Recent developments in mass-spectrometry-based targeted proteomics of clinical cancer biomarkers.

Routine measurement of cancer biomarkers is performed for early detection, risk classification, and treatment monitoring, among other applications, and has substantially contributed to better clinical outcomes for patients. However, there remains an unmet need for clinically validated assays of cancer protein biomarkers. Protein tumor markers are of particular interest since proteins carry out the majority of biological processes and thus dynamically reflect changes in cancer pathophysiology. Mass spectrometry-based targeted proteomics is a powerful tool for absolute peptide and protein quantification in biological matrices with numerous advantages that make it attractive for clinical applications in oncology. The use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methodologies has allowed laboratories to overcome challenges associated with immunoassays that are more widely used for tumor marker measurements. Yet, clinical implementation of targeted proteomics methodologies has so far been limited to a few cancer markers. This is due to numerous challenges associated with paucity of robust validation studies of new biomarkers and the labor-intensive and operationally complex nature of LC-MS/MS workflows. The purpose of this review is to provide an overview of targeted proteomics applications in cancer, workflows used in targeted proteomics, and requirements for clinical validation and implementation of targeted proteomics assays. We will also discuss advantages and challenges of targeted MS-based proteomics assays for clinical cancer biomarker analysis and highlight some recent developments that will positively contribute to the implementation of this technique into clinical laboratories.

Analytical Unreliability of 25 Hydroxy Vitamin D Measurements in Pre-Term Neonates.

BACKGROUND: Vitamin D supplementation is common practice for neonates and infants due to limited stores of vitamin D at birth. Although not commonly encountered, vitamin D toxicity can occur due to over-supplementation. However, toxic concentrations are often not included in method validation experiments, and assays often are not validated in the neonatal population. METHODS: We compared serial 25 hydroxy vitamin D [25(OH)D] measurements in pre-term neonates receiving 25(OH)D supplementation and identified 12 patients wherein concentrations of 25(OH)D were above 50 ng/mL (125 nM) that required additional investigations as the 25(OH)D results did not match the clinical picture. Available samples were compared across 4 immunoassay platforms (LIAISON XL, Roche Cobas e602, Abbott Alinity i, and Siemens Centaur XP) and LC-MS/MS. RESULTS: Concentrations of 25(OH)D observed on one individual immunoassay platform (LIAISON XL) fluctuated substantially between subsequent blood draws in select neonates with elevated concentrations. Serum samples from these patients showed variable agreement between LC-MS/MS and other immunoassay platforms. These fluctuations were not explained by the presence of 3-epimer-25(OH)D or 24,25(OH)2D. CONCLUSIONS: Although we were unable to identify a cause for the variable elevated results, our findings suggest that neonatal 25(OH)D measurements alone should not be used for assessment of nutritional monitoring, and that clinical correlation and other laboratory parameters including ionized calcium should be considered.

Highly sensitive tandem mass spectrometric measurement of serum estradiol without derivatization and pediatric reference intervals in children and adolescents.

OBJECTIVES: Monitoring estradiol (E2) is important for determining the onset of pubertal development as well as in the evaluation of girls with precocious puberty. However, E2 measurement remains an analytical challenge in children, who have lower circulating levels. We developed and evaluated a simple and sensitive LC-MS/MS procedure for serum E2 quantification in pediatric populations and established age- and sex-specific pediatric reference intervals. METHODS: Residual patient serum samples were used to evaluate the analytical performance of our in-house LC-MS/MS E2 assay. The evaluation included accuracy, precision, linearity, functional sensitivity (LLoQ), and method comparison. Age- and sex-specific pediatric E2 reference intervals were also established from a cohort of 405 healthy children (birth to 18 years) recruited with informed consent. Age- and sex-specific differences were assessed, and outliers were removed. Reference intervals were established using the robust method. RESULTS: The assay imprecision was <5.3 %. Assay linearity ranged from 13.7 to 1923.3 pmol/L. The LLoQ corresponding to a CV of 20 % was determined to be 8.9 pmol/L. Bland-Altman analysis revealed a mean bias of 29.3 pmol/L or 9.1 % between our LC-MS/MS E2 assay and an external reference laboratory measuring E2 by LC-MS/MS. CONCLUSIONS: Our LC-MS/MS E2 assay shows acceptable accuracy, precision, functional sensitivity (LLoQ), and linearity for E2 quantification. Our LC-MS/MS E2 assay also showed good agreement with an external reference laboratory measuring E2 by LC-MS/MS. In addition, using CALIPER samples, we established robust age- and sex-specific pediatric E2 reference intervals to improve accuracy of test result interpretation and clinical decision making.

Maternal Anti-Ro Antibody Titers Obtained With Commercially Available Immunoassays Are Strongly Associated With Immune-Mediated Fetal Heart Disease.

OBJECTIVE: Anti-Ro antibody-positive mothers are frequently referred for serial echocardiography due to the fetal risk of developing heart block and endocardial fibroelastosis. Little is known why only some and not all offspring develop these cardiac manifestations of neonatal lupus (CNL). This prospective study examined associations between anti-Ro antibody titers and fetal CNL. METHODS: Antibody-positive mothers referred since 2018 for fetal echocardiography at risk of CNL (group 1; n = 240) or with CNL (group 2; n = 18) were included. Maternal antibody titers were measured with a chemiluminescent immunoassay (CIA). Additional testing on diluted serum samples was used to quantify anti-Ro 60 antibody titers above the analytical measuring range (AMR) of the standard CIA (≥1,375 chemiluminescent units [CU]). RESULTS: Among 27 total mothers with a fetal diagnosis of CNL, all displayed anti-Ro 60 antibody titers that exceeded the AMR of the CIA at least 10-fold. Of 122 mothers in group 1 who underwent additional anti-Ro 60 antibody testing, event rates of CNL (n = 9) were 0% (0 of 45) among mothers with anti-Ro 60 antibody titers from 1,375-10,000 CU, 5% (3 of 56) among mothers with titers from 10,000-50,000 CU, but 29% (6 of 21) among mothers with titers >50,000 CU (odds ratio 13.1, P = 0.0008). Of mothers in group 2 with a primary diagnosis of CNL, 0% (0 of 18 mothers) had anti-Ro 60 antibody titers <10,000 CU, 44% (8 of 18 mothers) had titers from 10,000-50,000 CU, and 56% (10 of 18 mothers) had titers >50,000 CU. CONCLUSION: CNL is associated with substantially higher anti-Ro antibody titers than are obtained using a standard CIA. Enhancing the assay measuring range allows an improved specificity of identifying pregnancies at risk of CNL.

Determination of pediatric reference limits for 10 commonly measured autoantibodies.

OBJECTIVES: The objective of this study was to establish pediatric reference limits for autoimmune disease markers in the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) cohort of healthy children and adolescents to support their interpretation and clinical decision making. The CALIPER is a national study of healthy children aiming to close gaps in pediatric laboratory medicine by establishing a robust database of pediatric reference intervals for pediatric disease biomarkers (caliperdatabase.org). METHODS: Healthy children and adolescents (n=123, aged 1-19) were recruited to CALIPER with informed consent. Serum autoantibody testing conducted on the BIO-FLASH analyzer (Werfen, Barcelona, Spain) included anti-dsDNA IgG, anti-Sm IgG, anti-RNP IgG, anti-SSB/La IgG, anti-Ro60 IgG, anti-Ro52 IgG, anti-cardiolipin IgG, anti-MPO IgG, anti-PR3 IgG, and anti-tTG IgA. Pediatric reference limits representing 95th, 97.5th, and 99th percentiles were calculated using the non-parametric rank method according to Clinical Laboratory Standards Institute C28-A3 guidelines. RESULTS: The proportion of samples with results above the lower limit of the analytical measuring range were: anti-cardiolipin IgG 90%, anti-dsDNA 22%, anti-Sm 13%, anti-RNP 0.8%, anti-SSB/La 0%, anti-Ro60 0%, anti-Ro52 0%, anti-MPO 25%, anti-PR3 9%, and anti-tTG IgA 28%. Pediatric reference limits and associated 90% confidence intervals were established for all 10 markers. All autoantibodies could be described by one age range except for anti-cardiolipin IgG and anti-MPO. A sex-specific difference was identified for anti-tTG IgA. CONCLUSIONS: Robust pediatric reference limits for 10 commonly clinically utilized autoimmune markers established herein will allow for improved laboratory assessment and clinical decision making in pediatric patients using the BIO-FLASH assay platform worldwide.

Bias in IGF-1 concentrations and interpretation across three different clinical laboratory assays.

In this study, we compared the DiaSorin LiaisonXL IGF-1 immunoassay to both the Roche Elecsys IGF-1 immunoassay and to the liquid chromatography-high resolution mass spectrometry (LC-MS) IGF-1 assay. Our study shows a constant positive bias in DiaSorin compared to the Roche immunoassay (mean 42 µg/L, 24%), and a proportional positive bias in DiaSorin compared to the LC-MS method (mean 49 µg/L, 29%). Further, we demonstrate the potential clinical impact of this bias by evaluating 43 adult samples, collected over a 2-month period, which were shown to be discrepant based on a chart review. Despite the positive analytical bias in the Diasorin assay compared to the LC-MS assay, the Diasorin assay upper reference limits were lower than those of the LC-MS assay. This effect caused nine out of forty-three samples to show falsely elevated results when they were clinically diagnosed as negative for acromegaly. Discussed in the context of previous literature, our findings emphasize the importance of adjusting reference intervals for IGF-1 assays based on the clinical needs of a patient population.

Clinical, Methodological, and Practical Considerations for Algorithmic Testing in Autoimmune Serology.

BACKGROUND: Autoimmune serology tests are central to the classification, screening, diagnosis, and monitoring of a variety of autoimmune disorders. To improve the appropriateness of serologic evaluation and support laboratory resource utilization, reflex testing approaches have been proposed and implemented across clinical laboratories. Reflex testing involves a staged approach where an initial test result triggers subsequent tests based on prespecified rules. CONTENT: Various reflex testing approaches in the context of antinuclear antibody-associated rheumatic disease, antineutrophil cytoplasmic autoantibody-associated vasculitis, celiac disease, and myasthenia gravis are reviewed here. Clinical, analytical, and practical considerations of reflex testing implementation are addressed as well as associated limitations and challenges. SUMMARY: Serology reflex testing algorithms for the evaluation of autoimmune diseases can support clinical diagnosis and laboratory resource use but may be challenging to implement and are often applied variably across institutions. Assessments of evidence-driven guidelines, clinical impact, and impact on laboratory workflow are essential to this task.

MultiInflammatory Syndrome in Children: A View into Immune Pathogenesis from a Laboratory Perspective.

BACKGROUND: Multiinflammatory syndrome in children (MIS-C) is a novel and rare inflammatory disorder associated with severe acute respiratory syndrome coronavirus 2 infection in school-age children. Reports in the past year have suggested a multisystem pathophysiology characterized by hyperinflammation, gastrointestinal distress, and cardiovascular complications. Clinical laboratory investigations, including routine blood testing for inflammatory (e.g., C-reactive protein, ferritin) and cardiac (e.g., troponin, brain natriuretic peptides) markers have provided insight into potential drivers of disease pathogenesis, highlighting the role of the laboratory in the differential diagnosis of patients presenting with similar conditions (e.g., Kawasaki disease, macrophage activating syndrome). CONTENT: While few studies have applied high-dimensional immune profiling to further characterize underlying MIS-C pathophysiology, much remains unknown regarding predisposing risk factors, etiology, and long-term impact of disease onset. The extent of autoimmune involvement is also unclear. In the current review, we summarize and critically evaluate available literature on potential pathogenic mechanisms underlying MIS-C onset and discuss the current and anticipated value of various laboratory testing paradigms in MIS-C diagnosis and monitoring. SUMMARY: From initial reports, it is clear that MIS-C has unique inflammatory signatures involving both adaptive and innate systems. Certain cytokines, inflammatory markers, and cardiac markers assist in the differentiation of MIS-C from other hyperinflammatory conditions. However, there are still major gaps in our understanding of MIS-C pathogenesis, including T cell, B cell, and innate response. It is essential that researchers not only continue to decipher initial pathogenesis but also monitor long-term health outcomes, particularly given observed presence of circulating autoantibodies with unknown impact.

AACC Practical Recommendations for Implementing and Interpreting SARS-CoV-2 Emergency Use Authorization and Laboratory-Developed Test Serologic Testing in Clinical Laboratories.

BACKGROUND: The clinical laboratory continues to play a critical role in managing the coronavirus pandemic. Numerous US Food and Drug Administration emergency use authorization (EUA) and laboratory-developed test (LDT) serologic assays have become available. The performance characteristics of these assays and their clinical utility continue to be defined in real time during this pandemic. The AACC convened a panel of experts from clinical chemistry, microbiology, and immunology laboratories; the in vitro diagnostics industry; and regulatory agencies to provide practical recommendations for implementation and interpretation of these serologic tests in clinical laboratories. CONTENT: The currently available EUA serologic tests and platforms, information on assay design, antibody classes including neutralizing antibodies, and the humoral immune responses to SARS-CoV-2 are discussed. Verification and validation of EUA and LDT assays are described, along with a quality management approach. Four indications for serologic testing are outlined. Recommendations for result interpretation, reporting comments, and the role of orthogonal testing are also presented. SUMMARY: This document aims to provide a comprehensive reference for laboratory professionals and healthcare workers to appropriately implement SARS-CoV-2 serologic assays in the clinical laboratory and to interpret test results during this pandemic. Given the more frequent occurrence of outbreaks associated with either vector-borne or respiratory pathogens, this document will be a useful resource in planning for similar scenarios in the future.

Complex biological patterns of soluble cytokines and CD163 in childhood necessitating age-specific reference intervals for evidence-based clinical interpretation.

BACKGROUND: Cytokine measurements to support clinical laboratory and research investigations have become increasingly common in pediatrics. However, there is a paucity of accurate pediatric reference intervals (RIs) essential to the interpretation of cytokine results. To address this gap, here, we establish age- and sex-specific pediatric reference values for clinically relevant inflammatory markers including CD163, and the cytokines IL-1ß, IL-6, IL-10, IL-18, TNF-α, IFN-γ, and CXCL-9. METHODS: Healthy children and adolescents (n = 311, 1-19 years) were recruited as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) study. Multi-analyte measurements in plasma and analytical performance verification were conducted on the ProteinSimple® Ella™ automated immunoassay platform (Bio-Techne, MN, USA). Age- and sex-specific RIs were calculated based on Clinical and Laboratory Standards Institute guidelines. Additionally, 75th and 95th percentile cut-offs were determined. RESULTS: Three types of reference value distributions were observed: (a) consistent levels throughout age and sex: IL-6, and IFN-γ, (b) gradual decline in concentration with age: CD163, TNF-α, CXCL-9, and IL-10, (c) significantly higher concentrations during ages 4-14 years than earlier and later ages: IL-1ß and IL-18. Reference values for CXCL-9, IL-10, and TNF-α under 8 years of age differed significantly from older children. CD163, IL-18 and IL-1ß required three age partitions. CD163 demonstrated significant sex differences in ages 8-13 years. CONCLUSION: The circulating profile of cytokines in children is complex and can vary by age and sex. This necessitates careful interpretation of test results based on age and/or sex specific RIs facilitating more accurate clinical decision making.

The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Routine T4 No More? Reducing Excess Thyroid Hormone Testing at a Pediatric Tertiary Care Hospital.

OBJECTIVES: To promote resource stewardship in thyroid hormone testing at a pediatric tertiary care hospital. STUDY DESIGN: Quality improvement approaches generated 3 change ideas that were implemented simultaneously in the hospital electronic medical record: (1) a reflex free thyroxine (fT4), whereby fT4 is automatically reported if the thyroid-stimulating hormone is outside the normal range; (2) a forced-function for thyroid hormone ordering, whereby a provider must select an appropriate indication for ordering fT4 or triiodothyronine (T3); and (3) a clinical decision support message displayed at the time of ordering thyroid function tests. Laboratory data were audited to determine the mean number of fT4 and T3 tests performed per week as well as indications for testing. RESULTS: The mean number of fT4 and T3 tests processed per week decreased from 154 ± 21 and 11 ± 7, respectively, in the preintervention period, to 107 ± 12 (30% reduction) and 4 ± 3 (66% reduction) postintervention. These reductions were sustained for the full 20-week assessment period. Process and balancing measures revealed no unintended adverse consequences. Approximate cost savings were $43 000 per year. CONCLUSIONS: We describe the successful implementation of electronic medical record-based interventions (reflex fT4, forced-function selection of indication, decision support text) leading to sustained improvements in healthcare use, with significant associated cost-savings.

Measuring Gastric pH in Tube-fed Children With Neurologic Impairments and Gastroesophageal Disease.

OBJECTIVES: The aim of the study was to determine the extent of agreement between pH paper and handheld pH meter with a laboratory pH meter for gastric pH measurement in children with neurologic impairments and gastrostomy tubes who have gastroesophageal reflux disease (GERD). METHODS: In this prospective observational study, gastric contents were aspirated from gastric or nasogastric tubes and the pH measured using 3 techniques: pH paper, handheld pH meter, and laboratory pH meter (the gold standard). Agreement between techniques was assessed with intraclass correlation coefficient (ICC), Bland-Altman analysis, and kappa statistic. RESULTS: Among 43 patients contributing 67 gastric samples, the ICC was 0.75 (95% confidence interval [CI]: 0.69-0.97) between the handheld and laboratory meters, 0.69 (95% CI: 0.63--0.94) between the pH paper and laboratory meter and 0.69 (95% CI: 0.63-0.94) between the handheld meter and paper. The Bland-Altman analysis between the handheld and lab meters showed a mean difference of -0.03 pH units (limits of agreement: -0.52 to 0.47 pH units) and 0.17 pH units (limits of agreement: -0.99 to 1.33 pH units) between the paper and lab meter. The kappa coefficients for a pH ≥4 were 1.0 (95% CI: 1.0--1.0) between the handheld and lab meters and 0.9 (95% CI: 0.77--1.0) between the paper and lab meter. CONCLUSIONS: The findings suggest that both point-of-care tests, the pH meter and pH paper, correlate well with the gold standard for testing pH with a laboratory pH meter, indicating usefulness in point-of-care testing for monitoring gastric pH in tube-fed children with neurologic impairments and GERD.

Pathophysiology of COVID-19: Mechanisms Underlying Disease Severity and Progression.

The global epidemiology of coronavirus disease 2019 (COVID-19) suggests a wide spectrum of clinical severity, ranging from asymptomatic to fatal. Although the clinical and laboratory characteristics of COVID-19 patients have been well characterized, the pathophysiological mechanisms underlying disease severity and progression remain unclear. This review highlights key mechanisms that have been proposed to contribute to COVID-19 progression from viral entry to multisystem organ failure, as well as the central role of the immune response in successful viral clearance or progression to death.

Screening for Nonclassic Congenital Adrenal Hyperplasia in the Era of Liquid Chromatography-Tandem Mass Spectrometry.

CONTEXT: Screening for and diagnosing non classic congenital adrenal hyperplasia (NCCAH) uses serum 17-hydroxyprogesterone (17OHP) thresholds established from immunoassay data; however, a new liquid-chromatography tandem mass spectrometry (LC-MS/MS) method results in lower 17OHP values. The evolution of immunoassays is also challenging our diagnostic cut-off for glucocorticoid insufficiency and few data re-evaluate the utility of testing for glucocorticoid insufficiency in NCCAH. OBJECTIVE: (1) Evaluate the 17OHP threshold that predicts NCCAH in children using LC-MS/MS, and (2) determine the prevalence of glucocorticoid insufficiency in NCCAH. METHODS: A retrospective chart review of pediatric patients who underwent ACTH stimulation tests with cortisol and 17OHP measurements from 2011 to 2018 for assessment of NCCAH. Other adrenal pathologies were excluded. A cortisol < 415 nmol/L defined glucocorticoid insufficiency. Published correlation data determined a 17OHP of 3.3 nmol/L by LC-MS/MS was equivalent to 6 nmol/L by immunoassay. Data analysis was by measures of diagnostic accuracy. RESULTS: Of 188 patients included, 23 (12%) had NCCAH (21/23 had genetic confirmation); the remaining 2 had peak 17OHP > 30 nmol/L. Baseline 17OHP ≥ 6 nmol/L most accurately screened for NCCAH-sensitivity and specificity 96%. Almost all genetically confirmed NCCAH (20/21) had peak 17OHP > 30 nmol/L; all subjects with other diagnoses peaked < 30 nmol/L. Glucocorticoid insufficiency was present in 55% with NCCAH. CONCLUSIONS: Despite the increased specificity of LC-MS/MS, a baseline 17OHP ≥ 6 nmol/L most accurately screened for NCCAH; this supports current practice guidelines. This threshold identified all with glucocorticoid insufficiency, notably prevalent in our cohort and for whom glucocorticoid stress dosing should be considered.

Direct Detection of Monoclonal Free Light Chains in Serum by Use of Immunoenrichment-Coupled MALDI-TOF Mass Spectrometry.

BACKGROUND: Free light chain (FLC) quantification is the most analytically sensitive blood-based method commercially available to diagnose and monitor patients with plasma cell disorders (PCDs). However, instead of directly detecting monoclonal FLCs (mFLCs), FLC assays indirectly assess clonality based on quantifying κ and λ FLCs and determination of the к/λ FLC ratio. Often an abnormal FLC ratio is the only indication of a PCD, and confirmation by a direct method increases diagnostic confidence. The aim of this study was to develop an analytically sensitive method for direct detection of mFLCs. METHODS: Patient sera (n = 167) previously assessed by nephelometric FLC quantification and immunofixation electrophoresis (IFE) were affinity enriched for IgG, IgA, and total and free κ and λ light chains and subjected to MALDI-TOF MS. Relative analytical sensitivity of these methods was determined using serially diluted sera containing mFLCs. RESULTS: In sera with abnormal FLC ratios (n = 127), 43% of monoclonal proteins were confirmed by IFE, 57% by MALDI-TOF MS without FLC enrichment, and 87% with FLC enrichment MALDI-TOF MS. In sera with normal FLC ratios (n = 40), the FLC MALDI-TOF MS method identified 1 patient with an mFLC. Serial dilution and analysis of mFLC containing sera by IFE, nephelometry, and FLC MALDI-TOF MS demonstrated that FLC MALDI-TOF MS analysis had the highest analytical sensitivity. CONCLUSIONS: FLC immunoenrichment coupled to MALDI-TOF MS enables direct detection of mFLCs and significantly increases the confirmation of abnormal serum FLC ratios over IFE and MALDI-TOF MS without FLC enrichment, thereby providing added confidence for diagnosing FLC PCDs.

Novel insights into head and neck cancer using next-generation "omic" technologies.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous disease that develops via one of the two primary carcinogenic routes: chemical carcinogenesis through exposure to tobacco and alcohol or virally induced tumorigenesis. Human papillomavirus (HPV)-positive (HPV(+)) and HPV-negative (HPV(-)) HNSCCs represent distinct clinical entities, with the latter associated with significantly inferior outcome. The biologic basis of these different outcomes is an area of intense investigation; their therapeutic regimens are currently also being reevaluated, which would be significantly facilitated by reliable biomarkers for stratification. With the advent of the omics era and accelerated development of targeted therapies, there are unprecedented opportunities to address the challenges in the management of HNSCC. As summarized herein, side-by-side molecular characterization of HPV(+) versus HPV(-) HNSCC has revealed distinct molecular landscapes, novel prognostic signatures, and potentially targetable biologic pathways. In particular, we focus on the evidence acquired from genome-wide omics pertinent to our understanding of the clinical behavior of HNSCC and on insights into personalized treatment opportunities. Integrating, mining, and validating these data toward clinically meaningful outcomes for patients with HNSCC in conjunction with systematic verification of the functional relevance of these findings are critical steps toward the design of personalized therapies.

Integrated omic analysis of oropharyngeal carcinomas reveals human papillomavirus (HPV)-dependent regulation of the activator protein 1 (AP-1) pathway.

HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcomes relative to HPV-negative patients, but the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n = 27) and HPV-negative (n = 26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis, and immune response. The differential abundances of cortactin and methylthioadenosine phosphorylase were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p = 0.023 and p = 0.009, respectively). An additional 1,124 proteins were independently corroborated through comparison to a published proteomic dataset of OPC. Furthermore, utilizing the Cancer Genome Atlas, we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundances to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs. These proteins might contribute to reduced survival in these patients via their established role in radiation resistance. Through interrogation of Cancer Genome Atlas data, we demonstrated that activation of the ß1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of activator protein 1 transcription factor target genes are plausible consequences of elevated cortactin protein levels.