The objective of this registry is to collect data on real-life treatment conditions for patients for whom multiple organ dialysis with Advanced Organ Support (ADVOS) albumin hemodialysis is indicated.This registry was performed under routine conditions and without any study-specific intervention, diagnostic procedures, or assessments. Data on clinical laboratory tests, health status, liver function, vital signs, and examinations were collected (DRKS-ID: DRKS00017068). Mortality rates 28 and 90 days after the first ADVOS treatment, adverse events and ADVOS treatment parameters, including treatment abortions, were documented.This analysis was performed 2 years after the first patient was included on January 18, 2017. As of February 20, 2019, 4 clinical sites in Germany participated and enrolled 118 patients with a median age of 60  of whom 70 were male (59.3%). Patients had a median SOFA Score of 14  and a predicted mortality of 80%. The median number of failing organs was 3 .Four hundred twenty nine ADVOS treatments sessions were performed with a median duration of 17 hours . A 5.8% of the ADVOS sessions (25 of 429) were aborted due to device related errors, while 14.5% (62 of 429) were stopped for other reasons. Seventy nine adverse events were documented, 13 of them device related .3-) and base excess returned to the physiological range, while partial pressure of carbon dioxide (pCO2) remained unchanged. At the time of the analysis, 28- and 90-day mortality were 60% and 65%, respectively, compared to an expected ICU-mortality rate of 80%. SOFA score was an independent predictor for outcome in a multivariable logistic regression analysis.A significant reduction in serum creatinine (1.5 vs 1.2 mg/dl), blood urea nitrogen (24 vs 17 mg/dl) and bilirubin (6.9 vs 6.5 mg/dl) was observed following the first ADVOS treatment session. Blood pH, bicarbonate (HCOThe reported data show a high quality and completion of all participating centers. Data interpretation must be cautious due to the small number of patients, and the nature of the registry, without a control group. However, the data presented here show an improvement of expected mortality rates. Minor clotting events similar to other dialysis therapies occurred during the treatments. Tissues from affected organs are all perfused with blood, which results in the main target for these treatments. As such, therapies including toxin removal, correction of acid-base disturbances, balance of electrolyte and fluid, carbon dioxide (CO2) removal and, even oxygenation should be part of a unified system. Instead, modular hardware as add-on devices to a continuous renal replacement therapy (CRRT) circuit is currently common.,14 Since there is often a relation between an organ dysfunction and the impairment of other organs, a multidisciplinary management with a focus on MOF is needed.Currently, the term extracorporeal organ support (ECOS) is being employed to describe all forms of therapies entailing blood extraction and further processing it in specifically designed circuits and devices.–19 Additionally, thanks to a revolutionary dialysate recirculating and recycling circuit, the pH value and composition of the dialysate can be individualized for each patient, which allows acid-base balance control, including metabolic acidosis correction and CO2 removal.–22The Advanced Organ Support (ADVOS) therapy integrates kidney, liver, and lung support in 1 single device. In this ECOS procedure, a recirculating and recyclable albumin enriched solution serves as the primary dialysate fluid and is intended to remove protein-bound toxins from the blood. Thereby, in contrast to conventional dialysis procedures, not only water-soluble substances  are eliminated, but also albumin-bound substances  can be removed, as shown in preclinical and clinical studies.We present here the objectives and design of a noninterventional, multi-center, and nonrandomized patient registry. The objective of the study was to collect data on real-life treatment conditions for patients for whom multiple organ dialysis with the ADVOS device was indicated without any study-specific intervention, diagnostic procedures, or assessments. Collected data comprised safety and performance parameters, including outcome indicators . The criteria used to analyze safety and performance is depicted in Table 2This was a non-interventional, multi-center, non-randomized registry in post marketing surveillance. The objective was to collect data on real-life treatment conditions for patients for whom multiple organ dialysis is indicated without any study-specific intervention, diagnostic procedures, or assessments. This information should help to improve the treatment of these seriously ill patients in the future.In this registry, the hemodialysis device ADVOS multi, including applicable accessories , and fluids  was used for extracorporeal multiple organ dialysis to support liver and kidney function which is in line with the intended use being defined for this medical device in accordance with its CE mark.The registry evaluated the performance and safety of the procedure and recorded mortality rates at 28 and 90 days. The registry was intended to help to create recommendations for ADVOS treatments as well as to identify appropriate supportive and diagnostic measures. Finally, it was evaluated if registry data on adverse events, mortality rates and treatment under real-life practice conditions were comparable with published data from other trials.2.1The ADVOS multi  is a hemodialysis device indicated for patients with acute, chronic, and acute-on-chronic liver failure or renal failure. It is especially intended to remove water-soluble and protein-bound toxic substances, to normalize or improve composition of blood in case of electrolyte or acid-base disturbances, and to remove fluids in case of fluid overload.2 effective surface . The patient blood is then cleaned by passing it through two parallel high-flux dialyzers with a 1.9 m2.,21 The removed filtrate is continuously replaced by permeate  and alkaline and acidic concentrates.The dialysate results from mixing an alkaline concentrate , an acidic concentrate  and osmosis water. It consists of sodium, chloride, potassium, calcium, magnesium, phosphate, and bicarbonate. It contains 200 ml of 20% pharmaceutical grade albumin, which is sourced from the hospital's pharmacy. The ADVOS multi employs a recirculating procedure for the dialysate. For this purpose, the dialysate is divided into 2 paths, where the pH level is either increased  or decreased  to release anionic  or cationic  substances from albumin, respectively. This allows to restore the dialysate albumin binding capacity and to filter toxins by convection through 2 high-flux filters with an effective surface of 1.3 mBy adjusting the ratio of the alkaline and acidic concentrates, the pH of the dialysate can be adjusted within its ranges . Concentrate flows can be adjusted between 160 and 320 ml/minutes. All added and replaced fluids are continuously balanced, including the user-defined ultrafiltration (UF) rate. The permeate is provided in the movable container under the machine. In this container, the used filtrate (waste liquids) is accumulated in a separate bag.The ADVOS multi and its fluids are not intended for children, pregnant or nursing women, or persons with Creutzfeldt-Jakob disease.2.2This interim analysis was performed 2 years after the first patient was included on January 18, 2017. Adult patients  requiring Advanced Organ Support according to the indications for use (see above) and at discretion of the treating physician, were enrolled in the registry. No exclusion criteria was defined.As of February 20, 2019, 4 clinical sites in Germany participated and enrolled 118 patients. In detail, the University Hospital Hamburg-Eppendorf (UKE) enrolled 79 patients, the Mainz University Medical Center from the Johannes Gutenberg University, included 7 patients, the University Hospital in Essen registered 13 patients and the Weiden Clinic  contributed with 19 patients.2.3Data on clinical laboratory tests, vital signs, health status, liver function, and ADVOS multi treatment parameters were collected at different time points:-At hospital admission-At baseline (i.e. immediately before the first ADVOS treatment session)-After the first ADVOS treatment session-On Days 1, 3, and 7 after the first ADVOS treatment session, and once immediately after the last session of the same treatment cycle .-On days 28 and 90 after the first treatment session.If due to a worsening of the health condition during the observation period, a new ADVOS session was deemed appropriate on an enrolled patient who was not treated with ADVOS for >1 week, the patient was considered a new subject. This strategy pretended to homogenize the baseline characteristics of the patients. However, the patient was considered as a single subject for mortality rate analysis. Only 2 patients were on this situation.All patients who were exposed to the registry medical device were evaluated for Adverse Events (AEs) like catheter problems, bleeding, allergic reactions, clotting, electrolyte imbalances, and infections.2.4A fully web-based Trial software consisting of an Electronic Data Capturer (EDC), a Clinical Trial Management System (CMTS) and a Safety Control Center (SCC) was employed for data handling .Monitoring, project management, and data management was done by FGK Clinical Research  following standard operating procedures. FGK Clinical Research handled the data cleaning process, including logical check, and query processes.All required clinical data for this registry study were collected with an electronic Case Report Form (eCRF). Consistency checks were applied to verify correctness of data. An audit trail recorded all entries and corresponding changes.For patients who were unable or unwilling to provide informed consent and for patients whose data were collected retrospectively, data was anonymous  and could not be associated to a patient's name.2.5No specific primary or secondary endpoint was defined, as the objective was simply to collect data on real-life conditions during multiple organ dialysis. To evaluate the performance and safety, the assessments depicted in Table The settings  employed during ADVOS treatments were summarized for each patient after the last treatment session was conducted.2.6t test for paired samples was used to compare values before and after ADVOS sessions and treatments. Variables without homogeneous distribution were compared via Mann–Whitney U Test. A two-tailed P value lower than .05 was considered to indicate statistical significance. Mortality was assessed using Kaplan–Meier curves. Data were analyzed with IBM SPSS 24.0 for Windows. No statistical comparison was performed for binary variables.Continuous variables are reported as mean and standard error. Shapiro–Wilk and Levene tests were performed to assess the normal distribution of samples and the homogeneity of variance, respectively. The Student  Briefly, variables that were statistically associated with mortality in a bivariate analysis were considered as candidate variables in the multivariable model. A forward step-wise selection procedure with inclusion and exclusion cut-offs of 0.05 and 0.10, respectively, was followed. Adjusted odds ratios with 95% confidence intervals were reported. The cases with missing data were excluded. The Cox and Snell, and the Nagelkerke statistics were reported as a measure of variability explained by the model.A multivariable logistic regression model was developed to evaluate patient demographic and baseline clinical parameters (immediately before the first ADVOS session) associated with death, as previously done in other patient registries.2.7This study was approved by the Bavarian State Medical Association (Bayerische Landesärztekammer) on the 2nd of November 2016 and is registered in the German Registry for Clinical Studies (DRKS) and the International Clinical Trials Registry Platform from the World Health Organization (DRKS00017068). The registry poses no additional risks to the patient beyond those related to the collection and storage of the data. The patients were treated on-label, and their treating physician had already made the decision that treatment with a multiple organ dialysis was indicated.All patients who participated in the registry and for whom pseudonymous data collection was scheduled had to sign an informed consent form. For patients who did not consent, no personal registry-related data can be accessed due to the untraceable nature of the anonymous data. The necessary amendments were performed during the conduction of this registry in order to adapt to the new EU General Data Protection Regulation.Personal patient data is kept confidential in compliance with the European Data Protection Directive and other applicable international and national requirements.The costs for the registry study management and ethics committee fees were borne by ADVITOS GmbH.33.1Median age of registry participants was 60 years , and 70 of them were male (59.3%). Patients were critically ill with a median SOFA Score immediately before the first ADVOS session of 14  and a predicted mortality of 80%. The median number of failing organs was 3 IQR: 2, 4)  and base excess returned to the physiological range, while pCO2 remained unchanged  sessions per patient with a median duration of 17  hours , suggesting a better outcome when ADVOS is not used as a last line therapy Fig. . In fact4 This is reflected, among others, by the design of the study, the use of a clinical trial management tool provided with edit checks, by the possibility to perform audit trails and by the completeness of the data sheets with more than 80% of the required data fulfilled.Patients in the current registry were provided with ADVOS therapy according to its indications for use. The data summarized in this report provide real world evidence (RWE) on an emerging albumin hemodialysis device for multiple organ support. The obtained RWE is reliable as it holds enough quality based on its complete, transparent, generalizable, timely, and scalable documented data.4.1,28 Moreover, ADVOS therapy runs at low blood flows (median of 120 ml/minutes in this cohort), which reduces the risk of bleeding or hemolysis prevalent during other blood purification therapies, such as extracorporeal membrane oxygenation (ECMO) or extracorporeal CO2 removal (ECCO2R) with higher blood flows.,30Less than 6% of the treatments were aborted due to device errors, and only 13 clotting cases were described as adverse events related to the ADVOS therapy, none of them serious. According to the more than 6800 hours of ADVOS treatment in this study, the therapy can be considered safe. The significant reduction of platelet count seems to be not different to common dialysis treatments.All the electrolytes remained in the physiological range during treatments Table  but a si4.23-). These data correlate with those already presented in the pre-clinical trials,,17 and in clinical experiences.–20 Newer case-series currently being completed and already presented in scientific meetings show a similar trend.,31Three main benefits have been observed with ADVOS: a reduction in protein-bound disease markers , a removal of water-soluble substances  and a correction of acid-base parameters  with the ADVOS system. Huber recently reported ADVOS to be “the first integrated MOST device” (MOST = multiple organ support therapy).4.3The current cohort presents a heterogeneous population including patients with very different diagnosis and prognosis. First, patients with different organ dysfunctions and failures  including liver, kidney, coagulation, cardiovascular or respiratory systems were recruited Fig. . Further2 levels and 55% serum bicarbonate levels below commonly accepted physiological ranges.Therefore, these results might not be generalizable. In contrast, a subgroup analysis should be performed in order to identify different sub-cohorts. In these subgroups, controlled clinical trials are warranted to define the target population that could benefit most from the ADVOS therapy. Specially interesting could be the case of patients with acid-base disorders. In this report, 52.5% of patients had acidemia, 25% elevated pCO4.4First, a patient registry lacks the level of evidence of a randomized controlled trial (RCT). However, the real-world evidence obtained with these results are of valuable importance, as discussed previously.Second, only 118 patients without a control group were included. What is more, the type and number of patients included by each participating site was very different. This makes difficult to draw conclusions, especially in the centers with a lower number of patients. Nevertheless, as shown in Table Third, most of the data were obtained retrospectively. According to the study plan, this did not allow to a complete follow-up (FU)  unless the patient remained in hospital during the (FU) period. As a result, data on long-term outcome was not obtained.–39Fourth, even if the medical history was documented, an exact diagnosis was not reported. This issue hinders a sub-group analysis according to the underlying syndrome. In order to perform such analysis, surrogate parameters such as bilirubin, creatinine, lactate or acid-base parameters could be used instead. In fact, using a cut-off value of 16, huge differences are shown in mortality rates in our patients (SOFA <16: 47% vs SOFA ≥16: 72%). New studies comparing patients with different baseline severity of illness might be helpful to define a target population who could benefit most from the ADVOS therapy.Finally, as a consequence of the absence of consensus guidelines for the use of ADVOS, patients were probably treated too late. As shown by the high baseline SOFA Score before the ADVOS treatments (median of 14), multiple organ failure had already developed in most of the patients, which significantly reduced the chances of survival.5–21 On the other hand, the small number of device-related adverse events highlights the safety of the therapy. Moreover, a trend towards a mortality rate reduction was observed in each of the participating sites. Nevertheless, all the data provided in this report should be carefully interpreted due to the nature of patient's registries.This 2-year report shows that the Registry on Extracorporeal Multiple Organ Support with the ADVOS system is a feasible and valuable tool to increase the evidence on the safety and performance of the ADVOS therapy. On the 1 hand, data on removal of water-soluble and protein-bound substances, as well as acid-base correction correlate well with previous results.We want to acknowledge Prof. Wolfgang Huber for its valuable advice during the study design and the manuscript writing.VF and JL participated in the study design and conceptualization. AP and VF contributed with the statistical analysis and wrote the manuscript. AF, DJ, JL, and BT participated in data curation. JWM, AK, and SK provided critical review with important intellectual input. All authors are accountable for all aspects of the work, and all authors read and approved the final manuscript.Conceptualization: Valentin Fuhrmann, Jens Lutz.Data curation: Andreas Faltlhauser, Bartosz Tyczynski, Dominik Jarczak, Jens Lutz, Julia Weinmann–Menke.Formal analysis: Valentin Fuhrmann, Aritz Perez Ruiz de Garibay.Funding acquisition: Aritz Perez Ruiz de Garibay.Methodology: Valentin Fuhrmann.Supervision: Valentin Fuhrmann.Writing – original draft: Valentin Fuhrmann, Aritz Perez Ruiz de Garibay.Writing – review & editing: Valentin Fuhrmann, Aritz Perez Ruiz de Garibay, Andreas Faltlhauser, Bartosz Tyczynski, Dominik Jarczak, Jens Lutz, Julia Weinmann–Menke, Andreas Kribben, Stefan Kluge.


Maxicircles of all kinetoplastid flagellates are functional analogs of mitochondrial genome of other eukaryotes. They consist of two distinct parts, called the coding region and the divergent region (DR). The DR is composed of highly repetitive sequences and, as such, remains the least explored segment of a trypanosomatid genome. It is extremely difficult to sequence and assemble, that is why very few full length maxicircle sequences were available until now. Using PacBio data, we assembled 17 complete maxicircles from different species of trypanosomatids. Here we present their large-scale comparative analysis and describe common patterns of DR organization in trypanosomatids.  Trypanosomatids are unicellular parasitic organisms with a single large mitochondrion per cell ,2. MitocLeishmania tarentolae revealed that various repetitive elements are grouped into larger clusters, which, in turn, are repeats themselves. Most repetitive units within clusters are not perfect repeats, as they share 65–100% sequence identity [The DR was shown to be composed of repetitive sequence elements and, therefore, extremely hard to sequence and assemble ,27,28. Midentity . The DR identity . Trypanosoma lewisi, flanking 12S and ND5 genes, respectively, revealed that these loci have different patterns of repeats [With the development of sequencing technologies some maxicircles were sequenced completely ,30,31,32Leishmania spp. under various conditions [Arguably, an even more interesting observation is the ability of DR to evolve quickly. Massive structural rearrangements were observed in cultures of nditions ,34,35. ORepeated sequences, especially tandem repeats and large duplications, complicate the assembly process and result in errors even in the coding sequences. Nevertheless, sufficiently long reads can help to overcome this hurdle . In the Using our custom pipeline to assemble PacBio reads containing 12S and/or ND5 mitochondrial genes we recovered 17 full-length mitochondrial maxicircle sequences of different trypanosomatid species. Trypanosoma brucei (Lister 427), while the longest one of 47,384 bp is from T. cruzi (Dm28c). Trypanosoma spp., where cryptogenes undergo the most extensive RNA editing [Maxicircles have different sizes. The shortest sequence in this study of 23,201 bp belongs to i Lister 27, while editing , while t editing ,38,39.Leishmania spp., the DR length between the two strains of L. braziliensis and three strains of L. donovani vary by ≈3 kbps. The DRs of maxicircles are much more variable in size, reflecting the dynamics of this structure in evolution. This difference is over 20 kbps for maxicircles of different trypanosomatid species. Even within the phylogenetically compact group of Leishmania donovani strains and L. infantum is about 4500 bp, they cluster together on the PCA plot. The same is true for T. cruzi strains, which possess even more dramatic DR length difference. The most parsimonious explanation for this observation is that the level of repeat arrays is very unstable, despite the fact that the structure of small repetitive units of the DR is species-specific. Rearrangements at this level may account for the DR length variation and can happen frequently, due to the recombination between stable repetitive units.PCA  of triplet frequencies demonstrated that even on the level of very small sequence elements (triplets) the DR has species-specific composition and the clustering is generally consistent with phylogenetic relationships of the species . ImportaLeishmania amazonensis has 160 exact matches of 5’-TTAAATTAAATTAAATTAAATTAAA-3’ sequence in the 33,779 bp-long maxicircle, and this sequence is a pentamer of the ‘TTAAA’ block itself. The DR of L. pyrrhocoris H10 has only 45 perfect repeats of 5’-ATATTGAAAATAAAGTGCTAGATA-3’ sequence in its maxicircle of approximately the same length, and this sequence is not composed of smaller units. In the DRs of T. cruzi TCC maxicircles, the most frequent 24-mer is repeated only 25 times, implying more diffused organization of repeated units.Next, we analyzed the pattern of k-mer frequencies in maxicircles for small values of k . This anL. donovani strains have maxicircles, which are located close to each other on the PCA axis  [Previous studies of sequences, derived from the DRs of clusters ,40. In acopelti) . Here, wND5 and 12S genes, respectively. Both elements are composed of repeated sequences, but these sequences have a different length of the repeat (periodicity), structure, and pattern of repetitions. The P5 is a tandem repeat with a large period, but small number of repetitions. In contrast, the P12 element is composed of highly repetitive units with a small period, which are organized in repeat arrays of varying length. Arrays are frequently interspersed by repeat units of a radically different type, the I-elements, which are dissimilar to repeats forming the arrays  for the six species are shown in yanensis a or L. athiopica a. The mo. brucei f, in whiLeishmania infantum share over 95% identity in their sequences , while three Trypanosoma spp. and three monoxenous trypanosomatids analyzed here have less regular GC profiles of their P12 element.Of note, the GC profiles of most T. cruzi maxicircles . Inspecting coverage profiles in Integrated Genome Browser (IGB), we noticed that generally, but not always, less covered regions of the maxicircle were from the DR. We carefully examined all the assemblies and discarded one for L. donovani FDAARGOS_361 and L. pyrrhocoris H10) we used available Illumina paired-end sequencing reads to assess the quality of assemblies. We found that Illumina reads align well on both of our assemblies, supporting all nucleotides of the maxicircle sequences. For L. donovani strain FDAARGOS_361, mapping the paired-end reads (150 + 150 with average insert size of 180 bp) onto our assembly produced a profile with uneven coverage of the maxicircle sequence. For Leptomonas pyrrhocoris H10 we used the 150 + 150 paired-end reads with average insert size of 940 bp. In this case the coverage profile did not differ between the coding and divergent regions [L. donovani DR has more homogeneous repeat units and, as such, is a more difficult reference for the read mapping). For two species ( regions  (SupplemLeishmania donovani strain Pasteur with that from the GenBank (CP022652), which was produced by the HGAP3 assembly algorithm from the same PacBio reads. The GenBank sequence contains duplicated nucleotides on the 5’ end, which overlap with ≈4000 bp on the 3’ end of the assembly. After trimming, both sequences had nearly equal length and high level of sequence identity, though identity percent dropped slightly in the DR, indicating that the choice of the PacBio assembly method may influence the resultant sequence, especially in the low-covered regions. We also compared the maxicircle of Here we present the sequences of 17 new full trypanosomatid maxicircles. The big problem of maxicircle sequencing is the divergent region, which remains difficult to assemble even using the modern NGS techniques ,32. In tND5 and 12S genes, respectively. Previously, similar structures have been described for T. lewisi [Our analysis revealed the common architectural pattern of the DR organization. We described two regions with different structure, the P5 and P12 elements, according to their proximity to the . lewisi  as secti. lewisi . The sec12S-proximal clusters of the Leishmania spp. DRs were investigated previously [Trypanosoma brucei [The difference in DR length is connected to the massive rearrangements in the P12 element, which is built from the repetitive elements, further organized in arrays. Numbers and lengths of such arrays are species-specific and can change quickly. Sequence rearrangements in the eviously ,40 and ta brucei . The dri12S-proximal region is very dynamic, varying between phylogenetically close strains of Leishmania donovani and L. braziliensis. Inverted repeats are detected frequently in this region, possibly indicating its high recombination potential. Moreover, we detected a few PacBio reads bearing ND5 and/or 12S rRNA sequences, which were not included in the final assembly by the software used. These reads can originate from rare maxicircle variants, but current datasets have insufficient sequencing depth to confirm this.It has been also proposed that there can be different structural classes of maxicircles within the same pool of kDNA ,18,34, bLeishmania spp. it is present in a form of a GC-rich tandem repeat. In some species it can be reduced or absent, indicating that this structural pattern is not crucial for maxicircle replication or transcription.Repeat units in the P12 locus are both species-specific and conservative, strongly supporting the conclusion that these arrays are not junk DNA. It is plausible to suggest that these repeat arrays contain binding sites for some transcription factors or DNA maintenance proteins, which may stabilize such DNA-protein complexes. In this case, structural rearrangements of the P12 element can play a role in nucleoid remodeling, controlling access of proteins to the coding region loci. The P5 element appears to be more conservative in terms of organization, and in T. brucei P12 element [It has been suggested that a putative replication origin and promotor sequences are located in the DR ,22. For In order to assemble full-length maxicircles, we used the available PacBio datasets deposited in the SRA NCBI database. Information on accession numbers of sequences, used in this work, is summarized in 12S and ND5 maxicircle gene sequences, using sequences of Leptomonas pyrrhocoris, Trypanosoma cruzi, T. brucei, Leishmania amazonensis, L. tarentolae, and Crithidia expoeki as queries. Parameters for Blastn search were “-evalue 1e-10 -word_size 5 -outfmt 6”. Reads producing alignments with alignment length over 1000 were selected for further analysis. Assembly was performed with the Canu v1.8 assembler [Leishmania donovani and Leptomonas pyrrhocoris H10, we also tested the assembly protocol with different values of the “genomeSize” parameter (in a range of 20–100 k with a step of 10 k) and it did not influence the final contig sequence. To eliminate overlaps, the resultant sequences were trimmed using the nucmer tool from MUMmer v3.23 package [Reads were downloaded and converted to the fasta format. For quality control, all reads below 10 kbps were discarded. A local database for Blastn  was built from these reads and searched for the ssembler  with the package  with setND1, ND4, COI, Cyb, 9S, MURF2, and COII genes. These genes are not  edited and, therefore, are good markers for automated annotation. We concluded that all assembled maxicircles preserved gene order documented for trypanosomatid maxicircles [Assembled maxicircles were annotated in a semi-automatic mode. We checked the synteny of maxicircle genes using Blastn searches for icircles .12S and ND5 genes, followed by the manual curation. For the convenience of further analysis, we rotated all maxicircle sequences, so that each assembly started from the ND5 gene, followed be the DR, 12S, and rps12 genes. The best scoring match was used to determine the initial annotation coordinates for the Leishmania donovani FDAARGOS_361 and Leptomonas pyrrhocoris H10, the Illumina paired-end sequencing data were used to verify PacBio assemblies. Illumina reads were mapped with Bowtie2 v2.3.4.1 [Assemblies were checked by two criteria: (i) read coverage is sufficient throughout the assembly, and (ii) final maxicircle has perfectly assembled CR. The PacBio reads were mapped on the assembly with bwa mem algorithm of bwa v0.7.12 , with thv2.3.4.1  with thev2.3.4.1 .Frequency of each k-mer in a sequence was calculated using a custom python script. Repeated sequence regions were determined with the “repeat-match” from the MUMmer package. Regions with high level of sequence identity were found by the nucmer tool of the same package. Tandem repeats were identified using mreps v2.6  with “-rhttp://colorbrewer2.org/#type=diverging&scheme=Spectral&n=9), which sets red, green, and blue colors for values around 80%, 90%, and 100%, respectively. Dotplot diagrams of maxicircles were generated with mummerplot in MUMmer v3.23.Resulting outputs of repeat and homology search algorithms were plotted using Circos . Format We assembled and analyzed 17 complete maxicircles from different trypanosomatid species. Our analysis was focused on most arcane part of a maxicircle—the divergent region. This region is composed of repeated sequences, difficult to sequence and assemble, even with the modern NGS methods. Comparative analysis allowed us to infer the high-level architecture of the DR and describe some species-specific structural features. Our findings are consistent with previous works, which were focused on low-level sequence features, and shed light on how these low-level elements are organized and how they evolve.


Total RNA was extracted from the peripheral blood of mice, and samples were then subjected to RNA sequencing. When complete data were normalized, the high-dose group and low-dose group were screened for differentially expressed genes . DEGs were analyzed by gene ontology enrichment, and potential core genes were screened using protein–protein interaction (PPI) network and weighted gene co-expression network analysis (WGCNA). Moreover, the survival data in GSE65682 were used to observe the correlation between core genes and prognosis. A total of 967 DEGs were identified in the low-dose group, of which 390 were up-regulated and 577 were down-regulated. These genes were mainly enriched in white blood cell activation, lymphocyte activation, immune system response etc. LCK, ZAP70, ITK, CD247, and DOCK2 were found at the core of PPI network, while WGCNA found that interferon-inducible protein 35 (IFI35), ITGB3, and mediator complex subunit 25 (MED25) may be potential core genes. It was demonstrated that CD247, DOCK2, IFI35, ITK, and LCK core genes were positively correlated with prognosis based on GSE65682. CD247, DOCK2, IFI35, ITK, LCK, and MED25 might be important targets affecting the prognosis of sepsis.Sepsis is a common clinical disease with high mortality, and patients with sepsis have varied prognoses. Researchers need to explore the underlying mechanisms that determine the prognosis of sepsis. Hence, a mouse model was used to evaluate new potential prognostic markers of sepsis. Mice were randomly divided into low-dose group ( Sepsis is a common critical clinical illness with high mortality, and data show that its incidence is approximately 19 million patients per year in the United States ,3. The cThe experimental mice were obtained from the Experimental Animal Center of Southwest Medical University. Male Kunming mice aged 8–12 weeks, weighing 25–30 g were selected for the present study. The mice were housed in the Animal Laboratory of the Central Laboratory of Southwest Medical University, with a 12-h day–night cycle and an ambient temperature of 24 ± 2°C. Moreover, mice were raised in an ordinary rearing environment with free access to water and food. The experiments on animals began after 5 days of acclimatization to the environment. All animal experiments were approved by the Animal Ethics Association of Southwest Medical University. LPS was purchased from the American Sigma Inc (Fungus: O55: B5).n=3) and the death group (n=3). The survival group (low-dose group) received intraperitoneal injection of an equal volume of PBS solution and 20 mg/kg LPS, while the death group (high-dose group) received intraperitoneal injection of 40 mg/kg LPS. The body temperature and activity status of each group of mice were measured during the above time points. When the body temperature of the mouse in the death group reached 23°C (dying state), the blood from the eyeball was taken. At the same time point, one mouse from the survival group was randomly selected to have its eyeball blood taken, and the sample was treated with EDTA-2K for anticoagulation. During collection of blood samples, the survival group mice died of massive hemorrhage due to eyeball enucleation.The pre-experimental mice were randomly divided into four groups, each with ten mice, which summed up to a total of 40 mice. The mice were fasted for 10 h prior to the experiment and resumed eating 1 h after modeling. The mice from each group were administered with intraperitoneal injection of LPS and equal dose of PBS solution with drug concentrations of 10, 20, 40, and 50 mg/kg. Thereafter, their body temperature and activity status were measured 0, 2, 4, 8, 12, 24 h, 2, 3, 4, 5, and 7 days after injection, and the survival curve was plotted to determine the best modeling conditions. In the observation study, some mice died within 7 days due to the LPS intraperitoneal injection; those surviving over 7 days were killed by cervical spondylectomy. In the formal experiment, the mice were randomly divided into two groups: the survival group  were used to construct sequencing library. One microgram of the qualified test sample was taken. The test samples were prepared, the library was built, and the RNA-seq was sequenced according to the Illumina TruSeq™ RNA sample manual.In accordance with the instructions of Invitrogen, the TRIzol method was used to extract the total RNA from whole blood, and DNase I (TaKaRa) was used to eliminate the genomic DNA. The RNA quality was then measured by 2100 Bioanalyzer (Agilent) and then quantified using ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (ODhttp://bioinformatics.sdstate.edu/idep/) network tool based on R language [All clean sequencing data were compared using the GenBank mouse genome database, and subsequently, gene annotation was performed. To ensure the accuracy of gene expression levels, the fragments per kilobase of exon per million (FPKM) was employed to normalize gene expression levels. By performing log-uniform analysis on differentially expressed genes (DEGs), the differential genes in the survival and death groups caused by LPS were screened . Moreover, data visualization was carried out through integrated Differential Expression and Pathway analysis (iDEP)  online network tool is a popular tool for PPI analysis [The protein–protein interaction (PPI) network is a common method used in screening potential core genes, and the STRING  co-expression method was adopted for core gene screening . The purn=239) was the low-expression group, while the last half (n=240) was the high-expression group. As regarding GSE65682 data, GraphPad Prism 7.0 was adopted to plot the survival curve of the above core genes. Log-rank (Mantel–Cox) test was used to identify statistical differences between the two groups of data.Clinical data are the foundation of scientific research. In order to better screen genes related to prognosis and verify the relationship between core genes and prognosis, human sepsis data GSE65682  were useAfter an intraperitoneal injection of different concentrations of LPS, the mortality of mice has shown to be dose-dependent. In general, the higher the concentration, the higher the mortality rate A. FurtheAfter sequencing and gene annotation of peripheral blood cells of mice, the gene expression data were logarithmically processed B. As a rOn one hand, GO analysis revealed that the highly expressed genes in the survival group were mainly enriched in leukocytes, T cells, lymphocyte activation and differentiation, immune system processes, positive regulation of leukocyte adhesion, and peptide metabolic processes. On the other hand, genes highly expressed in the death group were mainly enriched in immune response, endogenous immune response, defense response, and response to external stimuli. The enrichment of these functions basically reflected the changes in biological processes in different states, as illustrated in In the present study, the PPI network analyzed by the STRING online tool included 67 core proteins, which were closely linked together. The proteins of LCK, ZAP70, JUN, ITK, CD247, DOCK2, and HCK were located at the core of the network, which might be involved in many different functions. In the present study, proteins involved in the immune system, cell surface receptor signaling pathways, cell proliferation regulation, cell differentiation, and cell death regulation were paid more attention, as illustrated in IFI35), ITGB3, ZHX2, USP25, FECH, KDM6B, and mediator complex subunit 25 (MED25) might be the potential core genes, as shown in Co-expression analysis is a common method of bioinformatics, which supplements the PPI analysis deficiency. We employed the WGCNA analysis method to construct a co-expression module for genes. A total of five modules were screened out in the present study, and two of them were mainly distributed. We visualized the first 25 genes of these two modules and discovered that interferon-inducible protein 35 (P<0.05), and the difference in the statistics of ITK survival curve was P=0.06. The expression value of MED25 demonstrated a negative correlation with the prognosis of patients (P<0.05), as shown in To further explore the correlation between genes, we screened the prognosis of clinical patients, and the genetic data of sepsis peripheral blood cells in GSE65682 were used for survival analysis. Finally, we found that the expression values of CD247, DOCK2, IFI35, and LCK manifested a positive correlation with the prognosis of patients (Sepsis is a common critical clinical condition, especially in patients in the ICU. Sepsis involves multidisciplinary and multisystem changes . ClinicaIn the present study, peripheral blood cells were sequenced and screened under bioinformatics technology. A total of 967 DEGs were screened out. Among them, 390 genes were up-regulated and 577 genes were down-regulated in the survival group. Through GO analysis, it was found that gene function enrichment in the survival group was mainly related to the activation and differentiation of white blood cells, lymphocytes, and T cells, which suggested that the body is establishing a second line of defense, or may participate in the establishment of endotoxin tolerance mechanisms. The gene function enrichment in the death group was mainly related to immune response and endogenous defense response, which indicated that the body produces a large number of inflammatory mediators and induces an inflammatory cascade amplification effect. This was consistent with our ‘inflammatory storm’ theory.In the method of screening core genes, we used PPI and WGCNA to screen the genes together , as far CD247, DOCK2, IFI35, LCK, MED25, and ITK. Among them, CD247, DOCK2, IFI35, LCK, and ITK showed positive correlation with prognosis, suggesting that they might be beneficial to patient survival. The MED25 gene was negatively correlated with the prognosis, which indicated that it plays a role in the promotion of death process of patients.To further increase the positive rate of bioinformatics screening, we included the clinical prognostic data of patients. Verification in the human body will make the conclusion more convincing. The present study screened six potential core genes: CD247 is a characteristic protein located in the T-cell membrane. Existing research revealed that it is mainly related to immune diseases , while tThe present study confirmed the prognostic correlation of the six core genes screened. However, reports on the screened genes and sepsis are limited, which suggests that they may be potential research targets, while their specific functions and mechanisms need to be further studied.


The purpose of this retrospective analysis was to build and validate nomograms to predict the cancer-specific survival (CSS) and overall survival (OS) of head and neck neuroendocrine carcinoma (HNNEC) patients.A total of 493 HNNEC patients were selected from the Surveillance, Epidemiology, and End Results (SEER) database between 2004 and 2015, and 74 HNNEC patients were collected from the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital (HCH) between 2008 and 2020. Patients from SEER were randomly assigned into training (N=345) and internal validation (N=148) groups, and the independent data group (N=74) from HCH was used for external validation. Independent prognostic factors were collected using an input method in a Cox regression model, and they were then included in nomograms to predict 3‐, 5‐, and 10‐year CSS and OS rates of HNNEC patients. Finally, we evaluated the internal and external validity of the nomograms using the consistency index, while assessing their prediction accuracy using calibration curves. A receiver operating curve (ROC) was also used to measure the performance of the survival models.The 3-, 5-, and 10-year nomograms of this analysis demonstrated that M classification had the largest influence on CSS and OS of HNNEC, followed by the AJCC stage, N stage, age at diagnosis, sex/gender, radiation therapy, and marital status. The training validation C-indexes for the CSS and OS models were 0.739 and 0.713, respectively. Those for the internal validation group were 0.726 and 0.703, respectively, and for the external validation group were 0.765 and 0.709, respectively. The area under the ROC curve (AUC) of 3-, 5-, and 10-year CSS and OS models were 0.81, 0.82, 0.82, and 0.78, 0.81, and 0.82, respectively. The C-indexes were all higher than 0.7, indicating the high accuracy ability of our model’s survival prediction.In this study, prognosis nomograms in HNNEC patients were constructed to predict CSS and OS for the first time. Clinicians can identify patients’ survival risk better and help patients understand their survival prognosis for the next 3, 5, and 10 years more clearly by using these nomograms. Neuroendocrine carcinomas are rare malignancies of the head and neck. HNNEC is rare, accounting for approximately <5% of all head and neck cancers. Most patients present with advanced disease at the time of diagnosis. This may be due to the previous lack of clear pathologic diagnostic criteria. In the previous literature, typical carcinoid and atypical carcinoid neuroendocrine carcinomas were exceedingly rare in HNNECs, perhaps because they had been classified under non-descriptive categories, such as “neuroendocrine carcinoma, not otherwise specified (NOS)”. These tumors often look similar to other sites’ neuroendocrine tumors, and are thus particularly difficult to distinguish . Early sIn the early years, the literature on head and neck NEC was limited to case reports –15 and sThe most common clinical manifestation of salivary gland NEC is a progressively expanding neck mass arising from the parotid or submandibular glands . The maiHNNEC is an exceedingly rare entity that is highly malignant, aggressively invasive, and has a high relapse rate and a poor prognosis. There is a paucity of data on the prognostic factors influencing survival in HNNECs; therefore, it often presents diagnostic uncertainty and therapeutic challenges, and patients may be initially misdiagnosed. As a result, larger sample sizes are needed to confirm the relationship between clinical factors and prognosis, and further research is required to explore the risk factors associated with HNNECs.There was also a need to construct a prognostic prediction model to accurately predict the survival of this cancer. For this reason, we used the Surveillance, Epidemiology, and End Results (SEER) database to construct a prognostic model related to CSS and OS in patients with HNNECs to adjust clinical practice and improve patient survival.The SEER database is supported by the National Cancer Institute as an authoritative source of information on population-based cancer incidence and survival, and it is considered the gold standard for cancer registry worldwide . This isThe SEER 22 database covers data from the years 2004 to 2015 from 22 cancer registries throughout the United States. Cases collected were done based on the International Classification of Diseases for Oncology, Third Edition (ICD-O-3) topography, and histology/behavior codes. Using the SEER database, 70 sites in the head and neck location were collected and analyzed. The following codes were included in this study. Oral cavity, oropharynx, and hypopharynx including malignant neoplasms from C01.9 (base of tongue) to C06.9 , C09.0 (tonsillar fossa) to C09.9 , C10.0  to C10.9 (oropharynx NOS), C12.9 (pyriform sinus) to C13.9 (hypopharynx NOS), C14.0 , and C14.8 . Nasopharynx included C11.0  to C11.9 . Nasal cavity and sinuses included C30.0  to C31.9 . Larynx included C32.0 (glottis) to C32.9 . Subsequently, we filtered the cases based on ICD-O-3 histology/behavior codes covering large-cell neuroendocrine carcinoma (8013/3), small-cell neuroendocrine carcinoma, NOS (8041/3), typical carcinoid (8240/3), neuroendocrine carcinoma, NOS (8246/3), and atypical carcinoid tumor (8249/3).Patients contained in this study had to satisfy the eligibility criteria: (a) The patients with HNNECs were enrolled between 2004 and 2015, (b) HNNEC was their first or only primary diagnosis, (c) The diagnosis was verified by histological examination, and (d) Complete and integrated follow-up data of patients could be available. The exclusion criteria included the following: (a) Patients were younger than 20, (b) Information about the tumor stage and the follow-up data was lacking,and(c) Death certificate and autopsy cases were also excluded. Finally, 493 eligible HNNEC’s patients were chosen from the SEER database. A random split‐sample approach was applied to separate the total eligible patients into a training cohort (n=345) and internal validation cohort (n=148) at a split ratio of 7:3, using the “caret” package in R version 3.6.3. At the same time, 74 patients with pathological diagnosis of HNNEC from our medical center between 2008 and 2020 were included in the external validation cohort. This retrospective validation was approved by the Ethics Committee of our hospital. .The detailed screening process is presented in We carried out the analysis of the above-mentioned factors descriptively. Overall survival (OS) measured the time from the date of diagnosis until the date of death from any cause or last follow-up, and the cancer special survival (CSS) was estimated from the interval between the HNNEC diagnosis until the cancer-associated death or the last follow-up. Then the Kaplan–Meier method was applied to plot survival curves, using the log-rank test to analyze the differences in survival. Both univariate and multivariate Cox regression modeling was employed to evaluate the significant prognostic factors of CSS and OS. By calculating the hazard ratios (HR) and 95% confidence intervals (95% CI), the relationship between the risk factors and survival could be assessed. All analyses were completed by the statistical software SPSS 22.0 and R version 3.6.3. The results of P<0.05 were selected as statistically significant.Using the independent prognostic factors identified in the multivariate analysis, nomogram models of CSS and OS were constructed. The calibration plot and concordance index (C-index) was conducted to evaluate the performance of the CSS and OS nomograms in the training, internal validation, and external validation cohorts successively. The C-index ranges from 0 to 1.0, with 1.0 expressing a perfect predictor and 0.5 indicating a completely random prediction. In addition, we plotted the ROC curves and computed the areas under ROC (AUC) to evaluate the model forecasting capability.In SEER, we obtained believable data on CSS and OS for 493 HNNECs patients. In total, 316(64.0%) patients in the overall cohort died by the time of the last follow-up. Among these patients, 261(52.9%) died due to HNNEC and 55(11.2%) died because of causes other than HNNEC. The OS rates of the overall cohort at 3, 5, and 10 years were 44.3%, 36.5%, and 26.2%, respectively. The CSS rates of the overall cohort at 3, 5, and 10 years were 50.9%, 43.0%, and 35.0%, respectively. Cumulative incidence curves by age at diagnosis, race, gender, stage, and treatment were presented in Univariate and multivariate analyses of characteristics associated with the CSS and OS were presented in Multivariate analysis was conducted to identify the independent predictors of HNNCE. Multivariate Cox proportional hazard regression models of OS showed that age≥80 years , being male , AJCC stage III , N2 , N3 , M1 , and not receiving radiotherapy  were risk factors associated with OS. Multivariate Cox analysis of CSS indicated that AJCC stage III , AJCC stage IV , N3 , M1 , and being widowed  were risk factors connected with CSS. 4.Age, gender, AJCC stage, N stage, M stage, radiation therapy, and marital status were the independent prognostic factors integrated to establish prognostic nomograms for evaluating the 3-, 5-, and 10-year OS and CSS of HNNEC patients Figure 3A total of 493 patients diagnosed with HNNEC were collected in the SEER database between 2004 and 2015. First, we developed and validated nomogram models for HNNEC based on clinical features and treatment modalities. Additionally, the calibration curves and the ROC curve showed a high prediction accuracy of the proposed nomograms. When external validation was performed on the Independent medical center cohort, the C-indexes for OS or CSS were all higher than 0.7. These results proved the high accuracy and excellent discrimination ability of our models’ survival prediction.Existing analysis of prognosis in HNNEC is usually achieved by comparison with the OS or CSS. In recent years, some studies using the SEER database have been published. Patel et al. reported on 201 SNEC cases in 2015. They compared DSS (CSS) between patients with SNECs. Their results indicated that some adverse prognostic factors influence the survival of SNEC, including tumor locations such as ethmoid and maxillary involvement, treatment plan such as radiotherapy without surgical resection, and AJCC stages III and IV . Ghosh eIn our study, an OS nomogram was developed based on six independent prognostic factors, including age, sex/gender, AJCC stage, N stage, M stage, and radiotherapy. A CSS nomogram was developed based on four independent prognostic factors: AJCC stage, N stage, M stage, and marital status. Every variable corresponds to a fraction on the logarithmic scale of each nomogram. The fractions for all variables were summed to obtain the total score, and a vertical line could be drawn straight from the total‐points scale to evaluate the chance of surviving for 3, 5, and 10 years.Our two nomograms included several prognostic variables that are used in routine clinical practice. Some research studies have found an inclination for the nomogram score to increase with the M classification , 47. ThiRetrospective research studies based on the SEER database found that survival for grade III or IV (20.5%) LNEC was significantly lower than that for grade I or II (60.2%). Low histological grades (I/II) were observed to have better 5-years CSS than grades III/IV, which is consistent with our results . AlthougAge and sex/gender are the secondary factors for OS but not CSS. This finding is reasonable because CSS pays more attention to cancer itself. In our analysis, older age indicated a worse prognosis. Previous literature has reported that the average age at the time of diagnosis was between 55 and 56 years for SNEC , 44. In Furthermore, we also found a male predilection in our study, with a male to female ratio of 1.97:1. The reason for this sexual discrepancy might be that a higher proportion of men smoke , 47. HowMoreover, we found that being married was a protective variable of CSS. It influenced the survival of patients with HNNEC. A report indicated that unmarried patients, including the widowed and divorced, were at higher risk of tumor metastasis, inadequate treatment, and death from cancer . The reaApparently, undergoing surgery could improve the survival rate significantly according to univariate analyses. However, this was not significant in multivariate analysis. In our two nomogram models, we found that those who underwent postoperative radiotherapy could obtain a lower score than those who didn’t. In other words, patients with postoperative radiotherapy showed better survival rates. This was confirmed in both univariate and multivariate analyses. This phenomenon was also reported in a meta-analysis that found that surgery plus radiotherapy with or without chemotherapy was associated with a lower risk of death .Our survival curve also indicated that both surgery and surgery combined with radiotherapy were significantly associated with improved outcomes in HNNEC patients . However, chemotherapy did not provide greater outcomes in these patients, and the application of chemotherapy did not improve the overall survival of these patients .We found that the most common locations for HNNEC were the salivary gland, nasal sinuses, and larynx, which is in agreement with previous reports. While univariate analysis reached statistical significance, multivariate Cox regression analysis showed that location may not be an independent prognostic factor. Thus, it was not included in our nomogram.An earlier study by Likhacheva et al. (2011) found that pathologic classification may not be a crucial factor in the clinical therapy of sinonasal NEC . HoweverAs far as we know, other nomograms have been built to predict the survival of head and neck cancer previously, including those for nasopharyngeal carcinoma, tongue squamous cell cancer, salivary gland cancer, and adenoid cystic cancer, among others. However, our analysis is the first to design a nomogram for HNNEC, and this nomogram could help physicians make clinical decisions. For example, there are two HNNEC patients in AJCC stage III: one is a 31-year-old married man who received radiotherapy after surgery, while the other patient is a 77-year-old widowed woman who underwent surgery only, without radiation therapy. According to the AJCC staging system, these two patients would yield a duplicate prognosis. However, applying our nomograms could produce a different result: the 3-, 5-, and 10-year OS rates of the former patient were 80%, 74%, and 62%, respectively, and those of the latter were 32%, 22%, and 12%, respectively. The corresponding CSS rates were 76%, 70%, 60%, and 42%, 31%, 19%, respectively.There were also some potential limitations to our study. Although the strength of the SEER database is its relatively greater ability to provide more data for studies of rare cancers, this is a retrospective study and lacks specific treatment details. For example, SEER data did not include the specific chemotherapy regimen and could not distinguish between sequential and concurrent chemoradiation. In addition, details such as local control rate, cigarette use, alcohol consumption, and toxic reactions are not available for research. We used the data of our medical center to externally validate the nomogram model, due to the rare incidence in our single hospital and the extremely small number of HNNEC patients, a multi-centered, large sample study is still required to further validation. In the future, we look forward to prospective studies to test nomograms to compensate for these limitations.In conclusion, we have established an easy-to-use visual nomogram with several clinical and pathological factors to predict the survival risk of HNNECs. A fraction corresponding to HNNEC patients’ prognostic factors would be acquired on the nomogram point scale. Adding these scores to the total on the bottom scale could predict the 3-, 5-, and 10-year OS and CSS of HNNEC patients.https://seer.cancer.gov/data-software/documentation/seerstat/.The datasets presented in this study can be found in online repositories: The studies involving human participants were reviewed and approved by Medical Ethics Committee of Hunan Cancer Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.OYY and FL contributed to conception and design, methodology and investigation, and study supervision. OYY and WJX contributed to data collection, data analysis and interpretation and writing and review. HBT contributed to data analysis and interpretation and the software. SNF and YZC contributed to the validation. All authors contributed to the article and approved the submitted version.This study was supported by grants from Hunan Provincial Science and Technology Department , Beijing Hope Run Special Fund of Cancer Foundation of China (No. LC2016W05 and LC2016W06), and Health Commission of Hunan Province (No. B2016048 and C2017044). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: The neutrophil-to-lymphocyte ratio (NLR) has been reported to be associated with survival in solid malignancies. The main goal was to evaluate the prognostic significance of the NLR in patients with p16-negative squamous cell carcinoma of unknown primary (SCCUP) in head and neck.Methods: The association between the NLR and clinical pathologic variables was evaluated by the chi-square test. The primary endpoint of interest was disease-specific survival (DSS). Univariate and Coxmodel analyses were used to evaluate prognostic factors.Results: A total of 153 patients were included in the analysis. Cancer cachexia was noted in 10 patients. The mean NLR value was 3.9 (range: 1.4–8.3). A high NLR was significantly associated with cancer cachexia development. The 5-year DSS rate was 58%. In patients with NLRs varying from 1.4 to 3.7, the 5-year DSS rate was 71%; in patients with NLRs varying from 3.7 to 6.0, the 5-year DSS rate was 57%; in patients with NLRs varying from 6.0 to 8.3, the 5-year DSS rate was 39%, and the difference was significant (p = 0.001). Further Cox model analysis confirmed the independence of the NLR in predicting survival.Conclusions: In patients with p16-negative SCCUP, an NLR ≥ 6.0 is significantly associated with worse prognosis. Squamous cell carcinoma (SCC) of the head and neck (SCCHN) is one of the relatively uncommon malignant epithelial tumors in the body and usually has neck lymph node metastasis at initial diagnosis. However, in the clinic, ~1–4% of metastatic nodes can occur without any evidence of primary disease even after a clinical examination by a head and neck specialist, cytology and biopsy, endoscopy and radiological investigations including ultrasound, CT, MRI, and PET/CT , 3. TherRecent basic studies present the important role of the systemic inflammatory response in tumor cell invasion by promoting tumor cell proliferation, microvascular regeneration, and tumor metastasis , 13. TheThe Zhengzhou University Institutional Review Board approved this retrospective study, and all patients had given written consent for medical research before initial treatment. All the procedures were consistent with the relevant requirements.From January 2000 to December 2016, the medical records of patients with surgically treated SCCUP were retrospectively reviewed. Enrolled patients had to meet the following criteria: first, no previous history of SCC of the head and neck; second, negative immunohistochemistry of p16 ; and thiIn our cancer center, investigations of clinical and endoscopic examination, ultrasound, CT or/and MRI, and PET-CT were always performed in patients with a neck mass after malignant disease was suggested by fine-needle aspiration biopsy. Neck dissection of level I-V was suggested for all patients, bilateral operation was performed if there was doubt of bilateral lesion. Functional neck dissection was usually the first choice unless there was N3 disease or apparent invasion of sternocleidomastoid muscle or internal jugular vein or accessory nerve. After therapy, the patients were examined every 3 months during the first year, every 6 months during the second year, and once per year after the second year.The NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count within 2 weeks before operation , 19, 20.p < 0.05 was considered significant.Patient demographic variables were compared across NLR values using the chi-square test. The primary endpoint of interest was disease-specific survival (DSS), and the survival time was calculated from the date of surgery to the event or the last follow-up. The Kaplan-Meier method (log-rank test) was used to plot the DSS curve. The factors of age, sex, smoking, drinking, neck lymph node stage, extracapsular spread, cancer cachexia, adjuvant treatment, and NLR were included for analyzing their association with DSS, and the variables that were significant in the univariate analysis were then analyzed in the Cox model to find the independent prognostic factors with the use of a forward stepwise (likelihood ratio) procedure. All statistical analyses were performed using SPSS 20.0, and A total of 153 patients  were enrolled for analysis, and the mean age was 62.7 (range: 28–73) years. Smoking and drinking status were reported in 57 and 43 patients, respectively. Cancer cachexia was noted in 10 patients. Thirty-one patients presented with pN1 disease, 53 patients with pN2a disease, 61 patients with pN2b disease, 6 patients with pN2c disease, and 2 patients with pN3 disease. Extracapsular spread (ECS) was reported in 37 patients. Disease grade was distributed as follows: well differentiation in 20 patients, moderate differentiation in 73 patients, and poor differentiation in 60 patients. The mean NLR value was 3.9 (range: 1.4–8.3).Unilateral neck dissection was performed in 147 patients, and bilateral neck dissection was performed in 6 patients. Radical neck dissection was performed in 15 patients, modified radical neck dissection was performed in 84 patients, and functional neck dissection was performed in 60 patients.p = 0.008). No significant relationship between the NLR and other variables was noted .The associations between the NLR and clinical pathologic variables are presented in The mean follow-up time was 79.5 (range: 15–221) months, 138 patients underwent adjuvant radiotherapy, and 64 patients underwent adjuvant chemotherapy. Recurrence occurred in 85 patients: 69 patients had loco-regional recurrence, and 16 patients had simultaneous loco-regional and distant recurrence. In these 85 patients, 14 cases received a second operation, 43 cases were treated by palliative chemotherapy combined with targeted drug, 28 cases refused any treatment.p = 0.001). Further Cox model analysis confirmed the independence of the NLR in predicting survival (A total of 73 patients died of the disease, and the 5-year DSS rate was 58%. In patients with an NLR varying from 1.4 to 3.7, the 5-year DSS rate was 71%; in patients with an NLR varying from 3.7 to 6.0, the 5-year DSS rate was 57%; in patients with an NLR varying from 6.0 to 8.3, the 5-year DSS rate was 39%, and the difference was significant (survival .The role of the NLR in predicting prognosis in SCCHN has been widely discussed –17, 22. The association between the NLR and clinical pathologic variables has been described by previous studies in head and neck cancer , 20, 23.It is still unclear whether the NLR plays the role of a non-specific marker of systemic stress that is associated with poor outcomes and whether the systemic inflammatory response is a reflection of a tumor that is unresponsive to treatment, similar to performance status. The exact association between a high NLR and poor outcome in cancer patients has not been fully elucidated. Some possible explanations can be mentioned based on previous evidence. The inflammatory microenvironment induces genotoxic stress via multiple mechanisms, including reactive oxygen species and the induction of activation-induced cytidine deaminase. The pretreatment NLR value reflects the status of the immune system and systemic inflammation. Elevation of neutrophils is a sign of local as well as systemic inflammatory responses. Several cytokines and angiogenic factors are produced by neutrophils, and these agents play an important role in promoting tumor development . AdditioCancer cachexia is mainly caused by diminished oral intake and catabolic factors secreted by tumors, including interferon-gamma, interleukins, and tumor necrosis factor . A recenPrognosis in SCCUP has been frequently analyzed, and widely accepted risk factors include advanced node stage, extracapsular spread, and adjuvant therapy –34. SimiThe limitations of this study must be acknowledged. First, it was a retrospective study, and the sample size was relatively small. A larger sample size in a prospective study is thus needed to clarify these questions. Second, although the NLR has been well-established as an indicator of prognosis in a variety of cancers, including breast, esophageal, renal, and liver cancers, the complexity of carcinogenesis is so high in malignancies that a single indicator may not be a reliable predictor to determine outcome; additionally, neutrophil and lymphocyte counts are non-specific parameters because they can be influenced by concomitant conditions, such as infections or inflammation.In summary, the NLR is significantly associated with prognosis in patients with p16-negative SCCUP.All data generated or analyzed during this study are included in this published article. And the primary data could be obtained from the corresponding author.The Zhengzhou University institutional research committee approved our study and all participants signed an informed consent agreement. All the related procedures were consistent with Ethics Committee regulations.All the material came from our cancer center, and the publish consent have been obtained from all the patients.CX, HL, WD, and QF: study design, manuscript writing, and manuscript revising: JY, WD, and QF: studies selecting and data analysis. JY, JW, XZ, and QF: study quality evaluating. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alopecia areata (AA) is an inflammatory disease with a genetic and autoimmune basis. Herein, it was aimed to study the efficacy and safety of an immunomodulatory therapeutic agent, diphenylcyclopropenone, while manifesting its association with histopathological features, prognostic factors, and side effects.In this retrospective study, 98 patients  with alopecia, who were referred to the Hair Disease Polyclinic at the Department of Dermatology, between 2011 and 2015, were included. Together with medical histories and dermatological examinations, a skin biopsy for histopathological examination was conducted for all of the patients prior to therapy. Therapeutic success was evaluated on the basis of the hair regrowth percentage.Regarding the overall treatment success, 33 (34%) patients had complete response, 16 (16%) had partial response (between 50% and 99%), 27 (28%) had minimal response (between 1% and 49%), and 22 (22%) were nonresponders. Both sexs were equally represented in the outcome.There was a significant relation between the severity of alopecia and the treatment outcome (P = 0.038). Patients with AA had significantly better response when compared to those with alopecia totalis and universalis. There was no statistically significant relation with other parameters, such as disease duration, age, sex, atopy history, age of onset, and histopathological features. Alopecia areata (AA) is an inflammatory disease with a genetic and autoimmune basis, leading to various degrees of nonscarring patchy hair loss of the scalp and whole body areas. The estimated prevalence of AA is between 0.1% and 0.2% [1]. The exact aetiology of AA is still unknown, but genetic disposition has been identified, with some genes like TRAF1/C5 locus. Autoimmunity also plays a major role, with accompanying diseases of T cell auto-reactivity, such as Hashimoto’s tiroiditis or vitiligo. According to some studies, having a history of atopy also increases the risk of AA [2]. Cytokines like interferon-gamma, interleukins, and tumor necrosis factor alpha are thought to play a major role in the pathogenesis of the disease.Clinically, patients present with asymptomatic, patchy hair loss, with normal appearing underlying skin. Spontaneous resolution can occur, especially for those with limited involvement. However many patients have a chronic and recurrent course, with many attacks over the years. Treatment of alopecia has always been a big challenge for physicians. There are many different treatment modalities, with variable efficacies, including topical, intralesional or systemic corticosteroids, cyclosporine-A, local or systemic phototherapy, interferon-α, photodynamic therapy, acupuncture, topical minoxidil, anthralin, and topical immunotherapy agents.Topical application of diphenylcyclopropenone (DPCP) was originally developed in 1978 by Happle et al. [3]. The exact mechanism of action of this treatment modality is not fully discovered. However, DPCP induces an allergic contact dermatitis and this is thought to decrease the T-cell-mediated immune reaction against the hair bulb. There is also evidence that it acts on the autoreactive T-lymphocytes within the follicular milieu to induce apoptosis. DPCP immunotherapy has a modulatory effect on the proinflammatory cytokines within the hair follicle, and also provides antigenic competition, to distract the lymphocytes from their primary target.In this study, the efficacy and safety of topical immunotherapy with DPCP were evaluated retrospectively. The favorable prognostic factors to predict the response to DPCP immunotherapy were also assessed.In this retrospective study, 98 patients  with alopecia areata, totalis, or universalis, who were referred to the Hair Disease Polyclinic at the Department of Dermatology of Cerrahpaşa Medical Faculty, from August 2011 to June 2015, were included. All of the patients had clinically diagnosed alopecia and for all of them, the diagnosis was confirmed with histopathological examination. The patients had been resistant to any other topical or systemic conventional therapies for at least 6 months. Prior to the therapy, the patients were informed about the efficacy, estimated duration, and possible side effects of the therapy, such as enlargement of the lymph nodes on the head and neck area; severe eczematous reactions, such as bullous eruptions or urticarial plaques limited to the scalp or extending to other body parts; fever, malaise and fatigue, especially within 2 days after the treatment; and sometimes anaphylaxis-like reactions. All of the patients gave their informed consent. If a patient was below 18 years of age, consent was taken from their family. The mean age of the patients was 23.5 years, ranging from 5 to 59 years. Exclusion criteria included current pregnancy or lactation, being a child under the age of 5, and having vitiligo, severe photosensitivity, or active systemic malignancies. Before initiation of the therapy, factors such as sex, duration of the disease, age of onset, age at the beginning of therapy, previous treatments, history of atopy, type and severity of hair loss (in forms of percentage), presence of ophiasis pattern, and eyebrow, eyelash, beard, and body hair involvement were recorded. On the first visit, a physical assessment was performed, with emphasis on grading the percentage of scalp involvement, type of alopecia, and nail involvement. Prior to the therapy, a skin biopsy of 4 mm in diameter was taken from all of the patients, from the affected site on the scalp and histopathological investigations were conducted. The following parameters were assessed during the histopathological investigations of the scalp biopsies: number of total follicular units, number of total hair follicles, number of follicular stelae, number of anagen, catagen and telogen hair follicles, degree of lymphocytic infiltration of the hair follicle, and degree of perifollicular fibrosis. The degree of fibrosis was evaluated numerically as 0: no fibrosis, 1: mild fibrosis, 2: moderate fibrosis, and 3: severe fibrosis. Lymphocytic infiltration was also assessed similarly, as 0: no lymphocytic infiltration; 1: mild lymphocytic infiltration, which represented the involvement of less than 10% of total number of hair follicular bulbus and the presence of less than 3 rows of lymphocytes around each hair follicle; 2: moderate lymphocytic infiltration, which represented involvement of 10%–50% of the hair follicular bulbus and the presence of 3 to 6 rows of lymphocytes around each hair follicle; and 3: severe lymphocytic infiltration, which represented the involvement of more than 50% of all hair follicular bulbus and the presence of more than 6 rows of lymphocytes around each hair follicle.At the end of the study, therapeutic success was evaluated on the basis of the percentage of hair regrowth on the scalp.Topical immunotherapy with DPCP was performed following a standard protocol of sensitization. This protocol comprised the application of a 2% concentration of DPCP solution diluted in acetone, over an area of 2 × 2 cm on the occipital region of the scalp. The patients were told to avoid water contact of the sensitized area for 2 days and avoid sun exposure by using a wig or protective hat. After 2 days, the patients were checked to detect whether or not sensitization to DPCP had occurred. There was a lag period of 2 weeks after the sensitization, and then the treatment began. The initial DPCP concentration was 0.001%. DPCP solution was applied to all of the affected areas on the scalp, together with the eyebrows, once every week. Again after each session, the patients were told to avoid water contact for 2 days, including excessive sweating and sun exposure. DPCP solution was left on the scalp for 48 h and then washed off with a mild shampoo. The concentration of the DPCP solutions were increased weekly, unless there were serious side effects, including irritant contact dermatitis and photoallergic reactions, and the final concentration of 2% was reached at the end of week 6. Concentrations of 0.001%, 0.01%, 0.1%, 0.2%, 0.5%, 1%, and 2% were applied sequentially.During follow-up visits, the side effects of the patients were recorded, as well as the grade of hair regrowth. Once complete or cosmetically acceptable hair regrowth (amount of growth that eliminated the need for using a wig or hat) was achieved, the intervals of the DPCP application were prolonged to 2 weeks, 3 weeks, and monthly. By this method, the DPCP immunotherapy was discontinued gradually. In the case of hair loss during this tapering-off period, therapy was restored at weekly intervals. If there was no obvious response at the end of 6 months, immunotherapy was considered as noneffective and discontinued.Statistical analyses were performed using SPSS 19.0 for Windows . The Mann–Whitney, Wilcoxon, and Kruskal-Wallis statistical tests were performed for each data set and P < 0.05 was considered as statistically significant.The mean age of the patients was 23.2 years, ranging from 5 to 59 years. The mean age of the male patients was 21.8 years, with a standard deviation of 12.4. The mean age of the female patients was 25.5 years, with a standard deviation of 15.6. The demographic and clinical data are shown in Table 1. The mean period of disease was 4.2 years before the DPCP treatment (Table 1).Among the 98 patients, 49 (50%) were diagnosed with AA, while 19 (19.4%) had alopecia totalis, and 30 (30.6%) had alopecia universalis. At the beginning of the immunotherapy, 9 (9.25%) of the patients had less than 25% hair loss, which according to the Alopecia Areata Investigational Assessment Guidelines, was categorized as S1, while 13 (13.25%) had between 25% and 50% (S2), 16 (16.25%) had between 50% and 75% (S3), and 11 (11.25%) had between 75% and 99% (S4). Of the patients, 49 (50%) had total hair loss on the scalp area (S5), with a diagnosis of alopecia totalis or universalis.Additionally, 20 (20.4%) patients had a previous history of atopy, including atopic dermatitis or allergic asthma.With regards to the previous treatments, 37 (37.7%) patients had used only topical treatment modalities, including topical corticosteroids, minoxidil (2% or 5% concentration), and intralesional corticosteroid injections. Of the patients, 61 (62.3%) had tried both topical and systemic treatment options, including systemic corticosteroids, cyclosporine, and ultraviolet therapy.The mean duration of DPCP immunotherapy in the analyzed cohort was 13.1 months, with a minimum of 4 months and a maximum of 44 months.Regarding the overall treatment success, 33 (34%) patients had complete response , 16 (16%) patients had partial response (between 50% and 99%) , 27 (28%) patients had minimal response (between 1% and 49%), and 22 (22%) patients were nonresponders . Both sexes were equally represented in the outcome.Among the 22 nonresponding patients, 8 responded by 100% and 7 responded by 75%–99% during the course of the therapy followed by a sudden and complete loss of hair.The Mann–Whitney U test was used to investigate the relation between the type of alopecia and the treatment result. Statistical analyses revealed a significant relation between the type and severity of alopecia and the treatment outcome (P = 0.038). AA patients had significantly better response to the treatment when compared to the alopecia totalis or alopecia universalis patients. On the other hand, there was no statistically significant difference between the success rate of the therapy and the other parameters, such as the disease duration before DPCP therapy, age, sex, atopy history, age of onset of alopecia, and age at start of DPCP immunotherapy.Histopathological parameters were also compared with the success rates of the treatment, but there were no significant relationships with regards to the presence of fibrosis, lymphocytic infiltration, number of follicular stelae, and number of catagen hair follicles  Figure 4A shows mild fibrosis, Figure 4B shows moderate fibrosis, and Figure 4C shows severe fibrosis. In the case of lymphocyte infiltration, Figure 4D shows mild lymphocyte infiltration, Figure 4E shows moderate lymphocyte infiltration, and Figure 4F shows intense lymphocyte infiltration.The total number of sessions and thus, duration of therapy, that the patients received were found to be positively and significantly correlated with the success rates, meaning that the more sessions a patient received and the longer the therapy continued, and the better their response was.During the sensitization process, almost all of the patients experienced minimal erythema, itching, and burning sensation over the affected area. However, during the course of the therapy, among the 98 patients, 56 (57%) showed no side effects and 38 (38%) complained about minimal erythema and sensitivity over the scalp, especially during the subsequent 2 days after the application. Other common side effects included pruritus, dermatitis, vesicles, bullae, and flu-like symptoms. Of the patients, 4 (4%) had serious side effects, such as lymph node enlargement, fever, and general malaise; 3 (3%) had irreversible hyperpigmentation of the head and neck area, and 2 (2%) developed vitiligo macules.AA is an organ-specific autoimmune disease, and the stem cells of the hair follicles are characteristically spared, which implies a potential for regrowth of the hair. Despite this fact, to date, there remains no curative treatment for AA and none of the existing treatment options have been able to alter the course of the disease. Phototherapy or photochemotherapy, topical and oral corticosteroids, and cyclosporine and biological agents, such as efalizumab and etanercept, have all been used with various success rates [4–8]. Among these treatment options, topical immunotherapy agents have revealed promising clinical success with low side effect profiles. Topical sensitizers, such as dinitrochlorobenzene, SADBE, or DPCP create an allergic contact dermatitis, via delayed-type (type IV) hypersensitivity reaction [9]. Dinitrochlorobenzene is a highly mutagenic and squaric acid dibutylester that can easily lose stability in the case of long-term use and high temperatures [10]. Thus, DPCP was the choice of topical agents for this study, as well as many others in the literature [11]. The exact mechanism of action for DPCP has still not been clarified; however, the likelihood of a regulatory-T lymphocyte mediated role within the follicular unit is high [12]. Although antigenic competition with the responsible antigens has been by far the most accepted theory, other remarkable effects over the cytokine system have included increased IL-10 secretion and low CD4+ to CD8+ T cell ratios, together with the induction of other immunomodulatory substances, such as CTLA4 .In many studies of DPCP topical immunotherapy, initially, the solution was applied to one half of the scalp and only after there was hair growth, was the treatment extended to the entire scalp [15]. This approach aimed to determine the efficiency of the treatment while minimizing the side effects. However in the current study, after the sensitization process, the solution was applied to all of the hairless areas of the scalp, so as to obtain cosmetically acceptable results sooner.In many studies, it was reported that combination therapy with minoxidil or other agents did not improve the success rates [16–18]. Therefore, any topical or systemic treatment other than DPCP was not given to the patient group.The studies have revealed a wide range of treatment efficacies, with a success rate of up to 85% for cosmetically acceptable hair regrowth. In a study of 148 patients, 100% regrowth rate was achieved for patients with less than 50% hair loss [19–24]. This wide range of results could have been due to differences in the treatment protocols, data sets, evaluation methodology, prognostic factors, absence of uniform terminology for evaluating the results, and differences between the statistical methods used for analyzing the results. Some studies, on the other hand, have shown no differences between topical immunotherapy and placebo or other treatment modalities, such as topical corticosteroids, especially for patients with limited alopecia lesions . These findings contradicted the previous data in the literature, as well as the current study, which clearly showed a better response in cases with limited involvement. According to the overall results of the studies in the literature, DPCP immunotherapy for AA is a powerful option with a limited side effect profile.According to the previous efficacy studies of DPCP, chronic and extensive disease, and accompanying nail changes were reported to be poor prognostic factors [20], while limited involvement for a short period of time, absence of nail changes, older age of onset, and a negative history of atopic dermatitis correlated with a more favorable prognosis .In the case series herein, the success rate and treatment response were only associated with the extent of hair loss and thus, the type of alopecia. Patients with limited AA had better prognosis and good response to therapy, while extensively involved alopecia totalis and alopecia universalis cases had worse a prognosis and resistant course. Other parameters, such as sex, duration of the disease, atopy history, age of onset of alopecia, and age at start of DPCP immunotherapy had basically no effect on the prognosis of the disease during DPCP immunotherapy. Many other studies have also supported this finding, with the severity of alopecia being the sole important factor for the outcome of the treatment .Many adverse events have been reported due to the use of DPCP, including eczematous reactions, urticaria, vitiligo, lymphadenopathy, hyperpigmentation, or erythema multiforme-like reactions [18]. In the current study, during the sensitization process, almost all of the patients experienced minimal erythema, itching, and burning sensation. Overall, the most commonly encountered side effect was erythema and itching, followed by the formation of papules, vesicles, bullae, and flu-like symptoms. Other less frequent side effects were lymph node enlargement, fever, general malaise, irreversible hyperpigmentation of the head and neck area, and vitiligo macules.The use of DPCP in children is still a controversial area, although some studies have shown good results with acceptable side effect profiles [29]. In the current study, children above 5 years of age were also included and the treatment results or safety parameters were similar to those of the adult patients.AA can have serious aesthetic and psychological consequences for patients due to its unpredictable course, consisting of spontaneous remissions and relapses. All of the conventional treatment options have a limited chance of success. Topical DPCP immunotherapy is a safe and effective alternative for patients with resistant disease and long-term results will provide more precise data about the success rate of this treatment modality.The study protocol received institutional review board approval from ethical committee of the Cerrahpaşa Medical Faculty and all participants provided informed consent.