Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.The budding yeast  Summary: We show, through biochemical reconstitution experiments and live-cell imaging, that functions and localization of Kar3 are dictated by the stage of the cell cycle and the activities of two Kar3 accessory binding partners – Cik1 and Vik1. Saccharomyces cerevisiae, MTs in vegetatively growing cells have only one essential function: to faithfully segregate sister chromatids to daughter cells during mitosis  generates spindle forces , properlDissecting the functions specific to each type of Kar3 heterodimer provided a unique opportunity to investigate a novel mode of kinesin regulation; however, this has been challenging. The activity of Kar3 heterodimers on MTs was first described as a minus-end-directed translocation with low processivity , a propein vitro assays of Kar3 function and regulation cited above did provide insights into possible Kar3 functions, but inconsistency of the results is also a source of confusion. While these studies are pioneering efforts to elucidate Kar3 function, they might have suffered from complications, such as use of heterogeneous assay constituents and innovative but, nevertheless, non-physiological recombinant fusion proteins to stably construct the two Kar3 heterodimers. These earlier reconstitution assays might also have suffered from a lack of post-translational modifications present in native yeast cells. Importantly, these modifications are likely to be specific to the cell cycle phase. Also missing from previous in vitro MT dynamics studies using purified proteins is the complete panoply of microtubule-associated proteins (MAPs) present within the cell and, thus, the possibility to uncover emergent properties of this system in its full complexity.The in vitro assay to reveal effects of two distinct Kar3 heterodimers on MT dynamics in the context of the complexity of a cell at each stage of the cell cycle. We also describe association of Kar3 heterodimers with, and locomotion along, MTs. In total, we show that Kar3Cik1 and Kar3Vik1 have similar but distinct motility profiles and functions that are regulated in the cell cycle and intrinsic to the respective heterodimer. Complementary in vivo studies demonstrate the applicability of these results to live cells.We have previously developed a biochemical reconstitution assay that uses yeast lysates and harnesses the genetic tractability of budding yeast while enabling imaging of assembly dynamics of single MTs, which is very difficult in living cells . This asS. cerevisiae strains that express GFP-Kar3 fusion protein from the native KAR3 locus. These strains also express mRUBY2-TUB1 , S phase (cdc7-1), metaphase (cdc23-1) or late anaphase (cdc15-2)  . Arrestecdc15-2) A. The tearyogamy . Cells aaryogamy B. When caryogamy . For 97.We quantified the GFP-Kar3 signal along spindles in S phase-, metaphase- and anaphase-arrested cells C. FluoreGFP-TUB1 and arrested the cells by using conditional cdc alleles, as above. Interestingly, we found that Cik1-mScarlet was not detectable in G1-arrested cells   paired wed cells D. By coned cells E, indicaed cells . Cik1-mSTo fully quantify these localization patterns, we also measured the relative fluorescence intensities of either Cik1-mScarlet or Vik1-mScarlet and compared them to the relative fluorescence intensity of GFP-Tub1 along the spindle in cells arrested in metaphase . The peaCIK1-AID or VIK1-AID allele into the tsGFP-KAR3 mRUBY2-TUB1 cdc strains used above. The AID motif is an auxin-inducible degron that triggers proteolysis of the tagged protein in the presence of 3-indole acetic acid (3-IAA)  . The AID (3-IAA) . The amo (3-IAA) . These sEffects of Vik1 depletion were consistent with the difference in heterodimer localization B. DuringTo confirm the change in Kar3 localization during metaphase upon depletion of each of the accessory proteins, we quantitatively analyzed the fluorescence intensity of the GFP-Kar3 signal along the spindle D. The vaCIK1-AID and VIK1-AID alleles together with GFP-KAR3 and mRUBY2-TUB1. This strain also contains the cdc23-1 allele, which enabled us to arrest cells in metaphase. When these cells were arrested and the Kar3-binding partners depleted as above, GFP-Kar3 was detected near the SPBs in 3.0% of cells or, when in association with the nuclear MTs, in only 7.3% of cells   A. Spindl1Δ cells , which i1Δ cells B, sugges1Δ cells B. Cells GFP-TUB1, an AID-tagged protein as well as temperature-sensitive CDC-mutant alleles that allows arrest at different cell cycle stages. We found that cells depleted of Kar3 had an increased number of cMTs in S phase and metaphase  in Cik1-depleted and Vik1-depleted lysates. MTs disassembled at a similar speed in Cik1-depleted lysates or faster when Vik1 was depleted  (−3 events s−1)  B. Globalctively) C. MTs innts s−1) . These dnts s−1)  and our nts s−1) D, which nts s−1) .In S phase-arrested lysates depleted of Kar3, Cik1 or Vik1, MT assembly and disassembly rates both increased relative to rates in lysates from S phase-arrested WT cells D,E. The −1) and disassembly (1.03±0.28 vs 1.51±0.91 µm min−1) rates both increased relative to rates in WT lysates  tracking the plus-end of a growing MT  inowing MT B, (3) moowing MT C, and  in Vik1-depleted lysates had motors moving in minus-end direction. These data suggest that the minus-end-directed motility observed in our assay depends on both Kar3 heterodimers being present in metaphase lysates.In metaphase-arrested lysates, GFP-Kar3 was observed tracking the plus-end in 79.5±0.6% of observed MTs. This number dropped to only 4.9±4.9% of MTs when Cik1 was depleted. However, GFP-Kar3 was observed tracking plus-ends in 85.3±0.5% of MTs in Vik1-depleted lysates. These observations demonstrate that Cik1 is also necessary for Kar3 to track plus-ends during metaphase. In WT extracts, GFP-Kar3 interacted with stabilized MT minus-ends in 34.4±2.8% of MTs but fewer (8.1±8.1% of MTs) instances were observed in Cik1-depleted lysates and even fewer (1.5±1.5%) in Vik1-depleted lysates. This observation matches our CIK1-AID) or from translocating towards the plus-end along the lattice. In contrast, Vik1 depletion decreased the number of MTs with GFP-Kar3 on the minus-end, GFP-Kar3 molecules with plus-end-directed motility, and static interactions of GFP-Kar3 along MT lattices. Additionally, diffuse GFP-Kar3 signals were now observed along the lengths of MTs in Cik1-depleted lysates during anaphase  in metaphase-arrested cell lysates, which is much slower than previously reported in vitro rates  reported by others for Kar3Cik1  at 25°C. Strains containing AID tag alleles were treated with a 1:2000 dilution of 0.5 M 3-indole acetic acid  in 100% DMSO  and buffered with 50 mM potassium phosphate buffer at pH 6.2 for the 30 min prior to harvesting or imaging. Lysates were prepared with a SPEX cryogenic grinder  as described by z-stack series spaced 0.5 µm apart were taken every 30 s for 10 min.Strains were grown overnight in rich medium at permissive temperature. They were then diluted to OD=0.1 in casamino acid medium  supplemented with amino acids and grown at permissive temperature to early log phase. Cells were then shifted to 37°C for 3.5 h. If necessary, 3-IAA was added in the last 30 min of arrest to concentrations listed above. Cells were then mounted onto coverslips coated with concanavalin A  and covered with pre-warmed medium. Microscopy was performed on a Nikon Eclipse Ti2 with a Nikon 60× Plan Apochromat objective and a 1.5× booster  using an Orca Flash 4.0 camera . Images were captured using Nikon Elements software. Temperature was maintained with a heated chamber and objective collar . GFP was excited using a 488-laser line. mScarlet-I and mRUBY2 were excited using a 561-laser line. Eleven slice Cleared lysates were prepared as described by 2O and left in a 2% Hellmanex III  solution for 2 h. Afterwards, the cover glasses were rinsed three times with ddH2O and dried with nitrogen gas. Cover glasses were then laid over a 50 µl drop of 100 mM HEPES, 1 mg ml−1 PLL-g-PEG and 1 mg ml−1 PLL-PEG-Biotin for 1 h in a humidity chamber. Cover glasses were then rinsed with 1× PBS for 2 min and then in ddH2O for 2 min. Finally, they were dried with nitrogen and stored at 4°C.Glass slides were washed once for 30 min with acetone and then washed once for 30 min with 96% ethanol, and left to air dry. Cover glasses were passivated by sonication in acetone for 30 min, followed by a 15-min soak in 96% ethanol. The cover glasses were then rinsed three times with ddHAssembly of flow chambers for TIRF imaging was performed as described by For MT dynamics assays, lysates were prepared as described by in vitro, lysates were prepared as above with the following modifications. To enrich the population of soluble Kar3, frozen lysate powder was not diluted with 10× PEM buffer. Instead, 0.5 µl of Protease Inhibitor Cocktail IV was added to 0.24 g of lysate powder. The mixture was thawed on ice for 10 min and then sonicated for 2 s  using a W-385 sonicator with a #419 microtip  prior to ultracentrifugation.For characterization of GFP-Kar3 on microtubules MT dynamics reconstitution assays were performed on a Nikon Eclipse Ti2 inverted TIRF microscope using a Nikon 60× Plan Apochromat TIRF objective (NA 1.49) with a 1.5× booster lens  in a temperature-controlled chamber  warmed to 28°C and captured with an Orca Flash 4.0 camera  operated by Nikon Elements software. Single-plane TIRF images of 488 nm and 561 nm illumination were taken every 5 s for 30 min.z-plane every 5 s for 20 min.Studies of motors on microtubules were performed on Nikon Eclipse Ti2 inverted microscope using a Nikon 60× CFI Apo TIRF objective (NA 1.49) and an Orca Fusion Gen III sCMOS camera  at 1.5× magnification using the Nikon NIS Elements software. Using a LUNF 4-line laser launch  and an iLas2 TIRF/FRAP module  total internal reflection fluorescence (TIRF) was performed to capture a single Image files were analyzed using Fiji (NIH). Statistics were calculated using Graphpad Prism . For finding an average spindle length, 50 spindles from either metaphase- or anaphase-arrested genotypes were counted. The average lengths of cytoplasmic MTs and the average number of cytoplasmic MTs per cell was calculated. This was done by counting and measuring the length and number of visible MTs outside the nucleus from 50 cells for each condition at the time-point zero. Statistical significance was determined by one-way ANOVA and using the Kruskal–Wallis test comparing the depleted strains to the WT. Fluorescence intensity along spindles was calculated by subtracting background fluorescence with a rolling ball average radius of 25 pixels, a sliding parabola and disabled smoothing. A 5-pixel wide line was used to measure SPB intensities from their center. Spindles were standardized to 100 points similar as described previously . Each poKymographs for measuring dynamics were constructed from all MTs whose entire length was trackable for the entire 30-min observation window after registration. Dynamics parameters and statistics were calculated as described by Click here for additional data file.10.1242/joces.260621_sup1Supplementary informationClick here for additional data file.


Arthroplasty is the final treatment option for maintaining mobility and quality of life in many primary degenerative and (post-) traumatic joint diseases. Identification of research output and potential deficits for specific subspecialties may be an important measure to achieve long-term improvement of patient care in this field.Using specific search terms and Boolean operators, all studies published since 1945 to the subgroups of arthroplasty listed in the Web of Science Core Collection were included. All identified publications were analysed according to bibliometric standards, and comparative conclusions were drawn regarding the scientific merit of each subgroup.Most publications investigated the subgroups of septic surgery and materials followed by approach, navigation, aseptic loosening, robotic and enhanced recovery after surgery (ERAS). In the last 5 years, research in the fields of robotic and ERAS achieved the highest relative increase in publications In contrast, research on aseptic loosening has continued to lose interest over the last 5 years. Publications on robotics and materials received the most funding on average while those on aseptic loosening received the least. Most publications originated from USA, Germany, and England, except for research on ERAS in which Denmark stood out. Relatively, publications on aseptic loosening received the most citations, whereas the absolute scientific interest was highest for the topic infection.In this bibliometric subgroup analysis, the primary scientific outputs focused on septic complications and materials research in the field of arthroplasty. With decreasing publication output and the least financial support, intensification of research on aseptic loosening is urgently recommended. Arthroplasty is the final treatment option for maintaining mobility and quality of life for patients with many primary degenerative and (post-) traumatic joint diseases. The steadily increasing number of primary and revision surgeries is based on continuous scientific advancements in this field . MinimalThe number of joint replacements worldwide is expected to continuously increase in the forthcoming years –11. FurtWithin the framework of a bibliometric study, the development of the scientific performance in individual research areas can be investigated in accordance with scientific standards. This involves recording all publications in a thematic field. Subsequently, by analysing the baseline data, conclusions are drawn about the quantity of published research papers. Further analyses allow conclusions on the quality of the research . This enables scientifically substantiated statements and comparisons to be made on the individual areas of research. Whereby the different research areas within arthroplasty related research should receive balanced scientific attention depending on the clinical relevance. Since only a balanced research effort can lead to complete coverage of all clinical questions.This study is based on the hypothesis that increasing publication pressure –19, diffThe data collection was carried out using the Web of Science Core Collection of the worldwide established multi-disciplinary search platform for bibliographic database Web of Science™ (WoS) –26.To include as many publications on the subject of arthroplasty as possible, the search terms arthroplast*, prostheti*, periprosth*, replacement* were combined with the joint specific searchterms: hip, shoulder, joint*, elbow, knee, ankle, femo*, humer*, tibia*, glenoid*.The research on arthroplasty was divided by the authors into the most clinically as well as scientifically relevant subgroups: Periprosthetic infections and septic surgery (infection), aseptic loosening, surgical approach (approach), enhanced recovery after surgery (ERAS), perioperative navigation and robotic assisted surgery (navigation/robotic), and material science . The most clinically and scientifically relevant keywords were used as search terms. The search terms, which can be obtained from We utilized the analysis function of the Web of Science to identify the total number of publications, publications by individual countries, institutes, and authors, as well as their respective publication counts. Additionally, we identified funding agencies associated with the publications and the journals in which they were published. The resulting data set was then transferred to an Excel table . To determine relative publishing performance, further statistical processing was performed using GraphPad PRISM v. 9.3.1. .To further investigate the funding agencies, they were manually assigned to either the private sector  or the non-private sector  according to their economic background which was determined by a manually online investigation.https://jcr.clarivate.com). For journals that are no longer published, the IF of the last year of publication was used. If the journal had been renamed in the meantime, the IF of the current journal was used. The geographical assignment was made according to the information in the Journal Citation Report.The journals were manually ranked regarding their current impact factor (IF) of the Journal Citation Report  followed by materials , navigation/robotics , approach , aseptic loosening  and ERAS (390 publications) accounted for the largest number of publications .Publications regarding materials dominated the research landscape until 2015. Since 2016, there has been an exponential increase in publications on the topic of infection .The topics of infection and materials each had the highest number of authors and institutes . The topic ERAS had the lowest number of authors and institutes .On average, more than 2 institutes participated in a publication, with the most collaborations for the ERAS topic area . Most co-authorships  were found on the topic of infection .A total of 1,065 different journals were used to publish on the various topics. Among these, the distribution was most diversified in absolute terms for infection and in relative terms for ERAS .The average impact factor of the journals in which research papers were published was highest for materials and lowest for navigation/robotic .In research on the topic of materials, publications received the highest average funding .In this context, the private sector provided varying proportions of funding. Publications on the topic of navigation/robotic were by far the most frequently funded by the private sector. Publications on the topic of ERAS received below-average private-sector funding  .Except for ERAS, where authors from Denmark published the most, authors from the United States published the most in all topics. The distribution of publications of the leading 10 countries can be seen in In absolute terms, publications on the topic of infection were cited most frequently. In relation to the number of publications, the topic of aseptic loosening received the most citations . Thus, despite low publication performance, aseptic loosening showed a higher h-index than the topics approach and navigation/robotic .There were marked differences regarding self-citations between the various subject areas. Specifically, only 5.23% of the citations on the topic of aseptic loosening were self-citations. In the field of infection, 43.24% of all publications were self-citations  .In this study, we were able to show for the first time that publication patterns differ remarkably between subgroups in the research field of arthroplasty. The topics of infection and materials received a comparatively higher level of attention, whereas aseptic loosening and ERAS had fewer publications dedicated to them. Even though the number of researchers and institutes involved in each of these areas was relatively equal, the different research fields received varying degrees of funding. In the topics infection and materials, approximately every second publication received funding. For the topics aseptic loosening and approach, on the other hand, not even every third publication received funding. Despite the lower publication output in aseptic loosening, these publications received on average the highest number of citations and were published in journals with comparatively high impact factors.For a considerable period, the research landscape has been dominated by publications focusing on new materials and types of prostheses. In the last 10 years, however, there has been a surge in the publication rate on the topic of periprosthetic infections. Most studies rank aseptic loosening as the main reason for revision surgery –33. HoweIn general, all authors strive to publish as many papers as possible in journals with the highest possible impact factors. The number of publications, the achieved citations and h-index increase the scientific reputation of the individual and ultimately determine academic success –37. SelfAs defined in Journal Citation Reports, a self-citation does not refer to an author citing their own work, but rather to a citation of a work from the same journal in which the author's research is published. Therefore, the high self-citation rate may be attributed to a more concentrated distribution of publications across fewer journals, leading to a higher rate of citations within a given journal. However, whether this is the sole reason for the significant discrepancy requires further investigation through self-citation-centered bibliometric studies. The relatively new research fields of ERAS and navigation/robotic have become increasingly relevant in recent years.Navigation/robotic, supported by the highest funding rate from the private sector, has recorded a distinct increase in publications. In addition to funding, this can certainly be explained by advances in computer technology and the general progress of digitization.The clinical and scientific landscape in the field of joint replacement is currently characterized by increasing technologization, as demonstrated in this study. The new digital possibilities are intended to increase intraoperative precision and improve outcomes . The incLike all bibliometric studies, this study is subject to some limitations. Unspecific search terms result in off-topic publications, while over-specific combinations can lead to the exclusion of certain relevant publications. Even though the Web of Science databases are among the most comprehensive databases, not all publications are represented and there are deficits especially with regard to non-English publications . The useIn this bibliometric subgroup analysis, the primary scientific outputs focused on septic complications and materials research in the field of arthroplasty, which is also reflected in a higher rate of financial funding in these topics. Even if publications on aseptic loosening were less frequently published and financially supported, the publications were cited more frequently on average. Even though a bibliometric work can never make absolute statements on the contents due to the underlying methodology, it can be concluded that the clinically highly relevant research field of aseptic loosening should receive increased financial support. This will promote the improvement of patient care in the long term.


To evaluate antibiotic prescribing in ambulatory oncology clinics and to identify opportunities to improve antibiotic use.Retrospective cohort of adult patients who received care at 4 ambulatory oncology clinics from May 2021 to December 2021. Patients were included if they actively followed with a hematologist-oncologist for a cancer diagnosis and received an antibiotic prescription for uncomplicated upper respiratory tract infection (URTI), lower respiratory tract infection (LRTI), urinary tract infection (UTI), or acute bacterial skin–skin structure infection (ABSSSI) at an oncology clinic. The primary outcome was receipt of optimal antibiotic therapy, defined as a composite of drug, dose, and duration according to local and national guidelines. Patient characteristics were described and compared; predictors of optimal antibiotic use were identified using multivariable logistic regression.P = .057).In total, 200 patients were included in this study: 72 (36%) received optimal antibiotics and 128 (64%) received suboptimal antibiotics. The proportions of patients receiving optimal therapy by indication were ABSSSI (52%), UTI (35%), URTI (27%), and LRTI (15%). The most common suboptimal prescribing components were dose (54%), selection (53%) and duration (23%). After adjusting for female sex and LRTI, ABSSSI  was associated with optimal antibiotic therapy. Antibiotic-associated adverse drug events occurred in 7 patients; 6 occurred patients who received prolonged durations and 1 occurred in a patient who received an optimal duration (Suboptimal antibiotic prescribing in ambulatory oncology clinics is common and mostly driven by antibiotic selection and dosing. Duration of therapy may also be an area for improvement as national oncology guidelines have not adopted short-course therapy. Antimicrobial stewardship is challenging in cancer patients due to uncertainties in infection diagnostics, complexities in immunocompromised population care, and the increased risk of infection and death due to multidrug-resistant organisms. Although contemporary cancer care has evolved from predominantly hospital-based practice to ambulatory settings , the majority of antimicrobial stewardship efforts and practice remain in hospital settings. Ambulatory antimicrobial stewardship efforts are less mature and represent an area of needed growth, particularly in high-risk populations such as those with cancer.Advances in cancer therapeutics, testing, and antimicrobials with activity against multidrug-resistant organisms have allowed patients with cancer to live longer, but ∼60% of cancer-related deaths are due to infection. In 2019, healthcare providers prescribed 251 million outpatient antibiotic prescriptions, and up to 50% were considered inappropriate. However, antibiotic prescribing practices in ambulatory oncology clinics are relatively unknown. This lack of data, challenges to prescriber education, fear of bad outcomes, just-in-case mentality, and the clinician perception of the patient demand for antibiotics are all drivers of suboptimal antibiotic use that are applicable to ambulatory oncology patients. Although more data are needed, current literature suggests that antimicrobial stewardship interventions developed and studied in general populations can be safely extrapolated to patients with cancer.Despite the high burden of infection, cancer patients are not well represented or are excluded from antimicrobial stewardship literature altogether, especially in ambulatory settings where antibiotic overuse is most common. In this study, we evaluated antimicrobial prescribing in ambulatory oncology across a healthcare system that caters to a diverse patient population to identify areas to improve antibiotic use. We hypothesized that most antibiotics prescribed would be suboptimal, which would also lead to a high incidence of adverse events.Forming a baseline understanding of antibiotic prescribing practices and adherence to antimicrobial stewardship guidelines in the ambulatory oncology population is necessary to design and implement meaningful antimicrobial stewardship interventions.This retrospective cohort study was conducted across 4 outpatient cancer clinics within Henry Ford Health in southeastern Michigan. The study received institutional review board approval (no. 15246-01) with waiver of consent.Patients were included in the study if they received an antibiotic prescription for an upper respiratory tract infection (URTI), lower respiratory tract infection (LRTI), urinary tract infection (UTI), or acute bacterial skin and skin structure infection (ABSSSI) from an ambulatory oncology clinic between May 1, 2021, and December 31, 2021. Additional inclusion criteria were age ≥18 years and active follow-up with a hematologist or oncologist for a cancer diagnosis at the time of care. Patients were excluded if they had multiple infections, were pregnant, were breastfeeding, were currently enrolled in a clinical trial, had been diagnosed with febrile neutropenia, or had had a previous treatment for infection of interest in the prior 30 days. Patients with a history of solid-organ or bone marrow transplant were excluded due to complexity in comparison to hematology-oncology populations and to prevent heterogeneity.Patient data were manually reviewed and extracted from the electronic health record (EHR) using the Epic Slicer Dicer data extraction tool . Data extraction parameters were set to identify patients who received an antibiotic prescription in an ambulatory oncology clinic during the prespecified timeframe. The identified patients were then randomized and screened for inclusion. Data collection included patient demographics, comorbid conditions including cancer diagnosis, infection characteristics , and antibiotic treatment. Patient outcomes included treatment failure, delayed chemotherapy, and adverse drug events. All data were captured by a single investigator using a standardized electronic case report form.S1). The cohort was divided according to receipt of suboptimal or optimal treatment. Infection types were categorized according to medical record documentation; LRTI included chronic pulmonary obstructive disorder (COPD) exacerbation and community-acquired pneumonia (CAP); URTI included pharyngitis, bronchitis, and sinusitis; ABSSSI included purulent and nonpurulent cellulitis, abscess, and infected bite injuries; UTI included lower urinary tract infections  and pyelonephritis. The full definitions of optimal regimens by disease state are listed in Supplementary Table 1. An analysis of prolonged antibiotic durations distinct from NCCN duration of therapy recommendations was performed and was defined as days of therapy exceeding institutional guideline recommended short-course durations (Supplementary Table S1).The primary end point of optimal antibiotic therapy was defined in accordance with the National Comprehensive Cancer Network (NCCN) guidelines and institutional antimicrobial stewardship guidelines was defined in accordance with the standard of care. Optimal treatment was defined as a composite of optimal drug, dose, and duration  while on antibiotic therapy. We defined 30-day treatment failure as a documented change in antibiotic due to a lack of clinical improvement or due to a documented antibiotic adverse drug event, hospital admission due to worsening index infection, or development of recurrent infection. Only ADEs that were suspected to be due to the antibiotics the patient received were captured, as documented by the treating provider in the electronic health record (EHR).All types of encounters including clinic visits, telephone encounters, and Epic MyChart EHR messages were reviewed for up to 30 days after antibiotics were initiated to find a documented secondary end point. Patients were presumed to be end point free at 30 days if they had no documented encounters.This study was designed to detect a 20% difference between any 2 variables associated with receipt of optimal antibiotics and assumed a baseline prevalence of 30% suboptimal antibiotic use. Assuming a type 1 error frequency of 5% and a power of 80%, a sample size of 200 patients was targeted. The assumption of 30% suboptimal antibiotic use was derived from prior internal data related to outpatient antibiotic prescribing at Henry Ford Health.2 or Fisher exact test, and continuous variables were compared using the Mann-Whitney U test. To determine variables independently associated with optimal antibiotic prescribing, variables associated with the outcome (P < .25) from bivariate analysis were entered into a multivariable regression model using a backward, stepwise approach. Variables included in the model were restricted to an event-to-variable ratio of 10:1; model fit was assessed using the Hosmer-Lemeshow goodness of fit test. A separate a priori analysis of optimal versus prolonged antibiotic duration of therapy was performed to assess associations with patient harm. All statistics were calculated using SPSS Statistics for Windows version 26.0 software .Descriptive statistics , were used to describe the cohorts of optimal and suboptimal antibiotic use. In bivariate analyses, categorical variables were compared using the Pearson χIn total, 200 patients were included in the study; 64% of patients received a suboptimal antibiotic course and 36% received an optimal antibiotic course. The most common infection types were UTI 41%), ABSSSI (31%), LRTI (17%), and URTI (11%). Cohort characteristics are listed in Table %, ABSSSIThe proportion of optimal and suboptimal antibiotic use by indication is depicted in Figure Among patients with LRTI indications, CAP was notably associated with suboptimal antibiotic use due to selection of monotherapy instead of a CAP with comorbidities combination regimen or respiratory fluoroquinolone. Additional components of suboptimal antibiotic use by infection type are listed in Table Based on the results of the bivariate analyses and clinical rational, the following variables were included in a multivariable logistic regression model: female sex, public insurance, LRTI, ABSSSI, and presence of central venous catheter Table . Other vP = .819). Also, there was no statistically significant difference in the proportion of patients that had a delay in chemotherapy , or any antibiotic-associated ADE  between the optimal and suboptimal groups.There was no statistically significant difference in the proportion of patients who experienced a 30-day treatment failure in the optimal and suboptimal antibiotic groups, respectively  included non–face-to-face encounter , female sex , ECOG score of 0 , history of infection or colonization with a multidrug-resistant organism within the previous year , history of IV antibiotic use within the previous 3 months , and ABSSSI . Outcomes associated with prolonged duration included recurrent infection within 30 days  and antibiotic-associated ADE .Of 200 antibiotic regimens, 47% consisted of a prolonged duration that was defined as a duration outside the local guideline recommended short-course therapy and was distinct from more permissive NCCN recommendations. Variables associated with prolonged duration from bivariate analysis  was expected because NCCN guidelines were utilized for the primary end point and had more lenient recommendations. The results of the present study are consistent with other literature that has evaluated antibiotic appropriateness in ambulatory oncology, in which prospective audit and feedback is not routinely performed. Future opportunities include use of EHR programming and decision support to optimize antibiotic selection, dose, and duration. Chew et al reported a low appropriateness of duration prescribed, which was attributed to the lack of a pre-set duration in the EHR. In response to these data, our institution implemented new EHR programming for pneumonia in patients with comorbidities, which prepopulates guideline-recommended regimens. Additional research is needed on short-course therapy. Existing randomized trials support short courses for febrile neutropenia. If shorter is better for the neutropenic host, we speculate that short-course therapy may also be safely applied to other oncology patients.Albeit limited, studies do exist that support stewardship efforts in immunosuppressed patients.This study had several limitations. The retrospective study design was appropriate given the nature of the research question, but data collection was dependent on information documented within the EHR and vulnerable to patient loss to follow-up. However, with the ease of accessibility of providers through messaging or telephone, this information was captured if patients were inclined to notify their provider about ADEs. There was no application of strict definition for infection or colonization when considering optimal prescribing, and additional opportunities may exist to reduce unnecessary antibiotics for asymptomatic bacteriuria, bronchitis, and sinusitis. The external validity of study findings could be limited by our institution’s demographics and antibiotic prescribing patterns.In conclusion, our findings suggest that opportunities for optimal antibiotic prescribing exist in ambulatory cancer care. Antimicrobial stewardship programs should leverage the EHR and existing clinical pharmacist infrastructure in ambulatory oncology clinics as a future direction for interventions to improve antibiotic prescribing and avoid patient harm. The design and implementation of meaningful and pragmatic antimicrobial stewardship interventions that promote appropriate short-course antibiotic therapy in ambulatory oncology clinics are an additional focus.


Operating-room audiovisual recording is increasingly proposed, although its ethical implications need elucidation. The aim of this systematic review was to examine the published literature on ethical aspects regarding operating-room recording.MEDLINE (via PubMed), Embase, and Cochrane databases were systematically searched for articles describing ethical aspects regarding surgical  recording from database inception to the present (the last search was undertaken in July 2022). Medical subject headings used in the search included ‘operating room’, ‘surgery’, ‘video recording’, ‘black box’, ‘ethics’, ‘consent’, ‘confidentiality’, ‘privacy’, and more. Title, abstract, and full-text screening determined relevance. The quality of studies was assessed using Centre for Evidence-Based Medicine grading and no formal assessment of risk of bias was attempted given the theoretical nature of the data collected.From 1048 citations, 22 publications met the inclusion criteria, with three more added from their references. There was evident geographical (21 were from North America/Europe) and recency  bias and an exclusive patient/clinician perspective (25 of 25). The varied methodology  and evidence level (14 level V and 10 level III/IV) prevented meaningful systematic grading/meta-analysis. Publications were narratively analysed for ethical thematic content  that was then grouped by the four principles of biomedical ethics of Beauchamp and Childress, accounting for 63 distinct considerations concerning beneficence , non-maleficence , justice , and autonomy . From this, a set of proposed guidelines on the use of operative data is presented.For a surgical video to be a truly valuable resource, its potential benefits must be more fully weighed against its potential disadvantages, so that any derived instruments have a solid ethical foundation. Universal, ethical, best-practice guidelines are needed to protect clinicians, patients, and society. Operating-room audiovisual recording is increasingly proposed, although its ethical implications need elucidation. The aim of this systematic review was to examine the published literature on ethical aspects regarding operating-room recording. Indeed, since the 19th century2, surgeons have performed operations specifically in rooms called ‘theatres’, initially in front of crowds either standing or in tiered seating. More recently, as modern surgery developed aseptic techniques and environments, as well as defined training curricula3, operating rooms (OR) often include viewing areas and screens for trainees and other observers to watch. With the broad move towards minimally invasive surgery with digital cameras and the capability for surgical video display, transmission, and recording, large audiences are now being reintroduced to surgery. Surgeries can now be watched back for surgical (including patient and public) education, training, and development4. In conjunction with artificial intelligence (AI) models, surgical video recordings enable the creation of OR black boxes (ORBB)6 that have the potential to further transform the field by providing richer detail than traditional operative notes, with AI promising automatic postoperative analysis and insights and potentially intraoperative decision support8. Thus, surgical videos now have more interest and value than ever before.The concept of surgery as a spectacle is not new. There is a long and continuing tradition of operative performance being independently viewed9–14, and both the European General Data Protection Regulation (GDPR)15 and US Health Insurance Portability and Accountability Act (HIPAA)16 provide legal frameworks to protect citizen privacy, but ethical considerations have been less formally addressed . There are methods, however, such as the framework of the principles of biomedical ethics of Beauchamp and Childress17 , which can help to identify, address, and ultimately solve ethical medical dilemmas.Yet, there are concerns over the ethical and legal implications of surgical video recording and the resulting data processing, both in terms of its sourcing and use. Some legal aspects have been discussed elsewhereThe aim of this systematic review was to examine the published literature on ethical aspects of OR data collected via video and/or audio recording, using the principles of Beauchamp and Childress to categorize perspectives, and consider their consequences for individuals  and also the general population. Like all systematic reviews, its purpose was to draw together and analyse the existing evidence base, to identify current best practice and gaps that need to be addressed in the future.19—CRD42022348406) was completed in accordance with the PRISMA20 guidelines.This systematic review . The references of included publications were also searched to ensure completeness.MEDLINE (via PubMed), Embase, and Cochrane databases were searched from database inception to the present (the last search was undertaken in July 2022). There were no limits or restrictions on the basis of date or language of publication at the time of searching; however, only articles available in English were included in the full-text review. Medical subject headings used in the search included ‘operating room’, ‘surgery’, ‘video recording’, ‘black box’, ‘ethics’, ‘consent’, ‘confidentiality’, ‘privacy’, and more  in the title or abstract in relation to surgical videos and data and medical device recorders. Original research  studies, as well as reviews and commentaries, published in peer-reviewed journals were included.Surgical audiovisual recording was defined as any recording from intracorporeal recording devices , as well as medical device recorders recording the OR and/or the procedure itself (panoramic video), including ORBBTable 1. The text and reference lists of included articles were manually searched for additional articles of interest, which were assessed according to the same outlined inclusion and exclusion criteria. Discrepancies that occurred at the title- and abstract-screening stages were resolved by automatic inclusion. Discrepancies at the full-text stage were resolved by consensus between the two reviewers and, if disagreement persisted, a third reviewer (R.A.C.) was consulted.After the removal of duplicates, titles and abstracts were screened for relevance by two reviewers (R.W. and E.C.K.). R.W. and E.C.K. then reviewed the full-text articles according to the inclusion and exclusion criteria as outlined in 33 grading and assessment followed the guidelines of the National Centre for Research Methods on conducting narrative synthesis35. A publication’s perspectives were grouped thematically under one or more of the four principles of biomedical ethics of Beauchamp and Childress: beneficence, non-maleficence, autonomy, and justice8–11. The total number of times each ethical principle was discussed was tallied, taking into account the fact that multiple principles could be discussed in each paper, and the data presented systematically. No formal assessment of risk of bias was attempted given the theoretical nature of the data collected.Relevant data were collected by a single reviewer (R.W.) using a predefined pro forma. Data were sought on the following items from each included article: study characteristics , study design, and ethical aspect. The quality of studies was assessed using Centre for Evidence-Based Medicine (CEBM)Fig. 1. Following the search strategy, 1048 potentially eligible publications were identified. After removing duplicates, unrelated fields, abstracts without full text, and non-relevant papers, 22 manuscripts met the inclusion criteria, with a further 3 publications being added from their references.A PRISMA flow diagram of the study selection process is presented in Table 2) of greatly varying CEBM quality . Given this diversity, it was not possible to formally grade the evidence or to undertake statistical analysis. Included publications were therefore synthesized narratively.Publications were primarily from North America (52 per cent) and Europe (32 per cent), but also came from Australia, Asia, and South America. Publication types included commentaries and surveys/interviews, as well as descriptive, opinion, and narrative reviews . Beneficence was discussed in 22 of 25 publications, comprising 35 per cent of total discussion detail, with non-maleficence, justice, and autonomy being discussed in 17 (27 per cent), 14 (22 per cent), and ten (16 per cent) papers respectively . Considerations over confidentiality (15 publications), ownership (11 publications), and consent (9 publications) were also raised frequently.Benefits discussed included performance (13 publications), education and research (11 publications), transparency and improved patient understanding (7 publications), quality improvement and safety (10 publications), and field advancement through AI (3 publications).post-hoc case review, improvement in technical skill, and the Hawthorne effect. Eight publications discussed the learning and reflective opportunity from re-watching operations49. Six discussed how such analysis can improve operative skill (including improved error detection) and one review discussed the use of motion-analysis software and AI to examine and aid improvement44. Six also discussed the Hawthorne effect , which is defined as a change in normal behaviour when individuals are aware they are being observed. Four publications52 acknowledged that this could have a positive or negative effect, with improvement being possibly attributed to increased accountability, attentiveness, and meticulousness, whereas a negative effect could be due to anxiety, stress, or theatrics. Enhanced performance due to improved intraoperative communication was also discussed55, as was the use of the recording to optimize OR dynamics46.Positive change in performance was attributed, in general, to 44, intraoperative engagement22, and targeted feedback21. Two articles discussed the benefits of recording for all theatre staff in learning OR dynamics55.All publications concerning education/research advocated that operative recording could benefit surgical trainees either generally or more specifically through video-based coaching and tele-mentoring56. Six proposed that such recording provides a clear objective record of what happened during surgery instead of relying on memory and self-reported dictation/notes, thus reducing bias in the OR by ensuring that there is a record of all operative steps that might otherwise be missed57. Four of the seven articles discussed improved patient understanding, which can lead to more informed health decisions46 and even help patients come to terms with the emotional trauma of surgery46. Recordings may also act as an aid to open disclosure and duty of candour when explaining complications to patients58.Seven articles discussed transparency and increased patient understanding as a key benefit of operative data recording54. Operative recording allows the types of errors assessed to be broken down into technical  and non-technical  errors, either alone  or in combination.Eight publications specified quality-improvement usefulness through the detection of OR errors, with two publications attributing this to improved documentation and audit36, with another stating that AI access to outcome-linked operative videos could allow for quality improvement, allow for new technique and technology development, and give us a better insight into surgery itself39. A further review delineated the use of motion-analysis software and AI pattern recognition, already in use in ophthalmic surgery44.One author group discussed how AI can be used to look for patterns associated with success and failure, to identify better surgeonsConcerns discussed related to privacy and confidentiality , storage and security (5 publications), and open discussion (3 publications).59 (15 publications) with reference to European and US privacy laws. A cross-sectional survey of operating-theatre staff showed that 45 per cent of respondents were concerned about OR recording data security. The personal privacy of both patients and OR staff themselves was considered in five publications. One study involving in-theatre recording using a Google Glass headset quantified this risk to patients as 0.4 exposures or potential privacy breaches per minute. Exposures included patients’ faces or any identifying information being accidentally recorded, with such an exposure occurring every 2.5 min of operating time43. Two publications offered data encryption38 and anonymization51 to mitigate these concerns.Patient confidentiality was the main area of discussion52. OR recording has the potential to limit engagement46, as questions may not be asked or answered so freely. Any unwillingness to engage in open discussion may have the added effect of reducing performance if intraoperative communication is silenced or censored52.Three publications discussed the potential harms of OR recordings and their consequences and concerns regarding education and surgical performance60. File names should not contain patient identifiers. For example38, research groups that need to associate performance analysis with surgical outcomes can use a two-way patient identifier hashing mechanism to encrypt medical record numbers and allow the association between specific patient videos and their outcomes without sharing any sensitive data with a third party47.The recommendations for OR recording security ranged from physical, under lock and key, to encryption techniques (two-way hashing mechanism), password protection, and anti-hacking or firewall software with storage media, including physical discs, software platforms, and cloud databasesRespect for patient autonomy was discussed in 10 of the included publications, including implications with regard to consent and data use (9 publications), as well as editing of data (5 publications).60 recommended that the surgical team also provide consent for recording. In cases where consent cannot be obtained, one study stressed that recording may not be pursued42, whereas two claimed that it can, provided that it is an emergency situation55 and/or consent is acquired before any use of the data52. Three publications suggested that the scope of the data, including potential uses , must be discussed at the time of consent and that each potential use should be explicitly stated52. In a survey, a majority of interviewees felt recordings should be restricted to medical professionals. Regarding withdrawal of consent, three publications stated this should be possible at any time52 and should not affect patient care52. No direct comparison was made between the opinions of patients and physicians.All of these publications recommended that consent is obtained from patients and three publications52 relies on edits, as otherwise the data would be too large and viewing would be too time-consuming39. However, editing and compressing an hours-long operation into a few minutes introduces bias47 and is likened by two authors to tampering with the medical record52 or hiding physician mistakes40. Two publications recommended that the original file be maintained52.Much of the utility of operative videos for education, publication, and evaluationThe principle of the fair treatment of individuals, as well as the equitable allocation of healthcare resources, was discussed in relation to data ownership (11 publications), cost (2 publications), credentialling (5 publications), and distributing surgical knowledge (3 publications).55, while one recommended that they are allowed to view the recording52. Four argued for its inclusion in the medical record and, as such, the ownership may fall to the institution in which it was created56, but this was considered not necessarily the case if the recording was for quality-improvement initiatives47. Three publications argued the opposite, stating that anonymized data should not be included in the medical record39 and two suggested no definitive owner54. One study argued that the surgeon may be entitled to an operative video given that the gestures of the surgery represent the culmination of the surgeon’s education and experience38. Two of the above recommended that it should be explicit during the consent process whose property the video recording will be52. Only one proposed shared data ownership between multiple stakeholders and raised the issue of ownership regarding potential monetization39.A key ethical and legal topic discussed was that of data ownership. Eleven publications debated who owned the surgical data acquired and their recommendation of stakeholder varied greatly. Two stated that patients own their own video recording40. One publication argued the opposite, stating that if recording improves performance and outcomes, then the system overall will be cost saving36, although no formal cost–benefit analyses were performed.In a cross-sectional survey, the cost of OR recording was raised by a minority of subjects61. This can also be extended to senior surgeons instead of traditional surrogates (for example outcomes or volumes)44. OR recording may also reduce derogatory OR behaviour (discussed in two publications)40.Improved credentialling of trainees (three publications), reduced discriminatory behaviours (two publications), and better assessment of surgical skill (two publications) were proposed. Four publications suggested that OR recording materially evidences performance and can be used to objectively track a trainee’s progress, reducing bias in career advancement62 to lower-income countries was discussed, with two publications suggesting that open use of video recording allows new procedures to be better disseminated and ultimately makes surgery better52. One study suggested that audiovisual recording coupled with tele-mentoring would allow expert surgical advice to be given at a distance43.The use of videos to allow for more even and equitable distribution of surgical knowledge63. Surgical videos, with their high frame number, contrast, and content, qualify as big data64, which can be analysed by AI65. It is hoped that such detailed machine analysis can provide new metrics and identify otherwise hidden patterns indicative of success and failure, giving better insight66. Surgery is not unique in looking to recordings to improve quality. For comparison, black box recorders were first made mandatory in the airline industry in 196067 and all new cars produced within the European Union from 2022 require ‘electronic data recorders’68. However, whereas historically medicine and medical ethics have taken a paternalistic approach to the doctor–patient relationship, this has changed dramatically in recent decades. Prompted in part by multiple controversies surrounding instances of this relationship being abused throughout the 20th century69–71 and previously , greater importance is now rightly placed on patient autonomy and shared decision-making. Therefore, it is important to consider deeply how surgical recording sits ethically in the modern era of clinical practice as much as the mere technological capability.Although the premise is old, understanding and advancing surgery through its observation has been recently portrayed as a new field and one which is rapidly expanding due to a new capability to aggregate and store recordings and use automated methods to analyse them72–74), quality-improvement measures more generally , and surgical-device development (including post-marketing surveillance) are clear areas where targeted quality initiatives addressing both technical and non-technical aspects of surgery can be augmented through the use of surgical videos, such use cases cannot be simply advocated in isolation from patient, practitioner, and societal rights, responsibilities, and obligations. Non-maleficence for instance challenges beneficence if privacy concerns are disrespected and autonomy and justice also need consideration regarding recording consent and the storing, editing, and fair and equitable use of a video, allocation of resources, and ownership respectively. Until all aspects are fully considered, it is premature to conclude, as several publications do47, that implementing surgical recording falls already and automatically under a surgeon’s professional duty of care. Overall, publications in this systematic review focus more heavily on positive potential and only on the individuals immediately involved (that is patient and clinician privacy) without considering the implications of widespread operative recording . Further, there seems to be a dichotomy between published opinions regarding privacy between patients and staff, with patients, but not staff, being concerned about their individual privacy, despite evident exposure risks. Also, importantly, all publications found by systematic search were published in the 21st century (and the majority since 2010) and also North America and Europe (84 per cent) were predominant as the origin (similar to a 2021 systematic review that found 94 per cent of 70 380 recordings were similarly originated78). This is possibly because the technological capability is often commercially provided and the perhaps greater potential for commercial exploitation by healthcare practitioners/providers in more technology-driven capitalist economies.To help with this, as in other areas, the pillars of Beauchamp and Childress can be usefully applied, weighing beneficence against the issues presented in the other pillars. While surgical training  may pose severe risks for this in the near future. Traditionally, consent for data recording should be able to be withdrawn at any stage, but more sophisticated analytic methods make processed data intrinsic to the methods, making complete removal of such data where the data has already been used  difficult if not impossible.It is clear from this systematic review that data management and ownership are crucial areas for further clarification and research. Legal frameworks that require researchers to make sure that personal data collected from patients and healthcare professionals are used fairly and lawfully, for limited and specifically stated purposes, in an adequate, relevant, and considered manner, and kept safe and secure and stored for no longer than is absolutely necessary80. Interestingly, privacy laws in Europe already include the caveat that data collected for healthcare quality improvement may not be required to be added to a patient’s medical record . If the same holds true for anonymized surgical recordings, a question arises over legal ownership and potential ‘secondary’-use restrictions. This is particularly important when the potential commercial use of these data is considered versus other important outputs that need weighing, such as public interest and societal good through better surgery. As the commercial value of ‘big data’ becomes apparent to the general population, controversies have arisen where medical data generated for research purposes were subsequently utilized for commercial purposes without the consent of the participants84. Certainly the idea of possession  ascribing ownership should not be automatically assumed. Another question that arises is whether patients should be allowed to view or hold a copy of their recording? There are many possible reasons why, alongside garnering evidence for potential medical malpractice, including increased transparency in ORs, understanding procedures and practices, and even patients’ or their loved ones’ recovery from the emotional trauma of surgery56. Patient ownership of such material naturally confers similar responsibilities (including in such matters as safe storage and restrictions regarding public sharing), as surgical videos may contain data belonging to others . There are also concerns regarding harm or the risk of misinterpretation in viewing surgical videos, especially perhaps when the outcome is known given that the standard is reasonable competence and not perfection and no precise definition as yet exists as to what represents error versus acceptable variation. To this end, some publications promote a shared ownership model  and/or a catalogued library of the operation, which may prove sufficient for many of the reasons operations may need to be viewed.Data ownership is a much debated concept and term, with many different aspects, ranging from ethical aspects, enshrined in the principles of autonomy, to overlapping legal aspects, ranging from privacy protection and personal rights to intellectual-property rights40 and knowledge distribution52 were discussed, overall, there was a paucity of literature pertaining to surgical video recording and the general population. Therefore, it was not possible to obtain an adequate estimation of the impact of the ethical implications of OR recording on the general population and society. Ethical considerations are not as amenable to the standard systematic review models predicated on measurable interventions, outcomes, and evidence, and, too often, there is overlap with legal aspects. Nonetheless, this systematic review generally serves the purpose of showing the gaps present in the current literature.There are a number of limitations to this systematic review, mostly relating to the quality of the publications, as discussed above. While societal costSurgical video recording is a growing reality with great potential, but which also presents ethical concerns. Certain general principles are already clear, yet to make this capability truly beneficial and fully operational, many issues still need to be addressed. With clarity, sincerity of purpose, and a responsible, balanced, proportional approach that takes into account practical realities, the value of this resource can be sustainably realized. While jurisdictions may differ, we as a surgical, scientific, and professional community have a responsibility, not only to apply existing ethical and legal frameworks, but to develop broadly applicable and workable guidelines. Given the complexity of the issues, and noting that many relevant aspects, such as liability and bias, still have to be covered, this will require close collaboration of interdisciplinary and inclusive teams, regulators, and society.zrad063_Supplementary_DataClick here for additional data file.


MET deregulation, primarily due to exon 14 skipping mutations or MET amplification. Some MET TKIs, including capmatinib and tepotinib, have proven to be highly effective in this molecularly defined subgroup of patients and are already approved for clinical use. Other similar agents are being tested in early-stage clinical trials with promising antitumor activity. The purpose of this review is to provide an overview of MET signaling pathways, MET oncogenic alterations primarily focusing on exon 14 skipping mutations, and the laboratory techniques used to detect MET alterations. Furthermore, we will summarize the currently available clinical data and ongoing studies on MET inhibitors, as well as the mechanisms of resistance to MET TKIs and new potential strategies, including combinatorial approaches, to improve the clinical outcomes of MET exon 14-altered NSCLC patients.In recent years, we have seen the development and approval for clinical use of an increasing number of therapeutic agents against actionable oncogenic drivers in metastatic non-small cell lung cancer (NSCLC). Among them, selective inhibitors, including tyrosine kinase inhibitors (TKIs) and monoclonal antibodies targeting the mesenchymal–epithelial transition (MET) receptor, have been studied in patients with advanced NSCLC with  EGFR) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) or rearrangements of anaplastic lymphoma kinase (ALK) or c-ros oncogene 1 (ROS1), consists of targeted therapies (www.nccn.org NCCN guidelines NSCLC version 3 2022 accessed on 14 May 2023) , immunotherapy has become the standard of care, as reported by updated ESMO guidelines, while a combination of chemotherapy and immunotherapy is recommended for most patients with negative or low PD-L1 expression .KRAS), the human epidermal growth factor receptor 2 (HER2), the mesenchymal-epithelial transition (MET) and rearrangements of the neurotrophic tyrosine receptor kinase (NTRK) and the rearranged during transfection (RET) genes, have gained significant attention because of the development of selective inhibitors, some of which have already received approval for clinical use in pretreated NSCLC patients [Recently, emerging biomarkers, including genetic alterations of the Kirsten rat sarcoma viral oncogene homolog  or MET amplification (MET-amp).MET-directed therapies, including tyrosine kinase inhibitors (TKIs) and monoclonal antibodies targeting MET, MET ligands, or the hepatocyte growth factor (HGF) ,6,7, havwww.FDA.gov accessed on 14 May 2023).Currently, two selective MET TKIs, capmatinib and tepotinib, have been demonstrated to be highly effective in this molecularly defined subgroup of patients, as showed in the GEOMETRY Mono-1 and VISION trials, respectively, and have obtained FDA and EMA approval. Moreover, other similar agents are being tested in early-phase clinical trials with promising anti-tumor activity ,8,9 . The extracellular portion of MET is made up of three types of domains: the N-terminal segment, folded to form a large semaphorin-like (SEMA) domain including the whole alpha subunit and part of the beta subunit; the plexin-semaphorin-integrin (PSI) domain which follows the SEMA and includes four disulfide bridges; in turn, the PSI is connected with four immunoglobulin-plexin-transcription factor (IPT) domains .The stability and degradation of the MET receptor are regulated by the intracellular juxtamembrane domain, encoded by exon 14 of the gene, containing a tyrosine residue (Y1003). The phosphorylated Tyr1003 works as a binding site for the casitas-B-lineage lymphoma (CBL) E3 ubiquitin ligase that is able to ubiquitylate MET leading to internalization of the receptor from the cell membrane to endocytic vesicles and subsequently to lysosome-mediated degradation ,14.The c-MET ligand was primarily identified both as a factor regulating motility and as a growth factor for hepatocytes, and only later recognized as the same molecule: the hepatocyte growth factor (HGF), also known as scatter factor (SF) . HGF actOther downstream pathways of c-MET are the PI3K/AKT, the STAT and the NF-κB pathways, involved in cell proliferation, differentiation, cell motility, invasion and survival. The p85 subunit of PI3K can bind c-MET directly or indirectly via GAB1, which in turn results in AKT/protein kinase B activation. This axis is responsible for c-MET signaling-mediated cell survival . MoreoveAlterations of the MET pathway are responsible for the tumorigenesis of many solid neoplasms. In NSCLC, this dysregulation occurs through mechanisms of gene mutation, gene amplification, rearrangement and protein overexpression .MET exon 14, such as point mutations, insertions and deletions have been identified in patients with NSCLC [Several alterations of th NSCLC . As commth NSCLC .MET exon 14 mutations was immunohistochemistry (IHC); however this has proved to be ineffective in clinical practice as it is characterized by a non-negligible rate of false negative results. FISH (fluorescence in situ hybridization) is used to identify copy number variations but, in the case of MET alterations, this method is burdened by a large laboratory-dependent variability. Real-time polymerase chain reaction (RT-PCR) is a low-cost method characterized by a high diagnostic sensitivity and therefore widely used in clinical practice to identify this mutation. However, the fragmentation of nucleic acids can invalidate the use of this technique in cases of scarce tissue samples. To date, the most useful method to detect MET exon 14 skipping mutations is next generation sequencing (NGS), which is characterized by a higher sensitivity. As for the detection of other gene alterations in NSCLC and also for MET exon 14 skipping, different NGS assays can be used, based on the analysis of DNA or RNA extracted from tumor tissue. Targeted amplicon-mediated DNA-based NGS is widely used in clinical practice; however, it may not be able to identify a part of the MET alterations, due to the high variety and specific localization of these mutations [MET exon 14 skipping than the amplicon/DNA-based, being able to identify this alteration even in a significant proportion of DNA-sequencing driver-negative cases [Point mutations are the most common alterations 49.5%) and are responsible for exon 14 skipping during pre-mRNA splicing, leading to the loss of the CBL-binding site and increasing the half-life of the MET receptor . Furtherutations . Anotherve cases . Despiteve cases . MET altve cases . However.5% and aMET exon 14 skipping undergoing NGS both on tumor tissue and on plasma samples, allowed the identification and classification of new variants of this alteration, using the in silico SpliceAI tool for skipping prediction. This method can help to define as skipping variants those with a delta score above 0.315 with a determination ratio of 0.92% [MET exon 14 skipping mutations and its variants, to allow for the best therapeutic choice.Finally, a recent retrospective analysis conducted on 4233 patients affected by NSCLC with MET exon 14 skipping mutation is more frequent in the adenocarcinoma histotype and in elderly patients (median age 77 years) with a history of smoking [KRAS, EGFR, ALK, ROS1, and RET and frequently coexist with the TP53 mutation [MET exon 14 skipping mutation has been associated with marked sensitivity to MET tyrosine kinase inhibitors (TKIs) [Regarding clinical and pathological features of MET-altered tumors, the  smoking . However smoking . De novomutation . The pres (TKIs) ,9.MET is amplification of the wild-type gene located on chromosome 7 (MET-amp). MET-amp can be evaluated by two different methods. The first is based on the identification of the gene copy number (GCN). MET-amp is defined as the presence of five or more copies of MET per cell (MET GCN ≥ 5). However, this method does not distinguish the amplification from polysomy [MET amplification (gene copy number increase) from MET polysomy (chromosome 7 copy number increase) [Another mechanism of oncogenic activation of polysomy . With thncrease) .MET-amp tumors, the MET/CEP7 ratio is high. The presence of one of these criteria identifies MET-amp tumors: MET/CEP7 ratio ≥1.8; >10% of cells with clusters of MET signals; or MET copy ≥15. Polysomy 7 was considered with MET/CEP7 ratio <1.8 and MET copy ≥5 but <15 [In a study by Noonan et al. there is a distinction between low (1.8–2.2), intermediate (2.2–5) and high (>5) ratios . Commonl but <15 .MET amplification depends on the platform used. In general, the sequenced gene of interest is normalized against a comparator such as archived sequences from normal samples [MET amplification is recognized as a de novo driver alteration in approximately 1–5% of NSCLC, and is usually typical of heavy-smoking patients [TP53, KRAS, and KEAP1) [With NGS, identification of  samples . However samples . Standar samples . MET amppatients . Moreoved KEAP1) .MET-amp is often associated with acquired resistance to common EGFR TKIs, in NSCLC patients with EGFR-sensitizing mutations [Finally, utations .MET overexpression has a variable incidence (about 35% to 72%) in NSCLC, depending on the antibody used for detecting it . With imMET creates a fusion gene with a wide variety of partners [TPR, KIF5B, HLA-DRB1 [MET fusions, but does not allow the identification of intragenic fusions. As far as IHC is concerned, the major limit is given by false positivity. Indeed, many cases, identified as IHC positivity, have been demonstrated to be FISH- and NGS negative, suggesting the hypothesis that IHC led to the misleading detection of mutations other than fusions  [MET gene fusions [MET fusions achieved benefits from treatment with MET inhibitors [One way for the oncogenic activation of the MET pathway is the formation of fusion genes through chromosomal translocation mechanisms. In NSCLC, the tyrosine kinase domain-encoding portion of partners , includiHLA-DRB1 ,50,51, mcations) . Althoug fusions . Despitehibitors .MET gene fusions could represent a mechanism of resistance to EGFR or ALK-TKIs [Moreover, although data are still limited given the rarity of this alteration, ALK-TKIs  , with Y1230, blocking catalytic activation. This type is also subdivided into type Ia and type Ib. Type Ia inhibitors, such as crizotinib, interact with MET by the solvent front residue G1163 and are also known as non-selective MET TKIs because this residue is not specific to MET. Type Ib inhibitors, such as capmatinib, tepotinib, savolitinib, and APL-101, selectively interact with MET alone, by the Y1230 residue. Type II MET TKIs, such as cabozantinib, foretinib, merestinib and glesatinib, are also ATP-competitive inhibitors. They fill the ATP-binding pocket and bind to the inactive “DFG out” conformation of MET. Therefore, their binding is independent of interactions with the A-loop. Type III MET TKIs are non-ATP competitive inhibitors, binding allosterically outside the ATP pocket ,23. An oTable 1ALK or ROS1 rearrangements [MET exon 14 skipping mutations decreases the viability of tumor cells and blocks related downstream signaling [MET exon 14 alteration have been performed [MET. A total of 69 patients with MET exon 14 skipping mutations were enrolled in the study and 65 of these were evaluable for tumor response. The objective response rate (ORR) was 32% (95% CI 21–45), median progression free survival (PFS) 7.3 months (95% CI 5.4–9.1) and median duration of response (DOR) 9.1 months (95% CI 6.4–12.7) [MET gene amplification by FISH (MET to CEP7 ratio ≥4) showed increased median PFS with crizotinib compared to patients characterized by intermediate- (MET/CEP7 ratio >2.2 to <4) or low- (MET/CEP7 ratio >1.8 to <2.2) level MET amplification  [MET exon 14 alterations with disease progression on or after platinum-based chemotherapy (www.fda.gov accessed on 14 May 2023). A phase II clinical trial, the METROS study, analyzing the efficacy of crizotinib in patients with MET exon 14 skipping mutations and amplification, showed limited clinical benefit, with an ORR of 27% (95% CI 11–47) and a median PFS of 4.4 months (95% CI 3.0–5.8) [MET alterations. In the c-MET ≥6 copies cohort, ORR was 16% and PFS 3.2 months while in all c-MET-mutations cohorts ORR was 10.7% and PFS 2.2 months [MET amplification and MET exon 14 skipping mutation and also in patients with NSCLC with ROS1 fusion .Crizotinib is an oral, small-molecule inhibitor of several kinases such as MET, ALK, ROS1 and RON. Currently, it is approved for the treatment of advanced NSCLC characterized by ngements ,57. Thisignaling . Severalerformed ,62,63,64ctively) . The mosctively) . TRAEs o3.0–5.8) . Anotherwww.fda.gov, accessed on 14 May 2023). The COSMIC-313 (NCT03937219) phase III clinical trial recently reported that among patients with previously untreated advanced renal-cell carcinoma and intermediate or poor prognostic risk, the combination of cabozantinib with nivolumab and ipilimumab led to significantly longer PFS compared to treatment with nivolumab and ipilimumab alone. However, it is important to note that the experimental group experienced a higher incidence of grade 3 or 4 adverse events compared to the control group [Cabozantinib (XL-184) is another small-molecule, non-selective type II MET TKI, able to target several kinases, including VEGFR1–3, RET, TIE2, FLT-3 and KIT. Currently, cabozantinib is approved for treatment of several cancers such as medullary thyroid carcinoma, renal cell carcinoma and hepatocellular carcinoma  compared to erlotinib alone  [MET alterations and it is still not now fully clear if cabozantinib treatment can play a role in patients with MET alterations. In an initial study enrolling patients with stage IV NSCLC with MET exon 14 skipping mutations and treated with crizotinib or cabozantinib, Paik et al reported a complete response in one patient treated with cabozantinib [Pre-clinical in vivo data have shown that cabozantinib can also be effective against other cancers such as lung cancer . Moreove1.7–2.2) . Howeverzantinib .MET alteration .Several phase II clinical trials are ongoing to evaluate the effectiveness and safety of cabozantinib in the treatment of metastatic NSCLC with MET exon 14 mutations (MET exon14 skipping and MET amplification) [MET exon 14 skipping mutations or advanced solid cancers with NTRK1–3 rearrangements (NCT02920996).Merestinib (LY2801653) is an oral ATP-competitive, multitargeted inhibitor of MET and other kinases involved in tumor cell growth, proliferation, and angiogenesis, such as AXL, ROS1, VEGFR2, FLT3, DDR1–2, MERTK and NTRK1–3. It was characterized by a manageable safety profile and potential antitumoral activity in pre-treated advanced cancer patients ,74. In pication) . An ongoMET mutations involving the residues D1228 and Y1230, which confer resistance to type I MET TKIs. Glesatinib was shown to be effective in tumor cell lines and exerted a significant regression of cell- and patient-derived tumor models harboring METex14 del mutation in vivo [Glesatinib is an oral ATP-competitive, multitargeted TKI of MET, VEGFR1–3, AXL, RON and TIE2. It is a unique type II MET inhibitor, and interacts with MET independently of the binding of A-loop, demonstrating its activity against  in vivo .MET alterations (mutation or amplification) has shown that the glesatinib safety profile was acceptable and the dosage of 750 mg twice daily (NCT00697632) [However, significant clinical data with this TKI are still missing. A phase I trial of glesatinib in advanced, pre-treated, NSCLC patients with activating genetic 0697632)  was seleCapmatinib (INC280) is an oral, ATP-competitive, type Ib MET TKI, which demonstrated high selectivity in MET-driven mouse tumor models .MET exon 14 alterations. Notably, enhanced antitumor activity of capmatinib was found in those models in which MET activation occurred concomitantly with other oncogenic alterations, such as EGFR mutations, when used in combination with EGFR TKIs [Significant antitumor activity was seen in cell-line- or patient-derived xenograft models with different mechanisms of MET activation, including GFR TKIs .MET exon 14 skipping mutation . This was based on the results of the GEOMETRY mono-1 (NCT02414139), a multicenter, non-randomized, open-label, multi-cohort, phase 2 trial evaluating capmatinib  in EGFR wild-type and ALK-negative NSCLC patients with MET alterations, including MET exon 14 skipping mutation or MET amplification. Patients were stratified based on MET status and prior lines of therapy. Increased ORR and PFS were observed in treatment-naïve patients compared to pretreated patients. Indeed, in the group of previously treated patients with MET exon 14 skipping mutations, the ORR and DoR were 41% and 9.7 months, respectively, while in the naïve patients’ group these were 68% and 12.6 months, respectively. In addition, an increased median PFS was found in naïve patients (12.4 months) compared to previously treated patients (5.4 months). This study also demonstrated the efficacy of capmatinib in patients with MET amplification and GCN ≥10 NSCLC, although to a lesser extent than in patients with MET exon 14 alterations.In May 2020 Capmatinib gained FDA accelerated approval for patients with metastatic NSCLC with In addition, this drug exhibits intracranial activity and an acceptable safety profile, as most of the TRAEs were of grade 1/2 and were represented by peripheral edema, nausea, vomiting, and increased serum creatinine levels. The treatment was discontinued in 11% of patients and a dose reduction occurred in 23% of patients.MET exon 14 mutation-positive NSCLC are ongoing . The phase 2 Geometry-N trial (NCT04926831) is evaluating the activity of capmatinib in adjuvant and neoadjuvant settings. Furthermore, a phase 3 study (NCT04427072) investigating capmatinib versus docetaxel in previously treated patients with MET exon 14 skipping NSCLC is still ongoing, while two clinical trials evaluating the safety and efficacy of capmatinib in combination with immunotherapeutic agents  were recently terminated due to lack of tolerability observed in the combination treatment group when compared to data from single agent studies .Several phase 2 clinical trials of capmatinib in patients with MET exon 14 skipping mutations based on the results of the phase II VISION trial. In this trial, the use of tepotinib in advanced NSCLC patients with MET alterations, including MET exon 14 skipping mutations and MET amplification, was correlated with an ORR of 46% (95% CI 36–57) based on an independent review, and a median PFS of 8.5 months (95% CI 6.7–11). Also, there were no significantly differences in terms of response between previously treated and naïve patients [MET exon 14-altered resulted similar to the other enrolled patients  [MET alterations.Tepotinib is an oral, type Ib MET inhibitor, acting as an ATP-competitive, and highly selective for MET. In vivo, tepotinib has been shown to be able to induce tumor regression in a human-cancer mice model, irrespective of whether or not MET activation was dependent on HGF ,81. In apatients . MoreoveMET exon 14 skipping alterations with progression disease after prior systemic therapy or who are unable to receive chemotherapy [MET exon 14 altered NSCLC or metastatic pulmonary sarcomatoid carcinoma (PSC) in Chinese patients [MET alterations are still ongoing (NCT04923945), also in combination with chemotherapeutic drugs (docetaxel) (NCT05777278) or immunotherapeutic agents  (NCT05374603). Additionally, savolitinib is being studied in patients with MET-driven resistance to EGFR TKIs in combination with osimertinib  and further in EGFR-mutant NSCLC patients with de novo MET positive alterations as first-line setting  .Savolitinib is an oral, ATP-competitive, type Ib MET TKI, with high selectivity for MET, approved on June 2021 in China for patients with advanced NSCLC with otherapy ,84. Thispatients . Savolitpatients ,86. To npatients . FurtherOther MET TKIs are being tested in ongoing clinical trials such as APL-101 , elzovantinib or TPX-0022 , and Glumetinib or SCC244 . To note, the first report of the data of the ongoing single-arm phase Ⅱ study trial GLORY (NCT04270591) showed promising results for Glumetinib (SCC244), with an ORR by BIRC of 60.9% overall, and 66.7% and 51.9% in treatment-naïve and previously treated patients, respectively. The median duration of response (DoR) was 8.2 months and the median PFS was 7.6 months [MET alterations. These antibodies inhibit the MET signaling pathway by blocking interactions between the MET receptor and its ligand HGF.In addition to MET TKIs, there are different antibodies targeting MET that have been studied or that are in development for the treatment of NSCLC patients with MET-positive NSCLC (defined as ≥50% of tumor cells with MET IHC scores of 2+ or 3+) resulted in early study termination, because it showed worse survival rates in the experimental group than in the placebo group, failing to demonstrate significant clinical efficacy [EGFR-mutated NSCLC demonstrated no difference in clinical outcomes in the intention-to-treat population [These antibodies can be categorized into anti-MET antibodies, including onartuzumab and emibetuzumab, and anti-HGF antibodies, such as rilotumumab and ficlatuzumab. The efficacy of specific anti-MET monoclonal antibodies has been shown to be unsatisfactory in clinical trials. A phase 3 study of erlotinib with or without onartuzumab in previously treated patients with advanced efficacy . A phasepulation . Howeverpulation . On the pulation .EGFR exon 20 insertion mutations showing intrinsic resistance to approved TKIs, for which the standard of care remains platinum-based chemotherapy with an associated reduced median overall OS of 16 months [EGFR exon 20 insertions who progressed on or after platinum-based chemotherapy (www.fda.gov accessed on 14 May 2023).Amivantamab is a monoclonal antibody with dual activity (anti-EGFR and anti-MET) but with a higher affinity for MET (40 pM) than EGFR (1.4 nM). In NSCLC with 6 months , amivant6 months . TherefoEGFR-mutant NSCLC, showed promising effectiveness and an acceptable safety profile . In a dose-expansion cohort including 43 patients with MET exon 14 skipping mutations, an ORR of 33%was reported. The response rate was impacted by prior therapy . The median PFS was 6.7 months [Results from the ongoing phase 1 CHRYSALIS study, evaluating amivantamab in combination with lazertinib (a third-generation EGFR TKI) for the treatment of osimertinib-relapsed, chemotherapy-naïve 7 months . A low i7 months .MET exon 14 skipping mutation and MET amplification  .In addition, other trials evaluating the combination of amivantamab plus lazertinib as a first-line  and subsequent-line  regimen or in combination with platinum-doublet chemotherapy after progression on osimertinib  are ongoing. Another trial is evaluating amivantamab and capmatinib combination therapy in NSCLC patients with MET alterations  (NCT04077099). Early results showed an acceptable safety profile and promising effectiveness. Grade ≥3 TRAEs occurred in 25% of patients (including pneumonia and pulmonary embolism). In total, 6 patients among the 36 who received the 2000 mg dose had a partial response (2 patients had MET exon 14 skipping mutations and 4 patients had MET gene amplification and/or overexpression) [Another bispecific antibody binding to two distinct epitopes of MET is REGN5093. This agent is being investigated in a phase 1/2 trial of NSCLC patients with MET amplification or exon 14 skipping mutation (NCT02648724) showed an ORR of 25% and DCR of 80%. For MET TKI-naïve patients, ORR was of 50% and DCR 100% while the median PFS was 6.5 months for the MET TKI-naïve group and 5.4 months for patients who received prior MET TKI therapy. All grade TRAEs occurred in 42.2% of patients, 13.3% of which were grade ≥3  [Another MET antibody is Sym015, an agent composed of 2 humanized antibodies. These antibodies bind non-overlapping epitopes on the SEMA-a domain of MET leading to MET receptor internalization and degradation avoiding the binding of HGF to MET . Early rl edema) .More recently, antibody–drug conjugates (ADCs) have entered the therapeutic NSCLC scenario with promising early results . AntibodEGFR-wild-type, nonsquamous NSCLC with MET overexpression, based on the preliminary results of an ongoing phase 2 study evaluating this agent in previously treated patients with MET overexpressing NSCLC (NCT03539536) [EGFR wild-type non-squamous subtypes . This agent also showed an acceptable safety profile, with the most common TRAEs being dermatitis acneiform, peripheral neuropathy dyspnea, fatigue and hypoalbuminemia [Telisotuzumab vedotin is a first-in-class ADC composed of a humanized MET-targeted antibody conjugated to the microtubule inhibitor monomethyl auristatin E (MMAE). On January 2022 the FDA assigned breakthrough therapy designation to telisotuzumab vedotin for advanced, previously treated, 3539536) . Recent uminemia ..Another ADC composed of a MET antibody conjugated to a novel microtubule inhibitor maytansinoid is REGN5093-M114. This compound is being evaluated in a phase 1/2 trial in patients with MET overexpressing advanced NSCLC and preclinical data are promising (NCT04982224) . An overTable 3MET-altered NSCLC.Limited and conflicting data for use of immunotherapy, in particular of anti-PD1/PD-L1 agents, have been reported in patients with MET-altered NSCLC patients (84%) compared with the wild-type group (59%) and especially high PD-L1 expression (50%) was found in the MET-altered NSCLC group compared to the wild-type group (60% versus 30%) [This specific molecularly defined subgroup of tumors has been associated with a higher percentage of PD-L1 expression ,101,102.MET alterations. This analysis also includes patients with other driver alterations such as KRAS (n = 271), EGFR (n = 125), BRAF (n = 43), HER2 (n = 29), ALK (n = 23), RET (n = 16), ROS-1 (n = 7). Compared to the group with MET alterations, ORR was found to be greater in the KRAS (26%), BRAF (24%), and ROS1 (17%) groups, while minor ORR was found in the other groups , while concerning PFS it was found to be lower in the other groups compared to the MET alterations group  [MET-altered NSCLC treated with ICIs, the ORR was 17% and the median PFS 1.9 months [METex14-altered versus wild-type tumors (3.8 mutations per megabase versus 7.0 mutations per megabase) [A multicenter, retrospective analysis demonstrated an ORR of 16% and a median PFS and OS of 3.4 and 18.4 months, respectively, for ICI therapy in 36 patients with for RET) . In anot9 months . In thisegabase) . Conversegabase) ,106.MET-altered NSCLC could be the influence of MET on the tumor microenvironment (TME). MET can modulate TME by making it more immunosuppressive, for example, through hepatocyte growth factor/c-MET signaling resulting in the accumulation neutrophils or by the inhibition of dendritic cells. Therefore, MET inhibition should control this immunosuppression [MET copy number and STING levels in patients with NSCLC treated with anti-PD1 therapy following progression on first-line chemotherapy, showed the worst response to treatment in patients with high MET copy numbers and low IFN. This suggested that MET amplification leads to reduced tumor immunogenicity thereby minimizing the response to ICI therapy [One reason underlying limited immunotherapy efficacy in pression ,107. Anopression . A retro therapy .This suggests a combination of a MET inhibitor with an ICI could potentially overcome resistance to immunotherapy. This strategy is being evaluated in several ongoing trials .Nevertheless, more data are needed to better assess the role of ICI therapy for treatment of patients with MET alterations, both alone and in combination with targeted drugs.Despite the initial enthusiasm for the results obtained with the use of MET TKIs in NSCLC patients with MET pathway alterations, both primary and secondary resistance mechanisms, limiting long-term benefit with these agents, have been described . From a MET alterations treated with MET TKIs and where resistance has been encountered [MET exon 14 skipping or MET amplification as their primary alteration, secondary mutations in the tyrosine kinase domain of the receptor were identified [Several cases of on-target mechanisms have been identified in patients with ountered . In mostentified , includientified .MET-altered NSCLC patients pretreated with crizotinib, potentially due to overlapping resistance mechanisms [The type of resistance mutation has been correlated to the type of MET inhibitor used. For example, in patients treated with type Ib inhibitors such as capmatinib, tepotinib, and savolitinib, the most frequent mutations involve codons D1228 and Y1230 , while fchanisms .Another strategy potentially able to bypass the development of this type of resistance is the use of drugs targeting the extracellular domain of MET. In this regard, a phase 1 study evaluated the efficacy of the monoclonal antibody Amivantamab in patients pre-treated with MET-TKIs, demonstrating efficacy rates in terms of RR 21.1% and clinical benefit in 57.9% .EGFR and KRAS and the cascade activation of downstream pathways. Preclinical studies, carried out on cell lines obtained from patients treated with MET-TKIs showing resistance due to KRAS mutations, evaluated a combination strategy with a MEK1/2 inhibitor (trametinib) and a MET inhibitor (crizotinib). Treatment with trametinib alone showed a low efficacy while the combination was more effective in controlling tumor growth. These results showed that mutation or amplification of KRAS can confer resistance to crizotinib treatment in pre-treated MET alterations cells and that the addition of trametinib can restore tumor inhibition [The off-target resistance mechanism is caused by the activation of signal transduction pathways alternative to that of MET. Genomic alterations most frequently associated with this resistance are the amplification of hibition .MET-exon14 skipping. These results were consolidated by preclinical studies on patient-derived cell lines treated with a combination of MET and PI3K inhibitors. This combination can inhibit the proliferation of cells harboring a METex14 mutation and a concurrent PIK3CA mutation or PTEN loss. As PIK3CA mutations and PTEN loss lead to activation of the PI3K pathway, these results suggest that PI3K pathway alterations cause resistance to MET TKIs [Finally, some studies have shown that the activation of the PI3K pathway can also lead to a resistance to MET-TKIs in patients with MET TKIs .No data are currently available on histological transformation as a mechanism of resistance to MET inhibitors.MET amplification has emerged as a known mechanism of resistance in a subset of patients (approximately 7–15%) with non-small cell lung cancer (NSCLC) harboring EGFR mutations and receiving treatment with EGFR tyrosine kinase inhibitors (TKIs) when experiencing disease progression [EGFR mutations, prolonged exposure to EGFR TKIs led to the emergence of MET amplification as a resistance mechanism. The cells with MET amplification showed sustained activation of the MET signaling pathway, leading to continued cell proliferation and survival even in the presence of EGFR inhibition. Similarly, another preclinical study by Turke et al [EGFR-mutant NSCLC cell lines with acquired resistance to EGFR TKIs and they found that MET amplification was present in these resistant cell lines. They also showed that the combination of EGFR and MET inhibitors effectively suppressed cell growth in these resistant cells, highlighting the importance of targeting both pathways to overcome resistance.gression . These rgression , showingke et al  demonstrMET amplification can arise as a known mechanism of resistance in EGFR-mutated NSCLC treated with EGFR TKIs, supporting the need for combination therapies targeting both EGFR and MET pathways to improve treatment outcomes.These preclinical studies provide evidence that EGFR T790M mutation status: 52% (12/23) in T790M-negative patients, 9% (2/23) in T790M-positive patients, and 40% (2/5) in patients with unknown T790M mutation status. Importantly, this combination exhibited a favorable safety profile [In the light of these findings, several studies have investigated the potential of combining MET and EGFR inhibitors. In a phase 1b study, the combination of gefitinib, an anti-EGFR agent, and savolitinib, a highly selective MET inhibitor, demonstrated encouraging objective response rates in patients with different  profile .In the currently ongoing SAVANNAH phase 2 trial, the efficacy of the combination of osimertinib plus savolitinib is being evaluated. Preliminary data have shown an ORR of 32%, a median DOR of 8.3 months and a median PFS of 5.3 months .EGFR-mutant, T790M-negative NSCLC with MET overexpression or MET amplification. Survival outcomes were similar between groups: the median PFS was 4.9 months in the tepotinib plus gefitinib group versus 4.4 months in the chemotherapy group. The median OS was 17.3 months in the tepotinib plus gefitinib group versus 18.7 months in the chemotherapy group. In patients with high (IHC3+) MET overexpression or MET amplification, PFS and OS were longer with tepotinib plus gefitinib than with chemotherapy. Respectively, the median PFS was 8.3 months versus 4.4 months and the median OS 37.3 months versus 17.9 months in the MET overexpression group, and the median PFS was 16.6 months versus 4.2 months and the median OS 37.3 months versus 13.1 in the MET amplification group [EGFR mutated NSCLC with MET-amp after progression on first-line osimertinib received tepotinib 500 mg (450 mg active moiety) + osimertinib 80 mg once daily. First results showed an ORR of 54.5% and the median DOR was not reached among patients with at least 9 months of follow up [The phase II trial INSIGHT investigated antitumor activity of the association of tepotinib plus gefitinib versus standard platinum-doublet chemotherapy in patients with on group . In the ollow up .n = 28). Of the EGFR-M+ patients, those who were c-Met high (n = 15) had an ORR of 52.6% [In a phase 1b, an antibody–drug conjugate targeting c-MET, Telisotuzumab vedotin (Teliso-V), was also evaluated in combination with erlotinib. The results were promising, with a median PFS of 5.9 months and ORR of 32.1% for EGFR-M+ patients . Two phase III studies are currently ongoing, evaluating the efficacy of the combination of anti-EGFR and anti-MET. In particular, the SAFFRON study is evaluating the efficacy and safety of savolitinib in combination with osimertinib versus platinum-based doublet chemotherapy in patients with mertinib . FinallyMET exon 14 skipping has become crucial in optimizing the therapeutic approach for advanced NSCLC. However, further data are still needed to determine the most effective sequential strategy among the available MET-TKIs, as well as therapeutic approaches to overcome or delay resistance. Standardizing the detection methods for MET alterations is also imperative to ensure consistency and accuracy in clinical practice.The role of the MET pathway in NSCLC has been extensively studied, highlighting its significance as both a targetable factor and a mechanism of resistance to other therapies. With advancements in targeted agents, the identification of MET-altered NSCLC. These areas require further investigation to develop effective treatment strategies and improve patient outcomes.Additionally, several clinical challenges need to be addressed, including the use of MET inhibitors for treating brain metastases and investigating the role of ICIs, either alone or in combination with anti-MET agents, in patients with