At all stages of f lowering, a decisive role is played by the family of MADS-domain transcription factors,the combinatorial action of which is described by the ABCDE-model of f lower development. The current volume ofdata suggests a high conservatism of ABCDE genes in angiosperms. The E-proteins SEPALLATA are the central hub ofthe MADS-complexes, which determine the identity of the f loral organs. The only representative of the SEPALLATA3clade in tomato Solanum lycopersicum L., SlMADS5, is involved in determining the identity of petals, stamens, andcarpels; however, data on the functions of the gene are limited. The study was focused on the SlMADS5 functionalcharacterization. Structural and phylogenetic analyses of SlMADS5 conf irmed its belonging to the SEP3 clade. Anin silico expression analysis revealed the absence of gene transcripts in roots, leaves, and shoot apical meristem,and their presence in f lowers, fruits, and seeds at different stages of development. Two-hybrid analysis showedthe ability of SlMADS5 to activate transcription of the target gene and interact with TAGL1. Transgenic plants Nicotianatabacum L. with constitutive overexpression of SlMADS5 cDNA f lowered 2.2 times later than the control; plantsformed thickened leaves, 2.5–3.0 times thicker stems, 1.5–2.7 times shortened internodes, and 1.9 times fewerf lowers and capsules than non-transgenic plants. The f lower structure did not differ from the control; however, thecorolla petals changed color from light pink to magenta. Analysis of the expression of SlMADS5 and the tobaccogenes NtLFY, NtAP1, NtWUS, NtAG, NtPLE, NtSEP1, NtSEP2, and NtSEP3 in leaves and apexes of transgenic and controlplants showed that SlMADS5 mRNA is present only in tissues of transgenic lines. The other genes analyzed werehighly expressed in the reproductive meristem of control plants. Gene transcripts were absent or were imperceptiblypresent in the leaves and vegetative apex of the control, as well as in the leaves and apexes of transgenic lines.The results obtained indicate the possible involvement of SlMADS5 in the regulation of f lower meristem developmentand the pathway of anthocyanin biosynthesis in petals. Throughout the plant’s life cycle, its root and shoot apicalmeristems maintain a pool of pluripotent stem cells, whichgive rise to new organs: roots and leaves respectively, duringvegetative development and flowers during reproductionstage. At the reproductive stage, the shoot apical meristemof the angiosperms turns into the inflorescence meristem,which forms determined flower meristems . In all aspects of flowering, the MADS-domain familyof transcription factors (TFs) plays a key role according to thewell-known ABCDE flower development model .The ABCDE model is based on genetic and molecularstudies, primarily of model species Arabidopsis thaliana (L.)Heynh., Antirrhinum majus L., and Petunia×hybrida hort.ex E. Vilm. . Accordingto the model, the identity of flower organs is determinedby five classes of genetic activities: A and E – sepals; A, Band E – petals; B, C and E – stamens; C and E – carpels; C, Eand D – ovules. At the molecular level, the ABCDE-model isexplained by the so-called “quartet” model, according to whichMADS-TFs of ABCDE classes in various combinations formtetramers: for example, C/C/E/E – to determine carpel identity,or A/B1/B2/E – to specify petal identity . These tetramers activate or suppresstranscription of target genes . The current data suggest a high structural andfunctional conservatism of A, B, C, D, and E genes in floweringplants .The genes of the E-class, A. thaliana SEPALLATA , which are involved in determiningthe identity of all floral organs, deserve special attention . The knockout of onlyone of the SEP genes does not have a significant effect on theA. thaliana flower, while the sep1 sep2 sep3 triple mutationtransforms all the flower organs into sepals; a new flowerwith the same development pattern is formed instead of thepistil . The quadruple sep1 sep2 sep3 sep4mutation leads to the replacement of all flower organs withleaf-like organs .SEP proteins are the central hub in the formation of MADSTFquartets . Among SEPs, SEP3 isthe most functionally pleiotropic and interacts with almostall MADS-TFs responsible for the identity of flower organs. SEP3 gene simultaneous ectopic expression expressionwith the A-, B-, or C-class genes transforms leavesinto flower organs .During plant evolution, SEP genes are believed to havearisen later than other flower-related MADS-box genes, butat the same time they became key players in the origin offlowering plants, as well as in the domestication and breedingof crops . Therefore,their study in cultivated plants can expand the understandingof the role of these genes in determining economicallyvaluable traits.The tomato Solanum lycopersicum L. is one of the mostimportant vegetables and, at the same time, a model forstudying the fleshy fruit development and ripening. Thetomato genome has been sequenced and annotated (https://www.solgenomics.net/), and contains several SEP genes:TAGL2 (Solyc05g015750.2.1), SlMADS6/TM29/LeSEP1(Solyc02g089200.2.1), RIPENING INHIBITOR ( MADSRIN)(Solyc05g012020.2.1), SlMADS98/SlCMB1 (Solyc04g005320.2.1), SlMADS1/ENHANCER-OF-JOINTLESS-2(Solyc03g114840.2.1), SlMBP21/JOINTLESS-2 (J2)(Solyc12g038510.1.1) and SlMADS5/TM5/TDR5/LeSEP3(Solyc05g015750.3.1) .In addition to determining the flower organ identity, SEPproteins, together with MADS-TFs of the FRUITFULL (FUL)and AGAMOUS (AG) subfamilies, are actively involvedin the regulation of fruit ripening. This is clearly demonstratedin tomato, the fruit ripening of which is controlled byFUL1/FUL2, TOMATO AGAMOUS 1 (TAG1)/TOMATOAGAMOUS-LIKE 1 (TAGL1) and MADS-RIN . At thesame time, FUL2 and TAGL1 have been shown to play anadditional role in pistil initiation and early fruit development, which is likely tobe performed in combination with the tomato SEP3 homolog,SlMADS5 .SEP1-like gene TAGL2 was shown to be expressed atstages I (anthesis) and II of the tomato fruit development. Suppression of SEP1-like TM29 causesthe development of parthenocarpic fruits and the flower reversion. Tomato SEP4-likeSlCMB1 regulates ethylene biosynthesis and the accumulationof carotenoids during fruit ripening; suppression of SlCMB1leads to a change in the inflorescence architecture and anincrease in the sepal size . SEP4-likeSlMADS1 acts as a negative regulator of fruit ripening . SEP4-like SlMBP21 specifies the sepal size mediated by ethylene and auxin signaling, as well as the abscissionzone formation .SEP4-like MADS-RIN is the main regulator of fruit ripening:gene knockout leads to the formation of an unripe fruit, includingthe absence of carotenoid accumulation .The only representative of the tomato clade SEP3, TFSlMADS5, is involved in determining the identity of theorgans of the three inner flower whorls ,interacting with MADS-TFs of the SEP and AG subfamilies. Despite the SEP3 significance, thisgene variability has been characterized in cultivated and wildtomato species, and the SlMADS5 expression was observedin some organs and tissues .The aim of the present study was to characterize the functionof S. lycopersicum SlMADS5. SlMADS5 structural, phylogeneticand expression analysis confirmed its belonging to theSEP3-clade. Analysis in the yeast two-hybrid GAL4-systemshowed the SlMADS5 TF activator properties and itsinteraction with C-class MADS-TF. Transgenic Nicotianatabacum L. plants with SlMADS5 constitutive overexpressionexhibited a pronounced phenotype of reproductive developmentsuppression.Tomato S. lycopersicum cv. Silvestre recordo and tobaccoN. tabacum cv. Samsun plants were used in the study. Tomatoaccessions were grown under controlled greenhouse conditionsuntil flowering. Roots, leaves, flowers and ripe fruits werecollected separately. Tissues were grounded in liquid nitrogenand stored at –70 °C. Tobacco accessions were grown in vitroon a sterile MS medium in a climatic chamber  until the formationof 4–6 leaves.Total RNA was isolated from tomato  and tobacco  tissues using the RNeasy Plant Mini Kit, and used for cDNA synthesis . Genomic DNA wasisolated from leaf tissues by the standard potassium acetatemethod  and used for PCR tests forthe presence of a transgene in the plant genome.Primers for gene amplification, sequencing, and expressionanalysis were generated based on the MADS-box transcriptsof S. lycopersicum cv. Heinz and tobacco N. tabacumgenes available in the NCBI (http://www.ncbi.nlm.nih.gov/); NtLEAFY; NtWUSCHEL; NtAG ;NtPLENA : NtSEP1; NtSEP3 ; NtSEP2 ) so that forwardand reverse primers are separated by at least one intron andmatch all possible transcripts for each of the analyzed genes(Table 1). The primer sequences were additionally verifiedusing Primer 3 and BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primers for CDS in-frame cloning intoplasmid vectors (GAL4 system) contained EcoRI and SalI  restriction sites at the 5′ end.Full-length SlMADS5, TAG1, and FUL2 cDNAs wereamplified using the cDNA, isolated from S. lycopersicum cv.Silvestre recordo flowers; PCR conditions: initial denaturationat 95 °C for 5 min; 30 cycles of denaturation (94 °C for 30 s), annealing (55 °C – 30 s) and synthesis (72 °C – 1 min); finalsynthesis (72 °C – 7 min). The PCR fragments of the expectedlength were purified using the MinElute Gel Extraction Kit, cloned into the pGEM®-T Easy plasmidvector  at EcoRI and Sal I sitesand sequenced (Core Facility “Bioengineering”). Further, theSlMADS5, FUL2, and TAGL1 CDSs were cloned into hybridvectors pAD-GAL4 and pBD-GAL4cam : each gene was ligated in frame with the activatordomain (pAD) and DNA-binding domain (pBD) of the yeastTF GAL4. Recombinant pJ69-4a strains carrying each pADgeneand pBD-gene construct separately, as well as in pairspAD-gene + pBD-gene, were obtained. For plant transformation,SlMADS5 cDNA was cloned in a sense orientation intoa binary vector based on pBin19, under the control of theenhanced cauliflower mosaic virus promoter 35S and nopalinesynthase (NOS) terminator. With this construct, a recombinantagrobacterial strain AGLØ was obtained.For sequence structural analysis, the NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), MEGA 7.0 and Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/) were used. Sequence phylogeny was assessed inthe MEGA7, using Maximum Likelihood method based onthe JTT model.Gene expression analysis was performed in silico , as well as by quantitative (q) real-time (RT) PCRin two biological and three technical replicates. The kit “Reactionmixture for carrying out qRT-PCR in the presence ofSYBR Green I and ROX”  and the CFX96Real-Time PCR Detection System  were applied. The qRT-PCR conditions were as follows:95 °C – 5 min; 40 cycles .The reference gene actin-7 (XM_016658880.1)  was used for normalizing the expressionof tobacco genes. Statistical processing of the results wascarried out usingthe GraphPad Prism v. 7.02 (https://www.graphpad.com).The analysis of SlMADS5 interactions with TAGL1 andFUL2 proteins was carried out in vivo in a two-hybrid GAL4-yeast system using the Saccharomyces cerevisiae Pj69-4astrain, according to the HybriZAP-2.1-Hybrid cDNA Two-Hybrid Synthesis Kit protocol (Stratagene).Leaf explants of tobacco (N. tabacum cv. Samsun) weretransformed using Agrobacterium tumefaciens strain AGLØ.To select transgenic regenerants, an MS medium containingkanamycin  for selection and carbenicillin(500 mg/L), which suppresses agrobacteria growth, was used.The rooted regenerants were adapted to the soil in greenhouseconditions and then tested for the presence of a transgene in thegenome by PCR with primers specific to the sequences of the5′ end of the transgene and the NOS-terminator (see Table 1).To confirm the conservatism of the SlMADS5 function intomato (cv. Silvestre recordo), an analysis of its interactionswith MADS-TFs TAGL1 and FUL2, the interaction withwhich was and was not, respectively, shown earlier , was carried out.Structural analysis of the SlMADS5 protein was carried outin comparison with the known tomato, tobacco, and A. thalianaSEP homologs. The presence of the main domains characteristicof MIKCc type MADS-TFs was confirmed, namelythe highly conserved MEF2-like MADS-domain (1–76 aa),an I-region (77–92 aa), a conserved keratin (K)-like domain(93–173 aa), and a variable C-region (174–241 aa) .The performed phylogenetic analysis testified the belongingof SlMADS5 to the SEP3 clade .To characterize TF SlMADS5 functionally, we analyzedthe expression of the SlMADS5 gene in various tomato organsand the ability of SlMADS5 protein to activate gene transcriptionand interact with MADS proteins of the C and A classes.Also, transgenic N. tabacum model plants with constitutiveoverexpression of SlMADS5 cDNA were obtained.In silico analysis of the SlMADS5 expression pattern wascarried out in roots, leaves, vegetative shoot meristem, flowermeristem, flower (from bud to fully open and anthesis stage),fruits (4–8 days after anthesis), fruit skin and pulp (stages:Immature Green (IMG); Mature Green (MG); Breaker (BR),color change; Orange (OR); Red Ripe (RR)), and in seeds . SlMADS5 transcripts were notfound in roots, leaves, and the vegetative apical meristem. Atthe same time, SlMADS5 expression was shown in flowers(maximum – at the anthesis stage), fruits, fruit peel (maximumat MG and BR stages), fruit pulp , and seeds (maximum at IMG stage) .In vivo analysis in the yeast two-hybrid GAL4 systemshowed that TF SlMADS5 has the property of activating thetranscription of target genes, interacts with the C-class MADSprotein TAGL1, but does not interact with the A-class MADSprotein FUL2 (Table 2).The characterization of transgenic tobacco plants withSlMADS5 constitutive overexpression was performed. Independentregenerants T0 35S::SlMADS5 (18 plants) wereadapted to the greenhouse, tested by PCR for the presence ofa transgene expression cassette in the genome, and comparedwith the control (non-transgenic tobacco plants) during development.In comparison with the control, 35S::SlMADS5plants  bloomed much later . Also, 35S::SlMADS5 phenotype was characterizedby a 2.5–3.0 times thicker stem, 2.0 times shortened internodes, thickened and darker leaves, and 2.5 times fewerflowers and capsules. The 35S::SlMADS5 flower structure didnot differ from the control.Seeds of two transgenic T0 lines (S5-16 and S5-17) witha pronounced phenotype were planted in a greenhouse.T1 plants, which gave a positive PCR signal for the presenceof a transgene in the genome, bloomed 1.3–1.5 times laterthan the control, had a 35S::SlMADS5 phenotype, and formedflowers with magenta-colored corolla petals, in contrast tolight pink petals in the control.Seeds of lines T1 S5-16-6, S5-16-7, S5-17-1 and S5-17-4were planted on MS medium (Km 50 mg/l); the 3:1 ratio of thenumber of Km-resistant to Km-sensitive seedlings indicated aheterozygous state of the transgene and one copy of it in thegenome of transgenic lines. In seedlings, internodes were nearabsent, and only T2 plants of the S5-16-7 line (14 accessions)formed a noticeable stem and were adapted to the greenhouse(the rest of the plants died after transfer to the soil). PlantsT2 S5-16-7 demonstrated the 35S::SlMADS5 phenotype: theybloomed 2.4 times later than the control; formed thickenedstems and leaves, shortened internodes, and 2.3 times lessseed capsules.In T1 lines S5-16-7 and S5-17-1, in comparison with thecontrol, we analyzed the SlMADS5 expression, as well as theexpression of tobacco genes associated with reproductivedevelopment: NtLFY, NtAP1 (plant transition to flowering), NtWUS , NtAG,NtPLE, NtSEP1, NtSEP2, NtSEP3 (key genes for the identityof the flower meristem and flower organs). For the analysis,we used tissues of leaves and apical meristems  of transgenic and control plants.Expression of the SlMADS5 transgene was present onlyin the tissues of S5-16-7 and S5-17-1 plants. The expressionpattern of the remaining analyzed genes was similar: theirmRNA was absent or was minimal in the leaves of the controland transgenic lines, as well as in the S5-16-7 and S5-17-1apexes of undefined status. At the same time, these genes werehighly transcribed in the reproductive meristems of controlplants .In this study, a functional analysis of the SlMADS5 gene, theSEP3 homolog in tomato, was carried out. Structural analysis confirmed that SlMADS5 belongs to the SEP3clade, which may indicate the conservatism of its role in the reproductive development of tomato, namely, its participationin determining the identity of petals, stamens, carpels,and ovules.It is known that SlMADS5 is not expressed in tomato leavesand roots and is expressed in flowers and fruits . Also, SlMADS5 mRNA is present in the meristemdomains that correspond to the future three inner whorls ofthe tomato flower, as well as during organogenesis and inthe corresponding mature organs .A detailed in silico analysis of the SlMADS5 expression patterncarried out in this study revealed that SlMADS5 mRNAis absent not only in roots and leaves, but also in the shootapical meristems and flower meristems at early stages of development. Gene transcription is activated late inthe development of the flower meristem, and reaches a peakin an open flower and in the peel of an immature fruit . This corresponds not only to the well-known role ofSEP3 homologs in determining the differentiation of flowermeristem cells corresponding to the three inner whorls oforgans , but also suggests the active participation of SlMADS5 in the aspects of development andripening of tomato fruits and seeds.To characterize the SlMADS5 function, transgenic tobaccoplants with constitutive overexpression of SlMADS5 cDNAwere obtained. The phenotype of transgene overexpressiondoes not determine its function; however, it may indicate asimilarity with the already characterized homologs. Earlier,the effect of heterologous overexpression of SEP3 homologsof different plant species was studied mainly using transgenicA. thaliana plants, but there are works with the useof Nicotiana spp. plants. Tobacco, like tomato, belongs tothe Solanaceae family and has the same flower structure;therefore, in this study, a heterologous expression system intobacco was selected.Various effects of overexpression of SEP3 homologs havebeen described. Thus, SEP3 constitutive expression in A. thalianasignificantly accelerates flowering .In these plants, the APETALA3 (B-class) and AG (C-class)genes are transcribed ectopically .Overexpression of the P. × hybrida SEP3-like gene FBP2leads to early flowering of the A. thaliana plants . Early flowering is caused by overexpression oftobacco SEP3-like gene NsMADS3 in N. sylvestris Speg. &Comes  and chrysanthemum SEP3-like geneCDM44 in N. tabacum .At the same time, no influence of overexpression of SEP3-homologous genes on the flowering time was also observed.Thus, homologous overexpression of FBP2 in P.×hybrida has no effect on plant vegetation period .Heterologous overexpression of Platanus acerifolia SEP3-likegenes in A. thaliana causes early flowering only in the caseof the PlacSEP3.2 gene, while overexpression of the secondgene, PlacSEP3.1, causes early flowering only in transgenictobacco plants .In the case of SlMADS5 constitutive overexpression, asignificant delay in flowering was observed, most likely associatedwith the incorrect development of the shoot apicalmeristem . Different effects of heterologous ectopicexpression of SEP3 homologs in transgenic plants maybe associated with structural differences in encoded proteinsequences responsible for binding to promoters of target genesor to partner proteins.Normally, traces of the A. thaliana SEP3 transcripts arefound in the inflorescence meristem, and gene expression isnoticeably activated only in the flower meristem parts, fromwhich petals, stamens, and carpels are subsequently formed. Therefore, thepresence of the TF SlMADS5 in tissues, where there shouldbe no tobacco SEP3 homologs, can lead to nonspecific proteinproteinand DNA-binding interactions of SlMADS5, whichcan disrupt the pattern of meristem development.To clarify the status of transgenic meristems S5-16-7 andS5-17-1, visually ready for flowering, we analyzed the expressionof genes whose activity is associated with the identity ofthe reproductive inflorescence and flower meristems (NtLFYand NtAP1) . Considering the results obtained , only the inflorescence meristem of thecontrol plant has reproductive status. The presence of a lowlevel of LFY expression in the vegetative apex of the controland in the S5-16-7 apex  suggests the initial stagesof the meristem transition to the reproductive state, since it hasbeen shown that in A. thaliana LFY begins to be expressed inthe flower meristem primordia at the periphery of the inflorescencemeristem .It is known that SEP3 is the central hub of the MADScomplexesin A. thaliana . TF SlMADS5also shows an exceptional ability to assemble tetrameric complexesof MADS TFs . The interactionof SlMADS5 with FUL2 and TAGL1 shown in this work (seeTable 2), as well as the role of FUL2 and TAGL1 in pistilinitiation and early fruit development , indicate the possible involvement ofSlMADS5 in determining the identity of the tomato pistil incomplex with FUL2 and TAGL1.One of the complexes, SEP3/SEP3/AG/AG, is requiredfor flower determination and completion of its development. This is due to a decrease in thenumber of stem cells because of the WUS gene suppressionwith the key participation of TF AG .Accordingly, in transgenic petunia plants with simultaneousoverexpression of SEP3-like FBP2 and D-class gene FBP11,where developmental arrest is observed at the cotyledonstage, transcription of AG-like FBP6 is activated and mRNAof WUS-like TERMINATOR is absent .This suggests the joint participation of SEP3, AG, and D-classgenes in the suppression of stem cells in the meristem.Taking into account the activation of AG expression inA. thaliana with SEP3 overexpression ,as well as the participation of SEP3 and AG in the suppressionof WUS transcription  and the interaction of TF SlMADS5 with theAG homolog TAGL1 (see Table 2), it can be assumed thatthe ectopically synthesized TF SlMADS5 is able to activatetranscription of the tobacco AG-like genes NtAG and NtPLEin transgenic shoot meristem. Subsequent formation of complexesSlMADS5/SlMADS5/NtAG/NtAG or SlMADS5/SlMADS5/NtPLE/NtPLE can lead to inhibition of meristemdevelopment due to the tobacco WUS-like gene NtWUSsuppression, since WUS plays a key role in determining thestem cell identity, the population of which is not supportedin plants with loss of WUS function .To test this possibility, we analyzed the expression ofSlMADS5, NtWUS, AG-like genes NtAG and NtPLE, as wellas SEP-like genes NtSEP1, NtSEP2, and NtSEP3. However,the presence of SlMADS5 ectopic expression did not leadto the activation of AG-like genes, and the expression ofNtWUS was significantly higher in the tissues of transgeniclines in comparison with the control (excluding the controlinflorescence meristem) . The latter can be a probablereason for the formation of significantly thickened, incomparison with the control, stem and leaves of transgenicplants of all 11 lines with the 35S::SlMADS5 phenotype  as a result of the increased number of stem cells andthe meristem overgrowth.It should also be noted that in transgenic plants, the anthocyanincolor of the flower corolla changed from pale pink(control) to magenta (35S::SlMADS5) . Previously,it was shown that the expression of the SEP-like geneMrMADS01 in Myrica rubra berries significantly increasesat the last stage of ripening, which allowed the authors tosuggest the involvement of this gene in the biosynthesis ofanthocyanins . Silencing the SEP-like genePaMADS7 in sweet cherry (Prunus avium) leads to a changein the content of anthocyanins in fruits . It canbe assumed that SlMADS5 is also involved in the regulationof anthocyanin biosynthesis in transgenic tobacco petals.Silencing of SlMADS5 gene leads to a change in the numberof flower whorls and the number of organs in whorls, as well asthe formation of green petals with signs of sepals, and sterileanthers and carpels with signs of sepals and petals, respectively, which may indicate the participation ofthe gene in determining the identity of tomato flower organs.Nevertheless, no complete homeotic transformation of certainflower organs was observed when SlMADS5 was suppressed.The data on the effect of SlMADS5 overexpression on thedevelopment of transgenic tobacco plants obtained in thisstudy also do not confirm the involvement of the gene indetermining the floral organ identity. 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The diagnostic performance of 18F-DCFPyL-PET/CT in localizing primary PCa within the prostate gland was assessed, allowing for PSMA-guided targeted-prostate biopsy.In primary prostate cancer (PCa) patients, accurate staging and histologic grading are crucial to guide treatment decisions. 18F-DCFPyL-PET/CT prior to robot-assisted radical prostatectomy (RARP). Two experienced and blinded nuclear medicine physicians assessed tumour localisation within the prostate gland on PET/CT, using a 12-segment mapping model of the prostate. The same model was used by a uro-pathologist for the RARP specimens. Based on PET/CT imaging, a potential biopsy recommendation was given per patient, based on the size and PET-intensity of the suspected PCa localisations. The biopsy recommendation was correlated to final histopathology in the RARP specimen. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for clinically significant PCa  were assessed.Thirty patients with intermediate-/high-risk primary PCa were prospectively enrolled between May 2018 and May 2019 and underwent 18F-DCFPyL-PET/CT were 61.4%, 88.3%, 68.1% and 84.8%, respectively.The segments recommended for potential targeted biopsy harboured csPCA in 28/30 patients (93%), and covered the highest Gleason score PCa segment in 26/30 patient (87%). Overall, 122 of 420 segments (29.0%) contained csPCa at final histopathological examination. Sensitivity, specificity, PPV and NPV for csPCa per segment using 18F-DCFPyL-PET/CT with the RARP specimens, an accurate per-patient detection (93%) and localisation of csPCa was found. Thus, 18F-DCFPyL-PET/CT potentially allows for accurate PSMA-targeted biopsy.When comparing the PCa-localisation on The online version of this article (10.1007/s00345-020-03490-8) contains supplementary material, which is available to authorized users. Prostate cancer (PCa) is the most common cancer in men in the Western world , 2. HistBesides conventional imaging modalities such as mpMRI, novel imaging techniques including prostate-specific membrane antigen-positron emission tomography/computed tomography (PSMA-PET/CT) have been introduced. PSMA is significantly overexpressed in malignant prostate cells, correlates with higher tumour grades and represents a marker of tumour aggressiveness , 10. PSM18F-DCFPyL (PSMA) PET/CT imaging for the primary detection of PCa. The primary aim was to assess the accuracy of 18F-DCFPyL-PET/CT to localise primary PCa within the prostate gland, by comparing imaging results from 18F-DCFPyL-PET/CT to final histopathology of the robot-assisted radical prostatectomy (RARP) specimen. The secondary objectives were to investigate the ability of 18F-DCFPyL-PET/CT to provide a recommendation for potential targeted biopsy and to assess the diagnostic accuracy of determining local tumour stage (pT).This is the first prospective study on the accuracy of This was a prospective, non-randomised study in patients with diagnosed primary PCa. Pre-operative imaging results were compared to histopathology following RARP. All subjects signed informed-consent for the collection of their clinical data. The study has been approved by the ethical review board of the Amsterdam University Medical Centre (AUMC) (review number 2017.543). Patients were enrolled consecutively between May 2018-May 2019 in Amsterdam UMC, location VUmc.Patients had histologically proven, intermediate or high-risk, PCa, for which they underwent RARP , 15. Of 18F-DCFPyL which was synthesised under Good Manufacturing Practices conditions, as described by Jansen et al. [18F-DCFPyL (IQR 299–324 MBq), and a median of 5.4 weeks (IQR 3.0–7.2) prior to surgery. Image-acquisitions were performed using a Philips Ingenuity TF -PET/CT system. No diuretics were administered prior to the scan. The scan trajectory included mid-thighs to skull-base, with 4 min per bed position. All PET scans were combined with a diagnostic CT scan , without contrast-enhancement. Images were corrected for decay, scatter, random coincidences, and photon attenuation.Patients were staged with n et al. , 18. PETImages were reconstructed with a BLOB-based Ordered-Subsets Expectations Maximization algorithm  ). The de18F-DCFPyL-PET/CT reading (> 300 scans), in consensus. The readers used the 12-segment mapping model to demarcate the image-detected tumour extent (Appendix-1 in ESM) [max) of the suspected lesions. Finally, the readers indicated if radiological extra-capsular extension  or invasion into the seminal vesicles (rT3b) was suspected.Scan interpretation was performed blinded for the pathology results and other imaging by two nuclear medicine physicians  with ample experience in  in ESM) . For all in ESM)   guidelines . All spe18F-DCFPyL-PET/CT was matched to the histopathology results and the sensitivity, specificity, positive predicting value (PPV), and negative predicting value (NPV) were calculated on a segment basis. Correlation of 18F-DCFPyL-PET/CT with histopathology was considered if exactly the same segment was demarcated . Since there are no anatomical landmarks to delineate the segments, artificial segmentation can occur, causing a mismatch between the PET/CT- and histopathological findings while both correspond with the same lesion. Therefore, a second analysis of diagnostic accuracy was performed, in which PET correlation was also considered if there was a discrepancy of up to 1 region in the coronal or sagittal plane  [18F-DCFPyL-PET/CT to differentiate locally advanced disease (> rT3a) from prostate-confined disease (rT2). Numerical variables were summarised with median values and interquartile ranges (IQR); categorical variables with proportions (%). To compare medians of non-parametric data, the Mann–Whitney-Wilcoxon test and the Kruskal-Wallis test were used (significance set at p < 0.05). Statistical analysis was performed with IBM® SPSS® Statistics for Windows®, version 26.The localisation of the detected prostate tumour by reement) , 24. RecA total of 30 patients was included in this study, having a median initial PSA-level of 11.1 ng/ml (IQR 5.8–22.4). According to EAU guidelines, 10/30 (33.3%) patients had intermediate-risk PCa and 20/30 (66.6%) had high-risk PCa . Pre-ope18F-DCFPyL-PET/CT to detect csPCa per segment with total agreement was 61.4% (95%CI 52.2–70.0%), 88.3% (95%CI 83.9–91.6%), 68.1% (95%CI 58.5–76.6%), and 84.8% (95%CI 80.2–88.5%), respectively (Appendix-2 in ESM). For near-total agreement, the sensitivity, specificity, PPV and NPV of 18F-DCFPyL PET/CT to detect csPCa per segment was 84.4% (95%CI 76.5–90.1%), 97.0% (95%CI 94.1–98.5%), 92.0% (95%CI 84.9–96.0%), and 93.8% (95%CI 90.3–96.1%), respectively. The area under the curve (AUC) of 18F-DCFPyL-PET/CT was 0.78 (95%CI 0.73–0.84) for the total agreement scores, and 0.85 (95%CI 0.80–0.90) for the near-total agreement scores (Appendix-3 in ESM). True positive segments had a median SUVmax of 8.26 (IQR 5.25–11.40), which was significantly higher than the median SUVmax of false-positive segments of 4.06 (IQR 3.56–5.10) (p = 0.02). The median SUVmax of true positive segments did not correlate with ISUP grade groups (p = 0.95) (Appendix-4 in ESM).All patients showed PSMA expression in the prostate. In 30 evaluated patients, 420 segments  could be used both for PET/CT and histopathological mapping evaluation. PCa was present in 129 of the 420 (30.7%) segments on histopathological examination, and csPCa was found in 122 of the 420 segments (29.0%) . The sensitivity, specificity, PPV and NPV of 18F-DCFPyL-PET/CT revealed csPCa in 28/30 (93.3%) patients. Moreover, it pinned the index PCa lesion in 26/30 (86.7%) patients. An example of potential 18F-DCFPyL-guided biopsy recommendation and concurrent histopathological examination of the RARP specimen is shown in Fig. max 8.62 (IQR 6.41–12.62). The recommendation for potential biopsy from the nuclear physicians that matched csPCA had a median SUVmax of 8.55 (IQR 6.34–13.79), and was significantly higher than the recommended potential biopsy segments that did not contain csPCa (median SUVmax of 3.10 (IQR 2.86–3.87) (p = 0.02).The primary potential biopsy recommendation by the nuclear medicine physician harboured csPCa in 24/30 (80.0%) patients and detected the index PCa lesion in 23/30 (76.6%) patients. When both the primary and secondary recommended segments would potentially be targeted, 18F-DCFPyL-PET/CT to detect locally advanced tumour growth (≥ pT3a) was 35.7% (95%CI 14.0–64.3%), 93.8% (95%CI 67.7–99.7%), 83.3% (95%CI 36.5–99.1%), and 62.5% (95%CI 40.8–80.4%), respectively (Appendix-5 in ESM). For the detection of pT3a sub-stage, the sensitivity, specificity, PPV and NPV of 18F-DCFPyL-PET/CT were 20.0% (95%CI 3.5–55.8), 100% (95%CI 80.0–100), 100.0% (95%CI 19.7–100), and 71.4% (95%CI 51.1–86.0), respectively. For the detection of pT3b sub-stage, the sensitivity, specificity, PPV and NPV of 18F-DCFPyL-PET/CT were 75.0% (95%CI 21.9–98.7), 92.3% (95%CI 73.4–98.7), 60.0% (95%CI 17.0–92.7), and 96.0% (95%CI 77.7–99.8), respectively.Final histopathological analysis revealed pT3a in 10/30 (33.3%) patients, and pT3b in 4/30 (13.3%) patients. The sensitivity, specificity, PPV and NPV of 18F-DCFPyL-PET/CT imaging was used to locate primary PCa within the prostate gland, exploring the diagnostic potential of PSMA-based targeted biopsies. A total of 30 patients diagnosed with intermediate and high-risk PCa that underwent 18F-DCFPyL-PET/CT prior to RARP was analysed. When using a prostate-mapping model, the potential 18F-DCFPyL-PET/CT-based targeted biopsy recommendation detected csPCa in 28/30 (93.3%) patients. Moreover, it detected the index PCa lesion in 26/30 (86.7%) patients. Potentially, this makes PSMA-targeted biopsy a diagnostic tool that may adequately guide precision prostate biopsy. In biopsy-naive patients at increased risk of metastatic spread, and in whom staging imaging is mandatory (e.g. PSA ≥ 20), 18F-DCFPyL-PET/CT could potentially be used simultaneously to stage patients and to target PSMA-avid prostatic lesions suspicious for PCa.This is the first prospective study in which 18F-DCFPyL-PET/CT imaging demonstrated a moderate per segment-based sensitivity for the detection of csPCa of 61.4%, at a 88.3% specificity. The moderate sensitivity indicates that 18F-DCFPyL-PET/CT was not able to detect all localised csPCa. Segmentation of the prostate gland is problematic, as no clear anatomical landmarks are available to delineate the different segments within the prostate (Appendix-1 in ESM). A tumour located on the border of the apex and middle part of the prostate could be classified in different segments by the nuclear medicine physician and uro-pathologist, while in fact, they detected the same lesion. Therefore, the near-agreement score was introduced to approximate clinical reality. The sensitivity of the near-agreement score of 18F-DCFPyL-PET/CT imaging for the detection of csPCa was higher at 84.4% with a specificity of 97.0%.68Ga-PSMA-PET/CT and mpMRI prior to RARP [MpMRI has found a prominent place in the identification and localisation of PCa . Few stu to RARP . This st18F- PSMA-1007-PET/CT and mpMRI with subsequent RARP. Nine of the men were diagnosed with MRI-TBx and 1 with systematic biopsy. Using a 36-segment mapping model, a similar assessment of agreement and near-total agreement was used. In 10 patients, 18F-PSMA-1007 PET/CT showed a high sensitivity , specificity , and accuracy  for the detection of csPCa. Although the specificity was similar, this study did however show lower sensitivity for PSMA-PET/CT compared to mpMRI . Above mentioned studies implicate that PSMA-PET/CT imaging performs at least equal to mpMRI to locate primary PCa. The rates of mpMRI might have been overestimated as at least a part of included patients in previously mentioned studies were diagnosed by MRI-TBx. Thus, selection bias may have been introduced.In another study, Kesch et al.  studied 18F-DCFPyL-PET/CT. So, similar to mpMRI, a substantial number of patients with ≥ pT3a was understaged by PSMA-based imaging [18F-DCFPyL-PET/CT of 93.8% is congruent with similar 68 Ga-PSMA studies (specificity > 90% for T3b disease) [There is therapeutic importance to distinguish between T2 and T3 disease  [disease) , 25, 26.18F-DCFPyL-PET/CT performs in a truly biopsy naïve cohort of patients. The present study was set up to evaluate the capability of PSMA-PET/CT to guide targeted prostate biopsy for the detection of csPCa, not with the goal to discriminate between those who should be or should not be biopsied. Unfortunately, not all patients received a pre-operative mpMRI, limiting direct comparison to 18F-DCFPyL-PET/CT. Moreover, some of the patients who received a mpMRI had a longer interval between the mpMRI and the PET/CT scans due to the mpMRI being performed at the referring centre. Finally, since the RARP-specimen will always change shape (due to organ slicing and shrinking artefacts) when it is removed from the body, no truly exact anatomical correlation is possible. Therefore, the partial agreement score was used to correct for this pitfall.Our study has inherent limitations. Since the PET/CT resolution is confined at 5 mm, limited diagnostic accuracy for small PCa-foci is to be expected. A selection bias has been introduced due to the selection of patients with biopsy-confirmed csPCa. It is thus unclear how 18F-DCFPyL-PET/CT with the RARP specimen using anatomical mapping, an accurate per-patient localization (93%) of csPCa was found within the prostate. 18F-DCFPyL-PET/CT proves promising for PSMA-targeted biopsy and provides a moderate local staging ability.When comparing the localisation of PCa on Supplementary file1 (JPG 61 kb)Supplementary file2 (JPG 138 kb)Supplementary file3 (JPG 79 kb)Supplementary file4 (JPG 70 kb)Supplementary file5 (JPG 177 kb)Below is the link to the electronic supplementary material.


Aegilops sharonensis, a wild relative of wheat, harbors diverse disease and insect resistance genes, making it a potentially excellent gene source for wheat improvement. In this study, we characterized and evaluated six wheat-A. sharonensis derivatives, which included three disomic additions, one disomic substitution + monotelosomic addition and two disomic substitution + disomic additions. A total of 51 PLUG markers were developed and used to allocate the A. sharonensis chromosomes in each of the six derivatives to Triticeae homoeologous groups. A set of cytogenetic markers specific for A. sharonensis chromosomes was established based on FISH using oligonucleotides as probes. Molecular cytogenetic marker analysis confirmed that these lines were a CS-A. sharonensis 2Ssh disomic addition, a 4Ssh disomic addition, a 4Ssh (4D) substitution + 5SshL monotelosomic addition, a 6Ssh disomic addition, a 4Ssh (4D) substitution + 6Ssh disomic addition and a 4Ssh (4D) substitution + 7Ssh disomic addition line, respectively. Disease resistance investigations showed that chromosome 7Ssh of A. sharonensis might harbor a new powdery mildew resistance gene, and therefore it has potential for use as resistance source for wheat breeding. Aegilops sharonensis Eig , a wild relative of wheat, is endemic to the coastal plains of Israel and southern Lebanon  substitution line (A. sharonensis 4Ssh (4D) substitution line by using a nullisomic backcrossing procedure. A. sharonensis amphiploid (genome AABBS1S1). A. sharonensis. A. sharonensis introgression lines. Recently, both A. sharonensis amphidiploids. However, there are very few reports on the isolation of wheat plants carrying individual A. sharonensis chromosomes. A. sharonensis, and produced three 1Ssh (1A) substitution lines, two 1Ssh (1B) substitution lines, three 1Ssh (1D) substitution lines and two 1Ssh (5D) substitution lines. Therefore, the set of wheat-A. sharonensis chromosome lines is still not complete, which greatly limits the mapping and utilization of excellent genes derived from this species in wheat.ion line . Later, A. sharonensis chromosome derivatives, including three disomic addition lines, one disomic substitution + monotelosomic addition line, and two disomic substitution + disomic addition lines, were identified by (Polymerase Chain Reaction) PCR-based landmark unique gene (PLUG) markers and fluorescence in situ hybridization (FISH) analysis. In addition, the infection types (ITs) of disease resistance, spike and grain characteristics of these wheat-A. sharonensis chromosome lines were also investigated to provide useful information for the possible subsequent development of wheat-A. sharonensis translocations for wheat genetic improvement.In this study, six wheat-Triticum aestivum cv. Jinan17 (JN17) and Jimai22 (JM22) were maintained at the Crop Research Institute, Shandong Academy of Agricultural Sciences in Jinan. T. aestivum cv. Chinese Spring (CS) was provided by Prof. Z. J. Yang, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu. The diploid A. sharonensis accession (TA1995) was provided by Mr. J. Raupp, Wheat Genetic and Genomic Resources Center, Kansas State University, United States. The CS-A. sharonensis amphiploid (JIC-31) and six unidentified CS-A. sharonensis chromosome lines  were kindly provided by Prof. S. M. Reader, John Innes Centre, United Kingdom.8 were synthesized by Chengdu Ruixin Biological Technology Co., Ltd. Probe sequences, the fluorochromes for probe labeling, FISH protocols and labeled DNA signal detection methods were according to 8 as a probe could be used to identify wheat chromosomes except 1A, 3D, 4D, 5D, and 6D, as described by EcoRI fragment isolated from bread wheat d JIC-31 . The karA. sharonensis chromosomes in lines JIC-32 to JIC-37 showed no signals associated with that probe. However, slightly different Oligo-pSc119.2-1 signals were found on both terminal regions of all A. sharonensis chromosomes , indicating that this pair of A. sharonensis chromosomes might be 1Ssh or 6Ssh.FISH using Oligo-pTa535-1 onto omosomes . In addie JIC-32 . MoreoveA. sharonensis chromosomes in JIC-32 to JIC-37, a total of 526 PLUG primer pairs were used to develop A. sharonensis chromosome-specific markers. As a result, fifty-one primer pairs could generate polymorphisms in A. sharonensis, the CS-A. sharonensis amphiploid, CS, JM22, and JN17. Among them, four, eight, nine, six, two, five, and seventeen belonged to chromosome homoeologous groups 1–7, respectively  substitution + 5SshL monotelosomic addition (A. sharonensis 6Ssh disomic addition (A. sharonensis 4Ssh (4D) substitution + 6Ssh disomic addition (sh (4D) substitution + 7Ssh disomic addition line  substitution + 5SshL monotelosomic addition, 6Ssh disomic addition, 4Ssh (4D) substitution + 6Ssh disomic addition and 4Ssh (4D) substitution + 7Ssh disomic addition lines were more elongated than that of CS. The CS-A. sharonensis 4Ssh disomic addition line (JIC-33) showed slightly elongated spikelets and overall longer spikes than that of CS. The CS-A. sharonensis 4Ssh (4D) substitution + 6Ssh disomic addition (JIC-36) showed shorter spikes and fewer spikelets per head than that of CS  showed slender grains and darker pericarp color than that of CS (sh disomic addition (JIC-33) showed smaller grains than those of CS.Grain morphologies of the six CS-at of CS  and the A. sharonensis 4Ssh (4D) substitution + 7Ssh disomic addition (JIC-37) was not obtained. The CS-A. sharonensis 4Ssh (4D) substitution + 7Ssh disomic addition (JIC-37) was nearly immune to powdery mildew, while CS and other CS-A. sharonensis chromosome lines tested were highly susceptible to powdery mildew (sh of A. sharonensis might carry powdery mildew resistant gene(s).Stripe rust, leaf rust, stem rust, and powdery mildew resistance tests showed that all the materials were moderately to highly susceptible to stripe rust, leaf rust, and stem rust  except ty mildew , suggestA. sharonensis chromosomes into wheat is difficult due to the presence of gametocidal (Gc) genes that control the preferential transmission of chromosome 4Ssh (A. sharonensis additions or substitutions (T. urartu-A. sharonensis amphiploid TA3398 in North Dakota in 1972 (unpublished). A. sharonensis chromosome 4S1 (some scientists defined the genome of A. sharonensis as S1S1). A. sharonensis Gc2 gametocidal gene (Gc2mut), which opened a way for introgression of genes from A. sharonensis into wheat. 1, 3S1, 5S1, 6S1, and 7S1 in a 4S1 (4D) background. A. sharonensis introgression lines which they then separated into six groups based on different substituted chromosomes belonging to definite homoeologous groups and different numbers of translocations. A. sharonensis amphiploid (genome AABBS1S1). A. sharonensis, and produced three 1Ssh (1A) substitution lines, two 1Ssh (1B) substitution lines, three 1Ssh (1D) substitution lines and two 1Ssh (5D) substitution lines.Transferring each pair of ome 4Ssh . Therefoitutions . Maan foA. sharonensis introgression lines are very rare. Furthermore, none to date has reported the production of wheat-A. sharonensis 2Ssh introgression lines. In this research, six wheat-A. sharonensis introgression lines were identified, including a CS-A. sharonensis 2Ssh disomic addition (JIC-32), a 4Ssh disomic addition (JIC-33), a 4Ssh (4D) substitution + 5SshL monotelosomic addition (JIC-34), a 6Ssh disomic addition (JIC-35), a 4Ssh (4D) substitution + 6Ssh disomic addition (JIC-36) and a 4Ssh (4D) substitution + 7Ssh disomic addition (JIC-37). Among these six introgression lines, four possessed chromosome 4Ssh, suggesting that chromosome 4Ssh of A. sharonensis was transmitted preferentially into wheat due to the gametocidal gene, which confirms the reports of preferential transmission of gametocidal chromosomes of earlier researchers  substitution + 5SshL monotelosomic addition (JIC-34) and the CS-A. sharonensis 4Ssh disomic addition line (JIC-33) were highly susceptible to powdery mildew, indicating that there were no powdery mildew resistance genes on chromosomes 4Ssh and 5SshL of A. sharonensis. However, the CS-A. sharonensis 4Ssh (4D) substitution + 7Ssh disomic addition (JIC-37) was nearly immune to powdery mildew (sh of A. sharonensis might carry new powdery mildew resistant gene(s).To date, more than 70 powdery mildew resistance genes have been permanently designated . Among ty mildew , suggestThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/CL conceived and designed the experiments. XW, ZHY, HW, JBL, and RH performed the experiments. WX, GL, and JG performed disease resistance testing. YZ, FL, DC, and AL analyzed the data. XW wrote the manuscript. HL, ZJY, JJL, and CL revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We describe a case of metastatic pulmonary calcification in a 71-year-old male, images with  He was on diuretic therapy for chronic kidney disease for 4 years, followed by oral adsorptive carbon therapy for 13 months. He had been on hormonal therapy for prostate cancer (cT2cN0M0) for 9 months. He was hospitalized elsewhere for poor oral intake and general weakness. Laboratory studies at that time revealed the following : calcium 15.5 mg/dL (8.2–10.2 mg/dL); phosphorus 7.8 mg/dL (2.5–4.5 mg/dL); creatinine 3.67 mg/dL (0.7–1.2 mg/dL); total 25-hydroxyvitamin D level 12.06 ng/mL (approx. 30 ng/mL); and parathyroid hormone 15.71 pg/mL (15–65 pg/mL). 18F-fluorodeoxyglucose (FDG) PET/CT was performed to exclude the possibility of malignancy-related hypercalcemia. 18F-FDG) PET/CT images were acquired 1 h after intravenous injection of 238 MBq of 18F-FDG. The PET/CT images showed an increase in FDG uptake in the bilateral lower lungs  bone scan. It also revealed significantly increased diffuse uptake in the bilateral lower lung fields (Further examination with a bone scan was therefore recommended. Since the bone scan is a sensitive test for diagnosing metastatic pulmonary calcification, we performed a bone scan to identify metastatic pulmonary calcification and determine whether bone metastasis existed . The patg fields . The sca18F-FDG PET/CT to detect metastatic pulmonary calcification [18F-FDG PET/CT findings in patients with metastatic pulmonary calcification. In conclusion, in patients with chronic kidney disease, when hypercalcemia is present and PET/CT shows ground-glass opacity with mild FDG uptake, metastatic pulmonary calcification can be considered one of the differential diagnoses, though this is rare.Metastatic pulmonary calcification is a frequently underdiagnosed disease. Because usual imaging modalities such as chest radiographs and CT scan findings are not specific ,9. Only fication ,11. Howe


Biliary tract cancers (BTCs) are rare tumours with regional differences. Prognostic factors are poorly understood. Gemcitabine + platinum (GP) is the standard first-line chemotherapy in metastatic patients. We aimed to search for prognostic factors in patients with advanced disease in a cancer centre in South America.We conducted a retrospective analysis of patients with advanced BTC treated with chemotherapy. Variables were age (< or ≥70 years), Eastern Cooperative Oncology Group (ECOG) performance status (0/1 versus 2/3), gender, primary site (intrahepatic (IHC), extrahepatic (EHC), gallbladder (GB)), staging , metastatic sites, albumin (>3.5 g/dL versus <3.5 g/dL), biliary obstruction and first-line chemotherapy . Cox regression method was used to explore factors.p = 0.007), albumin < 3.5 g/dL (p = 0.001), biliary obstruction (p = 0.006), 5FU-based (p = 0.006) and single-agent (p < 0.0001) were associated with worse OS. ECOG performance status 2/3 (p = 0.058) and bone metastases (p = 0.051) were marginally related. In multivariate analysis, male (p = 0.003), bone metastases (p = 0.023), biliary obstruction (p = 0.001), 5FU-based (p = 0.016) and single-agent (p = 0.023) were independently associated with inferior OS.From 2010 to 2017, 104 patients were included. Median age was 62 years (32–86) and 22.1% were older than 70 years. Most patients had ECOG performance status 0/1 (63.4%), were female (51.9%) and were metastatic (82.7%). Bone metastases were found in 19.2%. Primary IHC, EHC and GB were 54.8%, 36.5% and 8.7%, respectively. GP was used by 79.8%. Median follow-up was 32.4 months. Median overall survival (mOS) was 11.4 months. In univariate analysis, male (In this retrospective study, we observed that male patients, bone metastases, biliary obstruction and regimens other than GP had worse survival. Larger studies should be conducted to confirm our findings. Biliary tract cancers (BTCs) are a rare group of malignances . Main suWe conducted a retrospective analysis of patients treated at a cancer centre in Brazil, from 2010 to 2017. Patients were included if they were 18 years of age or older, had histologic diagnosis of metastatic, recurrent or locally advanced unresectable BTC . All patients were deemed eligible to receive first-line chemotherapy by their treating physician. Exclusion criteria were absence of histologic confirmation, absence of data regarding systemic treatment and mixed histologies (hepatocellular-cholangiocarcinoma). Medical files were used as source of information. This study was approved by the local Ethics Committee (2680/19).Descriptive statistics were used for main demographic characteristics. Survival curves were estimated using Kaplan–Meier method and compared with log-rank test. OS was defined as date of first chemotherapy cycle and death from any cause. Progression-free survival (PFS) was defined as date of first chemotherapy cycle and disease progression or death from any cause. Radiologic assessments of response and clinical benefit were performed according to local guidelines, typically by means of clinical examination every 2 weeks and computed tomography or magnetic resonance imaging every 8 weeks. Progressive disease was identified by the treating physician.2X tests were used to analyse categorical variables distributions between genders. To evaluate prognostic factors, univariate and multivariate analysis were performed using the Cox regression method. Age (<70 years versus >70 years), gender, staging , primary site (EHC versus IHC versus GB carcinoma), Eastern Cooperative Oncology Group (ECOG) performance status (0–1 versus 2–3), bone metastasis, baseline Carbohydrate Antigen 19-9 (CA19-9), albumin (<3.5 g/dL versus > 3.5 g/dL), biliary obstruction and first-line treatment (gemcitabine + platinum (GP) versus 5-Fluorouracil (5-FU) based versus monotherapy) were used in the univariate model. All tests were considered statistically significant with a two-sided p value of < 0.05. Statistical analysis was performed with IBM SPSS 20.Between July/2009 and March/2017, 104 patients were identified. Main demographics are shown in Regarding first-line chemotherapy , GP, 5-Fp = 0.32). Access to second-line was not statistically different among gender .Numerically, more female patients used GP as first-line . At time of analysis, seventy-six (73.1%) patients had died. Median OS (mOS) was 11.4 months (95% CI: 9.0–13.7) . Median p = 0.007), albumin < 3.5 g/dL (HR: 4.73 (95% CI: 1.96–11.4); p = 0.001), biliary obstruction (HR: 2.15 (95% CI: 1.24–3.71); p = 0.006), 5FU-based (HR: 2.64 (95% CI: 1.31–5.30); p = 0.006) and single-agent (HR: 6.27 (95% CI: 2.36–16.6); p < 0.0001) were associated with worse OS. Bone metastases (HR: 1.82 (95% CI: 0.99–3.34); p = 0.051) and ECOG performance status 2–3 (HR: 1.58 (95% CI: 0.98–2.53); p = 0.058) were marginally associated with prognosis.In univariate analysis, male patients (hazard ratio (HR): 1.88 (95% CI: 1.19–2.98); p = 0.003), bone metastases (HR: 3.53 (95% CI: 1.18–10.5); p = 0.023), biliary obstruction , 5FU-based (HR: 6.24 (95% CI: 1.41–27.6); p = 0.016) and single agent chemotherapy (HR: 7.22 (95% CI: 1.30–39.8); p = 0.023) were significantly and independently associated with inferior OS ; erior OS . mOS waserior OS .BTC is a rare gastrointestinal neoplasm but with increasing incidence over the past years, especially for IHC . SurgeryOur data bring relevant information about two aspects of metastatic BTC. The first is regarding the standard first-line treatment. Patients treated with GP regimens presented a more favourable outcome. GP was superior to Gemcitabine in the Phase III ABC-02 study and was established as the standard of treatment first-line therapy for metastatic biliary tract carcinomas . On the et al [p = 0.0031) and OS (13.0 versus 7.5 months (HR: 0.31 (95% CI: 0.15–0.63); p = 0.0013). However, in our study, 7.6% of patients were treated with FOLFOX in first-line and despite the small sample, outcomes were worse than those of patients treated with Gemcitabine combinations, reinforcing the role of GP. Clinical reasons not to employ GP were not accurately assessed in our analysis.Recently, the ABC-06 trial established FOLFOX as a new standard for second-line treatment of BTC . Based oet al  publisheSecond, female gender was significatively associated with better survival. We found that survival was almost doubled among female patients. In analogous tumours like metastatic pancreatic cancer, gender is a controversial prognostic factor , 14 . Hoet al [p = 0.037). Baton et al [p = 0.0002). Another retrospective large series from Korea with 740 BTC patients [p = 0.04).Clinical data endorses our findings. Bridgewater et al  describeon et al  publishepatients  also shop = 0.03) [Some aspects of BTC regarding access to therapeutics may help clarify this difference. Undergoing second and further lines of treatment is probably a strong prognostic factor. In a large study of second-line therapy, out of 378 patients that received first-line chemotherapy, only 96 patients (25%) received second-line. Female/male ratio was 31% and 21%, respectively ( = 0.03) . In our Currently, it is clear that there are striking differences in gastrointestinal cancer incidences among male and female patients. These differences are in part explained by exposition to risk factors such as smoking . NeverthDrug effects are also variable between genders. 5-FU based chemotherapy tends to be more toxic in women. A polled analysis of more than 28,000 patients, demonstrated that neutropenia and gastrointestinal toxicity were more likely to affect female patients . BenefitFinally, it is noteworthy that we have a growing body of evidence regarding molecular biology of BTC. Fibroblast Growth Factor Receptor (FGFR) fusions and Isocitrate Dehydrogenase (IDH) mutations are among targetable alterations in this scenario. Clinical trials have shown benefit of targeted therapies for such patients , 29. DesIn summary, our data identified prognostic factors related to outcome in metastatic BTC. Although FOLFOX has become the standard on the second-line, in our series patients receiving first-line treatment other than gemcitabine combination had worse prognosis. We also showed that bone metastasis, biliary obstruction and male gender were correlated with worse prognosis. These data could be useful for future trials in selecting patients with higher risk to more intensive and precise therapy. Further studies and multicentric collaborations are fundamental to validate our findings and also to understand the biology of this rare neoplasm.None.The authors declare that they have no conflicts of interest.Approved by local ethics committee (2680/19).Not applicable.All authors contributed to study concept and design; data acquisition; data analysis and interpretation; drafting of the manuscript; critical revision of the manuscript intellectual content and statistical analysis.


Bacillus cereus strain HT18, isolated from forest soil, was 5,333,415 bp long. The genome included 5,825 putative coding sequences and 35.2% GC content; the strain had 5 plasmids. Average nucleotide identity based on BLAST+ (ANIb) and digital DNA-DNA hybridization (dDDH) results showed that HT18 was 98.78% and 90.70% homologous, respectively, to B. cereus ATCC 14579T.The genome sequence of  Bacillus cereus group (phylum Firmicutes) comprises Gram-positive, spore-forming, facultative, anaerobic, rod-shaped bacteria with low-GC-content genomes . After colony isolation, the cells were cultured in LB broth at 37°C for 24 h. Then, genomic DNA was isolated using Marmur’s method .http://platanus.bio.titech.ac.jp/pltanus_trim)  (dnaA first was performed with DFAST version 1.4.0 (https://dfast.ddbj.nig.ac.jp) (http://jspecies.ribohost.com/jspeciesws/) and the Genome-to-Genome Distance Calculator version 3.0 (http://ggdc.dsmz.de/ggdc.php)  and decoded on a MiSeq instrument (Illumina). A total of 1,488,012 reads with 431,000,004 bases were decoded with an average insert size of 641 bp and spot length of 602 bp with 2 × 300-bp paired ends. Low-quality bases (Q scores of <15) were trimmed, and short reads (<25 bp) were removed using Platanus trim version 1.0.7 (gdc.php)  using thOne contig of the assembled genome sequence was 5,333,415 bp long (35.2% GC content) with a sequence depth of approximately 80. A total of 5,825 coding regions, 107 tRNAs, 42 rRNAs, and 3 CRISPR regions were annotated. Five plasmid DNA sequences were also assembled .B. cereus strains WPySW2 and AFA01, and 99.9% and 99.9% homologous to the foodborne pathogen FORC021 (https://figshare.com/articles/dataset/suppl_Table_pdf/17111096). Although strain HT18 showed high homology to B. thuringiensis serovar Berliner ATCC 10792T, it was closely related to B. cereus ATCC 14579T when it was compared to reference strains of the B. cereus group. Therefore, strain HT18 was identified as a B. cereus strain.Based on the ANIb (%) and dDDH (%) results, strain HT18 was 99.9% and 100% homologous to both PRJDB11181, BioSample number SAMD00278679, DRA number DRA011610, accession numbers AP024504 to AP024509, and SRA numbers DRX266213 (Illumina) and DRX266214 (ONT).The genome sequence and annotation data for strain HT18 were deposited in DDBJ/GenBank under BioProject number