Activated STAT3 is an essential factor in cancer development; therefore, blocking STAT3 may be of therapeutic benefit in multiple cancer types. Thanks to recent developments in the regulatory function of non-coding nucleic acids, a new branch of nucleic acid-based molecules with significant therapeutic potential has emerged. Therapeutics such as siRNA, shRNA, ASO, and ODN-decoy that target STAT3 found an application in cancer therapies. The main advantage of using nucleic acid-based therapeutics is their high specificity based on the complementarity of the therapeutic sequence with the target. Conversely, oligonucleotide therapeutics struggle with stability, toxicity, sensitivity to nucleases, specificity toward cell type, and cellular uptake, which hamper their applicability. Among the various solutions proposed to overcome these problems, embedding the oligonucleotide in the carrier is an interesting strategy. The review combines the knowledge about STAT3 biology in cancer, the application of anti-STAT3 oligonucleotide therapeutics in cancer therapy, and carriers for their delivery.High expression and phosphorylation of signal transducer and transcription activator 3 (STAT3) are correlated with progression and poor prognosis in various types of cancer. The constitutive activation of STAT3 in cancer affects processes such as cell proliferation, apoptosis, metastasis, angiogenesis, and drug resistance. The importance of STAT3 in cancer makes it a potential therapeutic target. Various methods of directly and indirectly blocking STAT3 activity at different steps of the STAT3 pathway have been investigated. However, the outcome has been limited, mainly by the number of upstream proteins that can reactivate STAT3 or the relatively low specificity of the inhibitors. A new branch of molecules with significant therapeutic potential has emerged thanks to recent developments in the regulatory function of non-coding nucleic acids. Oligonucleotide-based therapeutics can silence target transcripts or edit genes, leading to the modification of gene expression profiles, causing cell death or restoring cell function. Moreover, they can reach untreatable targets, such as transcription factors. This review briefly describes oligonucleotide-based therapeutics that found application to target STAT3 activity in cancer. Additionally, this review comprehensively summarizes how the inhibition of STAT3 activity by nucleic acid-based therapeutics such as siRNA, shRNA, ASO, and ODN-decoy affected the therapy of different types of cancer in preclinical and clinical studies. Moreover, due to some limitations of oligonucleotide-based therapeutics, the importance of carriers that can deliver nucleic acid molecules to affect the STAT3 in cancer cells and cells of the tumor microenvironment (TME) was pointed out. Combining a high specificity of oligonucleotide-based therapeutics toward their targets and functionalized nanoparticles toward cell type can generate very efficient formulations. The signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family . STAT3 hSTAT3 belongs to the transcriptional factors (TFs) family and consists of several domains critical for dimerization, transactivation, and DNA binding. The structure of STAT3 can be distinguished an N-terminal domain (NH2), a coiled coil domain (CCD), a DNA-binding domain (DBD), a linker domain (LD), an Src Homology 2 domain (SH2), and a transactivation domain (TAD) . The SH2Canonical activation of STAT3 can be induced by the stimulation of receptor tyrosine kinases (RTK), e.g., epidermal growth factor receptor (EGFR) or cytokine receptors, such as an interleukin-6 (IL-6) receptor ,13 Figu. IL-6 biCCND1), cyclin B1 (CCNB1), and cyclin-dependent kinase 1 (CDC2), is increased under the control of activated STAT3 -PEG[5K] was investigated [STAT3 or Chol-DsiSTAT3 led to tumor growth inhibition. However, particles loaded with Chol-DsiSTAT3 had a more tremendous potential to reduce STAT3 on the protein level than Chol-siSTAT3 in 4T1 breast cancer in vivo [In a study conducted by Ye et al., the therapeutic effect of 3′-cholesterol-modified stigated . In a mo in vivo . STAT3 siRNA and curcumin were administered to the B16F10 melanoma cells [STAT3 siRNA-loaded liposomes caused significant tumor growth inhibition in contrast with the treatment using control liposomes and naked STAT3 siRNA in the melanoma cancer model. Moreover, the effectiveness of liposome penetration through the skin increased after using iontophoresis. Iontophoresis is a method that transports ionic therapeutic agents through the skin using low-level electric current. Among the tested therapy variants, the most significant reduction in melanoma growth in vivo was observed after the intratumoral administration of liposomes containing curcumin and STAT3 siRNA and after using liposomes containing curcumin and STAT3 siRNA with iontophoresis. Although tumor reduction was observed in both cases, the most considerable tumor reduction was observed with the application of drugs by intratumoral injection [Cationic liposomes containing ma cells . The appnjection .STAT3 siRNA to B16 melanoma cells using these nanoparticles was very effective and caused a reduction in STAT3 expression and an induction of cell apoptosis [In a study conducted by Erdene-Ochir and co-workers, the nanoparticles based on alkylated cationic curdlan derivatives were used to treat melanoma . The obtpoptosis . STAT3 siRNA into B16.F10 melanoma cells by polyplexes made of oleic acid- and stearic acid-modified polyethylenimine (PEI- p-STAT3-siRNA) decreased STAT3 expression [STAT3 led to reduced VEGF expression and decreased cell viability [In another study, the delivery of pression . Furtheriability . STAT3 siRNA delivered by PEI vehicles effectively silenced STAT3 and inhibited melanoma cell proliferation in contrast with the application of naked STAT3 siRNA. Further in vivo study revealed that using microneedles that contained PEI carriers for STAT3 siRNA delivery in a mouse melanoma model resulted in STAT3 silencing and tumor growth inhibition [Pan et al. developed a method for delivering siRNA deep into the skin via dissolving microneedles made of dextran 40, polyvinylpyrrolidone (PVP 17), and HA to treat melanoma . Howeverhibition . STAT3 shRNA into A2780CP and A2780 ovarian cancer cells inhibited STAT3 gene expression [STAT3 shRNA carrying plasmid, the inhibition of cell proliferation and the induction of cell apoptosis were observed. This also led to a decrease in the BRIC5, BCL-2, and VEGF protein levels and an increase in cleaved caspase-3. However, for the research performed in vivo, they used the STAT3 silencing plasmid enclosed in cationic liposomes to deliver the cargo to the tumor efficiently. Treatment with shSTAT3/lipoplexes reduced STAT3 protein levels and inhibited tumor growth. Further studies showed increased apoptosis in tumors treated with shSTAT3/lipoplexes and that cancer cell proliferation and angiogenesis were inhibited compared with the control [Jiang and colleagues found that introducing pression . After t control . STAT3 shRNA transfected into H1650 lung cancer cells showed that construct decreased STAT3 on the mRNA and protein levels [STAT3 shRNA in vitro application resulted in the inhibition of cell proliferation and the induction of cell apoptosis. After the implantation of modified cancer cells in mice, tumor growth was significantly inhibited compared with the control. The authors also looked for a suitable carrier to help with in vivo STAT3 shRNA delivery into the cancer cells. In a comparison study, they tested chitosan, vitamin E succinate–chitosan–histidine (VCH), and polyethyleneimine as potential carriers. Based on low toxicity, high integration into A549 lung cancer cells, and the silencing of STAT3 expression as observed in vitro, they selected VCH particles for further research in cancer therapy [The in vitro research using n levels . The STA therapy . STAT3, BCL-xL, and CCND1. Furthermore, in vivo studies indicated that STAT3 inactivation by the intravenous injection of ultrasound microbubble vesicles carrying the STAT3 ODN-decoy treated with ultrasound irradiation was the most effective in inhibiting esophageal squamous cell carcinoma growth. A similar effect was observed in the mouse group treated with the STAT3 ODN-decoy loaded in liposomes plus ultrasonic irradiation. Still, its efficiency was lower than in the mice that received the STAT3 ODN-decoy loaded ultrasonic microbubble plus ultrasonic irradiation [Zhang et al. investigated methods for the efficient and safe delivery of the STAT3 ODN-decoy to esophageal squamous cell carcinoma . First, adiation . VEGF, MMP9, CDH2, and Vimentin (VIM) and elevated levels of CDH1. The study showed no toxic side effects caused by the SLN-STAT3 ODN-decoy in mice [Cationic solid lipid nanoparticles (SLNs) were an effective gene delivery system due to their high biocompatibility, stability, and low cytotoxicity . Zhang e in mice . BCL-2, MCL-1, BRIC5, and mucin 4 (MUC4) compared with the simultaneous TRAZ and STAT3 ODN-decoy application without practice. In the mouse breast cancer xenograft model, the treatment of CaP@HA loaded with the STAT3 ODN-decoy plus TRAZ inhibited tumor growth and induced tumor cell apoptosis to a greater extent than the application of a naked oligonucleotide therapeutic in combination with TRAZ or TRAZ alone. Moreover, mice treated with CaP@HA loaded with the STAT3 ODN-decoy and TRAZ had a higher survival rate than the other treated groups [The effect of the combinatory therapy based on trastuzumab (TRAZ) with the STAT3 ODN-decoy delivered by nanoparticles in breast cancer that overexpressed HER2 was investigated . Trastuzd groups . Zhang et al. conducted studies on the delivery of the STAT3 ODN-decoy (STAT3d) using gold nanoparticles (AuNPs) . The conAs indicated above, numerous in vitro and in vivo assays testing oligonucleotide-based agents that either decrease STAT3 expression or directly inhibit STAT3 DNA-binding ability were reported. These reports have shown encouraging results in preclinical models far beyond proof-of-concept studies. However, only a few nucleic acid-based approaches have advanced human clinical testing. Therefore, optimizing these nucleotide therapeutics’ specificity, potency, stability, and delivery is essential for enhancing their therapeutic benefits in the clinic. In STAT3 siRNA is currently recruiting patients [STAT3 siRNA CAS3/SS3 (CAS3/SS3) in combination with localized radiation therapy in the treatment of patients with relapsed or refractory NHL (NCT04995536). CAS3/SS3 is a molecule comprising a CpG oligonucleotide and a STAT3 siRNA that selectively targets the TLR9 receptor and STAT3 mRNA, respectively. They act together to interfere with the cancer cells’ growth ability. Radiotherapy utilizes high-energy X-rays to kill cancer cells and reduce the tumor’s volume. Accordingly, the administration of CAS3/SS3 combined with local radiotherapy may increase the number of inactivated tumor cells [STAT3 expression and STAT3 activation of downstream targets, and local or systemic immune responses are to be measured in the study. The estimated completion date of the study is 2024 [The only ongoing clinical trial using patients . The aimor cells . Eightee is 2024 .STAT3 ASO simultaneously with radiotherapy, chemotherapy, or immunotherapy. AZD9150 (Danvatirsen) is an antisense oligonucleotide (ASO) inhibitor of STAT3 that has shown clinical activity in several phase I/II clinical studies. Up to January 2023, thirteen studies on AZD9150 in cancer treatment were recorded in the ClinicalTrials.gov database . Among tOne of the first-in-human studies was a phase I/II clinical trial of the next-generation antisense oligonucleotide inhibitor of STAT3 . The trial was carried out in patients with solid and hematologic malignancies refractory to at least one prior systemic therapy (NCT01563302). Of the thirty patients enrolled in the study, including twenty-seven patients with diffuse large B cell lymphoma (DLBCL), ten received a dose of 2 mg/kg and twenty received a dose of 3 mg/kg. Both tested AZD9150 doses proved to be safe and well tolerated. In DLBCL patients, two partial and two complete responses with a median duration of response of 10.7 months occurred after AZD9150 administration at both tested dose levels . As no sAZD9150, given in combination with Acalabrutinib (kinase inhibitor), was analyzed in a phase Ib trial conducted in 2018–2021, focusing on treating hematological malignancies (NCT03527147). The study was carried out in thirty patients with relapsed or refractory aggressive non-Hodgkin’s lymphoma (NHL), and DLBCL reported no safety concerns about the proposed combination .Other studies on AZD9150 in combination with checkpoint immunotherapies were also conducted. A phase I/II trial of AZD9150 in combination with durvalumab (anti-PD-L1 antibody) in Japanese patients with advanced solid tumors (NCT03394144) was conducted in the years 2018–2019 . Eleven Another phase Ib multicenter trial tested durvalumab as a monotherapy and combined with AZD9150 in patients with relapsed or refractory DLBCL (NCT02549651). The combined treatment was generally safe in the group of patients with relapsed or refractory DLBCL; however, limited antitumor activity was indicated .PD-L1 and CD73 gene expression levels, were investigated. According to transcriptomic analysis, the durvalumab plus AZD9150-treated group did not reveal any changes related to immune cell function in peripheral blood. Therefore, further testing of these drugs in the model of resectable NSCLC is warranted [Attempts to treat early-stage non-small-cell lung cancer (NSCLC) with AZD9150 in combination with durvalumab were undertaken in 2019 as a phase II randomized trial (NCT03794544). The study reported the safety and clinical activity of the combined formulation. Outcome measures, such as tumor and microbiome biomarkers, as well as mRNA signatures in blood samples, including basic tumor arranted ,213. Moreover, five active, non-recruiting clinical trials concerning using AZD9150 for cancer treatment were found . One of In 2018, a phase Ib/II clinical trial was launched in the USA (NCT03421353). The safety, tolerability, pharmacokinetics, and preliminary antitumor activity of AZD9150 and durvalumab, with or without chemotherapy, will be analyzed in this study . SeventyAccording to ClinicalTrials.gov, only one clinical trial testing the use of STAT3 ASO is recruiting. It is a multicenter phase II trial conducted in patients suffering from metastatic NSCLC (NCT03334617). This study has a modular structure, which allows for a preliminary assessment of the effectiveness, safety, and tolerability of various treatment formulations, including durvalumab, in combination with AZD9150. Recruitment for this study began in 2017. The study is mainly aimed at patients who have progressed on an anti-PD-1/PD-L1 therapy .Among terminated studies, one item can be found. A study registered under the number NCT02417753 aimed to measure changes in immunological parameters in the malignant ascites in patients with advanced cancer after AZD9150 treatment. The study was closed because of the inability to find eligible patients .The first and only study of an ODN-decoy targeting STAT3 in humans was registered in 2008 as an early phase I trial NCT00696176) and completed in 2011 . In this and compInhibition of STAT3 activity in cancer is beneficial, as numerous reports indicate ,221,222.Carriers not only can protect the oligo from nucleases and safely deliver it into cells but also, upon functionalization, selectively target cancer cells or other cells within the TME. According to the basic concept of using nanoparticles in cancer treatment, their accumulation in the cancer tissue occurs due to the effect of increased permeability and retention (EPR). However, the passive accumulation of nanoparticles in the TME is rather limited since only approximately 0.7% of the administered nanoparticle doses reached the tumor . In addiThis report is focused on applying oligonucleotide therapeutics that directly target cancer cells. However, one should be aware that silencing STAT3 activity could also alter the functions of non-tumor cells within the TME. Due to the biology of STAT3 and its role in cancer development, therapies targeting this molecule in immune cells, cancer-associated fibroblast, or endothelial cells are also under investigation. Moreover, in vivo studies of STAT3 inhibition, independent of whether the drugs (including oligonucleotide-based ones) are delivered in naked or embedded form, target various cells within the TME unless active targeting is used. Additionally, the complexity of the problem increases due to the biology of STAT3. As a result of alternative splicing, two STAT3 isoforms are produced, STAT3α and STAT3β. Both isoforms perform different functions in physiological and pathological conditions. Moreover, STAT3 can be activated by both canonical and non-canonical pathways, resulting in various post-translational modifications that affect its activity. Since oligonucleotide-based therapeutics can not only exert therapeutic effects by inhibiting their target at the nucleic acid and protein levels but also by editing genes, their application to control the STAT3 structure is possible. Perhaps therapies based on selecting the most desirable variant of STAT3 showing therapeutic properties rather than blocking its activity are an interesting approach. The use of oligonucleotide therapeutics makes it possible to achieve such a goal. Combining a specific drug with active delivery in a carrier could increase the outcome and safety of such therapy.


Digital pathology offers the potential for significant benefits in diagnostic pathology, but currently the efficiency of slide viewing is a barrier to adoption. We hypothesised that presenting digital slides for simultaneous viewing of multiple sections of tissue for comparison, as in those with immunohistochemical panels, would allow pathologists to review cases more quickly.Novel software was developed to view synchronised parallel tissue sections on a digital pathology workstation. Sixteen histopathologists reviewed three liver biopsy cases including an immunohistochemical panel using the digital microscope, and three different liver biopsy cases including an immunohistochemical panel using the light microscope. The order of cases and interface was fully counterbalanced. Time to diagnosis was recorded and mean times are presented as data approximated to a normalised distribution.Mean time to diagnosis was 4 min 3 s using the digital microscope and 5 min 24 s using the light microscope, saving 1 min 21 s , using the digital microscope. Overall normalised mean time to diagnosis was 85% on the digital pathology workstation compared with 115% on the microscope, a relative reduction of 26%.With appropriate interface design, it is quicker to review immunohistochemical slides using a digital microscope than the conventional light microscope, without incurring any major diagnostic errors. As digital pathology becomes more integrated with routine clinical workflow and pathologists increase their experience of the technology, it is anticipated that other tasks will also become more time-efficient. Over the past few years, digital pathology has been deployed for primary diagnosis in a few flagship projects around the world. Our institution now routinely digitally scans 100%Of late, much of the research has been concerned with diagnostic accuracy as the introduction of a new technology must not compromise patient safety. Many single papers and a systematic reviewPreliminary work from our group showed that early whole slide imaging (WSI) viewers were 60% slower than the microscope,A more recent development of this software though allows for simultaneous viewing of multiple sections of tissue for comparison, something that is simply not possible with light microscopy. With as little as 61.9% of a pathologist’s time spent viewing an image when at the microscope,We therefore designed an experiment to compare the time with diagnosis using a digital system with the microscope. The digital system minimises the effort to manually switch between separate slides, instead offering the user a one-touch method of reviewing the case. It is hypothesised that the use of an appropriately designed digital pathology workstation can offer a reduction in time to reach a diagnosis without compromising diagnostic confidence.A purposive sampling strategy was employed to recruit 16 participants from within our institution: 8 senior trainee histopathologists and 8 consultant histopathologists from a range of subspecialist fields (not including liver pathology). Pathologists were recruited in person and those included were those who were approached and were willing to be involved. This study took place prior to the digitisation of our department resulting in a wide range of experience of using a digital microscope, from very little to many days’ cumulative experience.All participants were asked to view three cases using the digital microscope, and three different cases using the light microscope. The order of cases and interface was fully counterbalanced.All cases were liver needle core biopsies of tumours, with clinical details available in 10.1136/jclinpath-2021-207961.supp2Supplementary dataEach case contained one or more H&E-stained slides, as well as multiple slides stained with immunohistochemical stains that were relevant to the case. An example of the slides for a case can be seen in Before the experiment was undertaken, each participant was given a 15-minute training session using the digital microscope. A standard training session was divided into three sections: section one in which the researcher would show the participant how to use the software, section two where the participant would use the software themselves and the researcher would evaluate their use of it, and section three where they were asked to perform a diagnostic task.All trials took place in a quiet, windowless room in the histopathology department of our institution usually used for teaching purposes. The digital microscope was placed at one end of the room with a light microscope set up on an adjacent table. The only light source was a standalone lamp placed next to the door behind the participant, to standardise the effect of ambient lighting on the display as far as possible at 10 lux.A Dell Precision T5500 with AMD W5000 graphics card was used for this experiment. A Barco Coronis Fusion (6 MP) display  was used in conjunction with a Barco Nio (2 MP) display . The larger 30-inch screen was a split screen setup, the left side displaying the H&E slide (which was in constant display) and the right displaying the immunohistochemical slide. The smaller screen 21-inch on the right side of the participant displayed thumbnail images of all slides and highlighted which slide was currently in view. Viewing software was the Leeds Virtual Microscope.The light microscope was a Leica DMR microscope  with ×2.5, ×5, ×10, ×20, ×40 and ×100 objectives and ×10 eyepiece. The microscope lens was reset by the researcher to the lowest magnification at the start of each trial (×2.5 magnification). The participants were provided with a practice slide in order to familiarise themselves with the microscope prior to the experiment.The experiment was recorded using a three-video camera setup: one captured a ‘down-the-microscope’ view from the microscope camera mount, one captured the microscope work area (placed in the corner of the room furthest from the microscope) and the other directly captured the participants’ face and body from in front while at the digital workstation . Timing began when they picked up the first glass slide and finished when the set was completed. The video data were analysed to capture each instance of the participant interacting with the slide, panning, zooming, using the microscope condenser and writing notes. A screenshot of the participant video recording with the synchronised view down the microscope can be found in 10.1136/jclinpath-2021-207961.supp1Supplementary dataStatistical analysis was performed in Stata V.16. Data approximated a normal distribution and therefore are summarised by the mean and SD.As analyses indicated a wide variation in time to diagnosis according to the case, a normalised time to diagnosis  is reported as the primary outcome measure. We also report actual time to diagnosis for ease of understanding.Multiple linear regression was used to estimate the time to diagnosis in minutes adjusting for the binary fixed effects of experience level (trainee vs consultant) and interface . CIs were generated to the 95% level. A sensitivity analysis of variance (ANOVA) was also performed on normalised time with the within-subject factor being light microscope versus digital microscope and between-subject variable being experience level (trainee vs consultant).A summary of the main results can be seen in In terms of overall results, the mean time to diagnosis was 4 min 3 s using the digital microscope and 5 min 24 s using the light microscope, as shown in Overall, normalised mean time to diagnosis was 85% on the digital pathology workstation compared with 115% on the microscope; a relative reduction of 26% , as can be seen in When subcategorising the results by experience, the mean time to diagnosis for trainees using the digital microscope was 3 min 31 s, and 5 min 25 s using the light microscope. This equates to a time-saving using the digital microscope of 1 min 54 s . The mean time to diagnosis for consultants using the digital microscope was 4 min 35 s, as compared with 5 min 24 s using the light microscope. This results in a non-statistically significant time-saving using the digital microscope of 0 min 48 s . The normalised mean time to diagnosis for trainees was 74% and 116% compared with consultant times of 96% on the digital microscope and 114% on the light microscope, respectively. This equates to a reduction of 42%  for trainees, and again a non-statistically significant reduction of 18%  for consultants.When evaluating the results by set, the mean time to diagnosis for set A across all participants using the digital microscope was 3 min 13 s, and 5 min 11 s using the light microscope. This equates to a time-saving using the digital microscope of 1 min 58 s . The mean time to diagnosis for set B across all participants using the digital microscope was 4 min 53 s, and 5 min 38 s using the light microscope, a difference which was not statistically significant . The mean normalised time to diagnosis for set A was 77% compared with 123%, set B 93% compared with 107% on the digital microscope and the light microscope, respectively. This equates to a statistically significant reduction in normalised time to diagnosis per case for set A of 47% , but again a non-statistically significant difference of 14% .When combining the data across both interfaces, the data are summarised in The mean time to diagnosis by trainees was 4 min 29 s, as compared with consultants with a mean time of 5 min 0 s. Therefore, trainees were faster across both modalities by a mean time of 31 s, but this difference was not significant . Similarly, when combining data across both interfaces, the mean time to diagnosis for set A cases was 4 min 13 s, and set B was 5 min 16 s, which represents a non-significant mean difference of 1 min 6 s .Results of multivariable linear regression adjusting for experience level were largely the same as the unadjusted results presented above. Time to diagnosis on the digital microscope was 1 min 21 s faster than on the light microscope . Consultants took 31 s longer to reach diagnoses than trainees, but this was not statistically significant . There was an adjusted reduction in normalised time to diagnosis of 30% on the digital microscope as compared with the light microscope . There was an adjusted reduction in normalised time to diagnosis by trainees of 10% as compared with consultants but this was, again, non-significant . Similarly, there were no notable differences in the results of the sensitivity ANOVA performed on actual and normalised time, the details of which can be found in 10.1136/jclinpath-2021-207961.supp3Supplementary dataThere were no major diagnostic errors made on either interface. Two participants gave a discordant diagnosis of hepatocellular carcinoma in a case of probable metastatic carcinoma—in set B. Review of the case revealed some cytological features that might support such a diagnosis and further immunohistochemistry (IHC) might be required to entirely rule it out, making the true diagnosis ambiguous. Therefore, either diagnosis was acceptable for the purposes of this study.The significant difference in time to diagnosis demonstrates that for this set of diagnostic tasks, the digital microscope was quicker with a time-saving of 1 min 21 s per case, or a 26% relative reduction in time to diagnosis. As far as we are aware, this is the first study evaluating the impact of the digital microscope on time to diagnosis for cases involving immunohistochemically stained slides.A mean reduction of 1 min 21 s per case using the digital microscope is a considerable time-saving in a health service that is struggling under ever-increasing demand for services. Previous work from our group has estimated the number of cases in our institution requiring extra stains to be approximately 5%. Given that our institution handles approximately 60 000 surgical cases per year, the use of the digital microscope for these cases alone may result in a time-saving of 67.5 histopathologist hours over the course of a year within our institution. This saving is likely to be higher in institutions where immunohistochemical or special stains are used more frequently.The effect of increased experience of many of the specialist trainee participants with digital system was possibly reflected in the results, with the trainee cohort being just over 1 min quicker than the consultants on the digital pathology workstation per case, but this difference was not statistically significant . Alternatively, this may reflect the fact that trainee histopathologists were more familiar with liver specimens, as no consultant liver histopathologists were included in this study. However, no major diagnostic errors were made, thus suggesting unfamiliarity had no overall impact. Moreover, the use of only liver biopsies across the two interfaces will have prevented unfamiliarity with the type of case from biasing the results.The time to diagnosis for set B was longer than the time to diagnosis for set A, although not statistically significant . This may have been due to some diagnostic difficulty surrounding one case; a diagnosis of hepatocellular carcinoma was made twice during the trial and given as a differential diagnosis once. If this was given only on the digital microscope, this may pose questions regarding image quality and the regulations regarding the use of digital slides for diagnostic work. However, these diagnoses were made on both interfaces: once on the digital microscope and the second on the light microscope. This would lead us to believe that these mistakes were due to the diagnostic difficulty surrounding of that case, as opposed to issues regarding image fidelity on the digital microscope.Participants largely reported positive experiences using the digital microscope. A large proportion of participants used the system as intended and as was shown in the training session. However, some opted for a different technique, and rather than using the digital microscope at low power to identify an area of interest and then zoom, many participants used the digital microscope at very high magnification and scrolled the whole length of the image. This technique is inevitably time-consuming and may be due to unfamiliarity of the digital microscope for some participants; time reduction per case will likely increase with continued use and increased experience.Fourteen participants reported that they found the two-screen digital microscope very useful for comparing areas of tissue side by side and aiding a diagnosis. Two participants commented on the controls and the ergonomics of the mouse and keyboard design and felt that a less cumbersome method of panning and switching between slides needed to be implemented. One participant commented that this implementation of a digital microscope to look at immunohistochemical cases was the best they had used to date. It is unsurprising that comments were not completely unanimous regarding the digital microscope workstation; it is well known that there is not a ‘one-size-fits-all’ workstation for digital radiologists.The design features of the digital microscope are a major strength to this study. The use of medical grade displays with high technical specifications enables more tissue to be viewed on the screen at one time reducing the need for interaction by the histopathologist (pan/zoom) for this low power assessment task; an 8-megapixel display shows approximately the same amount of tissue as a ×10 light microscope eyepiece. A high luminance and contrast ratio increases, the ability of the pathologist to make a confident diagnosis at low magnification. This, in combination with a viewer that facilities fast viewing, is likely responsible for the considerable time-saving afforded by the digital microscope in this study as this reduces refreshing time. Additionally, side-by-side viewing reduces cognitive load and streamlines the process of referencing the H&E WSI to check an area for relevance; light microscopy demands that pathologists remove the IHC glass slide, load the H&E glass slide and navigate to the area of interest, which is challenging and time-consuming.Previous work from our group did not demonstrate a time-saving from increased screen resolution,et alet alet alAnother study by Hanna et al.The effect of working digitally does not just result in potential time-savings in time to diagnosis, but instead impacts the entire laboratory workflow. Although it is outside of the scope of this work to discuss the impact of a whole system evaluation of digitised pathology services, this has recently been addressed by the work of Baidoshvili Inevitably, there were several limitations to our work. First, this was a small study with known considerable user variation. Second, the time difference to diagnosis will be affected by the participant’s familiarity with the case type. Although this should not impact the primary outcome in this study (difference between the two interfaces), having participants diagnosing case types that they are familiar with may be more reflective of the time-savings observed in routine clinical practice. It should also be noted that many routine cases do not require immunohistochemical slides to review and therefore the observed time-saving may not be applicable to these cases. Third, it would be advantageous to spend longer familiarising participants with the digital microscope; familiarity with one interface and not the other will inevitably bias the results in favour of the familiar interface. Lastly, due to the prototype nature of the digital microscope, the usability could be improved. There were some issues regarding slide registration , which proved particularly problematic for participants who were less adept with the digital microscope. Further, time-saving will be likely observed as the digital microscope becomes more user-friendly.To conclude, the digital microscope reduced time per case by 1 min 21 s per case and a relative reduction of 26%, without any major diagnostic errors as compared with the light microscope. This is likely due to the ability to view multiple slides simultaneously, which is not possible using analogue systems. We anticipate that these time-savings will have a major improvement on pathologist productivity at a time where pathology services are strained, and serve as a point from which to build other user interfaces to enhance pathologist productivity.Digital pathology may offer benefits over microscope in viewing whole slide images—one unique capability is the ability to review side by side, with synchronised pan and zoom.We designed a pragmatic study looking at evaluation of liver biopsy cases including an immunohistochemical panel, where serial comparisons are needed.We used a custom viewer that allowed side-by-side viewing, and rapid review of immunohistochemical images in the sequence.We found that mean time was 5 min 24 s on the light microscope and 4 min 3 s on digital microscope, a reduction of 1 min 21 s  and a relative reduction of 26%. These benefits were seen with relatively little training and exposure to the system, and further work is needed to evaluate the real-world impact.


The interplay between genetic and environmental impacts on dental and facial morphology has been widely analyzed, but little is known about their relative contributions to airway morphology. The aim of this study was to evaluate the genetic and environmental influences on the cephalometric variables of airway morphology in a group of postpubertal twins with completed craniofacial growth.The materials comprised lateral head cephalograms of 94 pairs of twins  with completed craniofacial growth. Zygosity was determined using 15 specific DNA markers. The computerized cephalometric analysis included 22 craniofacial, hyoideal, pharyngeal structural linear and angular variables. Genetic analysis and heritability estimation were performed using maximum likelihood genetic structural equation modeling (GSEM). Principal component analysis (PCA) was used to assess the correlations between cephalometric measurement variables.2 = 0.64 and 0.5, respectively). Lower airway parameters showed only common and specific environmental determination . The relationship between the maxilla and the hyoid bone  showed very strong additive genetic determination. The size of the soft palate was affected by additive and dominant genes. Its length (SPL) was strongly influenced by dominant genes, while its width (SPW) showed a moderate additive genetic influence. Owing to correlations in the behavior of variables, the data could be expressed in 5 principal components that jointly explained 36.8% of the total variance.Upper airway dimensions showed moderate to high genetic determination . The airway, mode of breathing, and craniofacial formation are very closely interrelated during growth and development . It is kNasal breathing abnormalities may develop due to a variety of conditions, such as adenoid and tonsil hypertrophy, mandibular or maxillary retrognathism, a short mandibular body, and backward and downward rotation of the mandible, which may lead to upper airway stenosis, reduction of the pharyngeal airway space and even the development of obstructive sleep apnea (OSA) , 6. ObesThe prognosis of the success for orthodontic and dentofacial orthopaedic correction of malocclusion is determined by the extent to which a particular malocclusion can be influenced by therapeutic environmental intervention. Generally, malocclusions with a genetic cause are thought to be less amenable to treatment than those with an environmental cause. The same is truth for the success of airway morphology improvement by means of corrective orthodontics and orthopaedics . TherefoAlthough the use of comprehensive phenotype analysis in combination with large-scale genome-wide association studies maximizes the efficiency with which clinically relevant phenotype–genotype correlations can be detected, only a few correlations of this type have been discovered. Significant genetic contributions to variables such as the timing of dental maturation, incisor and canine crown diameters, missing or supernumerary teeth, arch dimensions and Class III malocclusion development have been established . HoweverTwin studies combined with advanced statistical methods provide an opportunity to determine the relative contributions of genetics and environment to dentofacial development , 11, 14.The aim of this study was to evaluate the genetic and environmental influences on the cephalometric variables of airway morphology in a group of postpubertal twins with completed craniofacial growth.The study was undertaken in the Department of Orthodontics, Lithuanian University of Health Sciences (LSMU). The sample consisted of 94 pairs of same-gender twins  selected from the register of the Twin Centre at LSMU. The protocol was approved by Kaunas Regional Ethical Committee (No. BE – 2–41). All twins had clinical consultations, and lateral cephalograms necessary for this study were performed. The CVM method was used to assess the completion of skeletal maturation .Inclusion criteria: twins of European origin, cervical vertebral maturation (CVM) stage 6 (active growth completed), high-quality cephalometric data available from both twins in the database.Exclusion criteria: previous orthodontic treatment, permanent tooth extractions, dental or facial trauma, systemic diseases or syndroms.All participating twins underwent DNA tests to determine their zygosity .Zygosity determination was carried out using a DNA test. The polymerase chain reaction set AmpFLSTR Identifiler  was used to amplify short tandem repeats, and 15 specific DNA markers  and the Amel fragment of the amelogenin gene were used for comparison of genetic profiles. Zygosity determination using this molecular genetic technique has 99.9% accuracy [The cephalometric analysis was used to measure airway and skeletal dimensions. The cephalograms were taken in centric occlusion under standard conditions using digital X-ray equipment. For standardized positioning, a cephalostat was used to stabilize the subject’s head in a constant position relative to the sensor. Lateral cephalometric (LC) radiographs were taken after swallowing. All lateral cephalograms had the same magnification. The radiographs were analyzed by using Dolphin Imaging v.11.7.Definitions of cephalometric landmarks, reference lines, and cephalometric measurements are presented in Fig. S, sella – the midpoint of the hypophyseal fossa; N, nasion – the anterior point at the frontonasal suture; A, point A – the deepest point in the curvature of the maxillary alveolar process; B, point B – the deepest point in the curvature of the mandibular alveolar process; ANS, point ANS – the anteriormost point of the anterior nasal spine; PNS, point PNS – the posteriormost point of the hard palate; Ad1, point Ad1 – the point of intersection of the posterior pharyngeal wall and line PNS-Ba; SPPW, point SPPW – the point of intersection of the posterior pharyngeal wall and the line that extends perpendicularly from the posterior pharyngeal wall to the center of the soft palate; SPP, point SPP – the point of intersection of the posterior margin of the soft palate and the line that extends perpendicularly from the posterior pharyngeal wall to the center of the soft palate; MPW, point MPW – the middle pharyngeal wall, located at the intersection of the posterior pharyngeal wall and the line extending perpendicularly from that surface to U; TPPW, point TPPW – the point of intersection of the posterior pharyngeal wall and the extension of line B-Go; LPW, point LPW – the point on the posterior pharyngeal wall from which a perpendicular line will pass through point V; PCV, point PCV – the point of intersection of the posterior pharyngeal wall and an extension of the lower edge of the second cervical vertebra; U – uvula, tip of the uvula; V, vallecula – the point where the epiglottis meets the base of the tongue; AH, anterior hyoid – the most anterior and superior point on the body of the hyoid bone, representing the inferior part of tongue; Gn, gnathion – the midpoint between Pogonion and Menton; Go, gonion – the mid-plane point at the gonial located by bisecting the posterior border lines of the mandible; Me, menton – the lowest mandible anterior point.Cephalometric points: PNS-Ad1 – distance between PNS and Ad1; SPPW-SPP – distance between SPPW and SPP; U-MPW – distance between U and MPW; PPW-TPP – distance between PPW and TPP; LPW-V – distance between LPW and V; PCV-AH – distance between PCV and AH; S-N – distance between S and N; N-Me – distance between N and Me; S-Go – distance between S and Go; PNS-ANS – distance between PNS and ANS; SPL – soft palate length; SPW – soft palate width; PNS-AH – distance between PNS and AH; ANS-AH – distance between ANS and AH; ANS-V – distance between ANS and V; Go-Gn – distance between Go and Gn; Ulip-E – distance between upper lip anterior border and E line; Llip-E – distance between lower lip anterior border and E line; Wits – distance perpendicular to points A and B onto the occlusal plane in mm; SNA – angle determined by points S, N and A; SNB – angle determined by points S, N, B; SN-MP – angle formed by Go-Me.Cephalometric variables: Intraobserver method error was checked on 20 randomly selected patients’ cephalograms with the method offered by Bland and Altman [Genetic structural equation modeling (GSEM) was performed using the “OpenX” package . Classichttps://cran.r-project.org/web/packages/nFactors/index.html). The principal components were rotated using varimax rotation. The number of components was determined using the “nScree” function from the “nFactors” package according to the optimal coordinates index. A variable belonged to a component if the absolute value of the component loading was larger than 0.5.Principal component analysis (PCA) was used to reduce the dimensionality of cephalometric measurement data and to check the correlations between variables. PCA was performed using the “principal” function from the “psych” package . P values below 0.05 were considered statistically significant.The results of error analysis found no significant differences between the initial and repeated measurements Table .2, c2, d2, e2) of the best-fitting model for each parameter was counted. The results of the model-fitting analysis are presented in Tables The AIC was calculated for each parameter, and the AIC values of each model were analyzed. Only the lowest values were chosen and considered to be the most suitable model for further analysis. The contribution of factors , with d2 = 0.51. Lower airway parameters were mostly determined by environmental factors. PPW-TPP, LPW-V, and PCV-AH showed only common and specific environmental dependency.Variables representing upper airway dimensions  showed moderate to high genetic determination (AE model), with aSkeletal variables were all dependent on genetics to some extent. Maxilla length and had high dominant genetic determination, and Go-Gn and S-Go showed additive genetic, common environmental, and specific environmental influences. N-Me length was affected by additive genetic factors and by common and specific environmental influences.The size of the soft palate was determined by additive and dominant genetic factors. Its length (SPL) was strongly influenced by dominant genetic factors, while its width (SPW) showed a moderate additive genetic influence.2 = 0.9 and 0.92, respectively.Variables reflecting the relationship between the maxilla and the hyoid bone  showed very strong additive genetic determination, with dThe parameters representing the sagittal position of the mandible and its relationship with the cranial base and lip position were all strongly influenced by genetics. Angles SNA and SNB fit best to the model determined by additive genes and specific environment. Angle SN-MP was determined by specific and common environmental factors, angle Ulip-E was determined by dominant genetic factors, and Llip-E was determined by additive genetic factors.According to the correlations in the behavior of the variables, the data were reduced to 5 principal components, which jointly explained 36.8% of the total variance Table . The firUnderstand upper airway morphology, assessing its heritability and knowing characteristics of its growth in a healthy population could help doctors identify persons at risk of breathing problems, such as snoring, OSA or mouth breathing, and even improve the treatments available to patients –25.Heritability was analyzed to understand how upper airway morphology was influenced by genetic factors. The results of our study showed that 19 of 23 cephalometric parameters were strongly determined by genetics, while the remaining parameters were strongly influenced by environmental factors or both genetic and environmental factors. The considerable influence of genetic factors on pharyngeal space variations has been studied by Billing et al. . The stu2 = 0.51). This might be explained by the fact that the nasopharyngeal area is surrounded by the body of the sphenoid bone, the basilar part of the occipital bone and the arch of the atlas on the posterior and superior sides; the morphology of these bony structures are strongly determined by genetic predisposition. On the other hand, the nasopharynx communicates with the oropharynx on the inferior side and the soft palate on the superior side, and these airways are necessary for speech, breathing and swallowing [2 = 0.19).These findings are in agreement with the results of our study: the nasopharyngeal airway measurement (PNS-Ad1) was influenced by genetic factors . The high heritability of this trait means that the oropharyngeal airway space is strongly influenced by genetic factors. This is in contrast to the results of previous studies, which have suggested that the oropharynx is more likely to be related to environmental factors, such as posture, than to genetic factors and that surrounding soft tissues are more influenced by environmental factors [The oropharyngeal airway space (U-MPW) was determined by additive genetic factors (a factors .2 = 0.22), which, although statistically nonsignificant in the overall sample, can also be crucial for some individuals. This might be because the oropharynx is surrounded by the tongue and the hyoid bone on the anterior side and the cervical vertebrae on the posterior side; these structures can change their positions [The oropharynx has an important role in orthodontic treatment planning. It has been reported that rapid maxillary expansion (RME) causes not only an increase in dental width but also changes in the oropharyngeal airway space . After oositions .The upper airway space has been studied by orthodontists for its close relation to the jaws and the craniofacial morphology. Some studies have revealed that the respiratory system is related not only to upper airway size but also to malocclusion type or craniofacial structures . In the Our results showed that hypopharyngeal structures are under environmental influence. It is known that there is a direct correlation between pharyngeal space and obesity . AccordiContrary to the environmental influence hypothesis, J. H. Kang showed that the structure of the hypopharynx has high heritability. This contradiction of our findings and the findings of J. H. Kang et al. could be due to inaccuracy of measurement because the vallecula can collect saliva, preventing initiation of the swallowing reflex. These measurements can also be influenced by head posture, cervical spine position and craniofacial angulation. Da Costa et al. stated that exact measurements of hyoid bone position through cephalometric analysis are difficult because even small deviations may generate apparent variation in the location of the hyoid .Some of the limitations that we encountered in this study are common for research of this nature. The most common limitation in twin studies is sample size . It is wSince most studies use two-dimensional cephalometry for orthodontic diagnosis and treatment planning, it is not surprising that some difficulties are encountered. The main problems that orthodontists face are difficulties in evaluating three-dimensional structures of the upper respiratory tract with two-dimensional cephalometric analysis, difficulties in identifying the landmarks, and overprojection , 44. TheThe dimensions of the upper airway are strongly determined by genes, while the parameters of the lower airway are mainly affected by environmental factors.


Although precision medicine using genetic information offers significant promise, its uptake and eventual clinical and economic impacts are uncertain. Health care payers will play an important role in evaluating evidence and costs to develop coverage and reimbursement policies.To elicit U.S. health care payer preference for genomic precision medicine to better understand trade-offs among clinical benefits, uncertainty, and cost.Using key informant interviewer discussions (N = 6 payers), we identified 6 key attributes of genetic tests important to payers: type of information the test provides (screening vs. treatment prediction), probability that the member has an informative genetic marker, expert agreement on changing medical care based on the marker, quality-of-life gains, life expectancy gains , and cost to the plan. We designed a stated preference discrete choice experiment using these attributes and administered a web survey to a sample of U.S. health care payers. We used effects coding and analyzed the data using an error component mixed logit modeling approach.The survey response rate was 58% (150 participants completed the survey). Approximately 53% of respondents had previous experience evaluating genetic tests for reimbursement, and 85% had more than 5 years of health care decision-making experience. Payers valued improvements in quality of life the most , followed by medical expert agreement on the treatment change . Payers placed a relatively lower value for genetic tests with lower marker probability . Payers mWTP was lowest for resolving uncertainty in quality of life  and life expectancy gains .Payers exhibited a strong preference for genetic tests that improved quality of life, had high expert agreement on changing medical care, and increased life expectancy. These findings suggest that payers will need evidence of clinical utility to support coverage and reimbursement of genomic precision medicine. What is already known about this subjectThe evolution of multigene sequencing has provided significant momentum for precision medicine.Lack of consistency in coverage and reimbursement policies and lack of consistent evidentiary standards are among the several key barriers identified for implementation of genetic precision medicine.What this study addsUsing a discrete choice experiment, we identified 6 attributes that were important to the U.S. payers for the evaluation of the genetic tests and demonstrated how payer preference varied according to the test’s attributes.Payers exhibited a strong preference for genetic tests that improved quality of life, had high expert agreement on changing medical care, and increased life expectancy.These preferences are consistent with payer demands for evidence of clinical utility and clinical consensus of genetic test utilization.1 The evolution of genetic testing from single-gene variant testing to multigene sequencing or even whole genomes has provided significant momentum for precision medicine because of its speed, accuracy, and low cost. However, coverage and reimbursement policies for precision medicine have been cited as a barrier to implementation.2Precision medicine—herein defined as the use of genetic information to guide clinical actions—can be used to implement preventative interventions based on disease risk or to tailor treatment regimens based on likely response or toxicity.3 In another study, Lu et al. (2018) found inconsistent coverage and reimbursement policies for clinical guideline–recommended pharmacogenomic tests (single-gene and multigene tests) for cancer.4 The variation in coverage and reimbursement policies was more prevalent for multigene tests compared with that of single-gene tests in oncology. Payers who covered multigene testing based their coverage policy on the clinical guideline recommendations and used evidence from a wide range of publications such as clinical validation studies, cohort studies, editorials, and reviews.5A recent study identified 5 key barriers for implementation of genetic testing, including 2 which focused on payers: the lack of consistency in coverage and reimbursement policies and lack of consistent evidentiary standards of clinical utility.6 Stated preference discrete choice experiments (DCEs) have been successfully used to estimate stakeholder preference about genomics. Specifically, DCEs have been used to evaluate patient preference for genetic counseling for cancer screening programs, cancer treatments, and the willingness to pay for different types of health care appointments among cancer patients.8 DCEs have also been used to evaluate provider preference, but there is a dearth of studies evaluating payer preference for genomic technologies despite their key role in determining access.3 The lack of data on payer preference for genetic precision medicine tests impedes test developer and researcher ability to develop and provide timely access to innovative tests.To inform appropriate decision making about complex health technology innovations like genetic testing, health care stakeholders must weigh a variety of factors including benefits, harms, costs, and uncertainties.The objective of our study was to elicit U.S. health care payer preference for covering precision medicine tests. We hypothesized that different characteristics of genetic tests would influence payer preference in reimbursement decision making. We chose to use DCEs for a preference-elicitation method to (a) rank the genetic tests attributes after estimating the relative importance of each attribute in reimbursement decision making and (b) determine payer marginal willingness-to-pay (mWTP) estimates for genetic testing attribute levels.We first identified attributes and attribute levels that payers deemed important for their genetic testing reimbursement decision-making process. We then estimated the relative importance of the attributes in payer decision making by asking payers to choose between 2 hypothetical genetic tests with different test attributes in multiple choice tasks.9 The identified attributes and their levels were confirmed for payers using payer interviews. We invited 17 payers for a qualitative interview in-person or over the phone to identify the attributes of a genetic test that are important in the reimbursement decision-making process. Of 17 payers invited, 6 payers provided consent and participated in the interviews and also completed a demographic questionnaire. The payers who consented were mostly from private for-profit plans (50%), and a majority served as medical director (83.3%). The qualitative interview, led by an experienced facilitator, was guided by available genetic test literature and expert opinion. Based on the qualitative interviews, we identified 6 attributes as important to payers for reimbursement of genetic tests: (a) type of information the test provides (screening vs. treatment prediction), (b) probability that the member has an informative genetic marker, (c) expert agreement on changing medical care based on the marker, (d) quality-of-life gains, (e) life expectancy gains , and (f) cost to the plan  for each choice task. An example choice task is presented in We used the D-optimal approach with informative priors to design the choice-based experiment, which resulted in 144 choice tasks. D-optimal designs maximize the precision of the estimated parameters given a specific number of choice tasks and given the specified prior on the beta estimates.12 The survey also included a detailed description of the attributes and levels for the better understanding of the choice scenarios by the participants. We pretested the survey with a payer to assess the response burden, the time taken to complete the survey, and to ensure that participants could understand the survey and choice tasks. We finalized the survey design based on the pretesting responses and any concerns that the participants raised during the pretesting phase.The DCE survey included demographic and background information. We followed the Professional Society for Health Economics and Outcomes Research good research practices for conjoint analysis applications in health care throughout our study.https://tinyurl.com/yd7urm9b). After outlining the concept of genetic testing, the instructions described the 6 attributes of genetic tests included in this study in detail. The participants were asked to consider these attributes as some of the most important characteristics that are typically considered when reimbursing genetic tests. Each respondent was then randomly assigned to 1 of 9 blocks that included 16 choice tasks each. Before the choice tasks, the payers were asked to rank the 6 attributes in order of importance to them when deciding which test to reimburse.We hired a professional survey organization to administer the survey to 150 U.S. health care payers. The U.S. payers were initially contacted through email using an Internet sampling panel. The participants who consented were then referred to a password protected website to complete the survey. Of 258 payers targeted, 150 (58%) payers completed the DCE. An introduction to the questionnaire and DCE was presented through a video education module that outlined the concept of PM and explained the choice task attributes , addresses the within-participant correlation across choice tasks, and accounts for unobserved preference heterogeneity.13 The coefficient on opt-out indicator was constrained to zero and was assumed to be random and follow a normal distribution. This allows for correlation between the opt-in scenarios. The cost of the test to the plan and genetic marker probability were included as continuous variable and all other attributes were effect-coded and included as categorical covariates.All the analyses for this study were performed using STATA 13 software . We used the error component mixed logit model to analyze the responses from the DCE.14Cost parameter was modeled as a continuous variable to allow the estimation of the mWTP. The sign (positive or negative) of coefficients indicated whether the attribute level was preferred (not preferred) by the participant. A higher value of a coefficient represented a stronger preference of the attribute level and a lower positive value represented a weaker preference. A negative value of the coefficient of attribute level indicated the least desirable option. Effects coding is a restriction where the sum of utility contributions across all levels within a given attribute is zero. The estimated attribute levels are thus still relative to each other.A total of 150 participants completed the survey . Forty-tMost participants (≈77%) self-reported their training and education as MD, and 21 (14%) participants reported holding a PharmD degree. Most of the payers (≈85%) who participated had more than 5 years of experience in health care decision making. Approximately half (≈53%) of participants had prior experience evaluating a genetic test for coverage and reimbursement .Part-worth utility values, which measure how much the respondent’s decision to make a specific choice was influenced by each attribute and attribute levels, for the attributes and levels are presented in All else equal, the payers had disutility if the genetic test was used for screening alone compared with the use of the genetic test for treatment alone or screening plus treatment combined. In other words, payers preferred tests that guided treatment decision rather than preventative actions. Payers had disutility for genetic tests that resulted in lower medical expert agreement for change in medical care compared with tests resulting in higher medical expert agreement. The payers had a higher preference for genetic tests that resulted in greater changes in quantity and quality of life. Payers also preferred genetic tests that had lower uncertainty around the quantity of life, for example, among 2 tests that resulted in a quantity of life improvements of 3 months, the payers preferred lower uncertainty about life expectancy gains compared with higher uncertainty. Payers preferred the tests with lower plan costs compared with the tests with higher plan costs.The relative importance of attributes over the range of levels included in this DCE is shown in We estimated payer mWTP by comparing the differences in the levels of attributes to the cost coefficient . Payer mPayer mWTP for a test that had all medical experts agreeing compared with a test that had few experts agreeing for a change in the medical treatment of the plan member was $3,489 . Payer mWTP for a reduction in statistical uncertainty from high to low for a 1-year survival gain was $1,537 , for a reduction in uncertainty for a 4-year survival gain was $1,183 , and a reduction in uncertainty for a 3-month survival gain was $536 (95% CI = $437-$693).We elicited the U.S. payer preference for attributes and levels representing the characteristics for genetic tests used for disease risk screening and treatment of diseases. We used a sequential mixed methods approach to identify the attributes and a DCE survey to elicit payer preference. We found that payers assigned the most importance to the life expectancy and quality-of-life gains of plan members. We also found that uncertainty and expert agreement were important attributes.Format for Formulary Submissions.This was the first study to elicit and quantify the preferences of U.S. payers for genetic testing used for disease risk screening and/or treatment decisions. The results of our study have several implications. Our findings indicate that payers value increased certainty and may be willing to trade off certainty for higher attribute levels in other areas. These findings could be used to enhance health technology assessment activities highlighting key areas for evidence gathering, generation, and presentation aligned with payer preference. This may, in turn, facilitate timely and fully informed coverage and reimbursement decisions. For example, the information and evidence about new genetic tests could be incorporated into existing guidance of coverage and reimbursement of genetic tests, for example, Palmetto GBA, and the AMCP The results of this study can be used along with patient and provider preferences to make predictions about genetic test diffusion. Predictions about test diffusion can be useful for test developers with regard to market planning, pricing, and evidence generation. It can further be used by payers to inform financial planning and future budget impact estimates. Finally, these data can be used to estimate the value of conducting additional research about a genetic test and inform coverage and reimbursement decisions to accept, deny, or seek additional data.In an interesting finding from our study, the payers stated higher utility  for a genetic test that had all medical experts agreeing compared with a test that had few experts agreeing for a change in the medical treatment of the plan member, but the stated utility for a genetic test that reduces statistical uncertainty in life expectancy  was relatively low  elicited preferences of general public and cancer patients for genetic test for guiding cancer treatment and found that patients and the general public preferred genetic tests with higher sensitivity (95% vs. 50%).16 The authors also estimated a willingness to pay of $1,331 for improvement in test sensitivity from 50% to 95% for an aggressively curable cancer. Buchannan et al. (2016) elicited patient preference for genomic screening in chronic lymphocytic leukemia in the United Kingdom.17There is limited evidence for provider preference for genetic tests used for screening purposes versus informing treatment decisions, although several studies have evaluated patient preference in this area. Weymann et al. (2018) elicited patient preference for next-generation sequencing for colorectal cancer risk.18 In summary, these studies suggest patients and payers have overlapping preferences, but payers are also interested in expert consensus and greater certainty in estimates of clinical benefit.The researchers found that patients prefer tests that are more effective, more reliable, cheaper, and quickly return results. Regier et al. (2009) evaluated preference of patients’ families for the use of genetic testing in developmental disability and found that families prefer tests with higher diagnostic yield and shorter wait times.Our study has several limitations. First, because of the generalized nature of the hypothetical genetic tests from which we asked the payers to choose, the results of our study may not be directly applicable to specific genetic tests used for screening or treatment.Second, the attributes selection process included qualitative data from patients and providers. Therefore, some attributes that may have been solely important for the payers may have not been selected.Third, we used a probabilistic sample of the payer population to recruit the respondents using an online professional survey organization and an online panel. This group may not be fully generalizable to the entire U.S. payer population, and there may be some bias due to unobservable payer characteristics.Fourth, additional attributes may also be important to payers, but the DCE survey methodology must balance the number of attributes included against the patient burden and cognitive complexity of the tasks.Fifth, we pretested our DCE survey on 1 payer only. The potential implication of having a small sample for pretesting is that the range of some attribute levels included may not capture the preferences of the respondents. However, we did not observe such phenomenon, and we observed that the attribute levels were sensitive to respondent preference.Sixth, we did not include the analysis of preference heterogeneity among payers based on their geographic location or size. Finally, the response rate of DCE survey was 58%, and we did not collect data for those who participated versus those who did not participate.We evaluated payer preference for precision medicine. We found that payer demand for precision medicine is driven by clinical benefits—improvements in life expectancy and quality of life, and, of importance, increases with reduction in uncertainty around these benefits and with higher expert agreement on recommended medical actions. Our findings suggest that payers will demand evidence of clinical utility in terms of patient comprehensive health outcomes as well as clinical consensus about appropriate use to support coverage and reimbursement policies favorable to precision medicine testing. Future work will be required to better understand evidence thresholds for specific types of precision medicine tests.


Fusarium head blight (FHB) is one of the most studied fungal diseases of wheat, causing massive grain yield and quality losses. This study aimed to extend previous studies on the physiological and biochemical responses of winter wheat to FHB stress in a controlled environment by focusing on the ascorbate-glutathione pathway (AsA-GSH), photosynthetic efficiency, and stress hormone levels, thus providing insight into the possible interactions of different defense mechanisms during infection. The activity of AsA-GSH metabolism was increased in FHB resistant varieties, maintaining the redox state of spikes, and consequently preserving functional photosystem II. Furthermore, carotenoids (Car) were shown to be the major pigments in the photosystem assembly, as they decreased in FHB-stressed spikes of resistant and moderately resistant varieties, compared to controls. Car are also the substrate for the synthesis of abscisic acid (ABA), which acts as a fungal effector and its elevated content leads to increased FHB susceptibility in inoculated spikes. The results of this study contributed to the knowledge of FHB resistance mechanisms and can be used to improve the breeding of FHB resistant varieties, which is considered to be the most effective control measure. Fusarium species causing the disease Fusarium head blight (FHB) are listed among the top ten pathogens globally based on their importance [Fusarium species, such as changes in seasonal phenology, population dynamics, and geographic distribution [About 20% of the world’s annual wheat grain yield and quality losses are due to various plant damages caused by fungal pathogens ,2. Amongportance . It is aportance . Althougportance ,5,6. Theportance ,8. Extreribution , while aribution ,11. Efferibution . Howeverribution . Consideribution .2•−), singlet oxygen (1O2), hydroxyl radical (OH•), and regenerate oxidized carotenoids (Car) or α-tocopherol [Reactive oxygen species (ROS) production is one of the earliest plant responses following pathogen recognition ,16. Unstcopherol . AsA alscopherol .a (Chl a) fluorescence are commonly used. Chl a fluorescence is a method that is a sensitive and non-invasive marker of photosynthetic efficiency and as such, is even used for early detection of pathogen infection when symptoms are not yet visible [Pathogen attack can alter many primary metabolic processes, such as photosynthesis . During  visible .Fusarium spp. [Fusarium graminearum [Phytohormones are known to play a crucial role in defending wheat against various pathogens, including ium spp. . Among tminearum ,29,30. Sminearum ,30,31,32minearum . SAR alsminearum , which aminearum . While tminearum ,36,37. Aminearum , especiaminearum . HoweverAs wheat plants are repeatedly exposed to various pathogens, they have developed sophisticated mechanisms to control responses to these pathogens. Different previous studies focused on the antioxidant response of winter wheat to FHB stress in field conditions, where, except for FHB, various kinds of abiotic and biotic stresses can interact. Furthermore, winter wheat response to FHB stress was studied in the controlled conditions, too. Significant attempts have already been made to pinpoint mechanisms included in wheat defense response to FHB. However, there is still much to clarify about these mechanisms. Therefore, this study aimed to extend previous studies on the physiological and biochemical responses of winter wheat to FHB stress in a controlled environment by focusing on the AsA-GSH pathway, photosynthetic efficiency, and stress hormone levels, thus providing insight into the possible interactions of different defense mechanisms during infection. Since these cultivars have already been characterized for FHB resistance under field conditions, in this study we hypothesize that varieties differing in resistance to FHB will also show differences in the above-mentioned stress hormones, AsA-GSH metabolism, and key parameters of chlorophyll fluorescence and photosynthetic pigments.In order to assess disease severity, the number of infected spikelets per spike was counted at 10 dpi (days post inoculation), before tissue sampling. Symptoms of the infection were not visible earlier than 7 dpi when the majority of spikes exhibited FHB symptoms. At 10 dpi, almost all varieties had clearly visible spike bleaching . Table 1GSH content in spikes of tested wheat varieties was significantly affected by FHB stress only in varieties Tika Taka and Golubica a. These All varieties tested showed an increasing trend of oxidized GSH (GSSG) in FHB treatment, compared to the corresponding controls, where the GSSG concentration increase was not significant only in the variety Vulkan b. The hiAPX activity was significantly increased in the FHB-stressed spikes of varieties Galloper, Kraljica, and Vulkan, compared to corresponding controls, with the highest increase recorded in varieties Kraljica (92.4%) and Galloper (73.4%) a. VarietFusarium infection significantly increased MDHAR activity of variety Kraljica (49.6%), compared to the control, while non-significant decreasing trend was detected in variety Vulkan.FHB stress significantly decreased MDHAR activity in spikes of four tested wheat varieties, compared to controls b. VarietDHAR showed a similar trend in its activity as MDHAR. Most of the varieties significantly decreased DHAR activity as a response to FHB stress, compared to the corresponding control c. HoweveFusarium inoculations significantly induced GR activity in spikes of five out of the six varieties tested, compared to controls .Chl or Chl a b. VarietCar concentration decreased significantly in FHB-stressed spikes of varieties Kraljica (12.9%) and Vulkan (20.2%) compared to control, while the decrease in variety Galloper was not significant. Varieties Golubica and El Nino significantly increased Car in inoculated spikes by 19.3% and 11.9%, respectively. Variety Tika Taka also increased Car but non-significantly c.a to chlorophyll b ratio (Chl a/Chl b) were found in varieties Tika Taka, Kraljica, and Vulkan (a/Chl b significantly increased in variety Tika Taka (22.7%) and decreased in varieties Kraljica (16%) and Vulkan (7%) compared to control ones.Significant changes in chlorophyll d Vulkan d. In FHBa + Chl b) significantly decreased only in varieties Galloper (4.6%) and Kraljica (13.6%), while in the rest of the varieties this ratio increased . The lowest increase in Car/Chl a + Chl b ratio was obtained in the case of variety Vulkan (2.3%).Carotenoids-to-total-chlorophyll ratio (Car/Chl ncreased e. The in0/ABS) in control and FHB-stressed spikes on four measurement points  of each variety separately are shown in 0/ABS in six investigated varieties  (0/ABS in FHB-stressed spikes was observed at 7 dpi and 10 dpi (93.7% and 96.4%). This was the same as for the Golubica variety, where a significant decrease in TR0/ABS was recorded in the inoculated spikes of variety Tika Taka at 7 dpi (96.3%) and 10 dpi (92.5%), compared to controls   and 10 dpi (49.3%) was recorded, compared to 3 dpi, too  a. When ccontrols b. Furthed 93.9%) c. Furthedpi, too d. Howevecontrols e. When o studied f. When cabs) in control and FHB-stressed spikes of each variety separately are presented in abs can be observed in almost all varieties studied, compared to the first two measurements  by 75.4%  and Tika Taka (453.9%), while the lowest increase was recorded for variety Kraljica (48.4%) ( (48.4%) a.Fusarium infections, compared to controls. Significant changes in SA content were observed only for variety El Nino, which showed a significant increase in SA content (54.6%), and variety Vulkan, which showed a significant decrease in SA content (34.2%) in FHB treatment, compared to controls  and 24.0% by the second principal component (PC2) . The firThe world population and accompanying food needs are constantly increasing, while wheat and other cereals contribute significantly to the global food supply . It is aFusarium infection up to the fifth day after FHB inoculation were reported to be asymptomatic. After a few days, as mycelia spread up and down the rachis and through the spikelets, the spike axis was degraded, and symptoms became visible as bleaching [The early stages of leaching ,41. Studleaching . These ileaching . Since tleaching , the samFusarium inoculations in the present study was conducted by measuring the content of the GSH and GSSG, as well as the activities of the enzymes of the AsA-GSH pathway . GSH, together with AsA, is one of the crucial factors in stress tolerance of different plants, including wheat [Fusarium inoculations, despite increased GR activity. Increased GSH contents in FHB susceptible varieties could also be due to de novo GSH synthesis, indicating increased oxidation of cytosol [Following fungus detection by different proteins and cell wall components, several defense mechanisms are induced, including activation of non-enzymatic and enzymatic antioxidants ,44. The ng wheat . Higher  cytosol .2O2 [2O2, AsA is oxidized to monodehydroascorbate (MDHA). MDHA is then enzymatically recycled back to AsA by the activity of the enzyme MDHAR [Fusarium inoculated spikes was mostly decreased. However, FHB moderately resistant Kraljica and resistant Vulkan increased its activity, which implies successful AsA recycling. Except for AsA, GSSG produced during the detoxification of ROS by APX needs to be recycled back to GSH. GSH is regenerated by the flavoprotein oxidoreductase GR, mainly localized in chloroplasts [F. graminearum and F. culmorum in two varieties differing in resistance level to FHB and in corresponding control tissue. Results showed that activities of all measured enzymes increased from the flowering stage until the milk stage in moderately resistant, compared to susceptible variety, in both treatments. Nevertheless, increases in the activity of APX are in accordance with the results obtained in our study, where APX activity increased in FHB-inoculated spikes of all moderately resistant and resistant varieties. Similar results were obtained in the study by Motallebi et al. [F. culmorum. Furthermore, the authors observed much earlier induction of the antioxidant enzymes in moderately resistant and resistant varieties, followed by a decrease at 6 dpi. Similar results were obtained in other studies too [The pools of GSH and AsA are strongly correlated with the activities of the enzymes of the AsA-GSH pathway. APX is a potent scavenger of H2O2 . In the 2O2 . During me MDHAR . In our me MDHAR . Therefome MDHAR . DHAR acroplasts . Contrarroplasts  measuredi et al. , where tdies too ,50,51. Ta were recorded in FHB-inoculated spikes of varieties Golubica and El Nino, while in the rest of the varieties, its concentration was decreased or non-significantly affected by stress induced by FHB inoculations. Similar results were obtained for Chl b, too. Chl b contents were increased in Golubica and El Nino, but also in the variety Kraljica. Recent studies suggest that chlorophylls respond differently to environmental stresses and are an important part of the signal transduction network associated with stress responses. More precisely, low-dose stress can inhibit chlorophyll degradation and stimulate chlorophyll biosynthesis [Fusarium inoculated spikes of FHB susceptible and moderately susceptible varieties Golubica, Tika Taka, and El Nino was observed in Car. In addition to an increase in chlorophyll, an increase in Car was also detected in response to low-dose stress [a/Chl b ratio showed different trends in our study when compared to Chl a and Chl b contents. However, this ratio was most pronounced in the FHB-resistant variety Vulkan.In addition to enzymatic and non-enzymatic systems, FHB inoculations can also alter photosynthetic pigments. It is a generally accepted fact that high oxidative damage reduces the synthesis and accumulation of chlorophylls and other photosynthetic pigments . In our ynthesis ,54, therynthesis . Car havynthesis ,57. The e stress . Howevere stress  who repoe stress , Car pla0/ABS and PIabs, severe FHB stress negatively affected photosynthetic efficiency in wheat spikes of almost all varieties tested, but especially of varieties susceptible to FHB. Varieties Tika Taka, Golubica, and El Nino had the highest decrease in TR0/ABS and PIabs at 7 and 10 dpi, when compared to corresponding controls. Similar results for tested varieties were obtained in the previous research [2 assimilation, and consequently, indirectly inhibits the activity of ribulose 1,5-bisphosphate carboxylase through altered ion fluxes [Chlorophyll fluorescence and photosynthetic parameters have already been used for the detection of fungal infections ,60. In aresearch , where tresearch . A possin fluxes .F. graminearum increased levels of endogenous hormones such as ABA and related metabolites, SA, jasmonic acid (JA), and auxins (IAA) 4 days after inoculation [Fusarium species is rare, Qui et al. [Fusarium inoculations.Considering endogenous stress hormones, ABA is primarily important for eliciting adaptive responses to different types of abiotic stresses, such as drought, low temperature, and salinity . Howeverculation . Althougi et al.  investigi et al. . These rFusarium inoculations. Almost all of the varieties studied increased SA concentration as a response to FHB infection, except varieties Tika Taka and Vulkan, which decreased it. It is still unclear how SA affects wheat defense mechanisms to control FHB [Fusarium species have the ability to metabolize SA through the enzyme salicylate hydroxylase [2O2, is complicated [Arabidopsis thaliana with exogenous SA resulted in increased H2O2 content. Some authors pointed out that the induced production of SA is particularly important in the very early stages of FHB infection of wheat [F. graminearum, where the SA and Ca2+ pathways were activated within 6 hours after inoculations, followed by the activation of the JA-mediated pathway about 12 h after inoculation. Similar findings were confirmed at the gene level by Ameye et al. [2O2 concentration in spikes of resistant winter wheat varieties in the early stage of FHB infection could be the reason of improved FHB resistance, considering the fact that H2O2 acts as a signal molecule for induction of SAR. These results could potentially explain why the SA content in the present study was lower or just slightly changed at 10 dpi.The SA-mediated signaling pathway is one of the crucial pathways in wheat resistance to FHB . Contrartrol FHB . Some stroxylase ,67. Thisroxylase ,68. Howeplicated . Rao et plicated  showed tof wheat ,71. Dingof wheat  reportede et al.  conclude0/ABS, PIabs and APX mostly showing the reduction in FHB resistant and moderately resistant varieties in FHB treatment, compared to controls. Those relations confirmed the previous statement about AsA-GSH pathway as one of the major components of the antioxidant response to FHB stress influencing functionality of photosystem II. The close distance between ABA and Car showed the importance of their mutual relation and involvement in FHB susceptibility, especially in FHB-inoculated spikes of FHB susceptible varieties. Still, it is not clear how SA influenced Chl a and Chl b, as we might assume that response of SA to FHB in wheat spikes might have occurred before 10 dpi.Correlation of different traits and varieties under FHB stress conditions presented by PCA clearly showed that Vulkan is the most FHB resistant variety. Further, GSSG, GSH, GR, and ABA were closely located to FHB susceptible and moderately susceptible varieties in FHB treatment. Those traits and varieties were in negative relation with TR−1, P2O5 (1/2 vol.): 100–150 mg L−1, K2O (1/2 vol.): 200–400 mg L−1) and placed in a greenhouse . During tillering stage (GS21), the conditions maintained in the greenhouse were 10/14 h light/dark photoperiod, 10–14/8–12 °C day/night temperature with the maximum light intensity of 250 μmol m−2 s−1. With the start of stem elongation stage (GS31), conditions were set up to be 12/12 h light/dark photoperiod and 15–18/11–14 °C day/night temperature, while before anthesis stage (GS51) and until the end of experiment conditions were set up to 14/10 h light/dark photoperiod, 21–24/17–20 °C day/night temperature with the maximum light intensity of 750 μmol m−2 s−1. During the whole experiment plants were irrigated with water as necessary, usually twice per week. Nitrogen fertilization was carried out at the two-leaf development stage (GS12) using calcium ammonium nitrate (CAN) (27% N) and one fertilization against pests with the insecticide Vantex  (GS31). When the anthers started to extrude and flowering stage appeared (GS61), plants were inoculated with a mixture of Fusarium graminearum and F. culmorum inoculum. Each treatment consisted of six replicates set up in a randomized complete block design, where each replicate contained 4 plants/pot. Untreated plants were used as controls. Ten days after inoculation spike tissue for determination of GSH and GSSG, activities of the enzymes of the AsA-GSH cycle, and photosynthetic pigments was sampled, frozen in liquid nitrogen, and stored at −80 °C prior to further analysis. Before extractions, wheat spike tissue was grounded in a 10 mL stainless steel jars containing a grinding ball for 1 min at 30 Hz using a TissueLyser . For the analysis of stress hormones, ABA and SA, sampled wheat spikes were frozen in liquid nitrogen and lyophilized.The total of six winter wheat varieties used in this research  originated from the Agricultural Institute Osijek. Seeds of each winter wheat variety were first sown in seedlings’ trays and placed at room temperature to germinate. Trays with five-day-old winter wheat seedlings were moved in a plant growth chamber for six weeks to undergo a period of vernalization . Plants were then transferred in 2.5 L pots filled with soil : 100–200 mg LFusarium species were used for wheat inoculation: F. graminearum (PIO 31), isolated from the winter wheat collected in the eastern part of Croatia, and F. culmorum (IFA 104) obtained from IFA-Tulln, Austria. The conidial inoculum of F. graminearum and F. culmorum was produced by a mixture of wheat and oat grains (3:1 v/v). Macroconidia were washed off the colonized grains, suspension was diluted, final conidial concentrations of both fungi were set to 5 × 104 mL−1 using a hemocytometer  and diluted suspension of F. graminearum and F. culmorum were mixed together. The 20 μL of prepared inoculum mixture was injected with an automatic pipette  in the middle spikelet of the spike of each plant. Plants were subjected to two inoculation events two days apart. To provoke infection, misting treatment started one hour after each inoculation and lasted for the next 36 hours, where foggers sprayed the water every hour for a period of two minutes.Two Type II resistance to FHB (resistance to disease spread within the spike) was evaluated by counting the number of infected spikelets in the inoculated spike of one plant/pot at 10 dpi.w/v) and centrifuged for 15 min at 16,000× g and 4 °C. The reaction mixture consisted of 10 μL of resulting supernatant, 0.03 mg mL−1 DTNB, 0.11 U mL−1 GR, 1 mM EDTA, and 100 mM phosphate buffer (pH 7.0) in a final volume of 0.21 mL. Following a 5-min equilibration period, NADPH in a final concentration of 0.04 mg mL−1 was added to initiate a reaction. The formation of TNB was continuously recorded at 412 nm for 5 min at 25 °C. The amount of tGSH was determined by a standard curve of GSH, and results were expressed as nmol g−1 of FW. For GSSG determination, 2% of vinylpyridine and 5% of triethanolamine were added to an aliquot of deproteinized supernatant, and the reaction mixture was incubated for one hour at room temperature. The measurements were performed the same way as for the tGSH. The content of GSSG was determined using a standard curve for GSSG, and the results were expressed as nmol g−1 of FW. From the difference between tGSH and GSSG, the GSH content was obtained and expressed as nmol g−1 of FW.Total (tGSH) and oxidized glutathione (GSSG) content were determined using a kinetic method based on a continuous reduction in 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB) by reduced glutathione (GSH), where GR and NADPH reduce the GSSG , modifieg and 4 °C. Aliquots of obtained protein extracts were stored at −80 °C until further analysis. Additionally, protein concentration in the enzyme extracts was determined using bovine serum albumin as a protein standard [The wheat spike tissue powder obtained by grounding was homogenized with a cold 100 mM phosphate buffer (pH 7.0) containing 1 mM EDTA. Homogenized samples were then incubated for 15 min on ice and centrifuged for 15 min at 19,000× standard . Measure2O2, and diluted protein extract in 50 mM potassium phosphate buffer (pH 7.0). After 3 min of incubation at room temperature, the decrease in absorbance was measured at 290 nm for 5 min every 15 s. The APX activity was calculated using the molar extinction coefficient (ε = 1.708 mM cm−1) and expressed in U g−1 of protein.APX (EC 1.11.1.11) activity was measured according to a method described by Nakano and Asada . The rea−1. The decrease in absorbance was monitored at 340 nm for 3 min. MDHAR activity was calculated using the molar extinction coefficient (ε = 3.7 mM cm−1) and expressed as U g−1 of protein.MDHAR (EC 1.6.5.4) was determined according to a method described by Hossain et al.  and adju−1) and expressed as U g−1 of proteins.DHAR (EC 1.8.5.1) activity was determined by a method based on monitoring the GSH-dependent reduction in DHA described by Ma and Cheng  and adju−1) and expressed in U g−1 of protein.GR (EC 1.6.4.2) activity was measured according to a method by Racker et al.  and adjug and 4 °C. The procedure was repeated until the plant material was uncolored. The chlorophyll and Car content was determined spectrophotometrically, and photosynthetic pigment concentrations were expressed as mg g−1 FW.Photosynthetic pigments were measured according to a method by Lichtenthaler . Fresh wa fluorescence (ChlF) measurements on spikes of inoculated and control plants were performed at four measurement points: 1 dpi, 3 dpi, 7 dpi, and 10 dpi. The OJIP fluorescence transients were measured with a Handy-PEA fluorimeter . For each of six varieties, six plants from controlled treatment and six plants from FHB inoculated treatment were analyzed by performing measurements on spikes. Before measurements, spikes were dark adapted for 30 min. ChlF transients were induced by pulse of red saturating light  and recorded for 1 s. The JIP-test was applied to analyze and compare ChlF transients [The Chl ansients ,81.6 (Sigma-Aldrich) and (+)-cis, trans ABA-d6 (Trc) . After vortexing, the samples were placed in a Mixer Mill (Roche) for 2 min at a 30 Hz frequency, after which they were homogenized for 1 h at 4 °C. The samples were then centrifuged  and the resulting supernatant was used for analysis. LC-MS/MS screening of target stress hormones was carried out using Agilent Technologies 1200 series HPLC system equipped with 6420 triple quadropole mass spectrometer with electrospray ionization source (ESI) . Chromatographic separation was performed on the Zorbax XDP C18 column  . The electrospray ionization source was operated in negative mode, and samples were detected in the multiple reaction monitoring (MRM) modes. All data acquisition and processing was performed using Agilent MassHunter software . ABA and SA concentrations were calculated and expressed as ng mg−1 of DW.Determination of stress hormones (ABA and SA) was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) as described earlier . In brie0/ABS and PIabs) was performed using two sample t-test (p < 0.05). Determination of differences among measurement points for TR0/ABS and PIabs within each treatment separately was done using one-way analysis of variance (ANOVA), followed by Fisher LSD post hoc test (p < 0.05). Data analysis was performed within the R environment version 4.1.2  [https://www.bioinformatics.com.cn/en (accessed on 21 October 2023), a free online platform for data analysis and visualization.The data for determination of GSH and GSSG content, activities of the enzymes of the AsA-GSH cycle, photosynthetic pigments, and parameters of photosynthetic efficiency were presented as mean of six  independent biological replicates ± standard deviations. Determination of differences among treatments within each variety separately or among treatments of the same measurement point within each variety separately (for TRAustria)  and Stat0/ABS and PIabs in inoculated spikes of FHB resistant or moderately resistant varieties, compared to controls. Furthermore, Car increased in FHB-stressed spikes of susceptible varieties and decreased in FHB-stressed spikes of resistant and moderately resistant varieties. This may indicate their role as the main pigments in photosystem assembly. Car were also found to play a role in the biosynthesis of ABA, which was significantly increased in FHB-inoculated spikes of susceptible varieties. Although the exact role of ABA in biotic stress response is still ambiguous, increasing evidence suggests its role as a fungal effector whose elevated levels lead to increased FHB susceptibility in inoculated spikes of winter wheat varieties. However, to obtain a clearer picture, future research should be directed on the earlier response of winter wheat to FHB inoculation in a time-course manner at several measurement points as well as on effect of Fusarium mycotoxins on the studied parameters.FHB resistance is a complex trait comprised of a complex network of defense responses and mechanisms. Although it is known that SA plays a crucial role in FHB resistance, our studies showed conflicting results. The inconsistent levels of SA at 10 dpi in our study may imply that SA as a main factor in wheat resistance to FHB is probably more important in the very early stages of FHB infection. In the present study, increased activities of the enzymes of AsA-GSH pathway were detected in the FHB-inoculated spikes of moderately resistant and resistant winter wheat varieties. This suggests that the AsA-GSH pathway is one of the major components of the antioxidant response to FHB stress, maintaining the redox state of spikes and preserving functional photosystem II, which could be seen as a lower reduction in TR