Analysis and Validation of Image Search Engines in Histopathology.

Searching for similar images in archives of histology and histopathology images is a crucial task that may aid in patient tissue comparison for various purposes, ranging from triaging and diagnosis to prognosis and prediction. Whole slide images (WSIs) are highly detailed digital representations of tissue specimens mounted on glass slides. Matching WSI to WSI can serve as the critical method for patient tissue comparison. In this paper, we report extensive analysis and validation of four search methods bag of visual words (BoVW), Yottixel, SISH, RetCCL, and some of their potential variants. We analyze their algorithms and structures and assess their performance. For this evaluation, we utilized four internal datasets (1269 patients) and three public datasets (1207 patients), totaling more than 200, 000 patches from 38 different classes/subtypes across five primary sites. Certain search engines, for example, BoVW, exhibit notable efficiency and speed but suffer from low accuracy. Conversely, search engines like Yottixel demonstrate efficiency and speed, providing moderately accurate results. Recent proposals, including SISH, display inefficiency and yield inconsistent outcomes, while alternatives like RetCCL prove inadequate in both accuracy and efficiency. Further research is imperative to address the dual aspects of accuracy and minimal storage requirements in histopathological image search.

High-risk human papilloma virus status & outcomes for penile squamous cell carcinoma: A single institution experience.

OBJECTIVES: There is a paucity of data on North American cohorts of patients with penile squamous cell carcinoma (pSCC). Herein, we aimed to assess the sensitivity of various modalities to identify human papillomavirus (HPV) status, determine the prevalence of high-risk HPV-positivity, and evaluate the prognostic impact of relevant clinicopathologic variables. METHODS: Patients with pSCC (n = 121) consecutively treated with partial/total penectomy (2000-2022) at a single institution were included. HPV status (based on immunohistochemistry [IHC], in situ hybridization [ISH], and panviral metagenomic sequencing [PMS]), histologic features, and outcomes were reviewed. Outcome events included death due to disease and progression. RESULTS: The majority of patients were white (105/121, 86.8%). Thirty-seven (30.6%) were high-risk HPV-positive, and morphologic evaluation had a sensitivity of 97.3% (95% confidence interval [CI], 86.2-99.5) for predicting high-risk HPV status compared to IHC/ISH/PMS. Disease progression was more common among high-risk HPV-negative compared to high-risk HPV-positive patients (HR 2.74, CI 1.12-8.23, P = 0.03). Moreover, among high-risk HPV-negative patients, those with moderate-poorly differentiated tumors had increased disease-specific mortality (32.6%, CI 17.1-48.1) compared to those with well-differentiated tumors (0%). Among high-risk HPV-positive patients, those with basaloid morphology had lower disease-specific mortality (0% vs 14.4%, CI 0.0-33.1). CONCLUSIONS: We demonstrate high-risk HPV-positivity in approximately one-third of patients with pSCC. Morphologic evaluation alone had a high sensitivity in correctly determining HPV status. Our results suggest that high-risk HPV status and morphologic features (differentiation in high-risk HPV-negative, and basaloid subtype in high-risk HPV-positive pSCC) may have prognostic value.

Genetic variants affecting mitochondrial function provide further insights for kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is a complex disorder that has become a high prevalence global health problem, with diabetes being its predominant pathophysiologic driver. Autosomal genetic variation only explains some of the predisposition to kidney disease. Variations in the mitochondrial genome (mtDNA) and nuclear-encoded mitochondrial genes (NEMG) are implicated in susceptibility to kidney disease and CKD progression, but they have not been thoroughly explored. Our aim was to investigate the association of variation in both mtDNA and NEMG with CKD (and related traits), with a particular focus on diabetes. METHODS: We used the UK Biobank (UKB) and UK-ROI, an independent collection of individuals with type 1 diabetes mellitus (T1DM) patients. RESULTS: Fourteen mitochondrial variants were associated with estimated glomerular filtration rate (eGFR) in UKB. Mitochondrial variants and haplogroups U, H and J were associated with eGFR and serum variables. Mitochondrial haplogroup H was associated with all the serum variables regardless of the presence of diabetes. Mitochondrial haplogroup X was associated with end-stage kidney disease (ESKD) in UKB. We confirmed the influence of several known NEMG on kidney disease and function and found novel associations for SLC39A13, CFL1, ACP2 or ATP5G1 with serum variables and kidney damage, and for SLC4A1, NUP210 and MYH14 with ESKD. The G allele of TBC1D32-rs113987180 was associated with higher risk of ESKD in patients with diabetes (OR:9.879; CI95%:4.440-21.980; P = 2.0E-08). In UK-ROI, AGXT2-rs71615838 and SURF1-rs183853102 were associated with diabetic nephropathies, and TFB1M-rs869120 with eGFR. CONCLUSIONS: We identified novel variants both in mtDNA and NEMG which may explain some of the missing heritability for CKD and kidney phenotypes. We confirmed the role of MT-ND5 and mitochondrial haplogroup H on renal disease (serum variables), and identified the MT-ND5-rs41535848G variant, along with mitochondrial haplogroup X, associated with higher risk of ESKD. Despite most of the associations were independent of diabetes, we also showed potential roles for NEMG in T1DM.

The Use of Natural Therapies in Patients With Skin Cancer: A Scoping Review.

Complementary and alternative medicine (CAM) use has become a field of growing interest in dermatology. However, the prevalence of CAM use is difficult to quantify as it varies based on many factors. Given the exploratory nature of the topic, a scoping review was conducted to identify studies that quantify biologically based CAM use in skin cancer patients. A comprehensive search of Embase, PubMed, and Web of Science databases from inception to August 28th, 2023, was performed. A total of 3,150 articles were identified through the database search. After article screening, 6 studies were suitable for inclusion in this review. Articles included were all questionnaire, survey, or interview style. Biologically based CAM use is prevalent in skin cancer patients. It can be associated with many factors such as location, stage of cancer, and age. CAM use can interact with conventional therapy; therefore, physicians should employ a culturally competent approach to inquiring about CAM use in order to improve patient outcomes and identify patterns and predictors of use.J Drugs Dermatol. 2024;23(5):322-326. doi:10.36849/JDD.8077.

Lipid turnover through lipophagy in the newly identified extremophilic green microalga Chlamydomonas urium.

Autophagy is a central degradative pathway highly conserved among eukaryotes, including microalgae, which remains unexplored in extremophilic organisms. In this study, we described and characterized autophagy in the newly identified extremophilic green microalga Chlamydomonas urium, which was isolated from an acidic environment. The nuclear genome of C. urium was sequenced, assembled and annotated in order to identify autophagy-related genes. Transmission electron microscopy, immunoblotting, metabolomic and photosynthetic analyses were performed to investigate autophagy in this extremophilic microalga. The analysis of the C. urium genome revealed the conservation of core autophagy-related genes. We investigated the role of autophagy in C. urium by blocking autophagic flux with the vacuolar ATPase inhibitor concanamycin A. Our results indicated that inhibition of autophagic flux in this microalga resulted in a pronounced accumulation of triacylglycerols and lipid droplets (LDs). Metabolomic and photosynthetic analyses indicated that C. urium cells with impaired vacuolar function maintained an active metabolism. Such effects were not observed in the neutrophilic microalga Chlamydomonas reinhardtii. Inhibition of autophagic flux in C. urium uncovered an active recycling of LDs through lipophagy, a selective autophagy pathway for lipid turnover. This study provided the metabolic basis by which extremophilic algae are able to catabolize lipids in the vacuole.

Integrated information decomposition unveils major structural traits of in silico and in vitro neuronal networks.

The properties of complex networked systems arise from the interplay between the dynamics of their elements and the underlying topology. Thus, to understand their behavior, it is crucial to convene as much information as possible about their topological organization. However, in large systems, such as neuronal networks, the reconstruction of such topology is usually carried out from the information encoded in the dynamics on the network, such as spike train time series, and by measuring the transfer entropy between system elements. The topological information recovered by these methods does not necessarily capture the connectivity layout, but rather the causal flow of information between elements. New theoretical frameworks, such as Integrated Information Decomposition (Φ-ID), allow one to explore the modes in which information can flow between parts of a system, opening a rich landscape of interactions between network topology, dynamics, and information. Here, we apply Φ-ID on in silico and in vitro data to decompose the usual transfer entropy measure into different modes of information transfer, namely, synergistic, redundant, or unique. We demonstrate that the unique information transfer is the most relevant measure to uncover structural topological details from network activity data, while redundant information only introduces residual information for this application. Although the retrieved network connectivity is still functional, it captures more details of the underlying structural topology by avoiding to take into account emergent high-order interactions and information redundancy between elements, which are important for the functional behavior, but mask the detection of direct simple interactions between elements constituted by the structural network topology.

Comparison of a modified staging system with 8th edition AJCC criteria in a North American cohort of pT2/pT3 HPV-negative penile squamous cell carcinoma.

The staging for pT2/pT3 penile squamous cell carcinoma (pSCC) has undergone major changes. Some authors proposed criteria wherein the distinction between pT2/pT3 was made using the same histopathological variables that are currently utilized to differentiate pT1a/pT1b. In this single-institution, North American study, we focused on (HPV-negative) pT2/3 pSCCs (i.e., tumors invading corpus spongiosum/corpus cavernosum), and compared the prognostic ability of the following systems: (i) AJCC (8th edition) criteria; (ii) modified staging criteria proposed by Sali et al. (Am J Surg Pathol. 2020; 44:1112-7). In the proposed system, pT2 tumors were defined as those devoid of lymphovascular invasion (LVI) or perineural invasion (PNI), and were not poorly differentiated; whereas pT3 showed one or more of the following: LVI, PNI, and/or grade 3. 48 pT2/pT3 cases were included (AJCC, pT2: 27 and pT3: 21; Proposed, pT2: 22 and pT3: 26). The disease-free survival (DFS) and progression-free survival (PFS) did not differ between pT2 and pT3, following the current AJCC definitions (p = 0.19 and p = 0.10, respectively). When the pT2/3 stages were reconstructed using the modified criteria, however, a statistically significant difference was present in both DFS and PFS between pT2 and pT3 (p = 0.004 and p = 0.003, respectively). The proposed staging system has the potential to improve the prognostication of pT2/pT3 tumors in pSCC. Each of these histopathologic variables has been shown to have a significant association with outcomes in pSCC, which is an advantage. Further studies are needed to demonstrate the utility of this modified staging system in patient populations from other geographic regions.

Hydroxycitrate delays early mortality in mice and promotes muscle regeneration while inducing a rich hepatic energetic status.

ATP citrate lyase (ACLY) inhibitors have the potential of modulating central processes in protein, carbohydrate, and lipid metabolism, which can have relevant physiological consequences in aging and age-related diseases. Here, we show that hepatic phospho-active ACLY correlates with overweight and Model for End-stage Liver Disease score in humans. Wild-type mice treated chronically with the ACLY inhibitor potassium hydroxycitrate exhibited delayed early mortality. In AML12 hepatocyte cultures, the ACLY inhibitors potassium hydroxycitrate, SB-204990, and bempedoic acid fostered lipid accumulation, which was also observed in the liver of healthy-fed mice treated with potassium hydroxycitrate. Analysis of soleus tissue indicated that potassium hydroxycitrate produced the modulation of wound healing processes. In vivo, potassium hydroxycitrate modulated locomotor function toward increased wire hang performance and reduced rotarod performance in healthy-fed mice, and improved locomotion in mice exposed to cardiotoxin-induced muscle atrophy. Our findings implicate ACLY and ACLY inhibitors in different aspects of aging and muscle regeneration.

A Deep Learning Approach to Estimate Multi-Level Mental Stress From EEG Using Serious Games.

Stress is revealed by the inability of individuals to cope with their environment, which is frequently evidenced by a failure to achieve their full potential in tasks or goals. This study aims to assess the feasibility of estimating the level of stress that the user is perceiving related to a specific task through an electroencephalograpic (EEG) system. This system is integrated with a Serious Game consisting of a multi-level stress driving tool, and Deep Learning (DL) neural networks are used for classification. The game involves controlling a vehicle to dodge obstacles, with the number of obstacles increasing based on complexity. Assuming that there is a direct correlation between the difficulty level of the game and the stress level of the user, a recurrent neural network (RNN) with a structure based on gated recurrent units (GRU) was used to classify the different levels of stress. The results show that the RNN model is able to predict stress levels above current state-of-the-art with up to 94% accuracy in some cases, suggesting that the use of EEG systems in combination with Serious Games and DL represents a promising technique in the prediction and classification of mental stress levels.

Exploring the role of GHRH antagonist MIA-602 in overcoming Doxorubicin-resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the rapid proliferation of mutagenic hematopoietic progenitors in the bone marrow. Conventional therapies include chemotherapy and bone marrow stem cell transplantation; however, they are often associated with poor prognosis. Notably, growth hormone-releasing hormone (GHRH) receptor antagonist MIA-602 has been shown to impede the growth of various human cancer cell lines, including AML. This investigation examined the impact of MIA-602 as monotherapy and in combination with Doxorubicin on three Doxorubicin-resistant AML cell lines, KG-1A, U-937, and K-562. The in vitro results revealed a significant reduction in cell viability for all treated wild-type cells. Doxorubicin-resistant clones were similarly susceptible to MIA-602 as the wild-type counterpart. Our in vivo experiment of xenografted nude mice with Doxorubicin-resistant K-562 revealed a reduction in tumor volume with MIA-602 treatment compared to control. Our study demonstrates that these three AML cell lines, and their Doxorubicin-resistant clones, are susceptible to GHRH antagonist MIA-602.

Democratizing Artificial Intelligence in Anatomic Pathology.

CONTEXT.­: Artificial intelligence is a transforming technology for anatomic pathology. Involvement within the workforce will foster support for algorithm development and implementation. OBJECTIVE.­: To develop a supportive ecosystem that enables pathologists with variable expertise in artificial intelligence to create algorithms in a development environment with seamless transition to a production environment. RESULTS.­: The development team considered internal development and vended solutions. Because of the extended timeline and resource requirements for internal development, a decision was made to use a vended solution. Vendor proposals were solicited and reviewed by pathologists, IT, and security groups. A vendor was selected and pipelines for development and production were established. Proposals for development were solicited from the pathology department. Eighty-four investigators were selected for the initial cohort, receiving training and access to dedicated subject matter experts. A total of 30 of 31 projects progressed through the model development process of annotating, training, and validation. Based on these projects, 15 abstracts were submitted to national meetings. CONCLUSIONS.­: Democratizing artificial intelligence by creating an ecosystem to support pathologists with varying levels of expertise can break down entry barriers, reduce overall cost of algorithm development, improve algorithm quality, and enhance the speed of adoption.

Creating an atlas of normal tissue for pruning WSI patching through anomaly detection.

Patching whole slide images (WSIs) is an important task in computational pathology. While most of them are designed to classify or detect the presence of pathological lesions in a WSI, the confounding role and redundant nature of normal histology are generally overlooked. In this paper, we propose and validate the concept of an "atlas of normal tissue" solely using samples of WSIs obtained from normal biopsies. Such atlases can be employed to eliminate normal fragments of tissue samples and hence increase the representativeness of the remaining patches. We tested our proposed method by establishing a normal atlas using 107 normal skin WSIs and demonstrated how established search engines like Yottixel can be improved. We used 553 WSIs of cutaneous squamous cell carcinoma to demonstrate the advantage. We also validated our method applied to an external dataset of 451 breast WSIs. The number of selected WSI patches was reduced by 30% to 50% after utilizing the proposed normal atlas while maintaining the same indexing and search performance in leave-one-patient-out validation for both datasets. We show that the proposed concept of establishing and using a normal atlas shows promise for unsupervised selection of the most representative patches of the abnormal WSI patches.

CD99 Modulates the Proteomic Landscape of Ewing Sarcoma Cells and Related Extracellular Vesicles.

Ewing sarcoma (EWS) is an aggressive pediatric bone tumor characterized by unmet clinical needs and an incompletely understood epigenetic heterogeneity. Here, we considered CD99, a major surface molecule hallmark of EWS malignancy. Fluctuations in CD99 expression strongly impair cell dissemination, differentiation, and death. CD99 is also loaded within extracellular vesicles (EVs), and the delivery of CD99-positive or CD99-negative EVs dynamically exerts oncogenic or oncosuppressive functions to recipient cells, respectively. We undertook mass spectrometry and functional annotation analysis to investigate the consequences of CD99 silencing on the proteomic landscape of EWS cells and related EVs. Our data demonstrate that (i) the decrease in CD99 leads to major changes in the proteomic profile of EWS cells and EVs; (ii) intracellular and extracellular compartments display two distinct signatures of differentially expressed proteins; (iii) proteomic changes converge to the modulation of cell migration and immune-modulation biological processes; and (iv) CD99-silenced cells and related EVs are characterized by a migration-suppressive, pro-immunostimulatory proteomic profile. Overall, our data provide a novel source of CD99-associated protein biomarkers to be considered for further validation as mediators of EWS malignancy and as EWS disease liquid biopsy markers.

Characterisation of fatty acyl reductases of sunflower (Helianthus annuus L.) seed.

Long and very long chain fatty alcohols are produced from their corresponding acyl-CoAs through the activity of fatty acyl reductases (FARs). Fatty alcohols are important components of the cuticle that protects aerial plant organs, and they are metabolic intermediates in the synthesis of the wax esters in the hull of sunflower (Helianthus annuus) seeds. Genes encoding 4 different FARs (named HaFAR2, HaFAR3, HaFAR4 and HaFAR5) were identified using BLAST, and studies showed that four of the genes were expressed in seed hulls. In this study, the structure and location of sunflower FAR proteins were determined. They were also expressed exogenously in Saccharomyces cerevisiae to evaluate their substrate specificity based on the fatty alcohols synthesized by the transformed yeasts. Three of the four enzymes tested showed activity in yeast. HaFAR3 produced C18, C20 and C22 saturated alcohols, whereas HaFAR4 and HaFAR5 produced C24 and C26 saturated alcohols. The involvement of these genes in the synthesis of sunflower seed wax esters was addressed by considering the results obtained.

Characterization of platelet rich plasma in feline immunodeficiency virus-infected cats: Cell, and PDGF-BB and TGF-ß1 growth factor analysis.

Autologous platelet-rich plasma (PRP) contains growth factors (GFs) that modulate the expression of inflammatory cells; thus, these products could be considered a good strategy to favor tissue regeneration in feline immunodeficiency (FIV) positive cats. However, there is no scientific documentation on obtaining PRP in FIV-positive cats. Authors hypothesized that PRP can be obtained in FIV cats following the PRGF®-Endoret® methodology. The objectives of this study were to compare the platelet, erythrocyte, and leukocyte concentration between whole blood (WB) and the PRP; and determine the concentration of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor ß1 (TGF-ß1) in FIV-positive cats. Sixteen adults FIV-positive asymptomatic cats were included in the study. WB samples were drawn and the PRP was obtained by centrifugation at 265g for 10 min. Erythrocyte and leukocyte, platelets, and mean platelet volume (MPV) were determined both in WB and in PRP. PDGF-BB and TGF-ß1 concentrations were additionally determined in PRP. Platelet concentration increased 1.1 times in PRP fraction compared to WB, but no significant differences were reported. MPV was statistically higher in WB than in PRP (p = 0.001). Erythrocytes and leukocytes counts were decreased by 99% and 92%, respectively in the PRP fraction (p < 0.001). Regarding TGF-ß1, a higher concentration was shown in the PRP (p < 0.02). Although the product obtained could not be classified as PRP according to the PRGF®-Endoret® methodology, based on the drastic reduction of RBC and WBC, the PLT concentrate is of high purity.

Epigenetic mechanisms driving the pathogenesis of systemic lupus erythematosus, systemic sclerosis and dermatomyositis.

Autoimmune connective tissue disorders, including systemic lupus erythematosus, systemic sclerosis (SSc) and dermatomyositis (DM), often manifest with debilitating cutaneous lesions and can result in systemic organ damage that may be life-threatening. Despite recent therapeutic advancements, many patients still experience low rates of sustained remission and significant treatment toxicity. While genetic predisposition plays a role in these connective tissue disorders, the relatively low concordance rates among monozygotic twins (ranging from approximately 4% for SSc to about 11%-50% for SLE) have prompted increased scrutiny of the epigenetic factors contributing to these diseases. In this review, we explore some seminal studies and key findings to provide a comprehensive understanding of how dysregulated epigenetic mechanisms can contribute to the development of SLE, SSc and DM.

Solid-Basaloid Adenoid Cystic Carcinoma of the Breast: An Aggressive Subtype Enriched for Notch Pathway and Chromatin Modifier Mutations With MYB Overexpression.

Adenoid cystic carcinoma (AdCC) is a rare triple-negative breast cancer analogous to its extramammary counterparts. Diagnosis of the more aggressive solid-basaloid variant of AdCC (SB-AdCC) can be challenging due to poorly defined histopathologic and molecular features. We characterized 22 invasive and in situ basaloid carcinomas by morphology, immunohistochemistry, genetics, and MYB status using multiple platforms and assessed clinical behavior and neoadjuvant chemotherapy responses. After consensus review, 16/22 cases were classified as SB-AdCC. All SB-AdCC had predominantly solid growth and at least focal myxohyaline stroma and were immune-poor. Eosinophilic squamoid cells (69%, 11/16) and basement membrane-like secretions (69%, 11/16) were common, and intercalated ducts (31%, 5/16) were less frequent. SB-AdCC typically expressed SOX10 (100%, 16/16) and luminal markers (100%, 16/16 CK7; 88%, 14/16 CD117; 93%, 13/14 CAM5.2). SMA (40%, 6/15) expression was less common, and SMM (27%, 3/11), GATA3 (20%, 3/15), and p63 (25%, 4/16) were mostly negative. MYB protein and/or MYB RNA overexpression was universal in evaluable cases (13/13), with RNA in situ hybridization (10/10) more reliable than immunohistochemistry (10/11, plus 4 excisions inconclusive). Fluorescence in situ hybridization and/or next-generation sequencing identified MYB rearrangements (20%, 3/15) and amplifications/copy gains (60%, 9/15) but no MYB::NFIB fusions. SB-AdCC often had aberrations in Notch pathway (60%, including 40% NOTCH1 and 20% NOTCH2) and/or chromatin modifier (60%, including 33% CREBBP) genes, with relatively infrequent TP53 mutations (27%). Unclassified invasive basaloid carcinomas lacking described histologic features of SB-AdCC (n = 4) and basaloid ductal carcinoma in situ (n = 2) showed similar immunoprofiles and genetics as SB-AdCC, including Notch aberrations and MYB overexpression with MYB rearrangements/amplifications. Overall, nodal (22%) and distant (33%) metastases were common, and 23% of patients died of disease (mean follow-up, 35 months; n = 22). Responses were poor in all 7 neoadjuvant chemotherapy-treated patients, without any achieving pathologic complete response. The data highlight the histopathologic spectrum of basaloid carcinomas including SB-AdCC and reveal shared genetics and MYB activation, which can be diagnostically useful. Aggressive behavior and poor treatment responses emphasize a need for additional treatment approaches.

Assessing the capacity of methylated DNA markers of cervical squamous cell carcinoma to discriminate oropharyngeal squamous cell carcinoma in human papillomavirus mediated disease.

OBJECTIVE: Early identification of human papillomavirus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) is challenging and novel biomarkers are needed. We hypothesized that a panel of methylated DNA markers (MDMs) found in HPV(+) cervical squamous cell carcinoma (CSCC) will have similar discrimination in HPV(+)OPSCC tissues. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tissues were obtained from patients with primary HPV(+)OPSCC or HPV(+)CSCC; control tissues included normal oropharynx palatine tonsil (NOP) and cervix (NCS). Using a methylation-specific polymerase chain reaction, 21 previously validated cervical MDMs were evaluated on tissue-extracted DNA. Discrimination between case and control cervical and oropharynx tissue was assessed using area under the curve (AUC). RESULTS: 34 HPV(+)OPSCC, 36 HPV(+)CSCC, 26 NOP, and 24 NCS patients met inclusion criteria. Within HPV(+)CSCC, 18/21 (86%) of MDMs achieved an AUC ≥ 0.9 and all MDMs exhibited better than chance classifications relative to control cervical tissue (all p < 0.001). In contrast, within HPV(+)OPSCC only 5/21 (24%) MDMs achieved an AUC ≥ 0.90 but 19/21 (90%) exhibited better than chance classifications relative to control tonsil tissue (all p < 0.001). Overall, 13/21 MDMs had statistically significant lower AUCs in the oropharyngeal cohort compared to the cervical cohort, and only 1 MDM exhibited a statistically significant increase in AUC. CONCLUSIONS: Previously validated MDMs exhibited robust performance in independent HPV(+)CSCC patients. However, most of these MDMs exhibited higher discrimination for HPV(+)CSCC than for HPV(+)OPSCC. This suggests that each SCC subtype requires a unique set of MDMs for optimal discrimination. Future studies are necessary to establish an MDM panel for HPV(+)OPSCC.