. On drawing the probes together an electrical power of 70 W was applied using an electrical surgical generator  causing the bacterial material to rapidly heat. The resulting aerosol was channelled to a XEVO G2-XS Q-ToF instrument . Prior to the entry of the analyte containing vapour to the MS instrument, it was mixed in a T-piece device with isopropanol  containing 0.01 g/L of leucine encephalin  as a lock mass compound, with a solvent flow rate of 0.2 mL/min. Negative ions were detected in sensitivity mode over the 50 to 2500 m/z range, at a scan time of 1 second, with a resolution of 22,000. A minimum of three individual measurements were acquired per isolate analysis.Prior to REIMS analysis, isolates were cultured on Colombia blood agar  for 24 hours at 37 °C in normal aerobic atmosphere. The 45 m/z 554.2516. Resulting spectra were normalised according to total ion count and binned to 0.1 m/z bins. The lockmass compound peak and its isotopes (555.2518 and 556.2485) were excluded from further statistical processing. Statistical analysis was carried out afterwards on the Metaboanalyst 3.0 platform40. Generalized logarithmic transformation and Pareto scaling (data mean-centring and division by the square root of standard deviation of each variable) were applied on the 5000 most intense median bin values. Principal component analysis (PCA), partial least square – discriminant analysis (PLS-DA) and hierarchical cluster analysis (HCA) were then completed. Supervised classification was performed using the machine learning scikit-learn package41 and Random Forest algorithm42. The F1 scores – harmonic mean of the precision (the fraction of the prediction that is accurate) and the sensitivity (the fraction of samples that are correctly predicted) – were calculated and used to assess the classification accuracy. Ions of interest were annotated using tandem mass spectrometry and fragment identification.Data pre-processing was performed using the Offline Model Builder (version 1.1.29.0) software  and included combining scan measurements per sample, background subtraction, and correction for mass drift using leucine-enkephalin negative ion P. aeruginosa isolates collected from CF patients were cultured on Colombia blood agar (Oxoid) for 24 hours at 37 °C, under normal aerobic conditions. For each isolate, a single colony from the agar culture plate was used to inoculate 10 mL of brain heart infusion broth (Oxoid) and then cultured overnight at 37 °C, under a normal aerobic atmosphere. After this, a 2 mL aliquot of culture media was subjected to centrifugation for three minutes at 21,000 × g. The supernatant was removed and the cell pellet used for DNA extraction using a Fast DNA SPIN kit , following the manufacturer’s standard instructions. Bead beating was performed in FastPrep 24 instrument  using three consecutive cycles of 30 seconds at 6.0 m/sec, each divided by cooling at −20 °C for 3 min. DNA was eluted into 100 µL of PCR grade water . The success of DNA extractions was determined through visualisation of 5 µL of eluted DNA after running on an 1.0% agarose gel for one hour at 100 volts in 1X tris acetic acid EDTA (TAE) buffer. All isolate extractions displayed an intense band of high molecular weight DNA (>20 kbp). To determine DNA concentration, 1 µL of a one in ten dilution, using PCR grade water (Roche), was used in Qubit fluorometery using the dsDNA Broad sensitivity kit  and a Qubit 3.0 fluorometer for DNA concentration. The diluted DNA was carried forwarded for whole genome sequencing.The 45 P. aeruginosa libraries were run alongside 339 other DNA sequencing libraries so that 384 libraries were sequenced on one run of the HiSeq. 2500 platform. Resulting DNA sequences were merged, trimmed for quality, and aligned to the P. aeruginosa reference genome as part of the MicrobesNG sequence analysis pipeline. From the whole genome sequence assemblies for each isolate, the relevant gene sequences for the seven housekeeping genes used in the MLST scheme43 were extracted and the PubMLST website used to complete strain match searches under standard operating parameters. Five novel MLST types were assigned a new ST number through the PubMLST website where the data have been deposited.Whole genome sequencing was completed by the MicrobesNG service at the University of Birmingham  using the HiSeq. 2500 platform with 2 × 250 bp sequencing. DNA sequencing libraries were prepared using the NexteraXT kit . As part of the MicrobesNG service, the 45 The datasets generated and analysed during the current study are available on the MetaboLights website, study number MTBLS644.Supporting Information


To compare volumetric arc therapy (VMAT) and helical tomotherapy (HT) plans in terms of dosimetric parameters in positron emission tomography- (PET-) computerized tomography- (CT-) based radiation therapy planning in unresectable malignant pleural mesothelioma (MPM). 1) and PTV2 were 45.0 Gy and 54 Gy in 1.8 Gy/fr, respectively. Each technique was evaluated in terms of target volume coverage and OAR doses.  CT and coregistered PET-CT data from seven patients with histologically-proven MPM were utilized for VMAT and HT plans. Target volumes and organs at risk (OARs) were delineated. The prescription doses for planning target volume 1 (PTVp=0.001) and mean (p < 0.001) doses of PTV1, and PTV2 (p < 0.001 for maximum and p=0.001 for mean doses) favored the HT technique over VMAT, both techniques efficiently covered the target volumes. Additionally, HT also provided more homogeneous dose distribution (p < 0.001) and numerically lower doses received by most OARs, but again both rotational techniques were successful in keeping the OAR doses below the universally accepted limits. The major disadvantage of the HT technique was the requirement for longer treatment times  to accomplish the intended treatment.  Although the maximum ( Results of this dosimetric comparison clearly demonstrated the possibility of safe hemithoracic irradiation of medically/technically unresectable MPM patients with either of the two rotational RT techniques, namely the VMAT and HT. Clinically, considering their poor prognosis, these promising findings may open a potential new window for curative treatment of unresectable MPM patients, if further confirmed by future clinical studies. Malignant pleural mesothelioma (MPM) is a rare, but commonly fatal tumor. Despite the fact that extrapleural pneumonectomy (EPP) was viewed as the surgical standard for MPM, results of the Mesothelioma and Radical Surgery (MARS) trial challenged this tradition in favor of the pleurectomy/decortication (P/D) procedure , 2. TaioUntil recently, barring the hemithoracic irradiation after EPP, the primary role of radiotherapy (RT) was limited to prophylactic irradiation of the drain sites and palliation of symptomatic lesions in MPM. RT planning (RTP) for post-EPP hemithoracic RT is relatively straightforward with two traditional opposing photon beam fields and electron boost fields if required, which permits better sparing of the contralateral intact lung. However, because of the high risk of severe or even fatal acute and late pulmonary toxicities, this technique is almost never applicable for unresectable MPM for patients with bilateral intact lungs. Induction chemotherapy may alternatively increase the resection rates on a hypothetical basis, yet considering the fact that the response to induction chemotherapy is usually far less than the prerequisites for any type of curative surgery, RT still stays to be the main potential for cure or palliation in most MPM patients . RTP witTo our best information, negating the patients' requirement for alternative RT options and great innovations in diagnostic and RTP methodologies, to date no studies have compared the promising VMAT and HT dosimetrically in the setting of post-P/D or unresectable MPM where both lungs remain in place. Therefore, we aimed to compare the VMAT and HT techniques dosimetrically with regards to the tumor coverage and OAR doses.2-4N0-3M0 MPM patients as per the AJCC-7 staging system [We reviewed our patients' records to identify stage Tg system  who weren, where n was an integer.Seven patients with unresectable MPM who underwent positron emission tomography-computerized tomography (PET-CT) imaging in the supine position with both arms raised over their heads utilizing the T-bar to mimic set-up conditions were selected. The PET-CT scans were performed in an integrated PET-CT system  using a previously published imaging procedure . PET-CT Target volumes were defined both on the diagnostic CT and integrated PET-CT images by one of the two radiation oncologists with specific experience in RTP of MPM. The window and level for the PET images were set according to a method previously described by Erdi et al. . In this30 (volume receiving 30 Gy) < 50% and mean dose < 28–32 Gy for the liver, V35 < 50%, V50 < 40%, and mean dose < 34 Gy for the esophagus, and mean dose < 26 Gy for the heart were allowed. The intended lung dose limitations were as follows: total mean lung dose ≤ 21 Gy, V20 ≤ 40%, and contralateral lung V20 ≤ 7%.Diagnostic CT data sets were utilized for delineation of OARs due to the difficulties in defining the anatomic borders of organs with PET. For each patient, the OARs included the right and left lungs, total lung, liver, heart, esophagus, spinal cord, and right and left kidneys. For planning purposes, the Quantitative Analyses of Normal Tissue Effects in Clinic (QUANTEC) radiation dose constraints were applied . Accordi1 and PTV2 were 45.0 Gy  and 54 Gy , respectively. The primary end point was to deliver the prescribed doses to ≥95% of the each PTV without sacrificing OAR dose constraints. For this purpose, the created dose volume histograms were utilized for comparisons between the VMAT and HT plans in terms of the PTV coverage and OAR doses. Comparisons between the VMAT and HT plans were performed by analyzing mean, V5 (volume receiving 5 Gy), V10, and V20 doses for each lung and mean and V20 doses for the total lung, mean dose for the heart, mean doses for each kidney, maximum dose for the spinal cord, and mean and V30 doses for the liver. Conformity index (CI), homogeneity index (HI), total monitor units, and total treatment times were calculated for further comparisons. The CI and HI were defined as follows:Imax: maximum dose; RI: prescribed dose for PTV.The prescription doses for PTV45 (p=0.81) and PTV54 (p=0.65). However, the respective PTV45 maximum  and mean  and PTV54 maximum  and mean  doses favored the HT plan over the VMAT. Likewise, albeit the CI of both plans were similar , the HT plan provided significantly more homogeneous dose distribution than did the VMAT plan .Patient characteristics are shown in p=0.007) and median V10  doses were significantly lower with HT than with the VMAT plan. The median contralateral lung V5 , V10 , and V20  doses were likewise significantly lower in HT plans. Similarly, the total lung mean  and V20  doses also favored the HT over the VMAT plans in a significant manner. However, HT plans appeared to be preferable, yet both plans were judged to be acceptable for clinical use as the lung constraints were met with the either technique with regards to the QUANTEC recommendations for curative lung irradiation.Considering the OAR doses, the ipsilateral lung mean ; heart mean (p=0.006), V25 (p=0.002), and V30 (p=0.001) doses; and esophageal mean (p=0.001), V35 (p=0.017), and V50 (p=0.024) doses were also significantly better with HT than with VMAT plans, as depicted in p < 0.001) and, therefore, longer treatment times (7.4 versus 2.5 minutes/fr) compared with the VMAT procedure.The other OAR doses, namely the maximum spinal cord (n = 7) with two intact lungs exhibited that, in spite of the fact that the ipsi-, contralateral, and total lung and most OAR radiation doses were altogether notably lower in HT than the VMAT designs, both rotational RT techniques were successful in creating RTPs applicable to clinical practice in terms of target volume coverage and OAR doses defined by the QUANTEC report. However, our encouraging results also revealed that these relative advantages of HT plans came at the cost of significantly longer irradiation times.The outcomes of the present dosimetric comparison in unresectable MPM patients (p < 0.05) [Past investigations clearly demonstrated that both the VMAT and HT procedures were highly efficient to create excellent dose distributions in numerous tumor types, including the MPM , 15, 16. < 0.05) . Kishan  < 0.05) . Moreove < 0.05) , 20.20 (29.3% for HT and 36.2% Gy for VMAT) of total lung, and contralateral lung V20 (0.5% for HT and 3.5% Gy for VMAT) doses observed here in both techniques meet the predefined risk of less than 20% symptomatic pneumonitis, separately. Similarly, these results are also accordant with the respective values of mean < 20 Gy and V20 ≤ 37% for total lung dose constraints specified in the landmark lung cancer dose escalation study of the RTOG 0617 protocol [Despite of the proven efficacy of IMRT in MPM patients with intact lungs, the disappointingly high fatal pulmonary toxicity rates, as high as 46%, hampers its routine use in radiation oncology clinics –24. Thesprotocol . MoreoveEscalated RT doses may potentially improve local control rates which may translate into a survival advantage in nonmetastatic MPM patients . Underscp < 0.001), its feasibility may be questioned by some authors for the case of MPM. Rationally, although the 7.4 minutes is a tolerable time for most patients in the sense of a general radiation oncology practice, it may yet be problematic for some patients with limited tolerance capacity. Furthermore, longer treatment times may be disadvantageous in heavily loaded radiation oncology clinics. Nonetheless, it is again rational to foresee or at least hope that this problem will also be soon negated with the rapid innovations in RT technologies.Despite of HT procedure's notable dosimetric superiority over VMAT in some measures, the HT technique also has two major drawbacks. First, because of the need for longer irradiation times, HT is without any doubt more vulnerable to inherent respiratory movement problems mandating more careful setup and online imaging procedures. However, we believe that this is only a matter of time which will probably be solved in the near future with the improvements in HT technology. And second, as the HT technique requires relatively longer treatment times for each RT fraction compared with VMAT (7.4 versus 2.5 minutes; Although HT appeared to provide better dosimetric measures than the VMAT procedure in some specific end goals, the results of this comparative dosimetric study clearly demonstrated the possibility of safe hemithoracic irradiation of medically or technically unresectable MPM patients with either of the two rotational RT techniques. Clinically, considering their poor prognosis, these findings may potentially be promising for curative treatment of MPM patients. However, the clinical translations of these promising dosimetric outcomes ought to be confirmed by further clinical studies before their recommendation for routine clinical use.


Lichen planus (LP) is a chronic inflammatory mucocutaneous disease that commonly affects the oral cavity. Previous reports have suggested a possible association between LP and thyroid gland diseases (TGDs). The purpose of this study was to investigate possible associations between oral lichen planus (OLP) and TGDs.N = 102), were classified according to clinical course (symptomatic/asymptomatic), type  and location of lesions. Data on TGDs was compared to age- and gender-matched controls (N = 102) without OLP. Diagnosis of any type of TGD and related medication for study and control groups was recorded from the medical files provided by patients’ physicians. Statistical analysis used Student’s t-test and Fisher’s exact test; significance was set at p < 0.05.Patients diagnosed with OLP, both clinically and histopathologically (p > 0.05). No significant associations were found between different characteristics of OLP and hypothyroidism or other TGD (p > 0.05).TGDs , hypothyroidism and related medications were found in 16.6, 12.7 and 12.7% of patients with OLP, respectively. These findings were similar to the control group: TGDs  -15.7%, hypothyroidism - 9.8% and thyroid gland disease-related medication - 9.8% (We found no significant associations between the co-existence of OLP and TGD or related-medications. Our findings are in agreement with some of the previously published similar studies but in controversy with others. Further well-designed, multicenter studies with large groups of patients and controls may help to establish the nature of the associations between OLP and TGDs. Oral lichen planus (OLP) is a chronic immune-related condition of the oral mucosa of unknown exact etiology . CurrentThe immune dysregulation is probably associated with T cell- and cytokine-mediated mechanisms –3, but mA possible association between OLP and thyroid gland diseases (TGDs), especially hypothyroidism, has been reported –10, whicIn view of the wide range of reported prevalence of TGDs among OLP patients, we further add our experience and discuss updated insights in the immunologic etiology of TGDs.This is a retrospective case-control study that has been independently reviewed and approved by the Institutional Review Board of Tel-Aviv University, July 2012.The study group included 102 patients who have been diagnosed, clinically and histopathologically, with OLP. All OLP patients were diagnosed and managed in the Oral Medicine Clinic of Tel Aviv University, between the years 2002–2012. The final diagnosis of OLP was based on diagnostic criteria proposed by vander Meij and colleagues . PatientOLP lesions were classified according to the clinical course, type and location. The clinical characterization was made based on the symptomatology: asymptomatic or symptomatic. OLP type was classified as reticular/plaque, atrophic and erosive forms. In case of a combination of different types, the lesions were defined based on the most severe clinical appearance.Based on the information supplied by the family physician regarding the diagnosis of TGD and related medications, patients with TGDs were divided into two main groups, namely hypothyroidism and all other TGDs. TGDs were diagnosed based on routine studies of blood tests, ultrasound and when needed biopsy procedures. All additional relevant data, including concomitant systemic diseases/conditions and habits, like smoking and alcohol consumption, were also obtained from the medical files of the patients.p < 0.05.Statistical analysis was performed using the SPSS software , version 17. Comparison of parameters of age and gender between the OLP and control groups was performed by T-test. Associations between OLP and TGD  were analyzed by Fisher’s exact test. Statistical significance was set at + 13.2 years and that of the control group was 54.1 + 14.6 years, with no significant difference (p = 0.393).One hundred twenty-five medical files of OLP patients were initially examined, out of which only 102 fulfilled the diagnosis of OLP criteria. Females predominated both study and control groups (70.6%). The mean age at diagnosis in the OLP group was 55.7 The most common type of OLP was the reticular (54.9%), followed by erosive (27.5%) and atrophic (17.6%) types. Although the clinical manifestation of OLP was noted at any site of the oral cavity, the most commonly involved was the buccal mucosa (87.6%). Other sites included the gingivae (63.8%), tongue (40.2%), floor of the mouth (7.8%), palate (6.9%) and lip mucosa (5.9%). Symptoms were recorded in 53.9% of the OLP patients. Skin involvement was found in only 6.9%.p = 0.659). No significant differences were found in the frequency of other TGDs, such as Hashimoto’s thyroiditis, between OLP group and the control group (p = 0.748). For all TGDs, the frequency was similar in OLP (16.6%) and control (15.7%) groups (p > 0.05). In addition, no differences were noted between these groups in regard to other co-existence of systemic conditions. Interestingly, smoking habit was significantly less common in the OLP group (11.8%) than in the control (24.5%) (p = 0.028); no statistically significant differences were found between groups regarding alcohol abuse (p = 0.621).The most common TGD was hypothyroidism Table . A medicp > 0.05) were found. The same characteristics of OLP were also investigated for associations with the diagnoses of other TGDs. These also yielded no statistically significant results (p > 0.05).Characteristics of OLP, including symptomatology, clinical type  and oral sites were analyzed for associations with the diagnosis of hypothyroidism Table . No signp > 0.05). In addition, no significant associations were found between the clinical characteristics of OLP and TGDs. Interestingly, we found that smoking habit was significantly less common in the OLP group (11.8%) than in control (24.5%).The present study aimed to investigate any possible association between OLP and TGDs based on data documented in an academic dental center. Diagnosis of OLP was supported both clinically and microscopically in all cases. Diagnosis of TGDs was always confirmed by the patients’ records of the Health Maintenance Organization, to which each regular Israeli citizen is registered. In the current study we found that the prevalence of TGDs  in OLP patients was not significantly different from that of an age- and gender-matched control group  and only 350 in Asia-Middle East-Caribbean-South America zone (zone 2) . LikewisUp to 15% of patients aged 65 years and over may have subclinical hypothyroidism  above 4.0 μ IU/mL and normal free T4 levels), with only few, if any, symptoms suggestive of hypothyroidism . The ratSome studies that investigated the association between OLP and TGDs also referred to the levels of thyroid gland hormones or thyroid auto-autoantibodies. In part of these studies, it was found that 84 to 95% of OLP patients had normal TSH levels , 16, witWe have shown no association between OLP patients and TGDs in a group of OLP patients with well-documented medical data and clinical assessment performed by specialists in oral medicine. The present study agrees with some previous similar studies and contradicts the findings of others, further emphasizing the unresolved issue of a possible association between OLP and TGDs. Associations between OLP and TGDs seems to be complex and multifactorial, probably influenced by both endogenous and exogenous factors, some protecting and others promoting their inter-relations. It is necessary to establish multi-centric collaborations with larger study groups and increased inter-disciplinary collaborations between the field of oral medicine with those of endocrinology and epidemiology in an attempt to provide accurate characterization of the associations between OLP and TGDs.


The impact of sex on prognosis of patients with esophageal squamous cell cancer (ESCC) who underwent definitive radiotherapy remained unclear. The present study aimed to determine the impact of sex on the prognosis of patients with ESCC underwent definitive radiotherapy.Between January 2009 and December 2015, patients with ESCC underwent definitive radiotherapy in Shantou Central Hospital were included in this study. The Progression-free survival (PFS) and overall survival (OS) rates were estimated using the Kaplan-Meier method. The PFS and OS were compared between female and male patients. The Cox regression model was used to identify prognostic factors. Propensity score-matched analysis was performed to balance baseline characteristics between female and male patients.P = 0.0001, HR = 0.688, 95% CI, 0.567–0.836) and OS . In the matched cohort, female patients still had a significantly longer median PFS (13.5 months vs 11.6 months) and OS (19.6 months vs 16.1 months). Multivariate analysis showed that sex was an independent prognostic factor for PFS  and OS .A total of 683 ESCC patients treated with definitive radiotherapy were included, with 497 male and 186 female patients. In the whole cohort, female patients had a significantly longer median PFS  contains supplementary material, which is available to authorized users. Esophageal cancer (EC) is one of the most common malignant tumors in the world with an estimated 477,900 new cases and the fourth most common cause of cancer-related deaths with 375,000 deaths annually . The incSeveral retrospective studies have attempted to explore the role of sex on prognosis in patients with ESCC that received definitive chemoradiotheray, but failed to show correlation between sex and prognosis , 7. TherMany studies have investigated the prognostic value of sex in EC and revealed a sex difference in the prognosis –12. MoreWe conducted a retrospective study to evaluate the prognostic value of sex in patients with ESCC underwent definitive radiotherapy using the propensity-scored matching method to adjust for the prognostic factors.This was a retrospective study including patients with ESCC who underwent definitive radiotherapy at the Department of Radiation Oncology, Shantou Central Hospital between January 2009 and December 2015, with follow-up until May 31, 2018. Patients that received definitive radiotherapy in this study included three ESCC populations—the patients with inoperable disease, the patients refused surgery for personal willingness, and the patients were not suitable for surgery because of the underlying disease. There weren’t any patients that refused surgery because of their performance status. The intention of treatment was for cure. Inclusion criteria were as followed: 1) pathologically proven ESCC; 2) patients underwent definitive radiotherapy or chemo-radiotherapy. Exclusion criteria were as followed: 1) Adenocarcinoma or other pathological types; 2) Patients with distant metastatic disease; 3) Patients underwent palliative or adjuvant radiotherapy;4) Patients with a poor KPS of 10–50; 5) Patients failed to complete therapy. This study was approved by the Ethics Committee of the Shantou Central Hospital. Patient were staged according to the 6th AJCC TNM classification .All patients underwent definitive radiotherapy delivered by three-dimensional conformal radiation therapy or intensity-modulated radiation therapy technique. The primary gross tumor volume (GTV) and the nodal gross tumor volume (GTVnd) were delineated based on CT, barium esophagogram, endoscopic examination or PET imaging. The clinical target volume (CTV) encompassed tumor clinical target volume (CTVt) and nodal clinical target volume (CTVnd). The CTVt included the GTV plus a radial margin of 0.5 to 1 cm and a proximal and distal margin of 2.5 to 3 cm. The CTVnd included the GTVnd and its expansion margin of 0.5 to 0.8 cm. When developing the radiotherapy plan, the CTV plus a 0.5 to 1 cm expansion margin was defined as the planning target volume (PTV), which the prescribed dose is given to. In this study, patients received a prescribed dose of at least 50.4 Gy. Some patients received chemotherapy consisted of two cycles of platinum-based chemotherapy combined with 5-fluorouracil and a taxane (docetaxel or paclitaxel) concurrently with radiotherapy.All patients were examined daily during radiotherapy to monitor the treatment toxicities. The first follow-up was 1 month after completing radiotherapy then every 3–6 months for 5 years. The follow-up evaluation included a physical examination, blood test, barium esophagogram, CT scan of the neck, chest, and abdomen, and PET-CT when available. Information on patients’ age, gender, work-up, treatment, and follow-up was extracted from their medical records.P value < 0.05 indicated a statistical significance. All P values were two-sided. The statistical software IBM SPSS v22.0  was used for all statistical analysis.Progression was defined as the radiographic evidence of relapse, death from any cause. OS was measured from the date of treatment to the date of death from any cause or last follow-up. Comparisons of patient characteristics were carried out with a chi-square test. PFS and OS rates were estimated by the Kaplan-Meier method, and log-rank test was performed to evaluate the survival difference. The Cox regression model was used to perform multivariate analysis to identify prognostic factors. The propensity score-matched analysis , and radiotherapy dose) was performed using the one-to-one nearest neighbor method. A Between January 2009 and December 2015, a total of 683 patients with ESCC underwent definitive radiotherapy were included in this study, with 497 men and 186 women Additional file P = 0.0001, HR = 0.688, 95% CI, 0.567–0.836, Fig. P = 0.0005, HR = 0.702, 95% CI, 0.575–0.857, Fig. Median follow-up was 16.6 months  in the whole cohort, with a median follow-up of 15.9 months  for men and 21.1 months  for women. In the whole cohort, women had a significantly longer median PFS time compared with men patients  for women and 11.6 months  for men , tumor location , T stage , N stage , TNM stage  and treatment modality  were independent prognostic factors for PFS (Table P = 0.007) as an independent prognostic factor for OS, as well as tumor location , T stage , RT dose , and treatment modality .Multivariate analysis showed that sex . A similar finding was observed for distant metastasis event, with fewer patients experiencing distant metastases in female group than in male group  followed by surgery may explained the different prognosis between female and male patients treated with definitive radiotherapy . After nOn the basis of our current data, we hypothesized that sex difference would affect the radio-sensitivity of patients with ESCC exposed to radiotherapy. Following efforts will be made to find out the relation between sex and radio-sensitivity in ESCC. Previous studies have shown that androgen exposure can facilitate the growth of human esophageal squamous cell carcinoma cells and activation of androgen receptors may promote progression of esophageal squamous cell carcinoma , 24. TheThe current study had several limitations. The primary limitation was that the information about lifestyle factors, socioeconomic status, radiotherapy target, and other prognostic risks was not available. In addition, the retrospective and nonrandomized design limited the strength of the results.Our study demonstrated that sex was an independent prognostic factor in Chinese patients with ESCC who underwent definitive radiotherapy, with a better prognosis in female patients than men patients. Efforts should be made to investigate the biological mechanisms for the sex difference in the prognosis of ESCC patients.Additional file 1:Table S1. Multivariate analysis of clinical factors associated with Progression-Free Survival and Overall Survival among patients with ESCC. (DOC 34 kb)Additional file 2:Flow chart of patients selection. (TIFF 32 kb)


Osteopathy is common in patients with chronic pancreatitis (CP), but previous studies carry several limitations. Vitamin K is essential for bone metabolism, but its role in this setting has never been investigated. Our aim is to assess the prevalence of osteoporosis and osteopenia in CP patients, and to investigate the association between osteopathy and CP features and nutritional parameters, especially vitamin D and K levels.Multicentre cross-sectional study on CP patients diagnosed according to M-ANNHEIM criteria. Bone density was evaluated by dual-energy X-ray absorptiometry and pancreatic function by faecal elastase. Nutritional evaluation included vitamin D and vitamin K. Differences between patients with or without osteopathy were evaluated. The association between investigated variables and bone density were analysed with logistic regression analysis.In total, 211 CP patients were enrolled at eight Centres . In total, 18% had advanced-marked CP, 56% suffered from pancreatic exocrine insufficiency and disease aetiology was alcoholic in 43%. Vitamin D and K were deficient in 56% and 32%, respectively. Osteopenia was diagnosed in 42% and osteoporosis in 22%. In the multivariate analysis, female sex (OR 2.78), age (OR 1.07 per year) and higher BMI (OR 0.84) were associated with the presence of osteoporosis. In male patients, the only factor associated with osteoporosis was vitamin K deficiency (OR 4.23).The present data confirm a high rate of osteopathy in CP patients and highlight the relevance of vitamin K deficiency as only factor associated with osteoporosis in male patients for the first time. The clinical presentation depends on disease stage, as in early phases episodes of abdominal pain are more frequent while, as the disease progresses, signs of pancreatic exocrine and endocrine insufficiency develop3. Malnutrition due to exocrine pancreatic insufficiency, as well as alcohol abuse, chronic inflammation, and tobacco use can contribute to systemic complications in CP patients, including bone alterations4.Chronic pancreatitis (CP) is a fibro-inflammatory syndrome of the pancreas characterised by irreversible morphological changes that typically cause abdominal pain and can eventually result in permanent loss of the pancreatic function5. It negatively affects quality of life and is associated with an increased susceptibility to fractures, which are accompanied by considerable morbidity and mortality. A number of pathological conditions, such as primary hyperparathyroidism, hyperthyroidism, chronic kidney failure, chronic liver disease, different digestive disorders and the use of drugs such as glucocorticoids are associated to accelerated bone loss and development of osteopenia and osteoporosis5.Osteoporosis is a preventable disease characterised by low bone mass and micro-architectural deterioration of bone tissue with a consequent increase in bone fragility7.In particular, low bone mineral density (BMD) is a common finding in chronic intestinal disorders associated with diarrhoea and malabsorption, such as inflammatory bowel diseases and coeliac disease4. The prevalence of osteoporosis in CP patients has been reported to range from 5 to 39%11, and a meta-analysis estimated that about 25 and 40% of CP patients are affected by osteoporosis and osteopenia, respectively12. Indeed, the recent European guidelines for the diagnosis and treatment of CP recommend regular bone density assessment by dual-energy X-ray absorptiometry (DXA), and measurement of serum vitamin D as part of CP patients' primary care1.Changes in BMD are, however, also frequent in CP patients16.BMD alterations of in CP patients might be caused by maldigestion and malabsorption of micronutrients and of fat-soluble vitamins like vitamin D and K, by the chronic inflammatory status and by excessive alcohol intake and smoking24. No clear correlation between pancreatic function, disease severity, nutritional parameters and lifestyle factors were found24. Finally, none of the previous studies investigated vitamin K levels.However, the pathogenesis of bone density alterations in CP patients is not well established as previous studies carry several limitations. First, most studies had a very small sample size (the largest including 100 patients); moreover, different and heterogeneous nutritional parameters were evaluated and pancreatic exocrine insufficiency (PEI) or severity of the disease were not consistently defined27. In particular, there have been no previous studies examining the association between vitamin K levels and bone metabolism in CP patients.Vitamin K is a fat-soluble vitamin and may therefore be malabsorbed in CP patients. However, only few studies evaluated vitamin K levels in CP patients with findings of vitamin K deficiency in more than 60% of cases29. Several studies have shown a key role of vitamin K deficiency in causing low BMD in patients with other pathological conditions, including inflammatory bowel disease31, but there are no studies investigating this association in CP patients. Hence, the aims of this study were to asses in a large and homogeneously defined cohort of patients with CP: (a) the prevalence of osteoporosis and osteopenia; (b) the association between bone mineral density and CP features, and nutritional parameters, especially vitamin D and K levels.Vitamin K is essential for bone metabolism since it is a cofactor for the enzyme gamma glutamyl carboxylaxe, which converts osteocalcin (OC) to its carboxylated form. Fully carboxylated OC has a high affinity for calcium and hydroxyapatite and binds calcium to the hydroxyapatite crystal in the extracellular bone matrixThis is a multicentre cross-sectional study conducted in Estonia, Germany, Italy, Poland, Spain, Sweden and the United Kingdom, from April 2015 to October 2016, as part of the “Pancreas 2000” educational program”. Cases of CP seen at the participating Centres during the study period were eligible for enrolment. Enrolled patients gave written informed consent. Local ethic committee approval was obtained at each Centre.32. Patients with a diagnosis of pancreatic cancer, liver cirrhosis, chronic renal failure, primary hyperparathyroidism, small bowel, gastric or pancreatic surgery, celiac disease or inflammatory bowel disease were excluded. Chronic corticosteroids use ( ≥ 7.5 mg/dl for > 3 months) was also considered an exclusion criterion. Patients were not enrolled during acute episodes of exacerbation of symptoms of CP leading to inpatient hospital stay. The severity of CP was defined according to the M-ANNHEIM scoring system32. Patients were questioned on epidemiological data, environmental risk factors and lifestyle, history and course of disease, occurrence of menopause, history of previous low fragility/trauma fractures, defined as those occurring at the spine, hip and distal radius, and not associated with traumatic events9 and use of pancreatic enzyme replacement therapy (PERT) by a medical doctor in their native language. In keeping with ADA guidelines, patients were diagnosed with diabetes mellitus if they had either: (a) a HbA1c value ≥ 6.5% or a fasting blood glucose ≥ 126 mg/dL, or a 2 h post-OGTT glucose level ≥ 200 mg/dl33. Diabetics were asked about the date of diabetes diagnosis. A consumption of at least 12.5 g (1 unit) of alcohol/month was needed to be considered a drinker. One glass of wine, 1 pint or can of beer, one shot of hard liquor were each considered approximatively equal to 1 unit. For smokers, a consumption of at least 100 cigarettes or > 6 months of smoking was needed to be considered a smoker. The total amount of smoking was evaluated as pack-years, defined as the product of packs smoked per day and the total years of smoking. Body mass index (BMI) was calculated as usual adult weight/height2 (kg/m2)34.Inclusion criteria were age > 18 years and a diagnosis of probable or definite CP according to the M-ANNHEIM criteria35.Standardized osteodensitometry by DXA and biochemical tests were performed at each participating Centre within 6 months from the enrolment visit. DXA evaluated the BMD in the lumbar spine (L1–L4) and in the left femoral neck (except in patients with a left hip prosthesis or former fracture). Bone mass measurements results are reported as T-score, reflecting the number of standard deviations above or below the mean for a young adult population. Osteopenia was defined as a T-score < −1 and osteoporosis was defined as a T-score < −2.5In the reported analysis for the outcome “osteoporosis” in the case of patients having both osteopenia and osteoporosis at different body sites, we considered the worst condition to classify the patient.36. Vitamin D , total and ionized calcium, magnesium, parathyroid hormone (PTH) and c-reactive protein (CRP) were measured locally at each Centre. Vitamin D deficiency was diagnosed when levels were < 20 ng/ml.PEI was estimated with faecal elastase-1 using a monoclonal enzyme-linked immunosorbant assay  at each Centre. Results were classified as normal pancreatic function (> 200 mcg/g), mild PEI (100 to 200 mcg/g) and severe PEI (< 100 mcg/g)38.Determination of vitamin K levels  in the serum samples was centralized in the laboratories of S. Andrea Hospital, University “Sapienza” . Blood specimens for vitamin K determination were taken from patients fasting from at least 12 h, and immediately centrifuged at 4000 revolutions per minute to obtain sera and frozen at –80 °C until analysis. Details regarding ultra high performance liquid chromatography (HPLC) methods of vitamin K measurement are provided as Supplementary material. The lower limit to define for vitamin K deficiency was set at 0.2 ng/ml considering the values between the 5th and the 95th percentile of the distribution of a group of 20 healthy controls previously employed to set the method. Notably, this cut-off of ng/ml resulted in line with those reported by otherst test for continuous variables.The prevalence of osteoporosis or osteopenia was calculated dividing the patients with such pathological condition by the total number of participants. Differences in terms of demographics, clinical characteristics, risk factors and nutritional factors between patients with or without osteoporosis or osteopenia were evaluated trough Fisher exact test for categorical variables and The influence of nutritional and clinical parameters on BMD was evaluated throughout a logistic regression analysis with enter method, and odds ratio (OR) and their 95% confidence intervals (CI). The outcome variables were either osteopenia or osteoporosis, and the explored explanatory variables were: age (per increasing year); female sex; BMI (per increasing unit); PTH (per increasing unit); vitamin D deficiency; vitamin K deficiency; CRP (per unit); faecal elastase (per increasing unit); diabetes; active smoking; alcoholic aetiology; disease duration (years), use of PERT.All analyses were corrected for the Centre of enrolment. Centre of enrolment was, indeed, considered as a categorical covariate in all logistic regression analyses, assigning each individual patient to a nominal category on the basis of the Centre where the patient was enrolled.P-values are two-sided. P-values < 0.05 were considered statistically significant. Statistical analysis was performed using MedCalc version 13 .As osteopathy has peculiar mechanisms that are related with hormonal factors, a subgroup analysis by either female or male sex was planned. The possible interaction between vitamin D and vitamin K deficiencies and the diagnosis of osteoporosis was explored. The possible correlation between continuous variables was assessed throughout a Pearson correlation test. All reported In total, 211 patients with CP were enrolled . in total,101 patients (56.42%) had PEI. Faecal elastase was between 200 mcg/g and 100 mcg/g in 29/179 (16.20%) and < 100 mcg/g in 72/179 (40.22%). Overall, 116/211 patients (54.97%) were receiving treatment with PERT. According to the M-ANNHEIM classification, 18% of patients were classified as advanced or marked CP.Overall, 135/211 patients had osteopathy (64%). Eighty-nine of the 211 (42.18%) patients had osteopenia at any site as the worst bone damage, while 46/211 (21.80%) had osteoporosis that was located at the lumbar spine in 26% of cases, at the femoral neck in 39% and at both sites in 35%. The rate of osteoporosis was 0% (0/15) in Estonia, 17% (5/29) in Sweden, 18% (2/11) in England, 21% (12/57) in Germany, 21.4% (6/28) in Spain, 23% (3/13) in Poland and 31% (18/58) in Italy.Previous low-trauma pathological fractures were reported by 13/196 (6.63%) of patients who were able to recall these events. The remaining 16 patients were unsure about these events.Vitamin D was quantified within 6 months from enrolment in 204/211 patients (96.68%). The mean value of vitamin D was 20.2 ng/ml, and 115/204 (56.37%) had vitamin D deficiency ( < 20 ng/ml). The rate of vitamin D deficiency was 73% (11/15) in Estonia, 54% (15/28) in Sweden, 90% (9/10) in England, 51% (29/57) in Germany, 61% (17/28) in Spain, 100% (11/11) in Poland and 40% (22/55) in Italy.Serum samples for Vitamin K levels analysis were taken within 6 months from enrolment in 178/211 (84.36%) patients. The median Vitamin K concentration was 0.38 ng/ml (range 0.008–7.8). In total, 56 of the 178 CP patients (31.46%) had Vitamin K deficiency (< 0.2 ng/ml). The rate of vitamin K deficiency was 40% (6/15) in Estonia, 12.5% (3/24) in Sweden, 38% (21/55) in Germany, 37% (10/27) in Spain, 55% (6/11) in Poland and 22% (10/46) in Italy.Of the 56 patients with vitamin K deficiency, 37 (66%) were male and 47% had alcoholic aetiology of the disease. Only 9% of patients with vitamin K deficiency had advanced or marked CP, while 48% of them had PEI.P=0.05).In addition, a combined deficiency of vitamin K and vitamin D was observed in 33 of 178 patients (18.53%). Patients with combined deficiency of both vitamin D and K did not differ from those without vitamin deficiencies or single vitamin deficiency, but had a higher rate of alcoholic aetiology  had magnesium deficiency and 24/193 (12.43%) calcium deficiency, 18.28% of patients (32/175) had PTH levels above the upper normal range and 70/115 (60.86%) had CRP levels above the upper normal range.There were no differences between patients with or without osteopenia in terms of sex, age, length and severity of disease, presence of PEI and vitamin D or K insufficiency rate.P = 0.05) and the rate of diabetes  were lower in CP patients with osteopenia  and their mean age was higher  compared with those without. In addition, all women with osteoporosis were post-menopausal compared with 70% of those without osteoporosis (P = 0.01).CP patients with osteoporosis were more frequently female . CRP levels were not different between patients with normal bone density, osteopenia or osteoporosis.The mean BMI was lower in patients with osteoporosis compared with those without  in patients with osteoporosis.Neither vitamin D nor vitamin K levels were statistically different between patients with and without osteoporosis, while the mean value of PTH was significantly higher  , a higher age , a higher BMI  and a higher PTH  and a diagnosis of diabetes . Age , a higher BMI  and a higher PTH  were maintained as significant factors in the multivariate analysis.A logistic regression analysis was run to investigate the possible association between the analysed variables and the diagnosis of osteopathy (either osteopenia or osteoporosis), osteopenia or osteoporosis. Factors significantly associated with osteopathy in the univariate logistic regression analysis were: female sex  and higher age  were associated with a higher risk, while a higher BMI  was associated with reduced risk.As far as osteoporosis, in the univariate analysis, female sex  and higher age  were associated with a higher risk of being diagnosed with osteoporosis while a higher BMI  was associated with reduced risk  was associated with a lower risk of being diagnosed with osteoporosis while age was only a borderline significant factor . In addition, vitamin K deficiency was the only variable significantly associated with a higher risk in the multivariate analysis  . This was not the case for isolated deficiency of vitamin D or for combined deficiency of vitamins D and K, which were not associated with the diagnosis of osteoporosis.To investigate whether there was a an interaction between vitamins D and K deficiencies and osteoporosis, we conducted a further analysis on male CP patients, investigating those with vitamin D or K deficiency only or their combined deficiency separately (see Supplementary Table P = 0.002)  is the most prevalent form in the human diet, while Menaquinone (vitamin K2) is mainly synthetized by the gut microbiome. Whether this “microbiome-generated” fraction is active in a similar manner to vitamin K1 is uncertain19. Similarly, a relation between faecal elastase levels and BMD has been reported in some previous studies24, but not in others21. This might be due to impaired micronutrient absorption being only one of several factors leading to osteopathy in CP patients, and to most cases with PEI receiving PERT. Further prospective studies including only newly diagnosed patients without prior medication might clarify this issue. On the other hand, the present finding of a lower BMI being associated with a lower BMD is in accordance with previous findings23.Furthermore, we investigated the possible correlation between levels of vitamins D and K, faecal elastase and the T-score at femur and spine, without significant results Fig. . The pre42. We, therefore, explored whether there was an interaction between vitamins D and K deficiencies and osteoporosis, analysing the rate of osteoporosis in male CP patients with vitamin D or K deficiency only or with combined deficiency separately (see Supplementary Table P = 0.005). This was not the case for isolated vitamin D deficiency or for combined deficiency of vitamins D and K. These findings do not support the hypothesis of an interaction between vitamin D and K deficiencies in determining an increased risk of osteoporosis in this setting, but this topic deserves further investigation.Interestingly, a number of experimental and observational studies have suggested that vitamins D and K might act synergistically on bone health, as vitamin D seems able to enhance vitamin K-dependent proteins43, but also as a marker of decreased bone density4. In the present study, however, CRP levels were not associated with BMD. This difference might be due to the fact that this previous study evaluated high sensitivity CRP levels, which better reflects low-grade chronic inflammation, rather than CRP as in the present one.A relevant role for CRP, a marker of acute phase reaction, has been recently reported not only as a prognostic variable associated with CP course44.As expected, PTH levels were higher in patients with osteopathy and associated with osteoporosis in the whole investigated population Tables  and 3, bThe present study has some strengths: (1) the sample size is by far the largest for studies of this kind; (2) data were collected in a homogeneous and standardised way, the diagnosis of CP and its severity being defined according to the M-ANNHEIM classification; (3) exocrine pancreatic insufficiency was investigated through measurement of faecal elastase levels, which is nowadays the most commonly employed test for this aim; (4) different clinical and biochemical variables potentially associated with osteopathy were analysed including a centralized quantification of vitamin K, which was investigated for the first time in this setting.45, it is difficult to investigate a sufficient sample size of these patients. Also, the cross-sectional study design precludes the possibility to establish any cause-effect relationship, and we can only report an association between certain parameters and the presence of osteopathy, useful to generate hypotheses that need validation. As far as regards the method employed for vitamin K measure, while HPLC is currently considered the elective technique to measure vitamin K46, this remains a complex determination and the measure of markers of vitamin K activity such as undercarboxylated OC or protein induced by vitamin K absence (PIVKA) might have been helpful. However, as OC levels are also influenced by vitamin D and PTH levels46, which can be altered in CP patients, the concomitant measure of both vitamin K and these markers should be considered in future studies. Finally, as vitamin K is mainly transported by triglyceride-rich lipoproteins, the lack of triglyceride measure in the present study is a limitation as it does not allow normalization of vitamin K values.There are, however, some limitations which are partly intrinsic to the study setting of a multicentre cross-sectional study. Some investigations, such as DXA, and measurement of all biochemical parameters but vitamin K were performed at each local institution, and this might have caused bias due to different methods or instruments, although the same reference standards for biochemical parameters were employed. Keeping this in mind, we adjusted our logistic regression analyses for the Centre of enrolment. Furthermore, a cohort of newly diagnosed CP patients without prior medical counselling regarding lifestyle or dietary interventions might reflect the investigated association better. However, given the relatively low incidence and prevalence of CPIn conclusion, findings of the present study confirm the high rate of osteopathy in a large cohort of patients with CP and reinforce the recommendation to screen CP patients with DXA, in order to prevent pathological fractures. The present findings also suggest, for the first time, that specifically in male patients, who typically represent the majority of CP subjects, vitamin K deficiency is the most relevant factor associated with osteoporosis. As this study can only demonstrate an association and not causality, further investigation on the role of vitamin K on bone turnover in the setting of CP are necessary. Future studies should also investigate the association between dietary patterns and vitamins intake and clinically relevant outcomes associated with nutritional status and vitamin levels, such as risk of hospitalization for fractures, infections and cardiovascular events, in CP patients.In patients with chronic pancreatitis (CP) osteopathy has been reported to be common but the reported prevalence is heterogeneous and previous studies carry several limitations.Vitamin K, which plays a key role in bone metabolism, has never been investigated among factors associated with osteopathy in CP.This is the largest study aimed at investigating the prevalence of osteopathy in patients with chronic pancreatitis with standardized criteria to assess disease severity and pancreatic exocrine insufficiency (PEI).A high rate of osteopenia and osteoporosis was confirmed, alongside vitamin D deficiency and PEI, that were not associated with osteopathy.Female sex, age and BMI were associated with of osteoporosis in the whole cohort, while in male patients, the only factor associated with osteoporosis was vitamin K deficiency.Supplementary InformationSupplementary Table 2Supplementary Table 2


ADIPOQ locus, in ADIPOR1, and ADIPOR2 have been associated with adiponectin concentrations and cardio-metabolic complications in diverse ethnicities. However, no studies have examined these associations in Mexican children. We recruited 1 457 Mexican children from Mexico City. Six genetic variants in or near ADIPOQ , ADIPOR1 (rs10920533), and ADIPOR2 (rs11061971) were genotyped. Associations between serum adiponectin, genetic variants, and cardio-metabolic traits were assessed using linear and logistic regressions adjusted for age, sex, and recruitment center. Serum adiponectin concentration was negatively associated with body mass index, waist to hip ratio, low-density lipoprotein cholesterol, total cholesterol, triglycerides, fasting glucose, fasting insulin, homeostatic model assessment of insulin resistance, dyslipidemia and overweight/obesity status (7.76 × 10−40 ≤ p ≤ 3.00 × 10−3). No significant associations between genetic variants in ADIPOQ, ADIPOR1, and ADIPOR2 and serum adiponectin concentration were identified . No significant associations between the six genetic variants and cardio-metabolic traits were observed after Bonferroni correction . Our study suggests strong associations between circulating adiponectin concentration and cardio-metabolic traits in Mexican children.The adipocyte-derived adiponectin hormone bridges obesity and its cardio-metabolic complications. Genetic variants at the  Several therapeutic options are available, however controlling the development of obesity and its resulting complications has proven challenging2. Chronic obesity in its more severe forms leads to major reductions in life expectancy, with most of the excess deaths due to heart disease, cancer, and T2D3. As a result, obesity imposes a heavy socio-economic burden in both high-income and developing countries4.In 2016, the World Health Organization reported that 1.9 billion adults and 381 million children were overweight/obese, resulting in an important global health concern. Obesity is associated with the development of comorbidities (insulin resistance (IR), type 2 diabetes (T2D), dyslipidemia, hypertension), collectively known as the metabolic syndrome5. The prevalence of obesity in Mexican school-aged children was 11.8% in girls and 17.4% in boys in 20125. The metabolic syndrome prevalence was 9.4% in Mexican adolescents in 20106. The rise of childhood obesity in Mexico is largely explained by a ‘nutritional transition’ which reflects changes in dietary patterns characterized by increased consumption of foods that are high in fat and/or sugar, as well as reduced physical activity7. Beyond modifiable factors, the elucidation of biological determinants of obesity and its cardio-metabolic complications is expected to improve prediction, prevention and care, including novel treatments adapted to genetic profiles of high-risk populations8.The Mexican population is a group at high risk for developing obesity and the metabolic syndrome, especially in childhood9. Genetic mouse models have shown that deficiency of adiponectin contributes to IR, while its overexpression in leptin-deficient obese mice promotes adipose tissue expansion and improves insulin sensitivity11. Adiponectin acts on two receptors (adiponectin receptors 1 and 2) encoded by ADIPOR1 and ADIPOR2 genes, both of which appear to show functional redundancy12. Simultaneous disruption of both AdipoR1 and AdipoR2 in the liver of leptin-deficient obese mice leads to IR and marked glucose intolerance12. In humans, adiponectin is abundantly found in the bloodstream where it makes up 0.01–0.05% of total plasma protein13. Low serum adiponectin has been associated with obesity, IR, T2D, dyslipidemia, hypertension and coronary heart disease in cross-sectional studies9. Adiponectin level was also negatively associated with incident development of insulin resistance, T2D, dyslipidemia, hypertension, and coronary artery disease9. The relationship between adiponectin level and subsequent weight gain has been a topic of interest due to its paradoxical nature, where levels of adiponectin decrease with the development of obesity14. Adiponectin was positively associated with weight gain in children, but not in adults in prospective studies16.Adiponectin, an adipocyte-derived secretagogue, may be considered as one of the key hormones bridging obesity and its cardio-metabolic complications18. Encoded by the ADIPOQ locus and found on chromosome 3q27, adiponectin is a 30 kDA protein with both a collagenous N and a globular C-terminus18. Candidate gene studies, and more recently genome-wide association studies (GWAS), fine-mapping or resequencing experiments have identified numerous common and rare variants at the ADIPOQ locus associated with serum adiponectin level and metabolic traits24. If common variants in the ADIPOR1 and ADIPOR2 genes have not been associated with serum adiponectin levels, they contribute to IR, T2D and cardiovascular disease risk26.If serum adiponectin levels are influenced by modifiable factors such as physical activity and diet, genetic factors account for 30–93% of variation in adiponectin levels in humans29. A few studies investigated the association of SNPs in ADIPOQ ADIPOR1, and ADIPOR2 with serum adiponectin levels and cardio-metabolic traits in Mexican and Mexican-American adults33. However, to date, no study has investigated these genetic associations in Mexican children. This prompted us to analyze in 1 457 Mexican children 1) the association of adiponectin levels with cardio-metabolic traits, 2) the association of six SNPs in ADIPOQ, ADIPOR1, ADIPOR2, and serum adiponectin levels, and 3) the association of the same SNPs with cardio-metabolic traits.While serum adiponectin levels negatively correlate with obesity, T2D and the components of the metabolic syndrome in Mexican children, high adiponectin concentrations are associated with a metabolically healthy but obese profile in Mexican adults2 and a SDS-BMI of 0.71 ± 1.09. Within the sample, 20.8% of children were overweight and 23.0% were obese. Insulin resistance was identified in 11.1% of children, 3.1% had hyperglycemia including one child with T2D. Hypertension and dyslipidemia were present in 1.5% and 34.9% of the sample, respectively. The mean serum adiponectin concentration was 5.26 ± 1.23 µg/mL.Anthropometric and biochemical characteristics of the 1 457 Mexican children  are summarized in Table −30), SDS-BMI , WHR , SDS-WHR , LDL cholesterol , total cholesterol , triglycerides , fasting glucose , fasting insulin , and HOMA-IR . Nominal associations (p < 0.05) between serum adiponectin concentration and SBP, DBP, SDS-SBP, SDS-DBP and HOMA-B were observed, but did not reach statistical significance after Bonferroni correction , normal weight vs. overweight , normal weight vs. obese , normal weight vs. overweight and obese participants . Associations of serum adiponectin concentration with hypertension, hyperglycemia, and IR were not statistically significant (p ≥ 0.07).When metabolic traits were classified as binary traits Table , serum a−3) and negatively associated with fasting glucose  and HDL cholesterol . Nominal associations (p < 0.05) between serum adiponectin concentration and fasting insulin, total cholesterol and insulin resistance were observed, but did not reach statistical significance after Bonferroni correction .Genotype distributions and allele frequencies of the six selected SNPs are presented in Supplementary Table ADIPOQ , ADIPOR1 (rs10920533), and ADIPOR2 (rs11061971) with serum adiponectin concentration under an additive model, adjusted for sex, age, and recruitment center .We subsequently tested the association of the aforementioned SNPs with cardio-metabolic traits, adjusted for sex, age, and recruitment center and with and without adjustment for serum adiponectin concentration Tables  and 5. W−3.Statistical power calculations are summarized in Supplementary Tables −3. For associations between serum adiponectin and binary cardio-metabolic traits, we conducted an example statistical power calculation for the association of serum adiponectin and insulin resistance. With 127 cases in a sample of 1 457 participants, we had at least 80% power to detect effect sizes/odds ratios of 1.40 or greater for p-value = 4.2 × 10−3.For associations between serum adiponectin and continuous cardio-metabolic traits, we conducted an example statistical power calculation for the association of serum adiponectin and SBP, for which we had at least 80% power to detect beta values of 0.9 or greater for p-value = 4.2 × 10−4. For associations between the six SNPs and binary cardio-metabolic traits, we also examined the association between SNPs and insulin resistance, for which we had at least 80% power to detect effect sizes/odds ratios of 1.3 or greater when MAF is 0.1 or greater for p-value = 6.9 × 10−4.For associations between the six SNPs and continuous cardio-metabolic traits, we also examined the association between SNPs and SBP, for which we had at least 80% power to detect beta values of 2.5 or greater when MAF is 0.2 or greater for p-value = 6.9 × 10ADIPOQ, ADIPOR1, and ADIPOR2 with adiponectin serum levels and cardio-metabolic traits in Mexican children. We also compared the SNP genotypic distributions between Mexican children and adults from the 1000G. We found strong negative associations for adiponectin levels with BMI, WHR, LDL cholesterol, total cholesterol, triglycerides, fasting glucose, fasting insulin, and HOMA-IR, as well as dyslipidemia, overweight and obesity status. Further adjustment for BMI removed most of these associations, to the exception of fasting glucose. The same adjustment resulted in significant association between serum adiponectin concentration, HDL cholesterol and HOMA-B. The 6 SNPs had similar genotypic distribution in Mexican children and adults. We did not find any association between these SNPs and serum adiponectin concentration. While nominal associations were found between ADIPOR1 rs10920533, ADIPOR2 rs11061971, and ADIPOQ rs182052 and cardio-metabolic traits, none remained significant after Bonferroni correction for multiple testing. Based on our statistical power calculations, our study was only modestly powered , possibly due to our strategy to recruit children within an urban setting. The prevalence of hypertension in our sample (1.5%) was lower than previously reported (4.7% to 14%)45; however, previous studies classify hypertension using percentiles rather than a threshold, making comparisons difficult. The prevalence of IR in our sample (11%) was also low. A cross-sectional study of Mexican children aged 7–18 estimated the prevalence of IR at 20.3%, while the National Health and Nutrition Examination Survey found 52.1% of obese Mexican-Americans aged 12–19 to have IR46. The gradual increase of insulin and glucose concentrations observed during adolescence may partially explain this discrepancy48. We report an exceptionally high prevalence of dyslipidemia in our sample (34.9%). While this high prevalence may be reflective of a diet rich in refined carbohydrates and animal fats but limited in fiber, we cannot exclude the possibility that it may stem from the employed definition of dyslipidemia within our study49. Dyslipidemia is routinely defined by abnormal concentrations of one or two lipids, however we used three lipids, thereby artificially increasing the prevalence of dyslipidemia in our sample. The mean serum adiponectin concentration in our sample was lower than in previous reports in Mexican children29. Differences in the prevalence of obesity, blood samples (i.e. serum vs. plasma) and laboratory tests (i.e. radioimmunoassay vs enzyme immunoassay) can significantly affect measured adiponectin concentrations, making comparisons challenging.The Mexican population is at high risk for developing obesity, IR, dyslipidemia and T2D due to genetic predisposition in combination with recent demographic, socioeconomic and nutrition transitions50. Adiponectin plays an important role in modulating glucose and lipid metabolism by activating AMP-dependent kinase signaling51. The relationship between low serum adiponectin and obesity, IR, T2D, dyslipidemia, hypertension, and cardio-vascular disease has been extensively studied in adults50. Adiponectin levels have been found to be lower in obese European and East Asian children53. Here, we extended the negative association between serum adiponectin level and childhood overweight/obesity status to the Mexican population. The associations between serum adiponectin and continuous cardio-metabolic traits have been previously investigated in Mexican children in modestly sized studies. Consistent with our results, Cruz et al. determined negative associations with plasma adiponectin and BMI, insulin concentrations and HOMA-IR in an independent sample29. More recently, plasma adiponectin was inversely associated with insulin concentrations, TG, and HOMA-IR in obese Mexican children with the metabolic syndrome27. Our results evidenced an inverse association with adiponectin and WHR, LDL-C, total cholesterol, and fasting glucose, which has previously been shown in Latino and Hispanic youth, but not in a Mexican population55. We also observed an inverse association with adiponectin and dyslipidemia which is consistent with previous reports in a multiethnic adult population and European children57. Further adjustment for BMI substantially modified the pattern of association between serum adiponectin and cardio-metabolic traits, confirming that adiponectin has an important role in the regulation of body weight58. Taken together, our results suggest that adiponectin levels may contribute to the link between obesity, IR, glucose homeostasis, and dyslipidemia at a young age.Adiponectin is an insulin-sensitizing hormone secreted from the adipose tissue and is negatively associated with obesity and T2D in epidemiological studiesADIPOQ locus appear to cause substantial changes in circulating adiponectin concentrations59. The most frequently studied ADIPOQ variants associated with altered adiponectin concentrations include rs17300539, rs266729, rs2241766, and rs150129918. The rs17300539 variant is strongly associated with increased circulating adiponectin due to enhanced ADIPOQ promoter activity60. Associations with rs266729 and serum adiponectin are inconsistent, however the general trend suggests a decrease in adiponectin concentration which is further evidenced by lower ADIPOQ promoter activity60. ADIPOQ rs2241766 is strongly associated with lower adiponectin levels, possibly due to differences in RNA splicing or stability and rs1501299 is mainly associated with lower adiponectin levels60.Several common and rare variants at the ADIPOQ variants and adiponectin levels have been investigated in various populations, however limited information exists in the Mexican population60. ADIPOQ rs17300539 was associated with higher adiponectin concentrations in a study of 1 153 Hispanic Americans from San Antonio61. In a cross-sectional study of 242 Mexican-Mestizo adults, a positive association with ADIPOQ rs1501299 and circulating adiponectin was observed31. In the present study, we did not identify any significant associations with the selected ADIPOQ SNPs and serum adiponectin concentration, possibly due to limited power, age- or ethnic-dependent effects. To our knowledge, this is the first study to examine the association of genetic variants in ADIPOQ with serum adiponectin levels in a pediatric Mexican population. Further investigation with a more exhaustive SNP selection and larger sample sizes is warranted.Associations with ADIPOQ SNPs and cardio-metabolic traits and found nominally significant inverse associations between rs182052 and BMI and obesity status. The association of ADIPOQ rs182052 with BMI is consistent with findings by Sutton et al. who found the A allele of rs182052 associated with lower BMI in 811 Hispanic adults from San Antonio62. However, Richardson et al. found a positive association with the G allele of ADIPOQ rs182052 and BMI in 439 Mexican American adults from San Antonio and a trend for increased obesity risk has been observed in a small sample of Mexican children63. Among Brazilians, the A allele of ADIPOQ rs182052 was associated with a greater BMI and risk of obesity64. However, studies in European adult populations were unable to identify associations with the A allele of ADIPOQ rs182052 and BMI66. These results suggest possible age-dependent associations of ADIPOQ SNPs in children with BMI which may be considered in future replication studies in Mexican children.We subsequently tested the associations of ADIPOR1 and ADIPOR2 and serum adiponectin which is consistent with previous studies. GWAS in diverse ethnic groups did not identify ADIPOR1 or ADIPOR2 loci as important contributors to serum adiponectin level variation68. Cohen et al. investigated the association of ADIPOR1 and ADIPOR2 with serum adiponectin levels in Caucasian and African-American women but failed to show an association69. Subsequently, Matther et al. did not find associations with ADIPOR1 and ADIPOR2 and serum adiponectin levels in the Diabetes Prevention Program26. More recently, a meta-analysis of 2 355 European-Australians failed to find an association with serum adiponectin and genetic variants in adiponectin receptors70. We studied these associations in a Mexican population for the first time and our results are in line with previous publications. We also identified nominally significant associations between ADIPOR1 rs10920533 and total cholesterol and ADIPOR2 rs11061971 and obesity risk. Very few studies have examined genetic variation in ADIPOR1 and ADIPOR2 in relation to these cardio-metabolic traits, making comparisons challenging. Previous work in adult European populations suggests associations with ADIPOR1 rs10920533 and ADIPOR2 rs11061971 and IR, but we were unable to confirm these associations in the present pediatric Mexican population71. Further investigation is needed to determine the validity of these associations.We did not observe an association between genetic variants in ADIPOQ variant, rs266729 alters ADIPOQ gene expression and has consistently been associated with lower serum adiponectin concentrations and increased risk of IR and T2D72. Future work in the Mexican population including GWAS for adiponectin levels and Mendelian randomization studies are needed to determine the causal links between this hormone and the development of cardio-metabolic diseases.Despite the strong association between adiponectin levels and cardio-metabolic traits, we failed to identify associations with the selected SNPs and cardio-metabolic traits after Bonferroni correction. A possible explanation is that the association between adiponectin and metabolic traits is not causal and can be explained by confounding. Observational epidemiology is prone to confounding, reverse causation, and other sources of bias, thus our results should be interpreted with caution. Adiponectin concentration is inversely associated with obesity and T2D, however it is not yet known whether altered adiponectin concentrations are causal or merely a disease marker. Combining genetic epidemiology with classic epidemiology is one way to strengthen causality. For example, the common ADIPOQ, ADIPOR1 and ADIPOR2, adiponectin concentrations, and cardio-metabolic traits in a pediatric Mexican population. Measures of serum adiponectin concentration were available, allowing us to investigate the effects of genetic variants on adiponectin levels in addition to diverse cardio-metabolic traits. Furthermore, our study combines classic and genetic epidemiology to strengthen our conclusions. Children represent a purer phenotype as they have less exposure duration to an obesogenic environment, relative to adults58. Studying these associations in children may therefore provide more insight into the early biological determinants of obesity and cardio-metabolic complications. Limitations include the selection of ADIPOQ, ADIPOR1, and ADIPOR2 SNPs which was not exhaustive and did not include more recent GWAS discoveries73. Our study is also modestly powered to identify genetic effects, especially after adjusting for multiple testing correction74. Study participants were randomly selected from Mexico City and is therefore representative of the urban population of central Mexico, not of the Mexican population as a whole. Furthermore, the Mexican population is admixed with Native American, European, and West African ancestries with proportions varying within different regions of the country. Because we did not have ancestry-informative markers, we could not adjust for potential population stratification. Also, due to the cross-sectional nature of this study, causality cannot be inferred from the associations between serum adiponectin level and cardio-metabolic traits. Some cardio-metabolic traits are also correlated with each other . Recruitment was done in collaboration with local public schools. The study started in July 2011 and is still ongoing. A trained pediatrician performed all the anthropometric measurements. Blood samples were collected for biochemical measurements and DNA extraction. Children who had diagnosis of infectious disease, gastrointestinal disorders, administration of antimicrobial agents (within 6 months prior to study), incomplete questionnaires or biological samples were excluded. The child’s assent and written informed consent from the parents/guardians was obtained prior to enrolment into the study. The study protocol was approved by the Mexican Social Security Institute National Committee and the Ethical Committee Board. All procedures were conducted in accordance with the relevant guidelines and regulations of the Declaration of Helsinki2, were converted to age- and gender- adjusted standard deviation scores  using the LMS method according to guidelines from the Centers for Disease Control (CDC)77. Waist circumference (WC) was measured at the midpoint between the lowest rib and the iliac crest after a normal exhalation with children in the standing position. Hip circumference was measured at the level of the greater trochanters. The waist to hip ratio (WHR) was also converted to age- and gender- adjusted standard deviation scores (SDS-WHR) using the LMS method and growth charts based on US National Health and Nutrition Survey, cycle III (NHANES III)78. BMI was used to classify children as underweight, normal weight, overweight, or obese, according to the Centers for Disease Control and Prevention CDC 2000 references. Systolic and diastolic blood pressure (SBP and DBP) were measured using a mercurial sphygmomanometer . Blood pressure readings were taken for each participant twice on the right arm in a sitting position with 5 minutes rest between each measurement and the mean of the two readings was determined. Age- and gender- adjusted standard deviations scores for SBP and DBP (SDS-SBP and SDS-DBP) were calculated using methods specified by the fourth report from the National High Blood Pressure Education Program (NHBPEP) in children and adolescents79. Hypertension was defined as average measured blood pressure above the American Heart Association’s recommendations (systolic ≥ 140 mmHg or diastolic ≥ 90 mmHg). Blood samples were obtained following a 12 hour fast and were analyzed for glucose, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) using the ILab 350 Clinical Chemistry System (Instrumentation Laboratory IL. Barcelona Spain). Dyslipidemia was defined as fasting TG ≥ 100 mg/dL (0–9 years of age) or TG ≥ 130 mg/dL (10–19 years of age) and/or HDL-C < 35 mg/dL and/or LDL-C ≥ 130 mg/dL, according to current recommendations81. Insulin (IU) was measured by chemiluminescence  and homeostatic model assessment of insulin resistance (HOMA-IR) and beta-cell function (HOMA-B) were calculated using the equation by Matthews et al.82. Due to the risk of blood hemolysis, fasting insulin values < 1 µU/mL were discarded from the study. Insulin resistance was defined as HOMA-IR ≥ 3.4 83. The 2003 ADA criteria for FPG were used to classify participants as having normal glucose tolerance (NGT), impaired fasting glucose (IFG), or T2D. In absence of oral glucose tolerance test (OGTT) 2-hour fasting plasma glucose value, we used the 2003 American Diabetes Association criteria to define normal fasting glucose , impaired fasting glucose , and T2D (FPG ≥ 7.0 mmol/L), as previously described85. Hyperglycemia was defined as FPG > 5.6 mmol/L. Total adiponectin (µg/mL) was determined by ELISA .All participants were weighed using a digital scale  and height was measured with a portable stadiometer . Height, weight and body mass index (BMI), calculated as weight (kg)/height (m)ADIPOQ , ADIPOR1 (rs10920533), and ADIPOR2  associated with cardio-metabolic traits in literature and harboring minor allele frequencies ≥10% in the Mexican population according to the HapMap database. Genotyping of the SNPs was performed using the TaqMan Open Array Real-Time PCR System , following the manufacturer’s instructions. Three SNPs  did not reach valid Open Array assay scores. The Open Array experiment involved 64 polymorphisms in total. From the initial sample of 1 559 participants, 102 were excluded from the current analysis because (i) no blood sample was collected for DNA extraction; (ii) DNA extraction was unsuccessful; (iii) the individual genotyping success rate of the Open Array experiment based on the 64 polymorphisms was <90.6% 6 genotypes missing). The current analysis included 1 457 children with both genotypic and clinical data available. Only one SNP out of seven did not pass the quality control criteria (rs17366568). The six remaining SNPs harbored a genotyping call rate between 97 and 99%, and no deviation from Hardy-Weinberg equilibrium was observed . We selected 10 SNPs in 86. For associations of serum adiponectin with cardio-metabolic traits, two-tailed p-values < 4.2 × 10−3 after Bonferroni correction (0.05/12) were considered statistically significant. For association of SNPs in ADIPOQ, ADIPOR1, and ADIPOR2 with serum adiponectin concentration, p-values < 8.3 × 10−3 (0.05/6) were considered statistically significant. For association of the same SNPs with quantitative traits, p-values < 6.9 × 10−4 (0.05/72) were considered statistically significant. QUANTO software was used for statistical power calculations, assuming normal distribution of quantitative traits, 80% power, and using p-values adjusted for multiple comparisons. Non-biological outlier data were discarded using a Cook’s distance test followed by an expert verification. Based on Shapiro-Wilk test  or R (version 3.1.2). We followed the strategy reported previously by Ronald J Feise and considered independent Bonferroni corrections for each question askedSupplementary online material