Use of Electron Microscopy for the Detection of Contaminant Endophytic Bacteria In Vitro.

The success of in vitro cultivation, particularly for micropropagation purposes, depends on the efficient control of contaminants. In this context, the sterilization of plant material constitutes a fundamental step in initiating cultures. Microbial contaminants can be found either on the surface (epiphyte) or inside plant explants (endophyte). However, the latter is generally challenging to detect and may not always be eradicated through surface sterilization alone. Endophyte contaminants, such as bacteria, can persist within plant material over several cultivation cycles, potentially interfering with or inhibiting in vitro establishment, growth, or recovery of cryopreserved materials. Therefore, microscopy techniques, such as electron microscopy, can yield valuable insights into bacterial endophytes' localization, tissue colonization patterns, and functions in in vitro plant culture. This information is essential for adopting effective strategies for eliminating, preventing, or harmonious coexistence with contaminants.

Quantification of DNA Methylation by ELISA in Epigenetic Studies in Plant Tissue Culture: A Theoretical-Practical Guide.

This chapter describes a step-by-step protocol for rapid serological quantification of global DNA methylation by enzyme-linked immunosorbent assay (ELISA) in plant tissue culture specimens. As a case study model, we used the coconut palm (Cocos nucifera), from which plumules were subjected to somatic embryogenesis followed by embryogenic calli multiplication. DNA methylation is one of the most common epigenetic markers in the regulation of gene expression. DNA methylation is generally associated with non-expressed genes, that is, gene silencing under certain conditions, and the degree of DNA methylation can be used as a marker of various physiological processes, both in plants and in animal cells. Methylation consists of adding a methyl radical to carbon 5 of the DNA cytosine base. Herein, the global DNA methylation was quantified by ELISA with antibodies against methylated cytosines using a commercial kit (Zymo-Research™). The method allowed the detection of methylation in total DNA extracts from coconut palm embryogenic calli (arising from somatic embryogenesis) cultivated in liquid or solid media by using antibodies against methylated cytosines and enzymatic development with a colorimetric substrate. Control samples of commercially provided Escherichia coli bacterial DNA with previously known methylation percentages were included in the ELISA test to construct an experimental methylation standard curve. The logarithmic regression of this E. coli standard curve allowed methylation quantification in coconut palm samples. The present ELISA methodology, applied to coconut palm tissue culture specimens, is promising for use in other plant species and botanical families. This chapter is presented in a suitable format for use as a step-by-step laboratory procedure manual, with theoretical introduction information, which makes it easy to apply the protocol in samples of any biological nature to evaluate DNA global methylation associated with any physiological process.

OCEANIA: real-world study of ovarian cancer treatment patterns across multiple lines of therapy in Argentina and Brazil.

Aim: To evaluate real-world data on treatment patterns in Argentina and Brazil in patients with ovarian cancer. Methods: This study evaluated de-identified antineoplastic exposure data from a private healthcare provider in Argentina and health claims database (Orizon) in Brazil from 2010 to 2019 and 2015 to 2020, respectively. Results: Platinum-based chemotherapy was the most common first-line therapy (Argentina: n =311 [87.6%]; Brazil: n = 1142 [79.3%]). The proportion of patients receiving platinum-based chemotherapy declined across both populations from first- to second-line, while use of non-platinum-based, targeted, and hormone therapies increased. Duration of platinum-based treatment and time to next treatment decreased from first- to fourth-line. Conclusion: There is an unmet need for effective therapies that can prolong time to next treatment in ovarian cancer in Argentina and Brazil.

[Box: see text].

Small-bodied males invest in larger testes when highly ornamented.

Sperm competition and male mating rate are two non-mutually exclusive key evolutionary pressures selecting for larger testes within and across animal taxa. A few studies have tried to test the role of mating rate in the absence of sperm competition. Under the mating rate hypothesis, particular phenotypes of a given population that are expected to gain more mates (e.g., more ornamented males) are expected to make higher investments in testes size (a proxy for sperm production). We test this prediction in Polistes simillimus, a neotropical paper wasp in which females are single mated (no sperm competition) and males can mate with multiple partners. Testes size was predicted by body size (positive association), sexual ornamentation (negative association), and their interaction (among small males, testes size was positively related to ornamentation, but the opposite pattern was observed among large males). We propose that small-bodied well-ornamented males may face the highest risk of sperm depletion. Small-bodied males make relatively higher investment in testes size when highly ornamented. This strategy might be less profitable to large males, as they have overall larger testes. Our results provide strong evidence for the mating rate hypothesis.

Immune response in paper wasp workers: Task matters more than age.

Workers of social hymenopterans (ants, bees and wasps) display specific tasks depending on whether they are younger or older. The relative importance of behavior and age in modulating immune function has seldom been addressed. We compared the strength of encapsulation-melanization immune response (hereafter melanotic encapsulation) in paper wasps displaying age polyethism or experimentally prevented from behavioral specialization. Foragers of Polybia paulista had higher melanotic encapsulation than guards, regardless of their age. Nevertheless, melanotic encapsulation decreased with age when wasps were prevented from behavioral specialization. Thus, in this species, worker melanotic encapsulation seems more sensitive to task than age. Foraging is considered one of the riskier behaviors in terms of pathogen exposure, so upregulating melanotic encapsulation in foragers can possibly improve both individual and colony-level resistance against infections.

Smelly shark, smelly ray: what is infecting you?

AIMS: Although elasmobranchs are consumed worldwide, bacteriological assessments for this group are still sorely lacking. In this context, this study assessed bacteria of sharks and rays from one of the most important landing ports along the Rio de Janeiro coast. METHODS AND RESULTS: Bacteria were isolated from the cloacal swabs of the sampled elasmobranchs. They were cultured, and Vibrio, Aeromonas, and Enterobacterales were isolated and identified. The isolated bacteria were then biochemically identified and antimicrobial susceptibility assays were performed. Antigenic characterizations were performed for Salmonella spp. and Polymerase Chain Reaction (PCR) assays were performed to identify Escherichia coli pathotypes. Several bacteria of interest in the One Health context were detected. The most prevalent Enterobacterales were Morganella morganii and Citrobacter freundii, while Vibrio harveyi and Vibrio fluvialis were the most prevalent among Vibrio spp. and Aeromonas allosacharophila and Aeromonas veronii bv. veronii were the most frequent among Aeromonas spp. Several bacteria also displayed antimicrobial resistance, indicative of Public Health concerns. A total of 10% of Vibrio strains were resistant to trimethoprim-sulfamethoxazole and 40% displayed intermediate resistance to cefoxitin. Salmonella enterica strains displayed intermediate resistance to ciprofloxacin, nalidixic acid and streptomycin. All V. cholerae strains were identified as non-O1/non-O139. The detected E. coli strains did not exhibit pathogenicity genes. This is the first study to perform serology assessments for S. enterica subsp. enterica isolated from elasmobranchs, identifying the zoonotic Typhimurium serovar. Salmonella serology evaluations are, therefore, paramount to identify the importance of elasmobranchs in the epidemiological salmonellosis chain. CONCLUSIONS: The detection of several pathogenic and antibiotic-resistant bacteria may pose significant Public Health risks in Brazil, due to high elasmobranch consumption rates, indicating the urgent need for further bacteriological assessments in this group.

A mixture of mesotrione and atrazine harms adults and larvae of the predatory wasp Polistes satan.

Herbicides have been intensively used for weed control, raising concerns about their potentially adverse effects on non-target organisms. Research on the effects of these common agrochemicals on beneficial insects and the ecosystem services they provide (e.g., predation and pollination) is scarce. Therefore, we tested whether a commercial formulation comprising a mixture of mesotrione and atrazine was detrimental to adult females and larvae of the Neotropical predatory social wasp Polistes satan, which is an effective natural enemy of crop pests. Wasps were individually fed syrups contaminated with different concentrations of the herbicide above and below the maximum label rate (MLR = 12 mL/L). Survival was assessed. The locomotor activity, immune response, and midgut morphology of adults as well as the immune response of the larvae were also studied. Herbicide concentrations far above the MLR (12, 40, and 100 times) caused adult mortality, whereas lower concentrations (0.5, 1, and 6 times) did not. Herbicide exposure at 0.5 to 12 times the MLR increased adult activity. Adult exposure at 0.1 or 0.5 times the MLR did not affect melanotic encapsulation of foreign bodies but led to changes in the morphology of the midgut epithelium and peritrophic matrix. In larvae, the ingestion of herbicide at 0.1 or 0.2 times the MLR (corresponding to 9.6 and 19.2 ng of herbicide per individual) did not cause mortality but decreased their melanization-encapsulation response. Increased locomotor activity in herbicide-exposed adults can affect their foraging activity. The altered midgut morphology of adults coupled with the decreased immune response in larvae caused by herbicide exposure at realistic concentrations can increase the susceptibility of wasps to infections. Therefore, herbicides are toxic to predatory wasps.

Association of the Osseodensification Technique with Soft Tissue Substitute in a Limitrophe Edentulous Area: A Clinical Case Report with 5-Year Follow-Up.

The osseodensification (OD) technique differs from conventional milling for dental implant installation in that it preserves the prepared bone and compacts it toward the apex and lateral walls of the socket, resulting in bone compaction. By enabling autografting, bone expansion, and high implant insertion torques, OD has become an increasingly popular option. The aim of this clinical case report is to demonstrate the predictability of combining OD with guided bone and tissue regeneration (GBR/GTR) techniques for bone expansion in the maxilla with reduced thickness, while avoiding other reconstructive surgeries. The report presents the treatment of a 32-year-old female patient who had cosmetic concerns regarding the anterior maxillary region. The patient was using an adhesive prosthesis with pontic on tooth 13 fixed between teeth 12 and 14. After the case was planned, it was decided that bone expansion in the region would be performed using the OD technique. The implant installation (AR Torque, 3.5 × 11.5 mm, Conexão®) and guided bone regeneration (GBR) were done with the assistance of L-PRF (Stick Bone, associated with L-PRF membrane). Following the osseointegration period, a provisional resin crown was fabricated, and a collagen matrix membrane (Mucoderm®) was used to increase vestibular soft tissue volume and shape the patient's gingival profile. After a period of 120 days, the final crown was created and observed for a span of 5 years. The results showed stability of the case along with maintaining its esthetic and satisfactory function. The use of the osseodensification technique coupled with a connective tissue graft substitute has been anticipated for a long time. It has proven to be an excellent alternative to autogenous grafts.

Blood-based multi-cancer detection: A state-of-the-art update.

The early detection of cancer is a key goal of the National Cancer Plan formally released by the National Institutes of Health's (NIH) National Cancer Institute (NCI) in April 2023. To support this effort, many laboratories and vendors are developing multi-cancer detection (MCD) assays that interrogate blood and other bodily fluids for cancer-related biomarkers, most commonly circulating tumor DNA (ctDNA). While this approach holds promise for non-invasively detecting early signals of multiple different cancers and potentially reducing cancer-related mortality, there is a dearth of prospective clinical data to inform the deployment of MCD assays for cancer screening in the general adult population. In this review we highlight differing technologies that underpin various MCD assays in clinical development, the importance of achieving adequate performance specifications for MCD assays, ongoing clinical studies investigating the utility of MCD assays in cancer screening and detection, and efforts by the NCI's Division of Cancer Prevention (DCP) to establish a network infrastructure that has the capacity to comprehensively address the scientific and logistical challenges of evaluating blood-based MCD approaches and other cancer screening tools.

A predatory social wasp does not avoid nestmates contaminated with a fungal biopesticide.

Fungus-based biopesticides have been used worldwide for crop pest control as a safer alternative to chemical pesticides such as neonicotinoids. Both agrochemicals can be lethal and may also trigger side effects on the behavioral traits of non-target social insects, which play a crucial role in providing essential biological pest control services in agroecosystems. Here, we evaluated whether a commercial formulation of the entomopathogenic fungus Beauveria bassiana or the neonicotinoid imidacloprid causes mortality in foragers of Mischocyttarus metathoracicus. These social wasps are natural enemies of caterpillars and other herbivorous insects and inhabit both urban and agricultural environments in Brazil. We also tested whether wasps discriminate between biopesticide-exposed and unexposed conspecifics. Through a combination of laboratory (survival assay) and field experiments (lure presentation), along with chemical analyses (cuticular hydrocarbon profiles), we showed that topic exposure to the label rate of each pesticide causes a lethal effect, with the biopesticide exhibiting a slower effect. Moreover, wasps do not discriminate biopesticide-exposed from unexposed conspecifics, likely because of the similarity of their cuticular chemical profiles 24 h after exposure. Overall, the delayed lethal time at the individual level, combined with the indistinctive chemical cues of exposure and the lack of discrimination by conspecifics suggests that the fungal biopesticide may ultimately pose a threat to the colony survival of this predatory wasp.

Uncertainty model for automated gunshot residue particle length measurements obtained from electron microscopy images.

Currently, the use of algorithms and computer vision systems for metrological purposes has increased in different areas of knowledge to reduce human error and process deviations, consequently increasing reliability and reducing measurement uncertainties. This study presents a model for estimating the uncertainty of Feret's diameter (DF ) measurements of scanning electron microscopy (SEM) images from regular and irregular gunshot residue (GSR) particles at different magnifications. The data were extracted using the automatic measurement algorithm developed by the Brazilian Institute of Metrology, Quality and Technology (Inmetro). The proposed uncertainty model was based on the recommendations of the guide to the expression of uncertainty in measurement (GUM). The gold standard technique to identify and detect GSR particles is the SEM coupled to energy dispersive X-ray spectroscopy (SEM/EDS), which was used in the study. The low uncertainty values obtained in this study are justified by the refinement of the measurements performed at each stage of digital image procedures. The proposed uncertainty model contributes in an innovative way to the metrological evaluation of regular and irregular GSR particles at different images magnifications. The correct morphometry definition of these particles allows to study their distinction from other possible sources of GSR and, above all, their correlation with the type of ammunition used when firing the firearm. These measurement uncertainty calculations can be applied to any object images acquired by SEM, which provides more confidence in the results of measurements of the object of interest.

Group 4 ITI Consensus Report: Patient benefits following implant treatment in partially and fully edentulous patients.

OBJECTIVES: The aim of Working Group 4 was to address patient benefits associated with implant dentistry. Focused questions on (a) dental patient-reported outcomes (dPROs), (b) improvement in orofacial function, and (c) preservation of orofacial tissues in partially and fully edentulous patients following provision of implant-retained/supported dental prostheses were addressed. MATERIALS AND METHODS: Three systematic reviews formed the basis for discussion. Participants developed statements and recommendations determined by group consensus based on the findings of the systematic reviews. These were then presented and accepted following further discussion and modifications as required by the plenary of the 7th ITI Consensus Conference, taking place in 2023 in Lisbon, Portugal. RESULTS: Edentulous patients wearing complete dentures (CD) experience substantial improvements in overall dPROs and orofacial function following treatment with either complete implant-supported fixed dental prostheses (CIFDP) or implant overdentures (IODs). With respect to dPROs, mandibular IODs retained by two implants are superior to IODs retained by one implant. However, increasing the number of implants beyond two, does not further improve dPROs. In fully edentulous patients, rehabilitation with CIFDP or IOD is recommended to benefit the preservation of alveolar bone and masseter muscle thickness. CONCLUSIONS: Completely edentulous patients benefit substantially when at least the mandible is restored using an CIFDP or an IOD compared to CD. In fully edentulous patients, implant prostheses are the best option for tooth replacement. The availability of this treatment modality should be actively promoted in all edentulous communities, including those with limited access and means.

Effect of dental implant therapy on the preservation of orofacial tissues: A systematic review and meta-analysis.

OBJECTIVE: Fundamentally, this review addresses the following question: In partially or fully edentulous patients, do implant-supported dental prostheses preserve orofacial tissues when compared to conventional prostheses or no therapy? MATERIALS AND METHODS: This study was conducted according to the 2020 PRISMA guidelines for systematic reviews. Electronic searches were conducted at PubMed and Embase databases followed by manual search. Clinical studies comparing the effect of implant-supported prostheses with conventional rehabilitation or no treatment on alveolar bone resorption, remaining teeth, and jaw muscle thickness were considered for inclusion. A qualitative synthesis was conducted with all included studies, and data from selected studies were pooled quantitatively to perform a meta-analysis. RESULTS: A total of 14 studies were selected for analysis. Six studies reported on the effect of implant therapy on alveolar bone resorption (n = 453), six on the remaining teeth (n = 1014), while four studies evaluated masseter muscle thickness (n = 158). The results of the meta-analyses assessing alveolar bone resorption in the posterior mandible and in the anterior area of the maxilla, both fixed and random effects models, yielded no benefit of rehabilitation with implant-supported prostheses when compared to conventional prostheses. For masseter bone thickness, however, a significant benefit for implant-supported prosthesis was observed. CONCLUSIONS: This systematic review and meta-analysis were unable to unequivocally answer the focus question. There are some indicators of the benefit of implant-supported prostheses over conventional prostheses or no therapy in preserving orofacial tissues, particularly for masseter muscle thickness. However, the evidence is still insufficient to confirm such perception.

One piece of the puzzle: Modeling vector presence and environment reveals seasonality, distribution, and prevalence of sandflies and Leishmania in an expansion area.

The recent geographic spread of Leishmania infantum along the borders of Argentina, Brazil and Paraguay has been highlighted. In our previous study, Lutzomyia longipalpis was found in 55 of 123 patches surveyed, and in some patches, sandflies were found at higher densities, forming hotspots. Based on the One Health approach, we investigated the seasonality of the vector, the presence of parasite DNA, and the environmental factors that contribute to vector and parasite dispersal in these previously described hotspots in Foz do Iguaçu, Brazil. Entomological surveys were conducted monthly for one year. Fourteen hotspots peridomicile and six intradomicile were sampled. PCR was used to assess the prevalence of Leishmania DNA in sandflies. Zero-inflated negative binomial regression was used to determine the association of micro- and mesoscale environmental variables with the occurrence and abundance of the three most abundant sandfly species sampled. A total of 3543 species were captured, with Lutzomyia longipalpis being the predominant species (71.78%) of the 13 species found. Evandromyia edwardsi, Expapillata firmatoi, Micropygomyia ferreirana and Pintomyia christenseni were reported for the first time in the region. NDVI, distance to water, precipitation, west-to-east wind, wind speed, maximum and minimum relative humidity, and sex were significant variables associated with vector presence/abundance in the environment. Vector presence/abundance in the peridomicile was associated with precipitation, altitude, maximum temperature, minimum and maximum relative humidity, west-to-east wind, wind speed, and sex. Leishmania DNA was detected in an average of 21% of Lu. longipalpis throughout the year. Vector abundance is concentrated in urban and peri-urban areas, with some specimens present in different parts of the city and some sites with high vector abundance. This distribution suggests that the risk of actual contact between humans and parasite vectors in urban areas during the epidemic period is associated with patches of peri-urban vegetation and then extends into urban areas.

Sex recognition does not modulate aggression toward nest intruders in a paper wasp.

During social interactions, the behavior of an individual often depends on the sex of its social partner. Many animal societies have males and females that play very different behavioral roles, although they coexist and interact non-sexually. At specific phases of the colony cycle, social wasp females and males are contemporaries within a nest, they often interact, although mating occurs mostly off the nest, therefore providing an opportunity to test sex discrimination in contexts other than classical sexual ones. We performed a lure presentation experiment to test if Mischocyttarus metathoracicus discriminate between conspecifics of the 2 sexes during on-nest social interactions. Female wasps discriminated conspecific sex during experimentally simulated nest intrusions. Visual and chemical cues may account for this sex discrimination. Despite sex discrimination (evidenced by differential inspective behavior from the nest females toward the female and the male lures), female wasps were as aggressive toward lures of both sexes. In the female-dominated hymenopteran societies, males are often subordinate and not aggressive on nest, resulting in females directing less aggression to them compared to other females. Instead, M. metathoracicus males and females are both aggressive toward nestmates, so they might be perceived as similar threat during on-nest social interactions.

Implant Esthetic Complications: Anatomical, Prosthetic, and Patient-Centered Considerations for Treatment.

Implant esthetic complications can negatively affect a patient's perception of implant therapy and their quality of life. This article discusses the etiology, prevalence, and strategies for the treatment of peri-implant soft tissue dehiscences/deficiencies (PSTDs). Three common scenarios of implant esthetic complications were identified and described, in which PSTDs could be managed without removing the crown (scenario I), with the surgical-prosthetic approach (crown removal; scenario II), and/or with the horizontal and vertical soft tissue augmentation and submerged healing (scenario III).

Accuracy of static computer-aided implant surgery (S-CAIS) using CAD-CAM surgical templates fabricated from different additive manufacturing technologies.

STATEMENT OF PROBLEM: Different 3D printers are available for guided implant surgery, but studies that evaluate their source of errors and their cost-effectiveness are lacking. PURPOSE: The purpose of this in vitro study was to compare the accuracy of different 3-dimensional (3D) printed surgical templates made using different additive manufacturing technologies and to evaluate the effect of implant location on the accuracy of fully guided implant placement. MATERIAL AND METHODS: Fifty partially edentulous maxillary typodonts with edentulous sites in the right second premolar (SP), right lateral incisor (LI), left central incisor (CI), and left first molar (FM) locations were scanned and printed from the standard tessellation language (STL) datasets. The study compared 5 groups for the fabrication of implant surgical templates: Varseo S-Bego (Bego), Polyjet-Stratasys (Poly), Low Force Stereolithography-FormLabs (LFS), P30+-Straumann (P30), and M2-Carbon (M2). After fully guided implant placement, the typodont was scanned, and the 3D implant positions were compared with the master model by superimposing the STL files. Descriptive statistics were calculated for groups and subgroups, and comparisons among the groups and subgroups were conducted via 2-way mixed analysis of variance, Tukey honest significant difference, and post hoc Bonferroni tests (α=.05). RESULTS: The results were site specific and not consistent within each group. For angle deviation, the within-group analysis for P30 demonstrated significantly lower values for implants positioned at site SP (1.4 ±0.8 degrees) than for sites LI (2.3 ±0.7 degrees; P=.001) and CI (2.3 ±0.8 degrees; P=.007). For 3D offset at base for implant CI, LFS was significantly higher than Bego (P=.002), Poly (P=.035), or M2 (P=.001); P30 was also significantly higher than Bego (P=.014) and M2 (P=.006). LFS had a significantly higher 3D offset at the tip than Bego (P=.001) and M2 (P=.022) for implant CI. CONCLUSIONS: The choice of 3D printer seemed to influence fully guided implant surgery in terms of the final implant position compared with initial implant planning. However, although statistically significant differences were present across groups, all additive manufacturing technologies were within clinically acceptable values.

Ingestion of polystyrene microparticles impairs survival and defecation in larvae of Polistes satan (Hymenoptera: Vespidae).

Microplastics (MPs) are widespread pollutants of emerging concern, and the risks associated with their ingestion have been reported in many organisms. Terrestrial environments can be contaminated with MPs, and terrestrial organisms, including arthropods, are predisposed to the risk of ingesting MPs. In the current study, the larvae of the paper wasp Polistes satan were fed two different doses (6 mg or 16 mg at once) of polystyrene MPs (1.43 mm maximum length), and the effects of these treatments on immature development and survival till adult emergence were studied. Ingestion of the two doses resulted in mortality due to impaired defecation prior to pupation. The survival of larvae that ingested 16 mg of MPs was significantly lower than that of the control. The ingestion of 16 mg of MPs also reduced the adult emergence (11.4%) in comparison to the control (44.4%). MPs were not transferred from the larvae to the adults that survived. These findings demonstrate that MP ingestion can be detrimental to P. satan, e.g. larval mortality can decrease colony productivity and thus the worker force, and that MPs can potentially affect natural enemies that occur in crops, such as predatory social wasps.

Frequent Telomerase Reverse Transcriptase Promoter and Fibroblast Growth Factor Receptor 3 Mutations Support the Precursor Nature of Papillary Urothelial Hyperplasia of the Urinary Bladder.

The precursor nature of papillary urothelial hyperplasia of the urinary bladder is uncertain. In this study, we investigated the telomerase reverse transcriptase (TERT) promoter and fibroblast growth factor receptor 3 (FGFR3) mutations in 82 patients with papillary urothelial hyperplasia lesions. Thirty-eight patients presented with papillary urothelial hyperplasia and concurrent noninvasive papillary urothelial carcinoma, and 44 patients presented with de novo papillary urothelial hyperplasia. The prevalence of the TERT promoter and FGFR3 mutations is compared between de novo papillary urothelial hyperplasia and those with concurrent papillary urothelial carcinoma. Mutational concordance between papillary urothelial hyperplasia and concurrent carcinoma was also compared. The TERT promoter mutations were detected in 44% (36/82) of papillary urothelial hyperplasia, including 23 (23/38, 61%) papillary urothelial hyperplasia with urothelial carcinoma and 13 (13/44, 29%) de novo papillary urothelial hyperplasia. The overall concordance of TERT promoter mutation status between papillary urothelial hyperplasia and concurrent urothelial carcinoma was 76%. The overall FGFR3 mutation rate of papillary urothelial hyperplasia was 23% (19/82). FGFR3 mutations were detected in 11 patients with papillary urothelial hyperplasia and concurrent urothelial carcinoma (11/38, 29%) and 8 patients with de novo papillary urothelial hyperplasia (8/44, 18%). Identical FGFR3 mutation status was detected in both papillary urothelial hyperplasia and urothelial carcinoma components in all 11 patients with FGFR3 mutations. Our findings provide strong evidence of a genetic association between papillary urothelial hyperplasia and urothelial carcinoma. High frequency of TERT promoter and FGFR3 mutations suggests the precursor role of papillary urothelial hyperplasia in urothelial carcinogenesis.

Clinical features of patients with MTAP-deleted bladder cancer.

Advanced urothelial carcinoma continues to have a dismal prognosis despite several new therapies in the last 5 years. FGFR2 and FGFR3 mutations and fusions, PD-L1 expression, tumor mutational burden, and microsatellite instability are established predictive biomarkers in advanced urothelial carcinoma. Novel biomarkers can optimize the sequencing of available treatments and improve outcomes. We describe herein the clinical and pathologic features of patients with an emerging subtype of bladder cancer characterized by deletion of the gene MTAP encoding the enzyme S-Methyl-5'-thioadenosine phosphatase, a potential biomarker of response to pemetrexed. We performed a retrospective analysis of 61 patients with advanced urothelial carcinoma for whom demographics, pathologic specimens, next generation sequencing, and clinical outcomes were available. We compared the frequency of histology variants, upper tract location, pathogenic gene variants, tumor response, progression free survival (PFS) and overall survival (OS) between patients with tumors harboring MTAP deletion (MTAP-del) and wild type tumors (MTAP-WT). A propensity score matching of 5 covariates (age, gender, presence of variant histology, prior surgery, and prior non-muscle invasive bladder cancer) was calculated to compensate for disparity when comparing survival in these subgroups. Non-supervised clustering analysis of differentially expressed genes between MTAP-del and MTAP-WT urothelial carcinomas was performed. MTAP-del occurred in 19 patients (31%). Tumors with MTAP-del were characterized by higher prevalence of squamous differentiation (47.4 vs 11.9%), bone metastases (52.6 vs 23.5%) and lower frequency of upper urinary tract location (5.2% vs 26.1%). Pathway gene set enrichment analysis showed that among the genes upregulated in the MTAP-del cohort, at least 5 were linked to keratinization (FOXN1, KRT33A/B, KRT84, RPTN) possibly contributing to the higher prevalence of squamous differentiation. Alterations in the PIK3 and MAPK pathways were more frequent when MTAP was deleted. There was a trend to inferior response to chemotherapy among MTAP-del tumors, but no difference in the response to immune checkpoint inhibitors or enfortumab. Median progression free survival after first line therapy (PFS1) was 5.5 months for patients with MTAP-WT and 4.5 months for patients with MTAP-del (HR = 1.30; 95% CI, 0.64-2.63; P = 0.471). There was no difference in the time from metastatic diagnosis to death (P = 0.6346). Median OS from diagnosis of localized or de novo metastatic disease was 16 months (range 1.5-60, IQR 8-26) for patients with MTAP-del and 24.5 months (range 3-156, IQR 16-48) for patients with MTAP-WT (P = 0.0218), suggesting that time to progression to metastatic disease is shorter in MTAP-del patients. Covariates did not impact significantly overall survival on propensity score matching. In conclusion, MTAP -del occurs in approximately 30% of patients with advanced urothelial carcinoma and defines a subgroup of patients with aggressive features, such as squamous differentiation, frequent bone metastases, poor response to chemotherapy, and shorter time to progression to metastatic disease.