Hormones, stress, and heart disease in transgender women with HIV in LITE Plus.

INTRODUCTION: Cardiovascular disease (CVD) is a leading cause of death among transgender women and people with HIV. Exogenous estrogen and psychosocial stressors are known risk factors for CVD. Yet, few studies have used biomarkers to examine the role of stress in CVD risk among transgender women with HIV (TWHIV). This analysis examined whether stress moderates relationships between gender-affirming hormone therapy (GAHT) duration and CVD risk among TWHIV. METHODS: This cross-sectional analysis of baseline data from an observational cohort of 108 Black and Latina TWHIV in Boston, New York, and Washington, DC, enrolled December 2020 - June 2022, measured socio-demographics, medical diagnoses, medications, smoking history, and perceived stress via interviewer-administered surveys. Physiological stress was measured with 14 biomarkers to calculate allostatic load indices (ALI). Forty participants provided saliva samples used to calculate cortisol awakening response and cortisol daily decline. The 2018 American College of Cardiology revised pooled cohort equation estimated 10-year CVD risk. Data were analyzed in 2024. RESULTS: GAHT duration was positively associated with CVD risk scores in bivariate regression. In multivariable linear regression models (adjusting for age, income, education), only age and ALI remained significantly associated with CVD risk scores [ß 1.13, CI: 1.05, 1.21]. No stress measure significantly interacted with GAHT duration to affect CVD risk scores. In visual plots, GAHT duration increased CVD risk scores only for TWHIV experiencing the highest ALI. CONCLUSIONS: Stress plays an important role in CVD in TWHIV. More research is needed on non-GAHT factors, which influence CVD health among transgender women.

Awareness, use and perception of patient versions of clinical practice guidelines - a national cross-sectional survey among patients with a cancer diagnosis and healthcare providers.

BACKGROUND: To investigate awareness, use, and perceptions of the patient guidelines (PGs) of the German Guideline Program in Oncology (GGPO) and to explore general preferences regarding cancer information among patients and healthcare providers (HCPs). METHODS: Two cross-sectional surveys among patients with cancer (November 2020-May 2021) and among HCPs (April -June 2021) were set up as anonymised, self-administered, semi-structured online surveys, including open-ended questions. Data were analysed with descriptive statistics and qualitative thematic analysis. Patients were recruited from national self-help organisations and certified cancer centres located all over Germany. HCPs were recruited from cancer centres, scientific medical societies and guideline groups. RESULTS: Of 816 participating patients, 45% were aware of the GGPO-PGs, while 55% of the 455 participating HCPs were aware of them. Of those aware of the GGPO-PGs, 65% of patients and 86% of HCPs perceived them as helpful, while 95% in both groups saw them as comprehensive. Seventy-five percent of patients and 85% of HCPs were satisfied with the GGPO-PGs, 22%/13% were partially satisfied, and 3%/2% were rather/not at all satisfied. In addition to self-help organisations, physicians and hospitals were perceived as central in distributing the GGPO-PGs. More patients (78%) than HCPs (56%) stated a preference for detailed information, although the wish for concise information - e.g. decision aids - was concurrently expressed by the majority of all participants. Thematic analysis showed that up-to-dateness, trustworthiness, and supportive messaging are important properties for PGs. CONCLUSIONS: HCPs found the GGPO-PGs helpful, but awareness was low, which suggests that dissemination should be improved. This is also true for patients; however, further research needs to be done to increase the helpfulness of PGs for patients. Oncological PGs seem to be needed in different formats according to patients' situational needs. Theory-driven research should investigate how to best frame patient information in a supportive way.

Individually customized gender affirming genital procedures: techniques and considerations.

PURPOSE: Gender-affirming surgery is being increasingly performed for transgender and gender-diverse individuals diagnosed with gender dysphoria. However, there is a group of patients who may seek outcomes that are either a combination of or altogether different from those of binary procedures such as penile inversion vaginoplasty or phalloplasty. METHODS: We describe surgical techniques for less commonly performed gender-affirming genital procedures, in order to introduce these procedures to the medical and surgical community. RESULTS: Operative techniques for phallus-preserving vaginoplasty, vagina-preserving phalloplasty, and removal of genitalia with creation of perineal urethrostomy are described. Demographic characteristics and complications of these procedures in 16 patients are reported. CONCLUSION: Individually customized gender-affirming genital procedures, such as phallus-preserving vaginoplasty, vaginal-preserving phalloplasty, and removal of genitalia and creation of perineal urethrostomy, may better affirm the identities of some gender-diverse patients, and may also preserve desired sexual function of natal genitalia.

Preoperative Temporal Summation is Associated with Impaired Recovery Following Hip Arthroscopy for Femoroacetabular Impingement Syndrome .

Objectives: Chronic pain is a risk factor for worse outcomes following hip arthroscopy for femoroacetabular impingement syndrome (FAIS). Pain sensitization involves the central nervous system perceiving previously innocuous stimuli as noxious. Temporal summation can provide a surrogate measure of sensitization, and may be a clinical tool to identify patients at a higher risk for poor post-hip arthroscopy outcomes. Therefore, we aimed to 1) identify the prevalence of temporal summation in patients undergoing hip arthroscopy for FAIS, 2) determine if there a difference in postoperative improvement between individuals with and without preoperative temporal summation, and 3) examine preoperative predictors of poor postoperative recovery. Methods: 51 participants undergoing hip arthroscopy for FAIS underwent preoperative temporal summation testing. Three months postoperatively, 38 participants completed the 12-item International Hip Outcome Tool (iHOT-12) and reported their overall symptomatic improvement (0% to 100%, with 100% being normal). Participants were categorized on the presence ( Numeric Pain Rating Scale; NPRS   2) or absence (  NPRS < 2) of temporal summation. A Mann-Whitney U test was used to determine the difference in improvement between groups (temporal summation: temporal summation (TS), no temporal summation (NTS), and a linear regression was used to explore predictors of improvement. Results: 23 (45.1%) of 51 participants displayed preoperative temporal summation. In participants with postoperative data, those with temporal summation reported less improvement than those without (TS: 62.8%   29.7%; NTS: 82.7%   13.9%; p = 0.01; Cohen's d = -0.86). Temporal summation (Beta = -0.48; 95% CI -36.6, -8.7) and mental health disorder (Beta = -0.30; 95% CI -28.0, -0.48) predicted 28.1% of the variance in postoperative improvement (p = 0.002). Conclusion: The presence of preoperative temporal summation is common and related to worse postoperative recovery after hip arthroscopy for FAIS.

The association of depression with all-cause and cardiovascular disease mortality risk among transgender and gender diverse and cisgender patients.

BACKGROUND: Transgender and gender diverse (TGD) individuals have elevated mental and physical health disparities and a greater mortality risk compared to their cisgender (non-TGD) counterparts. METHODS: We assessed differences in the association of depression with all-cause and cardiovascular disease (CVD) mortality among TGD and cisgender Veterans Administration patients. A sample of 8981 TGD patients, matched 1:3 with cisgender patients (n = 26,924) patients, was created from administrative and electronic health record data from October 1, 1999 to December 31, 2016. Cox proportional regression models stratified by gender modality (i.e., TGD and cisgender) were used to assess the hazard of all-cause and CVD mortality associated with a history of depression. RESULTS: Adjusted models demonstrated that depression was significantly associated with a greater hazard of all-cause mortality among both TGD (aHR:1.18, 95 % CI: 1.04-1.34) and cisgender (aHR:1.22, 95 % CI: 1.17-1.28) patients. Similar to all-cause mortality, depression was significantly associated with a greater hazard of CVD mortality among cisgender patients ≥65 years (aHR = 1.23, 95 % CI = 1.13-1.35). Findings for TGD patients showed a similar pattern, though results were not significant. LIMITATIONS: Hazards may be underestimated since depression may be underdiagnosed. Further, we were unable to adjust for other health-related risk factors tied to mortality (e.g., smoking). CONCLUSION: Overall, depression was associated with a greater hazard of all-cause mortality among both TGD and cisgender patients. Future work should assess the equity of reach, quality, and outcomes of treatment for depression for TGD populations given the lack of attention to addressing the needs of this important patient demographic.

Evaluation of radiotherapy planning approaches for head and neck patients with tumors close to the skin surface.

PURPOSE: In radiotherapy of the head and neck (H&N) it is common for the clinical target volume (CTV) to extend to the patient's skin. Adding a margin for set-up uncertainty and delivery creates a planning target volume (PTV) that extends beyond the patient surface. This can result in excessive fluence being delivered to the build-up region and therefore the skin. This study evaluates four different planning methods used when inverse-planning H&N radiotherapy treatments with CTV extending to the skin. The aim of the study was to determine which planning method gives superior plan quality. METHOD: Ten H&N cancer patients with a CTV contoured to the skin were inverse-planned using four planning methods. The planning methods compared were: cropping the optimization PTV back from the skin surface by 5.0, 3.0, and 0.0 mm and a virtual bolus method. For each planning method, the increased fluence at the skin surface was analyzed. The CTV coverage and skin doses were compared. Plan robustness was evaluated by applying an isocenter shift of ±3.0 mm in the major axes. RESULTS: The planning method cropping the PTV 0.0 mm from the skin surface results in an increased fluence in the build-up region. The average volume of CTV receiving 98% of the prescription dose was 89.6% ± 3.4%, 91.6% ± 2.4%, and 93.5% ± 1.7% when cropped 5.0, 3.0, and 0.0 mm, respectively, and 93.4% ± 2.1% for the virtual bolus method. Introducing plan uncertainty affects CTV coverage the most when cropping 5.0 mm. When plan uncertainties are considered the methods of cropping 5.0, 3.0 mm, and the virtual bolus method have the same average skin dose within ±0.3%. CONCLUSION: This study shows that a virtual bolus planning method results in no increased fluence at the patient's surface, improves CTV coverage, and is the most robust to changes in setup and patient anatomy.

Immunofilaments Are Well Tolerated after Local or Systemic Administration in Mice.

The invention of nanosized biomaterials has paved the way for novel therapeutics that can manipulate cells on a nanoscale. Nanosized immunofilaments (IFs) are synthetic filamentous polymers consisting out of polyisocyanopeptides, which have been recently established as a powerful platform to activate specific immune cells in vivo such that they raise an antitumor immune response. However, toxicological effects or immunogenicity toward the IFs have not yet been investigated. In this study, we evaluated potential toxic or immunogenic effects in C57BL/6 mice upon intravenous or subcutaneous injection of nonfunctionalized IFs or immunostimulatory IFs over 30 days. We here present a detailed analysis of the gross pathology, hematological parameters, blood biochemistry, histology, and antibody-response against the IF backbone. Our results demonstrate that IFs do not induce severe acute or chronic toxicity in mice. After 30 days, we only found elevated IgG-titers in intravenously injected but not subcutaneously injected mice. In summary, we demonstrate that IFs can be administered into a living organism without adverse side effects, thereby establishing the safety of IFs as a therapeutic intervention.

Generation of nanobodies from transgenic 'LamaMice' lacking an endogenous immunoglobulin repertoire.

Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as 'LamaMouse'. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

Novel markers of MCL1 inhibitor sensitivity in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer sub-type with limited treatment options and poor prognosis. Currently, standard treatments for TNBC include surgery, chemotherapy, and anti-PDL1 therapy. These therapies have limited efficacy in advanced stages. Myeloid-cell leukemia 1 (MCL1) is an anti-apoptotic BCL2 family protein. High expression of MCL1 contributes to chemotherapy resistance and is associated with a worse prognosis in TNBC. MCL1 inhibitors are in clinical trials for TNBC, but response rates to these inhibitors can vary and predictive markers are lacking. Currently, we identified a 4-member (AXL, ETS1, IL6, EFEMP1) gene signature (GS) that predicts MCL1 inhibitor sensitivity in TNBC cells. Factors encoded by these genes regulate signaling pathways to promote MCL1 inhibitor resistance. Small molecule inhibitors of the GS factors can overcome resistance and sensitize otherwise resistant TNBC cells to MCL1 inhibitor treatment. These findings offer insights into potential therapeutic strategies and tumor stratification for MCL1 inhibitor use in TNBC.

Mycobacterium tuberculosis produces D-serine under hypoxia to limit CD8+ T cell-dependent immunity in mice.

Adaptation to hypoxia is a major challenge for the survival of Mycobacterium tuberculosis (Mtb) in vivo. Interferon (IFN)-γ-producing CD8+ T cells contribute to control of Mtb infection, in part by promoting antimicrobial activities of macrophages. Whether Mtb counters these responses, particularly during hypoxic conditions, remains unknown. Using metabolomic, proteomic and genetic approaches, here we show that Mtb induced Rv0884c (SerC), an Mtb phosphoserine aminotransferase, to produce D-serine. This activity increased Mtb pathogenesis in mice but did not directly affect intramacrophage Mtb survival. Instead, D-serine inhibited IFN-γ production by CD8+ T cells, which indirectly reduced the ability of macrophages to restrict Mtb upon co-culture. Mechanistically, D-serine interacted with WDR24 and inhibited mTORC1 activation in CD8+ T cells. This decreased T-bet expression and reduced IFN-γ production by CD8+ T cells. Our findings suggest an Mtb evasion mechanism where pathogen metabolic adaptation to hypoxia leads to amino acid-dependent suppression of adaptive anti-TB immunity.

Disparities in cancer incidence by sexual orientation.

INTRODUCTION: Cancer risk factors are more common among sexual minority populations (e.g., lesbian, bisexual) than their heterosexual peers, yet little is known about cancer incidence across sexual orientation groups. METHODS: The 1989-2017 data from the Nurses' Health Study II, a longitudinal cohort of female nurses across the United States, were analyzed (N = 101,543). Sexual orientation-related cancer disparities were quantified by comparing any cancer incidence among four sexual minority groups based on self-disclosure-(1) heterosexual with past same-sex attractions/partners/identity; (2) mostly heterosexual; (3) bisexual; and (4) lesbian women-to completely heterosexual women using age-adjusted incidence rate ratios (aIRR) calculated by the Mantel-Haenszel method. Additionally, subanalyses at 21 cancer disease sites (e.g., breast, colon/rectum) were conducted. RESULTS: For all-cancer analyses, there were no statistically significant differences in cancer incidence at the 5% type I error cutoff among sexual minority groups when compared to completely heterosexual women; the aIRR was 1.17 (95% CI,0.99-1.38) among lesbian women and 0.80 (0.58-1.10) among bisexual women. For the site-specific analyses, incidences at multiple sites were significantly higher among lesbian women compared to completely heterosexual women: thyroid cancer (aIRR, 1.87 [1.03-3.41]), basal cell carcinoma (aIRR, 1.85 [1.09-3.14]), and non-Hodgkin lymphoma (aIRR, 2.13 [1.10-4.12]). CONCLUSION: Lesbian women may be disproportionately burdened by cancer relative to their heterosexual peers. Sexual minority populations must be explicitly included in cancer prevention efforts. Comprehensive and standardized sexual orientation data must be systematically collected so nuanced sexual orientation-related cancer disparities can be accurately assessed for both common and rare cancers.

Gender-Affirming Hormone Therapy and Cervical Cancer Screening Rates in Transgender Men and Nonbinary People: A Cross-Sectional Study from a Boston Community Health Center.

Purpose: Clinical monitoring for patients receiving gender-affirming hormone therapy (GAHT) has the potential to facilitate their receipt of preventive health services. We aimed to determine whether GAHT is associated with increased utilization of cervical cancer screening among transgender men (TM) and nonbinary persons assigned female at birth (NB-AFAB). Methods: We conducted a cross-sectional observational study of a single community health center in Boston. Persons of all gender identities eligible for cervical cancer screening during 2008-2019 were assessed. The outcome of interest was receipt of cervical cancer screening based on U.S. Preventive Services Task Force recommendations. We compared the proportion of persons who received cervical cancer screening by prescription of GAHT. Results: We identified 13,267 eligible persons. This cohort included 10,547 (79.5%) cisgender women, 1547 (11.7%) TM, and 1173 (8.8%) NB-AFAB persons. Among all persons eligible for cervical cancer screening, TM and NB-AFAB persons were less likely to receive screening than cisgender women (56.2% and 56.1% vs. 60.5% respectively; odds ratio [OR] = 0.84; 95% confidence interval [CI] = 0.75-0.93; OR = 0.84; 95% CI = 0.74-0.94, respectively). Among TM, those prescribed testosterone were more likely to receive cervical cancer screening than those not prescribed testosterone (57.9% vs. 48.2%, OR = 1.47; 95% CI = 1.14-1.92). Among NB-AFAB adults, those prescribed testosterone were more likely to receive cervical cancer screening than those not prescribed testosterone (61.9% vs. 51.5%, OR = 1.53; 95% CI = 1.21-1.93). Conclusions: The benefits of engagement in care to access GAHT may extend beyond the hormonal intervention to preventive health services.

Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

Dimensional accuracy and simulation-based optimization of polyolefins and biocopolyesters for extrusion-based additive manufacturing and steam sterilization.

Polyolefins exhibit robust mechanical and chemical properties and can be applied in the medical field, e.g. for the manufacturing of dentures. Despite their wide range of applications, they are rarely used in extrusion-based printing due to their warpage tendency. The aim of this study was to investigate and reduce the warpage of polyolefins compared to commonly used filaments after additive manufacturing (AM) and sterilization using finite element simulation. Three types of filaments were investigated: a medical-grade polypropylene (PP), a glass-fiber reinforced polypropylene (PP-GF), and a biocopolyester (BE) filament, and they were compared to an acrylic resin (AR) for material jetting. Square specimens, standardized samples prone to warpage, and denture bases (n = 10 of each group), as clinically relevant and anatomically shaped reference, were digitized after AM and steam sterilization (134 °C). To determine warpage, the volume underneath the square specimens was calculated, while the deviations of the denture bases from the printing file were measured using root mean square (RMS) values. To reduce the warpage of the PP denture base, a simulation of the printing file based on thermomechanical calculations was performed. Statistical analysis was conducted using the Kruskal-Wallis test, followed by Dunn's test for multiple comparisons. The results showed that PP exhibited the greatest warpage of the square specimens after AM, while PP-GF, BE, and AR showed minimal warpage before sterilization. However, warpage increased for PP-GF, BE and AR during sterilization, whereas PP remained more stable. After AM, denture bases made of PP showed the highest warpage. Through simulation-based optimization, warpage of the PP denture base was successfully reduced by 25%. In contrast to the reference materials, PP demonstrated greater dimensional stability during sterilization, making it a potential alternative for medical applications. Nevertheless, reducing warpage during the cooling process after AM remains necessary, and simulation-based optimization holds promise in addressing this issue.

Adjuvant dendritic cell therapy in stage IIIB/C melanoma: the MIND-DC randomized phase III trial.

Autologous natural dendritic cells (nDCs) treatment can induce tumor-specific immune responses and clinical responses in cancer patients. In this phase III clinical trial (NCT02993315), 148 patients with resected stage IIIB/C melanoma were randomized to adjuvant treatment with nDCs (n = 99) or placebo (n = 49). Active treatment consisted of intranodally injected autologous CD1c+ conventional and plasmacytoid DCs loaded with tumor antigens. The primary endpoint was the 2-year recurrence-free survival (RFS) rate, whereas the secondary endpoints included median RFS, 2-year and median overall survival, adverse event profile, and immunological response The 2-year RFS rate was 36.8% in the nDC treatment group and 46.9% in the control group (p = 0.31). Median RFS was 12.7 months vs 19.9 months, respectively (hazard ratio 1.25; 90% CI: 0.88-1.79; p = 0.29). Median overall survival was not reached in both treatment groups (hazard ratio 1.32; 90% CI: 0.73-2.38; p = 0.44). Grade 3-4 study-related adverse events occurred in 5% and 6% of patients. Functional antigen-specific T cell responses could be detected in 67.1% of patients tested in the nDC treatment group vs 3.8% of patients tested in the control group (p < 0.001). In conclusion, while adjuvant nDC treatment in stage IIIB/C melanoma patients generated specific immune responses and was well tolerated, no benefit in RFS was observed.

pH and ROS Responsiveness of Polymersome Nanovaccines for Antigen and Adjuvant Codelivery: An In Vitro and In Vivo Comparison.

The antitumor immunity can be enhanced through the synchronized codelivery of antigens and immunostimulatory adjuvants to antigen-presenting cells, particularly dendritic cells (DCs), using nanovaccines (NVs). To study the influence of intracellular vaccine cargo release kinetics on the T cell activating capacities of DCs, we compared stimuli-responsive to nonresponsive polymersome NVs. To do so, we employed "AND gate" multiresponsive (MR) amphiphilic block copolymers that decompose only in response to the combination of chemical cues present in the environment of the intracellular compartments in antigen cross-presenting DCs: low pH and high reactive oxygen species (ROS) levels. After being unmasked by ROS, pH-responsive side chains are exposed and can undergo a charge shift within a relevant pH window of the intracellular compartments in antigen cross-presenting DCs. NVs containing the model antigen Ovalbumin (OVA) and the iNKT cell activating adjuvant α-Galactosylceramide (α-Galcer) were fabricated using microfluidics self-assembly. The MR NVs outperformed the nonresponsive NV in vitro, inducing enhanced classical- and cross-presentation of the OVA by DCs, effectively activating CD8+, CD4+ T cells, and iNKT cells. Interestingly, in vivo, the nonresponsive NVs outperformed the responsive vaccines. These differences in polymersome vaccine performance are likely linked to the kinetics of cargo release, highlighting the crucial chemical requirements for successful cancer nanovaccines.

Gross failure rates and failure modes for a commercial AI-based auto-segmentation algorithm in head and neck cancer patients.

PURPOSE: Artificial intelligence (AI) based commercial software can be used to automatically delineate organs at risk (OAR), with potential for efficiency savings in the radiotherapy treatment planning pathway, and reduction of inter- and intra-observer variability. There has been little research investigating gross failure rates and failure modes of such systems. METHOD: 50 head and neck (H&N) patient data sets with "gold standard" contours were compared to AI-generated contours to produce expected mean and standard deviation values for the Dice Similarity Coefficient (DSC), for four common H&N OARs (brainstem, mandible, left and right parotid). An AI-based commercial system was applied to 500 H&N patients. AI-generated contours were compared to manual contours, outlined by an expert human, and a gross failure was set at three standard deviations below the expected mean DSC. Failures were inspected to assess reason for failure of the AI-based system with failures relating to suboptimal manual contouring censored. True failures were classified into 4 sub-types (setup position, anatomy, image artefacts and unknown). RESULTS: There were 24 true failures of the AI-based commercial software, a gross failure rate of 1.2%. Fifteen failures were due to patient anatomy, four were due to dental image artefacts, three were due to patient position and two were unknown. True failure rates by OAR were 0.4% (brainstem), 2.2% (mandible), 1.4% (left parotid) and 0.8% (right parotid). CONCLUSION: True failures of the AI-based system were predominantly associated with a non-standard element within the CT scan. It is likely that these non-standard elements were the reason for the gross failure, and suggests that patient datasets used to train the AI model did not contain sufficient heterogeneity of data. Regardless of the reasons for failure, the true failure rate for the AI-based system in the H&N region for the OARs investigated was low (∼1%).