BAT1706 is a proposed biosimilar of bevacizumab (Avastin®). We aimed to compare the efficacy and safety of BAT1706 with that of EU‐sourced reference bevacizumab (EU‐bevacizumab) in patients with advanced nonsquamous non‐small cell lung cancer (NSCLC).18). Clinical equivalence was demonstrated if the 90% confidence interval (CI) of the BAT1706:EU‐bevacizumab ORR18 risk ratio was contained within the predefined equivalence margins of 0.75–1.33 , or 0.73–1.36 (US Food and Drug Administration), or if the 95% CI of the ORR18 risk difference between treatments was contained within the predefined equivalence margin of −0.12 to 0.15 (EMA requirements).Patients were randomized 1:1 to BAT1706 plus paclitaxel and carboplatin (BAT1706 arm) or EU‐bevacizumab plus paclitaxel and carboplatin (EU‐bevacizumab arm) given every 3 weeks for six cycles, followed by maintenance therapy with BAT1706 or EU‐bevacizumab. The primary endpoint was overall response rate at week 18  received at least one cycle of combination treatment. The ORR18 was comparable between the BAT1706 and EU‐bevacizumab arms . The ORR18 risk ratio of 1.08 (90% CI: 0.94–1.24) and the ORR18 risk difference of 0.03 (95% CI: −0.04 to 0.11) were within the predefined equivalence margins, demonstrating the biosimilarity of BAT1706 and EU‐bevacizumab. The safety profile of BAT1706 was consistent with that of EU‐bevacizumab and no new safety signals were observed.In total, 649 randomized patients (BAT1706, In patients with advanced nonsquamous NSCLC, BAT1706 demonstrated clinical equivalence to EU‐bevacizumab in terms of efficacy, safety, pharmacokinetics, and immunogenicity. A biosimilar is a biological product that is highly similar to, and has no clinically meaningful differences from, an existing approved reference biologic.Two Phase I clinical studies conducted in New Zealand (NCT03030430) and China (BAT1706‐002‐CR) demonstrated that a single intravenous infusion of BAT1706 (1 mg/kg) was well‐tolerated in healthy participants.In this pivotal Phase III study (NCT03329911), we compared the efficacy and safety of BAT1706 and EU‐bevacizumab, given in combination with chemotherapy, as first‐line treatment for advanced nonsquamous NSCLC, in order to evaluate the clinical equivalence of BAT1706 and reference EU‐bevacizumab.22.1This multicenter, randomized, double‐blind, Phase III, parallel, 2‐arm study (NCT03329911) was designed to compare the efficacy and safety of BAT1706 plus paclitaxel and carboplatin (BAT1706 arm) vs EU‐bevacizumab plus paclitaxel and carboplatin (EU‐bevacizumab arm). Adult patients aged ≥18 years were eligible for inclusion if they had: histologically or cytologically confirmed Stage IV nonsquamous NSCLC or recurrent disease; tumors without EGFR mutations or ALK rearrangements; at least one measurable target lesion according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1); received no prior systemic therapy for metastatic disease; an ECOG performance status of 0 or 1 and a life expectancy of more than 3 months; and adequate hematological, hepatic, and renal function. Key exclusion criteria included: a diagnosis of small cell carcinoma of the lung; known receptor tyrosine kinase 1 (ROS1) positive tumors; tumor cavitation, tumor invading into large blood vessels or close to large vessels with an increased risk of bleeding; having received prior therapy with monoclonal antibodies or small molecule inhibitors against VEGF or VEGF receptor; known brain metastasis or other central nervous system (CNS) metastasis that was either symptomatic or untreated; a significant thrombotic or hemorrhagic event ≤6 months prior to screening; the need for permanent oral anticoagulation treatment ; history of poorly controlled hypertension; and history of active gastroduodenal ulcer or abdominal fistula within 6 months prior to screening.2.2Patients were randomized (1:1) in a blinded manner to receive either BAT1706 plus chemotherapy or EU‐bevacizumab plus chemotherapy using the Interactive Web Response System (IWRS). Randomization was stratified by the following factors: stage of NSCLC ; sex ; and ethnicity (Asian or non‐Asian). Patient numbers were assigned consecutively. Double‐blinding was maintained throughout the study; unblinding was only allowed in cases of an emergency for safety concerns.2.32, after adequate premedication and before carboplatin was given. For Chinese patients , paclitaxel could be given at a dose of 175 mg/m2. The initial dose of carboplatin (area under the curve of 6 mg/mL/min) was administered intravenously in accordance with local best practices or the package insert.BAT1706 or EU‐bevacizumab, plus paclitaxel and carboplatin, were given every 3 weeks (21 days) for up to six cycles of combination therapy. BAT1706 (15 mg/kg) or EU‐bevacizumab (15 mg/kg) were administered intravenously prior to paclitaxel and carboplatin, with the initial dose given over 90 min; if well‐tolerated, subsequent infusions were given over 60 min. Dose adjustments were not permitted. Paclitaxel was administered intravenously over 3 h (or according to the package insert) at an initial dose of 200 mg/mFor cycle 1, day 1, only BAT1706 or EU‐bevacizumab were given; on day 2, paclitaxel and carboplatin were administered. From cycle 2 (day 22) to cycle 6, BAT1706 or EU‐bevacizumab were administered first, followed by paclitaxel and then carboplatin on the same day. Any delay in chemotherapy necessitated a delay in BAT1706/EU‐bevacizumab administration, and vice versa, during the combination treatment.After six cycles of combination therapy, all patients with complete response (CR), partial response (PR), or stable disease received maintenance therapy with BAT1706 or EU‐bevacizumab for up to 12 months after week 18. Maintenance therapy was stopped if any of the following occurred: investigator‐assessed disease progression, excessive toxicity, investigator's judgment, withdrawal of consent, loss to follow‐up, death, start of a new anticancer therapy, or study termination by the Sponsor, whichever occurred first.After this 12‐month treatment period, patients still benefiting from treatment were able to enroll in a long‐term extension (LTE) study.2.418), calculated as the proportion of patients achieving a PR or a CR at week 18, as assessed by independent and blinded central imaging review (CIR) using RECIST v1.1 criteria. Patients who did not complete the ORR18 assessment were considered nonresponders if they had discontinued the study due to intolerance, disease progression, withdrawal of consent, loss to follow‐up, or death. Tumor assessments were carried out at weeks 6, 12, and 18, regardless of whether a treatment cycle was postponed, and were then performed every three cycles (approximately every 9 weeks [assessed within days 15 to 21 of the third cycle]). During the first 18 weeks, the maximum window for tumor assessment was 1 week. Contrast‐enhanced computed tomography or magnetic resonance imaging of the chest and abdomen  was obtained at screening and at all imaging time‐points. Brain computed tomography or magnetic resonance imaging scans, as well as bone scans or X‐rays, were performed at screening and during the treatment period, as clinically indicated. If the bone scan was positive at screening, it was repeated at least every 12 weeks. Plain X‐rays of bone lesions were used, as clinically indicated, between isotopic bone scans during regular tumor assessments. Until week 18, all imaging for tumor assessments were sent for CIR and used for the primary efficacy analysis.The primary endpoint was the ORR at week 18 (ORR6) and week 12 (ORR12); best ORR of confirmed responses at the end of the study; duration of response ; investigator‐assessed PFS and OS at 12 months; and investigator‐assessed duration of PFS and OS. At 12 months, additional comparative safety/immunogenicity data , as well as other efficacy data , were collected from those patients with disease control who continued to receive BAT1706 or EU‐bevacizumab maintenance therapy.Secondary efficacy endpoints included: CIR‐assessed ORR at week 6 (ORRAll adverse events (AEs) and serious AEs were evaluated for assessment of safety. The National Cancer Institute Common Terminology Criteria for AE version 4.03 was used to grade the severity of treatment‐emergent AEs (TEAEs), which were defined as events that emerged during treatment (having been absent pretreatment or worsened relative to the pretreatment state) and with onset dates occurring between the first dosing day of study treatment, and until 28 days after the last dose of study treatment. Clinical laboratory results, electrocardiogram parameters, vital signs, and physical examinations were also assessed. AEs of special interest included anaphylactic reactions, arterial and venous thromboembolic events, febrile neutropenia, gastrointestinal perforations, hypertension, proteinuria, pulmonary hemorrhage, other hemorrhages, and wound healing complications. Blood samples were also collected throughout the study for pharmacokinetic (PK) analysis.2.5A total of 632 patients were planned to be enrolled in this study. The intention‐to‐treat (ITT) population included all randomized patients based on the intended treatment arm, regardless of treatment received. The per‐protocol (PP) population included patients in either treatment arm who had received either at least three cycles of their allocated treatment, or less than three cycles of treatment , along with one tumor assessment and no major protocol deviations that could significantly impact on efficacy or safety outcomes. Patients who discontinued prior to week 18 for any reason were counted as nonresponders in the primary endpoint analysis for the ITT population. Efficacy analyses were carried out in both the ITT and PP populations. Safety analyses were carried out in the safety population, which included all randomized patients who received at least one dose of BAT1706 or EU‐bevacizumab, and who were assigned to that treatment.18 were analyzed to assess clinical equivalence. Clinical equivalence was demonstrated if the 90% confidence interval (CI) of the BAT1706:EU‐bevacizumab ORR18 risk ratio was entirely contained within the equivalence margins of 0.75–1.33, complying with NMPA requirements, or 0.73–1.36, complying with FDA requirements. Clinical equivalence was also demonstrated if the 95% CI , sex , and ethnicity (Asian or non‐Asian). Stratification factors were from an IWRS, and the CIs for the median were calculated according to Brookmeyer and Crowley.For time‐to‐event variables such as PFS, OS, and DoR, Kaplan–Meier curves were calculated and displayed. A stratified Cox regression model yielded estimated hazard ratios (HRs) with 95% CIs, with treatment as the explanatory variable, and nonsquamous NSCLC stage (recurrent disease or Stage IV), sex , and ethnicity (Asian or non‐Asian) as stratification factors; an HR <1.0 favored BAT1706. Statistical analyses were carried out using SAS Version 9.4®. PK parameters were analyzed using Phoenix® WinNonlin® Version 8.0, with PK figures generated using SAS Version 9.4®.33.1The first patient was enrolled on December 11, 2017 (study initiation date), and the results reported here are based on a data cutoff date of November 05, 2019, at which point all patients had either completed 12 months of treatment, or had withdrawn from the study.In total, 651 patients were randomized (325 in the BAT1706 arm and 326 in the EU‐bevacizumab arm) and comprised the ITT population . In total, 581 (89.2%) patients had Stage IV cancer at the time of initial diagnosis.Demographic and baseline characteristics of patients were similar for both treatment arms Table . Most pa3.2At data cutoff, exposure to BAT1706 and EU‐bevacizumab was similar between the treatment arms. The median duration of therapy was 29.1 weeks (range: 3.0–62.1 weeks) in the BAT1706 arm and 27.0 weeks (range: 3.0–53.9 weeks) in the EU‐bevacizumab arm. Overall, patients in each treatment arm received a median of nine infusions of BAT1706 or EU‐bevacizumab. The median cumulative doses of BAT1706 and EU‐bevacizumab were 138.4 mg/kg and 130.1 mg/kg, respectively. Delays in BAT1706/EU‐bevacizumab dosing was reported in 161 patients in the BAT1706 arm (49.5%) and 155 patients the EU‐bevacizumab arm (47.8%), with the median dose delay being 7 days for both treatments.Median duration of exposure to carboplatin at data cutoff was similar between the two treatment arms: 17.9 weeks (range: 3.0–25.3 weeks) in the BAT1706 arm, and 17.7 weeks (range: 3.0–27.4 weeks) in the EU‐bevacizumab arm. Likewise, the median number of carboplatin infusions was similar: six in the BAT1706 arm, and five in the EU‐bevacizumab arm. The median cumulative carboplatin dose was 3264.6 mg in the BAT1706 arm and 2992.8 mg in the EU‐bevacizumab arm. Delays in carboplatin dosing were reported by 123 (37.8%) patients in the BAT1706 arm and 117 (36.1%) patients in the EU‐bevacizumab arm, with the median dose delay being 5 days in each arm. Carboplatin dose‐modification was reported in 51 (15.7%) and 56 (17.3%) patients in the BAT1706 and EU‐bevacizumab arms, respectively. Similar results to carboplatin were found for paclitaxel exposure. Overall, there were no differences between the treatment arms in exposure to carboplatin or paclitaxel across the study.3.3Similar results were found in the ITT and PP populations for the primary endpoint; data reported here are for the ITT population. At data cutoff, all randomized patients had completed the week 18 visit or had been excluded from the disease evaluation period.18 was 48.0% (95% Cl: 42.5–53.6) in the BAT1706 arm and 44.5% (95% CI: 39.0–50.1) in the EU‐bevacizumab arm, as shown in Table 18 (BAT1706/EU‐bevacizumab) risk ratio was 1.08, with the 2‐sided 90% CI of 0.94–1.24 falling entirely within the equivalence margin of 0.75–1.33, in compliance with NMPA requirements, and 0.73–1.36, in compliance with FDA requirements. In addition, the multivariate‐adjusted risk ratio was 1.07, with a 90% CI of 0.93–1.22.In the ITT population, the ORR95% Cl: 4.5–53.6 i18 risk difference between BAT1706 and EU‐bevacizumab was 0.03, with the 2‐sided 95% CI of −0.04 to 0.11 falling entirely within the equivalence margin of −0.12 to 0.15, in compliance with EMA requirements. In addition, the multivariate‐adjusted risk difference was 0.03, with a 95% CI of −0.04 to 0.11.The ORRClinical equivalence of BAT1706 and EU‐bevacizumab was demonstrated using both the risk ratio and risk difference methods, regardless of the stratification factors, and was supported by the multivariate‐adjusted risk analyses Table .3.4Similar results were found in the ITT and PP populations for all secondary efficacy endpoints; data reported here are for the ITT population.p = 0.412) , with a median PFS of 8.1 (95% CI: 7.1–8.3) months for BAT1706 and 7.7 (95% CI: 6.2–8.3) months for EU‐bevacizumab, with no difference in the time to median PFS between treatment arms  ; median OS was 16.4  months for BAT1706 and 18.5  months for EU‐bevacizumab, with no difference in the time to median OS between treatment arms  , with median DoR being 6.9 (95% CI: 5.7–7.6) months and 7.1 (95% CI: 5.5–9.0) months, respectively, with no difference in the time to DoR between treatment arms  patients in the BAT1706 arm and 235 (72.5%) in the EU‐bevacizumab arm; TEAEs of Grade ≥4 were reported by 92 (28.3%) patients in in the BAT1706 arm and 93 (28.7%) in the EU‐bevacizumab arm. In total, 35 (5.4%) patients died due to a TEAE (18 patients in the BAT1706 arm and 17 in the EU‐bevacizumab arm). Among these, 11 (1.7%) patients died due to a TEAE that was considered by the investigator to be related to BAT1706 (eight patients) or EU‐bevacizumab (three patients), and a similar number of patients  died due to a TEAE that was considered by the investigator to be related to carboplatin or paclitaxel. TEAEs that led to discontinuation of treatment were reported by 53 (8.2%) patients (28 patients in the BAT1706 arm and 25 in the EU‐bevacizumab arm). Discontinuations considered by investigators to be related to BAT1706/EU‐bevacizumab and paclitaxel/carboplatin were similar in both arms.3.6Mean serum concentrations were comparable between BAT1706 and EU‐bevacizumab throughout the study, both pre‐ and post‐dose (data not shown). For both treatments, individual serum concentrations remained quantifiable throughout the sampling interval, with mean trough concentrations comparable between BAT1706 and EU‐bevacizumab (data not shown).3.7Overall, the incidence of positive ADA results was low (≤5%) and decreased over time in both treatment arms. A total of 22 patients (12 patients in the BAT1706 arm and 10 in the EU‐bevacizumab arm), had negative ADA results at baseline and positive ADA results at the post‐baseline visit. No patients had neutralizing ADAs detected during the study. Overall, similar immunogenicity results were observed in the two treatment arms.418 was demonstrated, as the 90% Cl for the ORR18 risk ratio (BAT1706/EU‐bevacizumab) was entirely contained within the prespecified equivalence margins stipulated by the NMPA and FDA. In addition, the 95% CI for the risk difference in ORR18 between BAT1706 and EU‐bevacizumab was entirely contained within the equivalence margin stipulated by the EMA. These equivalence analyses, along with similar results found in the ITT and PP populations for ORR18, support the claim of clinical biosimilarity between BAT1706 and EU‐bevacizumab. Furthermore, clinical equivalence of BAT1706 and EU‐bevacizumab was shown regardless of stratification factors, as demonstrated in a multivariate‐adjusted analysis in both the ITT and PP populations. Multivariate analysis of ORR18 in the ITT and PP populations showed consistent results regardless of stratification factors . Results for the secondary endpoints were consistent with those from the primary endpoint, in both the ITT and PP populations. For example, in the ITT population, median time to PFS and 12‐month PFS were similar between the two treatment arms, and notably, there was no meaningful difference in the number of patients with PFS‐related events . Overall, the BAT1706 and EU‐bevacizumab efficacy outcomes of this Phase III study are consistent with previously published Phase II and III data for bevacizumab used in combination with carboplatin and paclitaxel in treating patients with advanced NSCLC.This Phase III biosimilar clinical study was conducted to compare efficacy, safety, PK, and immunogenicity of BAT1706 and EU‐bevacizumab in patients diagnosed with advanced nonsquamous NSCLC. Demographic and baseline characteristics were well‐balanced between the two treatment arms. Based upon equivalence margin criteria for biosimilarity stipulated by three international regulatory agencies , this study demonstrated the biosimilarity of BAT1706 and EU‐bevacizumab. The equivalence between BAT1706 and EU‐bevacizumab in terms of ORRThere were no notable differences between the two treatment arms regarding the incidence and severity of TEAEs and serious TEAEs, as well as changes in other safety parameters . There were similar rates of BAT1706‐ and EU‐bevacizumab‐related TEAEs, along with comparable carboplatin‐ and paclitaxel‐related TEAEs, in the two treatment arms. Incidence rates of serious, Grade ≥3, and Grade ≥4 drug‐related TEAEs were also similar between treatment groups. PK and immunogenicity were also similar in the two treatment arms, with serum concentrations and exposure parameters comparable between BAT1706 and EU‐bevacizumab. Overall, the safety profile of BAT1706 was consistent with the known safety profile of reference bevacizumab published in the literature,p < 0.001).It is worth mentioning that the use of bevacizumab plus platinum‐based chemotherapy (paclitaxel and carboplatin) is recommended in clinical practice as a standard treatment option for advanced or metastatic NSCLC.The goal of clinical trials on biosimilars is to demonstrate the totality of evidence on clinical equivalence,18 assessed by CIR was selected as the primary efficacy endpoint, following the guidelines on biosimilar development set out by the FDA, EMA and NMPA. PFS and OS were assessed as secondary endpoints, with no significant difference found between the treatment arms in time to median PFS or OS. Secondly, after 18 weeks of study treatment, tumor assessments were performed by investigators only without central review. There may be some variation between investigators in this evaluation, but the randomized, double‐blind design of the study will minimize any variations between groups. Finally, ICIs play an important role in cancer treatment in current clinical practice, and the addition of ICIs to bevacizumab plus chemotherapy has been shown to significantly improve outcomes in patients with NSCLC. This combination regimen was not recommended as a standard treatment option in NSCLC when this study was initiated, and so ICIs were not included in the evaluated treatment regimen. The results of this study demonstrate that BAT1706 shows clinical equivalence to EU‐bevacizumab and add to the totality of evidence that BAT1706 is similar to reference bevacizumab. If BAT1706 receives regulatory approval as a bevacizumab biosimilar, physicians can be confident that BAT1706 will demonstrate clinical equivalence to reference bevacizumab in any approved combination regimen.This study does have some potential limitations. Firstly, OS and PFS were not assessed as primary endpoints; however, it is important to note that this was a pivotal biosimilar clinical study designed to evaluate the clinical equivalence of BAT1706 and reference EU‐bevacizumab, and these endpoints are not sensitive for such an evaluation. Instead, the ORR5This Phase III study has demonstrated that in patients with advanced nonsquamous NSCLC, BAT1706 shows clinical equivalence to EU‐bevacizumab in terms of efficacy, safety, PK, and immunogenicity, when administrated in combination with paclitaxel and carboplatin. These data add to the totality of evidence that BAT1706 is similar to reference bevacizumab.Li‐kun Chen: Conceptualization ; formal analysis ; investigation ; resources (lead); supervision ; writing – review and editing . Jose David Gomez Rangel: Data curation ; investigation ; resources ; writing – review and editing . Timucin Cil: Data curation ; investigation ; resources ; writing – review and editing . Xingya Li: Resources . Irfan Cicin: Resources . Yihong Shen: Data curation ; investigation ; resources ; writing – review and editing . Zhihua Liu: Data curation ; resources ; writing – review and editing . Ozgur Ozyilkan: Resources . Jun Chen: Resources . Zhendong Chen: Resources . Helong Zhang: Resources . Ziyi Fu: Formal analysis ; investigation ; supervision ; writing – original draft (lead); writing – review and editing . Qingfeng Dong: Formal analysis (lead); writing – review and editing (supporting). Shuqiang Song: Conceptualization ; supervision ; writing – review and editing (supporting). Jin‐Chen Yu: Conceptualization (supporting); supervision ; writing – review and editing . Li Zhang: Conceptualization (lead); data curation ; formal analysis ; investigation ; methodology ; writing – original draft ; writing – review and editing (lead). Bondarenko Igor: Data curation ; writing – review and editing .Ziyi Fu, Qingfeng Dong, Shuqiang Song and Jin‐Chen Yu are employees at Bio‐Thera Solution Ltd. All the other authors declared that they have no conflicts of interest.This study was approved by an Independent Ethics Committee or Institutional Review Board. Each participating patient provided informed consent. This study (including the informed consent process) was conducted according to the ethical principles stated in the Declaration of Helsinki , applicable International Council for Harmonization (ICH) Good Clinical Practice Guidelines (CPMP/ICH/135/95), and the applicable drug and data protection laws and regulations of the countries where the clinical study was conducted. Clinical trial registration number: NCT03329911.


Eucalyptus snout beetles are a complex of at least eight cryptic species (Curculionidae: Gonipterus scutellatus complex), native to mainland Australia and Tasmania, that defoliate Eucalyptus trees and are considered important pests. Since the 19th century, three species of the complex have been introduced to other continents. Here, we document the presence of Eucalyptus snout beetles in Ecuador. We used DNA data for species identification and unambiguously demonstrated that the Ecuadorian specimens belong to the species Gonipterus platensis, which has low genetic diversity compared with other species in the complex. We analyzed G. platensis' potential distribution in South America with ecological niche models and found several areas of high to intermediate climatic suitability, even in countries where the pest has not been registered, like Peru and Bolivia. Accurate identification of species in the G. scutellatus complex and understanding of their potential distribution are essential tools for improved management and prevention tactics. Gonipterus platensis, in Ecuador. Ecological niche modeling revealed areas of climatic suitability in South America where this species is present or could invade in the future.We report the presence of the Eucalyptus snout beetle,  These beetles are native to Eastern Australia and Tasmania. Species of the complex have been accidentally introduced into Western Australia (at an unknown date), New Zealand in 1890, Africa in 1916, South America in 1925, Europe in 1975 and North America in 1994  , the larval parasitoid Entedon magnificus (Hymenoptera: Eulophidae), and even entomopathogenic fungi, which could be used alone or in combination in biological control programs  of Pontificia Universidad Católica del Ecuador (PUCE), Quito, Ecuador.Specimens of the For DNA extraction, we left the legs in 500 μL of TE buffer for 24 h at 4°C. After rehydration, each softened leg was crushed with a pestle for 15 min, then 500 μL of CTAB‐PVP and 16 μL of Proteinase K were added to each sample and left shaking at 56°C for 24 h. We then added 650 μL of chloroform to each sample and mixed them to form an emulsion; subsequently, samples were centrifuged for 10 min at 13000 rpm. We removed the supernatant from every sample and saved each in different 1.5 μL tubes. Around 600 μL of 100% Isopropanol were added to every sample for DNA precipitation, and then centrifuged for 10 min at 13,000 rpm. The pellet was then washed with 70% ethanol and centrifuged again for 10 min at 13,000 rpm. The pellet was left to dry, and then resuspended in 25 μL of 0.1X TE Buffer. DNA concentration was quantified using a Nanodrop 1000 from Thermo Scientific.We amplified two fragments of the mitochondrial gene COI with the following pairs of primers: the forward primer Jerry (5′‐CAA CAT TTA TTT TGA TTT TTT GG‐3′) and the reverse primer Pat (5′‐TCC AAT GCA CTA ATC TGC CAT ATT A‐3′) . This alignment tool is versatile because it can detect, and automatically transform, the direction of the sequences. The final matrix consisted of 152 sequences, including two sequences of Oxyops sp. as outgroups, and it had a length of 420 bp. We used IQ‐Tree 1.6.12 , we calculated haplotype diversity (H) and nucleotide diversity (π) with the functions hap.div and nuc.div from the package pegas in R v3.6.3 . The occurrence dataset included few records from the native range of G. platensis in Australia . Therefore, we focused on characterizing the potential distribution of the species only using records from the invaded range in South America. All records corresponded to iNaturalist Research Grade observations . Occurrence records were then verified to ensure georeferencing accuracy, first, by excluding localities without spatial reference and duplicates, and then, by visual examination mapping using the packages sp and maptools .After confirming the records in Ecuador as Hijmans, . The 5 kHijmans, . The fin2.4G. platensis using an ensemble distribution modeling method. Second, we implemented a maximum entropy approach to develop ecological niche models (ENMs). Our goal was to evaluate the predicted distribution of G. platensis in its introduced range throughout South America, with a focus on Ecuador. We used climate data from 19 WorldClim variables summarizing temperature and precipitation features , kernel = radial, and default gamma = 1/(length(data) − 1).Ensemble ENMs were produced using the R package SSDM v0.2.8 and combined four algorithms, generalized linear model (GLM), Maxent, random forest (RF), and support vector machine  in the R package ENMeval2.0 . AUC is a threshold‐independent measure that varies from 0 to 1, where a score of 1 represents perfect discrimination and a score of 0.5 represents a model no better than random  , E. globulus (n = 162), E. grandis (n = 36), E. urophylla (n = 16), and E. viminalis (n = 34). Data are available for download in Zenodo (https://doi.org/10.5281/zenodo.7818068).Eucalyptus trees in South America using a community pSSDM approach in the R package SSDM v0.2.8 , central and southern parts of the Ecuadorian Andes. Comparison of climate envelopes of the four top contributing climatic factors and elevation for Ecuador and the rest of South America highlighted some variation in the occupied niches  m a.s.l.  of 0.96, and good individual model AUC values . This model showed the Andes from Venezuela to Bolivia hosting the highest species richness of Eucalyptus and closely matching the distribution of G. platnensis in that region . However, the data showed a weak fit to the regression model (R2 = .1204).ica FAO, . Our comn Figure . The low4Eucalyptus snout beetle in Ecuador, determine its taxonomic placement, and model potentially suitable habitat throughout South America. With this framework, we provide the first genetic evidence for the presence of G. platensis in Ecuador. Furthermore, our ecological niche model analysis suggests areas of suitable habitat throughout a broad range of climatic and elevational regimes that may have played a role in the establishment of populations of Eucalyptus snout beetles in South America and could promote its future expansion into new areas.We aimed to document the introduction of the G. scutellatus species complex. However, most of the samples analyzed in this study came from Australia and Tasmania, with few samples coming from Portugal, Spain, and South Africa   are the most different compared with the sequences of South America and Tasmania, as evidenced by their long branches in the gene tree  were available in open‐source databases from the native range of Australia (n = 9). Indeed, little research has been carried out on Eucalyptus snout beetles in their native range, probably because in Australia they are only minor pests  for invasive species  in South America, particularly in Colombia and Ecuador. In northern South America little research has been conducted on forestry of Eucalyptus species, whereas in Brazil and Chile, forestry research is quite active. In the particular case of Ecuador, Eucalyptus trees were planted in the late 19th century, mainly in volcanic soils, known as “cangagua,” in the inter‐Andean valleys ; data curation ; project administration (lead); writing – original draft (lead). J. Angel Soto‐Centeno: Conceptualization (supporting); data curation ; formal analysis ; methodology ; writing – original draft (supporting). C. Miguel Pinto: Conceptualization (supporting); formal analysis ; methodology ; writing – original draft (supporting). Ana Avilés: Data curation (supporting); writing – review and editing . Washington Pruna: Data curation (supporting); writing – review and editing . Claudia Terán: Methodology (supporting); writing – review and editing . Álvaro Barragán: Conceptualization (lead); data curation ; project administration (supporting); writing – review and editing .This work was supported by PUCE's Research Directorate to projects: “Presencia del gorgojo del eucalipto en Ecuador y riesgo potencial para América del Sur” and M13434. Work by JAS‐C was partly supported by a National Science Foundation grant DEB:2135257.The authors have declared that no competing interests exist.https://doi.org/10.5281/zenodo.7823745 and https://doi.org/10.5281/zenodo.7818068.This article has earned an Open Data badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at


Pelvic ring fractures requiring surgical stabilization are severe injuries. Surgical site infections occurring after stabilization of the pelvis are serious complications, requiring complex and multidisciplinary treatment.This is a retrospective observational study from a level I trauma centre. One hundred and ninety-two patients who underwent stabilization of closed pelvic ring injuries without signs of pathological fracture were selected for inclusion into the study. After excluding seven patients for having incomplete data, the final study group consisted of 185 patients (117 men and 68 women). Basic epidemiologic data and potential risk factors were recorded and analyzed by Cox regression, Kaplan–Meier curves, and risk ratios in 2 × 2 tables. Categorical variables were compared by Fisher exact tests and chi squared tests. Parametric variables were analyzed with Kruskal–Wallis tests with post hoc Wilcoxon tests.p = 0.0232) and concomitant urogenital trauma (p = 0.0104). The common risk ratio for both these factors was 212.59 (8.78–5148.68), p = 0.0010. No significant risk factors were identified in men despite younger men having a higher incidence of infection (p = 0.1428).Surgical site infections occurred in 13% of the study group (24 from 185). Eighteen infections occurred in men (15.4%) and six in women (8.8%). There were two significant risk factors in women: age over 50 years (Overall rate of infectious complications was higher than in the literature, but this might be caused by inclusion of all patients regardless of surgical strategy. Higher age in women and lower age in men were associated with higher infection rates. Concomitant urogenital trauma was a significant risk factor in women. Pelvic fractures requiring surgical treatment are severe injuries regardless of the patient’s age. They are usually the result of high-energy trauma, such as traffic accidents, falls from height, or impacts from heavy objects. In patients with osteoporosis pelvic fractures can occur following low-energy trauma, such as falls from standing height or less .The stabilization of unstable pelvic ring injuries remains challenging, because of the inadequate posterior soft tissue coverage, complex local anatomy, and biomechanics . Open rep = 0.8318). There were two open pelvic ring injuries, but none was complicated by infection (p = 1.0000). Complex pelvic fracture with Morel-Lavallee lesion was revealed in five patients, also in non-infection group (p = 1.0000). Superficial skin or subcutaneous tissue infection was revealed in nine patients, and none of them was indicated for revision surgery. These patients were not included in the infection group.Medical records from a level I trauma centre  between the years 2009 and 2019 were retrospectively screened to identify all patients who underwent operative treatment for pelvic ring injuries. In total, 192 patients were identified during this period. After excluding patients with pathological fractures and patients with incomplete follow-up data, a total of 185 patients were include into the analysis. There was no significant difference in sex ratio when comparing the total number of patients who underwent operative treatment for pelvic ring fractures  with the patients included in the study   requiring operative treatment. During the primary operation, prophylactic cephalosporin antibiotics were administered, and the duration of antibiotic prophylaxis was 24 to 48 h , most often Cefuroxime or Cefazoline. Twenty-four hours duration was used in patients without risky comorbidities, e.g., diabetes, age up 70, chronic immunosuppressive treatment, malnutrition. Longer prophylaxis (> 48 h) was administrated in case of open pelvic ring fractures. During treatment, we used antibiotics according to the recommendations of the microbiological centre—most often Oxacillin and Clindamycin for staphylococcal infections, Ceftriaxone for G-infections, and a combination of crystalline penicillin and Metronidazole for anaerobic infections.We did not address the issue of orthopaedic surgeon—one experienced surgeon for the operation, in the event that a less experienced one operates, so we considered it irrelevant to evaluate the number of infections according to the surgeon.The time dependence of cumulative incidence of SSI was graphically expressed using Holt’s linear trend, Sen’s slope, and Mann–Kendall test were used for trend analysis.Demographic and epidemiological data, including age, sex, and data on the number of pelvic fractures and infectious complications, were collected and analyzed. We then collected and analyzed data concerning the following potential risk factors: mechanism of injury, type of fracture according to the AO/OTA classification, presence of injuries in other body regions, presence of concomitant urogenital trauma, and the type of implants. Injuries were classified according to the injury severity score (ISS) and presence of trauma of other parts of the body as: monotrauma (injuries of only the pelvis with ISS ≤ 15), combined trauma , and polytrauma .p-value approximation using the z-test and also with the in cases with suitable amounts of data from the results of the log-rank test, when the p-value was calculated from the chi squared test. The Kaplan–Meier graphs show the time until surgical revision for infectious complications individually for each risk factor.Due to the disproportionality of the data, we used two models, one for each gender. The significance of the Cox regression model of each group as a whole was evaluated by an LL test approximated to chi-square test. Apart from the Cox regression model, each risk factor was evaluated graphically using a Kaplan–Meier graph and log-rank test. In cases where more than two risk factors were evaluated at once, we used multiple long-rank tests with post hoc comparison using Fisher’s exact test because Bonferroni correction with paired comparisons was too conservative in regard to the distribution of the individual data. The risk ratio for individual risk factors was calculated using 2 × 2 tables with p-value less than 0.05 as significant. All statistical analyses were performed in MS Excel 2019  using realstat statistical macro  . For performing the comparison of the incidence of infections, we calculated the percentage of infections using the equation: infectionn(infection) represents the number of infections and N represents the total number of patients in the studies. The infection rate was expressed as a percentage from one hundred. The metanalytic like comparison was performed using methods described by Neyeloff et al. 2012 [The groups of women and men were compared using Woolf’s heterogeneity test. Heterogeneity was calculated using common odds ratio . In addition to categorical distribution for age (up to 50 and over 50 years), we also evaluated age quantitatively using the Kruskal–Wallis test with post hoc pairwise comparisons using the Mann–Whitney-Wilcoxon exact test, including not only the effect of gender but also the type of injury according to the AO/OTA classification. We considered all al. 2012 .p = 0.2586 (Fisher exact test). The mean time to the first revision surgery (± 1 SD) was 24.3 ± 11.8 days (95% CI: 19.6–29.0), and all re-operations were performed within two months. The average number or re-operations was 1.9 ± 2 (95% CI: 1.0–2.8) in men vs. 1.2 ± 0.4 (95% CI: 0.8–1.5) in women, p = 0.6261.From the total cohort of 192 patients, 25 severe infections requiring revision surgery  occurred. From the 185 patients included into the study, 24 severe infections occurred . Eighteen of these infections occurred in the group of males (15.4%) and six in the group of females (8.8%), p = 1.0000) .The average annual number of surgical procedures was 17.2 ± 4.8 SD (95% CI: 14.3–20.0). The average annual number of severe infections was 2.3 ± 2.1 SD (95% CI: 1.0–3.5). During the study period, the annual number of infectious complications had ascending trend when using Holt’s linear trend, and this was in concordance with the increasing annual incidence of multiple trauma and in discordance with the total number of pelvic ring stabilization . In the female group, however, the p-value for the Cox regression model was 0.0027. Among the female patients, age over 50 (risk ratio 11.19 (1.39–89.98), p = 0.0232) and coincidence of urogenital trauma (risk ratio = 6.56 (1.56–27.63), p = 0.0104) were identified as significant risk factors in 2 × 2 tables. The coexistence of both these risk factors further increased the risk of infectious complications requiring surgical revision. Further repeated Cox regression analyses were then performed in the female patients, using a select smaller numbers of risk factors. The smallest p-value was obtained by using age (above and below 50 years), urogenital trauma, and AO/OTA classification of pelvic fracture, which generated a p-value of 0.0006. The common risk ratio for age over 50 years and presence of urogenital trauma according to Cox regression was 212.59 , which shows a significant interaction between the two risk factors  and the presence of urogenital injury  and men with (p = 0.0118) and without infections (p = 0.0316). In contrast, there were no significant differences in the age of men with and without infection (p = 0.3697) , age was also evaluated quantitatively as a parametric value in the Kruskal–Wallis test with post hoc comparison using paired Mann–Whitney-Wilcoxon tests in their exact form. Women with infections were significantly older than women without infections  Tables  and 3.Tap < 0.0001). Paired comparisons are shown in the Fig. The effect of age by sex and type of injury according to the AO/OTA classification was tested. Four comparisons were significant (for more details see Table p = 0.0232), Fig. p = 0.0058).In men, the anterior approach proved to be a significant risk factor . Similarly, initial external fixation of the pelvic ring was not associated with a significantly higher risk of serious infectious complications, which may be due to the relatively lower number of patients with external fixation . There was a relatively higher proportion of combined approaches and anterior plates with iliosacral screws in the group of patients with severe infections. Epidemiological data in these patients are shown in Table In women, infectious complications occurred only in cases where combined surgical approaches were used. However, when evaluating of the association of type of approach  with severe infectious, no significant differences were found (Cox regression for the approach p = 0.7410 and 161.7 ± 63.3 (90.0–233.3) min in women with infection vs. 99.5 ± 40.9 (82.0–117.0) with no infection; p = 0.0524.The primary surgical time did not differ significantly although this time was non-significantly longer in women with infection. The mean surgical time (± SD (95% CI)) was 90.0 ± 21.2 (60.6–119.4) min in men with infection vs. 107.6 ± 57.0 (89.5–125.8) without, p = 0.2809; 24.0 ± 6.0(19.2–28.8) in women with infection vs. 23.4 ± 5.6 (21.9–24.9); p = 0.6941).The mean incision length in cm (sum of anterior and posterior incision) did not significantly differ regardless to sex (20.8 ± 6.7 (17.7–23.9) in men with infection vs. 22.7 ± 6.0 (21.4–24.0); Concerning the microbiological agents cultivated from the infected wounds, the most frequent finding was multiple pathogens, and the most common of which were coagulase-negative staphylococci, followed by Gram negative rods. The most frequently cultured pathogens are in Table Soft tissue infections complicating the treatment of pelvic fractures can be classified according to location as occurring outside of the pelvic skeleton within the gluteal, pubic or inguinal regions, or as occurring inside the lesser and greater pelvis, where they may further be classified as extraperitoneal (pelvic pyomyositis) or intraperitoneal (pelveoperitonitis). Infections of the pelvic skeleton include exogenous osteomyelitis, septic sacroiliitis, secondary septic coxitis, and septic symphysitis , 7. One For diagnosing surgical site infection after the operative treatment of pelvic fractures, we rely on local signs of infections, the qSOFA score, laboratory signs , and imaging method findings . Similar diagnostic methods have been recommended by many other authors , 16, 28.Conservative treatment for mild infections consists of regular wound dressing changes with irrigation with antiseptic solutions, local antibiotics, removal of sutures, and excision of devitalized tissue in local anesthesia , 16, 17.Cases of infected non-union are a separate issue. In the anterior segment, in addition to decortication, resection of the non-union and vascularized bone grafts can be used. This is particularly useful for cases of septic destruction of the symphysis. Vascularized fibular autografts are a good option, but require cooperative with a plastic surgeon , 14, 16.The optimal length of antibiotic treatment for pelvic infections is a controversial topic. In our centre for bone and joint infections or secondary pelvic pyomyositis, we give intravenous antibiotics for three weeks and then peroral antibiotics for another three weeks. For isolated soft tissue infections, we give intravenous antibiotics for two weeks, followed by peroral antibiotics for another two weeks. Each case is individualized according to the results of the cultivation, and each case is consulted with a microbiologist  is also gross limitation of our study, but we did not have sufficient data set to assessed covariates such as significant comorbidities , age and sex that all could be associated with change in haemoglobin and albumin regardless of primary pelvic ring injury. Both severe initial anaemia and hypoproteinaemia might be coupled with a poorer functional outcome and with a significantly higher risk of infection. Routine vitamin D level screening was also missing in our study, sarcopenia and muscle weakness cloud be worsened by severe depletion of vitamin D, especially in elderlies. We screened only patient up 65 years—in case of hypovitaminosis, we addressed patients for peroral supplementation.On the other hand, the strengths of the study are its relatively large patient cohort, the inclusion of patients with all types of implants and surgical approach, the graphical evaluation of the results, and the analysis of the annual number of surgical procedures performed for pelvic ring injuries and severe infections requiring reoperations.A relatively high number of serious infectious complications occurred in our cohort of patients who underwent surgery for fractures of the pelvis. Evaluation of the results showed that in female patients being over 50 years of age and having concomitant urogenital trauma were independent risk factors for surgical site infections. The combined effect of having both these risk factors substantially increased the risk of infectious complications. Women in the 60–69 age group and men aged 30–39 were most at risk of infection. Even through type B and C pelvic injuries and cases polytrauma were more frequently associated with infectious complications, this finding did not reach statistical significance. Similarly, the energy of the trauma was also insignificant. Infectious complications occurring in men were more frequently associated with an open approach, while in women they were more frequently associated with a combined approach. Only prospective studies, ideally with multicenter cooperation will be able to definitively elucidate the risk factors for infectious complications after pelvic osteosynthesis and validate of our results. For the diagnosis and treatment of infectious complications after pelvic osteosynthesis, we strongly recommend a multidisciplinary and individualized approach.


Capra are canonical mountain dwellers. Among these is the Siberian ibex (Capra sibirica), which is regarded as a relic species whose intraspecific classification has been controversial so far. We collected 58 samples in Xinjiang, China, and analyzed the mtDNA genes to shed light on the intraspecific relationships of the C. sibirica populations and estimate the divergence time. Intriguingly, we found that the mtDNA sequences of C. sibirica split into two main lineages in both phylogenetic and network analyses: the Southern lineage, sister to Capra falconeri, consisting of samples from Ulugqat, Kagilik (both in Xinjiang), India, and Tajikistan; and the Northern lineage further divided into four monophyletic clades A–D corresponding to their geographic origins. Samples from Urumqi, Sawan, and Arturk formed a distinct monophyletic clade C within the Northern lineage. The genetic distance between the C. sibirica clades ranges from 3.0 to 8.6%, with values of FST between 0.839 and 0.960, indicating notable genetic differentiation. The split of the genus Capra occurred approximately 6.75 Mya during the late Miocene. The Northern lineage diverged around 5.88 Mya, followed by the divergence of Clades A–D from 3.30 to 1.92 Mya during the late Pliocene and early Pleistocene. The radiation between the Southern lineage and C. falconeri occurred at 2.29 Mya during the early Pleistocene. Our results highlight the importance of extensive sampling when relating to genetic studies of alpine mammals and call for further genomic studies to draw definitive conclusions.Maternal lineages of mitochondrial DNA (mtDNA) are recognized as important components of intra and interspecific biodiversity and help us to disclose the phylogeny and divergence times of many taxa. Species of the genus  Capra sibirica split into two main lineages in both phylogenetic and network analyses: the southern lineage, sister to Capra falconeri, and the northern lineage further divided into four monophyletic clades A–D corresponding to their geographic origins. The split of the genus Capra occurred approximately 6.75 Mya during the late Miocene. The northern lineage diverged around 5.88 Mya, following the divergence of Clades A–D from 3.3 to 1.9 Mya during the late Pliocene and early Pleistocene. The radiation between the southern lineage and C. falconeri occurred at 2.29 Mya during the early Pleistocene.Intriguingly, we found that the mtDNA sequences of  It consists of 10 distinct species, such as wild goat/bezoar , domestic goat (C. hircus), markhor , western tur , eastern tur , Iberian ibex , Nubian ibex , Walia ibex , alpine ibex , and Siberian ibex . They are found in areas from sea level to high mountainous habitats, some even at 6700 m above sea level in the Himalayas  gene and control region (CR) sequences derived from feces, skin, or tissue samples collected in Xinjiang, China, together with sequences retrieved from GenBank. We aimed to elucidate the phylogeny and genetic structure of C. sibirica populations. Our findings are significant in terms of identifying conservation management units and providing effective protection.Mitochondrial markers have been widely used in the phylogenetic studies of many taxa, including mammals , and that of muscle and skin samples was extracted using a Tiangen tissue/blood DNA extraction kit , following the manufacturer's instructions. After electrophoresis detection, DNA concentration and purity were measured by the Thermo Nanodrop 1000 and stored at 4°C for later use.cytb gene PCR amplification, CR‐F (5′‐ACCCACGCATACAAACACCC‐3′) and CR‐R (5′‐ACTGTGATGCTCG TGCCTAC‐3′) for CR gene were designed using Primer Premier v.5.0 software and were synthesized commercially. The PCR reactions were carried out in a total volume of 25 μL, containing 40–150 ng of DNA, 5 pmol of each primer, and 12.5 μL of Tiangen's 2× Taq PCR Master Mix, with a final volume of 25 μL adjusted with RNase‐Free dd‐water. The PCR thermal cycling conditions were as follows: pre‐denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 54°C (cytb)/60°C (CR) for 30 s, extension at 72°C for 45 s, and final extension at 72°C for 5 min. PCR products were run on 1% agarose gel electrophoresis to check relative purity and concentration and then were sent to Sangni Biotechnology (Shanghai) for sequencing. Raw sequences obtained were manually edited in SeqMan, aligned with MEGA v.6.0  and Cytb‐R (5′‐ACCAGTATATTAATTGTACTACAAAGAC‐3′) for mtDNA 2.3S), nucleotide diversity (π), haplotype diversity (h), pairwise population fixation indices (FST), Fu's FS and Tajima's D. The genetic distance was calculated using the Kimura two‐parameter model with 1000 bootstraps in MEGA v.6.0.We used MEGA v.6.0  of all Capra species , Himalayan tahr , Bharal , Tibetan antelope , and Gemsbok . The sequences were aligned by MUSCLE in MEGA v.6.0  = 0.095, M = 10.8 Mya); Alcalá & Morales, Divergence time estimation was performed based on mtDNA 0 Edgar, . The nuc0 Edgar, . To esti33.1cytb sequences we obtained was 1095 bp long, and the final dataset of 43 CR sequences was 935 bp long, with indels in some sequences. PCR amplification from a few samples failed , some genetic parameters were not calculated. Due to the fact that the sequences from Ulugqat were split into two distinct lineages in phylogenetic trees and networks, we calculated genetic parameters accordingly, namely separated into two groups  in Table cytb sequences and 20 haplotypes with 168 segregating sites in 43 CR sequences  sequences of other Capra species (their accession numbers were provided in the phylogenetic trees) retrieved from NCBI GenBank. Because the lengths of sequence from NCBI were different and to include more sequences from different locations, phylogenetic trees were constructed based on cytb gene datasets of 376, 679, and 1095 bp, respectively, using the Bharal (Pseudois nayaur) sequence as an outgroup. As a result, all trees estimated using ML and NJ inferences and different datasets yielded highly congruent topologies , clade B from Mongolia, Kazakhstan, and Altai (Russia), while clade C contained sequences from Urumqi, Sawan and Arturk and clade D from Ulugqat, Kyrgyzstan, and Kazakhstan. Intriguingly, more than half of the sequences from Ulugqat and sequences from Kagilik, Tajikistan, and India formed another distinct clade E, the Southern lineage, a monophyletic sister lineage to Markhor . The clustering pattern of haplotype sequences in network analyses was consistent with the phylogenetic topology , while the minimum value (0.839) was between clades A and B  in the Late Miocene epoch. The mean TMRCA of the Northern lineages (clades A–D) of C. sibirica was 3.30 Mya (95%HPD: 2.21–4.46) in the Late Pliocene epoch. The mean TMRCA of the Southern lineage (Clade E) of C. sibirica and C. falconeri was 2.29 Mya (95%HPD: 1.42–3.29) in the Early Pleistocene epoch. Inferred divergence times from 1095 bp cytb sequences were comparable to those from 679 bp cytb sequences, although estimates from 376 bp cytb sequences were marginally higher than those from the other two datasets  occurred in the early Pleistocene epoch  clustering with h Figure . The phy Figures  and 3 wa Figures , and the Figures . Thus, p Figures .C. sibirica has been continuously increasing, and it has recently been downgraded to National Category II from Category I . This species is mainly distributed in the Xinjiang, Inner Mongolia, Tibet, and Gansu provinces of China. In our analyses, samples were collected from five different geographic locations, clustering into three clades, namely clades C, D, and E. Samples from Urumqi, Sawan, and Arturk formed a distinct clade C ; investigation ; validation ; visualization ; writing – original draft (supporting). Pei‐Pei Dong: Formal analysis ; investigation ; validation ; visualization . Daisuke Hirata: Data curation ; formal analysis ; methodology ; validation ; visualization ; writing – original draft (supporting); writing – review and editing . Shamshidin Abduriyim: Conceptualization (lead); data curation ; formal analysis (supporting); funding acquisition (lead); investigation ; methodology ; project administration (lead); resources (lead); supervision (lead); validation (supporting); visualization (supporting); writing – original draft (lead); writing – review and editing .This study was funded by the National Natural Science Foundation of China (No. 32260328).The authors declare no conflict of interest.


Colletotrichum lupini, the causative agent of lupin anthracnose, affects lupin cultivation worldwide. Understanding its population structure and evolutionary potential is crucial to design successful disease management strategies. The objective of this study was to employ population genetics to investigate the diversity, evolutionary dynamics, and molecular basis of the interaction of this notorious lupin pathogen with its host. A collection of globally representative C. lupini isolates was genotyped through triple digest restriction site‐associated DNA sequencing, resulting in a data set of unparalleled resolution. Phylogenetic and structural analysis could distinguish four independent lineages (I–IV). The strong population structure and high overall standardized index of association (r̅d) indicates that C. lupini reproduces clonally. Different morphologies and virulence patterns on white lupin  and Andean lupin (Lupinus mutabilis) were observed between and within clonal lineages. Isolates belonging to lineage II were shown to have a minichromosome that was also partly present in lineage III and IV, but not in lineage I isolates. Variation in the presence of this minichromosome could imply a role in host–pathogen interaction. All four lineages were present in the South American Andes region, which is suggested to be the centre of origin of this species. Only members of lineage II have been found outside South America since the 1990s, indicating it as the current pandemic population. As a seedborne pathogen, C. lupini has mainly spread through infected but symptomless seeds, stressing the importance of phytosanitary measures to prevent future outbreaks of strains that are yet confined to South America. Colletotrichum lupini revealed four distinct lineages, of which one is globally spread, indicated clonal reproduction, and showed variability in the presence/absence of a minichromosome.Population genetics of  After filtering, a total of 9923 and 1863 biallelic and phylogenetically informative SNPs spread over 10 chromosomes were retained for each data set, respectively. The mean sequencing depth was 16 before filtering and 18 after filtering the complete data set  allowed for pairwise comparison between regions. Analysis of molecular variance (AMOVA) showed that most variance (70%) is explained within regions , explaining 99.88% of the observed genetic variance, whereas the observed genetic variance within lineages only accounted for 0.14%. No big difference was observed between clone‐corrected (mlg.filter: 0.05) and nonclone‐corrected (mlg.filter: 0.0008) data sets (Table FST) further support an almost complete genetic differentiation between the lineages (Table r̅d) of 0.662 (p = 0.001) was found for both the clone‐corrected and nonclone‐corrected data sets chromosome present in the reference genome RB211 , this strongly indicates that at least C. lupini lineage II is a clonal population. However, it is likely that C. lupini as a species reproduces purely clonally, but due to limited samples from the presumed centre of origin South America and lineages I, III, and IV, further research is needed to confirm this. In clonal species, recombination can occur but is scarce enough to maintain a pattern of a clonal population structure  and III (JA20) have been found in close proximity in fields near Carhuaz (Peru), which would make genetic exchange theoretically possible. Clonal populations have the ability to quickly spread as they do not need a mating partner to produce large numbers of offspring  described in the reference genome RB221, representing lineage II  at 22°C in the dark as working cultures. Isolates were stored in 25% glycerol at −80°C for long‐term storage. DNA of 2‐week‐old single‐spore cultures was obtained as described in Alkemade, Messmer, Voegele, et al.  for paired‐end (150 bp) sequencing in one lane using a TrueSeq DNA HT prep kit  on an Illumina Miseq instrument. A total of 16 technical replicates were included to detect technical errors.de Table . A total, et al.  using a , et al. . The DNA4.2C. lupini reference genome  for quality control and demultiplexed using process radtags from Stacks v. 2.41  is a measure of multilocus linkage disequilibrium based on the variance of pairwise distances between genotypes to test the null hypothesis of random mating. The IA and standardized index of association (r̅d), taking into account the number of loci, were calculated for both the clone‐corrected and the nonclone‐corrected data set using the ia function of poppr with 999 permutations. An r̅d close to 0 indicates random mating, whereas an r̅d close to 1 indicates clonal reproduction.The index of association (4.6C. lupini reference genome (RB221) were used to predict a potential role in host interaction. We used SignalP v. 6.0  of the 4.7C. lupini isolates JA01 (II), CBS19225 (I), RB121 (I), and JA23 (IV) through stem‐wound inoculation as described by  test (p ≤ 0.05) was applied for pairwise mean comparisons of the different strains within each lupin accession.Virulence tests were performed on white lupin cultivar Feodora and Andean lupin landraces LUP17 and LUP100 with Figure S1. Sequencing depth before filtering (a) and after filtering (b) of the complete data set.Click here for additional data file.Figure S2.Colletotrichum lupini morphology. RB121 and JA23, grouped in lineage I and IV, respectively, are from this study. Isolates indicated with an asterisk are from Alkemade, Messmer, Voegele, et al.  Akaike information criterion (AIC) and (b) Bayesian information criterion (BIC) analysis. Indicated is the most probable number of genetic groups using the SNAPCLUST function AIC and BIC analysis.Click here for additional data file.Figure S4. Discriminant analysis of principal components (DAPC) and clustering of Colletotrichum lupini isolates. (a) Scatterplot of the DAPC using the C. lupini data set. DAPC was conducted with three principal components and three discriminant functions. (b) Clustering of C. lupini isolates. Posterior probabilities are shown on the y‐axis and samples are shown on the x‐axis. Analysis is based on 1863 biallelic single‐nucleotide polymorphisms.Click here for additional data file.File S1. Single‐nucleotide polymorphisms (SNPs), lineages, and chromosome positions.Click here for additional data file.Table S1:Colletotrichum isolates used in this study.Click here for additional data file.Table S2: Sequence similarity between technical replicates.Click here for additional data file.Table S3: Virulence of Colletotrichum lupini on white and Andean lupin. Asterisks indicate isolates screened in this study, data from other isolates come from Alkemade, Messmer, Voegele, et al. (p < 0.05)., et al. . DiffereClick here for additional data file.Table S4: Morphology of Colletotrichum lupini. Asterisks indicate isolates screened in this study, data from other isolates come from Alkemade, Messmer, Voegele, et al. (p < 0.05)., et al. . DiffereClick here for additional data file.Table S5. Diversity statistics of the nonclone‐corrected Colletotrichum lupini data set.Click here for additional data file.Table S6. Diversity statistics of the clone‐corrected Colletotrichum lupini data set.Click here for additional data file.Table S7. Results of the analysis of molecular variance conducted with the Colletotrichum lupini clone‐corrected data set.Click here for additional data file.Table S8. Linkage disequilibrium analysis within Colletotrichum lupini and among local populations.Click here for additional data file.


To explore filtered diffusion‐weighted imaging (fDWI), in comparison with conventional magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI), as a predictor for long‐term locomotor and urodynamic (UD) outcomes in Yucatan minipig model of spinal cord injury (SCI). Additionally, electrical conductivity of neural tissue using D‐waves above and below the injury was measured to assess correlations between fDWI and D‐waves data.axial from fDWI at injury site, fractional anisotropy and radial diffusivity from DTI above the injury site along with measures of edema length and cord width at injury site from T2‐weighted images. Locomotor recovery was assessed pre‐ and weekly post‐SCI through porcine thoracic injury behavior scale (PTIBS) and UD were performed pre‐ and at 12 weeks of SCI. D‐waves latency and amplitude differences were recorded before and immediately after SCI.Eleven minipigs with contusion SCI at T8‐T10 level underwent MRI at 3T 4 h. post‐SCI. Parameters extracted from region of interest analysis included Dp < 0.0001) post‐SCI and were labeled “poor” and “good” recovery. D‐waves amplitude decreased below injury and increased above injury. UD outcomes pre/post SCI changed significantly. Conventional MRI metrics from T2‐weighted images were significantly correlated with diffusion MRI metrics. Daxial at injury epicenter was diminished by over 50% shortly after SCI, and it differentiated between good and poor locomotor recovery and UD outcomes.Two groups of pigs were found based on the PTIBS at week 12 (Similar to small animal studies, fDWI from acute imaging after SCI is a promising predictor for functional outcomes in large animals. Spinal Cord Injury (SCI) currently affects 17,000 people in the US annually.MRI is valuable in the evaluation of SCI. The presence of hemorrhage and length of edema are predictive of SCI outcomes.Following electrical stimulation aimed at the motor cortex, D‐waves reflect the connectivity from motor cortex to the spinal cord via corticospinal tract. D‐waves measured from the epidural space have received little attention as a potential electrophysiological marker of injury severity. D‐waves represent descending motor‐evoked potentials from activated fast‐conducting corticospinal tract following a transcranial electrical stimulus targeting motor cortex.The overall goal of the present pilot study was to use the Yucatan minipig model to explore fDWI, in comparison with conventional MRI and DTI, as a clinical predictor for long‐term locomotor and urodynamic outcomes. Additionally, the electrical conductivity of neural tissue using D‐waves above and below the injury was measured to assess corrections between fDWI and electrophysiological data.This study was approved by the University of Louisville (UofL) Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition and the Animal Welfare Act and Regulations. The UofL animal care program is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). Eleven 5‐month‐old female Yucatan miniature swine  with weights of 17–21 kg were used for this study.After acclimation, animals began positive reinforcement training with a clicker.Once the animals finished clicker and target training, sling training was started to assess urinary tract function with urodynamics.To induce the anesthesia, Ketamine (5 mg/kg IM), Dexmedetomidine (0.04 mg/kg IM), and Glycopyrrolate (0.01 mg/kg IM) were mixed and administered as described before.In ventral recumbency condition a dorsal midline incision was made between T8 and T13 to expose the spinous processes, laminae and pedicles. Laminectomy was performed at T10 level to induce contusion compression injury. In the pedicle two multi‐axial screws were inserted at T11 and T13. Two titanium rods were used to articulate the arm of the impactor and screws and fixed the vertebral segments. To induce contusion SCI a 50‐gm weight impactor dropped from variable heights onto the spinal cord with a mass of 100 gm to cause the compression. Fentanyl patches and Maropitant citrate continued for 3 days postoperatively from day of injury. Propofol was discontinued and replaced with isoflurane (1%–2%) prior to and during MRI imaging on the day of surgery.Following SCI, the animals were monitored continuously by critical care veterinary staff under the direct supervision of a veterinarian for approximately 7 days until the animals met criteria to return to regular housing, which included the ability to ambulate, intake food & water, maintain normal urine output, and maintain normal body temperature. The urinary catheters remained in place until animals voided urine around the urinary catheter . Analgesia was continued with fentanyl transdermal patches for 6 days post‐operatively and meloxicam IV or PO once daily for 3 days post‐operatively. Blood glucose and potassium were monitored and supplemented IV as needed. Cephalosporin antibiotics were continued for 7 days post‐operatively or until urinary catheters were removed.n = 11) . Each sequence was performed with a large sagittal field of view (FOV) to evaluate the whole spine, a sagittal small FOV for higher resolution to cover the site of injury, and several axial locations at the injury site.MRI was performed on a Siemens Skyra 3T at the MRI research facility at the University of Louisville with custom pulse sequence developed in collaboration with the Baylor College of Medicine and the Medical College of Wisconsin. Eleven minipigs were scanned after 4 h post thoracic contusion injury (short‐term) Fig. . Five an2, in addition to four b0 images interleaved into the diffusion encoding table . Each slice in the DTI acquisition was gated to an oxygen saturation signal (SpO2) to reduce motion artifact with 200 ms delay and an acquisition window of 700 ms, resulting in TR ~ 5 s. The DTI acquisition was repeated 5 times with a total acquisition time of ~40 min. fDWI was collected without multiband excitation with 6 axial slices centered at the injury site using the same acquisition parameters, except for TE = 81 ms due to a simplified filter‐probe diffusion encoding scheme . Diffusion directions for fDWI started with a vector perpendicular to the spinal cord (left–right) with b value of 1350 s/mm2, followed by four vectors tilting and increasing this initial vector towards parallel to the cord axis by incrementally adding diffusion weighing in the slice direction . Each fDWI acquisition consisted of (four possible combinations of) polarity reversed repetitions of this 5‐direction filter‐probe diffusion encoding, in addition to one b0 image . The fDWI acquisition was repeated 3–5 times with a total acquisition time of ~13–20 min. Five unique directions were used for fDWI with a simplified filter‐probe table, by slightly tilting a high b‐value vector perpendicular to the spinal cord towards the cord axis . The concept of fDWI is to use the high b‐value diffusion gradients perpendicular to the spinal cord to suppress extracellular water signals which reduces the confounding effects of vasogenic edema prominent in SCI . The diffusion directions applied with lower b‐values parallel to the cord are used to quantify the parallel/axial diffusivity that reflects intra‐axonal hindrances. This concept is illustrated graphically in a previous human study of SCI.DTI was collected superior to the injury site centered at the T3 vertebra, including 12 axial slices with 5 mm thickness each using an inner‐volume echo planar imaging sequencehttps://4dfp.readthedocs.io/en/latest/index.html) and the fDWI Daxial and Dradial maps were generated by in‐house MATLAB scripts. For DTI analysis, anterior white matter (WM), left and right corticospinal tract (CST), posterior column (PC), and gray matter (GM) regions of interest (ROIs) were manually defined based on mean b0 images, mean diffusion‐weighted images, and DTI FA maps. Then, the left and right CST ROIs were merged, and the DTI measures averaged across slices after confirming no statistically significant difference in the DTI measures among the left and right CST and across slices. For fDWI analysis, whole spinal cord regions of interest (ROI) were manually defined based on mean diffusion‐weighted images, which show clear boundary of the spinal cords. We first applied thresholding on the mean images and then manually modified the ROIs. To avoid partial volume effects, we excluded inconclusive boundary voxels (conservative segmentation). The medians of fDWI measures at each slice were used as representative measures, and the lowest Daxial value  across the slices was used for subsequent statistical analysis. Conventional MRI images were quantified in a manner consistent with the NIH CDE recommendations for spinal cord injury, which include: the extent and length of edema (mm), and cord width (mm).The DTI and fDWI image volumes were registered using 2D x‐ and y‐translations on each axial slice. Diff_4dfp generated the DTI maps of fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) in 4dfp tools (D‐waves were recorded before and 10 min after SCI.n = 8) and 12‐weeks post‐injury (n = 8) under awake, non‐sedated conditions , and Prism 9 .PTIBS outcomes were summarized as mean and standard deviation (SD). D‐wave outcomes (latency and amplitude above and below injury) were evaluated by mixed linear models with a random intercept to account for individual variability. Independent variables in the models were recovery type (poor/good), timepoint (pre/post), and their interaction. Least square means and standard errors were used to summarize D‐waves outcomes. Different comparisons were performed through linear contrasts built on the interaction term. The urodynamic parameters  were evaluated using paired p < 0.001)  and good locomotor recovery (>7 point). At 12 weeks post‐injury significant differences were observed between two group of pigs , displacement , impulse  and velocity .Based on PTIBS, injury information's are summarized in Table axial and Dradial values (n = 5) ranged from 1.95 to 2.54 and from 0.28 to 0.40 μm2/ms, respectively. Those at short‐term (n = 10) ranged from 0.81 to 2.49 and from 0.26 to 0.53 μm2/ms, while those at long‐term (n = 5) ranged from 0.88 to 2.05 and from 0.52 to 1.34 μm2/ms, respectively. For the 5 animals with longitudinal studies, Daxial was diminished on the injury site after injury (short‐term) and on all 6 acquired slices at long‐term  and cord width   and FBC (mL) , while no changes were found on the VE (%)  and AFR (mL/s) . The poor locomotor recovery animals show a significant increase on the FBC (mL) , while significant reduction on the VV (mL) , VE (%)  and AFR (mL/s)  were observed.The animals were divided in two separated groups based on the motor recovery (poor locomotor recovery and good locomotor recovery) and the urodynamic parameters were compared between groups. The good locomotor recovery animals show a non‐significant increase on the VV      Fig.  and cord44) Fig. . fDWI de59) Fig. . DTI met49) Fig. .p: 0.0059, R2: 0.59) and 1 unit increase in cord width of the same MRI was associated with a −3.95 change in post‐injury PTIBS . fDWI was also a predictor of PTIBS with 1 unit of Daxial being associated with a preservation of 6.78 in post‐injury PTIBS . The best predictor of PTIBS is conventional MRI cord width, explaining about 70% of variance, followed by Daxial explaining 64% of variance. However, further analysis showed that the 2 predictors were statistically comparable . These results are summarized in Table On the 4‐h post‐injury conventional MRI, a 1 mm increase in edema length was associated with a 0.32 decrease in post‐injury PTIBS  with filter is believed to be more reflective of axonal injury associated with focal varicosities or beading that occurs in injured axons.axial was significantly diminished 4 h after injury and strongly predicted locomotor and lower urinary tract (LUT) recovery (Fig. axial decreases expanded to all 6 slices at 12 weeks post injury, which suggests ongoing degeneration of axons although the presence of inflammatory cells such as activated microglia may influence Daxial as well in long‐term evolving degenerative pathologies. Dradial, which is obtained from the fDWI protocol but does not include the filtering and is therefore akin to RD derived from DTI, did not change much at short‐term but changed more at long‐term. This suggests that degeneration occurred mostly at the injury site during the first 4 h from the injury, and there was degeneration after 4 h from injury both at the injury site and beyond it. We did not compare fDWI with DTI at the injury site, but we did evaluate the effects of DTI 6 segments rostral to the injury epicenter. There were DTI changes above injury consistent with Wallerian degeneration in all WM tracts at 12‐weeks post‐injury, but not so at 4 h post‐injury. These are in agreement with other studies that report DTI changes at sites remote to injury epicenter.Despite numerous studies, DTI has yet to become a standard of care. In an acute SCI setting, the interpretation of DTI parameters such as fractional anisotropy is confounded by edema, hemorrhage, and inflammation. fDWI was developed to overcome some of these limitations of DTI in acute injury to effectively filter or suppress extracellular signals from vasogenic edema using high diffusion weighting perpendicular to the spinal cord axis. The subsequent detection of diffusion parallel to the cord (Dery Fig. . InteresStructural edema has always been considered a good predictor of outcomes after SCI.axial and cord width values obtained on the early stages after SCI correlates with the further urodynamic performance 12 weeks post‐injury. The persistence of neural pathways connecting the cerebral structures and the lumbosacral controlling the LUT can explain the good urodynamic performance in those animals with less damage. However, the underlying inflammation and tissue damage revealed MRI techniques can also play an important role on the plasticity of the spinal pathways controlling the lower urinary tract following SCI.The present study demonstrated the detrimental effects of SCI on urinary functions. It was also demonstrated how different grades of injury produce a different level of bladder recovery after 12 weeks Fig. . All theWe measured motor‐evoked potentials using D‐waves in the epidural space above and below the injury rather than in the muscle. In contrast to Jutzeler et al.Small sample size is a major limitation of the study. A limited number of long‐term animals were used to evaluate the fDWI information, and in good recovery pigs only 3 animals were considered for urodynamic output at week‐12 post‐injury condition. Moreover, in our study no complete injury was included.Using high‐resolution inner‐volume diffusion MRI, we have demonstrated the feasibility of high‐quality microstructural imaging in the thoracic spinal cord of Yucatan pig. However, acquiring interpretable DTI data at the injury site is still challenging, hence DTI was only evaluated at sites remote from the injury epicenter. Moreover, we tried to quantify spared WM/GM in either the axial anatomical or fDWI images at the injury epicenter. However, given the limited image resolution, it was not meaningful in our data.Histopathological findings, though highly valuable to demonstrate neuropathological changes in the WM and GM after injury and could be correlated with MRI and D‐wave findings, were not considered in this study since the goal was to predict later functional recovery from acute MRI.In acute imaging of a large animal SCI model, fDWI correlated strongly with, though not outperforming, known conventional MRI predictors of locomotor recovery and urodynamic outcomes. Our results serve as an important stepping stone to extend the evidence of fDWI in small animal SCI model to clinical application.A. R. U., K. J. W., D. S., S. L., N. M. J., B. R., U. B., H. C., B. M., X. J., and B. M., designed and conceptualized the study. A. S., G. D., K. C., A. D. M., A. R. U., S, M., N. M. J., and M. M., contributed in data collection. A. R. U., B. U. and A. D. M. statistically analyzed the data. A. R. U., A. D. M., K. J. W., U. B., H. C., B. M., X. J., and B. M., interpreted the data. B. M., acquired funding for the study. All authors critically reviewed and approved the final version of the manuscript.The authors declared no conflicts of interest with regards to the current study.Supplementary Fig. 1Click here for additional data file.


MAML2 and EWSR1 were investigated by fluorescent in situ hybridization. Targeted next‐generation sequencing of 161 genes was performed on eight cases. Most MECs had low histological grade , with the presence of TILs  and PD‐L1 combined positive score ranging from 10 to 20 . All cases showed EGFR and AREG overexpression and were fusion negative. Enrichment of genetic alterations was observed in PI3K/AKT/mTOR and cell cycle regulation pathways, while only one case harbored TP53 mutations. This is the first study providing extensive molecular data on breast MECs and the largest collection of cases available to date in the literature. Breast MECs lack TP53 mutations found in high‐grade forms of triple‐negative breast cancers and MAML2 or EWSR1 rearrangements pathognomonic of salivary MECs. Triple‐negativity and PD‐L1 positivity suggest a window of opportunity for immunotherapy in these patients. The EGFR/AREG axis activation, coupled with the mutational patterns in PI3K/AKT/mTOR and cell cycle pathways warrants caution in considering MECs as low‐risk neoplasms.Mucoepidermoid carcinoma (MEC) of the breast is an extremely rare salivary gland‐type tumor characterized by epidermoid, basaloid, intermediate, and/or mucinous cells arranged in solid and cystic patterns. Despite their triple‐negative phenotype, breast MECs are generally considered low‐risk malignancies but their biology is largely unexplored; therefore, guidelines for clinical management are lacking. Here, we sought to characterize the molecular landscape of breast MECs. Thirteen cases were histologically reviewed, characterized for tumor‐infiltrating lymphocytes (TILs), and were subjected to immunohistochemistry for programmed death‐ligand 1 , EGFR, and amphiregulin (AREG). Rearrangements in  Here, we provide the most comprehensive molecular landscape of the largest collection of breast mucoepidermoid carcinoma cases available in the literature to date, reporting the results of immunohistochemistry, next‐generation sequencing and fluorescent in‐situ hybridization. Our analyses confirm that not all these tumors should be regarded as low‐grade triple‐negative breast cancers and therefore warrant deep‐dive research to support individualized treatment strategies. In salivary MECs, the most frequent molecular alteration is the t translocation, resulting in the oncogenic CREB‐regulated transcription coactivator 1 , Milan and Fondazione IRCCS Policlinico San Matteo, Pavia, Italy between 1994 and 2021. Taken together, 13 breast MECs were included in this study and subsequently revised, re‐classified, and re‐graded according to the latest World Health Organization recommendations and the Nottingham histologic grading system, respectively.2.2Four‐micrometer‐thick sections were subjected to Alcian blue (pH 2.5) staining (to demonstrate the presence of intra‐ and extra‐cytoplasmatic mucopolysaccharides) and IHC using anti‐human antibodies against ER, PgR, Ki67, HER2, cytokeratin (CK)7, CK20, CK5/6, p40, p63, programmed death‐ligand 1 , EGFR, AREG, and mismatch repair proteins , as previously described.2.3The evaluation of TILs was performed on 4‐μm‐thick hematoxylins and eosin (H&E)‐stained sections at a magnification of ×200, based on the recommendations by the International TILs Working Group.2.4MAML2 using ZytoLight® SPEC MAML2 dual‐color break‐apart probe . This probe can detect MAML2 rearrangements irrespective of the fusion partner, including the CRTC1‐MAML2 and CRTC3‐MAML2 fusions.EWSR1 translocation. According to the manufacturer's protocols, the nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI), and samples were evaluated by fluorescence microscopy Zeiss Axio Imager Z2 (Zeiss) combined with Metafer4‐MetaCyte system version V 3.14.143 (MetaSystems), as an acquisition system.Four‐micrometer‐thick sections from 11 samples underwent FISH to identify breaks in 2.5n = 8) were used for the analyses. In seven of the eight cases (87.5%), manual microdissection was performed before nucleic acid isolation to enrich tumor cell content using a sterile scalpel. DNA was extracted using the Maxwell® RSC DNA FFPE Kit  following the manufacturer's instructions and then quantified by the QuantiFluor® ONE dsDNA System (Promega) on the Quantus™ Fluorometer (Promega). The mutational analyses were performed through the NGS panel Oncomine Comprehensive Assay (OCA) v3 System (ThermoFisher Scientific), which evaluates the mutational status (single‐nucleotide variants (SNV), insertions/deletions, and copy number variations (CNV)) of 161 cancer‐related and clinically actionable genes, as previously described.https://www.Thermofisher.com/order/catalog/product/A35805). Briefly, 10 ng of genomic DNA was used for the library preparation, and the subsequent Ion 540™ chips (ThermoFisher Scientific) loading was performed automatically on the Ion Chef™ System (ThermoFisher Scientific). Sequencing was performed using the Ion S5™ System (ThermoFisher Scientific) and data were analyzed using the Ion Reporter™ Software (v. 5.16) (ThermoFisher Scientific). Only mutations with an allele frequency ≥5% and with adequate quality metrics were reported. Mutations were classified as actionable/pathogenic based on the annotation in three different publicly available cancer genomics data sets , https://cancer.sanger.ac.uk/cosmic). Clinically relevant and borderline alterations were visually inspected using the Integrative Genomics Viewer (IGV) software . The median absolute pairwise difference (MAPD) metric was used to identify low‐quality samples at risk of generating false results and therefore needed to be excluded; only cases with MAPD of <0.5 were included.Seven unstained slides at 4‐μm‐thick sections from representative formalin‐fixed paraffin‐embedded (FFPE) tissue blocks  targeting seven monomorphic homopolymer biomarkers . At least five out of the seven biomarkers must be fully analyzed to consider the result of the test valid. To be considered as MSI, tumors should harbor alterations in >2 microsatellite loci.33.1n = 10, 77%). The IHC analysis for epithelial and myoepithelial markers confirmed the presence of both types of cells in all tumors. When present, the cystic areas were lined by mucous cells, smaller eosinophilic cells, and solid components  demonstrated basaloid cells gradually merged into epidermoid and mucous cells. All cases but one were Alcianophilic, showing intra‐ and extra‐cytoplasmic deposits of mucopolysaccharides . Despite the presence of squamous differentiation in 4 (31%) cases, no keratinization was observed in all cases, excluding the possible differential diagnosis of breast adenosquamous carcinoma. At the histopathological revision, 2 (15%) cases showed very faint expression of ER in less than 10% of the neoplastic cells and were re‐classified as ER‐low. Furthermore, 3 (23%) HER2‐negative cases displayed a HER2‐low phenotype, that is, IHC score 1+ or 2+ with no gene amplification. The clinicopathologic characteristics of the patients included in this study are summarized in Table All MECs displayed ill‐formed glands featuring mucoid, epidermoid, and intermediate cells and variable degrees of cystic growth patterns within a desmoplastic/fibroid myxoid stroma Figure . The majdes n = 1, 92%. De3.2MAML2 and/or EWSR1 fusions by FISH. No gene rearrangements were detected in the tested cases (n = 9), as shown in Figure To define whether primary breast MECs harbor the hallmark diagnostic biomarker of salivary MECs, we investigated the presence of 3.3MAML2 rearrangements. All cases with enough material for additional ancillary studies (n = 9) showed EGFR overexpression. Among these, 3 (33%), 4 (44%), and 2 (22%) cases were scored by IHC as 3+, 2+, and 1+, respectively. Furthermore, all cases were AREG‐positive   cases which included the recurrent hotspot mutations p.H1047R, p.M1043I, and p.T1025I , and Notch homolog 1, and translocation‐associated (NOTCH1) in 2 (25%) cases as well as AKT1, SWI/SNF‐related matrix‐associated actin‐dependent regulator of chromatin subfamily B member 1 (SMARCB1), KRAS, and BRCA2 in 1 (13%) case, respectively. A pathogenic TP53 mutation (p.C238Y) was observed in one case (#3), which was a high‐grade MEC with HER2‐low expression; this mutation was mutually exclusive with PIK3CA. The CNVs analysis revealed gains in well‐known cancer genes, such as cyclin‐dependent kinase (CDK)2, CDK4, fibroblast growth factor 19 (FGF19), MYC, and KRAS. All the genetic alterations along with the clinicopathological features of the cases included in this study are detailed in Figure AKT1, PIK3CA, serine/threonine kinase 11 (STK11), NF1, tuberous sclerosis complex 2 (TSC2), and FGF3/4, with mutation detected in the vast majority  of cases. The most frequently affected genes of the cell cycle regulation pathways identified in 88% of cases were CDK2, CDK4, cyclin D3 (CCND3), STK11, and MYC. Similarly, tumor suppressor genes, including TP53, STK11, BRCA2, and NF2 were also mutated in 88% of the cases analyzed. Additionally, a relatively high proportion of cases  harbored mutations in genes involved in DNA repair pathways . None of the cases, including those that harbored mutations in MMR genes, showed aberrations in the MMR system when assessed by IHC  cases had sufficient DNA quantity and/or quality for molecular profiling through a comprehensive NGS panel targeting 161 cancer‐related and clinically actionable genes. All cases harbored at least 2 molecular alterations , as shown in the heatmap presented in Figure % cases h5I Table . AdditioHC Table . Cases w4MAML2 and/or EWSR1. This observation confirms on one hand that these rearrangements are not reliable diagnostic biomarkers for breast MECs  and on the other hand it suggests that EGFR and AREG overexpression are not strictly dependent on such genetic changes. In addition, we investigated for the first time the tumor immune microenvironment features of breast MECs, showing TILs and PD‐L1 activation. Finally, we highlight recurrent somatic genetic alterations in cancer genes and highly oncogenic signaling pathways, positing that at least a subset of breast MECs can be more aggressive than other salivary gland‐like TNBCs.Here, we provide the most comprehensive molecular landscape of the largest collection of breast MECs available in the literature to date, delivering new data on this vanishingly rare and potentially underdiagnosed TNBC subtype. Our analyses confirm that not all breast MECs are low‐grade TNBCs suggesting that they should undergo a thorough multidisciplinary discussion for appropriate clinical management. Furthermore, we provide previously unavailable evidence that the EGFR/AREG axis can be activated in breast MEC in the absence of the oncogenic rearrangements occurring in As a group of tumors, TNBC consists of different histologic subtypes, with highly heterogeneous clinical behavior.MAML2 fusions, has been found only in three cases of breast MEC.MAML2 fusion‐negative and EWSR1 fusion‐positive salivary MECs carry a slightly better prognosis.PIK3CA mutations have been suggested to activate an EGFR/extracellular signal‐regulated kinase (ERK) paracrine signaling axis in TNBC.Histologically, breast MECs display extensive morphological similarities with those arising in the salivary glands.The role of the tumor immune microenvironment has not been previously explored in breast MECs. To bridge this gap of knowledge, we characterized and quantified the presence of stromal TILs and tested the PD‐L1 and MMR status. Taken together, our cases were largely characterized by the presence of a different amount of TILs, suggesting an immune activation and possible tumor suppressor mechanisms.TP53 and PIK3CA.PIK3CA to be the most frequently altered gene showing highly recurrent pathogenic mutations in hotspot regions, at variance with a recent report failing to detect TP53 or PIK3CA mutations in two breast MECs.PIK3CA occurring in intermediate‐ or high‐grade MEC but only exceptionally in low‐grade tumors. In both studies, TP53 was the most commonly mutated gene and increased in frequency with increasing MEC grade.PIK3CA alterations are widely detected in the invasive ductal carcinoma population and its triple‐negative subtypes.TP53 mutations were detected in pulmonary MEC, thereby demonstrating that TP53 variations can differ between salivary gland MEC and MEC at other sites.TP53 mutations typically found in high‐grade forms of TNBC and salivary gland MECs but the presence of pathogenic PIK3CA alterations, which in combination with EGFR overexpression could provide the evidence for a potential mechanism of tumorigenesis in breast MEC.Comprehensive molecular studies of TNBC have demonstrated a heterogeneous mutational landscape with frequent genetic alterations in PIK3CA, in most of the cases analyzed, genetic alterations were also detected in other genes of the PI3K/AKT/mTOR pathway suggesting its improper activation in breast MEC and its potential targetability with recently developed drugs.BRCA but also in MMR genes. However, our MMR immunohistochemical analyses, where only one case was found as MMR‐low, were in line with previous reports in TNBC that documented extremely low incidence rates.MMR genes might be mutated, this is not always reflected by MMR deficiency in IHC and/or MSI.Our pathways analysis showed that along with mutations in Considering the rarity of MECs arising in the breast, no widely adopted guidelines are currently available for their clinical management. However, the vast majority of cases reported in the literature have been diagnosed at early stages. In these patients, if high‐risk features are detected  surgery with sentinel lymph node are still recommended.Our study has some limitations. First, given the rarity of MECs arising in the breast, we could analyze a relatively small number of cases, still representing the largest cohort of breast MECs in the literature. Second, considering the limited availability of material, not all the cases were fully analyzed. Third, survival analyses have not been performed, due to the small series of patients investigated. Large prospective studies are needed to assess the clinical outcome of patients with mammary MECs as well as the potential prognostic and predictive value of the biomarkers that are found altered in these tumors. Finally, given the small sample size, we were not able to extend the analysis to immune gene expression profiles through the construction of their immune landscape. This study, however, should be considered hypothesis‐generating and additional multicentric studies, coupled with comprehensive immune analysis would be required to identify MECs immune signatures.MAML2 or EWSR1 rearrangements found in salivary MECs. The immune milieu compared to TNBCs of no special type suggest an immunoediting activation. Finally, the EGFR/AREG axis activation, coupled with the complex patterns of mutations in PI3K/AKT/mTOR and cell cycle regulation pathways militate against the widely accepted belief that MECs should be invariably considered low‐risk TNBCs.In conclusion, breast MECs lack the hallmark TP53 typically found in high‐grade subtypes of TNBC and Konstantinos Venetis: Data curation (lead); formal analysis ; investigation ; methodology ; software ; validation (lead); visualization ; writing – original draft ; writing – review and editing . Elham Sajjadi: Data curation (lead); formal analysis ; investigation ; methodology ; project administration (lead); validation ; visualization ; writing – review and editing . Mariia Ivanova: Formal analysis ; investigation ; methodology ; project administration ; visualization ; writing – review and editing . Silvia Andaloro: Data curation ; investigation ; writing – original draft . Simona Pessina: Formal analysis ; visualization . Chiara Zanetti: Formal analysis ; visualization . Alberto Ranghiero: Data curation . Gabriele Citelli: Investigation . Chiara Rossi: Formal analysis ; investigation . Marco Lucioni: Formal analysis ; investigation ; resources ; supervision . Umberto Malapelle: Methodology ; supervision ; writing – review and editing . Fabio Pagni: Supervision ; validation ; writing – review and editing . Massimo Barberis: Resources . Elena Guerini‐Rocco: Funding acquisition ; methodology ; supervision ; validation ; visualization ; writing – review and editing . Giuseppe Viale: Formal analysis ; methodology ; resources ; supervision ; writing – review and editing . Nicola Fusco: Conceptualization, Funding acquisition ; methodology ; supervision ; validation ; visualization ; resources ; writing – review and editing .This research was funded by the Italian Ministry of Health with Ricerca Corrente funds.U.M. has received personal fees (as a consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Eli Lilly, Diaceutics, Jannsen, Diatech, Hedera, GSK, Merck and AstraZeneca. M.B. from MSD Oncology, Roche/Genetech, Astra Zeneca, Thermofisher Scientific, and Illumina. E.G.R. from Thermo Fisher Scientific, Novartis, AstraZeneca, Roche, Biocartis, and Illumina. G.V. from MSD Oncology, Pfizer, Dako, Roche/Genetech, Astellas Pharma, Novartis, Bayer, Daiichi; Sankyo, Menarini, Ventana Medical Systems Dako/Agilent Technologies, Cepheid, and Celgene. N.F. from Merck Sharp & Dohme (MSD), Boehringer Ingelheim, Novartis, AstraZeneca, and Daiichi‐Sankyo. These companies had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and/or in the decision to publish the results. All other authors declare no potential conflicts of interest.This study is in line with the local ethical guidelines and was approved by the local Ethical Committee under protocol number #UID3472.Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Table S1.Table S2.Table S3.Table S4.Click here for additional data file.