The relationship between breast cancer (BC) and thyroid disease (TD) is still controversial. The aim of the study was to investigate the possible coexistence of TD in patients with newly diagnosed BC and its correlation with BC clinical presentation with regard to menopausal status and stage of disease.This is a retrospective cohort study of all patients treated for primary BC between 2014 and 2016 at the Breast Unit of Trieste University Hospital. Clinical charts and reports were reviewed for coexisting thyroid disorders  and menopausal status at the time of BC diagnosis. Biomolecular profile, stage, and grading of BC were also evaluated.p = 0.16), although TD was more common in women aged ≥60 years. Women with BC diagnosed at pre-menopausal age were more likely to have thyroid autoimmune diseases . No association was detected among BC molecular profiles with either thyroid autoimmunity (p = 0.26) or altered thyroid function (p = 0.63). High-grade BC was more frequent in women with hyperthyroidism , but the grading was independent from the presence of thyroid autoimmune disease (p = 0.87). BC stage was related to both thyroid autoimmunity (p = 0.04) and thyroid function (p < 0.001), with 55.2% of women affected by benign TD presenting with stage I BC and more aggressive BCs found in hypothyroid patients.A total of 786 women and 7 men were included in the study. Co-presence of TD was found in 161(20.3%) cases: of these, 151(19.4%) patients presented benign TD and 10(1.3%) patients presented thyroid carcinoma. Thyroid autoimmunity was found in 51(32%) patients. Regarding thyroid function, 88(55%) patients had hypothyroidism, 19(12%) hyperthyroidism, and 54(33%) normal thyroid function. No statistically significant correlation was found between age and TD (According our study results, patients with primary BC present a greater incidence of autoimmunity disorders, especially when diagnosed in the pre-menopausal setting. However, further prospective studies are required to definitively prove causality. Breast cancer (BC) is the most common malignancy in women and the second most common cancer overall. Over 2 million new cases were diagnosed in 2018, accounting for almost 25% of cancer cases among women . In thisA recent meta-analysis performed by Hardefeldt et al.  found thAim of this study was to evaluate the prevalence of TD in a consecutive series of patients treated for primary BC in order to assess any possible association in terms of clinical presentation, management, and oncologic outcome.pathological diagnosis of primary BC confirmed by fine needle aspiration (FNA), core biopsy (CB), or vacuum-assisted biopsy (VAB) with Mammotome®.presence of TD, including any form of benign thyroid disease, thyroid cancer, thyroid autoimmunity disorders, and/or altered thyroid function .Between January 2014 and December 2016, 823 patients were treated for BC at the Breast Unit of Trieste University Hospital. Clinical data and information recorded from patients charts as well as radiology and pathology reports were retrospectively reviewed to assess for the following inclusion criteria:Patients with recurrent BC, patients with metastatic BC, and patients with histopathological diagnosis of lymphoma or sarcoma of the breast were excluded from the analysis.According to these inclusion criteria, the study population consisted of 793 patients.Variables routinely documented included patient age, gender, menopausal status for the female sex, BC pathological size, stage, grading and biological profile. In agreement with NCCN Guidelines , menopauLuminal A: hormone-receptor (HR) positive, HER2 negative, low levels of Ki-67 protein;Luminal B: HR positive, with either positive or negative HER2 and high levels of Ki-67 protein;HER2 type: HR negative, HER2 positive;Triple negative: HR negative, HER2 negative.Immunohistochemical profiling of BC samples was routinely performed as part of clinical care. According to the American Joint Committee on Cancer (AJCC) staging system (7th Edition)  and the According to the above mentioned classification systems and current treatment guidelines, BC patients underwent surgery (either breast conserving surgery or mastectomy) and/or medical treatment .For every patient, the following variables were identified: presence of benign TD, presence and histotype of thyroid carcinoma, presence of autoimmunity TD, thyroid functional status.Thyroid function was checked in every patient by means of venous dosage of thyroid hormones  and thyroid antibodies (i.e. anti-TPO and anti-TG).Hyperthyroidism was defined as high synthesis and/or secretion of thyroid hormones, determining suppressed TSH values (< 0.40 μIU/mL). Hypothyroidism was defined as reduced secretion of thyroid hormones, determining increased TSH values (> 4.00 μIU/mL).Thyroiditis was defined by the presence of thyroid antibodies and/or by means of ultrasound features and/or on histological examination of surgical specimens. Multinodular goiter was defined by the demonstration of multiple follicular nodules on ultrasound examination and/or on histological examination of surgical specimens.A thyroid ultrasound was performed in every patient presenting with altered thyroid function. Thyroid scintigraphy was indicated in case of hyperfunctioning disease, whereas FNA was carried out in patients with thyroid nodules suspicious for malignancy. Definitive histological examination was obtained in patients undergoing thyroidectomy.Correlation between menopausal status and thyroid disease, autoimmune thyroid disease or thyroid function;Correlation between breast cancer biological profile and thyroid disease, autoimmune thyroid disease or thyroid function;Correlation between breast cancer staging and thyroid disease, autoimmune thyroid disease or thyroid function;Correlation between breast cancer grading and thyroid disease, autoimmune thyroid disease or thyroid function.Quantitative data were reported as mean, median, standard deviation and interquartile range. The qualitative variables were expressed as absolute frequencies and percentages. Chi-square test (or F-Fisher Exact test when appropriate) and Proportion Test were used to assess association between categorical variables:p value less than 0.05 was considered statistically significant.Statistical analysis was performed using R  software. A Between January 2014 and December 2016, 823 patients were referred for BC at the Breast Unit of Trieste University Hospital. Of these, 793 patients were eligible for the purposes of this study.The cohort consisted of 786 (99%) women (mean age 66 ± 14 years), and 7 (1%) men (mean age 71 ± 12 years). Among the female population, 30 patients presented bilateral BC. Overall, 716 (87%) patients underwent breast surgery and 107 (13%) patients were not considered for surgery because of severe comorbidities and thus medically treated with hormonal therapy. BC molecular profile was reported in 754 (95%) patients, while no information were available in 39 (5%) cases because BC diagnosis was made by on FNA cytology.Information about thyroid function was recorded in 779 (98%) BC patients. Of these, 161  presented both BC and TD, 151 (93.8%) of them showed a benign TD and 10 (6.2%) presented malignant TD. Concerning data for thyroid cancer, mean age at the time of diagnosis was 65 ± 13 years and tumor subtypes were classified as follows: 8 papillary thyroid carcinomas, 1 follicular thyroid carcinoma, and 1 medullary thyroid carcinoma.Among the 161 BC patients with concurrent TD, 88 (55%) patients had hypothyroidism, 19 (12%) patients showed hyperthyroidism, and 54 (33%) patients had normal thyroid function.Thyroid autoimmunity was identified in 51 (32%) patients out of 161 cases of BC with concurrent TD. Table n = 10) and men (n = 7) were excluded subgroup analysis.Analysis was conducted on a cohort of 754 BC patients. Patients with malignant TD (p = 0.16).At first, we investigated the correlation between menopausal status and benign TD. Among pre-menopausal women with BC, benign TD was found in 17% of patients. Similar percentage (21%) was found in BC women aged > = 60 years (p = 0.05). Moreover, pre-menopausal BC women were more frequently affected by autoimmune TD than post-menopausal BC ones, while benign TD was generally much more common in post-menopausal BC women (71% vs. 29%).The analysis of correlation between menopausal status and autoimmunity showed that autoimmune TD was diagnosed in 45% of pre-menopausal BC patients and in 29% of post-menopausal BC patients, with a borderline significance at statistical analysis (p = 0.49).Evaluation of correlation between menopausal status and thyroid function, showed no differences at statistical analysis (p < 0.001). BC molecular profile was not associated with benign TD (p = 0.85), nor with thyroid autoimmunity (p = 0.26) and not even with thyroid function (p = 0.63).Molecular profile of BC was determined on immunostaining for 754 (95%) patients. Different BC molecular profiles and their distribution according to menopausal status are represented in Figs. The analysis of a possible correlation between BC staging and benign TD was possible in a subgroup of 136 cases out of 151 (90%), with a median age at the time of diagnosis of 68 years.BC stage distribution related to benign TD is showed in Table Different distribution of BC stage according to thyroid function was recorded (p < 0.001). A major proportion of stage I patients was observed among women with normal thyroid function, whereas patients with higher BC stage were more frequently affected by hypothyroidism Table .Table 3p = 0,05). Data are reported in Table Analyzing the presence or absence of autoimmune TD, no difference was found in distribution for stage 0, I and II BC, while stage III BC was much more common among women with autoimmune TD .Analysis of correlation between BC grading and thyroid function demonstrated that G3 carcinomas were more represented in women with hyperthyroidism (52.9%) than in those with hypothyroidism (38.6%) or normal thyroid function (21.4%) (p = 0.87).BC grading was not related to the presence or absence of autoimmune TD . Similar results have been recently published by Chiappa et al. [Differences was observed regarding autoimmune thyroiditis, with pre-menopausal BC patients being more frequently affected by this pathology than post-menopausal BC patients , although no correlation was found with thyroid autoimmunity (p = 0.26). When considering thyroid functional status, in the current study 55% of patients presented with hypothyroidism, 12% with hyperthyroidism and 33% with normal thyroid function. At statistical analysis, no significant correlation was found with the menopausal status (p = 0.49). Regarding the possible relationship between BC staging and the presence of benign TD, this study found that patients with normal thyroid function presented predominantly AJCC stage I disease (67.5%), whereas patients with hypothyroidism presented more frequently AJCC stage II-III disease (12.2%). This could indicate that a reduced thyroid function might predispose to develop a more advanced stage of BC, while a condition of euthyroidism seems to correlate with lower BC stage. In this context, Sogaard et al. [In the present series, the well-known aggressive TN and Her2 positive BC subtypes were more frequently observed in pre-menopausal women . This result is surprisingly in contrast with literature data, because several Authors have reported a higher prevalence of thyroid peroxidase antibodies (TPO-Abs) in BC patients, underlining their potentially protective role in terms of oncologic prognosis [As far as the correlation between BC stage and the presence of autoimmune TD is concerned, the results of this study showed that there were no significant differences in the number of patients with autoimmune and non-autoimmune TD in BC stage 0, I and II. Conversely, BC stage III disease was significantly more common among women with autoimmune TD  cases, all women and with a greater predominance of papillary thyroid carcinoma, reflecting the high epidemiological frequency of this type of thyroid cancer.Last but not least, when assessing for a possible correlation between BC and benign TD, the present analysis showed that the vast majority (56.3%) of women with benign TD had G2 BC while G3 carcinomas were more represented among women with hyperthyroidism (52.9%), suggesting that an increased thyroid function may correlate with a higher grading of BC. Lastly, statistical analysis showed that BC grading is independent from the presence of autoimmune TD.The present study has a number of limitation. Being a single-center experience based on retrospective non-randomized analysis, the possibility of generalizing the results is potentially limited. Additionally, the sample size is not very large and the observation period might not be long enough. However, the study managed to confirm that there is a relationship between BC and TD, although further studies with prospective analyses and extended follow-up are required in order to elucidate the nature of this relationship. Future research might investigate the precise pathological mechanisms that correlate the two pathologies also by checking the presence of estrogen and progesterone receptors on histologic specimens of thyroid carcinomas in order to possibly identify a subgroup of patients with higher risk of developing BC.


We have sequenced the genome of the largest freshwater fish species of the world, the arapaima. Analysis of gene family dynamics and signatures of positive selection identified genes involved in the specific adaptations and unique features of this iconic species, in particular it’s large size and fast growth. Genome sequences from both sexes combined with RAD-tag analyses from other males and females led to the isolation of male-specific scaffolds and supports an XY sex determination system in arapaima. Whole transcriptome sequencing showed that the product of the gland-like secretory organ on the head surface of males and females may not only provide nutritional fluid for sex-unbiased parental care, but that the organ itself has a more specific function in males, which engage more in parental care. Arapaima gigas) has a most remarkable biology. This legendary fish can reach a body length of almost 3 meters placing it the largest freshwater fish, exhibits the fastest known growth rates, and – supporting its importance for aquaculture – has the best food conversion so far recorded in fish2. Known as pirarucu in Brazil and paiche in Peru, it belongs to the bonytongues (Order Osteoglossiformes), one of the most basally diverging lineages of the teleost fish. Its natural distribution covers most of the Amazon River basin in Peru and Brazil, and it has been introduced as an aquaculture species to other rivers in tropical South America. As an obligate air-breathing fish obtaining up to 95% of its oxygen uptake by breathing, it is able to tolerate extremely low oxygen levels in the water, and is less susceptible to ammonia or nitrite intoxication due to its degenerated gills.The Amazonian freshwater fish arapaima . Genome wide studies on this phenomenon are not available but are desirable for understanding this spectacular growth ability and they will allow comparisons with the S. formosus genome, both in ontogenetic and evolutionary contexts.Gigantism and muscle growth separate arapaima  from its relative, the much smaller Asian arowana 10 because occasional reproduction in ponds so far has not provided enough information to establish captive breeding and aquaculture11. Arapaima is a fractional spawner and maturation of the ovary is synchronous12. Only the left gonad is functional and the right one is atrophied, both in males and females13. Therefore, biopsies via the gonoduct are difficult to take and, with regard to the body size, stressful as well as labour intensive. Here, a sex marker would facilitate reproductive management substantially14.Sex determination and the regulation of sexual maturity onset (“puberty”) are still largely unknown for arapaima15. Present on the head of males and females, it shows no sex-specific morphological differences. During the reproductive period, the secretory organ secretes a milky fluid that is thought to provide nutrients to the fry16. Due to a relatively low protein content of the secretion further functions such as pheromonal gland have been discussed. As such, agglomeration pheromones for the juveniles as well as priming pheromones targeting the opposite sex can be hypothesized. However, males engage much more in parental care and stay with the offspring for up to 3 months, while females leave the father and the offspring after about one month to reproduce again with other males during the same season. Males guide the offspring swimming above his darkened head (which appears to provide camouflage) to nutrient-enriched feeding areas. Insights into the reproductive biology of arapaima will not only support the development of reproductive technologies in aquaculture but will also contribute a new understanding of the evolution of sex determination and sex differentiation in fish.A morphological specialization in relation to reproduction is the so-called “secretory organ”, which has been reported to function in parental care4, including several genomes and a wide spectrum of transcriptomes will be highly useful for a better understanding of not only of the biology of this spectacular fish, but will provide necessary information for establishing arapaima in recirculation aquaculture facilities and for supporting conservation measures and, importantly, the restoration of natural stocks. Here, we focused on the sex determination system and the function of the secretory field, hypothesizing a sex-specific function as pheromonal gland.The availability of extended genomic resources17.We used a whole genome shotgun approach with Illumina technology (Hiseq2500) to sequence the genome of one male and one female at 58.6 and 59.8x coverage, respectively Fig. . The DNA18, the completeness of the male assembly is estimated to being 96.1% and in that of the female 95.7%  method from the vertebrate database7% Table . Because7% Table , we pred7% Table .19. In male arapaima, TEs account for 16.21% of the male and 16.77% of the female genome , are always of interest with regards to their important role in the evolution of genes, gene networks, and genomesme Table . To inveme Table , arapaimme Table  and Asiame Table , as an ants Fig. . After t22. According to divergence time estimations using MCMCTree, the two bony tongue lineages split approximately 138.4 million years ago , 103 functional trace amine-associated receptors (TAAR) and 107 vomeronasal receptors (VR) from 18 vertebrates , while tas1r2, the receptor gene for sensing sweet, was lost. This is in accordance with adaptation to a diet that is mainly composed of prey and does not contain much saccharides.Because arapaima is predominantly carnivorous, we also investigated the gene family of taste receptors, an important factor in the development of carnivore habits. 29. Such genes may be recognized by dN/dS ratios >1. To identify PSGs characteristic of the arapaima lineage, we first screened 3,128 one-to-one orthologs that were retrieved from at least 8 of the 12 fish species shown in Fig. ostf1), cell growth, and cell division . GO enrichment of these genes revealed “cyclin-dependent protein kinase holoenzyme complex” to be enriched (Table Positively selected genes (PSG) are the result of adaptive evolution and often associated with new enhanced or selected functions of an organismed Table , which msrc; Bone morphogenetic protein 7, bmp7; Growth Arrest Specific 2, gas2; Runt Related Transcription Factor 2, runx2; Neuron Navigator 2, nav2 and Centrosomal Protein 295, cep295). GO term analysis on these genes showed that functions related to development, in particular, development of the musculo-skeletal system, are enriched in positively selected genes ) that had no sign of positive selection in the lineage of Asian arowana, European eel and the two most recent common ancestor | value . The top 5% (295) genes of the ranked list were selected, among which 118 genes showed higher arapaima-European eel pairwise dN/dS values and no sign of positive selection in the lineages of Asian arowana, European eel and the two most recent common ancestor . Among these genes, functions related to cell cycle are also enriched  from all 12 species for arapaima-unique residues and identified 4,677 arapaima-unique residues in 1,959 protein sequences. These arapaima-unique residues were scored based on the conservation of flanking residues, and the protein sequences, based on the score of their arapaima-unique residues . The higher the score of a residue, the more conservative the sequence region that harbors it, which gives such substitutions a higher significance. In extension, the higher the score of a protein sequence, the more unique substitutions of higher significance it harbors. Hence we ranked them respectively in a descending order, and kept the top 1% of each list . Another top-ranked gene is mfsd14a   and cd48 (CD48 antigen) to be contained in both datasets. It is interesting to note that these scaffolds are considerably different from the rest of the genome with respect to TE content . We thus searched the arapaima reference genome for regions paralogous to the male-specific scaffolds that had male-specific tags using LAST36 and found four scaffolds that are paralogous to putative Y-linked scaffolds . Such a situation has been reported for the Y-chromosomes from several other fish species  or even detrimental to the opposite sex (sex-antagonistic genes), can become linked to the sex-determining locus. To search for such genes, we looked for sex-biased gene expression patterns in testis, ovary, male and female secretory organs. First, to compare gene expression between testis and ovary, we plotted the 20,927 genes that have RNA-seq reads mapped either in testis or/and in ovary. Among them, 26 genes that are covered by more testis reads than ovary reads are located in male-related scaffolds  Fig. .To further identify genes that are differentially expressed between in testis and in ovary, we calculated and compared the expression level using DESeq2, those genes with read counts >500 in one gonad and unexpressed in the other, meanwhile, with |log2FC| >4 were identified as differentially expressed gene Table . The samGenes that are generally known to be involved in gonad functions and structure and show a sex-biased expression in other fishes, display the expected expression profile Fig. .16. Interestingly, the secretory organ genes expressed exclusively in males were enriched in several pathways, e.g. insulin signaling, glycolysis and gluconeogenesis, and ovarian infertility genes  of differentially expressed transcripts in male and female secretory organs, with 421 transcripts exclusively expressed in males but only 45 transcripts expressed only in females Fig.  and S19.We generated a draft genome sequence of good contiguity that provided a useful reference for phylogenomic and comparative genomic evolutionary analyses. Much more work can be done with these genomic resources to improve our understanding of the biology and evolutionary ecology as well as questions connected to aquaculture, fisheries and conservation management.To this end, we identified candidate genes that may be related to arapaima-specific traits, for instance fast growth and large body size, adaptations to a carnivorous lifestyle, and the function of the secretory organ. In this context, it was surprising to find male-specific gene expression in the secretory organ that assigns both a fry-nutrition function and also a pheromone-type signaling functioning to local females.For the first time, we inferred from the genomic data a possible genetic sex determining system of male heterogamety in this species that presents homomorphic chromosomes for both sexes. We identified male- (Y-)specific scaffolds that can be potentially useful to identify the master male sex determining gene(s) and to design molecular markers that are highly desirable for aquaculture breeding and wild stock management.Fish were sacrificed by state-of-the-art humane killing . The experiments were carried out in accordance with the European Directive 2010/63/EU and German national legislation . All experimental protocols that are part of this study were approved through an authorization  of the LAGeSo, Berlin, Germany.A. gigas DNA for Illumina shotgun sequencing was derived from fin tissue of a single adult female (ID F3) and from a single adult male (ID M14). Libraries were produced using the Truseq DNA Nano sample prep kit using the 550 pb insert size option. Libraries were sequenced on a Hiseq 2500 using rapid v2 PE 2*250 nt mode . All sequences were assembled with DISCOVAR (version 52488) de novo ((https://software.broadinstitute.org/software/discovar/blog/)) using default parameters.37, using Sbf1 as a single restriction enzyme, and sequenced on a single lane of Hiseq2500 using the v4 SR100nt mode. The resulting read file was then demultiplexed using the process_radtags.pl script of STACKS software version 1.4438 with default settings. Demultiplexed reads were analyzed with the denovo_map.pl STACKS script with the following settings: m = 3 (more than 3 reads are needed to build a stack of sequences), M = 0 , N = 0 ,–gapped not set (gapped assembly disabled), H not set ,–max_locus_stacks = 1 (ustacks max locus per stack set to 1), and–keep_high_cov set (ustacks lumberjack step deactivated). Zero values for the M and N setting produce stacks of reads with no polymorphism, each resulting stacks of reads containing single allele reads or non polymorphic stacks of reads.Genomic DNA was extracted from 90% ethanol-preserved fin clips using a classical phenol/chloroform protocol. The arapaima RAD-tag library was built according to standard protocolshttp://www.repeatmasker.org/RepeatModeler, version 1.0.4) to generate a library of known and model a de-novo repeats  to annotate and mask repeat elements from the genome assembly. For TE insertion repeats, we also used RepeatLandscape  to calculate Kimura distance as a measure of age, and to display a relative age profile.The genome assembly was inspected by RepeatModeler , D. rerio (zebrafish), L. chalumnae (coelacanth), P. marinus (sea lamprey), T. rubripes (torafugu), T. nigroviridis (spotted green pufferfish), G. aculeatus (three-spined stickleback), O. latipes (Japanese medaka), C. milii (elephant shark)39, and from S. formosus (Asian bonytongue)9, the closest known relative to arapaima. These sequences were then aligned to the repeat-masked genome sequence using exonerate2.2.0 (http://www.ebi.ac.uk/about/vertebrate-genomics/software/exonerate) and Genewise2-2-040, respectively, to predict potential gene structures. To improve the efficiency of Genewise, we also used genBlastA1.0.141 to roughly locate each protein on the genome sequence before Genewise was implemented. For gene evidence from de novo annotation, AUGUSTUS3.2.342 was used to predict genes on the repeat-masked genome sequence with ‘zebrafish’ as the parameter for –species flag. In parallel, we also used GeneMark-ES43 in unsupervised training model (-ES) for the de novo prediction. For the gene evidence from transcripts, we collected RNA-seq reads from eyes, gills, spleen, lung, ovary, liver, heart, muscle and secretory field from one female and testis and secretory field from one male, and assembled the transcripts with and without the reference genome independently. Tophat and cufflinks 2.1.144 were used for the with-reference assembly. For the without-reference assembly, we used Trinity 2.4.0 and PASA 2.2.046. All gene evidence concluded by exonerate, Genewise, AUGUSTUS,GeneMark-ES, Tophat and cufflinks, and Trinity and PASA were collected and transferred to EVidenceModeler1.1.147 to screen for high quality gene models that are supported by all lines of evidence. Those high-quality gene models were then used to train AUGUSTUS for improved gene predictions specifically for arapaima. Finally, the trained AUGUSTUS was run again with all previously obtained gene evidence as input, to predict the final set of gene models for arapaima. To access the quality of the annotation result, we mapped the resulting sequences to Pfam48 using InterProScan 549 to investigate the portion of results that can be annotated with a known protein domain. We also used BUSCO18 based on the vertebrata odb9 database to access the annotation completeness. To assign gene symbols, we compared those gene sequences to the UniProt database (www.uniprot.org/e) using BLAST with a criterion e-value of 1E-5 (blastp2.2.28+50), and took the symbol of the best hit.Genome annotation was done by combining gene evidence from homology annotation, pts Fig. . For homL. oculatus (spotted gar), O. latipes (Japanese medaka), X. maculatus (southern platyfish), O. niloticus (Nile tilapia), G. aculeatus (three-spined stickleback), T. rubripes (torafugu), G. morhua (Atlantic cod), D. rerio (zebrafish), L. chalumnae (coelacanth) (Ensembl release 87), A. anguilla (European eel) (http://www.zfgenomics.com/sub/eel), S. formosus (Asian bonytongue) and arapaima (our annotation). We then used blastp2.2.28+50 to compare the pool to itself. Based on the resulting raw score, we calculated an H-score as are51 of sequence distance for each pair of queries and hits, and clustered the proteins into groups by Hcluster_sg52. For each group, we used TreeBeST 0.5.153 to build a gene tree guided by NCBI taxonomy phylogeny relationships (https://www.ncbi.nlm.nih.gov/taxonomy) and to infer orthology relationships. Ortholog relationships were then categorized as one-to-one, one-to-many or many-to-many using an in-house Perl (https://www.perl.org) script.To form a protein pool, we unified 338,336 protein sequences from 12 species 54. These alignments were filtered by trimAl55 with the parameters “-gt 0.8 –st 0.001 –cons 60” and then concatenated into a huge alignment. Based on the concatenated alignment, we reconstructed the phylogenomic tree for the 12 species using RAxML 8.2.956 with PROTGAMMAAUTO parameter to select the optimal amino acid substitution model with coelacanth set as the outgroup and 100 bootstraps to test robustness. The fourfold degenerate site alignment corresponding to the concatenated protein alignment was then also subjected to RAxML through option “-f e” under the general time reversible (GTR) model to optimize the branch lengths for the phylogenomic tree. To confirm the topology of this tree, we also repeated the phylogenetic reconstruction using MrBayes 3.2.6, during which two simultaneous, independent runs were performed for 100,000 iterations of a Markov Chain Monte Carlo algorithm, with six simultaneous chains and sampling trees every 200 generations, resulting in 500 trees. The first 100 trees were “burned in”, and the average standard deviation of split frequencies remained ≤ 0.01 after the burn-in threshold. Divergence times along the phylogenomic tree was then estimated under relaxed clocks as implemented in MCMCTree57, with the CDS sequence alignment corresponding to the concatenated protein alignment used as input, including four fossil records: O. latipes–T. nigroviridis (~96.9–150.9 million years ago (Mya)), D. rerio–G. aculeatus (~149.85–165.2 Mya)58, A. gigas–S. formosus (~140–200 Mya)24 and sarcopterygians–actinopterygians (~400–500 Mya)23 as constraint. The MCMC process was run for 1,500,000 steps and sampled every 150 steps.One-to-one orthologs across the 12 species were aligned as protein sequences using MUSCLE 3.8.3159, in a maximum likelihood framework, using as input the gene group (family) size result from Hcluster_sg and the phylogenic tree from phylogenetic analysis. We instructed the program to search for the maximum likelihood value of birth and death rate (λ) following parameters “-p 0.01 -r 10000”. This means using 10,000 Monte Carlo random samplings to determine the probability of a gene family with the observed sizes and its birth and death rate, and then only of gene family with probabilities less than 0.01 to report the birth and death rate (λ). Before inputting to the CAFE program, gene families with no homology in the SWISS-PROT database or with multiple functional annotations were removed.Gene family expansion and contraction was analyzed with the program CAFE 3.0http://www.ensembl.org)62. The method to identify and classify odorant receptor (OR) genes was adapted from Niimura26. First, TBLASTN was conducted to search whole genome sequences. As query, we extracted 1593 functional odorant receptor genes from 11 non-mammalian chordate species27. Because multiple queries will map to the same genome region, for each non-overlap hit region we took the query with the highest raw score as the best query. We then extended the non-overlapping hit regions on both sides and compared them to their best queries using Genewise. Coding sequences were extracted and extended to the start (ATG) and stop codons. Finally, we translated the coding sequences and kept those with more than 250 amino acids and no premature stop codon as final functional OR-potential genes. To assign them into groups (α–λ), we blasted them to a database containing the sequences of 1,593 functional ORs and 59 Non-ORs50. After discarding results with more than 40% identical matches, we assigned each query to the group with the best hit.ORs were identified from the genomes of torafugu, Japanese medaka, grass carp, Wuchang bream, channel catfish, Mexican tetra, zebrafish, arapaima, Asian bonytongue, European eel, spotted gar, coelacanth, elephant shark, green anole and chicken | , we screened arapaima, Asian arowana, European eel and the two most recent common ancestors (MRCA) lineages, respectively, for positively selected gene candidates, using CodeML in the branch-site model (model = 2 & NSsites = 2), with model A (fix_omega = 0) compared with the null model (fix_omega = 1 & omega = 1). For each analysis, all multiple sequence alignments were completed using MUSCLE in protein model, followed by pal2nal.pl63 for the protein to CDS sequence translation, and then filtered by Gblocks 0.91b64 in CDS model (−t = c), with alignment length <150 results excluded. All statistical analyses  were carried out in R (https://www.r-project.org/).The dN/dS analysis was performed mainly with the CodeML program from the PAML packageSequences were aligned and gap-removed for 6052 homologs  retrieved from all 12 fish species. Alignments were analyzed by an in-house Perl script to identify and score the unique arapaima amino acid residues. The unique residue score was measured according to the variance of flanking residues across species:http://www.bioconductor.org), which employs Fisher’s exact test and 2 × 2 contingency tables to check for significant over-representation of GO terms in one set compared with another set. GO categories with p < 0.05 were considered significantly enriched. Whole arapaima gene were used as background.Enrichment of GO terms for the genes that were selected in dN/dS analysis and identification of proteins with arapaima-unique residues was determined using the TopGO package from Bioconductor . Then blat hits were filtered and hits having a match length greater than 100 bp, a mismatch rate (match length divided by mismatch length) lower than 3% and coverage (match length divided by query length) greater than 10% were retained.An R script was used to select all contigs from the first genome having no hits from the second genome covering them. This was performed for both genomes. The corresponding contigs were considered as male or female genome specific.Male-linked RAD-tags were blasted to the sex-specific scaffolds and the rest of the reference genomes , and only the best hit for each query were kept. To find possible duplications, scaffolds from the male specific collection were blasted to the remainder of the male reference genome.Total RNA was isolated using TRIzol Reagent  according to the supplier’s recommendation. RNA from eyes (RIN 8), gills, spleen (RIN 7.8), lung (RIN 7.9), ovary (RIN 7.1), liver (RIN 7.7), heart (RIN 7.6), muscle (RIN 7.2) and secretory field (RIN 8.5) from one female and testis (RIN 8.1) as well as secretory field (RIN 7.5) from one male were obtained from the broodstock of the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB). The settlings were imported from Neotropical Fauna E.I.R.L  in 2013 (CITES 1054487) and raised at the facilities of the IGB. Maturing fish were sacrificed by state-of-the-art humane killing . The experiments were carried out in accordance with the European Directive 2010/63/EU and German national legislation. RNA-Seq reads were used as transcriptomic evidence for genome annotation and sex-biased expression analysis. Custom sequencing  of TruSeq libraries generated 25–30 million 100 bp paired end reads for each sample on the Illumina Hiseq4000 platform.65 with default settings. Differentially expressed genes were detected using the Bioconductor package DESeq266. A gene was considered to be expressed, if at least ten reads were detected. A gene was considered to be differentially expressed for the comparison ovary vs. testis, if a gene was expressed with read counts >500 in one gonad and unexpressed in the other. For the comparison male vs. female secretory organ a gene was required to have an expression value of at least 500 in male or female and a log2FC > 4. These highly stringent criteria were chosen to account for the fact that there were no replicates. For functional enrichment analysis the web tool DAVID (https://david.ncifcrf.gov/) has been used with human as reference. Venn diagrams were drawn using the online tool Venny (http://bioinfogp.cnb.csic.es/tools/venny/).Genes were aligned to the arapaima transcripts using Bowtie2Supplementary informationDataset 1


Locally advanced or metastatic non-small cell lung cancer (NSCLC) that has progressed after first-line treatment has a poor prognosis. Recent randomized clinical trials (RCTs) have demonstrated survival benefits of alternative treatments to docetaxel. However, information is lacking on which patients benefit the most and what drug or regimen is optimal. We report a systematic review and network meta-analysis (NMA) of second-line treatments in all subgroup combinations determined by histology, programmed death ligand 1 (PD-L1) expression, and epidermal growth factor receptor (EGFR) mutation.MEDLINE, PubMed, EMBASE, Biosciences Information Service , Cochrane Library, and abstracts from scientific meetings were searched for RCTs published up to September 2015. Key outcomes were overall survival (OS) and progression-free survival (PFS). Bayesian hierarchical exchangeable NMAs were conducted to calculate mean survival times and relative differences for eight subgroups, using docetaxel as the reference comparator. For OS, the NMA was based on hazard ratios applied to a first-order fractional polynomial model fitted to the reference treatment. For PFS, a second-order fractional polynomial model was fitted to reconstructed patient-level data for the entire network of evidence.The search identified 30 studies containing 17 different treatment regimens. Docetaxel plus ramucirumab was associated with a significant improvement in OS and PFS, relative to docetaxel, regardless of patient type. Docetaxel plus nintedanib showed similar efficacy to docetaxel plus ramucirumab in the nonsquamous populations. EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib showed superior levels of efficacy in EGFR mutation-positive populations and the one PD-1 immunotherapy (nivolumab) studied showed superior efficacy in the populations exhibiting high PD-L1 expression.In the absence of head-to-head comparisons, we performed a mixed-treatment analysis to synthesize evidence of the efficacy of each treatment. Benefits are optimized by targeting specific treatments to individual patients guided by histology, PD-L1 expression, and EGFR mutation status.CRD42014013780 available at www.crd.york.ac.uk/PROSPERO).This review is registered in PROSPERO (registration number: The online version of this article (10.1186/s12885-019-5569-5) contains supplementary material, which is available to authorized users. Lung cancer is the most commonly occurring life-threatening cancer in the world, with an estimated 1.8 million new cases diagnosed in 2012  . ApproxiThe natural history of untreated advanced NSCLC is poor, with a median survival of 2 to 5 months from diagnosis . In the A number of novel agents targeting specific pathways associated with apoptosis, cell proliferation, angiogenesis, and other antineoplastic mechanisms have recently emerged. This research has led to the regulatory approval of regimens that offer improved clinical outcomes for patients with NSCLC, and these regimens are now reflected in current guidelines from the National Comprehensive Cancer Network in the United States  and fromThe inclusion of information on biomarkers such as epidermal growth factor receptor (EGFR) mutations and programmed death ligand 1 (PD-L1) expression in clinical trials and the evidence of how particular interventions behave according to tumor histology means that the evidence base in NSCLC is becoming increasingly complex. Previous meta-analytic studies have tended to either focus on the entire population such as Jansen , only onThe aim of this study was to provide comprehensive evidence on the efficacy of available second-line treatment options for patients with advanced NSCLC of clinically meaningful subtypes defined by histology, PD-L1 expression, and EGFR mutation. For this purpose, we conducted an NMA with Bayesian hierarchical exchangeable structures which allow treatment effects to vary by covariates representing these subtypes.2), ramucirumab (10 mg/kg), vinorelbine (any dose), and best supportive care. Two further interventions also were included from this literature review due to growing interest in the Asian market. These were S-1 (40 mg/m2) and bevacizumab (15 mg/kg). After the systematic literature review had been completed, the trial results for pembrolizumab [A systematic literature review was undertaken to identify all relevant publications of phase 2/3 randomized controlled trials that were conducted in adult patients who had locally advanced or metastatic NSCLC and whose disease had progressed after first-line chemotherapy through 2 September 2015. There were no date, language, or geographical restrictions on the medical reference database searches. The search strategy involved searches of MEDLINE (using PubMed platform), MEDLINE In-Process (using PubMed platform), Embase (using Elsevier platform), Biosciences Information Service  and the Cochrane Library (using the Wiley platform). Additionally, the American Society of Clinical Oncology, the European Society of Medical Oncology, and the International Association for the Study of Lung Cancer were searched for relevant new evidence. The search included regimens containing the following interventions: docetaxel (any dose), erlotinib (150 mg), gefitinib (250 mg), gemcitabine (any dose), nintedanib (200 mg), nivolumab (3 mg/kg), pembrolizumab (any dose), pemetrexed . Where mature data from a single trial were reported later, the most recent data were used in the analysis.2). Although clinical trials typically report hazard ratios and median survival times, the fitting of parametric models to survival data lends itself more readily to the estimation of mean survival. Mean survival times are of particular importance to cost-effectiveness analyses, which are part of the health technology assessment process. Mean survival was estimated as the area under the probability of survival curve with a horizon of 60 years. Relative differences were assessed and mean estimates with 95% credible intervals presented. In addition, significance tests were performed on the difference between two distributions. The minimum of the proportions greater or less than 0 was used as a probability and converted to a two-tailed test by multiplying it by 2 [P values from a frequentist analysis, although they do not have the same statistical definition. Rank probability charts, cumulative probability charts and surface under the cumulative ranking (SUCRA) were also derived following the methods described by Salanti et al. [The outcomes studied were OS and PFS, which were evaluated in terms of relative mean survival compared to the reference treatment  and where there was external supporting evidence for the treatment covariate interactions (EGFR TKIs with EGFR mutation status and pemetrexed with histology). Where there was sufficient evidence for subpopulation results within a trial, these were used instead of the overall study-level results. Subgroup data were used in the NMA only where we had a reason to suspect there should be a difference between subgroups, or there was supporting evidence in the literature, or a treatment had an indication for a subgroup. Studies that did not present the relevant proportion of patients with a given tumor subtype were not included in the NMA, i.e., pemetrexed and proportion of patients with nonsquamous tumors not reported, TKIs and proportion of patients with tumors that were EGFR mutation positive not reported, and PD-L1 immunotherapies with proportion of patients with PD-L1 expression not reported. The following study-level covariates were included one at a time to see if they improved model fit: time since publication, proportion of patients with nonsquamous tumors, mean age, proportion of patients with Eastern Cooperative Oncology Group (ECOG) ≥ 1, proportion of patients with metastatic disease, and proportion of Asian patients.Kaplan-Meier charts were digitized using the DigitizeIt software package , and the2 , which p2 , which d2 , which a2 . If a st2 . All the2 . Hierarc2 ; Lim et 2 ; Sun et 2 ; Urata e2 ; Vale et2 ). Furthe2 ; Scaglio2 ), ninted2 ), and ni2 ; Brahmer2 ). PredicNoninformative priors were used for treatment efficacy and for between-study variances. For the fractional polynomial model, four random-effects models were conducted: first-order fractional polynomial NMA with an additional heterogeneity parameter for scale, first-order fractional polynomial NMA with additional heterogeneity parameters for scale and shape, second-order fractional polynomial NMA with an additional heterogeneity parameter for scale, and second-order fractional polynomial NMA with additional heterogeneity parameters for scale and the two shape parameters. A range of power functions were fitted for first- and second-order fractional polynomial models, which followed that of Jansen .Posterior sampling was performed using Markov Chain Monte Carlo, with three chains of 70,000 iterations per chain, each with a burn-in of 30,000 iterations for the fractional polynomial models and a total of 120,000 iterations for the hazard ratio models. Convergence was assessed through iteration plots, the shape of the posterior distributions, and Gelman-Rubin diagnostics . BayesiaFurther analyses were required to estimate mean survival times. For the hazard ratio approach, all of the reconstructed patient-level data for the reference data were collated. Bayesian first-order and second-order fractional polynomial models were fitted to these data with heterogeneity parameters for all scale and shape parameters. The same number of iterations and chains was used as stated for the hazard ratio NMA. A grid search for the best-fitting model with up to two covariates was performed.The efficacy of study treatments in each tumor subgroup was estimated by linear extrapolation of the parameters available for each proportion of the relevant tumor type. If not all of the required information was available, the efficacy of a combination regimen was estimated using the separate information from monotherapy regimens.2) to the rest of the network. This study used erlotinib (150 mg) as the comparator in a patient population with 16% EGFR mutation-positive tumors in the erlotinib arm and 25% in the docetaxel arm. The treatment switching, therefore, was likely to have caused confounding by potentially increasing the perceived relative efficacy for docetaxel (60 mg/m2), although the use of constraints prevented this reversal from being observed. The data for OS, including hazard ratios, median survival times, and covariate data, are presented in Additional file We screened 2601 records and 30 trials evaluating 17 interventions for inclusion in the NMA  from the network of evidence. Details of this model are given in the Additional file An assessment of the proportional hazards assumption and heterogeneity and inconsistency is provided in the Additional file The fitting of fractional polynomial models to multiple treatment data can result in survival curves that flatten before reaching zero, especially for second-order models with time-varying treatment effects.  with gains of 2.3 and 2.6 months, respectively  with a gain of 2.0 and 5.5 months, respectively  with 2.6 months  with gains of 12.9, 2.3, and 2.6 months, respectively  for OS and PFS.For OS, nivolumab, docetaxel plus ramucirumab and docetaxel plus nintedanib showed a significant improvement in mean survival over docetaxel (75 mg/m2), with a gain of 8.0 and 2.0 months, respectively  for PFS.For OS, nivolumab and docetaxel plus ramucirumab were the only interventions to show a significant improvement in mean survival over docetaxel (75 mg/m2) : docetaxel plus erlotinib (13.4), erlotinib plus pemetrexed (8.0), erlotinib (7.4), gefitinib (4.4), docetaxel plus nintedanib (2.6), and docetaxel plus ramucirumab (2.3)  : docetaxel plus erlotinib (8.1), erlotinib plus pemetrexed (7.0), erlotinib (6.8), gefitinib (5.4), and docetaxel plus ramucirumab (1.2)  over gefitinib and non-TKI regimens for OS. Docetaxel plus erlotinib, erlotinib, and gefitinib showed significant superiority (P < 0.05) over docetaxel plus ramucirumab for PFS.For OS, the following treatments showed a significant gain over docetaxel  : docetaxel plus erlotinib (11.9), erlotinib (6.5), nivolumab (5.5), gefitinib (3.9), and docetaxel plus ramucirumab (2.0)  : docetaxel plus erlotinib (8.1), erlotinib (6.8), gefitinib (5.4), and docetaxel plus ramucirumab (1.2)  over gefitinib and docetaxel plus ramucirumab for OS. Docetaxel plus erlotinib showed significant superiority (P < 0.05) over non-TKI regimens for PFS.For OS, the following treatments showed a significant gain over docetaxel  : docetaxel plus erlotinib (13.4), nivolumab (12.9), erlotinib plus pemetrexed (8.0), erlotinib (7.4), gefitinib (4.4), docetaxel plus nintedanib (2.6), and docetaxel plus ramucirumab (2.3)  : docetaxel plus erlotinib (8.1), erlotinib plus pemetrexed (7.0), erlotinib (6.8), gefitinib (5.4), nivolumab (5.0), and docetaxel plus ramucirumab (1.2)  over docetaxel plus ramucirumab for OS. Docetaxel plus erlotinib, erlotinib, gefitinib, and nivolumab showed significant superiority (P < 0.05) over docetaxel plus ramucirumab for PFS.For OS, the following treatments showed a significant gain over docetaxel  : docetaxel plus erlotinib (11.9), nivolumab (8.0), erlotinib (6.5), gefitinib (3.9), and docetaxel plus ramucirumab (2.0)  (difference in months shown in parentheses): docetaxel plus erlotinib (8.1), erlotinib (6.8), nivolumab (5.7), gefitinib (5.4), and docetaxel plus ramucirumab (1.2)  over docetaxel plus ramucirumab for OS. Docetaxel plus erlotinib, erlotinib, nivolumab, and gefitinib showed significant superiority (P < 0.05) over non-TKI regimens and non-PD-1 immunotherapies for PFS.For OS, the following treatments showed significant gains over docetaxel  by subgroup, including the difference in mean survival. Sensitivity analyses for random-effects models also were performed, and these are presented in Additional file 2) in patients with EGFR mutation positive tumor types regardless of histology. However, in the PD-L1 high expression group nivolumab showed a similar benefit to the TKI based regimens in patients with EGFR mutation positive tumor types. For the nonsquamous, low PD-L1 expression EGFR mutation negative subgroups, ramucirumab plus docetaxel and nintedanib plus docetaxel provided the greatest benefit. Combination treatments such as docetaxel plus erlotinib appeared to give the greatest benefit in certain subgroups but there was a greater uncertainty in these predictions which was reflected in the large credible intervals.Table The results from this study provide further evidence that it is important to appropriately target specific treatments for particular patient groups to optimize outcomes. The mean survival times reported in this study also were in agreement with previous NMAs. The improvement in OS, using the hazard ratio model applied to a fractional polynomial model for the reference treatment in this study, matched closely those reported by Jansen  for a seThe main strengths of this study were that data were identified by a systematic literature review and that model flexibility allowed particular treatments to vary by histology, PD-L1 expression, and EGFR mutation. In addition, the survival curves for the reference treatment were validated using the data from Penrod et al. . HeterogThe main study limitation was the variable length of follow-up time between studies, which meant there was some uncertainty in the extrapolation of OS and PFS survival curves. For OS, the hazard ratios models were simply applied to the reference model by histology, so treatment effects were assumed to continue after follow-up. However, to ensure PFS survival curves were plausible, the hazard rates from the reference treatment were used once the time exceeded the maximum follow-up data for a given intervention. Interventions with shorter follow-up times but that appeared to have lower hazard rates than the reference treatments therefore may have been at a disadvantage. However, the approach used in this study did make full use of the available data. The proportion of patients tested for EGFR mutation status varied across studies and may have been a source of bias. EGFR mutation also covers different genetic markers and other factors, such as EGFR copy number, although these may not be independent predictive markers . The preP = 0.1194; PFS, P = 0.4700, as stated by Reck et al. [There were inconsistencies between the results for OS and PFS for nintedanib, nivolumab, and S-1 plus bevacizumab, which makes it difficult to be certain of the relative effectiveness of these interventions. The efficacy of docetaxel plus nintedanib and nivolumab were allowed to vary by histology in this study, although the actual trial results for these interventions had not shown there was a significant association with histology . If theA further more general limitation with NMAs conducted in NSCLC is the rapidly changing landscape of possible therapies. For example, the results from studies of two more PD-1 immunotherapies have been published since the completion of the literature review conducted for our study. Herbst et al.  presenteIn conclusion, the overall trends across OS and PFS indicated that there was always at least one intervention that performed better than single-agent docetaxel. Docetaxel plus ramucirumab gave a consistent significant benefit across all NSCLC subtypes. Docetaxel plus nintedanib showed a similar efficacy to docetaxel plus ramucirumab in the nonsquamous population. Superiority was observed for regimens containing erlotinib or gefitinib compared to non-TKI regimens when used in patients whose tumors have EGFR mutations, which was expected given the evidence in the literature , 32–35. New treatments for NSCLC are being developed and studied; these treatments often are specific to particular biomarkers. This will add further complexity to NMAs conducted in this disease area. However, the results from this study should help inform the decision-making process for prescribing currently available treatments and could be used to help power future trials. Results also may be used to serve as a reference for the efficacy of existing treatments for patients with a particular tumor type, where only mixed population evidence so far exists. As far as we know, this is the only NMA in which investigators have attempted to model the treatment covariate interactions present in NSCLC for second-line treatment after disease progression has occurred.Additional file 1:Risk of bias assessment, study characteristics, sensitivity analyses, subgroup results and Bayesian code. (DOCX 3820 kb)


To date, little research has examined the quality of life and cancer-associated needs of bladder cancer patients. The objective of the current study was to assess the quality of life (QoL), informational needs, and supportive care needs (SCN) in a large sample of muscle invasive (MIBC) and non-muscle invasive (NMIBC) bladder cancer survivors across the treatment trajectory .Questionnaires were distributed to a convenience sample of patients registered with Bladder Cancer Canada, the Princess Margaret Cancer Centre, or The Ottawa Hospital. Eligibility criteria included being > 18 years of age, English-speaking, and diagnosed with bladder cancer. The questionnaire included an adapted tool to measure informational needs, and validated measures for QoL  and SCN . QoL scores and unmet needs were calculated and compared between disease groups and cancer trajectory groups.Of the 1126 surveys distributed, 586 were completed (response = 52%). Mean age was 67.3 ± 10.2 years, and 401 participants (68.7%) were male. The mean QoL score (BUSS) for the sample was 78.1 ± 17.9 (median 81.7). Respondents with MIBC had significantly lower QoL scores compared to NMIBC. Further, scores differed across the cancer phase groups with the follow-up surveillance group having significantly higher QoL scores compared to the newly diagnosed and advance/recurrent disease groups. The ten most highly ranked informational needs were from the medical, physical, and practical domains. Eighty-eight percent (95% CI 85–91%) of respondents reported at least one SCN, with a median of 12. Over half of the participants  had at least one unmet need and 15% had ≥ 10 unmet needs. Newly diagnosed participants had the highest number of unmet needs.We found that the number of unmet supportive care needs and quality of life differed across cancer trajectory and disease groups. Future efforts should focus on the development and evaluation of tailored resources and programs to address the needs of people diagnosed and treated for BC.The online version of this article (10.1007/s00520-019-4649-z) contains supplementary material, which is available to authorized users. Bladder cancer (BC) is the second most common genitourinary cancer in North America and accounts for an estimated 4.5% of all new cancer cases worldwide . The larn = 43) in North America found that there were limited resources directed to BC survivors including support groups, survivorship programming, community services, and patient navigation/information, and that providers lacked awareness of available resources [The diagnosis and treatment of BC are stressful events associated with numerous supportive care needs (SCN) that continue beyond treatment –11. Howeesources . Furtheresources , 11. In esources –16. Specesources .There remains a need for larger, more comprehensive studies that can inform appropriate person-centered care activities for BC patients, including the development and tailoring of appropriate patient education and supportive care resources and services. Consequently, we conducted a large cross-sectional descriptive study to describe the QoL and informational and supportive care needs of NMIBC and MIBC survivors across the cancer care trajectory.BC patients were recruited from the Genitourinary Clinic at Princess Margaret (PM) Cancer Centre, the Urology Clinic at The Ottawa Hospital (TOH), and through Bladder Cancer Canada, a national non-profit organization that seeks to support BC survivors and increase awareness of BC among the general public and medical community. Patients were eligible if they were > 18 years of age, could understand and read English, and had been diagnosed with any stage of NMIBC/MIBC. The study was approved by Research Ethics Boards at the University Health Network and The Ottawa Hospital. Participants were informed that completion of the questionnaire implied consent.Princess Margaret Cancer Centre and The Ottawa Hospital: Patients diagnosed with BC attending clinical appointments were invited to the study and provided the questionnaire package to complete in clinic or return in a pre-paid envelope. Bladder Cancer Canada (BCC): Registered patient members were emailed information regarding the study with a link to the questionnaire that was hosted on FluidSurveys. The four-point survey approach based on Dillman’s Tailored Design Method , 19 was Participants completed a one-time questionnaire that was developed with input from the research team. The questionnaire package was field tested with five randomly selected BC patients attending routine visits to PM. The field test results were reviewed to assess the time it took participants to complete the questionnaire and to identify any issues with completion of the questions. Completion of the survey implied consent. The questionnaire included the following: (1) Demographic and disease and treatment information, which contained questions about age, sex, residence setting, employment status, and comfort with using the internet. Clinical information included type(s) of treatment received, time since diagnosis, stage and type of bladder cancer at diagnosis, and the phase of cancer treatment they identified with, which were categorized into newly diagnosed, follow-up surveillance, and receiving treatment for recurrent or metastatic disease; (2) Quality of life assessed using the Bladder Utility Symptom Scale (BUSS)  which isU test, and between cancer trajectory groups (newly diagnosed and undergoing treatment vs. follow-up surveillance vs. treatment for recurrence or metastatic disease) with Kruskal-Wallis test. The proportion of items rated “important” or “very important” in each informational need domain was alculated with 95% confidence interval (CI), and compared across disease groups and cancer phase groups using chi-squared analyses. Proportion (+ 95% CI) of total and domain-specific SCN  were calculated and examined. Proportion of “Unmet needs” was calculated for each item (No. answering “Need was not fully met”/No. who identified need (either met/unmet)). SCN total and domain-specific met or unmet (±SD) needs were non-normally distributed and compared across disease groups and cancer phase groups using Mann-Whitney U and Kruskal-Wallis tests. Data analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 21.0 with p values of < 0.05 considered statistically significant. Bonferroni correction was made for multiple comparisons.Descriptive statistics were calculated to provide summary information about the participants and for each outcome measure, and examined for non-normality (kurtosis and skewness). BUSS total scores were not normally distributed and were compared between disease groups (MIBC vs. NMIBC) using Mann-Whiney n = 204  from PM, n = 129  from TOH, and n = 253  from BCC. The QoL (p = 0.428), informational needs (across domains p = 0.1 to p = 0.95), and SCN scores (met and unmet needs) (p = 0.09) were not statistically different across the three samples . Consequently, the data was pooled together for analyses.A total of 586 surveys were completed between November 2014 and April 2016 including The majority of participants were male (68%), born in Canada (68%), had at least post-secondary education (65%), and were very comfortable receiving health information in English (94%). Fifty-seven percent of participants were initially diagnosed with NMIBC, 23% with MIBC, and 18% did not know their diagnosed disease type. At the time of the survey, 13% were newly diagnosed and receiving primary therapy, 66% had completed treatment and were in post-treatment follow-up, and 15% were receiving treatment for recurrent or metastatic disease. Demographic and clinical data are summarized in Table U = 9497, p < 0.001). Further, scores differed across the cancer phase groups with the follow-up surveillance group having significantly higher QoL scores (median 83.3) compared to the newly diagnosed (median 72.5) and the recurrent/metastatic groups (median 74.6) (H(2) = 20.71 p < 0.001).The mean QoL score (BUSS) for the sample was 78.1 ± 17.9 (median 81.7). Respondents with MIBC had significantly lower scores (median 75) compared to NMIBC (median 85) , physical , practical , emotional , social , and spiritual . The ten most highly ranked informational needs were from the medical  domains p = 0.046). Across the cancer phase groups, the informational needs were also similar, with the exception of the practical domain items. They were ranked less important by respondents with recurrent or metastatic disease compared to those with newly diagnosed disease (p = 0.031) or receiving follow-up surveillance (p = 0.035).The MIBC group and the NMIBC group had similar informational needs, with the exception of the practical domain needs, which were ranked higher in the NMIBC group (Eighty-eight percent (95% CI 85–91%) of respondents reported at least one SCN with a median of 12 and a mean of 15 ± 12.2 . The most common SCN were in the comprehensive cancer care (CCC) domain with 82% (95% CI 78.8–85.2%) of respondents reporting a need, followed by 65% (95% CI 61.0–68.9%) in the existential survivorship (ES) domain, 55% (95% CI 50.9–59.1%) in the information (IN) domain, 47% (95% CI 42.3–51.2%) in the quality of life (QOL) domain, and 35% (95% CI 31.1–38.9%) in the relationships (REL) domain. The top 10 SCN (met or unmet) are listed in Table The median number of unmet needs was 1.0 with a mean of 4.2 ± 6.7. Over half of the participants  had at least one unmet need . The number of unmet needs differed across the cancer journey groups (H(2) = 17.68, p < 0.001). Newly diagnosed participants had a median of 3.5 unmet needs; participants in post-treatment follow-up had a median of 0.0 unmet needs, and those in the metastatic or recurrent cancer had a median 1.5 unmet needs.The MIBC group had median of 1.0 unmet need (mean 4.6 ± 6.5), and the NMIBC group also had a median of 1.0 unmet need (mean of 4.2 ± 6.8) (p = 0.018) and had lower total BUSS score (60.3 ± 21.1) compared to respondents who had < 10 unmet SCN .Fourteen percent (82/586) of respondents had ≥ 10 unmet needs with a median of 16.0 unmet SCN (mean 17.4 ± 5.8). This group of patients were more likely to be newly diagnosed with  of the respondents reported ≥ 1 unmet need, and the mean number of unmet needs was lower than the average reported in other cancers , 32. TheThe findings of this study need to be interpreted in the context of its limitations. This was a cross-sectional study, which does not allow for the examination of cause-effect relationships. In addition, this study was restricted to English-speaking residents in Canada and may not be representative of non-English speaking individuals, or those outside of Canada. We did not address some demographic questions regarding income or caregiver status, which may have had impacts on needs and QoL of the participants. In addition, approximately 2/3 of the study population had a post-secondary education which may be due to the online nature and questionnaire-based nature of the study. This may impact the representation of these findings to the entire BC patient population and potentially result in an underestimation of need. Furthermore, the study cohort may have a larger proportion of younger patients due to the online approach for the BCC population, which can impact the representation of these findings to the entire BC patient population. The BCC group was younger than the PM and OCC cohorts. The use of both online and offline questionnaires may also partially explain the lower response rates seen in the online cohort, as there was no personal interaction and waiting time in the clinic with BCC respondents as there were in the TOH and UHN sites. However, since the informational needs, SCN, and QoL scores did not differ significantly across the three sites, the different response rates likely did not affect our analyses. Despite these limitations, this study provides valuable insight into the QoL, informational needs, and SCN of BC patients. It also emphasizes the need for the development of targeted educational resources, formalized education pathways, and screening for and addressing SCN, as they are common and significantly impact QoL.This work provides the initial groundwork to improve person-centered care provided to BC patients. Healthcare professionals play an important role in the provision of patient education and psychosocial support at all phases of treatment, as they are often the patients’ first point of contact. While there are clear benefits in providing appropriate and effective patient education and psychosocial care , 38, patESM 1(DOCX 21 kb)


The incidence and associated risk factors for premature death were investigated in a population-based cohort study in Iran.A total of 7245 participants (3216 men), aged 30–70 years, were included. We conducted Cox proportional hazards models to identify the risk factors for premature death. For each risk factor, hazard ratio (HR), 95% confidence intervals (95% CI) and population attributable fraction (PAF) were calculated.n = 126), cancer (n = 51), road injuries (n = 15), sepsis and pneumonia (n = 9) and miscellaneous reasons (n = 61). The age-standardized incident rate of premature death was 2.35 per 1000 person years based on WHO standard population. Hypertension , diabetes  and current smoking  were significant risk factors for premature mortality; corresponding PAFs were 12.3, 22.4 and 9.2%, respectively. Overweight (body mass index (BMI): 25–29.9 kg/m2)  and obesity (BMI ≥30 kg/m2)  were associated with decreased premature mortality. After replacing general adiposity with central adiposity, we found no significant risk for the latter . Moreover, when we excluded current smokers, those with prevalent cancer/cardiovascular disease and those with survival of less than 3 years, the inverse association between overweight  and obesity , generally remained unchanged; although, diabetes still showed a significant risk .After a median follow-up of 13.8 years, 262 premature deaths (153 in men) occurred. Underlying causes of premature deaths were cardiovascular disease (CVD) (Controlling three modifiable risk factors including diabetes, hypertension and smoking might potentially reduce mortality events by over 40%, and among these, prevention of diabetes should be prioritized to decrease burden of events. We didn’t confirm a negative impact of overweight and obesity status on premature mortality events. Globally 42% of all deaths caused by non-communicable diseases (NCD) occurred before the age of 70 years and this rate ranges from 28% in high income countries to 48% in low- and middle-income countries. Based on World Health Organization (WHO) estimates, total NCD deaths will escalate to 52 million in 2030 . Hence, Among the traditional risk factors for premature mortality, smoking was shown to be the predominant risk factor for premature mortality; although, obesity, hypertension and poor diet also showed substantial additional impacts . HoweverRecently we showed that incident premature cardiovascular disease (CVD) was 5.06 per 1000 person-years in a Tehranian population . As a sen = 18,555) of Tehranian participants aged ≥3 years were recruited during the first (1999–2001) and second phase (2002–2005)  × 100,P ×  and lower risk for premature mortality. Furthermore, the presence of diabetes [2.62 (1.84–3.72)] was also associated with increased risk of premature death.In a sensitivity analysis, as recently suggested by other researchers , 12, to Our study showed that the three major modifiable risk factors including diabetes, hypertension and current smoking, altogether, account for over 40% of premature deaths. Among them, the highest PAF was attributable to diabetes, the value of which was over two-fold that of prevalent CVD. Also, overweight and obesity were associated with a lower risk, even among non-smokers without prevalent CVD/cancer who had survived at least 3 years.Our finding of diabetes, hypertension and smoking being the independent risk factors of premature mortality, largely support previous findings . AlthougIt is well known that premature deaths are higher in people with diabetes than those without diabetes , a risk In this study, when we excluded current smokers, prevalent CVD/cancer as well as those with survival of less than 3 years, the presence of diabetes still showed about a three-fold [2.62 (1.84–3.72)] risk for premature mortality events. It has been shown that over 470,000 Iranian adults are currently unaware of their disease . Hence, The high prevalence  and inciUnfortunately, the current studies have shown an increasing trend in smoking among Tehranian adults , 25. In Our data showed that in both univariate and multivariate analysis, being overweight and obese were associated with lower risk for premature mortality, a phenomena called the “obesity paradox” . This isThe strength of this study is the use of the TLGS as a large population-based-cohort with a long follow-up data with reliable surveillance for death events and use of actual measurements of variables rather than self-reported data.Our study has some limitations; first, due to low incidence of premature death among participants, our sample size might lack sufficient statistical power to detect risk factors in a sex stratified manner or according to the specific cause of death. Second, as inherent to any prospective study, the level of risk factors was assessed only at the baseline examination and a possible change during the follow-up was not considered in our data analysis. As such, some degree of misclassification might have occurred, leading to the attenuation of the hazard ratios for different risk factors. Third, regarding socio-economic status of the study population we assessed only educational status and did not evaluate certain other important components such as income status and the accurate occupational position of study population. Last but not least perhaps, this study was conducted among Tehranian adult population, so our results cannot be extrapolated to the whole country or other Middle-Eastern populations.In the current study we showed an age-standardized incidence rate of 2.35 per 1000 person-years based on WHO standard population for premature mortality during more than a decade of follow-up. Controlling three modifiable risk factors including diabetes, hypertension and smoking could potentially reduce mortality events by over 40%, viz. the prevention and control of diabetes being prioritized by national policy makers to decrease the burden of premature mortality events. We didn’t confirm a negative impact of overweight and obesity on premature mortality among our populations.


To quantify the dosimetric parameters of different bone marrow sparing strategies and to determine the optimal strategy for cervical cancer patients undergoing postoperative intensity-modulated radiotherapy (IMRT).Fifteen patients with cervical cancer were selected for analysis. The planning target volume (PTV) and the organs at risks (OAR) including small bowel, bladder, rectum, femoral heads, os coxae (OC), lumbosacral spine (LS) and bone marrow (BM) were contoured. For each patient, four IMRT plans with different strategies were generated, including one plan without BM as the dose-volume constraint, namely IMRT (N) plan, and three bone marrow sparing (BMS-IMRT) plans. The three BMS-IMRT plans used the BM, OC, OC and LS respectively, as the BM OAR, namely as IMRT (BM), IMRT (OC) and IMRT (OC + LS) plans. Dose volumes for the target and the OARs were compared using analysis of variance (ANOVA).p < 0.05) in IMRT (BM) plans while the average volume receiving ≥20, ≥30, ≥40Gy decreased by 7.1–24.2%. The LS volume receiving 40Gy showed the highest decrease  in IMRT (OC + LS) plans. On the other hand, in comparison with IMRT (BM), IMRT (OC) reduced the dose volume of to the OC, but increased the dose to LS while IMRT (OC + LS) plans reduced both the OC and the LS volume at all dose levels. Specifically, the V20 of OC and LS in the IMRT (OC + LS) plan decreased by 11.5 and 11.2%, respectively.Compared with IMRT (N) plans, the dose to the small bowel, bladder, rectum and femoral heads showed no increase in the three BMS-IMRT plans. However, the irradiated dose to BM, OC and LS significantly decreased. In particular, the mean dose of BM, OC and LS decreased by about 5Gy (By introducing the os coxae and lumbosacral spine as the dose–volume constraints, the IMRT plans exhibited the best sparing of the bone marrow without compromising the dose to surrounding normal structures. Therefore, we recommend adding the os coxae and lumbosacral spine as the BM OAR in such plans. Adjuvant pelvic radiotherapy with concurrent chemotherapy is the standard treatment approach or cervical cancer patients who have previously undergone hysterectomy . HoweverStudies have shown the advantages of pelvic intensity-modulated radiation therapy (IMRT), including better dosimetric distribution, relatively lower irradiation dose to normal tissues and fewer acute side effects, compared with conventional forward planning techniques , 5. HoweStudies have demonstrated the occurrence of acute HT is associated with the volume of BM irradiated, especially the volume of lumbosacral spine (LS) and lower pelvis irradiated –10. ThusFifteen cervical cancer patients treated with pelvic IMRT in our institution between January 2018 and July 2018 were selected for this study. Inclusion criteria were (1) staged IB–IIB, (2) biopsy-proven squamous cell carcinoma and (3) undergoing postoperative pelvic radiation therapy. Patients with high-risk pelvic lymph nodes receiving simultaneous integrated boost were excluded. The mean and median age of eligible patients was 56.3 and 61 years , respectively.All patients underwent CT simulation on a helical CT scanner  with 3 mm slice thickness. The scans were collected from the L1 vertebra to the region of 5 cm below the ischial tuberosities. Patients were immobilized with a vacuum-formed cradle in the supine position with comfortably full bladder and no bowel preparation prior to simulation.Normal tissue included small bowel, bladder, rectum, femoral heads and bone marrow (BM). For each patient, the small bowel was contoured consisted of as the entire peritoneal cavity from L4-L5. The external contour of bone marrow was delineated Figs.  and 2, rFor consistency, all contours were delineated by a single radiation oncologist. The clinical target volume (CTV) encompassed the gross tumor volume and potentially microscopic disease, extending from the L4-L5 vertebra to the inferior border of the obturator foramen. In detail, the CTV was generally comprised of the upper vagina, parametrial tissues, uterus (if present), and regional lymph nodes . Considering organ motion and setup error, a uniform 7 mm margin was applied to the CTV to generate the planning treatment volume (PTV). The 7 mm margin was determined using the van Herk methodology  with theFor consistency, all the treatment planning procedures were developed by the same radiation physicist. The inverse planned dynamic IMRT plans were prepared using Eclipse treatment planning system version 13.5 . The analytic anisotropic algorithm (AAA) with the grid size of 2.5 mm was used for computing dose to the irradiated region. The machine parameter optimization method was used with 6 MV photon beams, sliding-window fields and a multileaf collimator leaf size of 5 mm. All plans in the study were designed to be executed using Varian iX linear accelerator  at a dose rate of 400 MU/min, with cone beam computed tomography (CBCT) scanners on-board for daily image guidance before treatment. The CBCT images were acquired with scan parameters 125 kV, 80 mA and 3 mm slice thickness in the half-fan mode using a half bowtie filter. An automatic rigid registration process of the CBCT to the planning CT was performed in three translational directions, including left–right, superior–inferior, and anterior–posterior. To improve the set-up accuracy, the trained radiation therapist first evaluated the automatic bony match in the pelvis clip box and made necessary manual adjustments to acquire satisfactory bony matching. Then, the radiation oncologist checked whether the CTV was covered by the PTV. When deviations exceeded 5 mm, we would considered repositioning the patient, performing CBCT again and repeating the aforementioned matching process. Generally, the first CBCT procedure was verified by the radiation oncologist online while subsequently ones were verified via the offline review.For each patient, four inverse IMRT plans were generated: IMRT (N) plan without BM as the sparing objective, IMRT (BM) plan using total BM as the sparing objective, IMRT (OC) plan using OC as the sparing objective and IMRT (OC + LS) plan using OC and LS as the sparing objective. The plans were optimized with equally spaced nine coplanar fields, with gantry angles of 180, 140, 100, 60, 20, 340, 300, 260 and 220. A dose of 50.4 Gy in 28 fractions was delivered to the PTV.In the optimization process, all plans were generated adopting an identical set of PTV/OAR dose–volume constraints to keep the results comparable. The optimization parameters are similar to the RTOG 0418 . In all mean), the minimum point dose (Dmin), the maximal point dose (Dmax), D98%, D50%, D2%, conformity index (CI) and homogeneity index (HI).Data from the dose volume histograms (DVHs) acquired for all contoured organs and the target volume was analyzed. For the PTV, dosimetric parameters were quantified, including the mean dose , the V10 (the percent of volume that received 10Gy), V20, V30, V40 and V50.For the OAR , a set of dosimetric parameters was obtained, including the mean dose . Analysis of variance (ANOVA) was used to test the differences of the DVH parameters obtained from different planning strategies and 3 . The PTV planning objectives were achieved with four different dose limitation strategies and all plans were normalized to cover 95% of the PTV with ≥100% of the prescribed dose. Table The PTV volume was 1061 ± 110 cmp < 0.05) while the minimal dose decreased by 4.0–7.4%(p < 0.05) for the BMS-IMRT plans. Compared with IMRT (BM), the maximal PTV dose increased and the minimal dose decreased significantly in the IMRT (OC), and IMRT (OC + LS) plans, with the differences within 3.7%. In terms of CI and HI, IMRT (BM) plan and IMRT (N) plan showed the slightly better conformity and homogeneity than did in IMRT (OC + LS) plans, but they resulted in a greater dose to the bone marrow. Typical dose distributions for the four strategies compared in this study are shown in Fig. The dose distribution in the PTV satisfied the clinical requirement of less than 2% of the PTV receiving more than 107% of the prescribed dose. As shown in Table Table 30 and V40 of femoral heads showed a decrease in the IMRT (BM) plan , which may be caused by the dose limitation of the adjacent pelvis bone marrow. Briefly, the three bone marrow-sparing plans showed no significant dose reductions to the small bowel, bladder and rectal dose compared with conventional planning methods. Furthermore, comparing these parameters for the IMRT (BM) plans with the IMRT (OC) and IMRT (OC + LS) plans, respectively, we found no significant differences.No significant differences among the four strategies for the dosimetric parameters of small bowel, rectum and bladder, meaning that any marrow sparing is not at the expense of other OAR sparing. Additionally, compared with the IMRT (N) plan, the VTable p < 0.05) in IMRT (BM) plans. As seen in Table p < 0.05 for each pairwise comparison with IMRT (N) plans). The average V20, V30 and V40 of OC reduced by 14.0, 18.3 and 12.0% while those of the lumbosacral spine reduced by 7.1, 18.4 and 24.2%, respectively (p < 0.05) in IMRT (BM) comparison with IMRT (N) plans. The changes in Dmean, V20, V30 and V40 in the BM, OC and LS were shown in Fig. p < 0.05).Compared with the IMRT (N) plans, the mean dose of BM, OC and LS decreased by about 5Gy (20 of OC and LS in IMRT (OC + LS) plan decreased by 11.5 and 11.2% compared to IMRT (BM) plan. However, the LSS received a higher radiation dose than the OC, most likely because of its proximity to the PTV.On the other hand, compared with IMRT (BM) plans, IMRT (OC) reduced the dose volume of OC at different levels, but increased the dose to LS. For this reason, the mean dose to BM were barely lower than that in IMRT (BM) plans Table . In contThe present study quantified the dosimetric parameters for different bone marrow sparing strategies by limiting the dose delivered to bone marrow and determined the optimal strategy for cervical cancer patients undergoing postoperative radiotherapy.Results of this study indicated that our proposed strategy, i.e. adding the os coxae and lumbosacral spine as the planning constraints better spared the bone marrow from excessive radiation without increasing the dose on other normal tissues. In comparison with conventional bone marrow sparing IMRT schemes , the proAlthough IMRT (OC + LS) plans reduced bone marrow radiation dose while maintaining adequate target volume coverage, the conformity and homogeneity of PTV might be comprised slightly. This may be attributed to the following reason. In these IMRT plans, the treatment load was dispersed in nine evenly spaced fields to satisfy the coverage of the PTV and reduce normal tissue irradiation. For patients with cervical cancer, most of the BM were located in the beam’s eye view . Thus, t20 were more likely to experience leukopenia. In particular, these similar findings (i.e. V10-V20 as best predictors) highlighted the strong sensitivity of bone marrow stem cells to radiation at low doses. Other groups, on the contrary, reported that the volume of pelvic BM receiving high-dose radiation  significantly correlated with HT [More attention has been devoted in recent years to assess the dose–volume relationship between the amount of pelvic bone irradiated at different dose levels and the risk of acute hematologic toxicity (HT). Mell et al.  found th with HT , 22. ForTo the best of our knowledge, the present work is the first to take into account the separate use of the OC and LS for IMRT plans optimization. In the comparative dosimetric study, some DVH statistical parameters of BM, OC and LS decreased significantly. Although possibly beneficial, several important points remain to be addressed.Firstly, the results showed that the dose delivered to the LS was significantly greater owing to its proximity to the PTV. Excessive dose constraints, even with the optimal BM sparing strategy, may adversely affect the sparing of other normal tissues or compromise the PTV coverage. In this case, other advanced techniques such as Tomotherapy  and VMATAnother challenge is that the entire bone anatomy is generally contoured and entered as dose–volume constraints in the standard IMRT plans, which overestimates the volume of active BM and constrains optimization. It is known, however, that BM is comprised of both active “red” marrow and inactive “yellow” marrow, which cannot be well visualized on computed tomography (CT)  and onlyFinally, the proposed strategy of plan optimization using the OC and LS instead of the whole BM has been found to be more effective in limiting the bone marrow irradiation, compared with the conventional BMS-IMRT scheme. However, it is still unclear to what extent the clinically meaningful reductions in HT could be achieved. Future work should focus on the evaluating the optimization strategy by long term follow-up.For patients suffering from cervical cancer, IMRT plans with the os coxae and lumbosacral spine as the dose–volume constraints exhibited the optimal sparing of the bone marrow without increasing the dose to other normal tissues. Such novel dose limitation strategy would be expected to be a promising treatment approach to reduce acute HT.


Granulomatosis with polyangiitis (GPA), a necrotizing granulomatous disease, very rarely involves the central nervous system (CNS), particularly the pituitary. Delayed treatment may cause permanent bilateral blindness. We report an isolated case of pituitary GPA that manifested as a progressive bilateral temporal visual field (VF) defect and was diagnosed via pituitary biopsy. Additionally, we review ocular, chiasmal and cranial nerve involvement in pituitary GPA.A 20-year-old Chinese man was referred for repeated fever, sudden headache, diplopia with a bilateral best-corrected visual acuity (BCVA) of 10/20, ptosis in both eyes and restricted abduction on the right side. VF tests showed bitemporal hemianopsia. Laboratory tests revealed hypothyroidism and were negative for autoimmune markers. Enhanced magnetic resonance imaging (MRI) showed pituitary enlargement. The diagnosis was lymphocytic pituitaritis. After intravenous (IV) dexamethasone treatment, full recovery occurred within 2 months. Two years later, the patient was readmitted for headache recurrence. With oral prednisone, the visual acuity in his right eye rapidly decreased to hand motion (HM) within one month. Enhanced MRI showed pituitary enlargement and a new, invasive suprasellar CNS lesion. All infection- and autoimmune-related tests were negative. The visual acuity in his right and left eye decreased to no light perception (NLP) after 6 days and 2 weeks, respectively. The biopsy results suggested GPA. After IV methylprednisolone treatment, complete remission of the symptoms occurred and was confirmed by MRI. The 15-month follow-up showed no signs of recurrence.GPA typically affects the respiratory tract, lungs and kidneys. To date, 50 cases with pituitary involvement have been reported. Chiasmal and cranial nerve involvement leading to visual acuity impairment are common. We found 2 cases with severe visual loss resembling our case and discuss certain similarities.The online version of this article (10.1186/s12886-018-0966-0) contains supplementary material, which is available to authorized users. Granulomatosis with polyangiitis (GPA), formerly known as Wegener’s granulomatosis (WG), is an anti-neutrophil cytoplasmic antibody (ANCA)-related necrotizing granulomatous disease. GPA typically affects the upper respiratory tract, lungs and kidneys, but all organs can be involved because the disease is a systemic form of granulomatous small-vessel vasculitis. The central nervous system (CNS), particularly the pituitary, is rarely involved . Until 29/L and a neutrophil count of 3.73 × 109/L (59.5%). His renal function was normal, with a creatinine (Cr) and urea level of 61.17 μmol/L and 4.05 mmol/L, respectively. The urine was negative for protein and red blood cells. The urine-specific gravity was normal, while endocrine tests revealed a thyroid-stimulating hormone (TSH) level of 0.04 μIU/mL, an adrenocorticotropic hormone (ACTH) level of 1.70 pg/mL and a testosterone level of < 20.0 pg/mL. Morning cortisol, prolactin (PRL), random blood glucose and glycosylated hemoglobin levels were normal  for 3 days, followed by a decreased dosage of dexamethasone  and then oral prednisone (60 mg qd). Two months later, the patient’s BCVA recovered to 100/100 bilaterally with a normal VF. His ocular movement was normal, and he reported no diplopia or headache. Additionally, the endocrine hormone levels were within normal limits  was 10/20 bilaterally, with a normal intraocular pressure (IOP). He showed ptosis in both eyes, with restricted abduction on the right side. A slit-lamp examination yielded normal results for both the anterior segment and the fundus, with no relative afferent pupillary defect (RAPD). VF testing revealed bitemporal hemianopsia. Laboratory tests showed a white blood cell (WBC) count of 6.26 × 10al Table  and b. Nts Table  and d. TIn Sept. 2014, he was readmitted to the local hospital due to headache recurrence with nausea and vomiting. Endocrine tests showed an elevated PRL level and hypothyroidism Table , indicatIn Oct. 2014, the patient’s headache worsened with severe nausea and vomiting, and the visual acuity in his right eye decreased to hand motion (HM), with 80/100 in the left eye and an IOP of 13/17 mmHg. His left eye displayed ptosis, but the ocular position and eye movement were normal. A slit-lamp examination showed no abnormal findings in the anterior chamber, with an equal pupil size, but the right eye was RAPD positive. The fundus examination was normal except for bilateral pale optic papillomas Fig. . OpticalErythrocyte sedimentation rate (ESR), CRP, cryptococcal antigen, serum 1,3-beta-D-glucan assay (BDG test), interferon gamma release assay for tubuerculosis (T-SPOT test) and a lymphocyte culture yielded negative results. An enhanced paranasal CT scan showed only bilateral ethmoid and left sphenoid sinus inflammation. No positive results were detected by chest X-ray or multiple-organ B-mode ultrasound examination. Moreover, there were no traces of red blood cells or protein in his urine. The patient’s renal function was also normal . A multidisciplinary consultation concluded that with 30 days of oral corticosteroid therapy and no signs of relief, a CNS infection should not be excluded; therefore, oral prednisone (35 mg qd) was continued. The visual acuity of the patient’s right eye decreased to NLP 6 days later, with headache aggravation, sudden nausea and vomiting, and a reduction in the visual acuity of the left eye to counting fingers (CF). An ophthalmological examination, including an assessment of eye position and movement and the anterior and posterior segments, yielded the same results as before. Repeated VF test showed a temporal hemifield and a superior nasal quadrant defect Fig. . RepeateThe pituitary tissue specimen was stained and tested at two institutions in China , with similar results. Repeated acid-fast staining showed negative results. Hematoxylin and eosin (H&E) staining Fig.  showed aThe diagnosis of GPA is based on the 1990 ACR classification criteria. The sensitivity and specificity of these criteria are 88.2 and 92.0%, respectively. GPA mainly involves systems and organs with multiple small vessels, although the involvement of nearly every organ has been previously reported. The CNS is relatively rarely affected . The pitThe first unique characteristic of our case is the isolated pituitary involvement. The only other similar case reported to date  occurredIn other cases with pituitary involvement, lung and skin involvement reportedly occurred within 3 weeks  or severhiCD4+ cells are abnormal in GPA patients, and these cells are unable to inhibit the PR3 conversion of T lymphocytes.The second unique characteristic of our case is the ANCA negativity both at the first onset and during recurrence. Our literature review showed that the rate of ANCA-positive results is approximately 83–94% among all GPA cases and 91.9% among cases with pituitary involvement. This rate is related to the disease severity , and an We reviewed all 51 reported cases (including our case) for ophthalmic manifestations and found an ophthalmic involvement rate of 35% (20/51). Nine cases exhibited decreased visual acuity and VF defects. Two cases showed episcleritis; there were 2 cases of conjunctivitis, 1 case of dry eye and 2 cases of palsy of cranial nerves III and VI. Our patient presented with visual loss, VF defects, ptosis and abduction dysfunction. Compared to other pituitary lesions, GPA is more likely to involve more parts of the visual system, including the optic chiasm, optic nerve, cranial nerves III and VI, sclera and conjunctiva, and the progression of visual loss is more rapid because GPA is a form of vasculitis and is usually invasive. When the lesion spreads and invades or encircles the optic nerve, optic chiasm or cavernous sinus, severe visual loss with multiple cranial nerve palsies can occur within a very short time. Uveitis usually accompanies systemic vasculitis due to the abundant blood supply. Conjunctivitis and dry eye may be related to metabolic disorders caused by anterior pituitary dysfunction.We analyzed the 9 cases that presented with visual loss and VF defects and found several similarities. First, 7 of 9 cases presented with both anterior lobe and posterior lobe dysfunction; 1 case presented with isolated anterior lobe dysfunction, and the adrenocortical pituitary hormone levels were not described in the remaining case. Anterior lobe involvement in these 9 cases was far greater than in all GPA cases (72%) , which mThe visual prognosis in some cases is extremely poor. Four of 9 patients presented with unilateral or bilateral blindness during disease development –15. ThesIn conclusion, we present a case of GPA with isolated pituitary involvement characterized by anterior lobe dysfunction, optic chiasm infiltration and ANCA negativity. Our review of the literature indicates that GPA with an ophthalmic manifestation is always caused by chiasmal invasion and is accompanied by anterior lobe dysfunction and other CNS lesions; furthermore, this type of case is more likely to be invasive. The onset of visual loss is usually rapid and severe during recurrence of the disease. In clinical work, the diagnosis of ANCA-negative GPA is usually difficult and relies solely on the pathological biopsy. Whether other organs will become involved or ANCA positivity will occur in this case remains to be seen.Additional file 1:Comparison of 2 isolated GPA cases. In this file, we compared ophthalmological and MRI changes, pituitary function and prognosis of the isolated case of pituitary GPA reported by GA Roberts and our case. (DOCX 15 kb)Additional file 2:Comparison of four GPA cases with bilateral visual loss. In this file, we compared ophthalmological and MRI changes, pituitary function and treatment of 4 GPA cases with bilateral visual loss reported in literature. (DOCX 21 kb)